METHOD 532.     DETERMINATION OF PHENYLUREA COMPOUNDS IN
                 DRINKING WATER BY SOLID PHASE EXTRACTION AND HIGH
                 PERFORMANCE LIQUID CHROMATOGRAPHY WITH UV
                 DETECTION
                                Revision 1.0

                                 June 2000
M. V. Bassett, S.C. Wendelken, T.A. Dattilio, and B.V. Pepich (IT Corporation)
D.J. Munch (US EPA, Office of Ground Water and Drinking Water)
Method 532, Revision 1.0 (2000)
               NATIONAL EXPOSURE RESEARCH LABORATORY
                  OFFICE OF RESEARCH AND DEVELOPMENT
                U. S. ENVIRONMENTAL PROTECTION AGENCY
                          CINCINNATI, OHIO 45268
                                  532-1

-------
                                     METHOD 532

   DETERMINATION OF PHENYLUREA COMPOUNDS IN DRINKING WATER BY
         SOLID PHASE EXTRACTION AND HIGH PERFORMANCE LIQUID
                     CHROMATOGRAPHY WITH UV DETECTION
1.      SCOPE AND APPLICATION

       1.1     This is a high performance liquid chromatographic (HPLC) method for the
              determination of phenylurea pesticides in drinking waters. This method is applicable to
              phenylurea compounds that are efficiently extracted from the water using a C18 solid
              phase cartridge or disk. Accuracy, precision, and method detection limit (MDL) data
              have been generated for the following compounds in reagent water and finished ground
              and surface waters:

                                               Chemical Abstracts Service
              Analyte                               Registry Number

              Diflubenzuron                          35367-38-5
              Diuron                               330-54-1
              Fluometuron                           2164-17-2
              Linuron                               330-55-2
              Propanil                               709-98-8
              Siduron                               1982-49-6
              Tebuthiuron                           34014-18-1
              Thidiazuron                           51707-55-2

       1.2     MDLs are compound, instrument, and matrix dependent. The MDL is defined as the
              statistically calculated minimum concentration that can be measured with 99%
              confidence that the reported value is greater than zero.(1)  Experimentally determined
              MDLs for the above listed analytes are provided in Section 17, Table 3.  The MDL
              differs from,  and is usually lower than (but never above), the minimum reporting limit
              (MRL) (Sect. 3.16).  The concentration range for target analytes in this method was
              evaluated between 1.0 ug/L and 30 ug/L for a 500 mL sample. Precision and accuracy
              data and sample holding time data are presented in Section 17, Tables 4-9.

       1.3     This method is restricted to use by or under the supervision of analysts skilled in solid
              phase extraction (SPE), and HPLC analysis.
                                         532-2

-------
2.     SUMMARY OF METHOD

       2.1     A 500 mL water sample is passed through a SPE cartridge or disk containing a
              chemically bonded C18 organic phase to extract the phenylurea pesticides and surrogate
              compounds. The analytes and surrogates are eluted from the solid phase with
              methanol, and the extract is concentrated to a final volume of 1 mL. Components are
              then chromatographically separated by injecting an aliquot of the extract into an HPLC
              system equipped with a C18 column and detected using a UV/Vis detector.
              Identification of target and surrogate analytes and quantitation is accomplished by
              comparison of retention times and analyte responses using external standard
              procedures. Sample extracts with positive results are solvent exchanged and confirmed
              using a second,  dissimilar HPLC column that is also calibrated using external standard
              procedures.

3.     DEFINITIONS

       3.1     EXTRACTION BATCH - A set of up to 20 field samples (not including QC samples)
              extracted together by the same person(s) during a work day using the same lot of solid
              phase extraction devices and solvents, surrogate solution, and fortifying solutions.
              Required QC samples include: Laboratory Reagent Blank, Laboratory Fortified Blank,
              Laboratory Fortified Matrix, and either a Field Duplicate or Laboratory Fortified
              Matrix Duplicate.

       3.2     ANALYSIS BATCH - A set of samples that is analyzed on the same instrument during
              a 24-hour period that begins and ends with the analysis of the appropriate Continuing
              Calibration Check standards (CCC). Additional  CCCs may be required depending on
              the length of the analysis batch and/or the number of Field Samples.

       3.3     SURROGATE  ANALYTE (SUR) - A pure analyte, which is extremely unlikely to be
              found in any sample, and which is added to a sample aliquot in known amount(s) before
              extraction or other processing and is measured with the same procedures used to
              measure other sample components. The purpose of the SUR is to monitor method
              performance with each sample.

       3.4     LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
              blank matrix that is treated exactly as a sample including exposure to all glassware,
              equipment, solvents, reagents, sample preservatives, and surrogates that are used in the
              extraction batch. The LRB is used to determine if method analytes or other
              interferences are present in the laboratory environment, the reagents, or the apparatus.

       3.5     LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or other
              blank matrix to  which known quantities of the method analytes  and all the preservation
                                          532-3

-------
       compounds are added. The LFB is analyzed exactly like a sample, and its purpose is to
       determine whether the methodology is in control, and whether the laboratory is capable
       of making accurate and precise measurements.

3.6    LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an envi-
       ronmental sample to which known quantities of the method analytes and all the
       preservation compounds are added in the laboratory. The LFM is analyzed exactly
       like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results.  The background concentrations of the analytes in the
       sample matrix must be determined in a separate aliquot and the measured values in the
       LFM corrected for background concentrations.

3.7    LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFMD) - A
       second aliquot of the Field Sample used to prepare the LFM which is fortified,
       extracted, and analyzed identically. The LFMD is used instead of the Field Duplicate
       to access method precision and accuracy when the occurrence of target analytes is low.

3.8    LABORATORY DUPLICATES (LD1  and LD2) - Two aliquots of the same sample
       taken in the laboratory and analyzed separately with identical procedures. Analyses of
       LD1 and LD2 indicate precision  associated with laboratory procedures, but not with
       sample collection, preservation, or storage procedures.

3.9    FIELD DUPLICATES (FD1 and FD2) - Two separate  samples collected at the same
       time and place under identical circumstances, and treated exactly the same throughout
       field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the
       precision associated with sample  collection, preservation, and storage.

3.10   STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing one
       or more method analytes prepared in the laboratory using assayed reference materials
       or purchased from a reputable commercial source.

3.11   PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution containing
       method analytes  prepared in the laboratory from stock standard solutions and diluted as
       needed to prepare calibration solutions and other needed analyte solutions.

3.12   CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
       standard solution or stock standard solution and the surrogate analytes. The CAL
       solutions are used to calibrate the instrument response with respect to analyte
       concentration.

3.13   CONTINUING  CALIBRATION CHECK (CCC) - A  calibration standard
       containing one or more of the method analytes, which is analyzed periodically to verify
       the accuracy of the existing calibration for those analytes.
                                   532-4

-------
       3.14   QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
              concentrations that is obtained from a source external to the laboratory and different
              from the source of calibration standards. It is used to check standard integrity.

       3.15   METHOD DETECTION LIMIT (MDL) - The minimum concentration of an analyte
              that can be identified, measured and reported with 99% confidence that the analyte
              concentration is greater than zero.  This is a statistical determination of precision (Sect.
              9.2.4).  Accurate quantitation is not expected at this level.(1)

       3.16   MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be
              reported as a quantitated value for a target analyte in a sample following analysis.  This
              defined concentration can be no lower than the concentration of the lowest continuing
              calibration standard for that analyte, and can only be used if acceptable quality control
              criteria for this standard are met.

       3.17   MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided by
              vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and
              reactivity data including storage, spill,  and handling precautions.

       3.18   PEAK GAUSSIAN FACTOR (PGF) - The peak gaussian factor is calculated using
              the equation in Section 10.2.3.1. It provides a quantitative measure of peak
              asymmetry.  A perfectly symmetric peak would have a PGF of 1. Poor peak symmetry
              can result in imprecise quantitation, degraded resolution and poor retention
              reproducibility. For this reason, columns and conditions that produce symmetric peaks
              are required.

4.     INTERFERENCES

       4.1    All glassware must be meticulously cleaned. Wash glassware with  detergent and tap
              water, rinse with tap water, followed by reagent water.  A final rinse with solvents may
              be needed. In place of a solvent rinse, non-volumetric glassware can be heated in a
              muffle furnace at 400 C for 2 hours.  Volumetric glassware should not be heated
              above 120 C.

       4.2    Method interferences may also be caused by contaminants in solvents, reagents
              (including reagent water), sample bottles and caps, and other sample processing
              hardware that lead to discrete artifacts and/or elevated baselines in the chromatograms.
              All items such as these must be routinely demonstrated to be free from interferences
              (less than V3 the MRL for each analyte) under the conditions of the  analysis by
              analyzing laboratory reagent blanks as described in Section 9.3.  Subtracting blank
              values from sample results is not permitted.

                                           532-5

-------
              4.2.1  An extraneous peak was noted that elutes very near fluometuron on the
                     confirmation column that can cause problems with quantitation if the
                     chromatography is not fully optimized. No interferences were observed for the
                     primary column analysis.

       4.3    Matrix interferences may be caused by contaminants that are co-extracted from the
              sample. The extent of matrix interferences will vary considerably from source to
              source, depending upon the nature and diversity of the matrix being sampled.  Water
              samples high in total organic carbon may have an elevated baseline and/or interfering
              peaks.

       4.4    Solid phase cartridges and disks and their associated extraction devices have been
              observed to be a source of interferences in other EPA organic methods.  The analysis
              of field and laboratory reagent blanks can provide important information regarding the
              presence or absence of such interferences. Brands and lots of solid phase extraction
              devices should be monitored to ensure that contamination will not preclude analyte
              identification or quantitation.

5.     SAFETY

       5.1    The toxicity or carcinogenicity of each reagent used in this method has not been
              precisely defined. Each chemical compound should be treated  as a potential health
              hazard, and exposure to these chemicals should be minimized.  The laboratory is
              responsible for maintaining an awareness of OSHA regulations  regarding the safe
              handling of the chemicals used in this method. A reference file  of MSDSs should also
              be made available to all personnel involved in the  chemical analysis. Additional
              references to laboratory safety are available.(2"4)

       5.2    Pure standard materials and stock standards of these compounds should be handled
              with suitable protection to skin and eyes,  and care should be taken not to breath the
              vapors or ingest the materials.

6.     EQUIPMENT AND SUPPLIES (All  specifications are suggested. Brand names and/or
       catalog numbers are included for illustration only.)

       6.1    SAMPLE CONTAINERS - 500 mL amber or clear glass bottles fitted with PTFE
              (polytetrafluoroethylene) lined screw caps.

       6.2    VIALS - Screw cap or crimp top glass autosampler vials with PTFE faced septa,
              amber or clear.

       6.3    VOLUMETRIC FLASKS - Class A, suggested  sizes include  l,5,and 10 mL.

                                            532-6

-------
6.4    GRADUATED CYLINDERS -Suggested sizes include 5,10, and 500 mL.

6.5    MICRO SYRINGES - Various sizes.

6.6    ANALYTICAL BALANCE - Capable of accurately weighing to the nearest 0.0001
       g-

6.7    DISPOSABLE SYRINGES - 1 mL (B-D cat.#: 309602 or equivalent) size, used to
       filter sample extracts before analysis.

6.8    FILTERS - Disposable filters to filter sample extracts before analysis (Gelman 0.45 um
       Nylon Acrodisk cat.#: 4426 or equivalent).

6.9    SOLID PHASE EXTRACTION (SPE) APPARATUS USING CARTRIDGES

       6.9.1   EXTRACTION CARTRIDGES - 6 mL, packed with 500 mg (40 um dp)
             silica bonded with C18 (Varian cat.#: 1210-2052 or equivalent).

       6.9.2  SAMPLE RESERVOIRS - (VWR cat.#: JT7120-3 or equivalent) These are
             attached to the cartridges and water samples are poured into them, although
             they hold only 75 mL at one time. An alternative is a transfer tube system
             (Supelco "Visiprep"; cat. #: 57275 or equivalent) which transfers the sample
             directly from the sample container to the SPE cartridge.

       6.9.3   VACUUM EXTRACTION MANIFOLD - With flow/vacuum control
             (Supelco cat.#: 57044 or equivalent). The use of replaceable needles or valve
             liners may be used to prevent cross contamination.

       6.9.4  REMOTE VACUUM GAUGE/BLEED ASSEMBLY - To monitor and
             adjust vacuum pressure delivered to the vacuum manifold (Supelco cat.#:
             5 7161 -U or equivalent).

       6.9.5   CONICAL CENTRIFUGE TUBES - 15 mL, or other glassware suitable for
             elution of the sample from the cartridge after extraction.

       6.9.6  An automatic or robotic system designed for use with SPE cartridges may be
             used if all quality control requirements discussed in Section 9 are met.
             Automated systems may use either vacuum or positive pressure to process
             samples and solvents through the cartridge. All extraction and elution steps
             must be the same as the manual procedure. Extraction or elution steps may not
             be changed or omitted to accommodate the use of the automated system.

                                  532-7

-------
6.10   SOLID PHASE EXTRACTION (SPE) APPARATUS USING DISKS

       6.10.1 EXTRACTION DISKS - 47 mm diameter, manufactured with a C18 bonded
             sorbent phase (Varian cat.#: 1214-5004 or equivalent). Larger disks may be
             used as long as the QC performance criteria outlined in Section 9 are met.

       6.10.2 SPE DISK EXTRACTION GLASSWARE - Funnel, PTFE coated support
             screen, PTFE gasket, base, and clamp used to support SPE disks and contain
             samples during extraction.  May be purchased as a set (Fisher cat# K971100-
             0047 or equivalent) or separately.

       6.10.2 VACUUM EXTRACTION MANIFOLD - Designed to accommodate
             extraction glassware (Varian cat. #: 1214-6001 or equivalent).

       6.10.3 CONICAL CENTRIFUGE TUBES - 15 mL, or other glassware suitable for
             collection of the eluent that drips from the disk extraction base.

       6.10.4 An automated or robotic system may be used as specified in Section 6.9.6.

6.11   EXTRACT CONCENTRATION SYSTEM - To concentrate extracts in 15 mL
       conical tubes, the bottoms of which are submersed in a 40C water bath, under a
       steady steam of nitrogen to the desired volume (Meyer N-Evap, Model IE,
       Organomation Associates, Inc. or equivalent).

6.12   LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
       maintain a minimum vacuum of approximately 25 cm (10 in.) of mercury for cartridges.
       A greater vacuum of approximately 66 cm (26 in.) of mercury  may be used with disks.

6.13   HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
       INSTRUMENTATION

       6.13.1 HPLC SYSTEM  - Capable of reproducibly injecting 20 or 10  uL aliquots,
             and performing binary linear gradients at a constant flow rate near 1.5 mL/min.

       6.13.2 HPLC DETECTOR - A UV detector capable of collecting data at 240-245
             nm. For the development of this method, a photodiode array detector was
             used. A LC/MS system may also be used.

       6.13.3 PRIMARY COLUMN  - An HPLC column (4.6  x  150 mm) packed with
             3.5 urn dp C18 solid phase particles (Waters cat. # WAT200632). Any
             column that provides adequate resolution, peak shape, capacity,  accuracy, and
             precision (Sect. 9) may be used.

                                 532-8

-------
              6.13.4 CONFIRMATION COLUMN - An HPLC column (4.6 x 150mm) packed
                    with 5 dp cyanopropyl stationary phase ( Supelco "Supelcosil LC-CN' cat. #
                    58221-U).  The secondary column must be chemically dissimilar to the primary
                    column and must yield a different elution order for some compounds which will
                    result in dissimilar retention times compared to the primary column.

              6.13.5 HPLC DATA SYSTEM - A computerized data system is recommended for
                    data acquisition and manipulation. The Waters Mllennium software system
                    was used to generate all primary column data contained in the Section 17
                    tables.

7.      REAGENTS AND STANDARDS

       7.1     REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be used
              in all tests.  Unless otherwise indicated, it is intended that all reagents shall conform to
              the specifications of the Committee on Analytical Reagents of the American Chemical
              Society, where such specifications are available. Other grades may be used, provided
              it is first determined that the reagent is of sufficiently high purity to permit its use without
              lessening the quality of the determination.

              7.1.1  REAGENT WATER - Purified water which does not contain any measurable
                    quantities of any target analytes or interfering compounds at or above 1/3 the
                    MRL for each compound of interest.

              7.1.2  ACETONITRILE - High purity, demonstrated to be free of analytes and
                    interferences (HPLC grade or better).

              7.1.3  METHANOL - High purity, demonstrated to be free of analytes and
                    interferences (HPLC grade or better).

              7.1.4  ACETONE - High purity, demonstrated to be free of analytes and
                    interferences (HPLC grade or better).

              7.1.5  PHOSPHATE BUFFER SOLUTION, 25 mM - Used for HPLC mobile
                    phase.  Add 100 mL 0.5 M potassium phosphate stock solution (Sect. 7.1.5.1)
                    and 100 mL of 0.5 M phosphoric acid stock solution (Sect. 7.1.5.2) to
                    reagent water and dilute to a final volume of 4 L .  The pH should be about 2.4
                    and should be confirmed with a pH meter.  Filter using a 0.45 um nylon filter.

                    7.1.5.1 POTASSIUM PHOSPHATE STOCK  SOLUTION (0.5 M) -
                           Weigh 68 g KH2PO4 (Monobasic Potassium Phosphate) and dilute to
                           1 L using reagent water.

                                         532-9

-------
              7.1.5.2 PHOSPHORIC ACID STOCK SOLUTION (0.5M) - 34.0 mL of
                    phosphoric acid (85%, HPLC grade in reagent water) diluted to 1 L
                    with reagent water.

       7.1.6   SAMPLE PRESERVATION REAGENTS

              7.1.6.1 CUPRIC SULFATE, CuSO4'5H2O ( ACS Grade or equivalent) -
                    Added as a biocide to guard against potential degradation of method
                    analytes by microorganisms (Sect. 8.1.2).

              7.1.6.2 TRIZMA PRESET CRYSTALS, pH 7.0 (Sigma cat# T 3503 or
                    equivalent) - Reagent grade. A premixed blend of Tris
                    [Tris(hydroxymethyl)aminomethane] and Tris HCL
                    [Tris(hydroxymethyl)aminomethane hydrochloride]. Alternatively, a
                    mix of the two components with a weight ratio of 15.5/1; Tris HCL/Tris
                    may be used. These blends are targeted to produce a pH of 7.0 at
                    25C in reagent water. Tris functions as a buffer, binds free chlorine in
                    chlorinated finished waters, and prevents the formation of a copper-
                    based precipitate.

7.2    STANDARD  SOLUTIONS - When a compound purity is assayed to be 96% or
       greater, the weight can be used without correction to calculate the concentration of the
       stock standard. Solution concentrations listed in this section were used to develop this
       method and are included as an example.  Standards for sample fortification generally
       should be prepared in the smallest volume that can be accurately measured to minimize
       the addition of organic solvent to aqueous samples.   Even though stability times for
       standard solutions are suggested in the following sections, laboratories should
       used standard QC practices to determine when their standards need to be
       replaced.

       7.2.1   SURROGATE ANALYTES (SUR), MONURON (CAS #150-68-5) and
              CARBAZOLE (CAS# 86-74-8) - Monuron and carbazole were chosen as
              surrogates. Monuron is a phenylurea no longer in use in the U.S. that elutes
              early in the chromatogram.  Carbazole elutes late in the chromatogram (Figure
              1) and has been used as a surrogate in other EPA drinking water methods.
              Alternate surrogates may be selected if there is a problem with matrix
              interferences or chromatography. However, if an alternate surrogate is used, it
              must have similar chemical properties (structure, solubility, C18 retention, etc.)
              to the phenylureas, be chromatographically resolved from all target analytes and
              matrix interferences, and be highly unlikely to be found in any sample.

              7.2.1.1 SUR STOCK SOLUTION (5 to 7 mg/mL) - Accurately weigh

                                  532-10

-------
              approximately 25 to 35 mg of the neat SUR to the nearest 0.1 mg into
              a tared, 5 mL volumetric flask.  Dilute to the mark with the appropriate
              solvent: methanol should be used for monuron and acetonitrile for
              carbazole.  Prepare each compound individually, as they will be
              combined in the SUR primary dilution standard.  Stock solutions have
              been shown to be stable for 6 months when stored at -10C or less.
              Laboratories should use standard QC practices to determine when their
              standards need to be replaced.

       7.2.1.2 SUR PRIMARY DILUTION STANDARD (500 ug/mL) - Prepare
              the SUR Primary Dilution Standard (PDS) by dilution  of the SUR stock
              standards.  Add enough of each of the SUR stock standards to a
              volumetric flask partially filled with methanol to make a 500 ug/mL
              solution when filled to the mark with methanol.  The PDS has been
              shown to be stable for 3 months when stored at -10C or less.

7.2.2   ANALYTE STOCK STANDARD SOLUTION - Prepare analyte stock
       standard solutions for all compounds in methanol except thidiazuron and
       diflubenzuron. Thidiazuron and diflubenzuron should be prepared in acetone
       due to their limited solubility in methanol.  Acetone elutes early in the
       chromatogram and should not interfere with compound quantitation as long as
       its volume is minimized as specified in this method. Method analytes may be
       obtained as neat materials or as ampulized solutions.  Stock solutions have been
       shown to be stable for 6 months when stored at -10C or less.

       7.2.2.1 For analytes in their pure form that are soluble in methanol, prepare
              stock solutions by accurately weighing 25 to 35  mg of pure material to
              the nearest 0.1 mg in a 5 mL volumetric flask. Dilute to volume with
              methanol.

       7.2.2.2 Thidiazuron and diflubenzuron should be dissolved in acetone.
              Accurately weigh neat material to the nearest 0.1  mg into volumetric
              flasks, but using smaller amounts than those used for other target
              analytes, approximately 10 to 12 mg. Thidiazuron is especially difficult
              to dissolve,  10 mg of pure material should dissolve in a 10 mL final
              volume of acetone.  Sonnication may be used to help dissolve these
              compounds.

7.2.3   ANALYTE PRIMARY DILUTION STANDARD (PDS, 200 ug/mL and 10
       ug/mL) - Prepare the Analyte PDS by dilution of the stock standards (Sect.
       7.2.2). Add enough of each stock standard to a volumetric flask partially filled
       with methanol to make a 200 ug/mL solution when filled to the mark with

                            532-11

-------
                     methanol.  Once prepared, a dilution of the 200 ug/mL solution may be used to
                     prepare a 10 ug/mL solution used for low concentration spiking.  The PDSs
                     can be used to prepare calibration and fortification solutions. Analyte PDSs
                     have been shown to be stable for 3 months when stored at -10C or less.

              7.2.4   CALIBRATION SOLUTIONS - At least 5 calibration concentrations will be
                     required to prepare the initial calibration curve (Sect. 10.2). Prepare at least 5
                     Calibration Solutions over the concentration range of interest, approximately
                     0.5-15 ug/mL, from dilutions of the analyte PDS in methanol. The lowest
                     concentration of calibration standard must be at or below the MRL, which will
                     depend on system sensitivity. In this method, 500 mL of an aqueous sample is
                     concentrated to a 1 mL final extract volume. The calibration standards for the
                     development of this method were prepared as  specified below.
PREPARATION OF CALIBRATION (CAL) CURVE STANDARDS
CAL
Level
1
2
3
4
5
6
PDS Cone.
(ug/mL)
10
10
200
200
200
200
Volume PDS
Standard
(uL)
25
50
5.0
25
50
75
Final Volume of
CAL Standard
(mL)
1
1
1
1
1
1
Final Cone, of
CAL Standard
(ug/mL)
0.25
0.50
1.00
5.00
10.0
15.0
Equivalent Cone.
in 500 mL sample
(ug/L)
0.50
1.00
2.00
10.0
20.0
30.0
8.
SAMPLE COLLECTION. PRESERVATION. AND STORAGE
              SAMPLE BOTTLE PREPARATION

              8.1.1   Grab samples must be collected in accordance with conventional sampling
                     practices(5) using 500 mL amber or clear glass bottles fitted with PTFE lined
                     screw caps.

              8.1.2   Prior to shipment to the field, 0.25 g of cupric sulfate and 2.5 g of Trizma
                     crystals ( Sect. 7.1.6) must be added to each bottle for each 500 mL of sample
                     collected. Alternately, the Tris buffer may be prepared by adding 2.35 g of
                     Tris HC1 and 0.15 g Tris to the sample bottle in addition to the 0.25 g of cupric
                                          532-12

-------
                 sulfate. Cupric sulfate acts as a biocide to inhibit bacteriological decay of
                 method analytes. Trizma functions as a buffering reagent, binds the free
                 chlorine, and helps to prevent the formation of a precipitate. Add these
                 materials as dry solids to the sample bottle. The stability of these materials in
                 concentrated  aqueous solution has not been verified.

  8.2     SAMPLE COLLECTION

       8.2.1      When sampling from a cold water tap, remove the aerator so that no air
                 bubbles will be trapped in the sample. Open the tap, and allow the system to
                 flush until the water temperature has stabilized (usually about 3-5 minutes).
                 Collect samples from the flowing system.

       8.2.2      When sampling from an open body of water, fill a 1 quart wide-mouth bottle or
                 1L beaker with sample from a representative area, and carefully fill sample
                 bottles from the container.  Sampling equipment, including automatic samplers,
                 must be free of plastic tubing, gaskets, and other parts that may leach interfering
                 analytes into the water sample.

       8.2.3      Fill sample bottles, taking care not to flush out the sample preservation
                 reagents.  Samples do not need to be collected headspace free.

       8.2.4      After collecting the sample, cap carefully to avoid spillage, and agitate by hand
                 for 1 minute.  Keep samples sealed from collection time until extraction.

8.3     SAMPLE SHIPMENT  AND STORAGE - All samples should be iced during shipment
       and must not exceed 10 C during the first 48 hours after collection. Samples should be
       confirmed to be at or below 10 C when they are received at the laboratory. Samples
       stored in the lab must be held at or below 6 C until extraction, but should not be frozen.

8.4     SAMPLE AND EXTRACT HOLDING TIMES - Results of the sample storage stability
       study of all method analytes indicated that all compounds are stable  for 14 days in water
       samples that are collected, dechlorinated, preserved, shipped and stored as described in
       Sections 8.2 and 8.3.  Samples must be extracted within 14 days. Sample extracts may
       be stored in methanol at 0C or less for up to 21 days after extraction. Samples that are
       exchanged into reagent water/acetonitrile (60/40) for confirmational analysis may be
       stored 7 days at 0 or less; however, the combined extract holding time may not exceed
       21 days.
                                      532-13

-------
9.    QUALITY CONTROL

     9.1    Quality control (QC) requirements include the Initial Demonstration of Capability, the
           determination of the MDL, and subsequent analysis in each analysis batch of a Laboratory
           Reagent Blank (LRB), Continuing Calibration Check Standards (CCC), a Laboratory
           Fortified Blank (LFB), a Laboratory Fortified Sample Matrix (LFM), and either a
           Laboratory Fortified Sample Matrix Duplicate (LFMD) or a Field Duplicate Sample.
           This section details the specific requirements for each QC parameter.  The QC criteria
           discussed in the following sections are summarized in Section 17, Tables 10 and 11.
           These criteria are considered the minimum acceptable QC criteria, and laboratories are
           encouraged to institute additional QC practices to meet their specific needs.

     9.2    INITIAL DEMONSTRATION OF CAPABILITY (IDC) - Requirements for the Initial
           Demonstration of Capability are described in the following sections and summarized in
           Section 17, Table 10.

           9.2.1     INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND -
                     Any time a new lot of solid phase  extraction (SPE) cartridges or disks is used,
                     it must be demonstrated that a laboratory reagent blank is reasonably free of
                     contamination and that the criteria in Section 9.4 are met.

           9.2.2     INITIAL DEMONSTRATION OF PRECISION - Prepare, extract, and
                     analyze  4-7 replicate LFBs fortified at 5 to 10 ug/L, or near the mid-range of
                     the initial calibration curve, according to the procedure described in Section 11.
                     Sample preservatives as  described in Section  8.1.2 must also be added to
                     these samples. The relative standard deviation (RSD) of the results of the
                     replicate analyses must be less than 20%.

           9.2.3     INITIAL DEMONSTRATION OF ACCURACY - Using the same set of
                     replicate data generated for Section 9.2.2, calculate average recovery. The
                     average recovery of the replicate values must be within  20% of the true value.
           9.2.4     MDL DETERMINATION - Prepare, extract and analyze at least 7 replicate
                     LFBs at a concentration estimated to be near the MDL over at least 3 days
                     (both extraction and analysis should be conducted over at least 3 days) using
                     the procedure described in Section 11.. This fortification level may be
                     estimated by selecting a concentration with a signal of 2 to 5 times the noise
                     level. The appropriate concentration will be dependent upon the sensitivity of
                     the HPLC system being used.  Sample preservatives as described in Section
                     8.1.2 must be added to these samples. Calculate the MDL using the equation


                                         532-14

-------
                        MDL   Sl( n . 1; ! . aipha = o.99)

                where
                        Vu-aipha = 0.99) = Student's t value for the 99% confidence level with
                        n-1 degrees of freedom
                        n = number of replicates, and
                        S = standard deviation of replicate analyses.

                NOTE: Do not subtract blank values when performing MDL calculations.
                        This is a statistical determination based on precision only.(1) If the
                        MDL replicates are fortified at a low enough concentration, it is likely
                        that they will not meet method precision and accuracy criteria.

       9.2.5     METHOD MODIFICATIONS - The analyst is permitted to modify HPLC
                columns, HPLC detector, HPLC conditions, evaporation techniques, and
                surrogate standards, but each time such method modifications are made, the
                analyst must repeat the procedures of the IDC (Sect. 9.2).

9.3    Minimum Reporting Level (MRL) - The MRL is the threshold concentration of an
       analyte that a laboratory can expect to accurately quantitate in an unknown sample. The
       MRL may be established at an analyte concentration either greater than three times the
       MDL or at a concentration which would yield a response greater than a signal to noise
       ratio of five. Although the lowest calibration standard for an analyte may be below
       the MRL, the MRL must never be established at a concentration lower than the
       lowest calibration standard

9.4    LABORATORY REAGENT BLANK (LRB) - A LRB is required with each extraction
       batch (Sect. 3.1) of samples to determine any background system contamination. If within
       the retention time window of any analyte, the LRB produces a peak that would prevent
       the determination of that analyte, determine the source of contamination and eliminate the
       interference before processing samples.  Background contamination must be reduced to
       an acceptable level before proceeding. Background from method analytes or
       contaminants that interfere with the measurement of method analytes must be below V3 the
       MRL. If the target analytes are detected in the LRB at concentrations equal to or greater
       than this level, then all data for the problem analyte(s) must be considered invalid for all
       samples in the extraction batch.

9.5    CONTINUING CALIBRATION CHECK (CCC) - A CCC is a standard prepared
       with all compounds of interest which is analyzed during an analysis batch to ensure the
       stability of the instrument initial calibration.  See  Section 10.3 for concentration
       requirements, frequency requirements,  and acceptance criteria.
                                     532-15

-------
9.6    LABORATORY FORTIFIED BLANKS - A LFB is required with each extraction
       batch.  The fortified concentration of the LFB should be rotated between, low, medium
       and high concentrations from day to day. The low concentration LFB must be as near as
       practical to, but no more than two times the MRL. Similarly, the high concentration
       should be near the high end of the calibration range established during the initial calibration
       (Sect. 10.2).  Results of the LFB for the low level fortification must be 50-150% of the
       true value.  The concentration determined for the medium and high LFBs must be 70-
       130% of the true value.  If LFB results do not meet these criteria for target analytes, then
       all data for the problem analyte(s) must be considered invalid for all samples in the
       extraction batch.

9.7    SURROGATE RECOVERY - The surrogate standards are fortified into the aqueous
       portion of all samples, LRBs, and LFMs and LFMDs prior to extraction.  They are also
       added to the calibration curve and calibration check standards. The surrogate is a means
       of assessing method performance from extraction to final chromatographic measurement.

       9.7.1     When surrogate recovery from a sample, blank, or CCC is <70% or >130%,
                check (1) calculations to locate possible errors, (2) standard solutions for
                degradation, (3) contamination, and (4) instrument performance. If those steps
                do not reveal the cause of the problem, reanalyze the extract.

       9.7.2     If the extract reanalysis meets the surrogate recovery criterion, report only  data
                for the reanalyzed extract.

       9.7.3     If the extract  reanalysis fails the 70-130% recovery criterion, the analyst should
                check the calibration by analyzing the most recently acceptable calibration
                standard. If the calibration standard fails the criteria of Section 9.7.1,
                recalibration is in order per Section 10.2. If the calibration standard is
                acceptable, extraction of the sample should be repeated provided the sample is
                still within the holding time.  If the sample re-extract also fails the recovery
                criterion, report all data for that sample as suspect/surrogate recovery.

9.8    LABORATORY FORTIFIED SAMPLE MATRIX AND DUPLICATE (LFM AND
       LFMD) - Analysis of LFMs are required in each extraction batch and are used to
       determine that the sample matrix does not adversely affect method accuracy.  If the
       occurrence of target analytes in the samples is infrequent, or if historical trends are
       unavailable,  a second LFM, or LMFD, must be prepared, extracted, and analyzed from  a
       duplicate of the field sample used to prepare the LFM to assess method precision.
       Extraction batches that contain LFMDs will not require the analysis of a Field Duplicate
       (Sect. 9.8). If a variety of different sample matrices are analyzed regularly, for example,
       drinking water from groundwater and surface water sources, method performance should

                                      532-16

-------
be established for each. Over time, LFM data should be documented for all routine
sample sources for the laboratory.

9.8.1      Within each extraction batch, a minimum of one field sample is fortified as a
          LFM for every 20 samples extracted. The LFM is prepared by spiking a
          sample with an appropriate amount of the appropriate Analyte PDS (Sect.
          7.2.3). Select a spiking concentration at least twice the matrix background
          concentration, if known.  Use historical data or rotate through the designated
          concentrations to select a fortifying concentration. Selecting a duplicate bottle
          of a sample that has already been analyzed aids in the selection of appropriate
          spiking levels.

9.8.2      Calculate the percent recovery (R) for each  analyte using the equation
          where
                  A = measured concentration in the fortified sample
                  B = measured concentration in the unfortified sample, and
                  C = fortification concentration.

9.8.3     Analyte recoveries may exhibit a matrix bias. For samples fortified at or above
          their native concentration, recoveries should range between 70 -130%, except
          for thidiazuron which should be recovered at 60 - 120%. For LFM
          fortification at the MRL, 50 to 150% recoveries are acceptable. If the accuracy
          of any analyte falls outside the designated range, and the laboratory
          performance for that analyte is shown to be in control in the LFB, the recovery
          is judged to be matrix biased.  The result for that analyte in the unfortified
          sample is labeled suspect/matrix to inform the data user that the results are
          suspect due to matrix effects.

9.8.4     If a LFMD is analyzed instead of a Field Duplicate, calculate the relative
          percent difference (RPD) for duplicate LFMs (LFM and LFMD) using the
          equation

                        m   LPM-LPMD
                               532-17

-------
                 RPDs for duplicate LFMs should fall in the range of + 30% for samples
                 fortified at or above their native concentration.  Greater variability may be
                 observed when LFMs are spiked near the MRL. At the MRL, RPDs should
                 fall in the range of + 50% for samples fortified at or above their native
                 concentration. If the RPD of any analyte falls outside the designated range, and
                 the laboratory performance for that analyte is shown to be in control in the
                 LFB, the recovery is judged to be matrix biased.  The result for that analyte in
                 the unfortified sample is labeled suspect/matrix to inform the data user that the
                 results are suspect due to matrix effects.

9.9    FIELD DUPLICATES (FD1 AND FD2) - Within each extraction batch, a minimum of
       one field duplicate (FD) or LFMD (Sect. 9.8) must be analyzed.  FDs serve as a check
       the precision associated with sample collection, preservation, and storage, as well as
       laboratory procedures. If target analytes are not routinely observed in field samples, a
       LFMD should be analyzed to substitute for this requirement. Extraction batches that
       contain LFMDs will not require the analysis of a Field Duplicate.

       9.9.1      Calculate the relative percent difference (RPD) for duplicate measurements
                 (FD1 and FD2)using the equation
                 RPDs for duplicates should fall in the range of + 30%. Greater variability may
                 be observed when analyte concentrations are near the MRL.  At the MRL,
                 RPDs should fall in the range of + 50%. If the RPD of any analyte falls outside
                 the designated range, and the laboratory performance for that analyte is shown
                 to be in control in the LFB, the recovery is judged to be matrix biased. The
                 result for that analyte in the unfortified sample is labeled suspect/matrix to
                 inform the data user that the results are suspect due to matrix effects.

9.10   QUALITY CONTROL SAMPLES (QC S) - Each time that new standards are prepared
       or a new calibration curve is run, analyze a QCS from a source different from the source
       of the calibration standards.  The QCS may be injected as a calibration standard, or
       fortified into reagent water and analyzed as a LFB. If the QCS is analyzed as a continuing
       calibration check, then the acceptance criteria are the same as for the CCC.  If the QCS
       is analyzed as a LFB, then the acceptance criteria are the same as for an LFB.  If
       measured  analyte concentrations are not of acceptable accuracy, check the entire
       analytical  procedure to locate and correct the problem source.

                                      532-18

-------
10.  CALIBRATION AND STANDARDIZATION

     10.1    After initial calibration is successful, a Continuing Calibration Check is required at the
            beginning and end of each analysis batch, and after every tenth sample (Sect. 10.3).  Initial
            calibration should be repeated each time a major instrument modification or maintenance
            is performed.

     10.2    INITIAL CALIBRATION

            10.2.1    Establish HPLC operating parameters equivalent to the suggested conditions in
                     Section 17, Table 1.  The system is calibrated using the external standard
                     technique. For this method, a PDA detector was used and the analyte
                     absorbance at 240 or 245 nm was used in order to maximize target compound
                     signal relative to the background interferences. Other HPLC conditions may be
                     used as long as all QC requirements in Section 9 are met.

            10.2.2    Prepare a set of at least 5 calibration standards as described in Section 7.2.4.
                     The lowest concentration of calibration standard must be at or below the MRL,
                     which will depend on system sensitivity.

            10.2.3    INJECTION VOLUME - Optimum inj ection volume for the primary column
                     may vary between HPLC instruments when a sample is dissolved in an organic
                     solvent such as methanol. Prior to establishing the initial calibration, the
                     injection volume must first be determined.  Peak asymmetry on the primary
                     column was occasionally noted on one of the two instruments used to develop
                     this method. This asymmetry only occurred with 20 uL injections.  This
                     phenomena is attributed to the difference in elutropic strength between the initial
                     mobile phase composition (phosphate buffer/acetonitrile; 60/40) and the extract
                     solvent composition (100% methanol). In all cases, the asymmetry was
                     eliminated by reducing the injection volume to 10 uL.  Prior to establishing the
                     initial calibration curve, acceptable chromatographic performance is determined
                     by calculating the Peak Gaussian Factor (PGF).

                     10.2.3.1 Section 17 (Table 3) lists MDLs on the primary column using two
                             injection sizes: 20 uL and 10 uL.  Inject a 20 uL aliquot of the medium
                             level calibration standard on the primary column using the suggested
                             conditions listed in Section 17, Table 1.  Determine the PGF for the
                             analyte fluometuron using the equation

                                         1.83 x  W0.5
                                PGF =	
                                            W0.i
                                          532-19

-------
                        where,
                        W o 5 is the peak width at half height, and
                         W 0 j is the peak width at tenth height.

                If fluometuron has not been included in the calibration standards, one of the
                surrogate compounds may be substituted.

                NOTE: Values for W O.s and W O.i can be attained via most data acquisition
                        software packages. If these values are manually measured, the analyst
                        should limit the retention time window to enlarge the peak of interest
                        allowing accurate determination of the PGF. Inaccurate
                        measurements may result when using a chromatogram of the entire
                        analysis run.

                10.2.3.2 If the PGF is in the range of 0.90 to 1.10, the peak shape is
                        considered acceptable, and a 20 uL injection may be used. If not,
                        injection volume should be reduced, and the PGF redetermined as
                        described in Section 10.2.3.1.

       10.2.4   Generate a calibration curve for each analyte by plotting the peak response
                (area is recommended) against analyte concentration.  Both instruments used
                during method development yielded linear curves  for the target analytes over
                the concentration range of interest.  However, data may be fit with  either a
                linear regression (response vs concentration) or quadratic fit (response vs
                concentration). Alternately, if the ratio of the analyte peak area to
                concentration (or response factor) is relatively constant (RSD < 30%) an
                average response factor may be used to calculate  analyte concentration.
                Siduron separates into two isomers (labeled A &  B in Sect. 17, Figure 1).
                The responses of the two isomers should be added together before plotting
                against the concentration.

       10.2.5   Repeat steps 10.2.1 through 10.2.4 for the confirmation column. Laboratories
                may choose to wait to establish an initial calibration curve for the confirmation
                column until they have samples with positive results that require confirmation;
                however, this step must be successfully completed prior to confirming sample
                results.

10.3   CONTINUING CALIBRATION CHECK (CCC) - Minimum daily calibration
       verification is as follows.  Verify the initial calibration at the beginning and end of each
       group of analyses, and after every tenth sample during analyses. (In this context, a
       "sample" is considered to be a Field sample.  LRBs, LFBs,  LFMs, LFMDs and CCCs

                                      532-20

-------
            are not counted as samples.) The beginning CCC each day should be at or below the
            MRL in order to verify instrument sensitivity prior to any analyses. Subsequent CCCs
            should alternate between a medium and high concentration standard.

            10.3.1    Inj ect an aliquot of the appropriate concentration calibration solution and
                     analyze with the same conditions used during the initial calibration.

            10.3.2    Calculate the concentration of each analyte and surrogate in the check
                     standard. The calculated amount for each analyte for medium and high level
                     CCCs must be  30% of the true value. The calculated amount for the lowest
                     calibration point for each analyte must be within 50% of the true value. If
                     these conditions do not exist, then all data for the problem analyte must be
                     considered invalid, and remedial action should be taken which may require
                     recalibration. Any field sample extracts that have been analyzed since the last
                     acceptable calibration verification should be reanalyzed after adequate
                     calibration has been restored.

11.         PROCEDURE

     11.1    Important aspects of this analytical procedure include proper preparation of laboratory
            glassware and sample containers (Sect. 4.1), and sample collection and storage (Sect. 8).
            This section details the procedures for  sample preparation, solid phase extraction (SPE)
            using cartridges or disks, and extract analysis.
     11.2   SAMPLE PREPARATION

            11.2.1    Samples are preserved, collected and stored as presented in Section 8. All
                     field and QC samples must contain the preservatives listed in Section 8.1.2,
                     including the LRB and LFB. Determine sample volume.  The sample volume
                     may be measured directly in a graduated cylinder to the nearest 10 mL. To
                     minimize the need to use a different graduated cylinder for each sample, an
                     indirect measurement may be done in one of two ways: by marking the level of
                     the sample on the bottle or by weighing the sample and bottle to the nearest 10
                     g. After extraction, proceed to Section 11.3.5 for final volume determination.
                     The LRB and LFB may be prepared by measuring 500 mL of reagent water
                     into an erlenmeyer flask.

            11.2.2    Add an aliquot of the SUR PDS (Sect. 7.2.1.2) to all samples and mix by
                     swirling the sample. Addition of 10 uL of a 500 ug/mL SUR PDS to a 500 mL
                     sample will result in a concentration of 10 ug/L.
                                           532-21

-------
       11.2.3    If the sample is a LFB or LFM, add the necessary amount of analyte PDS.
                Swirl each sample to ensure all components are properly mixed.

       11.2.4    Proceed with sample extraction. Refer to Section 11.3 if SPE cartridges are
                being used. Refer to Section 11.4 if SPE disks are being used.

11.3   CARTRIDGE SPE PROCEDURE - Proper conditioning of the solid phase can have a
       marked effect on method precision and accuracy. This section describes the SPE
       procedure using the equipment outlined in Section 6.9 in its simplest, least expensive mode
       without the use of the alternate transfer system or robotics systems (Sect. 6.9.2 and 6.9.6)
       This configuration was used to collect data presented in Section 17.

       11.3.1    CARTRIDGE CONDITIONING - Once the conditioning of the cartridge is
                started, the cartridge must not be allowed to go dry until the last portion of the
                sample is filtered through it. If the cartridge goes dry during the conditioning
                phase, the conditioning must be started over. However, if the cartridge goes
                dry during sample extraction, the analyte and surrogate recoveries may be
                affected. If this happens the analyst should make note of this as this sample
                may  require re-extraction due to low surrogate recoveries.

                11.3.1.1 CONDITIONING WITH METHANOL - Assemble the extraction
                        cartridges into the vacuum manifold. Rinse each cartridge with two, 5
                        mL aliquots of methanol, allowing the sorbent to soak in the methanol
                        for about 30 seconds by turning off the vacuum temporarily during the
                        first rinsing.  Do not allow the methanol level to go below the top of
                        the cartridge packing.

                11.3.1.2 CONDITIONING WITH REAGENT WATER - Follow the
                        methanol rinse with two,  5 mL aliquots of reagent water being careful
                        not to allow the water level to go below the cartridge packing.  Turn
                        off the vacuum. Add approximately 5 mL additional reagent water to
                        the cartridge, and  attach  a reservoir (or transfer tube - Sect. 6.9.2)
                        before adding sample to the cartridge.

       11.3.2    CARTRIDGE EXTRACTION - Prepare samples, including QC samples, as
                specified in Section 11.2. The samples may be added to the cartridge using
                either a large reservoir attached to the cartridge or using a transfer tube from
                the sample bottle to the  cartridge.

                11.3.2.1 SAMPLE ADDITION USING RESERVOIRS - Attach a reservoir
                        to the conditioned cartridge. Fill the reservoir (Sect. 6.9.2) with
                        sample and then turn on the vacuum. Adjust the vacuum so that the

                                      532-22

-------
                 approximate flow rate is about 20 mL/min (minus 9-10 in Hg.). Care
                 must be taken to add additional aliquots of sample to the reservoir to
                 keep the cartridge packing from going dry before all the sample has
                 been extracted.  Rinse the sample container with reagent water and
                 add to the reservoir after the last addition of sample, but before the
                 cartridge goes dry.  After all of the sample has passed through the
                 SPE cartridge, detach the reservoir and draw air through the cartridge
                 for 15 minutes at full vacuum (minus 10-15 in Hg). Turn off and
                 release the vacuum.

         11.3.2.3 SAMPLE ADDITION USING TRANSFER TUBES - If the sample
                 transfer tubes are employed, make sure the transfer tube is attached to
                 the conditioned cartridge before turning on the vacuum.  Adjust the
                 vacuum to a similar flow rate of approximately 20 mL/min (minus 9-
                 10 in Hg). Rinse down the sample container with reagent water as it
                 approaches dryness. After all of the sample has passed through the
                 SPE cartridge, detach the transfer tube and draw air through the
                 cartridge for 15 minutes at full vacuum (minus 10-15 in Hg). Turn off
                 and release the vacuum.

11.3.3    CARTRIDGE ELUTION - Lift the extraction manifold top and insert
         collection tubes into the extraction tank to collect the extracts as they are eluted
         from the cartridge. Add approximately 3 mL of methanol to the top of each
         cartridge. Pull enough of the methanol into the cartridge at low vacuum to soak
         the sorbent. Turn off the vacuum and vent the system.  Allow the sorbent to
         soak in methanol for approximately 30 seconds.  Start a low vacuum (minus 2-
         4 in Hg) and pull the methanol through in a dropwise fashion into the collection
         tube. Repeat this elution a second time with approximately 2 mL of methanol,
         and then a third time with approximately 1 mL.

11.3.4    EXTRACT CONCENTRATION - Concentrate the extract to about 0.5 mL
         in a warm water bath (at about 40OC) under a gentle steam  of nitrogen.
         Transfer to a 1 mL volumetric flask, rinsing the collection tube with small
         amounts of methanol.  Adjust to volume with methanol. Filter the sample using
         a 1 mL syringe and filter (Sects. 6.7 & 6.8) into an appropriate autosampler
         vial.

11.3.5    SAMPLE VOLUME DETERMINATION - If the level of the sample was
         marked on the sample bottle, use a graduated cylinder to measure the volume
         of water required to fill the original sample bottle to the mark made prior to
         extraction. Determine to the nearest 10 mL. If using weight to determine
         volume, weigh the empty bottle to the nearest 10 g and determine the sample

                              532-23

-------
                weight by subtraction of the empty bottle from the original weight (Sect.
                11.2.1). In either case, the sample volume will be used in the final calculations
                of analyte concentration (Sect. 12.4).

11.4   DISK SPE PROCEDURE - Proper conditioning of the solid phase can have a marked
       affect on method precision and accuracy. This section describes the SPE procedure using
       the equipment outlined in Section 6.10 in its simplest, least expensive mode without the
       use of robotics systems (Sect. 6.10.4). This configuration was used to collect data
       presented in Section 17.

       11.4.1    DISK CONDITIONING - Once the conditioning of the disk is started, the
                disk must not be allowed to go dry until the last portion of the sample is filtered
                through it. If the disk goes dry during the conditioning phase, the conditioning
                must be started over. However, if the disk goes dry during  sample extraction,
                the analyte and surrogate recoveries may be affected.  If this happens the
                analyst should  make note of this as this sample may require re-extraction due to
                low surrogate recoveries.

                11.4.1.1  CONDITIONING WITH METHANOL - Assemble the extraction
                         glassware in the vacuum manifold, placing the disks on a support
                         screen between the funnel and base. Rinse each disk with two, 10
                         mL aliquots of methanol to the funnel, allowing the sorbent to soak for
                         about 30 seconds by pulling approximately ImL through the disk and
                         turning off the vacuum temporarily during the first rinsing. Draw the
                         methanol through the disk until it is 3-5 mm above the disk surface,
                         adding more methanol if needed to keep the methanol from going
                         below this level.

                11.4.1.2 CONDITIONING WITH WATER - Follow the methanol rinse with
                         two, 10 mL aliquots of reagent water being careful to keep the water
                         level  at 3-5 mm above the disk surface.  Turn off the vacuum.

       11.4.2    DISK  EXTRACTION - Prepare samples, including QC samples, as specified
                in Section 11.2. Fill the extraction funnel containing the conditioned disk with
                sample and turn on the vacuum. Care must be taken to add additional aliquots
                of sample to the funnel to keep the disk from going dry before all the sample
                has been extracted. Rinse the sample container with reagent water and add to
                the funnel after the last addition of sample, but before the disk goes dry. After
                all of the sample has passed through the SPE disk, draw air through the disk for
                15 minutes at full vacuum (minus 10-15 in Hg). Turn off and release the
                vacuum.
                                     532-24

-------
       11.4.3    DISK ELUTION - Detach the glassware base from the manifold without
                disassembling the funnel from the base.  Dry the underside of the base. Insert
                collection tubes into the manifold to collect the extracts as they are eluted from
                the disk.  The collection tubes must fit around the drip tip of the base to ensure
                the collection of all of the eluent.  Reattach the base to the manifold.  Add
                approximately 5 mL of methanol to the top of each disk. Pull enough of the
                methanol into the disk to soak the sorbent. Turn off the vacuum and vent the
                system.  Allow the sorbent to soak in methanol for approximately 30 seconds.
                Start the vacuum and pull the methanol through.  Attempt to pull the methanol
                through in a dropwise fashion into the collection tube by slowly turning the valve
                (which controls the flow through the funnel) until the methanol  starts eluting into
                the collection tube.  Repeat this elution a second time with approximately 4 mL
                of methanol, and then a third time with approximately 3 mL.

       11.4.4    EXTRACT CONCENTRATION - Proceed with extract concentration as in
                Section  11.3.4.

       11.4.5    SAMPLE VOLUME DETERMINATION - Proceed with sample volume
                determination as in Section 11.3.5.

11.5   SOLVENT EXCHANGE FOR CONFIRMATION ANALYSIS - Samples that will be
       confirmed must be exchanged into reagent water/acetonitrile (60/40). To accomplish this,
       transfer the remaining 980 uL of the extract to a 1 mL volumetric (or other appropriate
       collection tube). Mark the sample volume.  Take the extract to dryness in a warm water
       bath (at ~ 40C) under a gentle steam of nitrogen. Reconstitute the residue with a mixture
       of reagent water/acetonitrile (60/40) to the mark made before the extract was taken to
       dryness. Care must be taken to redissolve the film as thoroughly as possible. Use of a
       vortex mixer is recommended.  Transfer to an appropriate autosampler vial.

                Note: Recovery experiments have been conducted to investigate the effect of
                drying time on compound recoveries by allowing the extract to sit an additional
                1,2, or 4 hours in the bath after being taken to dryness.  These studies indicate
                that: 1) the best recoveries are obtained when extracts are reconstituted
                immediately after the methanol is removed; 2) recoveries decrease with
                increasing time in the bath; and 3) carbazole recovery is the most  sensitive to
                the additional time.  In our experiments, carbazole had acceptable recovery
                criteria (Sect. 12.3) at 2 additional hours of drying, but had exceeded the
                acceptable range after 4 hours of additional time.

11.6   ANALYSIS OF SAMPLE EXTRACTS

       11.6.1    Establish operating conditions as summarized in Table 1 of Section 17 for the

                                     532-25

-------
                     HPLC system.  Confirm that retention times, compound separation and
                     resolution on the primary and secondary columns are similar to those listed in
                     Tables 1 and 2 and Figures 1 and 2, respectively.

            11.6.2   Determine the optimal injection volume. Establish a valid initial calibration
                     following the procedures outlined in Section 10.2 using the optimum injection
                     size.  Complete the IDC requirements described in Section 9.2.

            11.6.3   Establish an appropriate retention time window for each target and surrogate to
                     identify them in the QC and field samples.  This should be based on
                     measurements of actual retention time variation for each compound in standard
                     solutions analyzed on the HPLC over the course of time. Plus or minus three
                     times the standard deviation of the retention time for each compound while
                     establishing the initial calibration and completing the IDC can be used to
                     calculate a suggested window size;  however, the experience of the analyst
                     should weigh heavily on the determination of the appropriate retention window
                     size.

            11.6.4   Check system calibration by analyzing a CCC (Sect. 10.3) and begin to inject
                     aliquots of field and QC samples using the same injection volume and
                     conditions used to analyze the initial calibration.

            11.6.5   The analyst must not extrapolate beyond the established calibration range. If an
                     analyte peak area exceeds the range of the initial calibration curve, the extract
                     may be diluted with methanol. Acceptable surrogate performance (Sect.  9.7)
                     should be determined from the undiluted sample extract. If confirmation
                     analyses require dilution, this must be done using reagent water/acetonitrile
                     (Sect. 11.5) Incorporate the dilution factor into final concentration calculations.
                     Any dilutions will also affect analyte MRL.

12.  DATA ANALYSIS AND CALCULATION

     12.1   Identify the method analytes in the sample chromatogram by comparing the retention time
            of the suspect peak to retention time of an analyte peak in a calibration standard or the
            laboratory fortified  blank.  Surrogate retention  times should be confirmed to be within
            acceptance limits (Sect. 11.6.3) even if no target compounds are detected.

     12.2   Calculate the analyte concentrations using the initial calibration curve generated as
            described in Section 10.2.  Quantitate only those values that fall between the MRL and
            the highest calibration standard.  Samples with target analyte responses that exceed the
            highest standard require dilution and reanalysis (Sect.  11.6.5).
                                           532-26

-------
     12.3   Positive results should be confirmed on the confirmation column (Sect. 17, Table 2) that
            has been initially calibrated, and confirmed to still be in calibration by analyzing
            appropriate CCCs (Sect. 10.3) prior to the confirmation analysis.  Quantitated values for
            the targets and surrogates on the confirmation column should be 50 - 150% of the primary
            column result.  If so, report the more accurate primary column result.  If not, report the
            lower of the 2 values and mark the results as suspect/confirmation to inform the data user
            that the results are suspect due to lack of confirmation.  If values are taken from the
            confirmation column, both surrogates must meet recovery acceptance criteria.
            Note: The PDA spectra of these compounds do not have sufficient resolution to
            definitively identify each target compound in this method.

     12.4   Adjust  the calculated concentrations of the detected analytes to reflect the initial sample
            volume and any dilutions performed.

     12.5   Prior to reporting the data, the chromatogram should be reviewed for any incorrect peak
            identification or poor integration.

     12.6   Analyte concentrations are reported in ug/L. Calculations should use all available digits of
            precision, but final concentrations should be rounded to an appropriate number of
            significant figures.

13.  METHOD PERFORMANCE

     13.1   PRECISION, ACCURACY, AND MDLs - Method detection limits (MDLs) are
            presented in Table 3 and were calculated using the formula present in Section 9.2.4.
            Tables  for these data are presented in Section 17. Single laboratory precision and
            accuracy data are presented for three water matrices: reagent water (Table 5);
            chlorinated, "finished" surface water (Table 6); and chlorinated, "finished" ground water
            (Table  7).

     13.2   COMPOUND STABILITY - Thidiazuron and diflubenzuron are extremely sensitive to
            free chlorine.  LFM recoveries were found to drop to 0% for thidiazuron and about 5%
            for diflubenzuron in the time required to extract samples in the presence of free chlorine.
            Other analytes are sensitive to free chlorine over time, even within the recommended
            sample holding time of 14 days. For example, LFM recoveries for diuron drop to about
            12% on day 14 in a finished surface water with a residual free chlorine concentration of
            0.8 mg/L.  This illustrates the importance of proper sample preservation (Sect. 8).  During
            method development, it was experimentally determined that the 2.5 g Trizma preset
            crystals (Sect. 7.1.6.2) was sufficient to dechlorinate a 500 mL sample with up to
            approximately 10 mg/L free chlorine as measured colorimetrically.  This level is about 2.5
            times the maximum allowable free chlorine residual in municipal tap waters promulgated in
            Stage I of the Disinfectant/Disinfection By-product Rule(6) and so should be sufficient to

                                           532-27

-------
           reduce free chlorine in samples from public water systems.

     13.3   ANALYTE STABILITY STUDIES

           13.3.1    FIELD SAMPLES - Chlorinated finished surface water samples, fortified with
                     method analytes at 10 ug/L, were preserved and stored as required in Section
                     8.  The average of triplicate analyses, conducted on days 0, 2, 7, and 14, are
                     presented in Section 17, Table 8. These data document the 14-day sample
                     holding time.

           13.3.2    EXTRACTS - Extracts from the day 0 extract holding time study described
                     above were stored below 0 C and analyzed on days 0, 8,  14, and 21. The
                     data presented in Section 17, Table 9, document the 21-day extract holding
                     time.

14.  POLLUTION PREVENTION

     14.1   This method utilizes solid phase extraction technology to extract analytes from water. It
           requires the use of very small volumes of organic solvent and very small quantities of pure
           analytes, thereby minimizing the potential hazards to both the analyst and the environment
           as compared to the use of large volumes of organic solvents in conventional liquid-liquid
           extractions.

     14.2   For information about pollution prevention that may be applicable to laboratory
           operations, consult "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 NW, Washington, D.C., 20036.

15.  WASTE MANAGEMENT

     15.1   The analytical procedures described in this method generate relatively  small amounts of
           waste since only small amounts of reagents and  solvents are used.  The matrices of
           concern are finished drinking water or source water. However, the Agency requires that
           laboratory waste management practices be conducted consistent with  all applicable rules
           and regulations, and that laboratories protect the  air, water, and land by minimizing and
           controlling all releases from fume hoods and bench operations. Also, compliance is
           required with any sewage discharge permits and regulations, particularly the hazardous
           waste identification rules and land disposal restrictions.  For further information on waste
           management, consult "The Waste Management Manual for Laboratory Personnel" also
           available from the American Chemical Society at the address in Section 14.2.
                                          532-28

-------
16. REFERENCES

    1.     Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L., "Trace
           Analyses for Wastewaters," Enviroa Sci. Technol. 1981, 15, 1426-1435.

    2.     "OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational
           Safety and Health Administration, OSHA 2206, (Revised, Jan. 1976).

    3.     "Carcinogens-Working with Carcinogens," Publication No. 77-206, Department of
           Health, Education, and Welfare, Public Health Service, Center for Disease Control,
           National Institute of Occupational Safety and Health, Atlanta, Georgia, August 1977.

    4.     "Safety In Academic Chemistry Laboratories," 3rd Edition, American Chemical Society
           Publication, Committee on Chemical Safety, Washington, D.C.,  1979.

    5.     ASTM Annual Book of Standards, Part H, Volume 11.01, D3370-82, "Standard
           Practice for Sampling Water," American Society for Testing and Materials, Philadelphia,
           PA, 1986.

    6.     Federal Register,  December 16, 1998,  63 (241) 69390-69476.
                                          532-29

-------
17.  TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
    TABLE 1.     CHROMATOGRAPHIC CONDITIONS AND RETENTION TIME
                 DATA FOR THE PRIMARY COLUMN
Peak Number
(Figure 1)
1
2
O
4
5
6
7
8
9
10
11
Analyte
Tebuthiuron
Thidiazuron
Monuron (SUR)
Fluometuron
Diuron
Propanil
Siduron A
Siduron B
Linuron
Carbazole (SUR)
Diflubenzuron
Retention
Time (min.)
2.03
2.48
2.80
4.45
5.17
8.53
8.91
9.76
11.0
12.8
13.9
    Primary Column: Symmetry 4.6 x 150 mm packed with 3.5 um C18 stationary phase.
                                 Conditions:
Solvent A
Solvent B
40% B
linear gradient 40-50% B
linear gradient 50-60% B
linear gradient 60-40%B
Flow rate
Wavelength
25 mM phosphate buffer
acetonitrile
0-9.5 minutes
9.5-10.0 minutes
10.0-14.0 minutes
14.0-15.0 minutes
1.5 mL/min
245 nm
Equilibration time prior to next injection 15 minutes.
                                  532-30

-------
TABLE 2.      CHROMATOGRAPHIC CONDITIONS AND RETENTION TIME
              DATA FOR THE CONFIRMATION COLUMN
Peak Number
(Figure 1)
1
2
3
4
5
6
7
8
9
10
11
Analyte
Tebuthiuron
Monuron (SUR)
Thidiazuron
Fluometuron
Diuron
Siduron A
Propanil
Siduron B
Linuron
Carbazole (SUR)
Diflubenzuron
Retention
Time (min.)
2.56
3.98
4.93
5.94
7.67
9.53
10.1
10.8
12.2
14.3
15.2
      Confirmation Column: Supelcosil 4.6 x 150 mm packed with 5 um cyanopropyl
      stationary phase.
                              Conditions:
Solvent A
Solvent B
linear gradient 20% B
linear gradient 20-40% B
40% B
linear gradient 40-20% B
Wavelength
25 mM phosphate buffer
acetonitrile
0-11.0 minutes, 1.5 mL/min. flow rate
1 1.0-12.0 minutes, 1.5 mL/min flow rate
hold to 16 minutes at 1.5 mL/min. flow rate,
step to 2.0 mL/min flow rate at 16 min. and
hold to 20 min.
20.0-20.1 minutes, 2.0 mL/min. flow rate
240 nm
Equilibration time prior to next injection 15 minutes.
                                532-31

-------
TABLE 3.  METHOD DETECTION LIMITS FOR 2 INJECTION VOLUMES IN
          REAGENT WATER WITH CARTRIDGE AND DISK EXTRACTION
          TECHNIQUES ON BOTH THE PRIMARY AND CONFIRMATION
          COLUMNS
Table 3A. Cartridge Extraction, Primary Column
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Spiking Cone. (ug/L)
20 uL inj.
0.050
0.100
0.050
0.050
0.050
0.100
0.050
0.050
10 uL inj.
0.100
0.100
0.100
0.100
0.300
0.600
0.300
0.100
MDLa (ug/L)
20 uL inj.
0.032
0.035
0.013
0.010
0.023
0.024
0.062
0.014
10 uL inj.
0.071
0.047
0.027
0.026
0.084
0.091
0.067
0.033
Table 3B. Disk Extractions, Primary Column, 20 uL Injection
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Spiking Cone. (ug/L)
0.050
0.100
0.050
0.050
0.100
0.100
0.100
0.050
MDLa (ug/L)
0.046
0.047
0.028
0.018
0.071
0.067
0.032
0.035
    a Method detection limit samples extracted over 3 days for 7 replicates and analyzed using
    conditions described in Table 1.
                                   532-32

-------
Table 3C. Cartridge Extraction, Confirmation Column, 20 uL Injection
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Spiking Cone. (ug/L)
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
MDLb (ug/L)
0.145
0.143
0.065C
0.056
0.066
0.136
0.085
0.126
b Method detection limit samples extracted over 3 days for 7 replicates and analyzed using
conditions described in Table 2.
c MDLs for fluometuron were reinjected due to an interfering peak which tended to coelute with
fluometuron.
                                      532-33

-------
TABLE 4.  PRECISION, ACCURACY AND SIGNAL-TO-NOISE COMPARISON FOR
           TWO HPLC SYSTEMS FOR LOW LEVEL SPIKES IN REAGENT WATER
           EXTRACTED WITH CARTRIDGES AND ANALYZED USING THE
           PRIMARY COLUMN
Table 4. Precision, Accuracy and S/N Comparison, 10 uL Injection, Primary Column
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&Ba
Linuron
Diflubenzuron
Monuron (SUR)a
Carbazole (SUR)a
HPLC System #la
Concentration = 1.0 ug/L(n=7)
Mean
% Rec.c
100
103
99
102
96
100
108
100
105
97
RSD
(%)
1.8
2.4
2.2
1.8
3.6
3.0
1.6
2.0
2.8
2.7
S/N
Ratiod
84
33
43
44
28
16
12
22
NC
NC
HPLC System #2b
Concentration = 1.0 ug/L(n=7)
Mean
% Rec.
105
102
104
104
100
109
103
98
94
104
RSD
(%)
2.7
1.9
2.3
3.8
6.4
11
4.1
3.3
2.3
2.7
S/N
Ratio0
17
36
35
34
16
40
21
11
NC
NC
    NC: Not Calculated
    aHPLC System 1 was equipped with a photodiode array detector that employed a 1 cm path
    length.
    bHPLC System 2 was equipped with a photodiode array detector that employed a 5 cm path
    length.
    cSeven replicates of the low level reagent water spikes were processed through the cartridge
    extraction procedure and injected on both instruments.
    dSignal-to-noise ratios were calculated for each peak by dividing the peak height for each
    compound by the peak-to-peak noise for each peak, which was determined for each component
    from the method blank over a period of time equal to the full peak width.
                                       532-34

-------
TABLE 5.  PRECISION AND ACCURACY DATA IN REAGENT WATER
Table 5 A. Cartridge Extraction, Primary Column, 20 uL Injection
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)a
Carbazole (SUR)a
Concentration = 1 ug/L (n=7)
Mean
Recovery
(%)
107
106
106
107
105
106
104
107
100
96.7
Relative
Standard
Deviation (%)
2.4
1.1
0.9
1.0
1.7
1.9
1.6
1.2
1.8
1.0
Concentration = 30 ug/L (n=7)
Mean
Recovery (%)
97.9
96.7
97.6
97.5
97.2
97.8
97.4
96.0
100.
96.1
Relative
Standard
Deviation (%)
1.4
1.6
1.5
0.0
1.4
1.6
1.5
1.4
2.0
1.5
Table 5B. Disk Extractions, Primary Column, 20 uL Injection
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)a
Carbazole (SUR)a
Concentration = 1 ug/L (n=7)
Mean
Recovery (%)
104
99.8
100
104
101
110
99.2
102
102
96.9
Relative
Standard
Deviation (%)
2.2
4.8
4.2
5.9
2.7
5.0
4.8
4.2
2.6
3.3
Concentration = 30 ug/L(n=7)
Mean
Recovery
(%)
101
101
101
101
101
103
99.0
100
99.0
95.1
Relative
Standard
Deviation (%)
2.3
2.4
2.3
3.2
2.3
2.3
2.5
2.5
2.8
3.0
"Surrogate concentration in all samples is 10 ug/L. Chromatographic conditions are described in Table
                                       532-35

-------
1.
Table 5C. Cartridge Extraction, Confirmation Column, 20 uL Injection
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&Ba
Linuron
Diflubenzuron
Monuron (SUR)b
Carbazole (SUR)b
Concentration = 1 ug/L (n=7)
Mean %
Recovery
100
93.6
87.6
96.9
88.0
91.0
79.8
83.7
105
93.8
Relative
Standard
Deviation (%)
4.9
5.1
12.4
3.9
5.9
7.0
13.3
6.9
4.3
6.0
Concentration = 30 ug/L (n=7)
Mean %
Recovery
103
101
97.0
102
103
103
99.8
80.4
102
83.6
Relative
Standard
Deviation (%)
4.8
4.8
5.7
2.7
4.9
5.0
5.6
18.6
4.5
7.6
     "Total siduron concentration is twice the concentration of the other target analytes: 2.0 and 60.0
     ug/L.
     bSurrogate concentration in all samples is 10 ug/L. Conditions described in Table 2.
                                           532-36

-------
TABLE 6. PRECISION AND ACCURACY3 OF LOW AND HIGH LEVEL FORTIFIED
          CHLORINATED SURFACE WATER USING CARTRIDGES
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)b
Carbazole (SUR)b
Concentration = 1 ug/L (n=7)
Mean %
Recovery
108
81
109
110
104
102
102
109
103
98
Relative
Standard
Deviation (%)
1.6
2.9
1.0
1.7
1.8
1.8
1.3
1.5
2.3
2.7
Concentration = 30 ug/L (n=7)
Mean %
Recovery
96
84
96
96
96
96
96
95
97
94
Relative
Standard
Deviation (%)
1.7
1.9
1.5
1.6
1.5
1.6
1.6
1.7
2.3
1.9
    aAll data collected using a 20 uL injection volume on the primary column and conditions described
    in Table 1.
    b Surrogate concentration in all samples is 10 ug/L.
                                     532-37

-------
TABLE 7. PRECISION AND ACCURACY3 OF LOW AND HIGH LEVEL FORTIFIED
          CHLORINATED GROUND WATER USING CARTRIDGES
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)b
Carbazole (SUR)b
Concentration = 1 ug/L (n=7)
Mean %
Recovery
109
95
103
106
106
106
101
104
100
95
Relative
Standard
Deviation
1.5
2.9
1.9
2.1
1.9
4.2
2.7
2.6
1.3
1.3
Concentration = 30 ug/L (n=7)
Mean %
Recovery
99
94
97
99
99
99
98
98
99
89
Relative
Standard
Deviation
1.2
1.2
1.9
1.1
1.1
1.2
1.9
1.0
1.0
4.7
    aAll data collected using a 20 uL injection volume on the primary column and conditions described
    in Table 1.
    bSurrogate concentration in all samples is 10 ug/L.
                                      532-38

-------
TABLE 8.  SAMPLE HOLDING TIME DATA3 FOR SAMPLES FROM A
           CHLORINATED SURFACE WATER, FORTIFIED WITH METHOD
           ANALYTES AT 10 ug/L, WITH CUPRIC SULFATE AND TRIZMA (Sect. 8.1.2)
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)b
Carbazole (SUR)b
DayO
% Recovery
93
72
93
94
93
92
93
93
96
88
Day 2
% Recovery
95
76
95
97
97
96
97
95
101
96
Day 7
% Recovery
98
84
98
99
98
98
98
98
103
92
Day 14
% Recovery
96
77
97
97
98
98
99
97
102
95
aStorage stability is expressed as a percent recovery value. Each percent recovery value represents the
mean of 3 replicate analyses. Relative Standard Deviations ([Standard Deviation/Recovery] x 100) for
replicate analyses were all less than 7.0%.
Samples analyzed using a 20 uL injection volume and conditions described in Table 1.
bSurrogate concentration in all samples is 10 ug/L.
                                        532-39

-------
532-40

-------
TABLE 9.  EXTRACT HOLDING TIME DATA3 FOR SAMPLES FROM A
           CHLORINATED SURFACE WATER, FORTIFIED WITH METHOD
           ANALYTES AT 10 ug/L, WITH CUPRIC SULFATE AND TRIZMA (Sect. 8.1.2)
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)
Carbazole (SUR)
Initial Injection15
% Recovery
93
72
93
94
93
92
93
93
96
88
Day8
Reinjection
% Recovery
95
73
95
96
96
94
97
95
98
90
Day 14
Reinjection
% Recovery
93
71
93
94
93
93
93
93
96
89
Day 21
Reinjection
% Recovery
103
80
107
99
105
106
102
107
94
91
aStorage stability is expressed as a percent recovery value. Each percent recovery value represents the
mean of 3 replicate analyses. Relative Standard Deviations ([Standard Deviation/Recovery] * 100) for
replicate analyses were all less than 5.0%.
bSame as day 0 sample hold time analysis.
Extract stability study consisted of the analysis of day 0 extracts stored at < 0 C and reinjected.
Samples were analyzed using a 20 uL injection volume and conditions described in Table 1.
                                         532-41

-------
TABLE 10. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS
Method
Reference
Sect. 9.2.1
Sect. 9.2.2
Sect. 9.2.3
Sect. 9.2.4
Sect. 10.2.3
Requirement
Initial
Demonstration of
Low System
Background
Initial
Demonstration of
Precision (IDP)
Initial
Demonstration of
Accuracy
Method Detection
Limit (MDL)
Determination
Peak Gaussian
Factor (PGF)
Specification and Frequency
Analyze LRB prior to any other
IDC steps.
Analyze 4-7 replicate LFBs
fortified at 10 ug/L (or mid cal).
Calculate average recovery for
replicates used in IDP.
Over a period of three days,
prepare a minimum of 7 replicate
LFBs fortified at a concentration
estimated to be near the MDL.
Analyze the replicates through all
steps of the analysis. Calculate
the MDL using the equation in
Sect. 9.2.4.
Calculated prior to establishing
the initial calibration.
Acceptance Criteria
Demonstrate that all
target analytes are below
l/2 the intended MRL or
lowest CAL standard,
and that possible
interference from
extraction media do not
prevent the identification
and quantitation of
method analytes.
RSD must be < 20%.
Mean recovery + 20% of
true value.
Data from MDL
replicates are not required
to meet method precision
and accuracy criteria. If
the MDL replicates are
fortified at a low enough
concentration, it is likely
that they will not meet
precision and accuracy
criteria.
A PGF range of 0.90 to
1.10 is acceptable.
                               532-42

-------
TABLE 11. QUALITY CONTROL REQUIREMENTS (SUMMARY)
 Method
 Reference
Requirement
Specification and Frequency
Acceptance Criteria
 Sect. 8.4
Sample and
Extract Holding
Times
Properly preserved samples
must be shipped at or below
10C and may be held in the lab
at or below 6C for 14 days.
Extracts in methanol may be
stored at or below 0C for up to
21 days after extraction.
Samples exchanged into 60/40
reagent water/acetonitrile may be
held at or below 0C for 7 days,
with the combined extract hold
time not to exceed 21 days.
Do not report data for
samples or extracts that
have not been properly
preserved or stored, or
that have exceeded their
holding time.
 Sect. 9.4
Laboratory
Reagent Blank
(LRB)
Include LRB with each
extraction batch (up to 20
samples). Analyze prior to
analyzing samples and determine
to be free from interferences.
Each analysis batch (Sect. 3.2)
must include either a LRB or an
instrument blank after the initial
low level CCC injection.
Demonstrate that all target
analytes are below 1/3 the
intended MRL or lowest
CAL standard, and that
possible interference from
extraction media do not
prevent the identification
and quantitation  of method
analytes.
 Sect. 9.7
Surrogate
Standards
Surrogate standards are added
to all calibration standards and
samples, including QC samples.
Surrogate recovery must
be 70-130% of the true
value.
                                          532-43

-------
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 9.8
Laboratory
Fortified Sample
Matrix (LFM)
With each extraction batch
(Sect. 3.1), a minimum of one
LFM is extracted and analyzed.
A duplicate LFM, or LFMD,
should be extracted when
occurrence of target analytes is
low. Laboratory duplicate
analysis is not required for
extraction batches containing a
LFMD.
Recoveries not within 70-
130% of the fortified
amount may indicate a
matrix effect, with the
exception of thidiazuron
which should have
recoveries of 60-120%.

If a LFMD is analyzed
instead of a Laboratory
Duplicate, target RPDs
should be+ 30%.
Sect. 9.9
Field Duplicates
(LDlandLD2)
Extract and analyze at least one
duplicate with each extraction
batch (20 samples or less). A
Laboratory Fortified Sample
Matrix Duplicate may be
substituted for a Field Duplicate
when the occurrence of target
analytes is low.
RPDs should be+ 30%.
Sect. 9.10
Quality Control
Sample
Analyze either as a CCC or a
LFB.  Analyze at least quarterly,
with each initial calibration, or
when preparing new standards.
A QCS analyzed as a
CCC will have the same
acceptance criteria as a
CCC, while a QCS
analyzed as a LFB will
have the same criteria as a
LFB.
                                         532-44

-------
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 10.2
Initial Calibration
Use external standard calibration
technique to generate a
calibration curve with five
standards that span the
approximate range of 1 to 30
ug/L sample concentration.
Calibration curve fit options are
discussed in Sect. 10.2.4.
Analyze a QCS.
QCS must be 30% of
true value.

When each calibration
standard is calculated as
an unknown using the
calibration curve, the
results must be 70-130%
of the true value for all but
the lowest standard.  The
lowest standard must be
50-150% of the true value.
The lowest CAL standard
concentration must be as
low or lower than the
intended MRL.
Sect. 10.2.3
Peak Gaussian
Factor (PGF)
Calculated prior to establishing
the initial calibration.
A PGF range of 0.90 to
1.10 is acceptable.
Sect. 10.3
Continuing
Calibration Check
(CCC)
Verify initial calibration by
analyzing a low level CCC prior
to analyzing samples. CCCs are
then injected after every 10
samples and after the last
sample, rotating concentrations
to cover the calibrated range of
the instrument.
Recovery for each analyte
must be 70-130% of the
true value for all but the
lowest level of calibration.
The lowest calibration level
CCC must be 50-150% of
the true value.
Sect. 12.3
Confirmation
Column Results
Positive results should be
confirmed using a chemically
dissimilar column.
Quantitated target values
should be 50% of the
primary result.  Primary
column results should be
reported.  If reporting
confirmation results, both
surrogates must meet 
30% criteria.
                                         532-45

-------
                                                  fin
                                                   4*




                                                   M)
0.050-
0.045-
0.040-
0.035-
0.025-
0.020
0.015-
0.010-
0.005-
                                  o
                                  c
                                  o
                          o
                               o
 
-------
                                              g
                                             o
                                              M)
                                             
0.12-
0.11-
0.10-
                                                         o
                                                         N
                                                                                                                     %
                                                                                                                    CJ
0.09-
0.08-
0.07-
                                                   O
0.05-
0.04-
0.03-
0.02-
0.01-
                         a
                         o
                        H
                     I
                   2.00
                                  4.00
1    '    I    '    '    '    I
      8.00           10.00

            Minutes
  I
12.00
  I
14.00
                                                                  (N

-------