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Gas Chromatography with Pulsed Flame Photometric Detection PODHORNIAK ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 873 RESIDUES AND TRACE ELEMENTS Gas Chromatography with Pulsed Flame Photometric Detection Multiresidue Method for Organophosphate Pesticide and Metabolite Residues at the Parts-Per-Billion Level in Representative Commodities of Fruit and Vegetable Crop Groups LYNDA V. PODHORNIAK,JUAN F. NEGRON, and FRANCIS D. GRIFFITH,JR U.S. Environmental Protection Agency, Office of Pesticide Programs, Biological and Economic Analysis Division, Analytical Chemistry Branch, 701 Mapes Rd, Fort George G. Meade, MD 20755-5350 A gas chromatographic method with a pulsed reassess all food and feed tolerances according to the new flame photometric detector (P–FPD) is presented standards. To satisfy the new requirements of FQPA, EPA for the analysis of 28 parent organophosphate (OP) must conduct risk assessment including aggregate exposure pesticides and their OP metabolites. A total of from various sources and cumulative exposure from related 57 organophosphates were analyzed in 10 repre- chemical(s), which share a common mechanism of toxicity. sentative fruit and vegetable crop groups. The To properly conduct these risk assessments, EPA needs method is based on a judicious selection of known more pesticide residue data at lower levels. While the U.S. procedures from FDA sources such as the Pesti- Food and Drug Administration (FDA) currently provides pes- cide Analytical Manual and Laboratory Information ticide residue data to EPA, the amendments contained in Bulletins, combined in a manner to recover the FQPA require more detailed risk assessment, which in turn re- OPs and their metabolite(s) at the part-per-billion quire more data on residues at lower levels than routinely re- (ppb) level. The method uses an acetone extraction ported from FDA compliance monitoring samples. This multi with either miniaturized Hydromatrix column parti- residue method was developed as part of EPA’s senior man- tioning or alternately a miniaturized methylene agement commitment to provide the tools to FDA to generate dichloride liquid–liquid partitioning, followed by the necessary pesticide data in a timely manner. solid-phase extraction (SPE) cleanup with Because the organophosphate (OP) pesticides and their graphitized carbon black (GCB) and PSA car- metabolites are the Agency’s top priority chemicals, our pro- tridges. Determination of residues is by pro- posed method identifies EPA’s top priority grammed temperature capillary column gas chro- 15 organophosphate pesticides and their metabolites as well as matography fitted with a P–FPD set in the the 13 high priority organophosphate pesticides and their me- phosphorus mode. The method is designed so that tabolites. The top priority OPs include azinphos-methyl and a set of samples can be prepared in 1 working day its oxon, chlorpyrifos and its oxon, coumaphos and its oxon, for overnight instrumental analysis. The recovery dichlorvos, dimethoate and its oxon, dioxathion, diazinon and data indicates that a daily column-cutting proce- its oxon and hydroxy metabolites, ethion and its monooxon, dure used in combination with the SPE extract fenthion and its oxon, sulfoxide, and sulfone metabolites, mal- cleanup effectively reduces matrix enhancement at athion and its oxon, naled, phosmet and its oxon, the ppb level for many organophosphates. The pirimiphos-methyl, trichlorfon, and tetrachlorvinphos. The OPs most susceptible to elevated recoveries high priority OPs include acephate, methamidophos, around or greater than 150%, based on peak area chlorpyrifos-methyl and its oxon, fenamiphos and its calculations, were trichlorfon, phosmet, and the sulfoxide, sulfone, and desisopropyl metabolites, isofenfos metabolites of dimethoate, fenamiphos, fenthion, and its oxon and desisopropyl metabolites, methidathion, and phorate. oxydemeton-methyl, parathion and its oxon, methyl parathion and its oxon, phorate and its oxon, sulfoxide, and sulfone me- tabolites, profenofos, tribufos, and sulprofos and its oxon, he Food Quality Protection Act (FQPA) amended the sulfoxide, and sulfone metabolites. This new method will de- Federal Insecticide, Fungicide, and Rodenticide Act termine the magnitude of the OP residues to the Tand the Food, Drug, and Cosmetic Act, establishing 1 part-per-billion (ppb) level, with the limit of detection more stringent standards for pesticides in foods and feeds. It (LOD) almost an order of magnitude lower for fruits and veg- required the U.S. Environmental Protection Agency (EPA) to etables that were selected for this study. Commodities chosen for this project include primary food Received August 28, 2000. Accepted by JS December 22, 2000. items in the diets of infants and children such as apples, or- 874 PODHORNIAK ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 Figure 1. P–FPD chromatograms of 57 organophosphates subdivided into 4 mixed analytical standards showing the sensitivity of the P–FPD. PODHORNIAK ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 875 Figure 2. Flow chart of Hydromatrix partition. 876 PODHORNIAK ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 Figure 3. Flow chart of liquid–liquid partition. PODHORNIAK ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 877 anges, head lettuce, peaches, carrots, blackberries, and toma- Sartorius Research Model R160D (Brinkman Instruments, toes. Each of these crops is a representative commodity of a Westbury, NY), or equivalent. major food crop group as defined in 40 CFR 180.34. It is an- (o) TurboVap.—Closed cell concentrator, 500 and ticipated that validation of a method for recovery of OPs in the 200 mL (Zymark Corp., Hopkinton, MA). representative commodities initially validates the method for (p) Vials.—Clear, 2 mL, target DP, P/N 5182-0714 other commodities in the crop group. Method validation data (Hewlett Packard), or equivalent; amber, 2 mL, target, DP were also generated for grapes, cranberries, and strawberries, I-D, P/N 5182-0717 (Hewlett Packard), or equivalent; glass which are not included in any crop grouping. inserts, 250 µL, flat bottom, P/N 5181-3377 (Hewlett Packard), or equivalent. METHOD (q) Vial caps.—Target, DP blue caps, T/RR septa, seal, Apparatus kim snap, PTFE, blue, crimp seal, Teflon/rubber, P/N 5181-1210 (Hewlett Packard), or equivalent; amber snap, Tef- (a) Solid-phase extraction (SPE) col- lon/rubber, P/N 5182-0550 (Hewlett Packard), or equivalent. umns.—SupelcleanTM EnviCarbTM graphitized carbon black (r) Büchner funnel.—Coors, porcelain with fixed perfo- (GCB) SPE columns, 6 mL, 0.5 g, Cat. No. 57094 (Supelco, rated plate, plate diameter 14.5 mm, 64 mm high (Fisher Sci- Bellefonte, PA); primary/secondary amine (PSA) SPE col- entific), or equivalent. umns, 0.5 g, 3 mL, Cat. No. 1210-2042, (BOND ELUT, (s) Sharkskin paper.—11 cm (Schleicher & Schuell, Varian Sample Preparation Products, Harbor City, CA), SPE Keene, NH), or equivalent. vacuum manifold and SPE column adapters (Supelco), or (t) Pipets.—Class A, Pyrex, 0.5, 1.0, 2.0, 3.0, 4.0, 5, 6, 7, equivalent. 8, 9, and 10 mL (Fisher Scientific), or equivalent. (b) Gas chromatograph.—HP-6890 (Hewlett Packard, (u) Gases.—He (carrier), high purity grade (Air Products, Palo Alto, CA) equipped with a Model 6890 automatic liquid Allentown, PA) or equivalent; H2, high purity grade (Air sampler (ALS) and fitted with a pulsed flame photometric de- Products), or equivalent; air, ultra high purity, zero grade (Air tector Model 5380 in the phosphorus mode (P–FPD) with a Products), or equivalent. Model 5380 detector controller (OI Analytical, College Sta- (v) Gas filters.—Moisture trap. Model 7214 (Alltech As- tion, TX). Gas chromatographic (GC) control and data acqui- sociates, Deerfield, IL) for He and air; gas purifier: high ca- sition by an HP Chem Station (Hewlett Packard) for GC sys- pacity gas purifier Cat. No. 2-3800-U (Supelco), for He; OMI tems, P/N G2070AA, revision A.06.03. indicating purifier, Cat. No. 23906 (Supelco), for He and H2; (c) Printer.—HP Laser Jet 4000 (Hewlett Packard). OMI-2 tube holder, Cat. No. 23921 (Supelco), for He and H2; (d) GC columns.—30 m, 0.25 mm id, 0.25 µm FT DB-1 refillable hydrocarbon and moisture trap, 1/8 in. fitting, Cat. capillary (J&W Scientific, Folsom, CA), or equivalent; 30 m, No. 32575-024 (VWR Scientific Products, Philadelphia, PA) µ 0.25 mm id, 0.32 m FT DB-17 capillary (J&W Scientific), or for H2; adsorbent refill activated carbon with molecular sieve µ equivalent; and 15 m, 0.25 mm id, 0.25 m FT DB-1701 cap- 13X, 32575-032 for H2. illary (J&W Scientific), or equivalent. (w) Blender.—Waring, single speed, glass container, (e) Chromatography columns, plain (no frit).—22 mm id Model 700G (Fisher Scientific), or equivalent. × 500 mm with Teflon stopcock, Cat. No. 420530-0244 (x) Food processor.—Robot Coupe Model R 301 Ultra (Kontes, Vineland, NJ), or equivalent. (Robot Coupe USA, Inc., Ridgeland, MS) or equivalent. (f) Centrifuge tubes.—Pyrex, stoppered, conical, glass, (y) Brass sieve.—No. 30. graduated, 15 mL (Fisher Scientific, Pittsburg, PA), or equiva- (z) Wire gauze.—40 mesh stainless steel (Jelliff Corp., lent, calibrated to 0.5 or 1.0 mL. South Port, CT). (g) Nitrogen evaporator.—12-Sample nitrogen evapora- (aa) Combustor.—3 mm id P/N 282913 (OI Analytical). tor, 50–55EC, N-Evap-111 (Organomation Associates, Inc., (bb) Optical filter.—Phosphorus, GG-495 yellow, P/N Berlin, MA), or equivalent. 282921 (OI Analytical). (h) Graduated cylinder.—25, 50, and 250 mL, Kimax (cc) High Pressure Merlin Microseal septum.—P/N HP (Fisher Scientific), or equivalent. 5182-3442 (Merlin Instrument Co., Half Moon Bay, CA). (i) Long neck, flat round bottom flask.—500 mL, 24/40, (dd) Ferrules.—Vespel/graphite (90%/10%), 250 µm, Kimax (Fisher Scientific), or equivalent. P/N HP 5181-3323. (j) Separatory funnels.—125 mL; Kontes or equivalent. Reagents (k) Funnels.—Pyrex with fluted bowls, 75 mm top diame- ter, 75 mm stem length (Fisher Scientific), or equivalent.
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