536 -1 EPA Document #: 815-B-07-002

METHOD 536 DETERMINATION OF TR IAZINE PESTICIDES AND THEIR

DEGRADATES IN DRINKI NG WATER BY LIQUID

CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY (LC/ESI-MS/MS)

Version 1.0

October 2007

G.A. Smith and B.V. Pepich (Shaw Environmental, Inc.) D. J. Munch (U.S. EPA, Office of Ground Water and Drinking Water)

TECHNICAL SUPPORT CENTER

OFFICE OF GROUND WA TER AND DRINKING WATER

U. S. ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, OHIO 45268536 -2 METHOD 536

DETERMINATION OF TRIAZINE PESTICIDES AND THEIR DEGRADA TES IN DRINKING WATER BY LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY (LC/ESI-MS/MS)

1. SCOPE AND APPLICATION

1.1 This is a liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESIMS/MS) method for the determination of triazine pesticides and their degradation products in finished drinking waters. This method requires the use of MS/MS in Multiple Reaction Monitoring (MRM) mode to enhance selectivity . Precision and accuracy data have been generated for the detection of th e triazine pesticides and their degradates in reagent water and finished drinking water from both groundwater and surface water sources. The single laboratory Lowest Concentration Minimum Reporting Level (LCMRL) has also been determined in reagent water. 1 The following compounds can be determined using this

method:

Analyte Chemical Abstract Services Registry Number (CASRN)

Atrazine 1912-24-9

Atrazine-desethyl 6190-65-4

Atrazine-desethyl-desisopropyl 3397-62-4

Atrazine-desisopropyl 1007-28-9

Cyanazine 21725-46-2

Propazine 139-40-2

Simazine 122-34-9

1.2 The MRM mass spectrometry conditions describe d in this method were developed using a conventional LC/MS/MS system.

1.3 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets the Data Quality Objectives (DQOs), which are based on the intended use of this method. The single laboratory LCMRL is the lowest true concentrati on for which the future recovery is predicted to fall between 50-150% recovery with 99% conf idence. Single labora tory LCMRLs for the analytes in this method ranged from 0.020 – 0.14 μ g/L, and are listed in Table 4. The procedure used to determine the LCMRL is described elsewhere. 1

1.4

1.5 Laboratories using this method ar e not required to determine an LCMRL, but they will need to demonstrate that their laboratory Minimu m Reporting Level (MRL) for this method meets the requirements described in Section 9.2.4.

Detection limit (DL) is defined as the statistic ally calculated minimum concentration that can

be measured with 99% confidence that th e reported value is greater than zero. 2 The DL is dependent on sample matrix, fortification c oncentration, and instrument performance.536 -3 Determining the DL for analytes in this method is optional (Sect. 9.2.6). DLs for method analytes fortified into reagent water ranged from 0.010 – 0.043 μ g/L. These values are also provided in Table 4.

1.6 This method is intended for use by analysts skilled in the operation of LC/MS/MS instrumentation and the interpretation of the associated data.

1.7 METHOD FLEXIBILITY – In recognition of t echnological advances in analytical systems and techniques, the laboratory is permitted to modify the separation technique, LC column, mobile phase composition, LC conditions and MS conditions (Sect. 6.10, 9.4, 10.2.2, 10.2.3, 10.2.4, and 12.1). Changes may not be made to sample collection and preservation (Sect. 8) or to the quality control requirements (Sect. 9) . Method modifications should be considered only to improve method performance. Modifications that are introduced in the interest of reducing cost or sample processi ng time, but result in poor er method performance, may not be used. The analytes must be resolved chro matographically in order to avoid the effects of matrix suppression or enha ncement and to provide analyte specificity . In all cases where method modifications are propose d, the analyst must perform the procedures

outlined in the Initial Demons tration of Capability (IDC, S ect. 9.2), verify that all QC acceptance criteria in this method (Tables 9 a nd 10) are met, and verify method performance on a real sample matrix (Sect. 9.4).

2. SUMMARY OF METHOD

2.1 Samples are pH adjusted and dechlorinated with ammonium acetat e and protected from microbial degradation using sodium omadine dur ing sample collection. Prior to analysis, isotopically enriched analytes [Atrazine- d5(ethyl-d5), Atrazine-desethyl-d7, Atrazinedesethyl-desisopropyl( 13 C3), Atrazine-desisopropyl-d5(ethyl-d5), Cyanazine-d5(N-ethyl-d5), Propazine-d14(di-isopropyl-d14) , and Simazine-d10(diethyl-d10) ] are added to the samples as internal standards. An aliquot of the samp le is injected without cleanup or concentration onto a chromatographic column (C18). The analytes are separated using an ammonium acetate-buffered binary mobile phase and subs equently detected by positive electrospray ionization tandem mass spectrometry. The conc entration of each id entified component is determined using the internal standard technique.

3. DEFINITIONS

3.1 ANALYSIS BATCH – A sequence of samples, wh ich are analyzed within a 30-hour period and include no more than 20 field samples. Each Analysis Batch must also include all required QC samples, which do not contribute to the maximum fiel d sample total of 20. The required QC samples include: Laboratory Reagent Blank (LRB), Continuing Calibration Check Standards (CCCs), Laboratory Fortified Samp le Matrix (LFSM), and Laboratory Fortified Sample Matrix Duplicat e or Laboratory Duplicate (LFSMD or LD).

3.2 CALIBRATION STANDARD (CAL) – A solution of the target analytes prepared from the primary dilution standard solution(s) or stock sta ndard solution(s) and the internal standards.

The CAL solutions are used to calibrate the in strument response with respect to analyte

concentration.536 -4 3.3 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard containing the method analytes and internal standards which is analyzed periodically to verify the accuracy of the existing calibration.

3.4 DETECTION LIMIT (DL) – The minimum con centration of an analyte that can be identified, measured and repor ted with 99% confidence that the analyte concentration is greater than zero. This is a st atistical determination (Sect. 9.2 .6), and accurate quantitation is not expected at this level. 2

3.5 INTERNAL STANDARD (IS) – A pure compound adde d to all standard solutions and field samples in a known amount. It is used to measure the relative response of the method analyte. The internal standard must be a compound that is not a sample component.

3.6 ION SUPRESSION/ENHANCEMENT – An observable loss or increase in target analyte response in complex (field) samples as compar ed to the response obtained in standard

solutions.

3.7 LABORATORY DUPLICATES (LDs) – Two sample aliquots (LD 1 and LD 2 ), taken in the laboratory from a single sample bo ttle, and analyzed separately with identical procedures. Analyses of LD 1 and LD 2 indicate precision associated sp ecifically with the laboratory procedures by removing varia tion contributed from sample collection, preservation and storage procedures.

3.8 LABORATORY FORTIFIED BLANK (LFB) – An a liquot of reagent water or other blank matrix to which a known quantity of the method analytes is added. The LFB is analyzed exactly like a sample including the preservation procedures in Section 8. Its purpose is to determine whether the methodology is in control, and whether the labora tory is capable of making accurate measurements.

3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM ) – An aliquot of a field sample to which a known quantity of the method analytes is added. The LFSM is processed and analyzed exactly like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analyti cal results. The background concentr ation of the analytes in the sample matrix must be determined in a separa te aliquot and the measur ed value in the LFSM corrected for background concentrations.

3.10 LABORATORY FORTIFIED SAMPLE MATR IX DUPLICATE (LFSMD) – A second aliquot of the field sample used to prepar e the LFSM which is fortified and analyzed identically to the LFSM. The LFSMD is used instead of the Laboratory Duplicate to assess method precision and accuracy wh en the occurrence of the target analytes is infrequent.

3.11 LABORATORY REAGENT BLANK (LRB) – An a liquot of reagent water or other blank matrix that is treated exactly as a sample incl uding exposure to all stor age containers, buffers, preservatives and internal standa rds. The LRB is used to determine if the method analytes or other interferences are present in the laboratory environment, the reagents, or the apparatus.536 -5 3.12 LOWEST CONCENTRATION MINIMUM RE PORTING LEVEL (LCMRL) – The single laboratory LCMRL is the lowest true concentrati on for which the future recovery is predicted to fall between 50-150% recovery with 99% confidence. 1

3.13 MATERIAL SAFETY DATA SHEETS (MSDS) – Th ese sheets contain written information provided by vendors concerning a chemical’s toxi city, health hazards, physical properties, fire, and reactivity data including stor age, spill, and handling precautions.

3.14 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be reported by a laboratory as a quantified value fo r the target analyte in a sample following analysis. This concentration mu st meet the criteria defined in Section 9.2.4 and must be no

lower than the concentration of the lowest calibration standard for the target analyte.

3.15 MULTIPLE REACTION MONITORING (MRM) – A mass spectrometric technique in which a precursor ion is first isolated then subsequently fragmented into product ion(s). Quantitation is accomplished by monitoring a spec ific product ion. As described in Section 10.2.2, MS parameters must be optimized for each precursor ion (Sect. 3.16) and product ion (Sect. 3.18).

3.16 PRECURSOR ION – For the purpose of this method, the precursor ion is the protonated molecule ([M+H] + ) of the target analyte ([M]). In MS /MS, the precursor ion is mass selected and fragmented by collisionally activated dissociation to produce distinct ive product ions of

smaller m/z

.

3.17 PRIMARY DILUTION STANDARD SOLUTION (PDS) – A solution containing the method analytes prepared in the laboratory fr om stock standard solutions and diluted as needed to prepare calibration sta ndards and other analyte solutions.

3.18 PRODUCT ION – For the purpose of this method, a product ion is one of the fragment ions produced in MS/MS by collisionally activat ed dissociation of the precursor ion.

3.19 QUALITY CONTROL SAMPLE (QCS) – A solution containing the method analytes at a

known concentration which is obta ined from a source external to the laboratory and different

from the source of calibration standards. It is used to check laboratory performance with externally prepared test materials.

3.20 REAGENT WATER (RW) – Purified water whic h does not contain any measurable quantity of the target analytes or in terfering compounds at or above 1/3 the MRL for the target analytes.

3.21 STOCK STANDARD SOLUTION (SSS) – A concen trated solution containing one or more of the method analytes that is prepared in th e laboratory using assayed reference materials or purchased from a reputable commercial source, so that the concentration and purity of analytes are traceable to certificates of analysis.

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wash glassware thoroughly and rinse with

reagent water.536 -6 4.2 Method interferences may be caused by contaminan ts in solvents, reagen ts (including reagent water), sample bottles and caps, and other sample processing hardware. These interferences may lead to discrete artifacts and/or elevated baselines in the chromatograms. All laboratory reagents and equipment must be routinely dem onstrated to be free from interferences (less than 1/3 the MRL for the target analytes) under the conditions of the analysis. This may be accomplished by analyzing LRBs as described in Section 3.11. Subtracting blank values from sample results is not permitted.

4.3 Equipment used for sample collection a nd storage has the potential to introduce interferences. The potential for interferences from these devices must be investigated during the Initial Demonstration of Capability (Sect. 9.2) by preparing and analyzing a Laboratory Reagent Blank (LRB). This procedure should be repeated each time a new brand or lot of equipment is used to ensure that background contamination does not interfere with the identification and quantit ation of the analytes.

4.4 Matrix interferences may be caused by contamin ants that are present in the sample. The extent of matrix interferences will vary c onsiderably from source to source depending upon

the nature of the water.

4.4.1 ION SUPPRESSION/ENHANCEMENT – Field samples containing moderate-to-high levels of organic carbon or dissolved solids may cause s uppression or enhancement of instrumental response for one or more of the analytes. Suppression and enhancement effects are introduced during the ionization pr ocess in the electrospray source when a coeluting contaminant influences the ionization of the analyte of interest. MS/MS methods are not immune to suppression or enhancemen t effects because the fragmentation process occurs after ionization. Isotopically enriched internal standards can help mitigate the effect of matrix-induced suppression or e nhancement, but good performance is best achieved by optimizing the chromatographic separation.

4.4.2 INTERFERING COMMON IONS – While MS/MS detection offers increased selectivity

relative to MS mode detect ion, interference can still o ccur in complex samples if common ions are present in co-eluting co mponents. Due to a lack of LC/MS/MS reference spectral libraries, optimized chro matographic separation is required for mass resolution of the analytes of interest in the method.

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely

defined. Each chemical should be treated as a potential health hazar d, and exposure to these chemicals should be minimized. Each laboratory is responsible for maintaining an awareness of OSHA regulations regarding safe handling of chemicals used in this method. A reference

file of MSDSs should be made available to all personnel involv ed in the chemical analysis.

5.2 Pure standard materials and stock standard solutions of the method compounds should be handled with suitable protection for skin, eyes, etc. 3-5536 -7 6. EQUIPMENT AND SUPPLIES (References to specific brands or catalog numbers are included as examples only and do not imply endorsement of the product. This does not preclude the use of other vendors or suppliers.)

6.1 SAMPLE CONTAINERS – Pre-cleaned, amber vials (40 mL or larger) fitted with PTFE/silicone septa and polypropylene screw caps (Fisher Cat. No. 02-912-377 or equivalent).

6.2 VIALS – Amber 2-mL glass autosampler vials w ith PTFE/silicone septa for storing standards (Fisher Cat. No. 03-375-20B or equivalent).

6.3 MICRO SYRINGES – Suggested sizes include 10, 25, 50, 100, 250, and 500 μL.

6.4 VOLUMETRIC FLASKS – Class A, suggest ed sizes include 5, 10, 50, 100, 250, and 500 mL for preparation of reagents and standards.

6.5 VOLUMETRIC PIPETTES – Class A, suggest ed sizes include 1, 4, 5, 10, 20, and 25 mL.

6.6 ANALYTICAL BALANCE – Capable of weighing to the nearest 0.0001 g.

6.7 DESOLVATION GAS – High purity compressed ga s (e.g., nitrogen or zero-air) used for desolvation in the mass spectrometer. The sp ecific type of gas, purity and pressure requirements will depend on the instrument manufacturers’ specifications.

6.8 COLLISION GAS – High purity compressed gas (e.g., nitrogen or argon) used in the collision cell of the mass spectrometer. The specific type of gas, purity and pressure requirements will depend on the instrument manufacturer’s specifications.

6.9 DISPOSABLE PASTEUR PIPETS – 9” borosilicat e glass, used to transfer samples to autosampler vials (Fisher Cat. No. 13-678-20C or equivalent).

6.10 LIQUID CHROMATOGRAPHY ELECTROSPRAY TANDEM MASS SPECTROMETRY SYSTEM (LC/ESI-MS/MS)

6.10.1 LC COLUMN – LC column, Waters Xterra ® RP18, 2.1 x 150 mm, 3.5 μm d p (Waters Part No. 186000410 or equivalent). Any column that provides adequate resolution, peak shape, capacity (Sect. 10.2.3 and 10.2.4), accuracy and precision (Sect. 9), and does not result in suppression or enhancement of analyte response (Sect. 13.4.2) may be used.

6.10.2 LC SYSTEM –The LC system (Waters 2695 or equivalent) must provide consistent sample injection volumes and be capable of performing binary linear gradients at a

constant flow rate.

6.10.3 MASS SPECTROMETER –The MS (Micromass Qu attroMicro or equivalent) must be capable of providing electrospray ionizati on with positive ion detection. The system must be capable of performing MS/MS to produce unique product ions (Sect. 3.18) for the target analytes within sp ecified retention time windows. Data presented in Section 17, Tables 4 - 8 were collected using an ESI-MS/MS system.536 -8 6.10.4 DATA SYSTEM – An interfaced data system is required to acquire, store, and output MS data. The computer software should have th e capability of processing stored LC/MS/MS data by recognizing a chromatographic peak within a given retent ion time window. The software must allow integration of the ion abundance of any specific ion between specified times or scan number limits. The so ftware must be able to construct a linear regression or quadratic calibration curve, and calculate analyte concentrations using the internal standard technique.

7. REAGENTS AND STANDARDS

7.1 REAGENTS AND SOLVENTS – Reagent grade or better chemicals should be used. Unless otherwise indicated, it is intended that all reagents will conform to the specifications of the Committee on Analytical Reagents of the Amer ican Chemical Society (ACS), where such specifications are available. Other grades may be used, as long as the reagent is of sufficiently high purity to permit its use without lessening the quality of the determination.

7.1.1 METHANOL – (CH 3 OH, CAS# 67-56-1) – High purity, de monstrated to be free of analytes and interferen ces (Fisher GC Resolv grade or equivalent).

7.1.2 AMMONIUM ACETATE – (NH 4 C 2 H 3 O 2 , CAS# 631-61-8) – High purity, demonstrated to be free of analytes and interferences (Fisher Brand HPLC gr ade or equivalent).

7.1.3 5 mM AMMONIUM ACETATE MOBILE PHASE – To prepare 1 L of mobile phase, add 0.3854 g ammonium acetate to 1 L of reagent water (final pH ~ 6.5). This solution is prone to volatility losses and s hould be replaced every 48 hours.

7.1.4 2.0 M AMMONIUM ACETATE/REAGEN T WATER CONCENTRATED STOCK – Add 38.5 g of ammonium acetate to a 250-mL vo lumetric flask. Dilute to volume with reagent water. An aliquot of this concentr ated stock solution is diluted 1:100 in all calibration standards and samples to yield a final concentration of 20 mM (1.5 g/L).

7.1.5 SODIUM OMADINE ® (Sodium 2-pyridinethiol-1-oxide, CAS# 3811-73-2) – (C 5 H 4 NOSNa) – 40 wt.% in water (Omadine is a registered trademark of Arch Chemicals, Inc.).

7.1.6 SODIUM OMADINE CONCENTRATED ST OCK SOLUTION (32 g/L) – Add 20 mL

of the 40 wt.% sodium omadine in water to a 250-mL volumetric flask. Dilute to volume with reagent water. The concentrated stoc k solution is diluted 1:500 in all calibration standards and samples to yield a final concentration of 64 mg/L.

7.2 STANDARD SOLUTIONS – When a compound’s pur ity is assayed to be 96 percent or greater, the weight can be used without correction to calculate the concentration of the stock

standard. Solution concentrations listed in this s ection were used to develop this method and are included only as an example . Stock standard solutions are estimated to be stable for six months. Any fortified or dilute solutions made from the stock standards are stable for at least 60 days provided they are stored at a temperature < 6 o C and the stock standard solutions have not exceeded their six month stability period. Although estimated stability times for standard solutions are given, labora tories should use standard QC practices to

determine when their standa rds need to be replaced.536 -9 7.2.1 INTERNAL STANDARD SOLUTIONS – This me thod uses a separate isotopically enriched internal standard for each target analyte, specifically Atrazine-d5(ethyl-d5) (CAS# 163165-75-1), Atrazine-desethyl- d7, Atrazine-desethyl-desisopropyl( 13 C3), Atrazine-desisopropyl-d5(ethyl-d5), Cyan azine-d5(N-ethyl-d5), Propazine-d14(diisopropyl-d14), and Simazine-d10(diethyl-d10 ) (CAS# 220621-39-6). It should be noted

that all of the internal standards except atrazine -desethyl-desisopropyl ( 13 C3) are commercially available; the at razine-desethyl-desisopropyl ( 13 C3) internal standard used during method development was custom s ynthesized by Cambridge Laboratories.

7.2.1.1 INTERNAL STANDARD STOCK STANDARDS (ISSS) (500 μ g/mL, except as noted) – Prepare the stock standards indi vidually by weighing 2.5 mg of the solid

standards into tared 5-mL volumetric flasks and diluting to volume with methanol. Atrazine-desethyl-desisopropyl ( 13 C3) and Simazine-d10 (diethyl-d10) exhibit limited solubility in methanol and must be prepared as < 100 μ g/mL stock solutions. To prepare the 100 μ g/mL stock solutions, weigh 2.5 mg of the solid standards into

tared 25-mL volumetric flasks and dilute to volume with methanol. Sonication may be required to achieve full di ssolution of the standards.

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (PDS) (1.0

μ g/mL) – Prepare the internal standard PDS by adding enough of each ISSS to a volumetric flask partially filled with methanol to make the final concentrations near

1.0 μ g/mL when filled to the mark with methanol. During method development,

addition of 5 μ L of the internal standard PDS to each 1.0-mL sample produced a final concentration of 5 μg/L. Analysts are permitted to use other PDS concentrations and volumes provided all field samples and CAL standards contain the same amount of internal standard, the concentration of the internal standard added provides adequate signal to mainta in precision, and the volume added has a negligible effect on th e final concentration.

7.2.2 ANALYTE STANDARD SOLUTIONS – Obtain th e analytes listed in the table in Section 1.1 as neat standards. Prepare the Analyte Stock and Primary Dilution Standards

as described below.

7.2.2.1 ANALYTE STOCK STANDAR D SOLUTION (500 μ g/mL, except as noted) – Prepare the stock standards individually by weighing 2.5 mg of the solid standards

into tared 5-mL volumetric flasks and dilu ting to volume with methanol. Atrazinedesethyl-desisopropyl and Simazine exhibit lim ited solubility in methanol and must be prepared as < 100 μ g/mL stock solutions. To prepare the 100 μ g/mL stock solutions, weigh 2.5 mg of the solid standa rds into tared 25-mL volumetric flasks

and dilute to volume with methanol. Sonication may be required to achieve full

dissolution of the standards.

7.2.2.2 ANALYTE PRIMARY DILUTION SOLUTION (PDS) (2.5 – 5 μ g/mL) – The analyte PDS is prepared by dilution of the Analyte Stock Standard solutions and is used to prepare the calibration standard s and to fortify the LFBs, LFSMs and LFSMDs with the target analytes.536 -10 7.2.3 CALIBRATION (CAL) STANDARDS – Prepare a procedural calibration curve from dilutions of the analyte PDS in reagent water. Transfer the standard to a proper storage container and add appropriate amounts of the ammonium acetate and sodium omadine concentrated stocks (Sect. 7.1.4 and 7.1.6) as shown in the table below. The target analyte concentrations found in Tables 5-7 can be used as a starting point for determining the calibration range. An example of the dilu tions used to prepare the CALs that were

used to collect data in Sec tion 17 is shown below. The lo west concentration calibration standard must be at or below the MRL, which may depend on system sensitivity. The CAL standards may also be used as CCCs. If stored, the aqueous standards must be refrigerated in the same manner as the samp les. A constant amount of the internal standard is added to each prepared Calibra tion Standard This is accomplished for each standard by taking 1.0 mL of the final cal ibration standard containing the 20 mM ammonium acetate and 64 mg/L sodium omadin e, and placing it in a 2.0-mL autosampler vial and adding the appropriate volume of Internal Standard PDS (Sect. 7.2.1.2).

CAL

Level Analyte

PDS

Conc. (μg/mL) Analyte

PDS

Volume

(μL) Ammonium

Acetate Stock

Volume *
(mL) Sodium

Omadine Stock

Volume *
(mL) Final CAL

Std. Volume (mL) Final CAL

Std. Conc.

(μg/L)

1 5 5 1.0 0.2 100 0.25

2 5 10 1.0 0.2 100 0.50

3 5 20 1.0 0.2 100 1.00

4 5 20 0.5 0.1 50 2.00

5 5 25 0.5 0.1 50 2.50

6 5 10 0.1 0.02 10 5.00

* Ammonium acetate stock (2.0 M) and sodium om adine stock (32 g/L) are added after the

standards have been dilute d to volume and transferred to storage containers.

7.2.3.1 This is a procedural calibration technique. If an alternate set of dilutions are used to prepare the calibration standards, the analys t must ensure that the dilution factor used to prepare the calibration standards remains consistent with the dilution factor during sample collection (Sect. 8.1.1). The dilution factor is currently 101.2/100 in

the table above and in Section 8.1. The an alyst must also ensure that all CAL standards, QC and field samples contain the preservation reagents at their proper concentrations (Sect. 8.1.1).

8. SAMPLE COLLECTION, PRES ERVATION, AND STORAGE

8.1 SAMPLE COLLECTION

8.1.1. Prior to shipment to the field, ammonium acetate and sodium omadine must be added to

each amber bottle fitted with a PTFE-lined scre w cap. If using the suggested 40-mL vials to collect a 40-mL aliquot, add 400 μ L of the ammonium acetate concentrated stock (Sect. 7.1.4) and 80 μ L of the concentrated sodium omad ine stock (Sect. 7.1.6). If other collection volumes are used, adjust the amount of preservation reagen t so that the final

concentrations of ammonium acetate and sodi um omadine in the sample containers are 1.5 g/L and 64 mg/L, respectively. Cap the vial s to avoid evaporation of the preservation

reagents.536 -11 8.1.2 When sampling from a cold water tap, open the tap and allow the system to flush until the water temperature has stabilized (usually approximately 3 to 5 minutes). Collect a representative sample from the flowing system using a beaker of appropriate size. Use this bulk sample to generate individual sample s as needed. Transfer a volume of at least 40 mLs into each collection container, cap the container, and invert it several times to mix the sample with the preservation reagen ts. Care should be taken not to overfill the vial and flush out the preservation reagents. Samples do not need to be headspace free.

8.1.3 When sampling from an open body of water, fill a beaker with water sampled from a representative area. Use this bulk sample to generate individual samples as needed.

8.2 SAMPLE SHIPMENT AND STORAGE – Samples mu st be chilled 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 ar e received at the laboratory. Samples stored

in the lab must be stored at or below 6 °C until analysis. Samples should not be frozen.

8.3 SAMPLE HOLDING TIMES – Samples should be an alyzed as soon as possible. Samples

that are collected and stored as described in Sections 8.1 a nd 8.2 may be stored prior to analysis for a maximum of 28 days.

9. QUALITY CONTROL

9.1 Quality Control (QC) requirements include the In itial Demonstration of Capability (IDC) and ongoing QC requirements that must be met when preparing and analyzing field samples. This section describes each QC parameter, its required frequency, and the performance

criteria that must be met in order to meet EPA quality objecti ves. The QC criteria discussed in the following sections are summarized in Section 17, Tables 9 and 10. These QC requirements are considered the minimum acceptable QC criteria. Laboratories are encouraged to institute additional QC pr actices to meet their specific needs.

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – The IDC must be successfully performed prior to analyzing any field samples. Prior to conducting the IDC, the analyst must meet the calibration requirements outline d in Sections 10.2 and 10.3. Requirements for

the initial demonstration of la boratory capability are described in the following sections and

are summarized in Table 9.

9.2.1 DEMONSTRATION OF LOW SYSTEM BA CKGROUND – Analyze a LRB and

confirm that the blank is reas onably free of contamination and that the criteria in Section

9.3.1 are met.

NOTE: It is good laboratory practice to incl ude a blank in the calibration of any

instrument. The method should also be check ed for carry-over by analyzing a RW blank immediately following the highest CAL standard . If this RW sample does not meet the

criteria outline in Section 9.3.1, then carry-over is present and should be identified and

eliminated.

9.2.2 DEMONSTRATION OF PRECISION – Prepare and analyze 7 replicate LFBs. These samples should be fortified near the midr ange of the initial calibration curve.Ammonium acetate and sodium omadine must be added to the samples as described in Section 8.1. The percent relativ e standard deviation (%RSD) of the concentrations of the replicate analyses must be ≤ 20% for all target analytes.

100 ion Concentrat Average ions Concentrat Measured ofDeviation Standard RSD % × =

9.2.3 DEMONSTRATION OF ACCURACY – Using the sa me sets of replicate data generated for Section 9.2.2, calculate the av erage percent recovery. Th e average percent recovery of the replicate analyses must be within + 30% of the true value.

100 ion Concentra t Fortifie d ion Concentrat Measured Average Recovery % × =

9.2.4 MINIMUM REPORTING LEVEL (MRL) CONF IRMATION – Establish a target

concentration for the MRL based on the in tended use of the method. The lowest

calibration standard used to establish the initial calibration (as well as the low-level Continuing Calibration Check) must be at or below the concentration of the MRL. Establishing the MRL concentration too low may cause repeated failure of ongoing QC requirements. Confirm or validate the MR L following the procedure outlined below.

9.2.4.1 Fortify and analyze seven replicate LFBs at or below the proposed MRL concentration. All samples must be fo rtified and processed using the sample collection protocols described in Section 8.1. Calculate the mean ( Mean ) and standard deviation ( S ) for these replicates. Determine the Half Range for the Prediction Interval of Results ( HR PIR ) using the equation below

HR PIR = 3.963 S

where S is the standard deviation, and 3.963 is a constant value for seven replicates. 1

9.2.4.2 Confirm that the upper and lower limits for the Prediction Inte rval of Results ( PIR =

Mean + HR PIR ) meet the upper and lower recovery limits as shown below.

The Upper PIR Limit must be ≤ 150 percent recovery.

Mean HR

FortifiedConcentration PIR + × ≤ 100 150%

The Lower PIR Limit must be ≥ 50 percent recovery.

Mean HR

FortifiedConcentration PIR − × ≥ 100 50%

536 -12536 -13 9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the criteria

described above. If these criteria are not met, the MRL has been set too low and must be determined again at a higher concentration.

9.2.5 QUALITY CONTROL SAMPLE (QCS) – Analyze a mid-level Quality Control Sample (Sect. 9.3.7) to confirm the accuracy of the calibration curve fit.

9.2.6 DETECTION LIMIT DETERMINATION (optional)

– While DL determination is not a specific requirement of this method, it may be required by various regulatory bodies associated with compliance m onitoring. It is the responsib ility of the laboratory to determine if DL determination is required based upon the intended use of the data.

Analyses for this procedure should be done over at least 3 days. Prepare at least 7 replicate LFBs. Ammonium acetate and sodium omadine must be added to the samples

as described in Section 8.1. Use the solutio ns described in Secti on 7.2 to fortify at a

concentration estimated to be near the DL . This concentration may be estimated by selecting a concentration at 2-5 times the noi se level. Analyze the seven replicates through all steps of Section 11.

NOTE : If an MRL confirmation data set meets these requirements, a DL may be calculated from the MRL confirmation data, and no additional analyses are necessary.

Calculate the DL using the following equation:

DL = St (n-1,1- α = 0.99)

where

t (n-1,1- α = 0.99) = Student's t value for the 99% c onfidence level with n-1 degrees of freedom (for seven replicate dete rminations, the Student’s t value is 3.143 at a 99% confidence level), n = number of replicates, and S = standard deviation of replicate analyses.

NOTE: Do not subtract blank values when performing DL calculations.

9.3 ONGOING QC REQUIREMENTS – Th is section describes the ongoing QC criteria that must be followed when processing and analyzin g field samples. Table 10 summarizes these requirements.

9.3.1 LABORATORY REAGENT BLANK (LRB) – A LRB is analyzed during the IDC and is required with each Analysis Batch (Sect. 3.1) to confirm that background contaminants are not interfering with the identi fication or quantitation of the target analytes. If within the retention time window of any analyte, th e LRB produces a peak that would prevent the determination of that analyte, determin e the source of contamination and eliminate the interference before processing samples. The LRB must contain ammonium acetate, sodium omadine, and the internal standard at the same concentrations used to fortify all field samples and CAL standards. Background from target analytes or contaminants that

interfere with the measurement of target analytes must be < 1/3 the MRL. If the target analytes are detected in the LRB at concentrations equal to or greater than this level, then536 -14 all data for the problem analyte(s) must be considered invalid for all samples which yielded a positive result.

NOTE: Although quantitative data below the MRL may not be accurate enough for data reporting, such data are useful in determini ng the magnitude of background interference. Therefore, blank contamination levels ma y be estimated by extrapolation when the

concentration is below the MRL.

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – CCC standards are analyzed at the beginning of each Analysis Batch, after every ten field samples and at the end of the Analysis Batch. See Section 10.4 for concentr ation requirements and acceptance criteria.

9.3.3 LABORATORY FORTIFIED BLANK (LFB) – Si nce this method utilizes procedural calibration standards, which are fortified re agent waters, there is no difference between the LFB and the continuing calibration check st andard. Consequentl y, the analysis of a separate LFB is not required as part of the ongoing QC; however, the acronym LFB is used for clarity in the IDC.

9.3.4 INTERNAL STANDARDS (IS) – The analyst mu st monitor the peak areas of the internal standards in all inject ions of the Analysis Batch. The IS responses (as indicated by peak areas) for any chromatographic run must not deviate by more than ± 50% from the average areas measured during the initial calibration for the internal standards. If the IS area counts for a sample do not meet this criterion, inject a second aliquot of the suspect sample as part of the same or a new Analysis Batch.

9.3.4.1 If the re-injected aliquot produces an acceptable internal standard response, report results for that aliquot.

9.3.4.2 If the IS area counts of the re-injected aliquot still do not meet the IS criterion,

check the IS area of the most recent CCC. If the IS crit erion is met in the CCC but not the sample, report the sample results as suspect/matrix.

9.3.4.3 If the IS area criterion is not met in both the sample and the CCC, instrument maintenance such as sample cone cleaning may be necessary. Perform the appropriate instrument maintenance and then re-inject the sample in a subsequent analytical batch.

9.3.5 LABORATORY FORTIFIED SAMPLE MATRIX (L FSM) – Analysis of a LFSM (Sect. 3.9) is required in each Analysis Batch. The LFSM is processed and analyzed exactly like a sample, and its purpose is to determine whet her 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 a liquot and the measured values in the LFSM corrected for background concentr ations. If a variety of di fferent sample matrices are analyzed regularly, for example drinking water from groundwater and surface water sources, performance data should be collected for each source.

9.3.5.1 Within each Analysis Batch, a minimum of one field sample is fortified as an LFSM for every 20 samples analyzed. The LFSM is prepared by fortifying a sample with an appropriate amount of the analyt e PDS (Sect. 7.2.2.2). Select a spikingconcentration that is greater than or e qual to the native back ground concentration, if known. Selecting a duplicate a liquot of a sample that has already been analyzed aids in the selection of an appropriate spik ing level. If this is not possible, use historical data and rotate through low, medium and high calibration concentrations when selecting a fortifying concentration.

9.3.5.2 Calculate the percent recovery (%REC) using the equation:



## ( ) 100 C B-A %REC ×=

where A = measured concentration in the fortified sample,

B = measured concentration in the unfortified sample, and

C = fortification concentration.

9.3.5.3 Recoveries for samples fortified at concen trations near or at the MRL (within a

factor of two times the MRL con centration) should be within + 50% of the true value. Recoveries for samples fortified at all other concentrations should be within + 30% of the true value. If the accuracy for any analyte falls outside the designated range, and the laboratory performance for that analyte is shown to be in control in the CCCs, the recovery is judged to be matrix biased. The result for that analyte in the unfortified sample is labeled suspect/mat rix to inform the data user that the results are suspect due to matrix effects.

9.3.5.3.1 Field samples that have native analyte concentrations above the DL but below

the MRL and are fortified at concentrations at or near the MRL should be

corrected for the native levels in orde r to obtain meaningful %REC values. This example and the LRB (Sect. 9.3.1) ar e the only permitted use of analyte

results below the MRL.

9.3.6 LABORATORY DUPLICATE OR LABORA TORY FORTIFIED SAMPLE MATRIX DUPLICATE (LD or LFSMD) – Within each Analysis Batch, a minimum of one Laboratory Duplicate (LD) or Laboratory Fo rtified Sample Matrix Duplicate (LFSMD) must be analyzed. Laboratory Duplicates ch eck the precision associated with laboratory procedures. If target analyt es are not routinely observed in field samples, a LFSMD should be analyzed rather than a LD.

9.3.6.1 Calculate the relative percent differen ce (RPD) for duplicate measurements (LD 1 and LD 2 ) using the equation:



## ( ) 100 2/LDLD

LDLD RPD

2 1 2

1 ×
+ −

=

9.3.6.2 RPDs for Laboratory Duplicates should be ≤ 30%. Greater va riability may be observed when Laboratory Duplicates have an alyte concentrations that are within a factor of 2 x MRL. At these concentrations, Laboratory Duplicates should have

536 -15RPDs that are ≤ 50%. If the RPD of the analyte falls outside the designated range, and the laboratory performance for the analyt e is shown to be in control in the CCC, the precision is judged to be matrix in fluenced. The result from the unfortified sample is labeled suspect/matrix to inform the data user that the results are suspect

due to matrix effects.

9.3.6.3 If a LFSMD is analyzed instead of a La boratory Duplicate, ca lculate the relative percent difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the equation:



## ( ) 100 /2LFSMD LFSM LFSMD

LFSM RPD ×
+

−

=

9.3.6.4 RPDs for duplicate LFSMs should be ≤ 30%. Greater variability may be observed when fortified LFSMs have analyte concen trations that are within 2 x MRL.

LFSMs at these concentrations should have RPDs that are ≤ 50%. If the RPD of the analyte falls outside the designated range , and the laboratory performance for the analyte is shown to be in control in the CCC, the precision is judged to be matrix

influenced. The result from the unfortifie d sample is labeled suspect/matrix to inform the data user that the results are suspect due to matrix effects.

9.3.7 QUALITY CONTROL SAMPLE (QCS) – A QCS is required if an alternate commercial source is available for the target analytes. A QCS should be evaluate d as part of the IDC (Sect. 9.2.5) and each time new PDS solutions ar e prepared. If standards are prepared infrequently, analyze a QCS at least quarter ly. The QCS should be fortified near the midpoint of the calibration range and analyzed as a CCC. Th e acceptance cr iteria for the QCS are the same as the mid- and high-level CCCs (Sect. 10.4). If measured analyte concentrations are not of acceptable accuracy, check the entire analytical procedure to locate and correct the problem.

9.4 METHOD MODIFICATION QC RE QUIREMENTS - The analyst is permitted to modify the separation technique, LC column, mobile phase composition, LC conditions and MS

conditions.

9.4.1 Each time method modifications are made, the an alyst must repeat the procedures of the IDC (Sect. 9.2) and verify that all QC crite ria can be met in ongoing QC samples (Sect. 9.3).

9.4.2 The analyst is also required to evaluate and document method performance for the proposed method modifications in real matrices that span the range of waters that the laboratory analyzes. This additional step is required because modifications that perform acceptably in the IDC, which is conducted in reagent water, can fail ongoing method QC requirements in real matrices. This is pa rticularly important for methods subject to matrix effects, such as LC/MS-based methods . If, for example, the laboratory analyzes finished waters from both surface and groundwat er municipalities, this requirement can be accomplished by assessing precision and accuracy (Sects. 9.2.2 and 9.2.3) in a surface water with moderate to high total organic ca rbon ( e.g., 2 mg/L or greater) and a hard groundwater (e.g., 250 mg/L as CaCO 3 equivalent, or greater).

536 -16536 -17 9.4.3 The results of Sections 9.4.1 and 9.4.2 must be appropriately documented by the analyst and should be independently assessed by the la boratory’s QA officer prior to analyzing field samples.

9.4.3.1 When implementing method modifications, it is the responsibility of the laboratory to closely review the results of ongoing QC, and in particular, th e results associated with the LFSM (Sect. 9.3.5), LFSMD (S ect. 9.3.6), CCCs (Sect. 9.3.2), and the IS area counts (Sect. 9.3.4). If repeated failures are noted, the modification must be

abandoned.

10 CALIBRATION AND STANDARDIZATION

10.1 Demonstration and documentation of accepta ble MS calibration and initial analyte calibration are required before performing the IDC (Sect. 9.2) and prio r to analyzing field samples. After initial calibration is su ccessful, Continuing Calibration Check (CCC) standards are required at the beginning and th e end of each analysis batch, and after every tenth sample (Sect. 10.4). Verification of th e MS calibration and th e initial calibration should be repeated each time a major instrume nt modification or maintenance is performed.

10.2 LC/MS/MS CALIBRATI ON AND OPTIMIZATION

10.2.1 MASS CALIBRATION – Calibrate the mass spectrometer with the calibration compounds and procedures specified by the manufacturer.

10.2.2 OPTIMIZING MS PARAMETERS – This method requires the utilization of ESI and Multiple Reaction Monitoring as defined a bove (Sect. 3.15). LC/M S instruments have several instrumental parameters that must be optimized. Each LC/MS system will have different optimal conditions which are infl uenced by the source geometry and system design. Due to the differences in desi gn, the recommendations of the instrument manufacturer should be followed when tuning th e instrument. In the development of this method, instrumental parameters were optimized for the precursor and product ions listed in Section 17, Table 3. Literature resources to date describe similar precursor and product ions. 6,7 Product ions other than those lis ted may be selected; however, the analyst is cautioned to avoid using ions with lower mass an d/or common ions that may not provide sufficient discrimi nation between the analytes of interest and co-eluting

interferences.

10.2.2.1 Prepare 0.05 – 1.0 μg/mL solutions of the targ et analytes in the approximate mobile phase compositions that correspond to the elution mobile phase compositions. This step will need to be repeated and the pa rameters re-optimized if an analyte is prepared in a mobile phase composition th at is significantly different from the mobile phase composition at the time the analyte elutes from the column.

10.2.2.2 Optimize the response of the precursor ion (Sect. 3.16) for each target analyte by infusing the prepared solutions (Sect. 10.2.2.1) directly into the mass spectrometer as recommended by the instrument manufact urer. The LC should be operating with a mobile phase composition and flow rate comparable to the elution conditions. Vary the MS parameters (source voltages, source and desolvation temperatures, gas536 -18 flows, etc.) until optimal analyte responses are determined. The target analytes may have different optima, thus requiring some compromise. LC/MS systems are subject to drift, which can affect run-to-run precision. Acco rdingly, the optimal conditions may be those that yield good response while mi nimizing the potential for drift. See Section 17, Table 2 for the ESI+ MS conditions used during method development.

10.2.2.3 In all cases, a precursor ion must be isolated (e.g., in a quadrupole or an ion trap) prior to fragmentation. Optimize the re sponse of the product ion (Section 3.18) for each target by infusing the prepared solu tions (Sect. 10.2.2.1) directly into the mass spectrometer as recommended by the instrume nt manufacturer. The LC should be operating with a mobile phase composition a nd flow rate comparable to the elution conditions. The MS/MS parameters (collis ion gas pressure, collision energy, etc.) are varied until optimal analyte responses are determined. The triazine compounds listed in this method exhibited very sim ilar optimal MS/MS conditions. See Section 17, Table 3 for the MS/MS conditions used in method development.

10.2.3 LC INSTRUMENT CONDITIONS – LC operating conditions are described in Section 17, Table 1. Conditions different from those described (e.g., LC columns and mobile phases) may be used if the QC criteria in Sections 9.2 and 9.3 are met and chromatographic separation of the targ et analytes is achieved (resolution > 50%).

NOTE: Chromatographic separation as defi ned does not include the isotopically enriched internal standards, which are ma ss separated. Co-elution of the internal standards with their analogous target analyt es helps mitigate matrix suppression and/or

enhancement effects. 8

10.2.4 ESTABLISH LC/MS RETENTION TIMES AND MR M SEGMENTS – Inject a mid- to high-level CAL standard under optimized LC/M S conditions to obtain the retention times of each target analyte. Divide the chromatogr am into segments that contain one or more chromatographic peaks. For maximum sensitivity in subsequent MS/MS analyses, minimize the number of MRM (Sect. 3.15) trans itions that are simultaneously monitored within each segment.

10.3 INITIAL CALIBRATION – During method developmen t, daily calibrations were performed; however, it is permissible to verify the calib ration with daily CCCs. Calibration must be performed using peak areas and the internal standard technique. Calibration using peak heights or external calibration is not permitted.

10.3.1 CALIBRATION STANDARDS – Prepare a set of at least five CAL standards as

described in Section 7.2.3. The lowest concentration of the calibration standards must be

at or below the MRL. The MRL must be confirmed using the procedure outlined in Section 9.2.4 after establishing the initial calibrati on. Additionally, fiel d samples must be quantified using a calibration curve that span s the same concentration range used to collect the IDC data (Sect. 9.2).

10.3.2 CALIBRATION – The LC/MS system is calibrated using peak areas and the internal standard technique. Concentra tions may be calculated using th e data system software to generate a linear regression or quadratic calib ration curve. Forcing the calibration curve536 -19 through the origin is not recommended. The MS/MS instrument used during method development was calibrated using weighted (1/X ) quadratic curves. Data may be fit with either a linear regression (response vs. c oncentration) or quadratic fit (response vs. concentration) and weighting may be used, if necessary.

10.3.3 CALIBRATION ACCEPTANCE CRITERIA – The validation of the calibration is determined by calculating the concentration of the analyte from each of the analyses used to generate the calibration curve. Calibration points that are ≤ MRL should calculate to

be within + 50% of their true value. All other calibration points should calculate to be

within + 30% of their true value. If these crit eria cannot be met, the analyst will have difficulty meeting ongoing QC criteria. In this case, corrective action should be taken to reanalyze the calibration standard s, restrict the range of calib ration, or select an alternate

method of calibration.

10.4 CONTINUING CALIBRATION CHECKS (CCCs) – The CCC verifies the calibration at the beginning, after every tenth field sample and at the end of each Analysis Batch. CCCs are not counted as samples . The beginning CCC for each Analysis Batch must be at or

below the MRL . This CCC verifies instrument sensitivit y prior to the analysis of samples. Subsequent CCCs should alternate between a me dium and high concentration CAL standard.

10.4.1 Inject an aliquot of the appropriate concen tration CAL standard and analyze with the

same conditions used duri ng the initial calibration.

10.4.2 Determine that the absolute areas of the quantita tion ions of each of the internal standards have not changed by more than ± 50% from the average areas measured during the initial calibration. If IS areas have changed by more than + 50%, remedial action is necessary (Sect. 10.4.4). Recalibrate according to Secti on 10.3 and verify sensitivity by analyzing a

CCC at or below the MRL.

10.4.3 Calculate the concentration of each anal yte in the CCC. The CCC fortified at ≤ MRL

must calculate to be within + 50% of its true value. CCCs fortified at all other levels

must calculate to be within + 30%. If these conditions do not exist, then all data for the analyte must be considered invalid, and re medial action (Sect. 10.4.4) must be taken. The remedial action may require recalibration. Any field samples that have been analyzed since the last acceptable CCC that are still within holding time should be reanalyzed after adequate calibration has been restored.

10.4.4 REMEDIAL ACTION – Failure to meet CCC QC performance criteria requires remedial action. Maintenance such as cleaning an i on source probe, sample cone, ion lenses and/or replacing LC columns may be require d. Following major maintenance, the analyst

must return to the initial calibration step (Sect. 10.3).

11. PROCEDURE

11.1 Important aspects of this analytical procedur e include proper sample collection and storage (Section 8), ensuring that the instrument is properly calibrated (Section 10) and that all required QC are met (Section 9). This section describes the procedures for sample preparation and analysis.536 -20 11.2 SAMPLE PREPARATION

11.2.1 Samples are dechlorinated, preserved, collected and stored as desc ribed in Section 8.1. All field and QC samples must contain the d echlorinating and preser vation agents listed in Section 8.1.1, including the LRB. Allow field samples to reach room temperature prior to analysis.

11.2.2 Transfer a 1.0 mL aliquot of each field or QC sample to an autosampler vial; as appropriate, samples that will be chromatographed using small particle diameter LC columns (< 2 μm) may be filtered prior to analysis (see note below). Add the IS PDS (Sect. 7.2.1.2), cap and mix well. Larger amoun ts of sample may be used, but the amount of internal standard must be increased to gi ve a final concentration that is equal to the

CAL IS concentration.

NOTE: If filtering is incorporat ed as part of the sample preparation, the first lot of filters must be subjected to the procedures ou tlined in the IDC (Sect. 9.2) and meet the acceptance criteria defined in Table 9 to ensure that they do not intr oduce interferences or retain any of the target analytes. Verification of subsequent lots of filters can be accomplished by examining a LRB and duplicate sa mples of LFBs fortified at the MRL.

The LFBs should calculate to be within + 50% of the true value. If the LRB or the LFBs fail this evaluation, the full IDC will need to be repeated with the new lot of filters. CAL

standards and CCCs should not be filtered in or der to identify potenti al losses associated with the sample filtration devices.

11.3 SAMPLE ANALYSIS

11.3.1 Establish MS operating conditi ons equivalent to those su mmarized in Tables 1–3 of Section 17 as per the guidance in Section 10.2. Column choice and instrument parameters should be optimized pr ior to initiation of the IDC.

11.3.2 Establish an appropriate retention time wi ndow (Sect. 10.2.4) for each analyte to identify them in QC and field sample chromatograms. This should be based on measurements of

actual retention time variation for each com pound in standard solutions analyzed on the LC over the course of time. The suggested variation is plus or minus three times the

standard deviation of the rete ntion time for each compound for a series of injections. The injections from the initial calibration and from the IDC (Sect. 9.2) may be used to calculate a suggested window si ze. However, the experience of the analyst should weigh heavily on the determination of an appropriate retent ion window size.

11.3.3 Establish a valid initial calibration following the procedures outlined in Section 10.3 or

confirm that the calibration is still valid by running a CCC as described in Section 10.4. If establishing an initial calibration for the fi rst time, complete the IDC as described in Section 9.2 prior to analyzing field samples.

11.3.4 Analyze aliquots of field and QC samples at appropriate frequencies (Section 9) with the

LC/MS/MS conditions used to acquire the initial calibration.536 -21 11.3.5 At the conclusion of data acquisition, use the sa me software settings that were used in the calibration procedure to identif y peaks of interest in the predetermined retention time windows. Identify an analyte by comparison of its retention time with that of the corresponding analyte peak in a CAL standard or CCC.

11.3.6 EXCEEDING CALIBRATION RANGE – The anal yst must not extrapolate beyond the established calibration range. If an analyt e result exceeds the range of the initial calibration curve, the sample may be dilu ted using reagent water containing 20 mM ammonium acetate and 64 μg/L sodium omad ine and with the appropriate amount of

internal standard added to ma tch the original level. Re-inject the diluted sample. Incorporate the dilution factor into final concentration calculations. The resulting data

should be documented as a dilution and the dilution will affect analyte MRLs.

12. DATA ANALYSIS AND CALCULATIONS

12.1 While complete chromatographic resolution is not necessary for accurate and precise measurements of analyte concentrations using MS/M S in reagent water, it is important in real matrices. The analyst must avoid chromatogra phic modifications that result in suppression or enhancement of analyte responses due to co-e luting matrix components. This is especially a problem with samples that contain higher TO C levels (Sect. 13.4.2). In validating this method, concentrations were calculated using the product ions listed in Table 3 of Section 17. Other ions may be selected at the discretion of the analyst as discussed in Section 10.2.2.

12.2 Calculate analyte concentrations using the multipoint calibrati on established in Section 10.3. Report only those values that fall between th e MRL and the highest CAL standard. Samples with target analyte responses that exceed the highest CAL standard require dilution and reanalysis (Sect. 11.3.6).

12.3 Calculations must use all available digits of precision, but final reported concentrations should be rounded to an appropria te number of significant figure s (one digit of uncertainty), typically two, and not more th an three significant figures.

12.4 Prior to reporting the data, th e chromatograms should be reviewed for any incorrect peak identifications or poor integrations.

12.5 Prior to reporting data, the labor atory is responsible for assuri ng that QC requirements have been met or that any appropr iate qualifier is documented.

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY AND DETECTION LI MITS – Tables for these data are presented in Section 17. Instrumental conditi ons are presented in Tables 1 through 3. DLs and LCMRLs are presented in Table 4. LCMR Ls were determined and calculated using a procedure described elsewhere. 1 Single laboratory precision and accuracy data are presented for three water matrices: reagent water (Table 5); chlorinated (finished) surface water (Table 6); and chlorinated (finishe d) groundwater (Table 7). Figure 1 shows an overlaid chromatogram of the normalized MS/MS res ponses for the analytes obtained under the conditions employed during method development.536 -22 13.2 SECOND LABORATORY EVALUATION – The performance of this method was demonstrated by a second laboratory, with resu lts similar to those reported in Section 17. The authors wish to acknowledge the work of Dr. Jody Shoemaker of the U.S. EPA National Exposure Research Laboratory, Cincinnati , OH for conducting the second laboratory

evaluation.

13.3 ANALYTE STABILITY STUDIES – Chlorinated surface water samples, fortified with target analytes at 5 μg/L, were preserved as required in Section 8 a nd stored over a 28-day period. The accuracy and precision of three replicate analyses, conducted on day 0, 7, 14, 21 and 28, are presented in Section 17, Table 8. These data were used to determine the 28-day holding time.

13.4 PROBLEM COMPOUNDS – During method deve lopment and the second laboratory evaluation, the following problems were obs erved for Atrazine-d esethyl-desisopropyl (DACT):

13.4.1 DACT is subject to hydrolysis at both high (> 9) and low (< 5) pH but has acceptable stability in neutral aqueous solutions. DACT is also degraded in the presence of free available chlorine, but stable in the pres ence of chloramines. Ammonium acetate mitigates both of these modes of loss and has sufficient buffer capacity at 20 mM to buffer finished groundwaters with relatively high pH that also have buffer capacity.

13.4.2 DACT and its internal standard (DACT- 13 C) exhibited ESI matrix suppression in the second laboratory evaluation after being fortified into a chlorinated surface water containing high total organic carbon (T OC) levels (9.5 mg/L). The DACT- 13 C response was suppressed 26% relative to the calibration; however, si nce DACT was affected by the suppression in an equivalent manner, the DACT recovery was 99%. The matrix suppression occurred due to co-eluting TOC in the early part of the chromatographic run and DACT is the analyte most susceptible to matrix effects. As discussed in Section 4.4.1, good performance is achieved by optimizi ng chromatographic separation, and in particular, the separation of DACT from early eluting contaminants. Modifications that seek to shorten the chromatographic sepa ration without accounting for the need to separate the analytes from early eluting inte rferences may result in failure to meet QC

criteria.

14. POLLUTION PREVENTION

14.1 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 Depart ment of Government Relations and Science Policy, 1155 16th Street N.W., Wa shington, D.C., 20036, or on-line at http://www.ups.edu/community/storeroom/Chemical_Wastes/wastearticles.htm.

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 so lvents are used. The matrices of concern are finished drinking water or source water. However, the Ag ency requires that laboratory waste management practices be conducted c onsistent with all applicable rules and536 -23 regulations, and that laborato ries protect the air, wate r, and land by minimizing and controlling all releases from fu me hoods and bench operations. Also, compliance is required with any sewage discharge permits and regul ations, particularly the hazardous waste identification rules and land disposal restri ctions. For further information on waste management, see the publications of the American Chemical Society’s Laboratory Environment, Health & Safety Task Force on the Internet at http://membership.acs.org/c/ ccs/publications.htm. Additional waste management information can be found in “Laboratory Wa ste Minimization and Pollution Prevention,” Copyright © 1996 Battelle Seattle Research Center, which can be located at http://www.p2pays.org/ref/01 /text/00779/index2.htm.

16. REFERENCES

1. Winslow , S.D.; Pepich, B.V.; Martin, J.J.; Hallberg, G. R.; Munch D.J.; Frebis, C.P.; Hedrick, E.J.; Krop, R.A. Statistical Procedures for Determinat ion and Verification of Minimum Reporting Levels for Drinking Water Methods. Environ. Sci. Technol. 2006

, 40 , 281-288.

2. Glaser, J.A.; Foerst, D.L.; McKee, G.D.; Quave, S.A.; Budde, W.L. Trace Analyses for

Wastewaters. Environ. Sci. Technol. 1981, 15 , 1426-1435.

3. “Guidelines, Recommendations, and Regulations for Handling Antine oplastic Agents”, Center for Disease Control, National Institute fo r Occupational Safety and Health, http://www.cdc.gov/niosh/topics/antineoplastic/pubs.html#b.

4. “OSHA Safety and Health Standards, General Industry,” (29CFR1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

5. “Safety in Academic Chemistry Laboratories,” Am erican Chemical Society Publication, Committee on Chemical Safety, 3 rd Edition, 1979.

6. Steen, Ruud J.C.A.; Hogenboom, Ariadne C.; Leonard s, Pim E.G.; Peerboom, Renee A.L.; Cofino, Wim P.; Brinkman, Udo A.Th. Ultra-trace Level Determination of Polar Pesticides and Their Transformation Products in Surface and Estuarine Water Samples Using Column Liquid Chromatography-Electrospray Tandem Mass Spectrometry. J. Chromatogr. A 1999

, 857, 157-166.

7. Huang, S.; Mayer, T.J.; Yokley, R.A.; Per ez, R. Direct Aqueous Injection Liquid Chromatography/Electrospray Io nization-Mass Spectrometry/Mass Sp ectrometry Analysis of Water for Atrazine, Simazine, and thei r Chlorotriazine Metabolites. J. Agric. Food Chem

. 2006

, 54 , 713-

719.

8. Kloepfer, A.; Quintana, J.B.; Reemtsma, T. Opera tional Options to Reduce Matrix Effects in Liquid Chromatrography-Electrospray Ionization-Mass Spect rometry Analysis of Aqueous Environmental Samples. J. Chromatogr. A 2005

, 1067 , 153-160.536 -24 17. TABLES, DIAGRAMS, FLOWCHART S, AND VALIDATION DATA

TABLE 1. LC CONDITIONS a

Time (min.) %5 mM NH 4 OAc % Methanol

Initial 90 10

5.0 90 10

25.0 70 30

25.1 60 40

35.0 60 40

35.1 50 50

45.0 50 50

45.1 90 10

65.0 90 10

a Chromatographic conditions: Column: Waters Xterra ® RP18, 2.1 x 150 mm, 3.5 μ m particle

diameter; Injection Volume: 100 μ L; Flow Rate: 0.250 mL/min.

TABLE 2. ESI-MS/MS METHOD CONDITIONS

MS Parameter Setting Polarity Positive Ion Mode Capillary Voltage 0.5 kV Source Temperature 100 o C Desolvation Temperature 260 o C

Desolvation Gas Flow 616 L/hr

Cone Gas Flow 26 L/hr

Collision Cell Pressure 2.1 e-3 mbar

Collision Gas Argon536 -25 TABLE 3. LC/ESI-MS/MS RETENTION TIMES, PRECURSOR AND PRODUCT IONS,

AND COLLISION ENERGIES

MRM Segment

# Analyte Ret.

Time (min.) Precursor

Ion (m/z) Product

Ion (m/z) Collision Energy

(eV)

1 Atrazine-desethyl-desisopropyl- 13 C (IS) 3.78 149 105.5 22

1 Atrazine-desethyl-desisopropyl 3.78 146 103.5 22

2 Atrazine-desisopropyl-d5 (IS) 11.26 179 137 22

2 Atrazine-desisopropyl 11.57 174 132 22

3 Atrazine-desethyl-d7 (IS) 18.66 195 147 22

3 Atrazine-desethyl 19.09 188 146 22

4 Simazine-d10 (IS) 30.48 212 137 20

4 Simazine 30.96 202 132 20

4 Cyanazine-d5 (IS) 32.03 246 219 20

4 Cyanazine 32.20 241 214 20

5 Atrazine-d5 (IS) 37.03 221 179 23

5 Atrazine 37.37 216 174 23

6 Propazine-d14 (IS) 42.87 244 196 20

6 Propazine 43.51 230 188 20

TABLE 4. LC/ESI-MS/MS LOWEST CONCENTRATION MRLs AND DLs a

Analyte LCMRL Concentration Levels (μg/L) Calculated LCMRL (μg/L) Calculated DL (μg/L) Atrazine-desethyl-desisopropyl 0.104, 0.208, 0.260, 0.520 0.14 0.028 Atrazine-desisopropyl 0.102, 0.204, 0.255, 0.510 0.11 0.043 Atrazine-desethyl 0.021, 0.052, 0.104, 0.130 0.026 0.017

Simazine 0.020, 0.050, 0.100, 0.125 0.020 0.010 Cyanazine 0.019, 0.048, 0.096, 0.120 0.027 0.010

Atrazine 0.021, 0.052, 0.104, 0.130 0.037 0.010 Propazine 0.020, 0.050, 0.100, 0.125 0.028 0.010

a DLs calculated using data acquired on a single day at the lowest LCMRL concentration level (n=7 sample

replicates).536 -26 TABLE 5. LC/ESI-MS/MS PRECISION AND ACCURACY OF METHOD ANALYTES FORTIFIED AT 0.5 μg/L AND 5.0 μg/L IN REAGENT WATER (n=7 SAMPLES)

Fortified Conc. = 0.5 μg/L (n=7) Fortified Conc. = 5.0 μg/L (n=7) Analyte

Mean % Recovery Relative Standard Deviation (%RSD) Mean % Recovery Relative Standard

Deviation (%RSD) Atrazine-desethyl-desisopropyl 98.9 4.9 89.4 4.2 Atrazine-desisopropyl 100 3.4 96.1 3.7 Atrazine-desethyl 99.4 2.7 97.6 1.3

Simazine 101 3.0 101 1.9 Cyanazine 97.7 1.3 99.3 1.4

Atrazine 99.4 2.5 99.7 1.6 Propazine 103 2.9 99.0 2.0

TABLE 6. LC/ESI-MS/MS PRECISION AND ACCURACY OF METHOD ANALYTES FORTIFIED AT 0.5 μg/L AND 5.0 μg/L IN A CHLORINATED SURFACE

WATER a (n=7 SAMPLES)

Fortified Conc. = 0.5 μg/L (n=7) Fortified Conc. = 5.0 μg/L (n=7) Analyte

Mean % Recovery b Relative Standard Deviation (%RSD) Mean % Recovery b Relative Standard

Deviation (%RSD) Atrazine-desethyl-desisopropyl 102 8.4 103 2.7 Atrazine-desisopropyl 100 6.6 101 3.7 Atrazine-desethyl 95.5 1.5 97.9 0.9

Simazine 94.5 3.7 99.4 1.7 Cyanazine 95.3 4.2 99.4 1.7

Atrazine 97.4 6.1 101 0.9 Propazine 94.9 6.0 105 1.5

a Surface water physical parameters: pH=7.1; Hardness = 154 mg/L; Free chlorine = 1.5 mg/L (Hach).

b Recoveries corrected for native levels in the unfortified matrix: Atrazine-desethyl-desisopropyl (0.043 μg/L),

Atrazine-desisopropyl (0.001 μg/L), Atrazine-desethyl (0.0 31 μg/L), Simazine (0.054 μg/L), Cyanazine (0.018 μg/L), Atrazine (0.316 μg/L) and Propazine (0.038 μg/L).536 -27 TABLE 7. LC/ESI-MS/MS PRECISION AND ACCURACY OF METHOD ANALYTES FORTIFIED AT 0.5 μg/L AND 5.0 μg/L IN A CHLORINATED

GROUNDWATER a (n=7 SAMPLES)

Conc. = 0.5 μg/L (n=7) Conc. = 5.0 μg/L (n=7) Analyte

Mean % Recovery b Relative Standard

Deviation (%RSD) Mean % Recovery b Relative Standard Deviation (%RSD)

Atrazine-desethyl-desisopropyl 106 3.5 95.7 2.5 Atrazine-desisopropyl 99.6 5.2 103 3.0 Atrazine-desethyl 95.7 1.7 98.6 1.5

Simazine 94.6 3.1 98.0 1.5 Cyanazine 93.5 1.8 101 1.3

Atrazine 96.7 0.9 100 1.5 Propazine 91.7 3.7 99.7 1.4

a Groundwater physical parameters: pH = 7.5; Hardness = 360 mg/L; Free chlorine = 0.49 mg/L.

b Recoveries corrected for native levels in the unfortified matrix: Atrazine-desethyl-desisopropyl (0.032μg/L),

Atrazine-desisopropyl (not detected), Atrazine-desethyl (0.036 μg/L), Simazine (0.030 μg/L), Cyanazine (0.018 μg/L), Atrazine (0.057 μg/L), and Propazine (0.036 μg/L).536 -28TABLE 8. LC/ESI-MS/MS SAMPLE HOLDING TIME DATA FOR METHOD ANALYTES FORTIFIED AT 5.0 μg/L IN ACHLORINATED SURFACE WATERa(n=3 SAMPLES)AnalyteDay 0Day 7Day 14Day 21Day 28%Rec%RSD%Rec%RSD%Rec%RSD%Rec%RSD%Rec%RSDAtrazine98.04.197.42.999.41.194.37.099.30.8Atrazine-desethyl1072.51044.11111.71034.71081.5Atrazine-desethyl-desisopropyl95.97.793.96.21021.290.86.61033.6Atrazine-desisopropyl1053.589.87.695.41.193.57.91021.8Cyanazine99.34.794.01.294.81.687.84.789.50.5Propazine1122.91024.81092.51036.11091.6Simazine94.26.099.64.893.66.889.111.81002.5aSurface water physical parameters: pH = 7.0; Hardness=154 mg/L; Free chlorine = 1.2 mg/L (Hach).536 -29 TABLE 9. INITIAL DEMONSTRATION OF CAPAB ILITY (IDC) QUALITY CONTROL REQUIREMENTS

Method

Reference Requirement Specification and Frequency Acceptance Criteria

Section

9.2.1 Demonstration of Low System Background Analyze a LRB prior to any other IDC steps. Demonstrate that all target analytes are < 1/3 of the MRL and that possible interferences from reagents and glassware do not prevent the identification and quantitation of target analytes.

Section

9.2.1 Test For System Carryover Analyze a reagent water blank after the high CAL standard during the IDC

calibration. Demonstrate that the target analytes are < 1/3 of the MRL and that carry-over from previous samples does not prevent the identification and quantitation of target analytes.

Section

9.2.2 Demonstration of

Precision Analyze 7 replicate CCCs fortified near the midrange

concentration. %RSD must be ≤ 20%

Section

9.2.3 Demonstration of Accuracy Calculate average recovery for replicates used in

Section 9.2.2. Mean recovery within + 30%

of the true value.

Section

9.2.4 Minimum Reporting Limit (MRL)

Confirmation Fortify and analyze 7 replicate CCCs at the proposed MRL

concentration. Calculate

the mean and the Half Range (HR). Confirm that the Upper PIR and Lower PIR (Sect. 9.2.4.2) meet the recovery criteria. Upper PIR ≤ 150%

Lower PIR ≥ 50%

Section

9.2.5 Quality Control Sample (QCS) Analyze mid-level QCS sample. Results must be within + 30%

of the true value.536 -30 TABLE 10. ONGOING QUALITY CONTROL REQUIREMENTS

Method

Reference Requirement Specification and Frequency Acceptance Criteria

Section

8.3 Sample Holding

Time 28 days when processed and stored according to Sections

8.1 and 8.2. Sample results are valid only if samples are analyzed within the sample holding time.

Section

10.3 Initial Calibration Use the internal standard calibration technique to generate a linear or quadratic

calibration curve. Use at

least 5 standard

concentrations. Validate the

calibration curve as

described in Section 10.3.3. When each calibration standard

is calculated as an unknown using the calibration curve, the

lowest level standard should be

within + 50% of the true value. All other points should be

within + 30% of the true value.

Section

9.3.1 Laboratory Reagent Blank (LRB) Daily, or with each Analysis Batch of up to 20 field samples, whichever is more frequent. Demonstrate that all target analytes are below 1/3 the MRL, and that possible interference from reagents and glassware do not prevent identification and quantitation of target analytes.

Section

10.4 Continuing

Calibration Check (CCC) Verify initial calibration by analyzing a low-level CCC at the beginning of each Analysis Batch. Subsequent CCCs are required after every 10 field samples, and after the last field sample in a

batch. The lowest level CCC must be

within + 50% of the true value. All other points must be within

+ 30% of the true value.

Results for field samples that are not bracketed by acceptable

CCCs are invalid.536 -31 TABLE 10. ONGOING QUALITY CONTROL REQUIREMENTS (CONTINUED)

Method

Reference Requirement Specification and Frequency Acceptance Criteria

Section

9.3.4 Internal Standard (IS) Isotopically labeled internal

standards are added to all standards and samples. Peak area counts for each IS

must be within ± 50% of the average peak areas in the initial

calibration.

Section

9.3.5 Laboratory Fortified Sample Matrix (LFSM) Analyze one LFSM per Analysis Batch. Fortify the LFSM with target analytes at

a concentration close to but greater than the native concentrations (if known).

Calculate LFSM recoveries. For LFSMs fortified at

concentrations ≤ MRL, the

result should be within + 50%

of the true value. All other

LFSMs should be within + 30%

of the true value.

Section

9.3.6 Laboratory Fortified Sample Matrix Duplicate (LFSMD) or Laboratory Duplicate(LD) Analyze at least one LFSMD or LD with each Analysis Batch of up to 20 field samples. For LFSMDs or LDs, RPDs

should be ≤ 30%.

Sections

9.3.7 Quality Control Sample (QCS) Analyze mid-level QCS sample when new PDS solutions are prepared, or at least quarterly if PDS solutions are prepared less frequently. Results must be + 30% of the

true value.FIGURE 1. NORMALIZED, OVERLAID CHROMATOGRAMS OF LC/ESI-MS/MS TRANSITIONS FOR METHOD 536ANALYTES FORTIFIED AT 0.5 μg/L IN REAGENT WATERa0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00Time0100%3.82

DACT DIA DEA Simazine Atrazine

Cyanazine PropazineaThe chromatograms for the isotopically-enriched internal standards are not included because they co-elute with their analogoustargetanalytes.536 -32