Article pubs.acs.org/JAFC
Single-Laboratory Ruggedness Testing and Validation of a Modified QuEChERS Approach To Quantify 185 Pesticide Residues in Salmon by Liquid Chromatography− and Gas Chromatography−Tandem Mass Spectrometry Brittany Holmes,* Arlene Dunkin, Royal Schoen, and Chris Wiseman Chemical and Hop Laboratory, Washington State Department of Agriculture, Suite 106, 21 North First Avenue, Yakima, Washington 98902, United States S Supporting Information *
ABSTRACT: In this study, we successfully applied a previously modified version of the QuEChERS approach to quantify pesticide residues in samples of fresh salmon. Analysis was performed using a combination of liquid and gas chromatography− tandem mass spectrometry (LC-MS/MS and GC-MS/MS). The validated QuEChERS method used ethyl acetate for the extraction solvent and involved two freezing steps and a C18 dispersive solid phase extraction for removal of lipids. Of the 228 pesticides initially screened, only 185 passed the method validation criteria (103 on LC-MS/MS and 82 on GC-MS/MS). In a quantitative validation, acceptable performances were achieved with overall recoveries of 70−120% and 0.990 1×, 5×, and 10× LOQ 80−120% of the test concentrations 80−120% of actual concentration 50−150% at 2 × LOQ
calibration STD accuracy fortification recovery
%CV < 20%
analysis precision
calibration STD precision interday: fortification recovery interday: matrix-matched STD recovery
%CV < 30% %CV < value predicted by Horwitz
method LOQ
quantitative MS/MS transition qualitative MS/MS transition
>10:1 S:N ratio >3:1 S:N ratio
a
%CV = coefficient of variation, defined as a ratio of the standard deviation (s) of a set of numbers (n), to their average (x), expressed as a percentatge: s/x × 100%; LOQ = limit of quantitation, at least 10:1 signal: noise; Horwitz %CV = 2(1−0.5logC), where C is concentration. mL centrifuge tubes, and solid phase extraction (SPE) columns were purchased from UCT. Standards with valid certificates of analysis were obtained from the National Pesticide Standard Repository, EPA, and ChemService. All neat standards were stored in a freezer at −20 °C or lower, unless degradation occurred, in which case standard was stored at the recommended temperature. Individual stock solutions at 1.0 mg/mL were prepared in methanol or acetone depending on solubility and instrumentation. Mixed standard solutions containing about 140 pesticides for LC and 90 pesticides for GC were prepared from the stock standards in methanol and acetone, respectively. All standard solutions were stored in a freezer at 0 °C; all cold-storage devices were secured and monitored according to ANSI/ISO/IEC 17025. Anytime a neat standard or solution was removed from cold storage, it was first
MATERIALS AND METHODS
Reagents and Chemicals. Acetonitrile, ethyl acetate, toluene, acetone, and acetic acid (HPLC grade or better) and methanol and formic acid (LCMS grade Optima) were purchased from Fischer Scientific. Magnesium sulfate, anhydrous reagent grade (MgSO4), sodium chloride, reagent grade (NaCl), primary−secondary amine (PSA), 1 mL mini-centrifuge tubes, polypropylene (PPE) 15 and 50 B
DOI: 10.1021/jf5055276 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry brought to room temperature before being used. Individual standard solutions were good for 1 year, whereas mixed solutions were used for only 6 months and then remade accordingly. Sample Collection and Processing. Using a rigorous statistical design, PDP has developed extensive procedures that ensure samples are randomly selected from the national food distribution system and reflect what is typically available to the consumer. PDP State sample collectors and State testing facilities are trained to adhere to detailed program Standard Operating Procedures that provide criteria for site selection and specific instructions for sample selection, collection, shipping, and handling. Samples are collected year-round from markets, terminal markets, distribution centers, and retail sites selected by the National Agricultural Statistics Service (NASS) using probability proportional to size methodology so that the data reported are a statistically defensible representation of the U.S. food supply. State population figures are used to assign the number of samples scheduled for collection each month.1 These population and distribution network-based numbers result in the following monthly collection assignments for each state: California, 13; Colorado, 2; Florida, 7; Maryland, 4; Michigan, 6; New York, 9; Ohio, 6; Texas, 8; and Washington, 4. This schedule results in a monthly target of 59 salmon samples, for a target of 708 total samples to be collected beginning July 2013 and ending June 2014. Acceptable samples included farm-raised, wild, organic, conventional, domestic, and imported Atlantic or Pacific salmon for the following species: Chinook (King), Chum (Dog, Keta, Silverbite), Coho (Silver), Pink (Humpback), and Sockeye (Red). Each sample consisted of 1 pound fresh or frozen (raw, uncooked) fillets, nuggets, steaks, or strips. Salmon with guts and/or heads, breading, seasonings, or flavoring (i.e., smoked) were excluded, but both bone-in/out and skin/skinless were included in sampling. Sample collectors rely solely upon the label for identification of collected samples. To ensure samples arrived at the laboratory in a frozen or cold-to-the-touch condition, they were collected in pesticide-free polyethylene bags, frozen overnight, and then shipped the following day by next-day air with ample frozen cold packs and insulating materials surrounding all sample units. Any samples that were no longer frozen or cold-to-thetouch once they arrived at Yakima Laboratory were discarded, and a resample was requested. Frozen salmon samples were held in a freezer at 0 °C or lower until the sample could be homogenized. Samples could also be refrigerated overnight before cutting approximately 1 pound portions into approximately 1 in. squares and frozen in labeled ziplock bags in a −20 °C freezer until homogenization. Each sample was then homogenized in a Retsch food processor for approximately 45 s in reverse and forward mode at 2200−4500 rpm until homogeneous. Portions of the homogenized sample were transferred into two labeled 8 oz polypropylene (PPE) sample cups, and 20 ± 0.2 g (±1%) was weighed out into two labeled 50 mL PPE centrifuge tubes containing a ceramic homogenizer; exact weight was recorded. Homogenized samples were stored at −20 °C for up to 72 h and moved to a −40 °C freezer for long-term storage (>72 h). GC-MS/MS Instrumentation. The GC system (Agilent 7890A, Palo Alto, CA, USA) was equipped with an autosampler (Agilent 7693) and coupled to a quadrapole mass spectrometer (Agilent MSD 7000B). MassHunter software was used for instrument control and data acquisition. Analytes were separated using two HP-1 ms columns (15 m long × 0.25 mm i.d. × 0.25 μm df) (J&W Scientific, Folsom, CA, USA), connected through a backflush union for a total column length of 30 m. The column head pressure was set at 16.727 psi at a total flow rate of 34.6 mL/min, using helium as carrier gas. The injection volume was 1 μL in splitless mode at 220 °C with a 3.5 min solvent delay. The oven temperature was programmed as follows: oven held at 90 °C for 1 min, then raised at 50 °C/min to 150 °C, then 6 °C/min to 200 °C, then 16 °C/min to 280 °C, and held at 280 °C for 9 min. Total run time was 21.533 min. The ion source and transfer line and source temperatures were set at 300 °C. Electron multiplier voltage was set by automatic tuning. Nitrogen and helium were used as the collision gases for all MS/MS experiments, and the flow in the collision cell was set at 1.5 and 2.25 mL/min, respectively. For each
analyte a scheduled multiple reaction monitoring (MRM) was optimized for two ion transitions in a 60 s detection window with a 10 ms dwell time. Complete method details can be found in the Supporting Information. LC-MS/MS Instrumentation. The LC system (Acquity UPLC, Waters Corp., Milford, MA, USA) was coupled to a tandem quadrapole mass spectrometer (Xevo TQ-S, Waters Corp.). MassLynx software (version 4.1) was used for instrument control and data acquisition. Analytes were separated on a universal, silica-based bonded phase Acquity HSS T3 column (2.1 mm × 50 mm, 1.8 μm particle size, Waters Corp.) at 40 °C with a sample injection of 4 μL. A binary mobile phase was composed of 0.1% v/v formic acid and 10 mM ammonium formate in water (A) and in methanol (B). A mobile phase gradient at 0.45 mL/min started at 5% B and went to 100% B in 9.5 min, was held for 1.5 min, and concluded by column equilibration at initial conditions for 4 min for a total run time of 15 min. The MS source operated using positive electrospray mode with the following conditions: capillary voltage of 1000 V, source offset of 25 V, desolvation gas flow of 850 L/h, desolvation and cone temperatures of 550 and 150 °C (respectively), cone gas flow of 150 L/h, collision gas flow of 0.15 mL/min, and nebulizer gas of 5 bar. For each analyte, at least one scheduled MRM with a 60 s detection window was optimized for qualitative analysis, and two were optimized for quantitative analysis. Complete method details can be found in the Supporting Information. Sample Extraction. For reagent blank, 20 mL of deionized water was used. The previously weighed out 20 g samples were thawed at 4 °C overnight or in cold water on the day of extraction. Thirty milliliters of ethyl acetate was added using an automated liquid dispensing unit verified before use by gravimetric check. Samples were then shaken for 5 min on a SPEX 2000 Geno grinder (SPEX Sample Prep, LLC, Metuchen, NJ, USA) at 1200 rpm and then centrifuged (1000 rcf, 5 min, ambient temperature) to compact the contents, allowing for addition of extraction buffers (8 g of MgSO4 and 2 g of NaCl). Samples were shaken for another 5 min at the same speed and inspected for air or liquid pockets; for tubes with visible air or liquid pockets, a disposable PPE transfer pipet was used to disperse the pocket and ensure complete mixing of buffer salts with sample. Sealed tubes were placed into a −20 °C freezer for 30 min and then centrifuged (3000 rcf, 5 min, ambient temperature); the upper ethyl acetate layer was decanted through a funnel and filter paper into a calibrated 50 mL glass graduated centrifuge tube. Target recovery for this layer is 18 mL (A1); excess liquid was aspirated to waste, or if 30%), and 25 had poor peak shape or resolution between matrix coextractive peaks. These analytes were subsequently dropped from analysis, resulting in a final screen of 103 analytes by LC-MS/MS and 82 analytes by GC-MS/MS. Of the compounds that were not detected or had poor recovery at the lowest spike level, a pesticide group that stood out included the highly lipophilic pesticides (i.e., acephate, chlorthalonil, imazalil, oxidemeton methyl, methamidophos, thiabendazole). This group of pesticides is liable to partition into the fat layer9 that formed during extraction procedure and settled out of the final extract, as shown in Figure 2. Extract pH was not controlled in this method, thus
(2) Sample Cleanup. Due to the sensitivity of the instrumentation, samples were subjected to additional cleanup and dilution steps prior to analysis. For LC-MS/MS 1 mL of final extract was quantitatively transferred into a mini-centrifuge tube containing 150 mg of MgSO4, 50 mg of PSA, and 50 mg of C18, vortexed for 1 min, and centrifuged (1000 rcf, 2 min, ambient temperature). Samples were diluted 1:8 in the following fashion: 125 μL of clean sample extract, 20 μL of 1.55 ppm TPP (internal standard), 355 μL of acetonitrile, 500 μL of methanol, and 1000 μL of 0.67 mM formic acid. Samples were filtered through a nylon 0.2 mm syringe filter into a maximum recovery LC vial. Remaining extract in mini-centrifuge tubes was held in reserve at 0 °C or lower. Final LC-MS/MS sample concentration was 0.25 g/mL. For GC-MS/MS, 1 mL of final extract was quantitatively transferred to a conditioned (4 mL, 3:1 acetone/toluene) SPE column containing 500 mg of PSA. Each SPE column was washed three times with 4 mL of 3:1 acetone/toluene. After the last washing, the column was kept under pressure for about 1 min to completely elute extract from the column. Samples were then concentrated to 0.5 mL under a stream of nitrogen set at no more than 10 L/min with a water bath set at 50 °C. To completely displace the other solvents from the final extract, 3 mL of toluene was added, and samples were concentrated again to 0.5 mL. Final volume was adjusted to 1 mL with toluene, and the sample was vortexed for 30 s. About half of the extracts were transferred to GC autosampler vials with 0.5 mL glass inserts; remaining extract was held in reserve at 0 °C or lower. Final GC-MS/MS sample concentration was 4 g/mL. Standard matrix blank homogenate followed the same extraction procedure for LC-MS/MS samples, but for GC-MS/MS it was doubly concentrated during the cleanup step by transferring 2 mL instead of 1 to create an 8 g/mL matrix blank for use in preparing GC-MS/MS calibration standards. Pesticide Residue Quantitation. Following extraction and cleanup, samples were analyzed for pesticide residues using both LC- and GC-MS/MS instrumentation. For quantification, a matrixmatched standard of blank salmon extract was prepared at 1, 5, and 10 times the limit of quantification (LOQ) for each pesticide by adding appropriate volumes of mixed fortification standard to the blank sample extract (after each instrument’s specific cleanup). The peak area of the primary transition was used to generate an external, threepoint calibration curve using least-squares linear regression analysis. A matrix-matched standard at 2 × LOQ was used to assess the validity of the generated calibration curve; method recoveries were not background subtracted. Quality Assurance/Control. Confirmation of pesticides found in fortified and incurred samples was determined by comparing expected retention time and the ratio of the two transition results to matrixmatched standards, per PDP criteria for identification and acceptable recovery as shown in Table 1. The stability of chromatography was assessed by analyzing continuing calibration verification standards every 7−11 samples. The accuracy and precision of continuing calibration standards were typically within ±20% of expected values. The presence of artifact pesticide residues arising from laboratoryassociated procedures, reagents, and/or matrix was assessed through the analysis of laboratory reagent and matrix blanks. Additionally, interday accuracy and precision were monitored throughout the entire study by means of statistical process control charting and review.
Figure 2. Sample diversity is apparent throughout the extraction: not only did the color vary between salmon samples but varying amounts were recovered for A1 (top) and different amounts of fat were separated and removed from extract after solvent exchange from ethyl acetate to acetonitrile (middle) and at the conclusion of the second freezing step (bottom).
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RESULTS AND DISCUSSION Method Validation. First, different samples of salmon were extracted and analyzed to find the lowest contaminated matrix for use in blanks and fortified samples. The interday precision and accuracy of recoveries for 88 GC and 142 LC analytes were determined by spiking a matrix blank in triplicate with a mixture of these pesticides at concentrations of 1, 5, and 10 times the LOQ for each analyte. Spiked samples were then
compromising the stability of base-sensitive and/or acid-labile pesticides (i.e., thiodicarb, chlorthalonil, 1-naphthol). Complete details on the results of the interday experiment can be found in the Supporting Information (Table S3). The accuracy of this method was confirmed by a blind, internal proficiency sample using the same matrix material as the blank. Ideally, a standard reference material would be used, D
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Figure 3. Average recoveries of laboratory-fortified blank analyzed by LC-MS/MS (n = 18−19 depending on analyte). Error bars represent standard deviation for analyte.
Figure 4. Average recoveries of laboratory-fortified blanks by GC-MS/MS analysis (n = 16−17 depending on analyte). Error bars represent standard deviation for analyte. E
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Figure 5. Surrogate standard recovery for propoxur (P) and methyl chlorpyrifos (MC). Average and standard deviation were determined for each analysis set (n = 30 for each set, n = 24 complete sets).
but none that met the scope of method (different fish species, incomplete analyte list) were available at the time of study. Internal proficiency testing consisted of two blind samples; each contained a mixture of seven analytes at concentrations ranging from 0.020 to 0.080 μg/mL. These samples were included with a regularly scheduled analysis set and subjected to the same extraction procedure as a regular sample, including the addition of quality controls and analysis by LC- and GCMS/MS. Amounts found were reported to a quality control officer for evaluation; overall, the recoveries were 70−100% with a minimum recovery of 71% for parathion ethyl by GCMS/MS. Complete proficiency testing results are included in the Supporting Information (Table S4). Intraday precision and accuracy for the final screen of 185 analytes were determined by monitoring a sample fortified at 2 × LOQ throughout the entire study duration for a total of about 18 data points for each analyte. Analysis by LC-MS/MS, summarized in Figure 3, had higher variability (expressed as % CV) than GC-MS/MS, summarized in Figure 4. Despite this, some compounds had irreproducible peak shapes as a result of interfering matrix coextractives that resulted in a subjective analysis. Of these compounds, five were eventually dropped from the GC-MS/MS screen; they were bioallethrin, folpet, hydroprene, oxychlordane, and prothiofos. Even with the addition of a C18 cleanup step, the amount of coextractives in the final sample was enough to negatively impact the precision of recovery for cymoxanil, dichlorvos (DDVP), diuron, endosulfan sulfate, fluridone, flusilazole, indaziflam, methomyl, napropamide, Prebane, and pirimiphos methyl, as shown in Figures 3 and 4. Method Ruggedness. The goal of this study was to survey salmon for pesticide residues over the course of a year; the diversity of samples collected over such a long period was going
to be large. To account for varying matrix effects from different salmon species (Atlantic, Pacific, King, Chinook, etc.) and origin (import, domestic, farmed, wild), two internal standards were spiked into every sample at 5 × LOQ: methyl chlorpyrifos (MC) and propoxur (P) for GC and LC analysis, respectively. The observed sample diversity was apparent throughout the entire study as shown in Figure 2, with extract color, volume extracted, and visible fat content being the most discernible features. The samples shown in Figure 2 are from a single analysis set of 30 samples; observed extract color varied from clear and colorless, clear and colored, and opaque and colorless to opaque and clear and remained throughout the extraction process. However, all of the data sets analyzed with this method successfully met PDP’s criteria listed in Table 1 for interlaboratory precision with average set recoveries of 101 and 77% for P and MC, respectively. During the course of this study several lots of reagents, materials, consumables, and standards were used, and a total of six operators were included to test the overall robustness of the written procedure. The statistical process control chart in Figure 5 shows that as the study progressed, the process variation decreased from around 20%CV to 10%CV, and the moving mean stabilized for both analyses. Initially the moving mean changed significantly between sample sets and %CV was high; after investigation, it was found that the standard operating procedure contained steps that were either confusing or had engineering controls that were not clearly defined (i.e., nitrogen flow rate and temperature for N-Evap). The procedure was revised in January 2014 and implemented starting with the second October data set (10FS13B). Sets analyzed with the new written procedure had improved precision, thereby stabilizing the moving mean. This demonstrates the significance a clear, concise, and F
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trometry; PSA, primary−secondary amine; QuEChERS, quick, easy, cheap, effective, rugged, and safe; SPE, solid phase extraction; USDA, U.S. Department of Agriculture; FDA, U.S. Food and Drug Administration; EPA, U.S. Environmental Protection Agency
thoroughly documented procedure has on ensuring a method’s ruggedness. During the course of this study, the analytical instruments had decreased sensitivity and poor intra- and interbatch precision as a result of source (LC) and inlet (GC) contamination by the salmon matrix. To reduce contamination and improve stability of instrument between runs, the frequency of routine maintenance tasks such as changing the GC liner, trimming the GC column, and cleaning the LC-MS/ MS source components was increased from one to two times per month to daily at around the October data sets. Nonroutine maintenance tasks for the LC-MS/MS were also increased from one to two times per year to after every 200−300 injections of salmon matrix; this included cleaning the ion block heater and ion optics. This increased maintenance suggests that the final salmon extract should have been subjected to an additional cleanup or dilution step to minimize instrument downtime. The observed process variation throughout the study cannot be attributed to just unclear written directions or contamination of analytical instrumentation by matrix coextractives; additional testing to isolate each source of error is needed before a statement can be made. However, the ruggedness testing results presented here support the claim that these extraction and analysis methods are suitable for long-term use by different operators and analysts using different lots of reagents and materials. Before this method can be considered official, it is recommended that it should be compared with reference methods in side-by-side analysis of the same sample and an interlaboratory study should also be performed.
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(1) Punzi, J. S.; Lamont, M.; Haynes, D.; Epstein, R. L. Outlooks on Pest Management; Research Information Ltd.: Burnham, UK, June 2005; pp 131−137. (2) U.S. Department of Agriculture, Agricultural Marketing Service, Pesticide Data Program, Standard Operating Procedures Page; http:// www.ams.usda.gov/AMSv1.0/ams.fetchTemplateData.do?template= TemplateG==ScienceandLaboratories=PDPProgramSOPs= PDP+Standard+Operating+Procedures+(SOPs)=pestcddataprg (accessed May 14, 2014). (3) Forsberg, N. D.; Wilson, G. R.; Anderson, K. A. J. Agric. Food Chem. 2011, 59, 8108−8116. (4) Berntssen, M. H.G.; Maage, A.; Julshamn, K.; Oeye, B. E.; Lundebye, A.-K. Chemospere 2011, 83, 95−103. (5) Schenck, F.; Calderon, L.; Podhorniak, L. V. J. AOAC Int. 1996, 79, 1209−1214. (6) Anastassiades, M.; Lehotay, S. J. J. AOAC Int. 2003, 86, 412−431. (7) Lazartigues, A.; Wiest, L.; Baudot, R.; Thomas, M.; Feidt, C.; Cren-Olive, C. Anal. Bioanal. Chem. 2011, 400, 2185−2193. (8) Rawn, D. F. K.; Judge, J.; Roscoe, V. Anal. Bioanal. Chem. 2010, 397, 2525−2531. (9) Chamkasem, N.; Ollis, L. W.; Harmon, T.; Lee, S.; Mercer, G. J. Agric. Food Chem. 2013, 61, 2315−2329. (10) USDA-AMS-S&T National Science Laboratory, Gastonia, NC, USA. Pesticide Residue Analysis of Plant and Animal Tissues, MET-100, revision 04, 2010. (11) Norli, H. R.; Christiansen, A.; Deribe, E. J. Chromatogr., A 2011, 1218, 7234−7241. (12) Koesukwiwat, U.; Lehotay, S.; Mastovska, K.; Dorweiler, K. J.; Leepipatpiboon, N. J. Agric. Food Chem. 2010, 58, 5950−5958. (13) U.S. Department of Agriculture, Agricultural Research Service, 2014. USDA National Nutrient Database for Standard Reference, release 26; Nutrient Data Laboratory Home Page, http://www.ars. usda.gov/ba/bhnrc/ndl (accessed Feb 27, 2014).
ASSOCIATED CONTENT
S Supporting Information *
Files 1 and 2 are the complete method details for GC-MS/MS and LC-MS/MS analytical instrumentation, respectively; Table S3 lists the method range and precision results for each of the 228 pesticides included in initial screen, as well as the ruggedness testing at 2 × LOQ for final screen of 185 pesticides; Table S4 lists the complete results of the internal proficiency testing samples. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*(B.H.) E-mail:
[email protected]. Notes
Mention of brand or firm names does not constitute an endorsement by either the U.S. or Washington States Department's of Agriculture above others of a similar nature not mentioned. The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Scientific Laboratory, Gastonia, NC, USA, for their work on modifying the QuEChERS approach used in this study. Sample preparation and method optimization tests were supported through the technical assistance of Tammy Best, Justin Chambers, Jorge Contreras, and Karen Lowe of the WSDA Chemical and Hop Laboratory.
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ABBREVIATIONS USED GC-MS/MS, gas chromatography−triple-quadrapole mass spectrometer; LC-MS/MS, liquid chromatography−mass specG
DOI: 10.1021/jf5055276 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
