V>EPA  Method 1694: Pharmaceuticals and
        Personal Care Products in Water,
        Soil, Sediment, and Biosolids by
        HPLC/MS/MS

        December 2007

-------
December 2007                                                Method 1694
                U.S. Environmental Protection Agency
                           Office of Water
                  Office of Science and Technology
               Engineering and Analysis Division (4303T)
                   1200 Pennsylvania Avenue, NW
                       Washington, DC 20460
                         EPA-821-R-08-002
                          December 2007

-------
December 2007                                                                  Method 1694

                                      Introduction

EPA Method 1694 determines pharmaceuticals and personal care products (PPCPs) in environmental
samples by high performance liquid chromatography combined with tandem mass spectrometry
(HPLC/MS/MS) using isotope dilution and internal standard quantitation techniques. This method has
been developed for use with aqueous, solid, and biosolids matrices.
                                       Disclaimer

This method has been reviewed by the Engineering and Analytical Support Branch of the Engineering
and Analysis Division (EAD) in OST. The method is available for general use, but has not been
published in 40 CFR Part 136. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
                                        Contacts

Questions concerning this method or its application should be addressed to:
Brian Englert, Ph.D.
Environmental Scientist
Engineering & Analytical Support Branch
Engineering and Analysis Division (4303T)
Office of Science and Technology, Office of Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue NW
Washington, DC 20460
http: //www .epa. gov/waterscience
ostcwamethods(@,epa. gov

-------
December 2007                                                     Method 1694

                            Table of Contents
INTRODUCTION	Ill
DISCLAIMER	Ill
1.0    SCOPE AND APPLICATION	1
2.0    SUMMARY OF METHOD	2
3.0    DEFINITIONS AND UNITS OF MEASURE	3
4.0    INTERFERENCES	3
5.0    SAFETY	5
6.0    EQUIPMENT AND SUPPLIES	6
7.0    REAGENTS AND STANDARDS	10
8.0    SAMPLE COLLECTION, PRESERVATION, STORAGE, AND HOLDING TIMES	14
9.0    QUALITY ASSURANCE/QUALITY CONTROL	15
10.0   CALIBRATION AND STANDARDIZATION	19
11.0   SAMPLE PREPARATION	23
12.0   EXTRACTION AND CONCENTRATION	28
13.0   EXTRACT CLEANUP	32
14.0   LC/MS/MS ANALYSIS	32
15.0   SYSTEM AND LABORATORY PERFORMANCE	33
16.0   QUALITATIVE DETERMINATION	34
17.0   QUANTITATIVE DETERMINATION	35
18.0   ANALYSIS OF COMPLEX SAMPLES	37
19.0   POLLUTION PREVENTION	38
20.0   WASTE MANAGEMENT	39
21.0   METHOD PERFORMANCE	39
22.0   REFERENCES	39
23.0   TABLES AND FLOWCHART	42
                                                                         IV

-------
December 2007                                                         Method 1694




24.0   GLOSSARY	69

-------
December 2007                                                                 Method 1694

                                     Method 1694
       Pharmaceuticals and  Personal Care Products in Water, Soil,
                   Sediment, and Biosolids by  HPLC/MS/MS


1.0   Scope and Application

       1.1     Method 1694 is for determination of pharmaceuticals and personal care products (PPCPs)
              in multi-media environmental samples by high performance liquid chromatography
              combined with tandem mass spectrometry (HPLC/MS/MS).

       1.2    This method was developed for use in Clean Water Act (CWA) programs; other
              applications are possible. It is based on existing EPA methods (Reference 1) and
              procedures developed at Axys Analytical Services (Reference 2) as well as previous work
              on pharmaceuticals and personal care products (Reference 3).

       1.3    The target analytes and their corresponding Chemical Abstracts Service Registry
              Numbers (CASRNs) are listed in Table 1.

       1.4    The detection limits and quantitation levels in this method are usually dependent on the
              level of interferences rather than instrumental limitations. The method detection limits
              (MDLs; 40 CFR 136, appendix B) and minimum levels of quantitation (MLs; 68 FR
              11790) in Tables 3, 5, 7, and 9 are the levels at which the analytes can be determined in
              the absence of interferences.

       1.5    This method is restricted to use by or under the  supervision of analysts experienced in
              LC/MS/MS or under the close supervision of such qualified persons. Each laboratory
              that uses this method must demonstrate the ability to generate acceptable results using  the
              procedure in Section 9.2.

       1.6    This method is performance-based which means that you may modify the method to
              improve performance (e.g., to overcome interferences or improve the accuracy or
              precision of the results) provided that you meet all performance requirements in this
              method.  These  requirements for establishing equivalency of a modification are in
              Section 9.1.2. For Clean Water Act (CWA) uses, additional flexibility is described at 40
              CFR 136.6. Modifications that are not within the scope of Part 136.6, or in  Section 9 of
              this method may require prior review and approval.

       1.7    Some of the compounds in this method are controlled substances. Laboratories
              performing this method should have all appropriate licenses and certifications and obtain
              all needed standards and chemicals from licensed sources. For some of the compounds in
              this method it may be necessary for laboratories to obtain a DEA license.

-------
December 2007                                                                      Method 1694

2.0    Summary of Method

        The target analytes in this method are divided into four groups (1 through 4). Each group represents
        an LC/MS/MS run, as detailed in Tables 2 to 9 in Section 23. Tables 2 and 3 are specific to Group
        1. Tables 4 and 5 are specific to Group 2. Tables 6 and 7 are specific to Group 3.  Tables 8 and 9
        are specific to Group 4.

        Groups 1, 2, and 3 are extracted under acidic (pH 2) conditions.  Groups 1 and 2 are run in the
        positive electrospray ionization (ESI+) mode and Group 3 is run in the negative electrospray
        ionization (ESI-) mode. Group 4 is extracted under basic (pH 10) conditions and is run in the ESI+
        mode. Group 3 is specific to the tetracyclines.

        The general steps in this method are summarized in Section 2.1 to 2.7. A flow chart that
        summarizes procedures for sample preparation, cleanup, and analysis is shown in Figure 1.

        2.1     Aqueous samples absent visible particles and filtrate from samples with visible particles -
               The pH of a 1-L sample aliquot is adjusted to 2 with acid. The pH of a second 1-L
               aliquot of sample is adjusted with 10 with base.  Stable, isotopically labeled analogs of
               the analytes of interest are spiked into their respective acid or base fraction. The acid
               fraction is stabilized with tetrasodium ethylenediamine-tetraacetate dihydrate
               (NA4EDTA.2H2O2H2O).

        2.2     Solid and semi-solid samples, including biosolids and visible particles from aqueous
               samples - A phosphate buffer and an ammonium hydroxide solution are used to adjust
               the pH, respectively, of up to 1 g each of dry solids from a solid sample, or 1 g each of
               dry solids filtered from an aqueous sample. The labeled compounds are spiked into their
               respective acid and base fractions.  The acid fraction is ultrasonically extracted three
               times with a phosphate buffer/acetonitrile solution and the base fraction is ultrasonically
               extracted three times with a ammonium hydroxide/acetonitrile solution.  The solutions
               are concentrated to remove the acetonitrile and diluted with reagent water.  The acid
               fraction is stabilized with NA4EDTA.2H2O2H2O.

        2.3     Sample cleanup - The acid and base fraction solutions are separately cleaned up using
               solid-phase extraction (SPE) with hydrophilic-lipophillic balance (HLB) cartridges.
               After cleanup, the fractions are exchanged to methanol, labeled injection internal
               standards are added, and the final volume is adjusted to 4 mL with the LC elution solvent.

        2.4     Determination by LC/MS/MS - The acid extract is analyzed in two positive electrospray
               ionization (ESI+) LC/MS/MS runs and one negative electrospray ionization (ESI-) run,
               each specific to a subset of the analytes of interest. The base extract is analyzed in a
               single ESI+ run.  The analytes are separated by the LC and detected by a tandem (1000
               resolution) mass  spectrometer.  A daughter m/z for each compound is monitored
               throughout a pre-determined retention time window.

        2.5     An individual compound is identified by comparing the LC retention time and presence
               of the daughter m/z with the corresponding retention time and daughter m/z of an
               authentic standard.

-------
December 2007                                                                     Method 1694

       2.6     Quantitative analysis is performed in one of two ways, using selected ion current profile
               (SICP) areas:

               2.6.1   For a compound for which a labeled analog is available, the concentration is
                      determined using the isotope dilution technique and a multipoint calibration of all
                      the target analytes. Isotope dilution provides automatic correction of the target
                      analyte concentrations.

               2.6.2   For a compound for which a labeled analog is not available, the concentration is
                      determined using the internal standard technique and a multipoint calibration of
                      all the target analytes.  The labeled compounds are used to recovery correct
                      results of those analytes quantitated by the internal standard technique.

               2.6.3   Additional labeled compounds may be incorporated into this method, at the
                      user's discretion to determine the concentration of the native compound using the
                      isotope dilution technique provided that all performance requirements in this
                      method are met.  Requirements for establishing equivalency are given in Section
                      9.1.2, and additionally for CWA uses, at 40 CFR 136.6.

       2.7     The quality of the analysis is assured through reproducible calibration and testing of the
               extraction, cleanup, and LC/MS/MS systems.
3.0    Definitions and Units of Measure

        Definitions and units of measure are given in the glossary at the end of this method.


4.0    Interferences

        4.1     Solvents, reagents, glassware, and other sample processing hardware may yield artifacts,
               elevated baselines, matrix enhancement or matrix suppression causing misinterpretation
               of chromatograms.  Specific selection of reagents and purification of solvents by
               distillation in all-glass systems may be required. Where possible, reagents are cleaned by
               extraction or solvent rinse.

        4.2     Proper cleaning of glassware is extremely important, because glassware may not only
               contaminate the samples but may also remove the analytes of interest by adsorption on
               the glass surface.

               4.2.1   Glassware should be rinsed with solvent and washed with a detergent solution as
                      soon after use as is practical. Sonication of glassware containing a detergent
                      solution for approximately 30 seconds may aid in cleaning. Glassware with
                      removable parts, particularly separately funnels with fluoropolymer stopcocks,
                      must be disassembled prior to detergent washing.

               4.2.2   After detergent washing, glassware should be rinsed immediately, first with
                      methanol, then with hot tap water. The tap water rinse is followed by another

-------
December 2007                                                                      Method 1694

                      methanol rinse, then acetone, and then methylene chloride.

               4.2.3  Baking of glassware in a kiln or other high temperature furnace (300 - 500 EC)
                      may be useful after particularly dirty samples are encountered. The kiln or
                      furnace should be vented to prevent laboratory contamination by vapors. Baking
                      should be minimized, as repeated baking of glassware may cause active sites on
                      the glass surface that may irreversibly adsorb the compounds of interest.
                      Volumetric ware should not be baked at high temperature.

               4.2.4  After drying and cooling, glassware should be sealed and stored in a clean
                      environment to prevent any accumulation of dust or other contaminants. Store
                      inverted or capped with solvent rinsed aluminum foil.

       4.3     All materials used in the analysis must be demonstrated to be free from interferences by
               running reference matrix method blanks (Section 9.5) initially and with each sample
               batch (samples started through the extraction process on a given 12-hour shift, to a
               maximum of 20 samples).

               4.3.1  The reference matrix must simulate, as closely as possible, the sample matrix
                      under test.  Ideally, the reference matrix should not contain the analytes of
                      interest in detectable amounts, but should contain potential interferents in the
                      concentrations expected to be found in the samples to be analyzed.

               4.3.2  When a reference matrix that simulates the sample matrix under test is not
                      available, reagent water (Section 7.6.1) can be used to simulate water samples;
                      playground sand (Section 7.6.2) can be used to simulate soils; and peat moss
                      (Section 7.6.3) can be used to simulate biosolids.

       4.4     Interferences co-extracted from samples will vary considerably from source to source,
               depending on the diversity of the site being sampled. Interfering compounds may be
               present at concentrations several orders of magnitude higher than the analytes of interest.
               Because low levels of PPCPs are measured by this method, elimination of interferences is
               essential. The cleanup steps given in Section 13 can be used to reduce or eliminate these
               interferences and thereby permit reliable determination of the PPCPs at the levels shown
               in Tables 3, 5, 7, and  9.

       4.5     It may  be useful to number reusable glassware  is to associate that glassware with the
               processing of a particular sample. This will assist the laboratory in tracking possible
               sources of contamination for individual samples, identifying glassware associated with
               highly  contaminated samples that may require extra cleaning, and determining when
               glassware should be discarded.

       4.6     Contamination from personal care products used by laboratory staff that are also target
               analytes is possible. Target analytes also include commonly used medications.
               Therefore, it is important to take precautions to avoid contamination of the samples, for
               example wearing of protective gloves and clothing (see Section 5).

-------
December 2007                                                                      Method 1694
5.0    Safety
       The target analytes in this method have many beneficial uses as pharmaceuticals or over-the-
       counter products. While their safety is less of a concern than for many environmental
       contaminants, laboratory staff should avoid direct contact with samples and pure standards.
       General guidelines are provided below.

       5.1     The toxicity or carcinogenicity of each chemical used in this method has not been
               precisely determined; however, each compound should be treated as a potential health
               hazard.  Pure standards of the compounds should be handled only by highly trained
               personnel thoroughly familiar with handling and cautionary procedures and the
               associated risks. It is recommended that the laboratory purchase dilute standard solutions
               of the analytes in this method. However, if primary solutions are prepared, they should
               be prepared in a hood, and aNIOSH/MESA approved toxic gas respirator may be
               necessary when high concentrations are handled

       5.2     This method does not address all safety issues associated with its use.  The laboratory is
               responsible for maintaining a current awareness file of OSHA regulations regarding the
               safe handling of the chemicals specified in this method. A reference file of material
               safety data sheets (MSDSs) should also be made available to all personnel  involved in
               these analyses.  It is also suggested that the laboratory perform personal hygiene
               monitoring of each analyst who uses this method and that the results of this monitoring
               be made available to the  analyst. Additional information on laboratory safety can be
               found in References 4-7. The references and bibliography at the end of Reference 6 are
               particularly comprehensive in dealing with the general subject of laboratory safety.

       5.3     The pure PPCPs and samples suspected to contain high concentrations of these
               compounds should be handled with care.

               5.3.1   Facility - When  finely divided samples (dusts, soils, dry chemicals) are handled,
                      all operations (including removal of samples from sample containers, weighing,
                      transferring, and mixing) should be performed in a glove box demonstrated to be
                      leak tight or in a fume hood demonstrated to have adequate air flow. Gross
                      losses to the laboratory ventilation system must not be allowed.  Handling of the
                      dilute solutions normally used in analytical and animal work presents no
                      inhalation hazards except in the case of an accident.

               5.3.2  Protective equipment - Disposable plastic gloves (Latex or non-Latex (such as
                      nitrile)), apron or lab coat, safety glasses or mask, and a glove box or fume hood
                      should be used.  During analytical operations that may give rise to aerosols or
                      dusts, personnel  should wear respirators equipped with activated carbon filters.
                      Eye protection (preferably full face shields) should be worn while working with
                      exposed samples or pure analytical standards.  Latex or non-Latex (such as
                      nitrile) gloves  are commonly used to reduce exposure of the hands.

               5.3.3  Training - Workers must be trained in the proper method of removing
                      contaminated gloves  and clothing without contacting the exterior surfaces.

-------
December 2007                                                                    Method 1694

               5.3.4   Personal hygiene - Hands and forearms should be washed thoroughly after each
                      operation involving high concentrations of the analytes of interest, and before
                      breaks (coffee, lunch, and shift).

               5.3.5   Confinement - Isolated work areas posted with signs, segregated glassware and
                      tools, and plastic absorbent paper on bench tops will aid in confining
                      contamination.

               5.3.6   Waste handling - Good technique includes minimizing contaminated waste.
                      Plastic bag liners should be used in waste cans.  Janitors and other personnel
                      should be trained in the safe handling of waste.  See Section 20 for additional
                      information on waste handling and disposal.

       5.4     Biosolids samples may contain high concentrations of biohazards, and must
               be handled with gloves and opened in a hood or biological safety cabinet to
               prevent exposure. Laboratory staff should know and observe the safety
               procedures required in a microbiology laboratory that handles pathogenic
               organisms when handling biosolids samples.

6.0   Equipment and  Supplies

    Note: Brand names, suppliers, and part numbers are cited for illustration purposes only.  No
    endorsement is implied.  Equivalent performance may be achieved using equipment and materials
    other than those specified here. Demonstration of equivalent performance that meets the
    requirements of this method is the  responsibility of the laboratory.

       6.1     Sample bottles and caps

               6.1.1     Liquid samples (waters, sludges and similar materials containing 5 percent
                        solids or less) - Sample bottle, amber glass, 1 L minimum, with screw cap.

               6.1.2     Solid samples (soil, sediment, sludge, filter cake, compost, and similar
                        materials that contain more than 5  percent solids) - Sample bottle, wide mouth,
                        amber glass, 500-mL minimum.

               6.1.3     If amber bottles are not available, samples must be protected from light.

               6.1.4     Bottle caps-Threaded to fit sample bottles. Caps must be lined with
                        fluoropolymer.

               6.1.5     Cleaning - Bottles  are washed with detergent and water, then solvent rinsed
                        before use. Liners  are washed with detergent and water and rinsed with reagent
                        water before use.

       6.2     Compositing equipment - Automatic or manual compositing system incorporating glass
               containers cleaned per bottle cleaning procedure above. Only glass or fluoropolymer
               tubing must be used. If the sampler uses a peristaltic pump, a minimum length of
               compressible silicone rubber tubing may be used in the pump only.  Before use, the tubing

-------
December 2007                                                                     Method 1694

               must be thoroughly rinsed with methanol, followed by repeated rinsing with reagent water
               to minimize sample contamination. An integrating flow meter is used to collect
               proportional composite samples.

        6.3     Equipment for sample preparation

               6.3.1   Laboratory fume hood of sufficient size to contain the sample preparation
                      equipment listed below.

               6.3.2  Glove box (optional)

               6.3.3  Tissue homogenizer - VirTis Model 45 Macro homogenizer (American Scientific
                      Products H-3515, or equivalent) with stainless steel Macro-shaft and Turbo-shear
                      blade.

               6.3.4  Vortex mixer

               6.3.5  Ultrasonic mixer

               6.3.6  Oven - Capable of maintaining a temperature of 110V5 C

               6.3.7  Desiccator

               6.3.8  Balance, analytical - Capable of weighing 0.1 mg

               6.3.9  Balance, top loading - Capable of weighing 10 mg

        6.4     Apparatus for measuring pH

               6.4.1   pH meter, with combination glass electrode

               6.4.2  pH paper, wide range (Hydrion Papers, or equivalent)

        6.5     Apparatus for ultrasonic and solid-phase extraction

               6.5.1     Sonic disrupter - 375 watt with pulsing capability and 1A or % in. disrupter horn
                        (Ultrasonics, Inc., Model 375, or equivalent)

               6.5.2    Sonabox (or equivalent), for use with disrupter.

               6.5.3    Vac-Elute Manifold (Analytichem International, or equivalent)

               6.5.4    Vacuum trap:  Made from 500-mL sidearm flask fitted with single-hole rubber
                        stopper and glass tubing.

               6.5.5    Vacuum source - Capable of maintaining 25 in. Hg, equipped with shutoff valve
                        and vacuum gauge.

               6.5.6    Rack for holding 50-mL volumetric flasks in the manifold.

-------
December 2007                                                                      Method 1694
               6.5.7    SPE cartridge - Hydrophilic-Lipophilic-Balance (HLB) 60 mg, Waters Oasis, 20
                        cc/1 g LP, 60 jam, or equivalent, calibrated per the procedure in Section 10.6.

        6.6    Filtration apparatus

               6.6.1    Vacuum filtration apparatus - 1-L , including glass runnel, frit support, clamp,
                        adapter, stopper, filtration flask, and vacuum tubing. For wastewater samples,
                        the apparatus should accept 90- or 144-mm disks.

               6.6.2    Glass-fiber filter - Whatman GMF 150 (or equivalent),  1 micron pore size, to fit
                        the vacuum filtration apparatus.

               6.6.3    Pressure filtration apparatus - Millipore YT30 142 FiW, or equivalent.

               6.6.4    Whatman GF/A (1.6 jam), or equivalent, differing diameters, to fit the pressure
                        filtration apparatus.

               6.6.5    Millipore, 0.2 jam, or equivalent to fit the pressure filtration apparatus.

        6.7    Centrifuge - Capable of rotating 500-mL centrifuge bottles or 50-mL centrifuge tubes at
               5,000 rpm minimum, equipped with 500-mL centrifuge bottles (glass or polypropylene
               bottles) with screw-caps, and 50-mL centrifuge tubes with screw-caps, to fit centrifuge.

        6.8    Pipet apparatus and pipets

               6.8.1  Pipetter - variable volume

               6.8.2  Pipet tips, disposable polypropylene, sizes from 1-10 joL to 5 mL

               6.8.3  Disposable, Pasteur, 150-mm long x 5-mm ID (Fisher Scientific 13-678-6A, or
                      equivalent)

               6.8.4  Disposable, serological,  50-mL (8- to 10- mm ID)

        6.9    Rotary evaporator - Buchi/Brinkman-American Scientific No. E5045-10, or equivalent,
               equipped with a variable temperature water bath and a vacuum source with shutoff valve at
               the evaporator and vacuum gauge. A recirculating water pump and chiller are
               recommended, as use of tap water for cooling the evaporator wastes large volumes of water
               and can lead to inconsistent performance as water temperatures and pressures vary.

               6.9.1  Round-bottom flask - 100-mL and 500-mL or larger, with ground-glass fitting
                      compatible with the rotary evaporator

               6.9.2  Boiling chips

                      6.9.2.1   Glass or silicon carbide - Approximately  10/40 mesh, extracted with
                                methylene chloride and baked at 450 EC for one hour minimum

-------
December 2007                                                                     Method 1694
                      6.9.2.2  Fluoropolymer (optional) - Extracted with methylene chloride

       6.10   Water bath - Heated, with concentric ring cover, capable of maintaining a temperature
               within V 2 EC, installed in a fume hood.

       6.11    Nitrogen evaporation apparatus - Equipped with water bath controlled in the range of 30 -
               60 EC (N-Evap, Organomation Associates, Inc., South Berlin, MA, or equivalent), installed
               in a fume hood.

       6.12   Amber glass vials, 2- to 5-mL with fluoropolymer-lined screw-cap

       6.13   Clear glass vials, 0.3-mL, conical, with fluoropolymer-lined screw or crimp cap

       6.14   HPLC/MS/MS System

               6.14.1 HPLC  system with high pressure inlet, multi-segment gradient capability, and
                      post-column pump for admission of calibrant. The  system must be able to
                      produce the LC separations for the analytical runs detailed in Tables 3, 5, 7, and
                      9 under the instrument conditions detailed in Tables 2, 4, 6, and 8, and must meet
                      other HPLC requirements in this method (Waters 2690, 2795, or equivalent).

               6.14.2 LC columns

                      6.14.2.1 Cis - 10.0 cm, 2.1 mm i.d., 3.5 :m particle size (Waters Xtera C18MS,
                               or equivalent)

                      6.14.2.2 Hydrophilic - 10 cm. 2.1 mm i.d., 3.0 jam  particle size (Waters Atlantis
                               HILIC, or equivalent)

                      6.14.2.3 Alternative columns other than described above have not been tested and
                               are not allowed for this method. EPA may establish criteria for
                               equivalency in later versions of this method.

               6.14.3 MS/MS system

                      6.14.3.1 Tandem MS with the necessary pumps, collision cell, makeup gases,
                               high vacuum system, and  capability for positive and negative ion
                               electrospray ionization (ESI) of the effluent from the HPLC. (Waters
                               Quattro Ultima triple quadrupole MS, or equivalent).  The system must
                               be able to produce parent-daughter transitions for the groups of
                               compounds in the acid and base fractions of the PPCPs for the
                               analytical runs detailed in Tables 3,5,7, and 9.

                      6.14.3.2 Instrument control and data system - Interfaced to the HPLC and
                               MS/MS to control the LC gradient and other LC and MS/MS operating
                               conditions, and to acquire, store, and reduce LC/MS/MS data.  The data
                               system must be able to identify a compound by retention time and
                               parent-daughter m/zs, and  quantify the compound using linear or

-------
December 2007                                                                     Method 1694

                               quadratic multi-point relative responses and response factors by isotope
                               dilution and internal standard techniques.

       6.15   Miscellaneous labware - Beakers, 400- to 500-mL; Erlenmeyer flasks; volumetric flasks;
              pipets; syringes; stainless steel spatulas; etc.
7.0   Reagents and Standards

    Note: All reagents are ACS Reagent Grade unless specified otherwise.

       7.1     pH adjustment and solution stabilization

               7.1.1   Potassium hydroxide - Dissolve 20 g reagent grade KOH in 100 mL reagent water.

               7.1.2   Sulfuric acid - Reagent grade (specific gravity 1.84)

               7.1.3   Hydrochloric acid - Reagent grade, 6N

               7.1.4   Phosphoric acid (H3PO4) - Reagent grade (85%), Fisher, or equivalent

               7.1.5   Sodium chloride - Reagent grade, prepare at 5% (w/v) solution in reagent water

               7.1.6   Ammonium hydroxide (NFLjOH) - Reagent grade, Anachemia, or equivalent

               7.1.7   Sodium dihydrogen phosphate monohydrate - Reagent grade, J.T. Baker, or
                      equivalent

               7.1.8   Oxalic acid, anhydrous

       7.2     Prepurified nitrogen

       7.3     Solvents, reagents, and solutions

               7.3.1   Acetic acid, acetone, acetonitrile ammonium acetate, formic acid,  methanol,
                      methylene chloride, HPLC water, ammonium formate.

               7.3.2   Solvents and purchased solutions should be lot-certified to be free of interferences.
                      If necessary, solvents should be analyzed by this method to demonstrate that they
                      are interference free.

       7.4     Buffer and elution solutions

               7.4.1   Phosphate buffer (sodium phosphate monohydrate/phosphoric acid) - 0.14 M
                      NaH2PO4.H2O 785% H3PO4 (1.93 g NaH2PO4.H2O in 99 mL of reagent water + 1
                      mLof85%H3PO4)

               7.4.2   Tetrasodium ethylenediamine tetraacetate hydrate (Na4EDTA2H2O ~+99.5%
                                                                                            10

-------
December 2007                                                                      Method 1694

                      titration), Sigma, used as received

               7.4.3  Formic acid solutions - Alfa Aesar, >99 percent purity

                      7.4.3.1   2% v/v in methanol

                      7.4.3.2  0.1% v/v in methanol

                      7.4.3.3  Formic acid/ammonium formate (0.1%) in water - dissolve 4 mL of
                               formic acid and 4 g of ammonium formate in 4.0 L of HPLC water.
                               Mix thoroughly and sonicate for 5 min.

                      7.4.3.4  Formic acid (0.1%) in methanol: water (75:25) - add 4 mL of formic acid
                               to 3.0 L methanol premixed with 1.0 L HPLC-grade water. Mix
                               thoroughly and sonicate for 5 min.

               7.4.4  Acetonitrilemethanol (1:1)- mix 500 mL methanol and 500 mL of acetonitrile.
                      Sonicate for 5 min.

               7.4.5  Oxalic acid solution (5 mM) - dissolve 0.45 g anhydrous oxalic acid in 1.0 L of
                      HPLC water.  Mix thoroughly and sonicate for 5 min.

               7.4.6  Oxalic acid/acetonitrile/methanol (5 mM) - dissolve 0.45 g anhydrous oxalic acid
                      in 500 mL acetonitrile premixed with 500 mL methanol. Mix thoroughly and
                      sonicate for 5 min.

               7.4.7  Acetonitrile/water (90%) - Add 400 mL HPLC-grade water to 3600 mL of
                      acetonitrile. Mix thoroughly and sonicate for 5 min.

               7.4.8  Ammonium acetate/acetic acid, 1 mM (0.1%) in water - Add 4 g NH^OAC and 4
                      mL acetic acid to 4.0 L of HPLC-grade water. Mix thoroughly and sonicate for 5
                      min.

       7.5     Sodium iodide/cesium iodide mass calibration solution - 2 mg/mL Nal and 50 jog/mL Csl
               in (1:1) isopropyl alcohol:water (Waters 700000889, or equivalent)  or other based on
               manufacture's specifications.

       7.6     Reference matrices - Matrices in which the PPCPs and interfering compounds are not
               detected by this method

               7.6.1   Reagent water - Bottled water purchased locally, or prepared by passage through
                      activated carbon

               7.6.2  High-solids reference matrix - Playground sand or similar material.

                      7.6.2.1   Playground sand is used to simulate the base fraction of solids in this
                               method, including biosolids (see Section 7.6.3.1 for simulation of the
                               biosolids acid fraction) - Place 1 g  of sand in a 50-mL centrifuge tube.
                               Add 15 mL of reagent water and adjust the pH to  10  0.5 with M^OH.


                                                                                             11

-------
December 2007                                                                       Method 1694

                                Add 20 mL of acetonitrile and sonicate for 20 minutes.  Discard the
                                aqueous phase.

                       7.6.2.2   Extract with a second 20-mL portion of acetonitrile. Decant and discard
                                the acetonitrile. The sand is now ready for spiking (Section 11.5.4).

               7.6.3   Biosolids (sludge) reference matrix - Dry peat moss, purchase from local garden
                       center. Note: Store peat moss in closed container to prevent further drying. Sand
                       may be used for the acid fraction if QC acceptance criteria (Section 9) are met.

                       7.6.3.1   Peat moss is used to simulate the acid fraction of biosolids in this
                                method (see Section 7.6.2.1 for information on the biosolids base
                                fraction) - Place 1 g of peat moss in a 50-mL centrifuge tube.  Add 15
                                mL of phosphate buffer (Section 7.4.1) and vortex to mix.  Extract with
                                20 mL of acetonitrile and discard the aqueous phase.

                       7.6.3.2   Extract with a second 20-mL portion of acetonitrile. Decant and discard
                                the acetonitrile. The peat moss is now ready for spiking (Section
                                11.4.3).

               7.6.4   Other matrices - Other reference matrices of interest may be used if the results
                       from the tests given in Section 9.2 demonstrate acceptable performance.  Ideally,
                       the matrix should be free of the analytes of interest, but in no case must the
                       background level of the analytes in the reference matrix exceed the minimum
                       levels in Tables 3, 5, 7, and 9.  If low background levels of the analytes of interest
                       are present in the reference matrix, the spike level of the analytes used in Section
                       9.2 should be increased to provide a spike-to-background ratio of approximately 5
                       (Reference 8).

        7.7    Standard solutions - Prepare from materials of known purity and composition or purchase
               as solutions or mixtures with certification to their purity, concentration, and authenticity. If
               the chemical purity is 98 % or greater, the weight may be used without correction to
               calculate the concentration of the standard.  Observe the safety precautions in  Section 5.

               7.7.1   Preparation and storage of solutions - For preparation of stock solutions from neat
                       materials, dissolve an appropriate amount of assayed reference material in solvent.
                       For example, weigh  10 to  20 mg of Ampicillin to three significant figures in a 10-
                       mL ground-glass-stoppered volumetric flask and fill to the mark with methanol.
                       After the compound is completely dissolved, transfer the solution to a clean 15-mL
                       vial with fluoropolymer-lined cap. When not being used, store standard solutions in
                       the dark at less than -10 C in screw-capped vials with fluoropolymer-lined caps or
                       under a non-reactive gas (e.g., nitrogen) in a flame-sealed glass ampul. Place a
                       mark on the vial or ampul at the level of the solution so that solvent loss by
                       evaporation can be detected. Replace the solution if solvent loss has occurred.

               7.7.2   Native (unlabeled; authentic) compound spiking solution - Separately prepare
                       Group 1 to Group 4 native compounds at the concentrations  shown in column 3 of
                       Table 10 in methanol, or purchase prepared solutions. If additional native
                       compounds are to be determined, include these compounds in this stock solution.


                                                                                               12

-------
December 2007                                                                      Method 1694

                       Stock solutions should be prepared at a frequency necessary to preclude
                       degradation from affecting the analysis. For example, it may be necessary to
                       prepare the tetracycline compounds weekly if concentrations drop more than 30 %
                       of their original concentration. Stock solutions should also be checked for signs of
                       degradation prior to preparation of calibration or performance test standards.

               7.7.3   Labeled compound spiking solution - Prepare Group 1 to Group 4 labeled
                       compounds at the concentrations shown in column 3 of Table  10 in methanol, or
                       purchase prepared solutions. If additional labeled compounds are to be used,
                       include these compounds in this solution. Note: The Group 2,  acid extracted
                       positive ESI (tetracyclines) contains the same labeled compounds as for Group 1
                       and 3, acid extracted positive and negative ESI, yet the only labeled compounds
                       used in determination of the Group 2 are Thiabendazole-d6 and  13C3-Atrazine.
                       This minimizes the work required to prepare solutions. Some of those surrogates
                       are used to quantify the Group 1 and 2 and some Group 3 in separate runs of the
                       same extract. This is not a requirement.

               7.7.4   Labeled injection internal standard spiking solutions - For the labeled injection
                       internal standards for Groups 1 and 2, prepare 13C-Atrazine in methanol at the
                       concentration shown column 3 of Table 10.  For the labeled injection internal
                       standard for Group 3, prepare 13C6-2,4,5-Trichlorophenoxyacetic acid (TCPAA) in
                       methanol at the concentration shown in column 3 of Table 10. For the labeled
                       injection internal standards for Group 4, prepare 13C3-Atrazine and Continine-d3 in
                       methanol at the concentrations shown in column 3  of Table  10.  If additional
                       labeled injection internal standards are to be used, include these  compounds in
                       these solutions.

               7.7.5   Calibration  standards - Combine and dilute the solutions in  Sections 7.7.1 and
                       7.7.2 to produce the calibration solutions in Table 11 or purchase prepared
                       standards for the CS-1 to CS-5 set of calibration solutions. These solutions permit
                       the relative response (labeled to native) and response factor to be determined as a
                       function of concentration.  The CS-3 standard is used for calibration verification
                       (VER).

        7.8    QC Check Sample - A QC Check Sample should be obtained from a source independent of
               the calibration standards.  Ideally, this check sample would be a Standard Reference
               Material (SRM) from the National Institute of Standards and Technology (NIST)
               containing the compounds of interest in known concentrations  in a sample matrix similar to
               the matrix of interest.  If no SRM is available, a certified reference material (CRM)  may be
               used or a QC check  sample may be prepared from materials from a source or lot of
               standards separate from those used for calibration and spiked into a clean reference matrix.

        7.9    Stability of solutions - standard solutions used for quantitative purposes (Sections 7.7.2 -
               7.7.5) should be assayed periodically (e.g., every 6 months) against SRMs from NIST (if
               available), or against certified reference materials from a source that will attest to the
               authenticity and concentration, to assure that the composition and concentrations have not
               changed.
                                                                                               13

-------
December 2007                                                                     Method 1694

8.0   Sample Collection, Preservation, Storage, and Holding Times

       8.1     Collect samples in amber glass containers following conventional sampling practices
               (Reference 9).

       8.2     Aqueous samples

               8.2.1   Samples that flow freely are collected as grab samples or in refrigerated bottles
                      using automatic sampling equipment. Collect 1-L each for the acid and base
                      fractions (2 L total).  If high concentrations of the analytes of interest are expected,
                      collect two smaller volumes (e.g., 100 mL each) in addition to the 1-L samples.
                      Do not rinse the bottle with sample before collection.

               8.2.2   If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. Any
                      method suitable for field use may be employed to test for residual chlorine.
                      Ascorbic acid has also been used by a number of other groups as a preservative for
                      a number of pharmaceuticals however it has not been tested for all of the
                      Pharmaceuticals covered under this method (Reference 10).

               8.2.3   Maintain aqueous samples in the dark at <6 EC from the time of collection until
                      receipt at the laboratory (see 40 CFR 136.6(e), Table II). If the  sample will be
                      frozen, allow room for expansion.

       8.3     Solid, mixed-phase, and semi-solid samples, including biosolids

               8.3.1   Collect samples as grab samples using wide-mouth jars. Collect a sufficient
                      amount of wet material to produce a minimum of 10 g of solids.

               8.3.2   Maintain solid, semi-solid, and mixed-phase samples in the dark at <6 EC from the
                      time of collection until receipt at the laboratory. Store solid, semi-solid, and
                      mixed-phase samples in the dark at less than -10 EC.

       8.4     Store sample extracts in the dark at less than -10 EC until analyzed. Analyze extracts within
               40 days of extraction.

       8.5     Holding times

                      EPA has not conducted formal holding time studies for these analytes to date. Use
               the information below as guidance. Exceeding these default holding times does not
               invalidate the sample results.

               8.5.1   Aqueous samples - Anecdotal evidence suggests that some may degrade rapidly in
                      aqueous samples. Therefore, begin sample extraction within 7 days of collection
                      (within 48 hours is strongly encouraged). Extracts should be analyzed within 40
                      days of extraction. Freezing of aqueous samples is encouraged  to minimize
                      degradation, in which case, samples  should be extracted within 48 hours of
                      removal from the freezer.
                                                                                             14

-------
December 2007                                                                      Method 1694

               8.5.2  Biosolid, solid, mixed-phase, and semi-solid samples - Anecdotal evidence
                      suggests that some may degrade rapidly in these samples. Therefore, begin sample
                      extraction within 7 days of collection (within 48 hours is strongly encouraged).
                      Extracts should be analyzed within 40 days of extraction. Freezing of biosolids,
                      mixed phase and semisolid samples is encouraged to minimize degradation, in
                      which case, samples should be extracted within 48 hours of removal from the
                      freezer..

               8.5.3  If extraction within 48 hours is not practical, samples should be frozen to increase
                      the holding time to seven days.

               8.5.4  If the sample will not be extracted within 48 hours of collection, the laboratory
                      should adjust the pH of aqueous samples to 5.0 to 9.0 with a sodium hydroxide or
                      sulfuric acid solution. Record the volume of acid or base used.  If aqueous samples
                      are stored frozen, extraction should begin within 48 hours of removal from the
                      freezer.
9.0    Quality Assurance/Quality Control

        9.1     Each laboratory that uses this method is required to operate a formal quality assurance
               program (Reference 11). The minimum requirements of this program consist of an initial
               demonstration of laboratory capability, analysis of samples spiked with labeled compounds
               to evaluate and document data quality, and analysis of standards and blanks as tests of
               continued performance. Laboratory performance is compared to established performance
               criteria to determine if the results of analyses meet the performance characteristics of the
               method.

               If the method is to be applied to sample matrix other than water (e.g., soil, sediment, filter
               cake, compost) the most appropriate alternate reference matrix (Sections 7.6.1 - 7.6.4) is
               substituted for the reagent water matrix (Section 7.6.1) in all performance tests.

               9.1.1   The laboratory must make an initial demonstration of the ability to generate
                      acceptable precision and recovery with this method. This demonstration is given in
                      Section 9.2.

               9.1.2  In recognition of advances that are occurring in analytical technology, and to
                      overcome matrix interferences, the laboratory is permitted certain options to
                      improve separations or lower the costs of measurements.  These options include
                      alternate extraction, concentration, and cleanup procedures, and changes in
                      columns and detectors (see also 40 CFR 136.6). Alternate determinative
                      techniques, such as the substitution of spectroscopic or immunoassay techniques,
                      and changes that degrade method performance, are not allowed. If an analytical
                      technique other than the techniques specified in this method is used, that technique
                      must have a specificity equal to or greater than the specificity of the techniques in
                      this method for the analytes of interest.

                      9.1.2.1   Each time a modification is made to this method, the laboratory is

-------
December 2007                                                                      Method 1694

                                required to repeat the procedure in Section 9.2. If the detection limit of
                                the method will be affected by the change, the laboratory is required to
                                demonstrate that the MDLs (40 CFR Part 136, Appendix B) are lower
                                than one-third the regulatory compliance level or the MDLs in this
                                method, whichever are greater. If calibration will be affected by the
                                change, the instrument must be recalibrated per Section 10.  Once the
                                modification is demonstrated to produce results equivalent or superior to
                                results produced by this method as written, that modification may be
                                used routinely thereafter, so long as the other requirements in this
                                method are met (e.g., labeled compound recovery).

                       9.1.2.2   The laboratory is required to maintain records of modifications made to
                                this method. These records include the following, at a minimum:

                                9.1.2.2.1   The names, titles, addresses, and telephone numbers of the
                                           analyst(s) that performed the analyses and modification, and
                                           of the quality control officer that witnessed and will verify
                                           the analyses and modifications.

                                9.1.2.2.2   A list of compounds (s) measured, by name and CAS
                                           Registry number.

                                9.1.2.2.3   A narrative stating reason(s) for the modifications.

                                9.1.2.2.4   Results from all quality control (QC) tests comparing the
                                           modified method to this method, including:

                                           a)  Calibration (Section 10).
                                           b)  Calibration verification (Section 15.2).
                                           c)  Initial precision and recovery (Section 9.2).
                                           d)  Labeled compound recovery (Section 9.3).
                                           e)  Analysis of blanks (Section 9.5).
                                           f)  Accuracy assessment (Section 9.4).

                                9.1.2.2.5   Data that will allow an independent reviewer to validate
                                           each determination by tracing the instrument output (peak
                                           height, area, or other signal) to the final result. These data
                                           are to include:

                                           a)  Sample numbers and other identifiers.
                                           b)  Extraction dates.
                                           c)  Analysis dates and times.
                                           d)  Analysis sequence/run chronology.
                                           e)  Sample weight or volume (Section 11).
                                           f)  Sample or extract volume prior to each cleanup step
                                               (Section 12).
                                           g)  Extract volume after each cleanup step (Section 12).
                                           h)  Final extract volume prior to injection (Section 12).
                                           i)  Injection volume (Sections 10.2.1 and 14.2).


                                                                                               16

-------
December 2007                                                                       Method 1694

                                           j)  Dilution data, differentiating between dilution of a
                                               sample or extract (Section 17.5).
                                           k)  Instrument and operating conditions.
                                           1)  Column (dimensions, material, particle size, etc).
                                           m) Operating conditions (flow rates, elution solvents,
                                               gradient, flow rates).
                                           n)  Detector (type,  operating conditions, etc).
                                           o)  Chromatograms, printer tapes, and other recordings of
                                               raw data.
                                           p)  Quantitation reports, data system outputs, and other data
                                               to link the raw data to the results reported.

               9.1.3   Analyses of method blanks are required to demonstrate freedom from
                       contamination (Section 4.3).  The procedures and criteria for analysis of a method
                       blank are given in Sections 9.5 and 15.4.

               9.1.4   The laboratory must spike all samples with labeled compounds to monitor method
                       performance. This test is described in Section 9.3.  When results of these spikes
                       indicate atypical method performance for samples, the samples are diluted to bring
                       method performance within acceptable limits. Procedures for dilution are given in
                       Section 17.5.

               9.1.5   The laboratory must, on an ongoing basis,  demonstrate through calibration
                       verification and the analysis of the ongoing precision and recovery standard (OPR)
                       and blanks that the analytical system is in control.  These procedures are given in
                       Sections 15.1 through 15.4.

               9.1.6   The laboratory should maintain records to  define the quality of data generated.
                       Development of accuracy statements is described in Section 9.4.

        9.2    Initial precision and recovery (IPR) - To establish the ability to generate acceptable
               precision and recovery, the laboratory must perform the following operations.

               9.2.1   For aqueous samples containing less than 1% solids, analyze four 1-L aliquots of
                       reagent water (7.6.1) each for the acid and base fractions according to the
                       procedures in Sections 11 through 18. For an alternate sample matrix, four aliquots
                       each for the acid and base fractions of the alternate reference matrix (Sections
                       7.6.2-7.6.4) are used.  All sample processing steps that are to be used for
                       processing samples, including preparation  (Section 11), extraction (Section 12),
                       and cleanup (Section 13), must be included in this test.

               9.2.2   Using results of the set of four analyses, compute the average percent recovery (X)
                       of the concentration of each compound in each extract and the relative standard
                       deviation (RSD) of the concentration for each compound, by isotope dilution for
                       compounds with a labeled analog, and by internal standard for compounds without
                       a labeled analog and for the labeled compounds.

               9.2.3   For each native and labeled compound, compare RSD and X with the
                       corresponding limits for initial precision and recovery in Table 12. If RSD and X


                                                                                                17

-------
December 2007                                                                      Method 1694

                      for all compounds meet the acceptance criteria, system performance is acceptable
                      and analysis of blanks and samples may begin. If, however, any individual RSD
                      exceeds the precision limit or any individual X falls outside the range for recovery,
                      system performance is unacceptable for that compound.  Correct the problem and
                      repeat the test (Section 9.2).

        9.3     To assess method performance on the sample matrix, the laboratory must spike all samples
               with the Labeled spiking solution (Section 7.7.3).

               9.3.1   Analyze each sample according to the procedures in Sections 11 through 18.

               9.3.2   Compute the percent recovery of the labeled compounds using the internal standard
                      method (Sections 10.4 and 7.2).

               9.3.3   The recovery of each labeled compound must be within the limits in Table 12. If
                      the recovery of any compound falls outside of these limits, method performance is
                      unacceptable for that compound in that sample. Additional cleanup procedures
                      must then be employed to attempt to bring the recovery within the normal range.  If
                      the recovery cannot be brought within the normal range after all cleanup
                      procedures have been employed, water samples are diluted and smaller amounts of
                      soils, sludges, sediments, and other matrices are analyzed per Section 18.

        9.4     Recovery of labeled compounds from samples should be assessed and  recorded.

               9.4.1   After the analysis of 30  samples of a given matrix type (water, soil, sludge, pulp,
                      etc.) for which the labeled compounds pass the tests in Section 9.3, compute the
                      average percent recovery (R) and the standard deviation of the percent recovery
                      (SR) for the labeled compounds only. Express the assessment as a percent recovery
                      interval from R ! 2SR to R + 2SR for each matrix. For example, if R = 90% and SR
                      = 10% for 30 analyses of biosolids, the recovery interval is expressed as  70 to
                      110%.

               9.4.2   Update the accuracy assessment for each labeled compound in each matrix on a
                      regular basis (e.g., after each 5-10 new measurements).

        9.5     Method blanks - A reference matrix method blank is analyzed with each sample  batch
               (Section 4.3) to demonstrate freedom from contamination. The matrix for the method
               blank must be similar to the sample matrix for the batch, e.g., a 1-L reagent water blank
               (Section 7.6.1), high-solids reference matrix blank (Section 7.6.2), biosolids reference
               matrix blank (Section 7.6.3) or alternate reference matrix blank (Section 7.6.4).

               9.5.1   Process the method blank(s) along with the IPR or batch of samples according to
                      the procedures in Sections 11 through 18. Analyze the blank immediately after
                      analysis of the OPR (Section 15.4) to demonstrate freedom from contamination.

               9.5.2   If any compound of interest (Table 1) is found in the blank at greater than the
                      minimum level (Tables  3, 5, 7, or 9) or one-third the regulatory compliance limit,
                      whichever is greater; or if any potentially interfering compound is found in the
                      blank above the minimum level for each native compound in Tables 3, 5, 7, or 9


                                                                                              18

-------
December 2007                                                                      Method 1694

                      (assuming a response factor of 1 relative to the quantitation reference in Tables 3,
                      5, 7, or 9 for a potentially interfering compound; i.e., a compound not listed in this
                      method), analysis of samples must be halted until the sample batch is re-extracted
                      and the extracts re-analyzed, and the blank associated with the sample batch shows
                      no evidence of contamination at these levels. All samples must be associated with
                      an uncontaminated method blank before the results for those samples may be
                      reported or used for permitting or regulatory compliance purposes.

        9.6     QC Check Sample - If available, analyze the QC Check Sample (Section 7.8) periodically
               to assure the accuracy of calibration standards and the overall reliability of the analytical
               process. It is suggested that the QC Check Sample be analyzed at least quarterly.

        9.7     The specifications contained in this method can be met if the apparatus used is calibrated
               properly and then maintained in a calibrated state.  The standards used for calibration
               (Section 10), calibration verification (Section 15.2), and for initial (Section 9.2) and
               ongoing (Section 15.4) precision and recovery should be identical, so that the most precise
               results will be obtained. A LCMSMS instrument will provide the most reproducible results
               if dedicated to the settings and conditions required for determination of PPCPs by this
               method.

        9.8     Depending on specific program requirements, field replicates may be collected to determine
               the precision of the sampling technique, and spiked samples may be required to determine
               the accuracy of the analysis when the internal standard method is used.
10.0   Calibration and Standardization

        10.1    Establish the LC/MS/MS operating conditions for the Group 1 through Group 4
               compounds, as suggested in Tables 2, 4, 6, and 8, to meet the retention times in Tables 3, 5,
               7, and 9, respectively.  The LC conditions may be optimized for compound separation and
               sensitivity.  Once optimized, the same conditions must be used for the analysis of all
               standards, blanks, IPR and OPR standards, and samples.

        10.2   Retention time calibration for the native and labeled compounds

               10.2.1 Inject the volume of CS-3 calibration standard (Section 7.7.5 and Table 11) listed
                      in Table 2, 4, 6, or 8, or other volume appropriate to system optimization.
                      Establish the beginning and ending retention times for the parent-daughter
                      descriptors in Tables 3,5,7, and 9.  Descriptors other than those listed may be used
                      provided the MLs in those tables are met.  Store the retention time (RT) for each
                      compound in the data system.

               10.2.2 The absolute retention time of last-eluted compound in each of the four Groups
                      must be equal  to or greater than its retention time in Tables 3,5,7, or 9; otherwise,
                      the LC operating conditions must be adjusted and this test repeated until this
                      minimum retention time criterion is met.

        10.3   Mass spectrometer calibration and optimization
                                                                                               19

-------
December 2007
Method 1694
               10.3.1 Mass calibration - The mass spectrometer must undergo mass calibration
                      according to manufacture's specifications to ensure accurate assignments of m/z's
                      by the instrument. This mass calibration must be performed at least annually to
                      maintain instrument sensitivity and stability. It must be repeated after performing
                      major maintenance on the mass spectrometer.

                      In the absence of vendor-specific instructions and acceptance criteria, the following
                      procedure may be used.

                      10.3.1.1  Introduce the NaCsI calibration solution (Section 7.5) to the MS at the
                                flow rate necessary to produce a stable aerosol spray (e.g., 10 |oL/min).

                      10.3.1.2  Scan the MS/MS over the mass range from 20 to 3000 Daltons.  Adjust
                                the source parameters to optimize peak intensity and shape across the
                                mass range.  The exact m/z's for NaCsI calibration are:
Calibration Masses (Daltons)
22.9898
132.9054
172.8840
322.7782
472.6725
622.5667
772.4610
922.3552
1072.2494
1222.1437
1372.0379
1521.9321
1671.8264
1821.7206
1971.6149
2121.5091
2271.4033
2421.2976
2571.1918
2721.0861
2870.9803

                       10.3.1.3  Mass calibration is judged on the basis of the presence or absence of the
                                exact calibration masses, e.g., a limit of the number of masses that are
                                "missed."  Absent vendor-specific instructions, all of the masses from
                                22.9898 to 1971.6149 must be present. If peaks above 1971 are missing
                                or not correctly identified, adjust the MS/MS and repeat the test. Only
                                after the MS/MS is properly calibrated may standards, blanks, and
                                samples be analyzed.

               10.3.2  Mass spectrometer optimization - Prior to measurements of a given analyte
                       Group (Table 2, 4, 6, or 8), the mass spectrometer must be separately optimized
                       for that Group.

                       10.3.2.1  Using the post-column pump (Section 6.14.1), infuse the CS-3
                                calibration solution (Table 11 a, b, or c) for the Group of interest.

                       10.3.2.2  Optimize sensitivity to the daughter m/z's for the high mass compounds
                                in each Group (Table 3, 5, 7, or 9).
                                                                                               20

-------
December 2007                                                                     Method 1694
                      10.3.2.3 After MS calibration and optimization and LC/MS/MS calibration
                               (Sections 10.4 and 10.5), MS and LC/MS/MS conditions may not be
                               altered without verifying calibration (Section 15.2).

        10.4   Calibration by isotope dilution - Isotope dilution is used for calibration of each native
               compound for which a labeled analog is available.  The reference compound for each
               native compound is its labeled analog, as listed in Tables 3, 5, 7, and 9. A 5-point
               calibration encompassing the concentration range is prepared for each native compound.
               The calibration solutions are listed in Table 11.

               10.4.1 To calibrate the analytical system by isotope dilution, inject calibration standards
                      CS-1 through CS-5 (Section 7.7.5 and Table 11).  Use the volume shown in
                      identical to the volume chosen in Section 10.2.1, the procedure in Section 14, and
                      the optimized operating conditions from Sections 10.1 - 10.3.

               10.4.2 For the compounds determined by isotope  dilution, the relative response (RR)
                      (labeled to native) vs. concentration in the  calibration solutions (Table 11) is
                      computed over the calibration range according to the procedures below.
                      Determine the response of each compound relative to its labeled analog using the
                      area responses of the daughter m/zs specified in Tables 3, 5,  7, and  9. Use the
                      labeled compounds listed in the tables as the quantitation reference  and the
                      daughter m/zs of these labeled compounds for quantitation. The area of the
                      daughter m/z for the native compound is divided by the area of the daughter m/z
                      of the labeled quantitation reference compound.

Note: Other quantitation references and procedures may be used provided that the results produced are
as accurate as results produced by the quantitation references  and procedures described in  this method.

               10.4.3 Calibrate the native compounds with a labeled analog using the following
                      equation:

                                        A   r
                                  RR = -
                                        A,Cn

                      Where:
                           An   =  The area of the daughter m/z for the native compound
                           A!   =  The area of the daughter m/z for the labeled compound.
                           Ci   =  The concentration of the labeled compound in the calibration
                                   standard (Table 11) (ng/mL).
                           Cn   =  The concentration of the native compound in the calibration
                                   standard (Table 11) (ng/mL).

               10.4.4 Compute the average (mean) RR, and the standard deviation and relative
                      standard deviation (RSD) of the 5 RRs.
                                                                                             21

-------
December 2007                                                                     Method 1694

               10.4.5 Linearity - If the RR for any compound is constant (less than 20% RSD), the
                      average RR may be used for that compound; otherwise, the complete calibration
                      curve for that compound must be used over the calibration range.

        10.5   Calibration by internal standard - Internal standard calibration is applied to determination
               of the native compounds for which a labeled compound is not available, and to
               determination of the labeled compounds for performance tests and intra-laboratory
               statistics (Sections 9.4 and 15.4.4). The reference compound for each native compound
               is listed in Table 3, 5, 7, or 9. For the labeled compounds,  calibration is performed at a
               single concentration using data from the 5 points in the calibration (Section 10.4).

               10.5.1 Response factors - Internal standard calibration requires the determination of
                      response  factors (RF) defined by the following equation:
                                          AISCn
                          Where:
                           An   =  The area of the daughter m/z for the native compound
                           Ais   =  The area of the daughter m/z for the internal standard.
                           C;s   =  The concentration of the internal standard (Table 11) (ng/mL).
                           Cn   =  The concentration of the native compound in the calibration
                                   standard (Table 11) (ng/mL).

               10.5.2 To calibrate the analytical system for compounds that do not have a labeled
                      analog, and for the labeled compounds, use the data from the 5-point calibration
                      (Section 10.4 and Table 11).

               10.5.3 Compute and store the response factor (RF) for all native compounds that do not
                      have a labeled analog. Use the labeled compounds and daughter m/zs listed in
                      Tables 3,5,7, and 9 as the quantitation references.

               10.5.4 Compute and store the response factor (RF) for the labeled compounds using the
                      labeled injection internal standard as the quantitation reference, as given in
                      Tables 3, 5, 7, and 9.

               10.5.5 Linearity - If the RF for any native compound without a labeled analog or for
                      any labeled compound is constant (less than 35% RSD), the average RF may be
                      used for that compound; otherwise, the complete calibration curve for that
                      compound must be used over the calibration range.

        10.6   SPE cartridge performance check

               In order to be used for extraction of aqueous samples or cleanup of solid-sample extracts,
               the performance of the HLB SPE cartridges must be checked at least once for each
               manufacturer's lot of cartridges. This performance check is accomplished by processing
               a spiked reagent water sample through the extraction procedure is Section 12 and
               analyzing the extract. Separate checks are performed for the acid and base fractions.
               Labeled compounds are not added to these check samples before extraction because the
                                                                                             22

-------
December 2007                                                                    Method 1694

              recovery correction inherent in isotope dilution will mask problems with the cartridges.
              Cartridge performance is acceptable if the recoveries of the native analytes are within the
              QC acceptance criteria for the OPR in Table 12. Perform this cartridge check as outlined
              below. Note - This performance check is performed when a new lot number of cartridges
              is purchased.

              10.6.1  Acid fraction - Acidify a 1.0-L aliquot of reagent water to pH 2.0  0.5.  Add 500
                      mg N34EDTA (Section 7.4.2) and spike with the Group 1, 2, and 3 native
                      compounds (Section 7.7.2 and Table 10). Do not spike the labeled compounds.
                      Process the solution through the SPE HLB procedure for the acid fraction in
                      Section 12. After processing, spike the solution with the Group 1, 2, and 3
                      labeled compounds (Section 7.7.3 and Table 10) and complete the analysis per
                      Sections 12 - 15. Recovery of the native compounds must be within the  QC
                      acceptance crieria for the OPR in Table 12. If the compounds are not recovered
                      in this range, adjust the elution volumes or reject the cartridge batch.

              10.6.2  Base fraction -  AdjustthepHa 1.0-L aliquot of reagent water to pH 10.00.5
                      and spike with the Group 4 native compounds (Section 7.7.2 and Table 10). Do
                      not spike the labeled compounds. Process the solution through the SPE HLB
                      procedure  for the base fraction in Section 12.  After processing, spike the extract
                      with the Group 4 labeled compounds (Section 7.7.3 and Table 10) and complete
                      the analysis per Sections 12 -  15. Recovery of the native compounds must be
                      within the  QC acceptance criteria for the OPR in Table 12. If the compounds are
                      not recovered in this range, adjust the elution  volumes or reject the cartridge
                      batch.
11.0  Sample Preparation

       Sample preparation involves modifying the physical form of the sample so that the analytes can
       be extracted efficiently. In general, the samples must be in a liquid form or in the form of finely
       divided solids in order for efficient extraction to take place. Table 13 lists the phases and
       suggested quantities for extraction of various sample matrices. For samples known or expected to
       contain high levels of the analytes, the smallest sample size representative of the entire sample
       should be used (see Section 18).

       Biosolids and solid samples are prepared per Section 11.4, extracted  per Sections 12.3 and 12.4,
       and cleaned up using SPE HLB cleanup in Sections 12.1 and 12.2.

       Aqueous samples - Because the analytes may be bound to suspended particles, the preparation of
       aqueous samples is depends on the presence of visible particles. Aqueous samples absent visible
       particles  are prepared per Section 11.3 and processed using SPE HLB cleanup in Sections 12.1
       and 12.2.

       Aqueous samples with visible particles - If visible particles can be seen in aqueous samples they
       should be filtered and the solids and aqueous portions of these samples should be extracted and
       combined prior to clean up as follows. Filtration of particles - assemble a clean filtration
       apparatus (Section 6.6). Apply vacuum to the apparatus, and pour the entire contents of the
                                                                                            23

-------
December 2007                                                                     Method 1694

       sample bottle through the filter, swirling the sample remaining in the bottle to suspend any
       particles.  Rinse the sample bottle twice with approximately 5 mL portions of reagent water to
       transfer any remaining particles onto the filter. Rinse any particles off the sides of the filtration
       apparatus with small quantities of reagent water. Weigh the empty sample bottle to 1 g.
       Determine the weight of the sample by difference. Save the bottle for further use.  Prepare and
       extract the filtrate using the procedure in Section 11.3. Prepare and extract the filter containing
       the particles using the same procedure for biosolids and solid samples in Section  11.4, Sections
       12.3 and 12.4, and Sections 12.1 and 12.2.  These extracts should be combined prior to analysis
       (Section 14) or results of separate analysis combined. It should be noted that the judgment of the
       analyst must be used to determine the need to analyze samples with visible particles that compose
       less than 1 % of the sample weight per Section 11.1.

       Procedures for grinding, homogenization, and blending of various sample phases are given in
       Section 11.5.

Note: Each sample batch (Section 4.3) is accompanied by a blank and an OPR. If the acid fraction
(Groups 1,  2,  and 3) only is to be analyzed then 1 acid blank and OPR must be used. If both the  acid
(Groups 1,  2,  and 3) and base (Group 4) fractions are to be analyzed, 1 acid blank and OPR as  well as 1
base blank and OPR must accompany the batch.  If the base fraction (Group 4) only is to be analyzed,  a
base blank and OPR must accompany the base batch.
        11.1    Determination of solids content

        The solids content of the bulk sample is determined from a subsample that is used only for the
        solids determination.  Separate procedures are used for the solids determination, based on the
        sample matrix, as described below.

               11.1.1 Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase.

                      11.1.1.1 Dry a GF/A filter (Section 6.6.4) and weigh to three significant figures.
                               Mix the bulk sample in the original container (e.g., cap the bottle and
                               shake) and take a 10.0 V 0.2 mL aliquot. Filter that aliquot through the
                               filter. Dry the filter in an oven for a minimum of 12 hours at 110 V 5
                               EC and cool in a dessicator.

                      11.1.1.2 Weigh the filter and calculate  percent solids as follows:

                                _ Weight of sample aliquot after drying (g) - weight of filter (g)
                        /o oOHClS                                                         X L\)\)
                                                           10g

               11.1.2 Non-aqueous liquids, solids, semi-solid  samples, and multi-phase samples in
                      which the main phase is not aqueous

                      11.1.2.1 Weigh 5 to 10 g of the bulk sample to three significant figures in a
                               tared beaker, weighing pan, or other suitable container. Dry for a
                               minimum of 12 hours at 110 V 5 EC, and cool in a dessicator.
                                                                                             24

-------
December 2007                                                                      Method 1694

                       11.1.2.2 Weigh the dried aliquot and calculate percent solids as follows:

                                        _  Weight of sample aliquot after drying (g)
                                          Weight of sample aliquot before drying (g)

        11.2   Estimation of particle size

               Extraction of a sample matrix is affected by the size of particles in the sample.  Ideally,
               the particles should be 1 mm or less.  The particle size can be estimated using the sample
               aliquot filtered or dried in Sections 11.1.1 or 11.1.2.  Spread the aliquot on a piece of
               filter paper or aluminum foil in a fume hood or glove box. Visually estimate the size of
               the particles in the sample.  If the size of the largest particles is greater than 1 mm, use
               one of the procedures in Section 11.5 to reduce the particle size to 1 mm or less prior to
               extraction. If the largest particles are 1 mm or less, proceed with sample preparation,
               using the procedures in Section 11.4

        11.3   Preparation of aqueous samples absent visible particles and corresponding QC  samples.

               Two separate sample aliquots are required to analyze all of the target analytes in this
               procedure: one aliquot is adjusted to pH 2  0.5 (Section 11.3.3.1) and the other aliquot
               is adjusted to pH 100.5 (Section  11.3.4.1). Following this pH adjustment, both
               aliquots are filtered separately, and the two filtrates are extracted using the SPE HLB
               cartridge per Section 12.

               11.3.1  Mark the original level of the sample on each of the two sample bottles.
                       Designate one bottle for the acid fraction and the other for the base fraction.
                       Weigh each sample plus bottle to the nearest 1  g. If only one sample bottle was
                       provided, and both the acid and base fractions are to be analyzed, split  the sample
                       in half and place each new aliquot in a separate clean container.

               11.3.2  For each sample batch (Section 4.3) to be extracted during the same 12-hour
                       shift, transfer four 1-L aliquots of reagent water to clean sample bottles or flasks.
                       Two of these aliquots will serve as method blanks (one for the acid fraction and
                       one for the base fraction) and the other two aliquots will be used to prepare the
                       OPR samples (one acid and one base).  (If both acid and base fractions are not
                       required, prepare only the reference matrix aliquots appropriate for the fraction of
                       interest.)

               11.3.3  Acid fraction - typically 500 mL to  1 L

                       11.3.3.1   Acidify the filtrate for the acid fraction to pH 2.0  0.5 with HC1
                                  while swirling or stirring the water. Re-adjust the pH as necessary to
                                  achieve pH 2.0  0.5. Maintain the pH above 1.95 to preclude
                                  deuterium-hydrogen exchange on the deuterium-labeled compounds.

                       11.3.3.2   Spike the acid fraction (Group 1, 2, and 3) native compounds
                                  (Section 7.7.2 and Table 10) into the reagent water aliquot that will
                                  serve as the acid fraction OPR.  Acidify the OPR aliquot and the
                                                                                               25

-------
December 2007                                                                      Method 1694

                                 blank aliquot in the same manner as the acid fraction of the field
                                 sample (11.3.3.1).

                      11.3.3.3   Spike the acid fraction (Group 1, 2, and 3) labeled compounds
                                 (Section 7.7.3 and Table  10) into the acid fractions of the samples
                                 and QC aliquots.

                      11.3.3.4   Add 500 mg NA4EDTA.2H2O (Section 7.4.2) to each of the acid
                                 fraction samples and QC aliquots.  Cap the bottles and mix by
                                 shaking. Allow the sample and QC aliquots to equilibrate for 1 to 2
                                 hours, with occasional shaking. Proceed to Section 12 for sample
                                 extraction.

               11.3.4 Base fraction - typically 500 mL to 1 L

                      11.3.4.1   Adjust the pH of the  second of the two sample bottles to pH 10.0 
                                 0.5 with NH/tOH while swirling or stirring the water. Re-adjust the
                                 pH as necessary to achieve pH 10.0  0.5.

                      11.3.4.2   Spike the base fraction (Group 4) native compounds (Section  7.7.2
                                 and Table 10) into the reagent water aliquot that will serve as the
                                 base fraction OPR. Adjust the pH of the OPR aliquot and the blank
                                 aliquot in the same manner as the base fraction of the field sample
                                 (11.3.4.1).

                      11.3.4.3   Spike the base fraction (Group 4) labeled compounds (Section 7.7.3
                                 and Table 10) into the base fractions of the samples and QC aliquots.

                      11.3.4.4   Cap the bottles and mix by shaking. Allow the sample and aliquots
                                 to equilibrate for 1  to 2 hours, with occasional shaking. Proceed to
                                 Section 12 for sample extraction.

        11.4   Preparation of solid samples and samples from filtered particles and corresponding QC
               samples.

               Filtered solids from aqueous samples are  treated as solid matrices, regardless of whether
               they are pourable liquids or solid materials. Two separate aliquots are required to
               analyze all of the target analytes in this procedure.  If the particle size estimated in
               Section 11.2 exceeds 1 mm, use one of the six size-reduction procedures in Section 11.5
               first. Following addition of buffer solutions, one aliquot is adjusted to pH 2  0.5 and the
               other aliquot is adjusted to pH 11  0.5.  Following pH adjustment, each aliquot is
               extracted separately per Section 12.

               11.4.1 Homogenize the sample in its original container, by shaking samples that are
                      pourable liquids, or by stirring solids  in their original container with a clean
                      spatula, glass stirring rod, or other suitable implement.
                                                                                              26

-------
December 2007                                                                     Method 1694

               11.4.2 Using the percent solids data collected in Section 11.1, collect two aliquots of the
                      well-mixed sample sufficient to provide 1.0 g of dry solids, but do not exceed a
                      maximum of 5 g wet weight. For biosolids, do not exceed 0.25 g of wet solids.
                      Place the two sample aliquots in separate clean 50-mL disposable centrifuge
                      tubes.  Designate one of the samples as the acid fraction, the other the base
                      fraction.

               11.4.3 For each sample batch (Section 4.3) to be extracted during the same 12-hour
                      shift, transfer two 1-g aliquots of peat moss (Section 7.6.3) to clean sample
                      bottles or flasks. These two peat moss aliquots will be used for the method blank
                      and the OPR sample for the acid fraction. Transfer two 1-g aliquots of clean
                      sand (Section 7.6.2) to clean sample bottles or flasks. These two clean sand
                      aliquots will be used for the method blank and the OPR sample for the base
                      fraction.  (If both acid and base fractions are not required, prepare only the
                      reference matrix aliquots appropriate for the fraction of interest.)

               11.4.4 Acid fraction

                      11.4.4.1  Add 15 mL of pH 2  phosphate buffer (Section 7.4.1) to the sample,
                                blank, and OPR. Vortex each for 5 min. Check and adjust the pH to
                                2.0  0.5 with buffer, vortexing the mixture after each addition.
                                Maintain the pH above 1.95 to preclude deuterium-hydrogen exchange
                                on the deuterium-labeled compounds.

                      11.4.4.2  Spike the acid fraction (Group 1, 2, and 3) native compounds (Section
                                7.7.2 and Table 10) into the peat moss aliquot that will serve as the
                                acid fraction OPR. Acidify the OPR aliquot and the blank aliquot in
                                the same manner as the acid fraction of the field sample (11.4.4.1).

                      11.4.4.3  Spike the acid fraction (Group 1, 2, and 3) labeled compounds
                                (Section 7.7.3 and Table 10) into the acid fractions of the samples and
                                QC aliquots.

                      11.4.4.4  Vortex the samples and QC aliquots. Proceed to Section 12.3 for
                                extraction of the solids acid fraction.

               11.4.5 Base fraction

                      11.4.5.1 Add 15 mL of reagent water to the sample, blank, and OPR. Vortex
                               each for 5 min.  Adjust the pH of the sample, blank, and OPR aliquots
                               to 10.0  0.5 by adding NFLjOH solution dropwise. Vortex for 5 min.
                               Check and adjust the pH to  10.0  0.5 with NFL^OH solution, vortexing
                               the mixture after each addition.

                      11.4.5.2 Spike the base fraction (Group 4) native compounds (Section 7.7.2 and
                               Table 10) into one of the QC aliquots. This aliquot will serve as the
                               OPR.  The other will serve as the blank.
                                                                                             27

-------
December 2007                                                                    Method 1694

                      11.4.5.3 Spike the base fraction (Group 4) labeled compounds (Section 7.7.3
                               and Table 10) into the samples and QC aliquots.

                      11.4.5.4 Vortex the samples and QC aliquots.  Proceed to Section 12.4 for
                               extraction of the solids base fraction.

       11.5   Sample grinding, homogenization, or blending

               Samples with particle sizes greater than 1 mm (as determined in Section 11.2) are
               subjected to grinding, homogenization, or blending.  The method of reducing particle size
               to less than 1 mm is matrix-dependent. In general, hard particles can be reduced by
               grinding with a mortar and pestle.  Softer particles can be reduced by grinding in a Wiley
               mill or meat grinder, by homogenization, or in a blender.

               11.5.1 Each size-reducing preparation procedure on each matrix must be verified by
                      running the tests in Section 9.2 before the procedure is employed routinely.

               11.5.2 The grinding, homogenization, or blending procedures must be carried out in a
                      glove box or fume hood to prevent particles from contaminating the work
                      environment.

               11.5.3 Grinding - Amorphous and other solids can be ground in a Wiley mill or heavy
                      duty meat grinder. In some cases, reducing the temperature of the sample to
                      freezing or to dry ice or liquid nitrogen temperatures can aid in the grinding
                      process. Grind the sample aliquots  in a clean grinder.  Do not allow the sample
                      temperature to exceed 50 EC. Also grind the blank and OPR reference matrix
                      aliquots using a clean grinder.

               11.5.4 Homogenization or blending - Particles that are not ground effectively, or
                      particles greater than 1 mm in size after grinding, can often be reduced in size by
                      high speed homogenization or blending.  Homogenize and/or blend the particles
                      or filter for the sample, blank, and OPR aliquots.

               11.5.5 After size reduction, return to Section 11.4 for preparation of the sample and QC
                      aliquots.
12.0  Extraction and  Concentration

This method employs solid-phase extraction (SPE) procedures to extract the target analytes from aqueous
samples.  Solid samples are extracted using ultrasonic extraction with acetonitrile. The extracts from
solid samples contain significant amount of coextracted interferences which can be removed through the
use of the same SPE procedure employed for the aqueous samples.

       12.1   Extraction of aqueous samples absent visible particles, and cleanup of extracts from filtered
              solids, solids and biosolids samples.

              Extraction of both the acid and base fractions of aqueous samples involve many of the same
                                                                                            28

-------
December 2007                                                                      Method 1694

               steps, beginning with the conditioning of the SPE cartridges.

               12.1.1 Assemble the SPE extraction apparatus and attach the SPE HLB cartridges
                      (Section 6.5.7).

               12.1.2 Condition an SPE HLB cartridge by eluting it with 20 mL of methanol, and 6 mL
                      of reagent water. Discard these eluants. When extracting the base fraction of a
                      sample, the conditioning steps stop here.  Do not let the cartridge go dry at any
                      point during the conditioning process.

               12.1.3 When extracting the acid fraction of a sample, complete the cartridge conditioning
                      step by eluting the cartridge with 6 mL reagent water at pH 2.0  0.5. Discard this
                      eluant.

               12.1.4 Using the SPE cartridge appropriate for the sample fraction (acid or base), load the
                      sample prepared as described in Sections 11.3.3.4  or 11.3.4.4 onto the cartridge at a
                      flow rate of 5-10 mL/min.  Extraction of a 1-L aqueous sample will take 100-200
                      minutes, thus use of a multi-position extraction manifold is desirable.

               12.1.5 Once the entire sample has passed through the cartridge, wash the acid fraction
                      cartridge with 10 mL of reagent water to remove the EDTA.  Do not wash the
                      cartridge for the base fraction.

               12.1.6 Dry the cartridges for either fraction under vacuum for approximately 5 min.

        12.2   Cartridge elution

               12.2.1 Acid fraction

                      12.2.1.1 Elute the analytes with 12 mL methanol.  Initiate the elution by vacuum
                               and complete the elution by gravity. Collect the eluant in a clean
                               centrifuge tube.

                      12.2.1.2 If triclocarban and triclosan are analytes  of interest, elute these two
                               analytes with 6 mL of acetone methanol  (1:1).  Combine with the
                               methanol eluant.

                      12.2.1.3 Proceed with concentration of the extract (Section  12.5).

               12.2.2 Base fraction

                      12.2.2.1 Elute the analytes with 6 mL methanol followed by 9 mL of 2% formic
                               acid solution (Section 7.4.3.1).  Initiate the elution by vacuum and
                               complete the elution by gravity.  Collect the eluant in a clean centrifuge
                               tube.

                      12.2.2.2 Proceed with concentration of the extract (Section  12.5).

        12.3   Acid extraction of solid samples


                                                                                               29

-------
December 2007                                                                      Method 1694
               12.3.1  Add 20 mL acetonitrile to the solid sample and the QC aliquots, sonicate for 30
                       min, and centrifuge for approximately 5 min at approximately 3000 rpm.

               12.3.2  Decant the extracts (supernatants) of the sample and the QC aliquots into separate,
                       clean 250-mL round-bottom flasks.

               12.3.3  Add 15 mL of phosphate buffer (Section 7.4.1) to the sample and the QC aliquots.
                       Adjust to pH 2.0  0.5 with HC1. Vortex to resuspend the solids. Check and
                       adjust the pH to 2.0  0.5 with buffer, vortexing the mixture after the addition.

               12.3.4  Perform a second extraction by repeating Sections 12.3.1 and 12.3.2, adding the
                       extracts to their respective flasks.

               12.3.5  Forthe third extraction, add 15 mL of acetonitrile only to each of the tubes.
                       Sonicate and centrifuge the tubes, and decant the supernatants into their respective
                       round-bottom flasks.

               12.3.6  If particles are visible in the extract, filter through a 110-mm or larger GF/A filter.
                       Using squeeze bottles, rinse the filter three times with reagent water, followed by
                       three rinses with acetonitrile.

               12.3.7  Proceed with concentration of the acid extract (Section 12.6) followed by SPE in
                       12.1 and 12.2.

        12.4   Base extraction of solid samples

               12.4.1  Add 20 mL acetonitrile to the solid sample and QC aliquots, sonicate for 30 min,
                       and centrifuge for approximately 5 min at approximately 3000 rpm.

               12.4.2  Decant the extracts (supernatants) of the sample and QC aliquots into separate,
                       clean 250-mL round-bottom flasks.

               12.4.3  Add 15 mL of reagent water to the sample and QC aliquots. Add NF^OH
                       dropwise to the sample and QC aliquots to pH 10.0  0.5. Vortex to resuspend the
                       solids. Check and adjust the pH to  10.0  0.5 with NFL.OH, vortexing the
                       mixture after the addition.

               12.4.4  Perform a second extraction by repeating Sections 12.4.1 and 12.4.2, adding the
                       extracts to their respective flasks.

               12.4.5  Forthe third extraction, add 15 mL of acetonitrile only to the centrifuge tubes.
                       Sonicate and centrifuge the tubes, and decant the supernatants into the round-
                       bottom flasks.

               12.4.6  If particles are visible in the extract, filter through a 110-mm or larger GF/A filter.
                       Using squeeze bottles, rinse the filter three times with reagent water, followed by
                       three rinses with acetonitrile.
                                                                                                30

-------
December 2007                                                                       Method 1694
               12.4.7  Proceed with concentration of the base extract (Section 12.6) followed by SPE in
                       Section  12.1 and 12.2.

        12.5   Concentration of aqueous sample extracts

               Extracts from the acid and base fractions of aqueous samples are concentrated separately to
               near dryness and the solvent exchanged to methanol, as described below. This same
               procedure is used to concentration the extracts of solid samples after they have been
               subjected to the SPE HLB cleanup procedure in Sections 12.1 -12.2.

               12.5.1  Concentrate the extract to near dryness under a gentle stream of nitrogen in a water
                       bath held at 50  5 C.

               12.5.2  Add 3 mL of methanol to the concentrated acid and base extracts, including the
                       blank  and OPR aliquots.

               12.5.3  Spike  the acid extracts with the labeled injection acid internal standards and the
                       base extracts with the labeled injection base internal standard (Table 10).

               12.5.4  Bring  the acid and base  extracts to a final volume of 4.0  0.1 mL with 0.1%
                       formic acid solution (Section 7.4.3.2). Vortex to mix.

               12.5.5  If visible particles are present in the extract, or if the extract is cloudy, filter
                       through  a 0.2-|o,m filter (Section 6.6.5.

               12.5.6  Transfer 1  mL of each extract to an LC/MS/MS autosampler vial for analysis.
                       Store the remaining 3 mL of extract as backup in a refrigerator. (Other proportions
                       of the  extract may be used as long as sensitivity is not compromised).

               12.5.7  Proceed to Section 14 for analysis.

        12.6   Concentration of the solid sample extracts

               Extracts from the acid and base fractions of solid samples are concentrated separately prior
               to cleanup and the extracts are reconstituted into aqueous solutions that are processed
               through the aqueous sample SPE HLB extraction procedures (Sections 12.1 -12.2) as a
               cleanup step.

               12.6.1  Concentrate the extracts from the acid and base fractions of the solid samples and
                       QC aliquots separately, to a final volume of 20 - 30 mL by rotary evaporation at 50
                       C.  Do not allow the extracts to go dry.

               12.6.2  Immediately after concentration, add 200 mL of reagent water and 500 mg of
                       NA4EDTA.2H2O to the acid fraction extract. Swirl to mix.

               12.6.3  Immediately after concentration, add 200 mL of reagent water to the base fraction
                       extract.  Check that the pH is 10.0 0.5. If necessary, adjust dropwise with
                                                                                                31

-------
December 2007                                                                     Method 1694

                      NH/tOH solution. Swirl to mix.

               12.6.4 Proceed to Section 13 for cleanup of the extracts of all solid samples and associated
                      QC aliquots.
13.0   Extract Cleanup

As noted in Section 12.6, the extracts from all solid samples are subjected to cleanup using the same SPE
procedure used to extract aqueous samples.  In essence, the solvent extract is reconstituted with reagent
water and the pH adjusted to that appropriate for the analytes of interest. The reconstituted sample is
processed through the same  SPE procedure and the final extract is concentrated and prepared for
instrumental analysis. Because the volume of the reconstituted solid sample extract is about 200 mL, the
SPE cleanup will take significantly less time than the extraction of a 1-L water sample. Therefore, it is not
recommended that aqueous sample extractions and cleanup of solid sample extracts be performed
simultaneously on the same  extraction manifold.

        13.1    The acid fraction extract of each  solid sample in Section 12.6.2 is processed through the
               SPE procedure, beginning at Section 12.1.1 and proceeding through Section 12.2.1.3.
               Process the associated QC aliquots (blank and OPR) through the cleanup procedure as well.

        13.2   The base fraction extract of each solid sample in Section 12.6.3 is processed through the
               SPE procedure, beginning at Section 12.1.1 and proceeding through Section 12.1.6, and
               12.2.2.1 through 12.2.2.2, but omitting Sections 12.1.3 and 12.1.5.  Process the associated
               QC aliquots (blank and OPR) through the cleanup procedure as well.

        13.3   After completing the SPE cleanup, concentrate the acid and base extracts of solid samples
               and QC aliquots separately per Section 12.5 and proceed to Section 14 for analysis.
14.0   LC/MS/MS Analysis

        14.1   Establish the same operating conditions established and optimized in Section 10.1 - 10.3
               for the calibration appropriate to the fraction and Group to be analyzed. Analysis is
               performed using positive electrospray ionization (ESI+) for the acid fraction Group 1 and 2
               analytes and the base fraction Group 4 analytes. Analysis is performed by ESI- for the acid
               fraction Group 3 analytes. Retention times (RTs), parent-daughter transitions,
               quantitation references, method detection limits, and minimum levels of quantitation for
               Groups 1, 2, 3, and 4 are given in Tables 3, 5, 7, and 9, respectively.

        14.2   Inject the volume of the concentrated  extract specific to the Group into the LC/MS/MS
               instrument. The volume injected must be identical to the volume chosen in Section
               10.2.1 and used for calibration in Section 10.3.1.

               14.2.1 Start the gradient according to the program appropriate for the Group (see Table
                      2, 4, 6, or 8  for recommended conditions).  Start data collection prior to elution
                      of the first analyte.
                                                                                              32

-------
December 2007                                                                    Method 1694

               14.2.2 Monitor the daughter m/z's for each analyte throughout its retention time
                      window. Where known, monitor m/z's associated with interferents expected to
                      be present.

               14.2.3 Stop data collection after elution of the last analyte in each Group. Return the
                      gradient to the initial mixture for analysis of the next sample extract or standard.
15.0  System and Laboratory Performance

       15.1   At the beginning of each 12-hour shift during which analyses are performed, LC/MS/MS
              system performance and calibration are verified for all native and labeled compounds.
              For these tests, analysis of the CS-3 calibration verification (VER) standard (Section
              7.7.5 and Table 11) must be used to verify all performance criteria. Adjustment and/or
              recalibration (Section 10) must be performed until all performance criteria are met. Only
              after all performance criteria are met may samples, blanks, IPRs, and OPRs be analyzed.

       15.2   Calibration verification

              15.2.1  Inject the VER (CS-3) calibration standard (Table 10) for the Group being
                      analyzed using the procedure in Section 14.

              15.2.2  The LC peak representing each native and labeled compound in the VER
                      standard must be present with a S/N of at least 10; otherwise, the LC/MS/MS
                      system must be adjusted and the verification test repeated.

              15.2.3  Compute the concentration of the native compounds that have labeled analogs by
                      isotope dilution and the concentration of the native compounds that do not have
                      labeled analogs and of the labeled compounds by the internal standard technique.
                      These concentrations are computed based on the calibration data in Section 10.

              15.2.4  For each compound, compare the concentration with the calibration verification
                      limit in Table 12. If all compounds meet the acceptance criteria, calibration has
                      been verified and analysis of standards and sample extracts may proceed.  If,
                      however, any compound fails its respective limit, the measurement system is not
                      performing properly.  In this event, prepare a fresh calibration standard or correct
                      the problem and repeat the verification (Section 15.2) tests, or recalibrate
                      (Section 10).

       15.3   Retention time

              15.3.1 The retention times of the native and labeled compounds in the verification test
                     (Section 15.2) must be  within  15 seconds of the respective retention times in the
                     most recent calibration verification standard.

              15.3.2 If the retention time of any compound is not within the limits specified, the LC is
                     not performing properly.  In this event, adjust the LC operating conditions and
                                                                                            33

-------
December 2007                                                                    Method 1694

                     repeat the verification test (Section 15.3) or recalibrate (Section 10), or replace the
                     LC column and either verify calibration or recalibrate.

       15.4  Ongoing precision and recovery

              15.4.1  Analyze the extracts of both the acid and base fractions of the ongoing precision
                      and recovery (OPR) aliquots prior to analysis of samples from the same batch.

              15.4.2  Compute the percent recovery of each native compound with a labeled analog by
                      isotope dilution (Section 10.4). Compute the percent recovery of each native
                      compound without a labeled  analog and of each labeled compound by the
                      internal standard method (Section 10.5).

              15.4.3  For the native and labeled compounds, compare the recovery to the OPR limits
                      given in Table 12. If all compounds meet the acceptance criteria, system
                      performance is acceptable and analysis of blanks and samples may proceed.  If,
                      however, any individual concentration falls outside of the range given, the
                      extraction/concentration processes are not being performed properly for that
                      compound.  In this event, correct the problem, re-prepare, extract, and clean up
                      the sample batch and repeat the ongoing precision and recovery test (Section
                      15.4).

              15.4.4  If desired, add results that pass the specifications in Section 15.4.3 to initial and
                      previous ongoing data for each compound in each matrix.  Update QC charts to
                      form a graphic representation of continued laboratory performance.  Develop a
                      statement of laboratory accuracy for each compound in each matrix type by
                      calculating the average percent recovery (R) and the standard  deviation of
                      percent recovery (SR). Express the accuracy as a recovery interval from R ! 2SR
                      to R + 2SR.  For example, if R = 95% and SR = 5%, the accuracy is 85 to  105%.

       15.5  Blank - Analyze the method blank extracted with each sample batch immediately
              following analysis of the OPR aliquot to demonstrate that there is no contamination or
              carryover from the OPR analysis. If native compounds will be carried from the OPR into
              the method blank, analyze one or more aliquots of solvent between the OPR and the
              method blank.  Results of analysis of the method blank must meet the specifications in
              Section 9.5.2 before sample analysis may begin.
16.0  Qualitative  Determination

       A native or labeled compound is identified in a standard, blank, or sample when the criteria in
       Sections 16.1 through 16.2 are met.

       16.1    The signal-to-noise ratio (S/N) at the LC peak maximum for each native compound at its
               daughter m/z must be greater than or equal to 2.5 for each compound detected in a
               sample extract, and greater than or equal to 10 in CALs and VER samples for parent to
               daughter transition except S/N of 3 in CS-1.
                                                                                            34

-------
December 2007                                                                   Method 1694

       16.2   The retention time of the peak for a native compound must be within 15 seconds of its
              RT in the most recent CS-3 standard (Table 11).

       16.3   Because of compound RT overlap and the potential for interfering substances, it is
              possible that all of the identification criteria (Sections 16.1 - 16.2) may not be met. If
              identification is ambiguous, an experienced spectrometrist (Section 1.5) must determine
              the presence or absence of the compound.

       16.4   If the criteria for identification in Sections 16.1 - 16.2 are not met, the compound has not
              been identified and the result for that compound may not be reported or used for
              permitting or regulatory compliance purposes. If interferences preclude identification, a
              new aliquot of sample must be analyzed. Refer to Section 18 for guidance.
17.0  Quantitative Determination

       17.1   Isotope dilution quantitation

              17.1.1 By adding a known amount of a labeled compound to every sample prior to
                     extraction, correction for recovery of the native analog of that compound can be
                     made because the native compound and its labeled analog exhibit similar effects
                     upon extraction, concentration, and chromatography. Relative responses (RRs)
                     are used in conjunction with the calibration data in Section 10.4 to determine the
                     concentration in the final extract, as long as labeled compound spiking levels are
                     constant.

              17.1.2 Compute the concentration of each compound in the extract using the RRfrom
                     the calibration data (Section 10.4) and following equation:


                      Cex(ng/mL)=A"C'
                                    A,RR
                     Where:
                     Cex= Concentration of the compound in the extract , and the other terms are as
                           defined in Section 10.4.3

       17.2   Internal standard quantitation and labeled compound recovery

              17.2.1 Compute the concentration of each native compound that does not have labeled
                     analog and each labeled compound using the RF from the calibration data
                     (Section 10.5) and the following equation:

                                          Ce(ng/mL)=
                     Where:
                     Cex= Concentration of the compound in the extract, and the other terms are as
                           defined in Section 10.5.1
                                                                                           35

-------
December 2007                                                                     Method 1694

               17.2.2 Using the concentration in the extract determined above, compute the percent
                      recovery of the labeled compounds using the following equation:

                     T,         ,n/s   Concentration found (ng I mL)   ,_
                     Re cov ery (%) =		 x 100
                                     Concentration spiked (nglmL)

        17.3   The concentration of a native compound in the solid phase of the sample is computed
               using the concentration of the compound in the extract and the weight of the solids, as
               follows:
                                                                 C V
                            Concentration in solid sample (nglkg) = 
                                                                   w,
               Where:
               Cex= Concentration of the compound in the extract.
               Vex = Extract volume in mL.
               Ws = Sample weight (dry weight) in kg.

               If desired, divide the concentration by 1000 to convert ng/kg to (ig/kg.

        17.4   The concentration of a native compound in the aqueous phase of the sample is computed
               using the concentration of the compound in the extract and the volume of water extracted,
               as follows:

                             Concentration in aqueous phase (ng/L) = 1000 x ( x V^)
                                                                             Vs

               Where:
               Cex= Concentration of the compound in the extract.
               Vex = Extract volume in mL.
               Vs = Sample volume in liters.

        17.5   If the SICP area at the daughter quantitation m/z for any compound exceeds the
               calibration range of the system, dilute the sample extract by the factor necessary to bring
               the concentration within the calibration range, adjust the concentration of the labeled
               injection internal standard to the original concentration in the extract, and analyze an
               aliquot of this diluted extract. If the compound cannot be measured reliably by isotope
               dilution, dilute and analyze an aqueous sample or analyze a smaller portion of a solid, or
               other sample.  Adjust the compound concentration, detection limit, and minimum level of
               quantitation to account for the dilution.

        17.6 Reporting of results

             17.6.1 Reporting units and levels

                    17.6.1.1  Aqueous samples - Report results in ng/L (parts-per-trillion).

                    17.6.1.2  Samples containing solids (aqueous samples containing visible
                              particles, solids, soils, sediments, filter cake, compost) - Report results
                              in (ig/kg (parts-per-billion) based on the dry weight of the sample.


                                                                                             36

-------
December 2007                                                                    Method 1694

                              Report the percent solids so that the result may be converted to aqueous
                              units.

             17.6.2 Reporting level

                    17.6.2.1  Report the result for each compound in each sample, blank, or standard
                              (VER, IPR, OPR) at or above the minimum level of quantitation (ML;
                              Table 3, 5, 7, or 9) to 3 significant figures. Report the result below the
                              ML in each sample as 1 ng/L; >1 mg/kg) of the compounds of
               interest, interfering compounds, and/or polymeric materials. The concentration of
               analytes and/or interferences in some extracts may overload the LC column and/or mass
               spectrometer.

       18.2    Analyze a smaller aliquot of the sample (Section 17.5) when the interferences preclude
               analysis of the full sample volume or amount. If a smaller aliquot of a solid, biosolid, or
               mixed-phase sample is analyzed, attempt to assure that the smaller aliquot is
               representative.
                                                                                            37

-------
December 2007                                                                     Method 1694

        18.3   Perform integration of peak areas and calculate concentrations manually when
               interferences preclude computerized calculations.

        18.4   Signal suppression - Coextracted interferences in the sample may suppress signals for the
               compounds of interest. To detect signal suppression, the labeled injection internal
               standard(s) must be monitored in the analysis.  If the signal for the labeled injection
               internal standard is suppressed by more than 30%, as compared to the average signal for
               the labeled injection internal standard in the 5-point calibration, the sample must be
               further cleaned up and reanalyzed.  If the sample cannot be cleaned up further, the sample
               or extract must be diluted, and a diluted sample or extract must be analyzed (Section
               17.5).

        18.5   Recovery of labeled compounds - For most samples, recoveries of the labeled
               compounds will be similar to those  from reagent water or from the alternate matrix
               (Section 7.6 and Table 12).

               18.5.1  If the recovery of any of the labeled compounds is outside of the normal range
                      (Table 12),  a diluted sample must be analyzed (Section 17.5).

               18.5.2 If the recovery of any of the labeled compounds in the diluted sample is outside
                      of normal range, the calibration verification standard (Section 7.7.5 and Table
                      11) must be analyzed and calibration verified (Section 15.2).

               18.5.3 If the calibration cannot be  verified, a new calibration  must be performed and the
                      original sample extract reanalyzed.

               18.5.4 If calibration is verified and the diluted sample does not meet the limits for
                      labeled compound recovery, this method does not apply to the sample being
                      analyzed and the result may not be reported or used for permitting or regulatory
                      compliance purposes. In this case, alternate extraction and cleanup procedures in
                      this method or an alternate  LC column must be  employed to resolve the
                      interference. If all cleanup  procedures in this method and an alternate LC
                      column have been employed and labeled compound recovery remains outside of
                      the normal range, extraction and/or cleanup procedures that are beyond this scope
                      of this method will be required to analyze the sample.


19.0   Pollution Prevention

        19.1    Pollution prevention encompasses any technique that reduces or eliminates the quantity
               or toxicity of waste  at the point of generation. Many opportunities for pollution
               prevention exist in laboratory operation. EPA has established  a preferred hierarchy of
               environmental management techniques that places pollution prevention as the
               management option of first choice.  Whenever feasible, laboratory personnel should use
               pollution prevention techniques to address waste generation. When wastes cannot be
               reduced at the source, the Agency recommends recycling as the next best option.

        19.2   The compounds in this method are used in extremely small amounts and pose little threat
               to the environment when managed properly. Standards should be prepared in volumes
                                                                                             38

-------
December 2007                                                                  Method 1694

              consistent with laboratory use to minimize the disposal of excess volumes of expired
              standards.

       19.3   For information about pollution prevention that may be applied to laboratories and
              research institutions, consult Less is Better: Laboratory Chemical Management for Waste
              Reduction, available from the American Chemical Society's Department of Governmental
              Relations and Science Policy, 1155 16th Street NW, Washington DC 20036, 202/872-
              4477.
20.0  Waste Management

       20.1   The laboratory is responsible for complying with all Federal, State, and local regulations
              governing waste management, particularly the hazardous waste identification rules and
              land disposal restrictions, and to protect the air, water, and land by minimizing and
              controlling all releases from fume hoods and bench operations. Compliance is also
              required with any sewage discharge permits and regulations. An overview of
              requirements can be found in Environmental Management Guide for Small Laboratories
              (EPA233-B-98-001).

       20.2   Samples at pH <2, or pH >12 are hazardous and must be neutralized before being poured
              down a drain,  or must be handled as hazardous waste.

       20.3   The compounds in this method decompose above 500 EC. Low-level waste such as
              absorbent paper, tissues, animal remains, and plastic gloves may be burned in an
              appropriate incinerator. Gross quantities (milligrams) should be packaged securely and
              disposed of through commercial or governmental channels that are capable of handling
              toxic wastes.

       20.4   For further information on waste management, consult The Waste Management Manual
              for Laboratory Personnel and Less is Better-Laboratory Chemical Management for
              Waste Reduction, available from the American Chemical Society's Department of
              Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C.
              20036.
21.0  Method  Performance

       Method 1694 was validated and preliminary data were collected in a single laboratory (Reference
       2). Performance data are given in Table 14.
22.0  References

       1      EPA Methods 610, 1668A, and 8321A

       2      "Analytical Procedure for the Analysis of Pharmaceutical Compounds in Solid and
              Aqueous Samples by LC-MS/MS," Axys Analytical Services proprietary.
                                                                                         39

-------
December 2007                                                                    Method 1694
       3      Previous work on pharmaceuticals and personal-care products

              3a Castiglioni at al, A Multiresidue Analytical Method Using Solid-Phase Extraction and
                  High-Pressure Liquid Chromatography Tandem Mass Spectrometry to Measure
                  Pharmaceuticals of Different Therapeutic Classes in Urban Wastewaters. J.
                  ChromatogrA 1092 (2005), 206-215.

              3b Dana W. Kolpin, Edward T. Furlong etc., Pharmaceuticals, Hormones, and Other
                  Organic Wastewater Contaminants in US Streams,  1999-2000: A National
                  Reconnaissance, Environ.Sci.Technol. 2002, 36,1202-1211.

              3c Michele E. Lindsey, Michael Meyer, and E.M.Thurman, Analysis of Trace Levels of
                  Sulfonamide and Tetracycline Antimicrobials in Groundwater and Surface Water
                  Using Solid-Phase Extraction and Liquid Chromatography/Mass Spectrometry, Anal.
                  Chem. 2001, 73, 4640-4646.

              3d Roman Hirsch, Tomas A. Ternes,  etc., Determination of Antibiotics in Different Water
                  Compartments via LC/ESI-MS/MS, Journal of Chromatography A, 815(1998) 213-
                  223.

              3e Fiese, E.F., and Steffen, S.H., Comparison of the acid stability of azithromycin and
                  erythromycin A, J. Antimicrobial  Chemotherapy, 25 Suppl. A(1990) 39-47.

       4      "Working with Carcinogens," Department of Health, Education,  & Welfare, Public
              Health Service, Centers for Disease Control, NIOSH, Publication 77-206, August 1977,
              NTIS PB-277256.

       5      "OSHA Safety and Health Standards, General Industry," OSHA  2206, 29 CFR 1910.

       6      "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety,
              1979.

       7      "Standard methods for the Examination of Water and Wastewater," 18th edition and later
              revisions,  American Public Health Association, 1015 15th St, N.W., Washington, DC
              20005, 1-35: Section 1090 (Safety), 1992.

       8      Provost, L.P., and Elder, R.S., "Interpretation of Percent Recovery Data," American
              Laboratory, 15: 56-83, 1983.

       9      " Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, 1916
              Race Street, Philadelphia, PA 19103-1187, 1980.

       10     A) Paul E. Stackelberg, Jacob Gibs, Edward T.  Furlong, Michael T. Meyer, Steven D.
              Zaugg, R. Lee Lippincott. Science of the Total Environment 377 (2007)  255-272. B)
              Zhengqi Ye and Howard S. Weinberg and Michael T. Meyer. Anal. Chem., 79 (3), 1135 -
              1144,2007.

       11     "Handbook of Analytical Quality Control in Water and Wastewater Laboratories,"


                                                                                           40

-------
December 2007                                                               Method 1694




             USEPA EMSL, Cincinnati, OH 45268, EPA-600/4-79-019, March 1979.
                                                                                     41

-------
December 2007
Method 1694
23.0  Tables and Flowchart

Table 1. Names and CAS Registry numbers for pharmaceuticals and personal-care products (PPCPs)
determined by isotope dilution and internal standard HPLC/MS/MS
Compound
CAS Registry
Labeled analog
CAS Registry

Acetaminophen
Albuterol
Ampicillin
Anhydrochlortetracycline (ACTC)
Anhydrotetracycline (ATC)
Azithromycin
Caffeine
Carbadox
Carbamazepine
Cefotaxime
Chlortetracycline (CTC)
Cimetidine
Ciprofloxacin
Clarithromycin
Clinafloxacin
Cloxacillin
Codeine
Cotinine
Dehydronifedipine
Demeclocycline
Digoxigenin
Digoxin
Diltiazem
1 ,7-Dimethylxanthine
Diphenhydramine
Doxycycline
Enrofloxacin
4-Epianhydrochlortetracycline
(EACTC)
4-Epianhydrotetracycline (EATC)
4-Epichlortetracycline (ECTC)
4-Epioxytetracycline (EOTC)
4-Epitetracycline (ETC)
Erythromycin
Erythromycin anhydrate
Flumequine
Fluoxetine
Gemfibrozil
103-90-2
18559-94-9
69-53-4
4497-08-9
4496-85-9
83905-01-5
58-08-2
6804-07-5
298-46-4
63527-52-6
57-62-5
51481-61-9
85721-33-1
81103-11-9
105956-97-6
61-72-3
76-57-3
486-56-6
67035-22-7
127-33-3
1672-46-4
20830-75-5
42399-41-7
611-59-6
58-73-1
564-25-0
93106-60-6
158018-53-2
4465-65-0
14297-93-9
14206-58-7
23313-80-6
114-07-8
59319-72-1
42835-25-6
54910-89-3
25812-30-0
1 3 C2-1 5N-Acetaminophen
Albuterol-d3




13C3-Caffeine





13C3-15N-Ciprofloxacin




Cotinine-d3
















































13C2-Erythromycin anhydrate

Fluoxetine-ds
Gemfibrozil -d6



                                                                                   42

-------
December 2007
Method 1694
Compound
Ibuprofen
Isochlortetracycline (ICTC)
Lincomycin
Lomefloxacin
Metformin
Miconazole
Minocycline
Naproxen
Norfloxacin
Norgestimate
Ofloxacin
Ormetoprim
Oxacillin
Oxolinic acid
Oxytetracycline (OTC)
Penicillin V
Penicillin G
Ranitidine
Roxithromycin
Sarafloxacin
Sulfachloropyridazine
Sulfadiazine
Sulfadimethoxine
Sulfamerazine
Sulfamethazine
Sulfamethizole
Sulfamethoxazole
Sulfanilamide
Sulfathiazole
Tetracycline (TC)
Thiabendazole
Triclocarban
Triclosan
Trimethoprim
Tylosin
Virginiamycin
Warfarin
CAS Registry
15687-27-1
514-53-4
154-21-2
98079-51-7
657-24-9
22916-47-8
10118-91-8
22204-53-1
70458-96-7
35189-28-7
82419-36-1
6981-18-6
66-79-5
14698-29-4
79-57-2
87-08-1
61-33-6
66357-35-5
80214-83-1
98105-99-8
80-32-0
68-35-9
122-11-2
127-79-7
57-68-1
144-82-1
723-46-6
63-74-1
72-14-0
60-54-8
148-79-8
101-20-2
3380-34-5
738-70-5
1401-69-0
11006-76-1
81-81-2
Labeled analog
13C3-Ibuprofen



Metformin-de


13C-Naproxen-d3
















1 3 Ce-Sulfamethazine

1 3 Ce-Sulfamethoxazole



Thiabendazole-d6
1 3 C6-Triclocarban
13Ci2-Triclosan
1 3 C3 -Trimethoprim


Warfarin-d5
CAS Registry





































Other standards
Unlabeled compound spiked into sample and used for recovery correction
Meclocycline



Labeled injection internal standard spiked into sample extract prior to injection into LC/MS/MS




13C3-Atrazine

13C6-2,4,5-Trichlorophenoxyacetic acid
(13C6-TCPAA)
                                                                                 43

-------
December 2007
Method 1694
Table 2.  Group 1 - Acidic extraction, positive electrospray ionization (ESI+) instrument conditions
Instrument
LC Column
Ionization
Acquisition
Injection Volume
Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro
Ultima MS/MS
Waters Xtera C18, 10.0 cm, 2.1 mm i.d., 3.5 urn particle size
Positive Ion Electrospray
MRM mode, unit resolution
15 ul
LC Gradient Program
Time
(min)
0.0
4.0
22.5
23.0
26.0
26.5
33.0
Flow Mixture1
95% Solvent A
5% Solvent B
95% Solvent A
5% Solvent B
12% Solvent A
88% Solvent B
100% Solvents
100% Solvents
95% Solvent A
5% Solvent B
95% Solvent A
5% Solvent B
LC Flow Rate
(mL/min)
0.150
0.250
0.300
0.300
0.300
0.150
0.150
Gradient
1
6
6
6
6
6
6
General LC Conditions
Column Temp
Flow Rate
Max Pressure
Autosamplertray
temperature
40 C
0.15-0.30
mL/min
345 Bar
4C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
140C
350C
80L/hr/
400 L/hr
   Solvent A = 0.3% Formic Acid and 0.1% Ammonium Formate in HPLC water
  Solvent B = 1:1 Acetonitrile:Methanol
                                                                                              44

-------
December 2007
Method 1694
Table 3.  Group 1 acidic extraction, positive electrospray ionization (ESI+) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation.
Analyte
Group 1
Native compounds
Sulfanilamide
Cotinine
Acetaminophen
Sulfadiazine
1 ,7-Dimethylxanthine
Sulfathiazole
Codeine
Sulfamerazine
Lincomycin
Caffeine
Sulfamethizole
Trimethoprim
Thiabendazole
Sulfamethazine
Cefotaxime
Carbadox
Ormetoprim
Norfloxacin
Sulfachloropyridazine
Ofloxacin
Ciprofloxacin
Sulfamethoxazole
Lomefloxacin
Enrofloxacin
Sarafloxacin
Clinafloxacin
RT (min)
Parent-
daughter
m/zs
Quantitation reference
Detection limits and minimum levels
Water (ng/L)
MDL
ML
Other (ng/kg)
MDL | ML
Extract (ng/
-------
December 2007
Method 1694
Analyte
Digoxigenin
Oxolinic acid
Sulfadimethoxine
Diphenhydramine
Penicillin G
Azithromycin
Flumeqine
Ampicillin
Diltiazem
Carbamazepine
Penicillin V
Erythromycin
Tylosin
Oxacillin
Dehydronifedipine
Digoxin
Fluoxetine
Cloxcillin
Virginiamycin
Clarithromycin
Erythromycin anhydrate
Roxithromycin
Miconazole
Norgestimate
RT (min)
12.6
13.1
13.2
14.5
14.6
14.8
15.2
15.3
15.3
15.3
15.4
15.9
16.3
16.4
16.5
16.6
16.9
16.9
17.3
17.5
17.7
17.8
20.1
21.7
Parent-
daughter
m/zs
391.2-355.2
261.8-243.8
311.0-156.0
256.8-168.1
367.5 - 160.2
749.9-591.6
262.0 - 173.7
350.3 - 160.2
415.5 - 178.0
237.4 - 194.2
383.4-160.2
734.4-158.0
916.0-772.0
434.3 - 160.1
345.5-284.1
803.1-283.0
310.3-148.0
469.1-160.1
508.0-355.0
748.9 - 158.2
716.4-158.0
837.0-679.0
417.0-161.0
370.5 - 124.0
Quantitation reference
13C3-Trimethoprim
13C3-Trimethoprim
1 3C6-Suhcamethoxazole
13C3-Trimethoprim
13C3-Trimethoprim
13C3-Trimethoprim
13C3-Trimethoprim
13C3-Trimethoprim
13C3-Trimethoprim
13C3-Trimethoprim
13C3-Trimethoprim
1 3C2-Ery thromy cin
13C2-Ery thromy cin anhydrate
13C3-Trimethoprim
13C3-Trimethoprim
13C3-Trimethoprim
Fluoxetine-ds
13C3-Trimethoprim
13C3-Trimethoprim
13C2-Ery thromy cin anhydrate
13C2-Ery thromy cin anhydrate
13C2-Ery thromy cin anhydrate
13C3-Trimethoprim
13C3-Trimethoprim
Detection limits and minimum levels
Water (ng/L)
MDL
5.7
0.6
0.1
0.4
2.4
1.3
2.7

0.6
1.4
4.4

13
3.3
0.6

3.7
4.3
3.6
1.0
0.4
0.2
1.3
2.5
ML
20
2
1
2
10
5
5
5
2
5
20
1
50
10
2
50
10
10
10
5
2
1
5
10
Other (ng/g)
MDL
9.4
0.62
0.55
0.66
13
1.6
1.4

0.30
1.6
19

8.1
9.4
0.41

2.8
9.2
3.4
1.2
0.46
0.22
0.90
1.4
ML
20
2
2
2
50
5
5
5
2
5
50
2
50
20
2
100
10
20
10
5
2
1
5
10
Extract (ng/OL)
MDL
1.4
0.2
0.03
0.1
0.6
0.3
0.7

0.2
0.4
1.1

3.2
0.8
0.2

0.9
0.1
0.9
0.3
0.1
0.05
0.3
0.6
ML
5
0.5
0.25
0.5
2.5
1.25
1.25
1.25
0.25
1.25
5
0.25
5
2.5
0.5
12.5
1.25
2.5
2.5
1.25
0.25
0.25
1.25
2.5
Labeled compounds spiked into each sample
Cotinine-d3
1 3C2-: 5N- Acetaminophen
13C3 Caffeine
Thiabendazole-de
13C3-Trimethoprim
13C6 Sulfamethazine
13C3 15N-Ciprofloxacin
1 3C6-Sulfamethoxazole
1 3C2-Ery thromy cin
2.8
4.5
9.3
9.8
10.0
10.1
10.9
11.2
15.9
180.0-79.9
155.2-111.0
198.0 - 140.0
208.1-180.1
294.0-233.0
285.1 - 162.0
336.1-318.0
260.0 - 162.0
736.4 - 160.0
13C3 Atrazine
13C3 Atrazine
13C3 Atrazine
13C3 Atrazine
13C3 Atrazine
13C3 Atrazine
13C3 Atrazine
13C3 Atrazine
13C3 Atrazine






















































                                                                                                                46

-------
December 2007
Method 1694
Analyte
Fluoxetine-ds
13C2-Erythromycin anhydrate
RT (min)
16.8
17.7
Parent-
daughter
m/zs
315.3-153.0
718.4-160.0
Quantitation reference
13C3 Atrazine
13C3 Atrazine
Detection limits and minimum levels
Water (ng/L)
MDL


ML


Other (Og/g)
MDL


ML


Extract (ng/
-------
December 2007
                                                            Method 1694
Table 4. Group 2 - Acidic extraction positive electrospray ionization (ESI+) instrument conditions
  Instrument
  LC Column
  Ionization
  Acquisition
  Injection Volume
Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro Ultima MS/MS
Waters Xtera C18, 10.0 cm, 2.1 mm i.d., 3.5 urn particle size
Positive Ion Electrospray
MRM mode, unit resolution
5uL
LC Gradient Program
Time
(min)
0.0
1.0
18.0
20.0
24.0
24.3
28
Flow Mixture1
10% Solvent A
90% Solvent B
10% Solvent A
90% Solvent B
40% Solvent A
60% Solvent B
90% Solvent A
10% Solvents
90% Solvent A
10% Solvents
10% Solvent A
90% Solvent B
10% Solvent A
90% Solvent B
LC Flow
Rate
(mL/min)
0.20
0.20
0.23
0.23
0.23
0.20
0.20
Gradient
1
6
6
6
6
6
6
General LC Conditions
Column Temp
Flow Rate
Max Pressure
Autosamplertray
temperature
40 C
0.20-0.23
mL/min
345 Bar
4C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
120C
400C
70L/hr/
450 L/hr
  Solvent A = 1:1 acetonitrile:methanol, with 5 mM Oxalic Acid
  Solvent B = HPLC H2O, with 5 mM Oxalic Acid
                                                                                            48

-------
December 2007
Method 1694
Table 5. Group 2 acidic extraction positive electrospray ionization (ESI+) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation.
Analyte
Group 2
Native compounds
Minocycline
Epitetracycline
Epioxytetracycline (EOTC)
Oxytetracycline (OTC)
Tetracycline (TC)
Demeclocycline
Isochlortetracycline (ICTC) l
Epichlortetracycline (ECTC) l
Chlortetracycline (CTC)
Doxycycline
Epianhydrotetracycline (EATC)
Anhydrotetracycline (ATC)
Epianhydrochlortetracycline (EACTC)
Anhdrochlortetracycline (ACTC)
RT
(min)
Parent-
daughter
m/zs
Quantitation reference
Detection limits and minimum levels
Water (ng/L)
MDL
ML
Other (ng/g)
MDL
ML
Extract (ng/|j,L)
MDL
ML
Analytes Extracted Under Acidic Conditions and Analyzed Using Positive Electrospray Ionization (+) ESI.

5.1
8.1
8.6
9.4
9.9
11.7
11.9
12.0
14.1
16.7
17.0
18.8
20.7
22.1

458.0-441.0
445.2-410.2
461.2-426.2
461.2-426.2
445.2-410.2
465.0-430.0
479.0 - 462.2
479.0 - 444.0
479.0 - 444.0
445.2 - 428.2
426.8 - 409.8
426.8 - 409.8
461.2-444.0
461.2-444.0

Thiabendazole-d6
Thiabendazole-de
Thiabendazole-d6
Thiabendazole-d6
Thiabendazole-de
Thiabendazole-de
Thiabendazole-de
Thiabendazole-de
Thiabendazole-d6
Thiabendazole-d6
Thiabendazole-de
Thiabendazole-d6
Thiabendazole-d6
Thiabendazole-de

51
3.6
4.1
2.1
1.9
6.6
1.7
7.7
1.2
2.8
7.7
4.6
28
5.2

200
20
20
20
20
50
20
50
20
20
50
50
200
50


8.6
18
2.2
2.8
7.9
3.5
26
2.3
2.3
14
7.1
23
11

200
20
50
20
20
50
20
100
20
20
50
50
200
50

13
0.9
1.0
0.5
0.5
1.7
0.4
1.9
0.3
0.7
1.9
1.2
7.0
1.3

50
5
5
5
5
12.5
5
12.5
5
5
12.5
12.5
50
12.5
Labeled compound spiked into each sample
Thiabendazole-d6
7.0
208.1-180.1
13C3 Atrazine






Injection internal standard
13C3 Atrazine
10.5
219.5-176.9
(134.0)
External standard






1. Isochlortetracycline (ICTC) is reported as the sum ICTC + ECTC due to a common transition ion.
                                                                                                                                   49

-------
December 2007
                                                           Method 1694
Table 6. Group 3 - Acidic extraction negative electrospray ionization (ESI-) instrument conditions
  Instrument
  LC Column
  Ionization
  Acquisition
  Injection Volume
Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro Ultima  MS/MS
Waters Xtera C18MS, 10.0cm, 2.1 mm i.d., 3.5 urn particle size
Negative Ion Electrospray
MRM mode, unit resolution
15 uL
LC Gradient Program
Time
(min)
0.0
0.5
7.0
12.5
12.7
16.0

Flow Mixture
60% Solvent A,
40% Solvent B
60% Solvent A,
40% Solvent B
100% Solvents
100%SolventB
60% Solvent A,
40% Solvent B
60% Solvent A,
40% Solvent B

LC Flow
Rate
(mL/min)
0.2
0.2
0.2
0.2
0.2
0.2

Gradient
1
6
6
6
6
1

General LC Conditions
Column Temp
Flow Rate
Max Pressure
Autosamplertray
temperature
40C
0.200 mL/min
345 Bar
4C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
100C
350C
50L/hr/
300 L/hr
1.  Solvent A = 0.1% Ammonium Acetate and 0.1% Acetic Acid in HPLC water
   Solvent B = 1:1 MethanolAcetonitrile
                                                                                          50

-------
December 2007
Method 1694
Table 7. Group 3 acidic extraction negative electrospray ionization (ESI-) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation
Analyte
Group 3
Native compounds
Naproxen
Warfarin
Ibuprofen
Gemfibrozil
Triclocarban
Triclosan
RT
(min)
Parent- daughter
m/zs
Quantitation reference
Detection limits and minimum levels
Water (ng/L)
MDL
ML
Other (Og/g)
MDL
ML
Extract (ng/OL)
MDL
ML
Analytes Extracted Under Acidic Conditions and Analyzed Using Negative Electrospray Ionization (-) ESI.

6.7
7.1
8.4
9.5
9.6
9.7

228.9 - 168.6
307.0-117.0
205.1-161.1
249.0-121.0
312.9-159.7
286.8-35.0

13C-Naproxen-d3
Warfarin-ds
13C3-Ibuprofen
Gemfibrozil-d6
13C6-Triclocarban
13C12-Triclosan

3.9
0.9
6.0
0.8
2.1
92

10
5
50
5
10
200

6.1
1.6
11
1.2
2.7
56

20
5
50
5
10
200

1.0
0.2
1.5
0.2
0.5
23

2.5
1.25
12.5
1.25
2.5
50
Labeled compounds spiked into samples
13C-Naproxen-d3
Warfarin-ds
13C3-Ibuprofen
Gemfibrozil-d6
13C6-Triclocarban
13C12-Triclosan
6.6
7.0
8.5
9.5
9.6
9.7
232.9 - 168.6
312.0-161.0
208.2-163.1
255.0-121.0
318.9-159.7
298.8-35.0
13C6-TCPAA
13C6-TCPAA
13C6-TCPAA
13C6-TCPAA
13C6-TCPAA
13C6-TCPAA




































Injection Internal Standard
13C6-TCPAA
4.9
258.8 - 200.7
External standard






                                                                                                                                  51

-------
December 2007
                                                            Method 1694
Table 8. Group 4 - Basic extraction positive electrospray ionization (ESI+) instrument conditions
  Instrument
  LC Column
  Ionization
  Acquisition
  Purge Solvent
  Injection Volume
Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro Ultima MS/MS
Waters Atlantis HILIC, 10 cm, 2.1 mm i.d., 3.0 urn particle size
Positive Ion Electrospray
MRM mode, unit resolution
100% CH3CN (changed from H2O)
2.0 u.L
LC Gradient Program
Time
(min)
0.0
5.0
12.0
12.5
16.0


Flow Mixture1
2% Solvent A
98% Solvent B
30% Solvent A
70% Solvent B
30% Solvent A
70% Solvent B
2% Solvent A
98% Solvent B
2% Solvent A
98% Solvent B


LC Flow
Rate
(mL/min)
0.25
0.25
0.25
0.25
0.25


Gradient
1
6
6
6
6


General LC Conditions
Column Temp
Flow Rate
Max Pressure
Autosamplertray
temperature
40 C
0.25 mL/min
345 Bar
4C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
120C
350C
70L/hr/
400 L/hr
1.  Solvent A = 0.1% Acetic Acid/Ammonium Acetate Buffer
   Solvent B = Acetonitrile
                                                                                           52

-------
December 2007
Method 1694
Table 9. Group 4 basic extraction positive electrospray ionization (ESI+) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation
Analyte
Group 4
RT
(min)
Parent-
daughter
m/zs
Quantitation reference
Detection limits and minimum levels
Water (ng/L)
MDL
ML
Other (ng/g)
MDL
ML
Extract (ng/OL)
MDL
ML
Analytes Extracted Under Basic Conditions and Analyzed Using Positive Electrospray Ionization (+) ESI
Native compounds
Cimetidine
Albuterol
Ranitidine
Metformin
6.9
9.4
10.3
11.0
253.1-159.0
240.0 - 148.0
315.0-175.9
131.1-60.1
Albuterol-d3
Albuterol-d3
Albuterol-d3
Metformin-de
0.6
0.9
0.7
23
2
2
2
100
0.78
0.39
1.1
38
2
2
2
100
0.2
0.2
0.2
5.8
0.5
0.5
0.5
25
Labeled compounds spiked into samples
Albuterol-d3
Metformin-de
9.4
11.0
243.0-151.0
285.1 - 162.0
Cotinine-d3
Cotinine-d3












Injection internal standard
Cotinine-d3
13C3-Atrazine
5.9
2.0
180.0-79.9
219.5-176.9
(134.0)
External standard
External Standard












                                                                                                                                  53

-------
December 2007
Method 1694
Table 10. Nominal concentrations of native compounds, labelled compounds, and instrument internal
standard solutions :
Compound Name
Native compound spike solutions for acid
extracted analytes (Groups 1 and 3)
Acetaminophen
Azithromycin
Caffeine
Carbodox
Carbamazapine
Cefotaxime
Clarithromycin
Cloxacillin
Codeine
Cotinine
Dehydronifedipine (Oxidized Nifedipine)
Diphenhydramine
Diltiazem
Digoxin
Digoxigenin
Erythromycin
Flumequine
Fluoxetine
Lincomycin
Miconazole
Norgestimate
Ormetoprim
Oxacillin
Oxolinic acid
Penicillin G
Penicillin V
Roxithromycin
Sulfachloropyridazine
Sulfadiazine
Sulfadimethoxine
Sulfamerazine
Sulfamethazine
Sulfamethizole
Sulfamethoxazole
Sulfanilamide
Spiking solution
concentration (iig/mL)

100
2.5
25
2.5
2.5
10
2.5
5
5
2.5
1
1
0.5
25
10
0.5
2.5
2.5
5
2.5
5
1
5
1
5
5
0.5
2.5
2.5
0.5
1
1
1
1
25
Typical amount spiked
into sample
(ng)
(Typical spiking volume
into sample: 30 uL)
3000
75
750
75
75
300
75
150
150
75
30
30
15
750
300
15
75
75
150
75
150
30
150
30
150
150
15
75
75
15
30
30
30
30
750
                                                                                          54

-------
December 2007
Method 1694
Compound Name
Sulfathiazole
Thiabendazole
Trimethoprim
Tylosin
Virginiamycin
1 ,7-Dimethylxanthine
Ampicillin
Ciprofloxacin
Clinafloxacin
Enrofloxacin
Lomefloxacin
Norfloxacin
Ofloxacin
Sarafloxacin
Gemfibrozil
Ibuprofen
Naproxen
Triclocarban
Triclosan
Warfarin
Native compound spike solutions for
tetracyclines (Group 2)
Tetracycline (TC)
Oxytetracycline (OTC)
Doxycycline
Chlortetracycline (CTC)
Anhydrochlortetracycline (ACTC)
Anhydrotetracycline (ATC)
4-Epianhydrochlortetracycline (EACTC)
4-Epianhydrotetracycline (EATC)
4-Epichlortetracycline (ECTC)
4-Epioxytetracycline (EOTC)
4-Epitetracycline (ETC)
Isochlortetracycline (ICTC)
Demeclocycline
Minocycline
Native compound spike solutions for base
extracted analytes (Group 4)
Albuterol
Cimetidine
Spiking solution
concentration (iig/mL)
2.5
2.5
2.5
10
5
250
2.5
8.75
10
5
5
25
2.5
22.8
2.5
25
5
5
100
2.5

0.5
0.5
0.5
0.5
1.25
1.25
5
1.25
1.25
0.5
0.5
0.5
1.25
5

1
2
Typical amount spiked
into sample
(ng)
75
75
75
300
150
7500
75
263
300
150
150
750
75
684
75
750
150
150
3000
75
(Typical spiking volume
into sample: 200 uL)
100
100
100
100
250
250
1000
250
250
100
100
100
250
1000
(Typical spiking volume
into sample: 15 uL)
15
30
                                                                                 55

-------
December 2007
Method 1694
Compound Name
Metformin
Ranitidine
Labeled compound solutions for acid
extracted analytes (Groups 1 , 2 and 3)
Meclocycline
d10-Carbamazepine-10,11-epoxide
ds-Cotinine
ds-Fluoxetine
d6-Gemfibrozil
13C2, 15N-Acetaminophen
13C6-Sulfamethoxazole
13C, d3-Naproxen
13C6-Triclocarban
13C3-Trimethoprim
d6-Thiabendazole
13C3-Caffeine
13C2-Erythromycin
13Ci2-Triclosan
ds-Warfarin
13C6-Sulfamethazine
13C3, 15N-Ciprofloxacin
13C3-lbuprofen
Labeled compound solutions for base
extracted analytes (Group 4)
d3-Albuterol
d6-Metformin
Instrument internal standard solutions for
acid extracted analytes (Groups 1 , 2 and 3)
13C3-Atrazine
13C6-2,4,5-Trichlorophenoxyaceticacid
Instrument internal standard solutions for
base extracted analytes (Group 4)
13C3-Atrazine
d3-Cotinine
Spiking solution
concentration (iig/mL)
100
2

8
2
2
1
1
4
1
3
0.5
1
1
3
1
4
1
1
4
4

1
4

2.5
2.5

2
2
Typical amount spiked
into sample
(ng)
1500
30
(Typical spiking volume
into sample: 100 uL)
800
200
200
100
100
400
100
300
50
100
100
300
100
400
100
100
400
400
(Typical spiking volume
into sample: 100 uL)
100
400
(Typical spiking volume
into extract: 80 uL)
200
200
(Typical spiking volume
into extract: 100 uL)
200
200
1.  See Sections 7.8 - 7.9 for solution details
                                                                                               56

-------
December 2007
Method 1694
Tables lla-c. Concentrations of calibration solutions (ng/mL)

Table 1 la  Concentrations of calibration standards for Group 1 and Group 3 compounds (ng/mL) (Acid
extraction, positive and negative ESI). CS=calibration standard.
Compound
Acetaminophen
Azithromycin
Caffeine
Carbadox
Carbamazapine
Cefotaxime
Clarithromycin
Cloxacillin
Codeine
Cotinine
Dehydronifedipine (Oxidized Nifedipine)
Diphenhydramine
Diltiazem
Digoxin
Digoxigenin
Erythromycin
Erythromycin anhydrate
Flumequine
Fluoxetine
Lincomycin
Miconazole
Norgestimate
Ormetoprim
Oxacillin
Oxolinic acid
Penicillin G
Penicillin V
Roxithromycin
Sulfachloropyridazine
Sulfadiazine
Sulfadimethoxine
Sulfamerazine
Sulfamethazine
Sulfamethizole
Sulfamethoxazole
Sulfanilamide
CS-1
50
1.25
12.5
1.25
1.25
5
1.25
2.5
2.5
1.25
0.5
0.5
0.25
12.5
5
0.25
0.25
1.25
1.25
2.5
1.25
2.5
0.5
2.5
0.5
2.5
5
0.25
1.25
1.25
0.25
0.5
0.5
0.5
0.5
12.5
CS-2
150
3.75
37.5
3.75
3.75
15
3.75
7.5
7.5
3.75
1.5
1.5
0.75
37.5
15
0.75
0.75
3.75
3.75
7.5
3.75
7.5
1.5
7.5
1.5
7.5
15
0.75
3.75
3.75
0.75
1.5
1.5
1.5
1.5
37.5
CS-3 (VER)
750
18.7
187.
18.7
18.7
75
18.7
37.5
37.5
18.7
7.5
7.5
3.7!
187
75
3.7!
3.7!
18.7
18.7
37.5
18.7
37.5
7.5
37.5
7.5
37.5
75
3.7!
18.7
18.7
3.7!
7.5
7.5
7.5
7.5
187.5
CS-4
2500
62.5
625
62.5
62.5
250
62.5
125
125
62.5
25
25
12.5
625
250
12.5
12.5
62.5
62.5
125
62.5
125
25
125
25
125
250
12.5
62.5
62.5
12.5
25
25
25
25
625
CS-5
10000
250
2500
250
250
1000
250
500
500
250
100
100
50
2500
1000
50
50
250
250
500
250
500
100
500
100
500
1000
50
250
250
50
100
100
100
100
2500
                                                                                            57

-------
December 2007
Method 1694
Compound
Sulfathiazole
Thiabendazole
Trimethoprim
Tylosin
Virginiamycin
1,7-Dimethylxanthine
Ampicillin
Ciprofloxacin
Clinafloxacin
Enrofloxacin
Lomefloxacin
Norfloxacin
Ofloxacin
Sarafloxacin
Gemfibrozil
Ibuprofen
Naproxen
Triclocarban
Triclosan
Warfarin
Labeled compounds
ds-Cotinine
ds-Fluoxetine
de-Gemfibrozil
13C2, 15N-Acetaminophen
13Ce-Sulfamethoxazole
13C-d3-Naproxen
13C6-Triclocarban
13C3-Trimethoprim
de-Thiabendazole
13C3-Caffeine
13C2-Erythromycin
13Ci2-Triclosan
ds-Warfarin
13C6-Sulfamethazine
13C3, 15N-Ciprofloxacin
13C3-lbuprofen
CS-1
1.25
1.25
1.25
5
2.5
125
1.25
4.4
5
2.5
2.5
12.5
1.25
11.4
1.25
12.5
2.5
2.5
50
1.25

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100
CS-2
3.75
3.75
3.75
15
7.5
375
3.75
13.1
15
7.5
7.5
37.5
3.75
34.2
3.75
37.5
7.5
7.5
150
3.75

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100
CS-3 (VER)
18.7
18.7
18.7
75
37.5
1870
18.7
65.6
75
37.5
37.5
187
18.7
171
18.7
187
37.5
37.5
750
18.7

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100
CS-4
62.5
62.5
62.5
250
125
6250
62.5
218.
250
125
125
625
62.5
570
62.5
625
125
125
2500
62.5

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100
CS-5
250
250
250
1000
500
25000
250
875
1000
500
500
2500
250
2280
250
2500
500
500
10000
250

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100
                                                                                 58

-------
December 2007
Method 1694
Compound
Instrument internal standards
13C3-Atrazine
13Ce-2,4,5-Trichlorophenoxyacetic acid
CS-1

50
50
CS-2

50
50
CS-3 (VER)

50
50
CS-4

50
50
CS-5

50
50
Table 1 Ib  Concentrations of calibration standards for Group 2 compounds (ng/mL) (Acid extraction,
positive ESI). CS=calibration standard.
Compound name
Tetracycline (TC)
Oxytetracycline (OTC)
Doxycycline
Chlortetracycline (CTC)
Anhydrochlortetracycline (ACTC)
Anhydrotetracycline (ATC)
4-Epianhydrochlortetracycline (EACTC)
4-Epianhydrotetracycline (EATC)
4-Epichlortetracycline (ECTC)
4-Epioxytetracycline (EOTC)
4-Epitetracycline (ETC)
Isochlortetracycline (ICTC)
Demeclocycline
Minocycline
Labeled compounds
ds-Cotinine
ds-Fluoxetine
de-Gemfibrozil
13C2, 15N-Acetaminophen
13Ce-Sulfamethoxazole
13C, ds-Naproxen
13C6-Triclocarban
13C3-Trimethoprim
de-Thiabendazole1
13C3-Caffeine
13C2-Erythromycin
13Ci2-Triclosan
ds-Warfarin
13C6-Sulfamethazine
13C3, 15N-Ciprofloxacin
13C3-lbuprofen
Instrument internal standards
CS-1
5
5
5
5
12.5
12.5
50
12.5
12.5
5
5
5
12.5
50

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100

CS-2
12.5
12.5
12.5
12.5
31.25
31.25
125
31.2
31.2
12.5
12.5
12.5
31.2
125

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100

CS-3 (VER)
25
25
25
25
62.5
62.5
250
62.5
62.5
25
25
25
62.5
250

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100

CS-4
50
50
50
50
125
125
500
125
125
50
50
50
125
500

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100

CS-5
150
150
150
150
375
375
1500
375
375
150
150
150
375
1500

50
25
25
100
25
75
12.5
25
25
75
25
90
25
25
100
100

                                                                                           59

-------
December 2007
Method 1694
Compound name
13C3-Atrazine1
13C6-2,4,5-Trichlorophenoxyacetic acid
CS-1
50
50
CS-2
50
50
CS-3 (VER)
50
50
CS-4
50
50
CS-5
50
50
1. Note: The Group 2, acid extracted positive ESI (tetracyclines) contains the same labeled compounds as
for Group 1 and 3,  acid extracted positive and negative ESI,  yet the only  labeled compounds used in
determination  of the Group 2 are Thiabendazole-d6 and  13C3-Atrazine.   This  minimizes the work
required to prepare solutions. Some of those surrogates are used to quantify the Group 1 and 2 and some
Group 3 in separate runs of the same extract. This is not a requirement.
Table lie  Concentrations of calibration standards for Group 4 (ng/mL) compounds (Base extraction,
positive ESI). CS=calibration standard.
Compound name
Albuterol
Cimetidine
Metformin
Ranitidine
Labeled compounds
ds-Albuterol
de-Metformin
Instrument internal standards
13C3-Atrazine
ds-Cotinine
CS-1
0.25
0.5
25
0.5

25
100

50
50
CS-2
0.75
1.5
75
1.5

25
100

50
50
CS-3 (VER)
3.75
7.5
375
7.5

25
100

50
50
CS-4
12.5
25
1250
25

25
100

50
50
CS-5
50
100
5000
100

25
100

50
50
                                                                                            60

-------
December 2007
Method 1694
Table 12. QC acceptance criteria for PPCPs in VER, IPR, OPR, and samples.
Compound
Acetaminophen
Albuterol
Ampicillin
Anhydrochlortetracycline (ACTC)
Anhydrotetracycline (ATC)
Azithromycin
Caffeine
Carbadox
Carbamazepine
Cefotaxime
Chlortetracycline (CTC)
Cimetidine
Ciprofloxacin
Clarithromycin
Clinafloxacin
Cloxacillin
Codiene
Cotinine
Dehydronifedipine
Demeclocycline
Digoxigenin
Digoxin
Diltiazem
1 ,7-Dimethylxanthine
Diphenhydramine
Doxycycline
Enrofloxacin
4-Epianhydrochlortetracycline (EACTC)
4-Epianhydrotetracycline (EATC)
4-Epichlortetracycline (ECTC)
4-Epioxytetracycline (EOTC)
4-Epitetracycline (ETC)
Erythromycin hydrate
Flumequine
Fluoxetine
Gemfibrozil
Ibuprofen
Isochlortetracycline (ICTC)
Lincomycin
Lomefloxacin
Metformin
Miconazole
Minocycline
VER
(%)
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
IPR
RSD
(%)
30
30
70
30
30
30
30
30
30
36
31
47
30
30
37
30
30
30
30
30
30
45
48
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
60
33
30
30
30
X
(%)
55 - 108
55 - 120
6- 180
55-121
8-127
36 - 108
55- 111
36-130
23 - 123
9- 168
49 - 155
6-108
55 - 108
8- 139
6-180
6-180
37-116
55-112
47 - 108
6- 180
8-165
6-133
13 - 108
55 - 124
53 - 108
24 - 149
55- 113
20 - 108
6-180
55-135
55 - 127
55 - 156
55 - 142
39 - 180
54- 112
55 - 108
55 - 108
6- 180
6-108
19-180
55-134
29 - 108
6-159
OPR
(%)
50 - 120
50-133
5-200
50 - 135
7-141
33 - 120
50 - 124
33 - 144
21-137
8-186
45 - 172
5-120
50 - 120
8-154
5-200
5-200
34 - 129
50 - 124
42 - 120
5-200
8-183
5-148
11-120
50 - 138
48 - 120
22 - 166
50 - 125
18 - 120
5-200
50 - 150
50 - 142
50 - 173
50-158
36 - 200
49 - 125
50 - 120
50 - 120
5-200
5-120
17 - 200
50 - 149
27 - 120
5-176
Labeled compound
recovery in samples (%)











































                                                                                    61

-------
December 2007
Method 1694

Naproxen
Norfloxacin
Norgestimate
Ofloxacin
Ormetoprim
Oxacillin
Oxolinic acid
Oxytetracycline (OTC)
Penicillin V
Penicillin G
Ranitidine
Roxithromycin
Sarafloxacin
Sulfachloropyridazine
Sulfadiazine
Sulfadimethoxine
Sulfamerazine
Sulfamethazine
Sulfamethizole
Sulfamethoxazole
Sulfanilamide
Sulfathiazole
Tetracycline (TC)
Thiabendazole
Triclocarban
Triclosan
Trimethoprim
Tylosin
Virginiamycin
Warfarin

1 3C2-: 5N- Acetaminophen
Albuterol-d3
13C3-Caffeine
13C3-15N-Ciprofloxacin
Cotinine-d3
13C2-Erythromycin hydrate
Fluoxetine-d5
Gemfibrozil-d6
13C3-Ibuprofen
Metformin-d6
13C-Naproxen-d3
1 3C6-Suhcamethazine
1 3C6-Suhcamethoxazole
Thiabendazole-d6 (A Pos)
Thiabendazole-d6 (TCY)
13C6-Triclocarban

70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130

70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
70 - 130
IPR
30
30
30
30
30
30
30
30
30
30
41
30
32
30
30
30
30
30
30
30
71
30
30
30
30
30
30
30
33
30

30
30
46
34
84
30
30
30
30
30
30
30
30
30
30
30
55 - 108
55-121
39 - 108
55 - 180
55 - 108
6- 180
46-112
55 - 165
6- 180
6-180
26 - 144
42 - 108
18 - 180
55 - 180
6- 180
55 - 108
55-133
55 - 128
55 - 108
55 - 108
6-170
45 - 108
55 - 139
55 - 108
55 - 108
55 - 108
55-114
17-134
6- 170
55 - 108

6-180
38 - 109
6-180
6-180
6- 108
55 - 108
55-113
42- 110
31 -109
6-127
37-118
6-141
55- 131
55-132
55 - 108
6- 155

50 - 120
50 - 135
36 - 120
50 - 200
50 - 120
5-200
42 - 124
50 - 183
5-200
5-200
24 - 160
38 - 120
17 - 200
50 - 200
5-200
50 - 120
50 - 148
50 - 142
50 - 120
50 - 120
5-189
41 - 120
50-155
50 - 120
50 - 120
50 - 120
50 - 126
16 - 149
5-189
50 - 120

5-200
35-121
5-200
5-200
5-120
50 - 120
50 - 126
38 - 122
28 - 122
5-141
34-131
5-157
50 - 146
50 - 146
50 - 120
5-172
































19-200
39-141
31-200
37-181
5-145
23 - 120
40 - 148
21-123
29 - 127
5-200
14-132
12-120
40 - 129
32-140
30-132
5-147
                                                                                 62

-------
December 2007
Method 1694

13C12-Triclosan
1 3C3-Trimethoprim
Warfarin-d5

70 - 130
70 - 130
70 - 130
IPR
30
30
30
6-151
55 - 162
55 - 159

5-168
50 - 180
50 - 177

5-153
50 - 172
50 - 200
                                                                                 63

-------
December 2007
Method 1694
Table 13. Suggested sample quantities to be extracted for various matrices'
Sample matrix2
Example
Percent
solids
Phase
Quantity
extracted
Single-phase
Aqueous
Solid
Drinking water
Groundwater
Treated wastewater
Dry soil
Filter cake
Compost
No visible
particles
>20
Aqueous
Solid
1000 mL
lg
Multi-phase
Liquid/Solid
Aqueous/solid2
Wet soil
Untreated effluent
Municipal sludge
1-30
1 -5
1-30
Aqueous and
solid
lg
0.25 g
1.   The quantity of sample to be extracted is adjusted to provide 1 g of solids (dry weight). One liter of aqueous
    samples containing 0.1% solids will contain 1 gram of solids. For aqueous samples containing greater than
    0.1% solids, a lesser volume is used so that 1 gram of solids (dry weight) will be prepared.
2.   1 g of solids (0.25 g for biosolids), or 5 g wet weight if solids content is <20%.
                                                                                                    64

-------
December 2007
Method 1694
Table 14. Performance Data from single laboratory validation.
Analyte
Solid-Based on 5 samples
Solids
Average
Recovery
Solids
Standard
Deviation
Solids
Relative
Standard
Deviation
Reagent Water-Based on 5 samples
Water
Average
Recovery
Water
Standard
Deviation
Water
Relative
Standard
Deviation
Biosolids-Based on 6 samples
Biosolids
Average
Recovery
Biosolids
Standard
Deviation
Biosolids
Relative
Standard
Deviation
Group 3 acidic extraction ESI-
Warfarin
Ibuprofen
Gemfibrozil
Naproxen
Triclocarban
Triclosan
d5- Warfarin
13C3-lbuprofen
d6-Gemfibrozil
13C-d3-Naproxen
13C6-Triclocarban
13012-Triclosan
90.76
103.16
97.94
96.57
100.60
97.52
113.44
59.25
65.37
65.52
20.36
42.75
5.96
3.63
2.84
6.23
2.36
6.78
8.21
2.34
2.85
5.45
1.38
3.75
6.57
3.52
2.90
6.45
2.34
6.95
7.23
3.95
4.36
8.31
6.78
8.77
86.52
97.43
98.11
95.41
106.09
93.14
143.13
90.20
94.94
98.95
100.55
108.28
3.59
3.70
2.45
5.03
4.81
3.35
8.22
8.04
3.16
3.99
3.10
5.80
4.15
3.80
2.50
5.27
4.53
3.60
5.74
8.91
3.33
4.03
3.09
5.36
119.64
93.82
78.35
99.94
265.00
359.73
145.03
74.30
65.80
55.18
54.18
71.62
13.67
7.96
21.19
10.10
190.08
500.34
23.97
21.35
28.35
20.45
36.10
34.48
11.42
8.48
27.05
10.10
71.73
139.09
16.52
28.74
43.09
37.06
66.62
48.15
Group 1 acidic extraction ESI+
Acetaminophen
Azithromycin
Caffeine
Carbadox
Carba maze pine
Cefotaxime
Ciprofloxacin
Clarithromycin
Clinafloxacin
Cloxacillin
Codeine
Cotinine
Dehydronifedipine
Diphenhydramine
Diltiazem
Digoxin
Digoxigenin
Enrofloxacin
Erythromycin-H2O
Flumequine
Fluoxetine
Lincomycin
Lomefloxacin
Miconazole
Norfloxacin
Norgestimate
104.50
66.08
96.41
107.42
98.84
122.83
95.76
54.67
172.32
261 .54
97.65
96.04
84.14
66.76
66.96
92.33
126.01
97.44
136.67
151.31
88.09
55.95
179.94
51.31
101.34
58.06
3.53
11.99
10.08
3.78
6.85
4.32
3.54
4.07
31.99
15.78
1.79
3.79
6.80
2.94
3.47
13.28
1.96
11.24
3.12
7.43
16.86
15.38
32.28
4.73
6.89
3.52
3.38
18.15
10.45
3.52
6.93
3.52
3.70
7.44
18.57
6.03
1.83
3.94
8.09
4.40
5.19
14.39
1.55
11.54
2.28
4.91
19.14
27.49
17.94
9.23
6.80
6.06
100.85
60.95
99.14
69.50
59.14
71.75
99.66
106.90
76.12
60.77
64.66
102.27
66.50
68.99
55.23
52.22
64.36
96.37
113.95
91.15
85.89
17.70
106.07
73.81
114.79
48.26
1.17
6.80
6.38
7.48
4.92
23.92
2.20
2.64
5.13
3.90
5.36
9.75
7.17
8.42
20.89
18.64
8.55
7.08
3.09
4.08
4.27
1.70
6.19
6.50
5.07
3.61
1.16
11.15
6.44
10.76
8.32
33.33
2.21
2.47
6.74
6.42
8.29
9.53
10.78
12.20
37.83
35.69
13.28
7.34
2.71
4.48
4.97
9.58
5.83
8.81
4.41
7.48
94.25
86.53
86.08
52.91
91.50
173.69
73.93
69.53
171.73
166.24
141.78
92.34
126.82
103.43
160.04
22.60
79.57
108.72
100.45
92.35
100.48
198.99
79.59
55.79
63.02
49.20
4.97
25.39
5.95
13.50
19.27
15.86
69.08
12.17
24.34
11.42
11.87
4.56
13.52
20.40
114.83
18.11
11.63
6.71
7.46
14.79
19.64
13.38
9.63
17.71
7.17
7.61
5.27
29.34
6.91
25.52
21.06
9.13
93.44
17.50
14.17
6.87
8.38
4.94
10.66
19.73
71.75
80.13
14.61
6.17
7.42
16.02
19.55
6.72
12.10
31.74
11.37
15.47
                                                                                          65

-------
December 2007
Method 1694
Ofloxacin
Ormetoprim
Oxacillin
Oxolinic Acid
Penicillin G
Penicillin V
Roxithromycin
Sarafloxacin
Sulfachloropyridazine
Sulfadiazine
Sulfadimethoxine
Sulfamerazine
Sulfamethazine
Sulfamethizole
Sulfamethoxazole
Sulfanilamide
Sulfathiazole
Thiabendazole
Trimethoprim
Tylosin
Virginiamycin
1 ,7 DimethylXanthine
1 3C2-1 5N-Acetaminophen
13C3-Caffeine
d3-Cotinine
1 3C3-N 1 5-Ciprofloxacin
1 3C2-Erythromycin-H2O
d5-Fluoxetine
13C6-Sulfamethazine
1 3C6-Sulfamethoxazole
d6-Thiabendazole
13C3-Trimethoprim
166.98
64.83
168.38
96.72
214.04
195.93
61.28
146.84
158.30
158.51
78.65
115.08
119.60
75.61
103.21
99.94
59.06
106.47
103.24
60.99
116.39
100.64
258.79
203.04
28.08
66.74
97.49
92.68
54.80
85.21
92.75
121.40
26.78
2.86
15.50
2.48
14.66
9.43
3.61
25.89
8.72
17.49
3.44
13.12
5.59
8.69
2.97
29.94
3.39
1.44
3.23
9.93
10.65
16.81
19.66
50.76
9.20
18.81
7.73
8.40
111
9.29
8.63
12.12
16.04
4.41
9.20
2.56
6.85
4.81
5.89
17.63
5.51
11.03
4.37
11.40
4.67
11.49
2.88
29.96
5.74
1.35
3.13
16.28
9.15
16.70
7.60
25.00
32.76
28.19
7.93
9.07
14.17
10.90
9.30
9.98
127.81
66.94
60.02
69.17
58.83
61.80
85.57
87.70
115.36
80.11
87.00
90.48
100.67
93.86
88.17
20.71
76.73
99.83
80.82
103.48
43.62
95.73
112.20
115.82
3.20
1 44.50
86.25
103.69
105.70
1 1 1 .77
117.76
1 44.35
12.16
3.44
6.22
6.30
6.77
7.81
2.23
4.25
3.88
2.33
2.95
1.01
6.19
4.52
3.63
0.95
3.22
1.92
5.65
9.59
15.26
7.16
4.48
7.57
0.35
16.99
2.83
6.05
11.12
9.32
4.64
9.96
9.51
5.14
10.36
9.10
11.51
12.63
2.60
4.84
3.36
2.91
3.39
1.12
6.15
4.82
4.12
4.59
4.20
1.93
6.99
9.27
34.99
7.48
3.99
6.54
10.92
11.75
3.28
5.84
10.52
8.34
3.94
6.90
78.44
78.76
163.73
108.07
99.09
157.80
83.70
108.36
90.24
107.55
67.87
136.01
103.35
70.14
102.56
130.84
92.10
81.89
98.81
47.80
172.33
137.15
137.17
119.47
68.57
132.12
54.62
94.67
50.78
72.67
66.75
94.08
34.96
5.96
11.69
12.09
14.24
9.85
19.61
8.57
9.86
12.80
9.14
9.46
11.96
3.00
12.66
8.89
8.07
6.03
5.35
14.56
42.36
38.02
28.54
9.50
19.19
13.74
13.12
35.78
7.28
10.63
5.19
12.15
44.57
7.56
7.14
11.19
14.37
6.24
23.42
7.91
10.92
11.90
13.46
6.96
11.57
4.28
12.34
6.79
8.76
7.36
5.42
30.46
24.58
27.72
20.80
7.95
27.99
10.40
24.03
37.79
14.35
14.63
7.78
12.91
Group 4 basic extraction ESI+
Albuterol
Cimetidine
Metformin
Ranitidine
d3-Albuterol
d6-Metformin
100.43
37.37
115.61
79.99
91.52
94.38
7.12
10.69
9.40
13.90
8.47
13.05
7.09
28.60
8.13
17.37
9.25
13.83
90.04
64.93
103.72
103.66
63.83
51.66
14.09
12.83
13.16
22.67
2.41
6.86
15.65
19.75
12.69
21.87
3.78
13.28
96.58
52.77
89.06
71.15
105.42
161.13
1.88
14.30
3.32
7.22
19.25
67.48
1.95
27.09
3.72
10.15
18.26
41.88
Group 2 acidic extraction ESI+
Chlortetracycline
4-Epichlortetracycline
Anhydrochlortetracycline
4-Epianhydrochlortetracycline
Isochlortetracycline
Demeclocycline
Doxvcvcline
121.24
112.41
92.62
53.57
65.88
54.53
67.83
6.98
8.71
10.43
1.87
5.01
1.96
2.96
5.75
7.75
11.27
3.49
7.61
3.59
4.36
95.24
96.83
102.22
82.28
149.37
136.58
119.65
22.32
15.65
11.51
13.31
16.06
3.18
1.01
23.44
16.16
11.26
16.18
10.75
2.33
0.85
1 1 4.43
95.59
50.40
33.88
91.65
76.03
87.03
45.67
32.60
21.73
8.30
25.51
31.01
34.42
39.91
34.11
43.12
24.49
27.83
40.79
39.55
                                                                                 66

-------
December 2007
Method 1694
Oxytetracycline
4-Epioxytetracycline
Tetracycline
4-Epitetracycline
4-Epianhydrotetracycline
Anhydrotetracycline
d6-Thiabendazole
112.85
119.40
93.41
138.95
70.11
50.21
77.07
3.12
6.94
3.95
3.42
6.87
4.13
4.76
2.77
5.81
4.23
2.46
9.80
8.23
6.18
148.84
122.38
1 24.79
102.11
170.82
98.14
64.80
5.76
6.25
4.69
4.02
22.25
2.50
3.21
3.87
5.11
3.76
3.94
13.02
2.55
4.95
74.46
83.55
77.98
97.37
67.87
86.20
89.82
16.46
18.09
19.24
37.03
23.27
34.27
15.10
22.10
21.65
24.68
38.03
34.29
39.76
16.81
                                                                                 67

-------
December 2007
                                                                                      Method 1694

Aqueous
Filte
Centrifuf

> Sample
r, or
e + filter

Solid Portion (If required) L

Aqueous Portion
Up to 1000 ml






Solid
Up to 1 g dry equivalent, not exceeding 5 g wet


15 ml pH 2.0
Phosphate Buffer
-
.
Add labeled
compounds, vortex


15 ml water
NH4OHtopH 10.0
1
Add labeled
compounds, vortex
                                                       15 ml pH 2.0 Phosphate Buffer
                                                          20 ml CH3CN, vortex
                                                           sonicate, centrifuge
                                                                   15 ml water NH4OH to pH 10.0
                                                                       20 ml CH3CN, vortex
                                                                       sonicate, centrifuge
                                                           Rotary evaporation
                                                                Dilute
                                                          (200 ml reagent water),
                                                            no pH adjustment
                                                            500 mg Na4EDTA
                 SPE HLB 20 cc/1 g
                      condition
                                                      SPE HLB 20 cc/1 g
                                                            condition
   Acid
      Load,
  wash 10 ml water,
     dry 5 min,
elutewith 12 ml MeOH
      Load,
     dry 5 min,
elute with 6 ml MeOH,
9 ml 2% FA in MeOH
Elute with 6 ml acetone:MeOH 1
1
                     N2 blowdown,
                reconstitute in 3 ml MeOH,
              add injection internal standards,
             dilute to 4 ml with 0.1 % FA buffer,
                       vortex
                                                                                                    Base
                                                          N2 blowdown,
                                                      reconstitute in 3 ml MeOH,
                                                    add injection internal standards,
                                                   dilute to 4 ml with 0.1% FA buffer,
                                                             vortex
Figure 1   Flow chart for determination of Pharmaceuticals and personal-care products by LC/MS/MS
                                                                                                                    68

-------
December 2007                                                                     Method 1694
24.0   Glossary
       These definitions and purposes are specific to this method but have been conformed to
       common usage to the extent possible.

       24.1    Units of weight and measure and their abbreviations

               24.1.1  Symbols

                       EC  degrees Celsius
                       OL  microliter
                       Om  micrometer
                       <    less than
                       >    greater than
                       %   percent

               24.1.2  Abbreviations (in alphabetical order)
                       cm  centimeter
                       g    gram
                       h    hour
                       ID   inside diameter
                       in.   inch
                       L    liter
                       M   Molecular ion
                       m   mass or meter
                       mg  milligram
                       min  minute
                       mL  milliliter
                       mm  millimeter
                       m/z  mass-to-charge ratio
                       N   normal; gram molecular weight of solute divided by hydrogen
                            equivalent of solute, per liter of solution
                       OD  outside diameter
                       pg   picogram
                       ppb  part-per-billion
                       ppm part-per-million
                       ppq  part-per-quadrillion
                       ppt  part-per-trillion
                       psig pounds-per-square inch gauge
                       v/v  volume per unit volume
                       w/v  weight per unit volume

       24.2    Definitions and acronyms (in alphabetical order)

               Analyte - A pharmaceutical or personal-care product tested for by this method.
               The analytes are listed in Table 1.

               Calibration standard (CAL) - A solution prepared from a secondary standard
               and/or stock solution and used to calibrate the response of the HPLC/MSMS
                                                                                      69

-------
December 2007                                                                     Method 1694
               instrument.

               Calibration verification standard (VER) - The mid-point calibration standard (CS-
               4) that is used to verify calibration. See Table 4.

               CS-1, CS-2, CS-3, CS-4, CS-5, CS-6 - See Calibration standards and Table 4.

               Field blank - An aliquot of reagent water or other reference matrix that is placed in
               a sample container in the field, and treated as  a sample in all respects, including
               exposure to sampling site conditions, storage, preservation, and all analytical
               procedures. The purpose of the field blank is to determine if the field or sample
               transporting procedures and environments have contaminated the sample.

               GPC - Gel permeation chromatograph or gel  permeation chromatography

               HPLC - High performance liquid chromatograph or high performance liquid
               chromatography

               Labeled injection internal standard - A labeled spiked into the concentrated extract
               immediately prior to injection of an aliquot of the extract into the LC/MS/MS.

               Internal standard - a labeled compound used as a reference for quantitation of other
               labeled compounds and for quantitation of a native compound other than the
               compound of which it is a labeled analog. See Internal standard quantitation.

               Internal standard quantitation - A means of determining the concentration of (1) a
               naturally occurring  (native) compound by reference to a compound other than its
               labeled analog and (2)  a labeled compound by reference to another labeled
               compound.

               IPR - Initial precision  and recovery; four aliquots of a reference matrix spiked with
               the analytes of interest and labeled compounds and analyzed to establish the ability
               of the laboratory to  generate acceptable precision and recovery.  An IPR is
               performed prior to the  first time this method is used and any time the method or
               instrumentation is modified.

               Isotope dilution quantitation - A means of determining a naturally occurring
               (native) compound by reference to the same compound in which one or more
               atoms has been isotopically enriched. In this method, labeled are enriched with
               deuterium to produce 2H labeled analogs or carbon-13 to produce 13C-labeled
               analogs. The labeled analogs are spiked into each sample to allow identification
               and correction of the concentration of the native compounds in the analytical
               process.

               Labeled compound - A molecule in which one or more of the  atoms is isotopically
               entriched, thereby increasing the mass of the molecule

               Laboratory blank -  See method blank

               Laboratory control sample (LCS) - See Ongoing precision and recovery standard
               (OPR)
                                                                                       70

-------
December 2007                                                                      Method 1694



               Laboratory reagent blank - See method blank

               May - This action, activity, or procedural step is neither required nor prohibited.

               May not - This action, activity, or procedural step is prohibited.

               Method blank - An aliquot of reagent water that is treated exactly as a sample
               including exposure to all glassware, equipment, solvents, reagents, internal
               standards, and surrogates that are used with samples. The method blank is used to
               determine if analytes or interferences are present in the laboratory environment, the
               reagents, or the apparatus.

               Method detection limit (MDL) - The lowest concentration at which an analyte can
               be detected under routine operating conditions (see 40 CFR 136, appendix B).
               MDLs are listed in Table 3, 5, 7, and 9.

               Minimum level (ML) - The greater of a multiple of the MDL or the lowest
               calibration point (see 68 FR 11790, March 12, 2003.) MLs are listed in Tables 3,
               5, 7, and 9.

               MS - Mass spectrometer or mass spectrometry

               Must - This action, activity, or procedural step is required.

               Native compound - A molecule in which the atoms all have naturally occuring
               isotopic abundances

               OPR - Ongoing precision and recovery standard (OPR); a method blank spiked
               with known quantities of analytes. Also known as a "laboratory control sample"
               (LCS). The OPR is analyzed exactly like a sample.  Its purpose is to assure that the
               results produced by the laboratory remain within the limits specified in this method
               for precision and recovery.

               Preparation blank - See method blank

               Quality control check sample (QCS) - A sample containing all or a subset of the
               analytes at known concentrations.  The QCS is obtained from a source external to
               the laboratory or is prepared from a source of standards different from the source of
               calibration standards. It is used to check laboratory performance with test
               materials prepared external to the normal preparation process.

               Reagent water - water demonstrated to be free from the analytes of interest and
               potentially interfering substances at the method detection limit for the analyte.

               Relative standard deviation (RSD) - The standard deviation times 100 divided by
               the mean.  Also termed  "coefficient of variation."

               RF - Response factor. See Section 10.5

               RR- Relative response. See Section 10.4
                                                                                        71

-------
December 2007                                                                       Method 1694
               RSD - See relative standard deviation

               Signal-to-noise ratio (S/N) - The height of the signal as measured from the mean
               (average) of the noise to the peak maximum divided by the width of the noise.

               Should - Although this action, activity, or procedural step is suggested and not
               required, you may be asked to explain why you changed or omitted this action,
               activity, or procedural step.

               SICP - Selected ion current profile; the line described by the signal at an exact
               m/z.

               SPE - Solid-phase extraction; an extraction technique in which an analyte is
               extracted from an aqueous solution by passage over or through a material capable
               of reversibly adsorbing the analyte. Also termed liquid-solid extraction.

               Stock solution - A solution containing an analyte that is prepared using a reference
               material traceable to EPA, the National Institute of Science and Technology
               (NIST), or a source that will attest to the purity and authenticity of the reference
               material.

               VER - See Calibration verification.
                                                                                         72

-------