v>EPA United States Environmental Protection Agency METHOD 561: DETERMINATION OF ENDOTHALL, GLYPHOSATE, GLUFOSINATE, AND AMINOMETHYLPHOSPHONIC ACID IN DRINKING WATER BY DIRECT AQUEOUS INJECTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY ------- Technical questions concerning this analytical method should be addressed to: William A. Adams, Ph.D. U.S. EPA, Office of Ground Water and Drinking Water, Standards and Risk Management Division, Technical Support Branch, 26 W. Martin Luther King Dr. Cincinnati, OH 45268 Phone:(513)569-7656 adams.william@epa.gov Questions concerning this document or policy should be addressed to: safewater@epa.gov Office of Water (MS-140) EPA Document No. 815-R-25-013 EPA contract 68HERC22C0058 August 2025 Authors Alan Zaffiro, APTIM (Cincinnati, OH) and Leah Villegas Ph.D., APTIM (Cincinnati, OH) Contractor's role did not include establishing Agency policy. Anthony Giovengo, Ph.D., U.S. EPA (Cincinnati, OH) and William A. Adams, Ph.D., U.S. EPA (Cincinnati, OH) Acknowledgements Laura Rosenblum, Ph.D., APTIM (Cincinnati, OH) The following organizations completed a validation study in their laboratories using this method, provided valuable feedback on the method procedures and reviewed the draft method manuscript: Shodex - Resonac America, Inc. (New York, NY) Eurofins Eaton Analytical, LLC (South Bend, IN) Merit Laboratories, Inc. (East Lansing, Ml) Agilent Technologies, Inc. (Santa Clara, CA) Disclaimer This analytical method may support a variety of monitoring applications. Publication of the method, in and of itself, does not establish a requirement, although the use of this method may be specified by the EPA or a state through independent actions. Terms such as "must" or "required," as used in this document, refer to procedures that are to be followed to conform with the method. References to specific brands and catalog numbers are included only as examples and do not imply endorsement of the products. Such reference does not preclude the use of equivalent products from other vendors or suppliers. 561-i ------- Table of Contents 1 Scope and Application 1 2 Method Summary 2 3 Definitions 2 4 Interferences 4 5 Safety 6 6 Equipment and Supplies 6 7 Reagents and Standards 8 8 Sample Collection, Preservation, and Storage 12 9 Quality Control 12 10 Calibration and Standardization 18 11 Procedure 21 12 Data Analysis and Calculations 22 13 Method Performance 23 14 Pollution Prevention 24 15 Waste Management 24 16 References 24 17 Tables, Figures and Method Performance Data 25 561-ii ------- Tables Table 1. Preparation of Synthetic Matrix Solution 10 Table 2. Preparation of Internal Standard Stocks from Neat Materials 10 Table 3. HPLC Method Conditions for Sample Injections 25 Table 4. HPLC Conditions for EDTA Pre-lnjections and Gradient Recycle 25 Table 5. ESI Method Conditions 26 Table 6. Analyte RTs, MRMs, and MS/MS Method Conditions 26 Table 7. LCMRL Results 26 Table 8. Precision and Accuracy Data for Reagent Water 27 Table 9. Precision and Accuracy Data for a Ground Water Matrix 27 Table 10. Precision and Accuracy Data for a Surface Water Matrix 27 Table 11. Precision and Accuracy Data Synthetic Sample Matrix 28 Table 12. Precision and Accuracy Data for a Finished Surface Water with Orthophosate Anti-Corrosive Agent 28 Table 13. Aqueous Sample Holding Time Data for a Ground Water Matrix 29 Table 14. Aqueous Sample Holding Time Data for a Surface Water Matrix 29 Table 15. Initial Demonstration of Capability (IDC) Quality Control Requirements 30 Table 16. Ongoing Quality Control Requirements 31 Figures Figure 1. Relative Retention of Early Eluting Preservatives to the Method Analytes in High-Hardness Water 32 Figure 2. Relative Retention of Late Eluting Preservatives to the Method Analytes in High-Hardness Water 33 561-iii ------- 1 Scope and Application Method 561 is a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of endothall, glyphosate, glufosinate, and aminomethylphosphonic acid in drinking water using direct aqueous injection. This method gives instructions for a single bimodal LC column. At the time of publication, the authors could not identify other columns capable of separating the method analytes with acceptable efficiency and resolving these analytes from matrix interferences. A procedure is provided in this method for demonstrating acceptable performance for alternate columns as they become available. Method 561 requires the use of MS/MS in Multiple Reaction Monitoring (MRM) mode to enhance selectivity. Accuracy and precision data have been generated in reagent water, high-hardness synthetic matrix, and drinking water for the compounds included in the analyte List. Method performance data were generated with an injection volume of 2 piL to minimize matrix effects. This method is intended for use by analysts skilled in the operation of LC-MS/MS instrumentation and the interpretation of the associated data. Analyte List Analyte Abbreviation CASRN Aminomethylphosphonic acid AM PA 1066-51-9 Endothall END 145-73-3 Glufosinate GLU 51276-47-2 Glyphosate GLY 1071-83-6 1.1 Lowest Concentration Minimum Reporting Limits The lowest concentration minimum reporting level (LCMRL) is the lowest concentration for which the future recovery is predicted to fall between 50 and 150% with high confidence (99%). Single-laboratory LCMRLs determined for the method analytes during method development are reported in Table 7. The procedure used to determine the LCMRL is described elsewhere.- Laboratories using this method are not required to determine LCMRLs, but they must demonstrate that they are able to meet the minimum reporting level (MRL) (Sect. 3.11) for each analyte per the procedure described in Section 9.1.4. 1.2 Method Flexibility 1.2.1 General Flexibility Requirements At a minimum, one isotopically labeled analogue must be used as an internal standard for each analyte. Changes may not be made to sample preservation and the quality control (QC) requirements. Automated sample preparation techniques were not investigated during method development. However, the laboratory may use automated sample preparation provided all quality control requirements are met. 561-1 ------- 1.2.2 Chromatographic Flexibility Requirements The authors identified only one column capable of separating the method analytes and resolving them from the preservatives (sodium omadine and ethylenediaminetetraacetic acid - EDTA) and common anions in drinking water (chloride, nitrate, and sulfate). If the laboratory selects an LC column or LC conditions different from those used to develop the method, the laboratory must demonstrate that the preservatives and common anions elute within the analytical run, preventing them from building up on the column. The procedure to identify retention times for the preservatives and matrix components is provided in Section 9.3 to assist the user in meeting this requirement. The analyst must then perform the procedures outlined in the Initial Demonstration of Capability (IDC, Sect. 9.1), verify that all QC acceptance criteria in this method (Sect. 9.2) are met, and verify method performance in synthetic matrix and a representative sample matrix (Sect. 9.3.3). 2 Method Summary Drinking water samples are collected in 120 mL bottles containing sodium carbonate and the preservative, sodium omadine. Sodium omadine reduces residual chlorine and, in excess, serves as a biocide to prevent microbial growth during sample storage. Sodium carbonate raises sample pH to approximately 11. Calcium precipitates at this pH as insoluble calcium carbonate. This step prevents interference from the calcium-trisodium ethylenediaminetetraacetic acid (EDTA) complex that elutes near the endothall-d6 internal standard. Prior to analysis, the samples are filtered to remove precipitated calcium carbonate. EDTA is added to the filtered aliquot to prevent chelation of the method analytes with metals, including alkaline earth metals. Finally, isotopically labeled analogues of the method analytes are added to the samples as internal standards. An aliquot of the filtered sample is injected onto the bimodal LC column, with anion exchange properties, specified in Section 6.9 that separates the method analytes from the following common anions (matrix components) in drinking water: chloride, sulfate, and nitrate, and the method preservatives: EDTA, EDTA complexed with Ca, EDTA complexed with Mg, and sodium omadine, the latter eluted as the deprotonated pyrithione anion. Prior to each sample injection, the column is conditioned with a 5 piL injection of 5 mmol EDTA in aqueous buffer and then recycled to starting mobile phase conditions. This technique prevents band broadening and fronting of the glyphosate peak caused by metals in the HPLC flow path. The matrix components and preservatives in the column eluate are diverted to waste; the analytes of interest are directed into the ESI-MS/MS system. The method analytes are qualitatively identified via retention time and a unique mass transition. The concentration of each analyte is calculated using the integrated peak area and the internal standard technique. 3 Definitions 3.1 Analysis Batch A set of samples that are analyzed on the same instrument during a 24-hour period that begins and ends with the analysis of the appropriate Continuing Calibration Check (CCC) standards. Additional CCCs may be required depending on the length of the Analysis Batch and the number of field samples. 3.2 Continuing Calibration Check (CCC) A calibration standard that is analyzed periodically to verify the accuracy of the existing calibration. 561-2 ------- 3.3 Field Duplicates (FD) Separate samples collected at the same time and sampling location, shipped, and stored under identical conditions. Method precision, including the contribution from sample collection procedures, is estimated from the analysis of Field Duplicates. Field Duplicates are used to prepare Laboratory Fortified Sample Matrix and Laboratory Fortified Sample Matrix Duplicate QC samples. 3.4 Internal Standards Isotopically labeled analogues of the method analytes that are added to the sample at a consistent concentration prior to LC-MS/MS analysis. 3.5 Internal Standard Quantitation Technique An analytical technique for measuring analyte concentration using the ratio of the peak area of the native analyte to that of an isotopically labeled analogue added to the original sample in a known amount. 3.6 Laboratory Fortified Blank (LFB) A volume of reagent water, containing method preservatives, to which known quantities of the method analytes are added. The LFB is used during the IDC (Sect. 9.1) to verify method performance for precision and accuracy. 3.7 Laboratory Fortified Sample Matrix (LFSM) An aliquot of a field sample to which known quantities of the method analytes are added. The purpose of the LSFM is to determine whether the sample matrix contributes bias to the analytical results. 3.8 Laboratory Fortified Sample Matrix Duplicate (LFSMD) A Field Duplicate of the sample used to prepare the LFSM that is fortified and analyzed identically to the LFSM. The LFSMD is used instead of the Field Duplicate to assess method precision when the method analytes are rarely found at concentrations greater than the Minimum Reporting Levels. 3.9 Laboratory Reagent Blank (LRB) An aliquot of reagent water, including preservatives fortified with the internal standards and processed identically to a field sample. An LRB is included in each Analysis Batch to determine if the method analytes or other interferences are introduced from the laboratory environment, the reagents, or glassware. 3.10 Lowest Concentration Minimum Reporting Level (LCMRL) The single-laboratory LCMRL is the lowest spiking concentration such that the probability of spike recovery in the 50% to 150% range is at least 99%.- 3.11 Minimum Reporting Level (MRL) The minimum concentration that may be reported by a laboratory as a quantified value for a method analyte. For each method analyte, the concentration of the lowest calibration standard must be at, or below, the MRL and the laboratory must demonstrate its ability to meet the MRL per the criteria defined in Section 9.1.4. 561-3 ------- 3.12 Precursor Ion The gas-phase species corresponding to the method analyte that is produced in the electrospray ionization interface. During tandem mass spectrometry, or MS/MS, the precursor ion is mass selected and fragmented by collision-activated dissociation to produce distinctive product ions of smaller mass to charge (m/z) ratio. For this method, the precursor ion is usually the deprotonated molecule ([M - H]~) of the method analyte. 3.13 Primary Dilution Standard (PDS) A solution that contains method analytes (or internal standards) prepared from stock standards. PDS solutions are used to fortify QC samples and diluted to prepare calibration standards. 3.14 Procedural Calibration Standard A solution of the method analytes, internal standards, and method preservatives prepared in reagent water. The procedural calibration standards are used to calibrate the instrument response with respect to analyte concentration. 3.15 Product Ion One of the fragment ions that is produced in MS/MS by collision-activated dissociation of the precursor ion. 3.16 Quality Control Sample (QCS) A solution containing the method analytes at a known concentration that is obtained from a source external to the laboratory and different from the source of calibration standards. The purpose of the QCS is to verify the accuracy of the primary calibration standards. 3.17 Stock Standard Solution A concentrated standard that is prepared in the laboratory using assayed reference materials or that is purchased from a commercial source with a Certificate of Analysis. 3.18 Synthetic Sample Matrix A synthetic matrix representing a solution of high concentrations of common anions (chloride, nitrate, and sulfate) in drinking water using alkaline earth metal salts (calcium and magnesium) prepared in reagent water with prescribed concentrations of sample preservatives. Procedural calibration standards, prepared by fortifying the synthetic sample matrix with the method analytes, are used to verify method performance when analyzing high ionic content samples with alternate LC columns. Instructions for preparing the synthetic sample matrix are provided in Section 7.9. 4 Interferences The information in this section was informed by the authors' experience with the Shodex VT-50 2D column (Sect. 6.9), used to develop the method and collect method performance data. However, the potential negative effects of high ionic concentrations of synthetic sample matrix components and preservatives must be equally evaluated and demonstrated when using alternate column technologies. 561-4 ------- 4.1 Labware, Reagents and Equipment Method interferences may be caused by contaminants in solvents, reagents, including reagent water, sample bottles and caps, and other sample processing hardware that may lead to discrete artifacts or elevated baselines in the chromatograms. Laboratories must demonstrate that these items are not contributing to interference by analyzing LRBs as described in Section 9.2.1. 4.2 Interference from Matrix Anions In addition to the method analytes, anion exchange columns, such as the Shodex VT-50 2D, retain anionic species in drinking water, including the method preservatives which are anionic in the basic mobile phase. Anionic complexes of EDTA with Ca and Mg are also retained. Figure 1 and Figure 2 are chromatograms showing the elution order of the method analytes and these matrix anions. High concentrations of matrix anions could cause instrument response suppression, band broadening, and low recovery. However, for the bimodal Shodex column, none of these effects were observed at the synthetic matrix concentrations listed in this section in the presence of the method preservatives (sodium omadine, sodium carbonate, and EDTA) and an injection volume of 2 piL. The authors verified method performance for matrix anions using synthetic matrix containing the three most common anions in drinking water: chloride at 390 mg/L, nitrate at 44 mg/L, and sulfate at 250 mg/L. These concentrations represent, or exceed, the EPA Primary Drinking Water Standard maximum contaminant level (MCL) for nitrate and the Secondary Drinking Water Standard MCLs for chloride and sulfate. To represent the worst case for high-hardness water, the authors consulted drinking water surveys for water hardness in North American drinking water (e.g., Morr 2006- and Azoulay 2001-) and hardness measurements made on local drinking water from aquifer sources. Morr reported up to 350 mg/L hardness as calcium carbonate. The authors measured between 330 mg/L and 350 mg/L in a local drinking water prepared from a groundwater source. With a formula weight of 100 mg/mmol for calcium carbonate, 350 mg/L equals 3.52 mmol, corresponding to 141 mg/L of Ca (atomic weight = 40 mg/mmol), assuming the hardness is entirely due to Ca and no Mg is present. The Azoulay survey found up to 48 mg/L (2.0 mmol) as Mg, atomic weight 24.3 mg/mmol. Therefore, synthetic matrix used to verify method performance contained 141 mg/L Ca and 48 mg/L Mg. The combined hardness of this synthetic matrix is 547 mg/L based on calcium carbonate equivalents. 4.3 Interference from Metals in the HPLC System and Samples 4.3.1 Analyte Complexes with Metals Glyphosate is known to form complexes with metals in aqueous solution, including alkaline earth metals, Ca and Mg-. As stated in EPA Method 548.1-, endothall complexes with Ca and Mg in drinking water and source waters. To prevent formation of these undesired complexes, EDTA, as trisodium ethylenediaminetetraacetic acid hydrate, is added to the samples after filtration in the laboratory at a concentration of 2.5 mmol, the molar equivalent to 250 mg/L water hardness expressed as calcium carbonate. 2.5 mmol EDTA is sufficient to complex the highest levels of Mg expected in drinking water (2.0 mmol, 48 mg/L) and any Ca not removed during the precipitation step of the sample preparation procedure. 561-5 ------- 4.3.2 Chromatographic Interference from Ca-EDTA Complex For the Shodex VT-50 2D column, the calcium-EDTA complex elutes near endothall and glyphosate under the chromatographic conditions of this method and produces an MRM transition identical to that used for the endothall-d6 internal standard. Although baseline separated, very high levels of Ca result in a broad calcium EDTA peak that interferes with the endothall internal standard peak. Because there is no alternate transition for endothall-d6, calcium must be removed during sample preparation by adding sodium carbonate to field samples and subsequent filtration in the laboratory. 4.3.3 Magnesium-EDTA Complex Calcium is effectively removed as insoluble calcium carbonate during the sample filtration step; however, magnesium hydroxide forms a colloidal solution that is not removed during the filtration step. At 48 mg/L (2.0 mmol) as Mg and a 2 piL injection volume, the authors observed no suppression of analyte response when using the Shodex VT-50 2D column. 4.3.4 Effect of Metals on Glyphosate Peak Shape The authors observed deterioration of glyphosate peak shape, revealed as bad broadening and fronting, for new and aged Shodex VT-50 2D columns. Glyphosate may be sensitive to metals in the HPLC flow path. The authors demonstrated that an EDTA pre-injection eliminates this degradation in column efficiency. This method requires an injection of EDTA prior to each sample injection. 4.4 Organic Matrix Interferences Matrix interferences may be caused by natural organic contaminants that are co-injected from the sample. The extent of organic matrix interferences was assessed using drinking water from a surface water source fortified with the method analytes. With an injection size of 2 piL, acceptable recoveries were observed in the presence of total organic carbon (TOC) greater than 2 mg/L C. 5 Safety Each chemical should be treated as a potential health hazard 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 safety data sheets should be made available to all personnel involved in the chemical analysis. 6 Equipment and Supplies References to specific brands and catalog numbers are included as examples only and do not imply endorsement of the products. Such reference does not preclude the use of equivalent products from other vendors or suppliers. 6.1 Sample Containers 120 mL (4 oz.) amber glass bottles with Teflon-lined septa. 6.2 Autosampler Vials Glass with Teflon-lined closures. 561-6 ------- 6.3 Sample Vials Glass with Teflon-lined septa, 15 mL size recommended. 6.4 Micro Syringes Liquid or gas tight syringes sized to transfer stock standards that cannot be measured using micro pipettes with polypropylene tips. 6.5 Pipets Automatic or manual micro pipettes with polypropylene tips may be used to prepare calibration standards, add internal standards, and fortify samples to prepare quality control samples. Capable of weighing to the nearest 0.0001 g. 6.6 pH Meter Used to adjust the pH of the aqueous ammonium bicarbonate mobile phase. 6.7 Syringes and Filters To filter field and QC samples, procedural calibration standards, and laboratory reagent blanks. 6.7.1 Plastic Syringes Polypropylene syringes with rubber-tipped plungers, 10 mL size recommended. 6.7.2 Syringe Filters, 0.45 [am glass fiber GE Whatman 25 mm glass fiber, 0.45 micrometer pore size, Cat. No. 6894-2504, or equivalent. 6.8 LC-MS/MS System 6.8.1 LC System The LC system must provide consistent sample injection volumes and be capable of performing binary linear gradients at a constant flow rate. To improve the inertness of the sample path, the authors replaced the metal mobile phase delivery tubing from the autosampler to the guard column with 0.0050 inch i.d. polyetheretherketone (PEEK) tubing. 6.8.2 Bimodal Guard Column Shodex (Resonac America, Inc., New York, NY) HILICpak VT-50G 2A, 10 x 2.0 mm i.d., 5 micrometer particle size, PEEK housing, polyvinyl alcohol substrate with quaternary ammonium group. 6.8.3 Guard Column Coupler PEEK tubing: 0.010 inch i.d. used during method development. 6.8.4 Bimodal Analytical Column Shodex (Resonac America, Inc., New York, NY) HILICpak VT-50 2D, 150 x 2.0 mm i.d., 5 micrometer particle size. PEEK housing, polyvinyl alcohol substrate with quaternary ammonium group. The maximum recommended column pressure for the bimodal column is 1450 psig. If the operating pressure 561-7 ------- at 40 °C is close to this limit, identify the i.d. of the post-column tubing and replace with tubing of a greater diameter. The authors replaced the 0.0040 inch i.d. capillary, supplied by the instrument manufacturer, between the column exit and the post-column divert valve with 0.010 inch i.d. PEEK tubing to prevent the column from exceeding the 1450 psig pressure limit. 6.8.5 Electrospray Ionization Tandem Mass Spectrometer (ESI-MS/MS) The mass spectrometer must be capable of electrospray ionization in the negative ion mode. The system must be capable of performing MS/MS to produce unique product ions for the method analytes within specified retention time windows. A minimum of 10 scans across the chromatographic peak is needed to ensure adequate precision. The LC-MS/MS must have the capability to program multiple divert windows to direct the column eluate to waste when the analytes are not eluting. 6.8.6 MS/MS Data System An interfaced data system is required to acquire, store, and output MS data. The computer software must have the capability of processing stored data by recognizing a chromatographic peak within a given retention time window. The software must allow integration of the abundance of any specific ion between specified time or scan number limits. The software must be able to construct a linear regression or quadratic regression calibration curve and calculate analyte concentrations using the internal standard technique. 7 Reagents and Standards HPLC or LC-MS-grade reagents must be used, if available. Otherwise, all reagents must conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society (ACS), where such specifications are available. Other grades may be used if the reagent is demonstrated to be free of analytes and interferences and all requirements of the IDC are met when using these reagents. The concentrations of analyte stocks, internal standard stocks, PDS solutions, and calibration standards listed in this section were used to develop this method and are included only as examples. Preparation instructions for mobile phases assume the use of the Shodex VT-50 2D column and a binary HPLC pumping system. Quaternary LC pumps may be used. Laboratories are responsible for developing appropriate mobile phase systems for alternate column technologies. 7.1 Reagent Water Purified water which does not contain any measurable quantities of any method analytes or interfering compounds greater than one-third of the MRL for each method analyte. 7.2 Acetonitrile H3CN, CASRN 75-05-8, HPLC or LC-MS grade. 7.3 1% Acetonitrile Solution For use diluting stock standards to prepare PDS (spiking) solutions and to reconstitute internal standards if purchased as the neat materials. Combine 10 mL of pure acetonitrile with 990 mL of reagent water. 561-8 ------- 7.4 Ammonium Bicarbonate NH4HCO3, CASRN 1066-33-7, HPLC grade, molecular weight equals 79.056 g/mol. 7.5 Ammonium Hydroxide NH4OH, CASRN 1336-21-6, approximately 56.6% in water as ammonium hydroxide (w/w), approximately 28% in water as ammonia, approximately 14.5 N (Fisher Scientific, Cat. No. A669, Certified ACS Plus grade, or equivalent). 7.6 Mobile Phase Preparation 7.6.1 50 mmol Ammonium Bicarbonate, pH 9.2 (Mobile Phase A) To prepare 1 L, add 3.95 g ammonium bicarbonate to 1 L of reagent water. Adjust pH to 9.2 with ammonium hydroxide. This mobile phase must be replaced every 2 weeks. Analyte and matrix component retention times increase as the buffer ages. 7.6.2 55% Acetonitrile (Mobile Phase B) To prepare 0.50 L, separately measure 275 mL of acetonitrile and 225 mL reagent water. Combine in a mobile phase bottle. This mobile phase may be used until exhausted. 7.7 Preservatives 7.7.1 Sodium Omadine- C5H4NNaOS, CASRN 3811-73-2. Sodium omadine is added to sample bottles to reduce residual chlorine and acts as a microbial growth inhibitor in drinking water samples. Reference number six provides information on the properties of sodium omadine. 7.7.2 Sodium Carbonate, Anhydrous Powder Na2C03, CASRN 497-19-8. Sodium carbonate is added to sample bottles to raise sample pH to approximately 11. 7.8 Ethylenediaminetetraacetic acid trisodium salt hydrate CASRN 85715-60-2, molecular weight equals 376.2 mg/mmol. Ethylenediaminetetraacetic acid (EDTA) complexes with metals, including alkaline earth metals, that could otherwise complex with endothall, glyphosate, AMPA, and glufosinate. EDTA is added to samples in the laboratory after filtration. EDTA is also used to prepare the pre-injection solution (Section 7.8.2). 7.8.1 EDTA Preservative, 45 mg/mL Dissolve 4,500 mg ethylenediaminetetraacetic acid trisodium salt hydrate in 100 mL reagent water. 7.8.2 EDTA Pre-injection Solution, 5.0 mmol Dissolve 188 mg of ethylenediaminetetraacetic acid trisodium salt hydrate in 100 mL of 50 mmol ammonium bicarbonate, pH 9.2 buffer. The pre-injection solution is used to condition the HPLC column between each run. This treatment narrows the width and prevents fronting of the glyphosate peak. 561-9 ------- 7.9 Synthetic Sample Matrix Preparation Procedural calibration standards prepared in synthetic matrix are used to confirm acceptable analyte recovery for alternate LC columns in the presence of common matrix ions in drinking water. Section 4.2 provides an explanation for the selection of the concentrations prescribed for the synthetic matrix. Obtain common forms of the following salts at 99% purity, or greater: sodium nitrate, calcium chloride, ammonium sulfate, and magnesium chloride. Table 1 gives the required concentration of each species in the synthetic matrix solution and other information helpful for preparing this solution. Table 1. Preparation of Synthetic Matrix Solution COMPOUND Mass of Salt Used Concentration (Formula Weight, mg/mmole) I\I03" (62) from NaN03 (85) 60.7 mg NaN03 44 mg/L CI" (35.45) from CaCI2 • 2H20 (147) 518 mg CaCI2-2H20 250 mg/L CI" (35.45) from MgCI2 (95.21) 188 mg MgCI2 140 mg/L Total Cl" 390 mg/L Ca2+ (40) from CaCI2-2H20 (147) From same addition above 141 mg/L (3.5 mmol) Mg2+ (24.3) MgCI2 (95.21) From same addition above 48 mg/L (2.0 mmol) S042" (96) from (NH4)2S04 (132) 344 mg 250 mg/L 7.10 ESI Interface Gases 7.10.1 Nitrogen Nebulizer Gas Nitrogen, used as a nebulizer gas in the ESI interface and as collision gas in some MS/MS platforms, should meet or exceed the instrument manufacturer's specifications. 7.11 Argon Used as collision gas in MS/MS instruments. Argon should meet or exceed instrument manufacturer's specifications. Nitrogen may be used as the collision gas if recommended by the instrument manufacturer. 7.12 Analyte Stock Standards The solution concentrations listed in this section were used to develop this method and are included only as examples. Obtain certified solutions of the method analytes in water. Typical concentrations are 1000-2000 ng/mL. 7.13 Internal Standards Stocks During method development the internal standards required for use with this method were acquired as neat materials. Table 2 lists the labeled analogues selected by the authors. Other labeled analogues may be substituted, although the analyst must ensure that the QC requirements defined in Section 9.2.7 are met. If purchased as neat materials, reconstitute with 1% acetonitrile in reagent water with sonication. The authors took the additional step of rinsing the 1.5 mL ampoule with pH 9.2 ammonium bicarbonate buffer (Sect. 7.6.1) several times to ensure complete transfer of the solid material into a 60 mL vial; then brought to 40 mL final volume with 1% acetonitrile. Table 2. Preparation of Internal Standard Stocks from Neat Materials 561-10 ------- Compound Vendor Cat. No. Mass Used Volume3 Stock, |o.g/mL AMPA -d2 CDNb D-8135 10 mg 40 mL 250 Glufosinate- CDN D-7962 10 mg 40 mL 250 as the ds- HCI hydrochloride Endothall-c/6 CDN D-7289 10 mg 40 mL 250 as the monohydrate hydrate (MW=210) Glyphosate-c/2 CDN D-8030 10 mg 40 mL 250 a Reconstituted in water plus 1% acetonitrile with sonication. b CDN Isotopes, Inc., or equivalent. 7.14 Analyte PDS The analyte PDS is used to prepare the calibration standards and to fortify LFBs, LFSMs and LFSMDs with the method analytes. Prepare the analyte PDS by combining and diluting the analyte stock standards in water with 1% acetonitrile added. Select nominal analyte concentrations for the PDS such that at least 10 piL of the PDS are used to fortify samples and prepare standard solutions. More than one PDS concentration may be necessary to meet this requirement. If preparing calibration standards and QC samples in 120 mL bottles (Sect. 7.16), an analyte PDS prepared at 20,000 ng/L for AMPA, glufosinate, and glyphosate and 10,000 ng/Lfor endothall is appropriate. The user may modify the relative concentrations of the individual analytes based on the confirmed MRLs and the desired monitoring range. 7.15 Internal Standard PDS Prepare the internal standard PDS by combining and diluting the internal standard stocks in water with 1% acetonitrile added. Select nominal analyte concentrations for the PDS such that at least 10 piL of the PDS are used to fortify samples and prepare standard solutions. During method development, the internal standard PDS was prepared at a single concentration of 15 ng/mL. For collection of performance data, an 80 piL aliquot of the internal standard PDS was added to 12 mL of filtered sample to give a concentration of 100 ng/Lfor each internal standard. 7.16 Procedural Calibration Standards Prepare a series of calibration standards of at least five levels by diluting the analyte PDS into reagent water in 120 mL bottles containing the method preservatives: 7.5 mg/sodium omadine (75 mg/L) and 530 mg anhydrous sodium carbonate (0.050 M, 5.3 g/L). The order of addition is as follows: Weigh the preservatives, sodium carbonate (530 mg) and sodium omadine (7.5 mg) into the sample bottles. Add 100 mL reagent water. Mix until the solids are dissolved. Add an appropriate aliquot of the analyte PDS (Sect. 7.1.4) to establish each calibration level. Filter, add EDTA, and fortify with internal standards as instructed in Sections 11.4 through 11.6. The lowest calibration standard must be at, or below, the MRL for each analyte. The calibration standards may also be used as Continuing Calibration Checks (CCCs). For the collection of method performance data, the concentration of the internal standards was 100 Hg/L. For AMPA, glufosinate, and glyphosate, the analyte calibration ranged from 10 ng/L to 400 ng/L and for endothall, 5.0 ng/L to 200 ng/L. 7.17 Storage Temperatures for Standards Solutions Refrigerate stock standards and PDS solutions unless the vendor recommends otherwise. Do not freeze. During method development, no change in analyte concentrations was observed over a period of 6 to 12 561-11 ------- months. Warm PDS and stock standards to room temperature prior to use. Calibration standards may be used for 6 months if evaporation is avoided. 8 Sample Collection, Preservation, and Storage 8.1 Sample Bottle Preparation 8.1.1 Sample Containers Amber glass bottles with PTFE-lined screw caps, 120 mL (4 oz.) volume recommended. If recycling bottles, include a rinse with tap water and a few drops of acetic acid to remove scale formed by precipitation of calcium carbonate. When recycling sample containers, use new septa to avoid contamination with method analytes during sampling. 8.1.2 Addition Of Preservatives Prior to shipment to the field, add sodium omadine and anhydrous sodium carbonate to the sample containers to produce concentrations of 75 mg/L and 5.3 g/L (0.050 M), respectively, in the field samples. For example, a 100 mL sample requires 7.5 mg of sodium omadine and 530 mg of sodium carbonate. 8.1.3 Collection Procedure Open the tap and allow the system to flush until the water temperature has stabilized. Collect samples from the flowing system. Samples do not need to be collected headspace free. After collecting the sample, cap the bottle and agitate by hand until the sodium carbonate is dissolved. If using 120 mL bottles, 4 oz., estimate the 100 mL level and fill approximately to that mark. 8.1.4 QC Samples Collect enough Field Duplicates to satisfy the requirement of analyzing one Field Duplicate in each Analysis Batch of 20 samples. Collect enough samples in triplicate to satisfy the requirement of at least one LFSM and LFSMD in each Analysis Batch of 20 samples. 8.2 Sample Shipment and Storage Samples must be shipped on ice. Samples are valid if any ice remains in the cooler when it is received at the laboratory or bottles are received within 2 days of collection and below 10 °C. Once at the laboratory, samples must be stored at, or below, 6 °C until analysis. Samples must not be frozen. 8.3 Sample Holding Time Analyze samples as soon as possible. Samples must be in contact with the sodium carbonate used to precipitate out metals for at least an hour, which includes both field samples and laboratory prepared QC samples. Samples must be analyzed within 28 days of collection. 9 Quality Control QC procedures include the IDC and ongoing QC requirements. This section describes each QC parameter, its required frequency, and the performance criteria that must be met in order to satisfy method objectives. The QC criteria discussed in the following sections are summarized in Table 15 and 561-12 ------- Table 16. These QC requirements are considered the minimum for an acceptable QC program. Laboratories are encouraged to institute additional QC practices to meet their specific needs. 9.1 Initial Demonstration of Capability The IDC must be successfully performed prior to analyzing field samples. The IDC must be repeated if changes are made to analytical parameters not previously validated during the IDC. This may include, for example, changing the injection volume, selecting alternate quantitation ions, substituting internal standards, and extending the calibration range. Prior to conducting the IDC, the analyst must meet the calibration requirements outlined in Section 10. The same calibration range used during the IDC must be used for the analysis of field samples. 9.1.1 Demonstration of Low System Background Prepare an LRB following the steps in Section 11.2. Filter, add EDTA, and fortify with internal standards as instructed in Sections 11.4 through 11.6. Analyze the LRB in a valid Analysis Batch immediately after injecting the highest calibration standard in the selected calibration range. Confirm that the blank is free from contamination as defined in Section 9.2.1. If the LRB results fail, the carryover or system contamination must be eliminated. Adjust the rinse settings of the sample introduction system or reduce the concentration of the highest standard in the calibration range. Perform an initial calibration and repeat the demonstration of low system background. 9.1.2 Demonstration of Precision 9.1.2.1 Prepare Seven Laboratory Fortified Blanks (LFBs) Collect 100 mL of reagent water in 120 mL bottles containing the method preservatives, sodium carbonate and sodium omadine (Sect. 8.1.2). Fortify near the midpoint of the initial calibration curve with an appropriate volume of Analyte PDS (Sect. 7.14). Filter, add EDTA, and fortify with internal standards as instructed in Sections 11.4 through 11.6. 9.1.2.2 Evaluate Precision Analyze seven replicate LFBs in a valid Analysis Batch (seven LFBs and an LRB). The percent relative standard deviation (%RSD) of the concentrations of the replicate analyses must be less than 20% for all method analytes. 9.1.3 Demonstration of Accuracy Using the same set of replicate data generated for Section 9.1.2, calculate the average percent recovery. The average recovery for each analyte must be within a range of 70-130%. 9.1.4 Minimum Reporting Level (MRL) Confirmation Establish a target concentration for the MRL (Sect. 3.12) based on the intended use of the method. If there is a programmatic MRL requirement, the laboratory MRL must be set at, or below, this level. Establishing the MRL concentration too low may cause repeated failure of ongoing QC requirements. Perform an initial calibration following the procedures in Section 10.3. The lowest calibration standard used to establish the initial calibration (as well as the low-level CCC) must be at, or below, the MRL. Confirm the laboratory's ability to meet the MRL following the procedure outlined below. 561-13 ------- 9.1.4.1 Prepare and Analyze MRL Samples Prepare seven LFBs at, or below, the proposed MRL concentration. Collect 100 mL of reagent water in 120 mL bottles containing the method preservatives, sodium carbonate and sodium omadine (Sect. 8.1.2). Fortify with an appropriate volume of Analyte PDS (Sect. 7.14). Filter, add EDTA, and fortify with internal standards as instructed in Sections 11.4 through 11.6. Analyze the LFBs in a valid Analysis Batch. 9.1.4.2 Calculate MRL Statistics Calculate the mean and standard deviation for each analyte in these replicates. Determine the Half Range for the Prediction Interval of Results (HRpir) using the following equation: HRpir = 3.963S Where, 5 = the standard deviation and 3.963 is a constant value for seven replicates.- Calculate the Upper and Lower Limits for the Prediction Interval of Results (PIR = Mean ± HRpir) as shown below. These equations are only defined for seven replicate samples. Mean + HRp,r Upper PIR Limit = —- x 100 Fortified Concentration Mean — HRpir Lower PIR Limit = — —— x 100 Fortified. Concentration 9.1.4.3 MRL Acceptance Criteria The laboratory's ability to meet the MRL is confirmed if the Upper PIR Limit is less than, or equal to, 150%; and the Lower PIR Limit is greater than, or equal to, 50%. If these criteria are not met, the MRL has been set too low and must be confirmed again at a higher concentration. 9.1.5 Calibration Verification Analyze a QCS (Sect. 9.2.10) to confirm the accuracy of the primary calibration standards. 9.2 Ongoing QC Requirements This section describes the ongoing QC elements that must be included when processing and analyzing field samples. 9.2.1 Laboratory Reagent Blank (LRB) A new LRB must be prepared for each Analysis Batch. Each lot of syringes and filters must be checked for interferences. If a new lot is required to complete an Analysis Batch, an additional LRB must be prepared utilizing the new lot. Background concentrations of method analytes must be less than one-third the MRL. If method analytes are detected in the LRB at concentrations greater than or equal to this level, then all positive field sample results (i.e., results at, or above, the MRL) for those analytes are invalid for all samples in the Analysis Batch. Subtracting blank values from sample results is not permitted. 561-14 ------- 9.2.2 Estimating Background Concentrations Although quantitative data below the MRL may not be accurate enough for data reporting, such data are useful in determining the magnitude of background interference. Therefore, the analyte concentrations in the LRB may be estimated by extrapolation when results are below the MRL. 9.2.3 Evaluation of Background when Analytes Exceed the Calibration Range After analysis of a sample in which method analytes exceed the calibration range, one or more LRBs must be analyzed (to detect potential carryover) until the system meets the LRB acceptance criteria. If this occurs during an automated sequence, examine the results of samples analyzed following the sample that exceeded the calibration range. If the analytes that exceeded the calibration range in the previous sample are detected at, or above, the MRL, these samples are invalid. If the affected analytes do not exceed the MRL, these subsequent samples may be reported. 9.2.4 Continuing Calibration Check (CCC) Analyze CCC standards at the beginning of each Analysis Batch, after every tenth field sample, and at the end of the Analysis Batch. See Section 10.4 for concentration requirements and acceptance criteria for CCCs. 9.2.5 Laboratory Fortified Blank Because this method utilizes procedural calibration standards, which are fortified reagent waters, there is no difference between the LFB and the Continuing Calibration Check standard. Consequently, the analysis of a separate LFB is not required as part of the ongoing QC; however, the term "LFB" is used for clarity in the IDC. 9.2.6 Internal Standard Areas The analyst must monitor the peak areas of the internal standards in all injections of the Analysis Batch. The internal standard responses (as indicated by peak area) in any chromatographic run must be within 50-150% of the average area measured during the initial calibration. If an internal standard area for a sample does not meet these criteria, analyze the original filtered sample in a subsequent Analysis Batch. Alternately, a fresh aliquot of the original field sample may be filtered and fortified with internal standards and EDTA for the repeat analysis. 9.2.7 Laboratory Fortified Sample Matrix (LFSM) Within each Analysis Batch, analyze a minimum of one LFSM. The native concentrations of the analytes in the sample matrix must be determined in a separate field sample and subtracted from the measured values in the LFSM. If various sample matrices are analyzed regularly, for example, drinking water processed from ground water and surface water sources, collect performance data for each source. 9.2.7.1 Prepare the LFSM Prepare the LFSM by fortifying a Field Duplicate with an appropriate amount of the analyte PDS (Sect. 7.14). Filter, add EDTA, and fortify with internal standards as instructed in Sections 11.4 through 11.6. Generally, select a spiking concentration that is greater than, or equal to, the native concentration for the analytes. Selecting a duplicate aliquot of a sample that has already been analyzed aids in the selection of an appropriate spiking level. If this is not possible, use historical data when selecting a fortifying concentration. 561-15 ------- 9.2.7.2 Calculate the Percent Recovery Calculate the percent recovery (%R) using the equation: 04-5) o/0 R = x 100 Where, A = measured concentration in the fortified sample, B = measured concentration in the unfortified sample, and C = fortification concentration. To obtain meaningful percent recovery results, correct the measured values in the LFSM and LFSMD for the native levels in the unfortified samples, even if the native values are less than the MRL. 9.2.7.3 Evaluate Analyte Recovery in the LFSM Results for analytes fortified at concentrations near or at the MRL (within a factor of two times the MRL concentration) must be within 50-150% of the true value. Results for analytes fortified at all other concentrations must be within 70-130% of the true value. If the accuracy for any analyte falls outside the designated range, and the performance for that analyte in the CCCs of the Analysis Batch is shown to be in control, the recovery is judged matrix biased. Report the result for the corresponding analyte in the unfortified sample as "suspect-matrix". 9.2.8 Laboratory Fortified Sample Matrix Duplicate (LFSMD) or Field Duplicate (FD) Within each Analysis Batch, analyze a minimum of one Field Duplicate or one Laboratory Fortified Sample Matrix Duplicate. If the method analytes are not routinely observed in field samples, analyze an LFSMD rather than an FD. 9.2.8.1 Calculate the RPD for the LFSM and LFSMD If an LFSMD is analyzed instead of a Field Duplicate, calculate the RPD using the equation: | LFSMD - LFSM \ RPD ~ (LFSMD + LFSM)/2 * 10° 9.2.8.2 Acceptance Criterion for the RPD of the LFSM and LFSMD RPDs for duplicate LFSMs must be less than, or equal to, 30% for each analyte. Greater variability may be observed when the matrix is fortified at analyte concentrations near or at the MRL (within a factor of two times the MRL concentration). LFSMs at these concentrations must have RPDs that are less than, or equal to, 50%. If the RPD of an analyte falls outside the designated range, the precision is judged matrix influenced. Report the result for the corresponding analyte in the unfortified sample as "suspect- matrix". 9.2.8.3 Calculate the RPD for Field Duplicates Calculate the relative percent difference (RPD) for duplicate measurements (FD1 and FD2) using the equation: |FDi — FD21 RPD = ' ^ ' x 100 (FDi + FD2)/2 9.2.8.4 Acceptance Criterion for Field Duplicates RPDs for Field Duplicates must be less than, or equal to, 30% for each analyte. Greater variability may be observed when Field Duplicates have analyte concentrations that are near or at the MRL (within a factor 561-16 ------- of two times the MRL concentration). At these concentrations, Field Duplicates must have RPDs that are less than, or equal to, 50%. If the RPD of an analyte falls outside the designated range, the precision is judged matrix influenced. Report the result for the corresponding analyte in the unfortified sample as "suspect-matrix" 9.2.9 Calibration Verification using QCS A QCS (as defined in Sect. 3.17) must be analyzed during the IDC, and then every 3 to 6 months thereafter. Collect 100 mL of reagent water in a 120 mL bottle containing the method preservatives, sodium carbonate and sodium omadine (Sect. 8.1.2). Fortify near the midpoint of the calibration range with an appropriate volume of the QCS stock standards. Filter, add EDTA, and fortify with internal standards as instructed in Sections 11.4 through 11.6. The acceptance criterion for the QCS is 80-120% of the true value. If the accuracy for any analyte fails the recovery criterion, prepare fresh standard dilutions and repeat the QCS evaluation. 9.3 Alternate Column QC Requirements The analyst is not permitted to modify the chromatographic conditions for the column specified in this method. These conditions were optimized during method development for the Shodex VT-50 2D column to ensure that matrix ions and method preservatives elute from the analytical column each run—and these components are separated from the method analytes. If an alternate column is chosen, the laboratory must demonstrate that the alternate column is capable of similar performance. The following steps are required to confirm acceptable performance for alternate columns. 9.3.1 Determine RTs of Analytes and Matrix Components Prepare calibration standards in both reagent water and in synthetic matrix (Sect. 7.9). Optimize separation of the method analytes by analyzing calibration standards. Using calibration standards prepared in synthetic matrix, determine the retention times of m/z 35 and 37 CI"; m/z 62 N03~; m/z 97 and 99 S042~; m/z 291 uncomplexed EDTA; m/z 329 EDTA complexed with Ca; m/z 313 EDTA complexed with Mg and omadine (detected as the pyrithione anion, m/z 126). Adjust the gradient as necessary to ensure that the matrix components listed above are separated as much as possible from the method analytes. Complete separation may not be possible; however, the column may not be used if the co- elution of matrix components causes QC failures during the IDC (Sect. 9.3.2). 9.3.2 Repeat the IDC Establish an acceptable initial calibration (Sect. 10.3) using the alternate column and the modified conditions. Repeat the procedures of the IDC (Sect. 9.1). 9.3.3 Document Performance in Synthetic Matrix and Representative Sample Matrices The analyst is required to evaluate and demonstrate precision (Sect. 9.1.2) and accuracy (Sect. 9.1.3) for the alternate column in synthetic matrix (Sect. 7.9) and 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, could fail ongoing method QC requirements in real matrices. This is particularly important for methods subject to matrix effects, such as LC-MS/MS-based methods and for ion chromatography columns that retain ionic matrix components. For a laboratory that routinely analyzes finished drinking water from municipal treatment plants that process ground water, surface water, or a blend of surface and ground water, finished drinking waters derived from a 561-17 ------- surface water with moderate to high total organic carbon (e.g., 2 mg/L or greater) and from a hard ground water (e.g., 250 mg/L calcium carbonate equivalent, or greater) are recommended. 10 Calibration and Standardization Demonstration and documentation of acceptable MS calibration and initial analyte calibration are required before performing the IDC and prior to analyzing field samples. The initial calibration should be repeated each time a major instrument modification or maintenance is performed. 10.1 MS/MS Optimization 10.1.1 Mass Calibration Calibrate the mass spectrometer with the calibration compounds and procedures specified by the manufacturer. 10.1.2 MS Parameters During the development of this method, instrumental parameters were optimized for the precursor and product ions listed in Table 6. Product ions other than those listed may be selected; however, the analyst should avoid using ions with lower mass or common ions that may not provide sufficient discrimination between the analytes of interest and co-eluting interferences. 10.1.2.1 Precursor Ion Optimize the response of the precursor ion ([M - H]~) for each analyte following manufacturer's guidance. Analyte concentrations of 5 ng/mL introduced to the ESI-MS/MS via split infusion into mobile phase were used for this step during method development. Vary the MS parameters (source voltages, source and desolvation temperatures, gas flows, etc.) until optimal analyte responses are determined. The electrospray parameters used during method development are listed in Table 5 and Table 6. The analytes may have different optimal parameters, requiring some compromise on the final operating conditions. 10.1.2.2 Product Ion Optimize the product ion for each analyte following the manufacturer's guidance. See Table 6 for MS/MS collision energies used to collect method performance data. 10.2 Chromatographic Conditions, Shodex VT-50 2D Column Establish LC operating parameters for analyte separation as specified in Table 3. Create a second gradient method for the EDTA pre-injections as specified in Table 4. The authors optimized these conditions for the Shodex VT-50 2D column to ensure that common matrix ions and method preservatives elute from the analytical column each run—and these components are separated as much as possible from the method analytes. Figure 1 and Figure 2 show the location of the matrix anions and preservatives relative to the method analytes. For the Shodex VT-50 SD column, it is recommended that laboratories utilize the entire gradient specified in Table 3. including the 17 minute hold time at 90% mobile phase A after the analytes elute. As shown in Figure 2 , the hold time is necessary to elute uncomplexed EDTA, detected as m/z 291 [H4- EDTA- H]~, introduced onto the column from the sample injection and the EDTA pre-injection. Figure 2 561-18 ------- also gives the location of EDTA complexed with Ca, detected as m/z 329 [H4-EDTA - 3H + Ca]~ and EDTA complexed with Mg, detected as m/z 313 [H4-EDTA - 3H + Mg]~ relative to the method analytes. If a laboratory chooses to use different chromatographic conditions see the flexibility requirement described in Section 1.2.2. 10.2.1 Equilibrate the Column Begin heating the column to 40 °C. Establish a flow of 0.10 mL/min at 90% A (50 mmol ammonium bicarbonate, pH 9.2) and 10% solvent B (55 % acetonitrile). When the system pressure stabilizes, increase the flow rate to 0.20 mL/min. 10.2.2 EDTA Pre-injections The laboratory must Inject 5 piL of the 5.0 mmol EDTA pre-injection solution (Sect. 7.8.2). Method development used the LC conditions listed in Table 4. This gradient recycles the column to 10% A in preparation for sample injections. If a laboratory chooses to use different chromatographic conditions see the flexibility requirement described in Section 1.2.2. The EDTA pre-injection must be run prior to each standard, field sample, and QC sample in the sequence. 10.2.3 Establish LC-MS/MS Retention Times, MRM Windows, and Divert Windows Inject a mid- to high-level calibration standard under the LC conditions specified in Table 3. 10.2.3.1 Retention Times Determine the retention times of each method analyte. The retention times observed during collection of the method performance data are listed in Table 6. 10.2.3.2 MRM Windows During method development, the chromatogram was divided into two windows—also called segments or functions. AMPA and glufosinate were monitored during the first MRM window; glyphosate and endothall were monitored in the second MRM window. 10.2.3.3 Matrix Divert Windows The instructions in this subsection apply to the Shodex VT-50 2D column and any alternate column technology. Divert column eluent to waste when analytes are not eluting. Enough time should be allowed for the baseline to stabilize between the valve switch that begins each analyte elution window and the appearance of the subsequent analyte signal. If the valve switch is too close to the analyte, the starting point of the analyte peak may be difficult to distinguish from the baseline disruption, especially for low analyte concentrations. Verify that analyte signals have returned to baseline before the valve switch that begins the following matrix divert window. 10.3 Initial Calibration Prepare a set of at least five procedural calibration standards as described in Section 7.16 to generate linear or quadratic calibration curves. The analyte concentrations in the lowest calibration standard must be at, or below, the MRL. Field samples must be quantified using a calibration curve that spans the same concentration range used to collect the IDC data (Sect. 9.1), i.e., analysts are not permitted to use a restricted calibration range to meet the IDC criteria and then use a larger dynamic range during analysis of field samples. 561-19 ------- 10.3.1 Calibration Acceptance Criteria Evaluate the initial calibration by calculating the concentration of each analyte as an unknown against its regression equation. For calibration levels that are less than, or equal to, the MRL, the result for each analyte should be within 50-150% of the true value. All other calibration points should be within 70- 130% of their true value. If these criteria cannot be met, the analyst could have difficulty meeting ongoing QC criteria. In this case, corrective action is recommended such as reanalyzing the calibration standards, restricting the range of calibration, or performing instrument maintenance. If the cause for failure to meet the criteria is due to contamination or standard degradation, prepare fresh calibration standards and repeat the initial calibration. 10.4 Continuing Calibration Analyze a CCC to verify the initial calibration at the beginning of each Analysis Batch, after every tenth field sample, and at the end of each Analysis Batch. The beginning CCC for each Analysis Batch must be at, or below, the MRL for each analyte. This CCC verifies instrument sensitivity prior to the analysis of samples. Alternate subsequent CCCs between the mid and high calibration levels. Verify that the CCC meets the criteria in the following sections. 10.4.1 Internal Standard Responses in CCCs The absolute area of the quantitation ion for each internal standard must be within 50-150% of the average area measured during the initial calibration. If these limits are exceeded, samples analyzed since the last acceptable CCC are invalid and corrective action is necessary (Sect. 10.5). 10.4.2 Analyte Responses in CCCs Calculate the concentration of each method analyte in the CCC. Each analyte fortified at a level less than, or equal to, the MRL must be within 50-150% of the true value. The concentration of the analytes in CCCs fortified at all other levels must be within 70-130%. If these limits are exceeded, then all data for the failed analytes must be considered invalid. Any field samples analyzed since the last acceptable CCC that are still within holding time must be reanalyzed after an acceptable calibration has been restored. 10.4.2.1 Exception for High Recovery If the CCC fails because the calculated concentration is greater than 130% (150% for the low-level CCC) for a method analyte, and field sample extracts show no concentrations above the MRL for that analyte, non-detects may be reported without re-analysis. Corrective action (Section 10.5) evaluating and correcting the high bias for the CCCs is required when this high-bias CCC occurs. 10.5 Corrective Action Failure to meet the CCC QC performance criteria requires corrective action. Following a minor remedial action, such as servicing the autosampler or flushing the column, check the calibration with a mid-level CCC and a CCC at the MRL, or recalibrate according to Section 10.3. If internal standard and calibration failures persist, maintenance may be required, such as servicing the LC-MS/MS system or replacing the guard column. These latter measures constitute major maintenance, and the analyst must return to the initial calibration step (Sect. 10.3). 561-20 ------- 11 Procedure The following instructions are based on filtering 12 mL of the original sample volume of 100 mL. Other filtering volumes may be used. 11.1 Prepare Fortified Matrix Samples Fortify LFSMs and LFSMDs with an appropriate volume of Analyte PDS (Sect. 7.14). Use separate duplicate field samples for the LFSM, LFSMD, and an unfortified sample to determine native analyte concentration. Fortify analytes into the original sample before the filtration step. 11.2 Prepare the LRB Collect 100 mL of reagent water in a 120 mL bottle containing the method preservatives, sodium carbonate and sodium omadine (Sect. 8.1.2). 11.3 Field Duplicates Select a Field Duplicate pair associated with the samples in the Analysis Batch. Subsampling from a single field sample is not permitted. 11.4 Filter Samples Shake stored samples and then separately filter exactly 12 mL of the field samples, LRB, field duplicates, and LFSMs into 15 mL vials. See Section 6.7 for syringe and filter specifications. 11.5 Add EDTA Add 250 piL of the 45 mg/mL EDTA solution (Sect. 7.8.1) to the 12 mL of filtered sample. The concentration of trisodium EDTA hydrate is 950 mg/L (2.5 mM). 11.6 Addition of Internal Standards Add 80 piL of the internal standard PDS (Sect. 7.15) to each sample, then cap and invert to mix. The concentration of each internal standard is 100 ng/L. The concentration of the internal standards must be the same in the samples as in the calibration standards. 11.7 Sample Analysis The EDTA pre-injection must be run prior to each standard, field sample, and QC sample in the sequence (Sect. 10.2.2). 11.7.1 Establish LC-MS/MS Operating Conditions Establish MS/MS operating conditions per the procedures in Section 10.1 and chromatographic conditions per Section 10.2. Establish a valid initial calibration following the procedures in Section 10.3 or confirm that the existing calibration is still valid by analyzing a low-level CCC. If establishing an initial calibration for the first time, complete the IDC prior to analyzing field samples. 11.7.2 Verify Divert Windows The analyst must ensure that each analyte and internal standard peak elutes entirely within the assigned window during each Analysis Batch. Make this observation by viewing the quantitation ion for each analyte in the CCCs analyzed during an Analysis Batch. If an internal standard or analyte peak drifts out 561-21 ------- of the assigned window into the divert window, then data for that analyte is invalid in all injections acquired since the last valid CCC. Retention times increase by approximately one minute as the buffer ages over two weeks. The authors recommend verifying divert windows after analyzing the low CCC to begin each Analysis Batch and before running the LRB and subsequent samples. 11.7.3 Sample Analysis Analyze field and QC samples in a properly sequenced Analysis Batch as described in Section 11.8. 11.8 Analysis Batch Sequence An Analysis Batch is a sequence of samples, analyzed within a 24-hour period, of no more than 20 field samples and includes all required QC samples (LRB, CCCs, the LFSM and LFSMD (or FD)). The required QC samples and the EDTA pre-injections are not included in counting the maximum field sample total of 20. LC-MS/MS conditions for the Analysis Batch must be the same as those used during calibration. 11.8.1 Analyze Initial CCC After a valid calibration is established, begin every Analysis Batch by analyzing an initial low-level CCC at, or below, the MRL. This initial CCC must be within 50-150% of the true value for each method analyte and must pass the internal standard area response criterion (Sect. 10.4.1). The initial CCC confirms that the calibration is still valid. Failure to meet the QC criteria may indicate that recalibration is required prior to analyzing samples. 11.8.2 Analyze Field and QC Samples After the initial CCC, continue the Analysis Batch by analyzing an LRB, followed by the field samples and QC samples. Analyze a mid- or high-level CCC after every ten field samples and at the end of each Analysis Batch. Do not count QC samples (LRBs, FDs, LFSMs, LFSMDs) when calculating the required frequency of CCCs. 11.8.3 Analyze Final CCC The last injection of the Analysis Batch must be a mid- or high-level CCC. The acquisition start time of the final CCC must be within 24 hours of the acquisition start time of the low-level CCC at the beginning of the Analysis Batch. More than one Analysis Batch within a 24-hour period is permitted. 11.8.4 Initial Calibration Frequency A full calibration curve is not required before starting a new Analysis Batch. A previous calibration can be confirmed by running an initial, low-level CCC followed by an LRB. If a new calibration curve is analyzed, an Analysis Batch run immediately thereafter must begin with a low-level CCC and an LRB. 12 Data Analysis and Calculations 12.1 Identify Peaks of Interest At the conclusion of data acquisition, use the same software settings established during the calibration procedure to identify peaks of interest in the predetermined retention time windows. Confirm the identity of each analyte by comparison of its retention time with that of the corresponding analyte peak in a recent CCC. 561-22 ------- 12.2 Calculate Analyte Concentrations Calculate analyte concentrations using the multipoint calibration. Report only those values that fall between the MRL and the highest calibration standard. 12.3 Significant Figures Calculations must use all available digits of precision, but final reported concentrations should be rounded to an appropriate number of significant figures (one digit of uncertainty), typically two, and not more than three significant figures. 12.4 Exceeding the Calibration Range The analyst must not extrapolate beyond the established calibration range. If an analyte result exceeds the range of the initial calibration curve, the filtered sample may be diluted using reagent water containing the appropriate amount of internal standard added to match the original level. Re-inject the diluted sample. Incorporate the dilution factor into final concentration calculations. The resulting data must be annotated as a dilution, and the reported MRLs must reflect the dilution factor. 13 Method Performance Method performance data were collected using the Shodex bimodal LC column specified in Section 6.9 and an injection volume of 2 piL. 13.1 Precision, Accuracy, and LCMRL Results Tables for these data are presented in Section 17. Single-laboratory LCMRLs are presented in Table 7. Single-laboratory precision and accuracy data are presented for five water matrices: reagent water (Table 8). high-hardness matrix from a ground water source (Table 9). high-TOC matrix from a surface water source (Table 10). synthetic sample matrix prepared per the formulation in Section 7.9 (Table 11). and finished drinking water from a surface water source, containing an orthophosphate anti-corrosive agent (Table 12). 13.2 Analyte Stability Study in High-Hardness Matrix Samples from a high-hardness matrix from a ground water source were inoculated with microbial-rich water from an impacted surface source and fortified with the method analytes. These samples were stored as required in this method. The percent change from the initial analyzed concentration observed after 7, 14, 21, 28, and 42 days is presented in Table 13 . 13.3 Analyte Stability Study in High-TOC Matrix Samples from a high-TOC matrix from a surface water source were inoculated with microbial-rich water from an impacted surface source and fortified the method analytes. These samples were stored as required in this method. The percent change from the initial analyzed concentration observed after 14, and 35 days is presented in Table 14. 561-23 ------- 14 Pollution Prevention For information about pollution prevention applicable to laboratory operations described in this method, consult: Less is Better, Guide to Minimizing Waste in Laboratories, a publication available from the American Chemical Society (accessed January 2024) at www.acs.org. 15 Waste Management Laboratory waste management practices should be 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. In addition, compliance is required with any sewage discharge permits and regulations, particularly the hazardous waste identification rules and land disposal restrictions. 16 References 1. US EPA. Technical Basis for the Lowest Concentration Minimum Reporting Level (LCMRL) Calculator; EPA 815-R-11-001; Office of Water: Cincinnati, OH, December 2010. 2. Morr, S.; Cuartas, E., M.D.; et al. How Much Calcium is in your Drinking Water? A Survey of Calcium Concentrations in Bottled and Tap Water and their Significance for Medical Treatment and Drug Administration. HSS Journal. 2006, 2, 130-135. 3. Azoulay, A.; Eisenberg, M. J.; et al. Comparison of the Mineral Content of Tap Water and Bottled Waters. J. Gen. Internal Medicine. 2001, 16, 168-175. 4. Thelem, K.D.; Jackson E.P; and Penner, D. The Basis for the Hard-Water Antagonism of Glyphosate Activity. Weed Science. 1995, Volume 43, 541-548. 5. U.S. EPA. Method 548.1: Determination of Endothall in Drinking Water by Ion-Exchange extraction, Acidic Methanol Methylation and Gas Chromatography/Mass Spectrometry, Revision 1.0. 1992 6. Sodium Omadine™ 40%, Product Fact Sheet, https://monsonco.com/wp- content/uploads/2019/09/Sodium-Omadine-40.-TDS.pdf, accessed January 2023. 7. US EPA. Statistical Protocol for the Determination of the Single-Laboratory Lowest Concentration Minimum Reporting Level (LCMRL) and Validation of Laboratory Performance at or Below the Minimum Reporting Level (MRL); EPA 815-R-05-006; Office of Water: Cincinnati, OH, November 2004. 561-24 ------- 17 Tables, Figures and Method Performance Data Table 3. HPLC Method Conditions for Sample lnjections°'b'c'd Time, min percentage of 50 mmol ammonium bicarbonate, pH 9.2 percentage of 55% acetonitrile Initial 10 90 0.1 10 90 7.0 50 50 10 50 50 10.01 55 45 15 55 45 18 80 20 18.01 90 10 35 90 10 a Guard Column = Shodex HILICpak VT-50G 2A, 10 x 2.0 mm i.d. 5 pirn dp b Analytical Column = Shodex HILICpak VT-50 2D, 150 x 2.0 mm i.d. 5 pim dp c Method performance data were collected under these conditions at an injection volume of 2 piL. d Column Flow = 0.2 mL/min, Column Temperature = 40 °C Table 4. HPLC Conditions for EDTA Pre-lnjections and Gradient Recycle° Time (min) percentage of 50 mmol ammonium bicarbonate, pH 9.2 percentage of 55% acetonitrile Initial 90 10 3.0 10 90 8.0 10 90 a Method performance data were collected under these conditions using an EDTA injection volume of 5 \il. 561-25 ------- Table 5. ESI Method Conditions ESI Conditions3 Polarity Negative ion Capillary needle voltage 2.5 kV Cone gas flow 200 L/hour Cone voltage (all analytes) 20 V Nitrogen desolvation gas 800 L/hour Desolvation gas temperature 400 °C a Method performance data were collected using a Waters (Milford, MA) Xevo TQ Absolute. Table 6. Analyte RTs, MRMs, and MS/MS Method Conditions Analyte Segment Retention Time Precursor Ion (m/z) Product lonc [m/z) Collision Energy (V) AMPA 1 9.13 110 63 12 AM PA-d2 1 9.00 112 63 12 GLU 1 10.67 180 85 18 GLU-c/s 1 10.50 188 89 18 END 2 17.37 185 141 14 END-de 2 17.30 191 147 14 GLY 2 17.47 168 63 16 GLY-d2 2 17.33 170 63 16 a An LC-MS/MS chromatogram of the analytes obtained using these parameters and a Waters (Milford, MA) Xevo TQ Absolute is shown in Figure 1 and Figure 2. b Precursor and product ions listed in this table are nominal masses. c Argon used as collision gas. Table 7. LCMRL Results Analyte LCMRL Fortification Levels (|ag/L) Calculated LCMRL (ng/L) AMPA 0.0, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10 1.4 Glufosinate 0.0, 0.50, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10 2.1 Endothall 0.0, 0.50, 1.0, 2.0, 2.5, 3.0, 4.0, 5.0 1.6 Glyphosate 0.0, 0.50, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10 2.2 561-26 ------- Table 8. Precision and Accuracy Data for Reagent Water Analyte Low Fortification (Hg/L) Mean %Ra (n=7) %RSDa High Fortification (Hg/L) Mean %R (i=5) %RSD AM PA 10 81.2 8.1 150 98.9 2.4 Glufosinate 10 98.8 4.2 150 101 1.6 Endothall 5 92.8 2.9 75 102 3.8 Glyphosate 10 81.5 4.2 150 100 3.6 a %R = percent recovery; %RSD = percent re ative standard deviation Table 9. Precision and Accuracy Data for a Ground Water Matrix° Analyte Low Fortification (Hg/L) Mean %Rbc (i=5) %RSDb High Fortification (Hg/L) Mean %R (i=5) %RSD AM PA 10 86.9 4.8 150 101 1.0 Glufosinate 10 97.2 2.0 150 103 1.9 Endothall 5.0 90.8 6.4 75 101 0.86 Glyphosate 10 89.7 2.6 150 99.1 1.7 a Ground water matrix was sampled after the clarifier and prior to water softener within the drinking water treatment plant. Hardness = 338 mg/L as CaC03, pH = 8.2 at 20 °C, Free Cl2 = 0.10 mg/L, Total Cl2 = 0.17 mg/L. b %R = percent recovery, corrected for native concentration; %RSD = percent relative standard deviation. c Corrected for native concentration Table 10. Precision and Accuracy Data for a Surface Water Matrix° Analyte Low Fortification (Hg/L) Mean %Rbc (i=5) %RSDb High Fortification (Hg/L) Mean %R (i=5) %RSD AM PA 10 85.9 6.0 150 99.6 2.4 Glufosinate 10 101 2.0 150 102 2.8 Endothall 5 92.7 2.6 75 99.7 2.3 Glyphosate 10 92.2 4.1 150 101 1.8 a Surface water matrix was sampled after the clarifier and prior to granular activated carbon within the drinking water treatment plant and chlorinated in our laboratory. Hardness = 122 mg/L as CaC03, pH = 8.4 at 20 °C, Free Cl2 = 0.82 mg/L, Total Cl2 = 1.26 mg/L, Total Organic Carbon (TOC) = 2.7 mg/L C. b %R = percent recovery; %RSD = percent relative standard deviation. c Corrected for native concentration. 561-27 ------- Table 11. Precision and Accuracy Data for a Synthetic Sample Matrix° Analyte Fortification (ng/L) Mean %Rb'c(n=5) %RSDb AM PA 80 103 6.6 Glufosinate 80 107 6.7 Endothall 40 103 4.9 Glyphosate 80 99.9 5.8 a Synthetic matrix was prepared with 141 mg/L Ca2+, 48 mg/L Mg2+, 44 mg/L N03 , 390 mg/L CI , 250 mg/L S042". b %R = percent recovery; %RSD = percent relative standard deviation. c Corrected for native concentration. Table 12. Precision and Accuracy Data for a Finished Surface Water with Orthophosphate Anti- Corrosive Agent" Analyte Fortification (ng/L) Mean %Rb,c (n=5) %RSDb AM PA 40 100 1.7 Glufosinate 40 101 1.6 Endothall 20 101 1.9 Glyphosate 40 104 1.5 a Surface water matrix was sampled after the clarifier and prior to granular activated carbon within the drinking water treatment plant and chlorinated in our laboratory. Hardness = 117 mg/L as CaC03, pH = 8.62 at 20 °C, Free Cl2 = 1.22 mg/L, Total Cl2 = 1.38 mg/L, Total Organic Carbon (TOC) = 2.1 mg/LC. b %R = percent recovery; %RSD = percent relative standard deviation. c Corrected for native concentration. 561-28 ------- Table 13. Aqueous Sample Holding Time Data for a Ground Water Matrix°'b Analyte Fortified Cone. (Hg/L) Day Zero Mean (Hg/L) Day Zero %RSD Day 7 %Changeb Day 7 %RSD Day 14 %Change Day 14 %RSD Day 21 %Change Day 21 %RSD Day 28 %Change Day 28 %RSD Day 42 %Change Day 42 %RSD AM PA 150 151 1.0 0.52 2.6 0.58 2.8 0.79 1.6 1.0 1.9 0.87 1.9 Glufosinate 150 155 1.9 -1.6 1.7 -1.2 0.71 -0.89 1.7 -1.2 1.2 -1.4 1.9 Endothall 75 75.7 0.86 -0.10 0.61 1.9 1.3 0.65 0.85 0.76 1.4 1.0 0.52 Glyphosate 150 149 1.7 -1.9 2.1 2.7 0.45 2.6 1.2 3.4 1.4 4.0 0.92 a Ground water matrix was sampled after the clarifier and prior to the water softener within the drinking water treatment plant. Hardness = 338 mg/L as CaC03, pH = 8.2 at 20 °C, Free Cl2 = 0.10 mg/L, Total Cl2 = 0.17 mg/L, n=5. b %Change = percent change from Day Zero calculated as follows: (Day X mean concentration - Day Zero mean concentration) / Day Zero mean concentration * 100%, where X is the analysis day. Table 14. Aqueous Sample Holding Time Data for a Surface Water Matrixa,b Analyte Fortified Cone. (|ig/L) Day Zero Mean (Hg/L) Day Zero %RSD Day 14 %Change Day 14 %RSD Day 35 %Change Day 35 %RSD AM PA 150 149 2.2 -3.1 12 -5.6 13 Glufosinate 150 152 2.5 -4.8 8.3 -6.8 10 Endothall 75 74.8 2.3 0.93 1.4 -1.4 4.1 Glyphosate 150 151 1.6 -4.2 12 -3.4 14 a Surface water matrix was sampled after the clarifier and prior to granular activated carbon within the drinking water treatment plant and chlorinated in our laboratory. Hardness = 122 mg/L as CaC03, pH = 8.4 at 20 °C, Free Cl2 = 0.82 mg/L, Total Cl2 = 1.26 mg/L, Total Organic Carbon (TOC) = 2.7 mg/L C, n=5. b %Change = percent change from Day Zero calculated as follows: (Day X mean concentration - Day Zero mean concentration) / Day Zero mean concentration * 100%, where X is the analysis day. 561-29 ------- Table 15. Initial Demonstration of Capability (IDC) Quality Control Requirements Method Reference Requirement Specification and Frequency Acceptance Criteria Section 10 Establish LC-MS/MS conditions, establish retention times and divert windows Prior to conducting the IDC Each analyte and internal standard peak must elute entirely within the MRM window (Sect. 11.4.2). 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. Evaluate the calibration curve as described in Section 10.3.1. When each calibration standard is calculated as an unknown using the calibration curve, the lowest level standard should be within 50-150% of the true value. All other levels should be within 70-130% of the true value. Section 9.1.1 Demonstration of low system background Analyze a Laboratory Reagent Blank (LRB) after the highest standard in the calibration range. Demonstrate that the method analytes are less than one-third of the Minimum Reporting Level (MRL). Section 9.1.2 Demonstration of precision Analyze 7 replicate Laboratory Fortified Blanks (LFBs) near the mid-range concentration. Percent relative standard deviation must be <20%. Section 9.1.3 Demonstration of accuracy Calculate mean recovery for replicates used in Section 9.1.2. Mean recovery within 70-130% of the true value. Section 9.1.4 MRL confirmation Fortify and analyze 7 replicate LFBs at the proposed MRL concentration. Confirm that the Upper Prediction Interval of Results (PIR) and Lower PIR meet the recovery criteria. Upper PIR <150% Lower PIR >50% Section 9.1.5 Calibration Verification Analyze mid-level QCS. Results must be within 80-120% of the true value. 561-30 ------- Table 16. Ongoing Quality Control Requirements Method Reference Requirement Specification and Frequency Acceptance Criteria 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. Evaluate the calibration curve as described in Section 10.3.1. When each calibration standard is calculated as an unknown using the calibration curve, the lowest level standard should be within 50-150% of the true value. All other levels should be within 70-130% of the true value. Section 10.4 Continuing Calibration Check (CCC) Verify initial calibration by analyzing a low-level CCC (concentrations at, or below, the MRL for each analyte) at the beginning of each Analysis Batch. Subsequent CCCs are required after every tenth field sample and to complete the batch. The lowest level CCC must be within 50-150% of the true value. All other levels must be within 70-130% of the true value. Verify that each internal standard and analyte peak elutes entirely within the MRM window (Sect. 11.4.2). Section 9.2.1 Laboratory Reagent Blank (LRB) With each Analysis Batch, analyze one LRB for each lot number of filters used to prepare samples. Demonstrate that all method analytes are below one- third the Minimum Reporting Level (MRL), and that possible interference from reagents and glassware do not prevent identification and quantitation of method analytes. Section 9.2.7 Internal standards Internal standards are added to all standards and samples. Peak area counts for each internal standard in samples and CCCs must be within 50-150% of the average peak area in the initial calibration. Section 9.2.8 Laboratory Fortified Sample Matrix (LFSM) Include one LFSM per Analysis Batch. Fortify the LFSM with method analytes at a concentration close to but greater than the native concentrations (if known). For analytes fortified at concentrations <2 x the MRL, the result must be within 50-150% of the true value; 70-130% of the true value if fortified at concentrations greater than 2 x the MRL. Section 9.2.9 Laboratory Fortified Sample Matrix Duplicate (LFSMD) or Field Duplicate (FD) Include at least one LFSMD or FD with each Analysis Batch. For LFSMDs or FDs, relative percent differences must be <30% (<50% if analyte concentration <2 x the MRL). Section 9.2.2 Field Reagent Blank (FRB) Analyze the FRB if any analyte is detected in the associated field samples. If an analyte detected in the field sample is present in the associated FRB at greater than one-third the MRL, the results for that analyte are invalid. Section 9.2.10 Calibration Verification using QCS Perform a Calibration Verification at least quarterly. Results must be within 80-120% of the true value. 561-31 ------- Figure 1. Relative Retention of Early Eluting Preservatives to the Method Analytes in High-Hardness Watera'b 1.0- 0.8- 0.6- 0.4- 0.2- 0.0 Chloride Sodium Omadine AMPA Nitrate Glu 8 9 10 Time (min) a Acquired using a Waters (Milford, MA) Xevo TQ Absolute. AMPA, GLU, and GLY at 40 ng/L; END at 20 ng/L. b Sodium omadine was observed as the pyrithione anion at m/z 126. 561-32 ------- Figure 2. Relative Retention of Late Eluting Preservatives to the Method Analytes in High-Hardness Water° 1.0- EDTA-Ca - EDTA-Mg Gly End Sulfate Free EDTA Time (min) Acquired using a Waters (Milford, MA) Xevo TQ Absolute. AMPA, GLU, and GLY at 40 ng/L; END at 20 ng/L. 561-33 ------- |