V>EPA Method 1694: Pharmaceuticals and
Personal Care Products in Water,
Soil, Sediment, and Biosolids by
HPLC/MS/MS
December 2007
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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
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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
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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
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December 2007 Method 1694
24.0 GLOSSARY 69
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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.
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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.
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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
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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).
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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.
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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
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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.
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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
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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
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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 (Na4EDTA»2H2O ~+99.5%
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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.
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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.
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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.
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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.
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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
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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).
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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
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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
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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
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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).
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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.
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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
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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 10.0±0.5
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
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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.
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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 10±0.5 (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
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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.
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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.
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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
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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
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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.
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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
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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.
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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
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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.
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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
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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.
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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
4°C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
140°C
350°C
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
4°C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
120°C
400°C
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
40°C
0.200 mL/min
345 Bar
4°C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
100°C
350°C
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
4°C
MS Conditions
Source Temp
Desolvation Temp
Cone / Desolvation
Gas Rate
120°C
350°C
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)
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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
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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.
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