ANALYTICAL METHODS FOR PESTICIDES AND PLANT GROWTH REGULATORS Edited by Gunter Zweig / Joseph Sherma ptt SYNTHETIC PYRETHROIDS PI) OTHER PESTICIDES Edited by Gunter Zweig / Joseph Shermai CONTENTS. 4, Fenpropathrin YOSHIYUKI TAKIMOTO, SEIYA YAMAMOTO, HIROHIKO YAMADA, AND JUNSHI MIYAMOTO 1. General IL. Analysis - References .. 5. Fluvalinate W.L, Fic, C. C. HELISTEN, I. M. VISSER, AND W. W. MILLER I. General... 2.22... IL. Analysis : References . 6, Permethrin H. SWAINE AND M. J, TANDY I. General Tl. Analysis... References . 7. Pydrin® SHELL DEVELOPMENT COMPANY I. General . I. Analysis... References . 8. Sumithrin® ‘YOSHIYUKI TAKIMOTO, SEIYA YAMAMOTO, HIROHIKO YAMADA, AND JUNSHI MIYAMOTO: L. General ......... IL Analysis - References . 69 70 B 121 124 131 133 134 146,80 W. L, FITCH ET AL, D. Biological Properties 1, BIOLOGICAL ACTIVITY Fluvalinate is an insecticide/acaricide of the photostable pyrethroid class with broad-spectrum pesticidal activity giving good control of pests of field crops and orchards, as well as vegetables and ornamentals (Henrick et al., 1980; Pike and Drake, 1980). While fluvalinate’s pesticidal spectrum is comparable with that of synthetic pyrethroids currently in use, it is superior to many of them as an acaricide and as an aphicide. In addition to these attributes, residues of the compound are nontoxic and nonrepellent to honeybees. 2. TOXICITY The acute oral LD gq of technical fluvalinate is 280 mg/kg for male rats and 260 mg/kg for female rats. Fluvalinate is moderately irritating to skin and eyes. Lifetime feeding studies are under way in the rat and mouse. At this lime no chronic toxic effects have been observed for fluvalinate. Fluvalinate is toxic to fish, Birds are tolerant to fluvalinate at levels comparable to mammals. The following summarizes the wildlife data: Animal LD yo" Mallard duck* > $620 ppm Bobwhite quail >2510 mg/kg Water flea (48 hours) 74 ppb Bluegill (96 hours) 0.89 ppb Rainbow trout (96 hours} 2.9 ppb "Or LC ® Bight-day dietary study. E. History Fluvalinate was first synthesized in the laboratories of Zoecon Corp. The pesticidal composition is covered by U.S. Patents No. 4260633 and 4243819 (Anderson ef al., 1981; Henrick and Garcia, 1981) and other patents pending. United States registration for use of fluvalinate on ornamentals was received in February 1983, F. Physical Properties Purified fluvalinate as a mixture of two diastereomers is a colorless oil having the following physical properties: Solubility (25°C): Freely soluble in many organic solvents, especially aromatic hydrocarbons, ether, dichloromethane, and alcohols, Water solubility is less than 5 ppb.5. FLUVALINATE 81 Vapor pressure: <1 x 1077 Torr at 25°C, n-Octanol-water partition coefficient: > 7000. G. Chemical Properties 1, METHOD OF SYNTHESIS Synthesis of racemic and partially resolved fluvalinate have been published (Henrick et a/., 1980). In these procedures valine (or d-valine) is converted to a-bromoisovalerate, which is coupled with a,x,2-trifluoro-p- toluidine. An ortho-chlorine substituent is then introduced into the aromatic ting with N-chlorasuccinimide. Finally, the diphenyl ether portion of the molecule is introduced with 2-cyano-3-phenoxybenzyl alcohol. 2. STABILITY a. Photochemical Stability Field results indicate excellent foliar stability under all conditions. Thin films of fluvalinate, either in glass or on silica gel, exposed to California sunlight in April, have a half-life of approximately 2 days. An aqueous emulsion containing 1.6 gm/liter fluvalinate active ingredient exhibited a half-life of approximately 12 days in a Pyrex vessel. b, Thermal Stability No significant degradation occurs after more than 3 hours at 100°C or 18 months at 42°C in glass. c. Hydrolytic Stabitity Aradiochemical study at the maximum concentration in water indicated excellent resistance to hydrolysis at low pH. The ester moiety and, ultimately, the trifluoromethyl group, are labile at high pH. HALF-LIFE OF FLUVALINATE IN Days pH 28°C arc 3 0 35 6 ww 8 9 1-2 1 H. Formulations Fluvalinate is available in petroleum distillate and water-based emulsi- fiable concentrate formulations.82 W. L, FITCH ET AL IL. ANALYSIS A. Formulation Analysis |, RECOMMENDED METHOD a. Principle The fluvalinate content of 50° technical and emulsifiable concentrates is determined by high-performance liquid chromatography (HPLC) using an internal standard method. The internal standard is ZR-2791 [3-phenoxy- benzyl 2-(4-(trifluoromethy|)anilino)-3-methylbutanoate]. b. Reagents and Apparatus Fluvalinate, standard of known purity (Zoecon Corp.). ZR-2791, internal standard, at least 95% purity. The chemistry of ZR-2791 is very similar to fluvalinate, but the two compounds completely resolve during HPLC (Zoecon Corp.). Methanol-isopropanol solvent sohetion, spectrograde methanol and spec- trograde isopropanol (80:20). 0.175 M Acetic acid solution: dissolve 10 ml glacial acetic acid, and make up to I liter with distilled water. High-performance liquid chromatograph, equipped with UV detector set at 254 nm. c. HPLC Conditions Soiveni composition (isocratic): 75% methanol-isopropanol solvent mixture, 25% 0.175 M acetic acid solution. Flow rate: 2 ml{minute. Column temperature: 35°C. Column: Brownlee 5-m Spheri-5, 25 em x 4.6 mm i.d. or equivalent (Brownlee Labs, Santa Clara, California), Guard column; Brownlee 5-m Spheri-5, 3 cm x 4.6 mm id. or equiv- alent d. Analysi Weigh a portion of sample or standard equivalent to 100 mg of fluvalin- ate into a 100-m] volumetric flask. Dilute to volume with solvent mixture to which 1% acetic acid has been added. Mix thoroughly. Pipette a 5-ml aliquot of the above solution into a 25-ml volumetric flask, and add a 5-ml aliquot of internal standard solution (1.0 mg/ml in methanol-isopropanol).5. FLUVALINATE 83 L_ A 8 Fic. |. HPL chromatograms from the analysis of fluv Peak 1, ZR-2791; peak 2, fluvalinate, (A) Standard; (B) sample. inate emulsifiable concentrate. Dilute to volume with 75% solvent mixture and 25% 0.175 M acetic acid solution. Make triplicate injections of 10-yl aliquots of samples and standards. Record peak areas. A typical chromatogram is shown in Fig. 1. e. Calculations Calculate fluvalinate content as follows: acti . ACEF % active ingredient = [—— BDG where A = area of sample peak, B = area of reference standard peak, C = area of internal standard from standard solution, 2 = area of internal standard from sample solution, £ = weight of standard, F = purity of standard, and G = weight of sample.84 W. L. FITCH ET AL. B. Residue Analysis 1, REVIEW OF METHODS The recommended method of analysis for fluvalinate is gas-liquid chromatography (GLC) with a fused silica capillary column and an electron capture detector (ECD). Capillary GLC-ECD was selected as the preferred method of analysis because of its greater sensitivity, selectivity, and conven- ience of operation over that of HPLC. Packed columns could not be used for GLC analysis, because fluvalinate is very unstable on these columns. Similar stability problems have been reported in the analysis of other pyrethroids on packed GLC columns (Kikta and Shierling, 1978). 2, RECOMMENDED METHOD a, Principle Fluvalinate residues in environmental and agricultural samples can be determined down to 0.01 ppm with capillary GLC and ECD. Samples are extracted with appropriate solvents and extraction apparatus. Cleanup steps required for various samples include solvent partitions, Florisil chromatography, and gel permeation chromatography (GPC). To improve accuracy and precision in the analysis of fluvalinate residues, the internal standard ZR-2791 is used to fortify each sample prior to analysis. The internal standard possesses properties very similar to those of fluvalinate. The internal standard follows fluvalinate throughout the entire method of analysis, but the two compounds completely resolve during GLC, thus allowing quantitative measurement of both compounds. b. Reagents Solvenis, glass distilled or equivalent; ensure that diethyl ether contains no more than 0.01%, alcohol (Burdick & Jackson Labs, Inc., Muskegon, Michigan, or equivalent). Fluvalinaie, technical, of known purity (95% or better), with no im- purities greater than 0.1% coeluting the internal standard (Zoecon Corp.). ZR-2791 internal standard, technical, of known purity. There should be no impurities greater than 0.1°%% cocluting with the active ingredient (Zoecon Corp.). Florisil, 60-100 mesh, PR grade (Wilshire Chemical Co., Gardena, California).5. FLUVALINATE 85 i. Heat Activation of Florisil Pour the Florisil into a glass dish to a depth not greater than 5 em Heat ina clean oven overnight (15—20 hours) at 150 +5°C. Ensure that the oven is free of contaminants, including plastic or rubber seals, that may contaminate the Florisil during heating. Remove the dish from the oven, and immediately pour the Florisil into a glass-stoppered bottle for storage. Cool at least 1 hour but not more than 2 days prior to water deactivation, ii, Deactivating Florisit Place the Florisil in a glass-stoppered container, and deactivate with distilled water to 6% water (water—Florisil, 6:94 w/w). Add water dropwise to Florisil while rotating the flask, Seal with a glass stopper, and rotate lightly: to break clumps. Mix gently but thoroughly for about 10 minutes, relieving pressure periodically if necessary. Allow to equilibrate at least 3 hours prior to use. Do not store for more than I month. It is best to make Florisil in batches of not more than 250 gm/lot (15 gm water/235 gm Florisil), since this amount is most effectively mixed in a 1-liter glass-stoppered bottle. c, Apparatus High-speed blender, Waring, explosion-proof power unit, with ex- plosion-proof switch, equipped with container assembly, all stainless steel with screw cover and Teflon gaskets (Eberbach Corp., Ann Arbor, Michigan). Food processor, with rotating steel blades and Lexan bowl: Cuisinart food processor, Medel CFPSA (Cuisinart, Inc., Greenwich, Connecticut), or equivalent. Wiley mili or electric coffee grinder, Moulinex, Type 228.1.00, with s-curved steel blades in a metal container. Concentrator tubes, used in GLC analysis of final extract, 10 ml (0 to 1 ml x 0.1 ml, accurately graduated) (Kontes Glass Co., Vineland, New Jersey). Gas chromatograph, suitable for use with capillary columns and equipped. with a capillary injection system and a linear-pulsed “Ni ECD. A Varian Model 3700 is suitable. Other equivalent instrumentation may be used but may require modification of the operating conditions in order to obtain adequate resolution and response. The capillary injection port should contain a packed injection port liner similar to that described by Jennings (1980). This 2-mm id. glass insert should be packed with a 0.5- to I-cm precolumn containing 3% OV-101 on Chromosorb W between glass wool plugs. Capillary column, J & W Durabond DB-1, 0.1-ym film thickness fused silica capillary, 0.25 mm diameter. Cut evenly to LO m with a sharp file (J & W Scientific, Inc, Rancho Cordova, California).86 W. L. FITCH ET AL GPC apparatus, automated gel permeation system, An 85:15 (v/v) mixture of cyclohexane—methylene chloride is used as the packing and eluting solvent, with a flow rate of 5 ml/minute. The column is 2.5 em i.d., and is packed with 50 gm SX-3 Bio-Beads, 200-400 mesh (Analytical Biochemistry Laboratories, Inc., Columbia, Missouri). GPC precolumn filter, disposable filter assembly with a 0.45-zm PTFE membrane, or equivalent (Gelman Science, Ann Arbor, Michigan). d. Sample Compositing Procedures To prepare the samples for extraction, first partially thaw frozen sample. Mix thoroughly for homogeneity. A method of particle size reduction, such as chopping or grinding, is chosen such that the subsequent extraction step with solvent thoroughly pulverizes the sample. The sample must be pulver- ized to ensure that the fluvalinate extraction efficiency is always consistently at least 90 (minimum) to 100%. Samples with tough connective tissue, such as tissue, organs, and fat from animals, and fruits, vegetables, and fibrous plant tissue, are first chopped to a suitable size for the grinding process. Fibrous plant tissue is conveniently ground in a Wiley mill, which contains rotating steel knives. Other samples may be conveniently ground with rotary steel knife blades in a food processor with an inert plastic Lexan bowl. If the sample is soft and spongy, sprinkle small amounts of dry ice into the processor to solidify for smoother chopping and grinding. Grind particles to ~2 mm for animal tissue and fat, and ~5 mm for plant tissue. Transfer a large sample of the ground composite to a glass jar. Seal the jar by covering the top with alumi- num foil, and screw lid shut. Store in the freezer at 0-S°C. As a more desirable alternative, a Hobart food chopper may be used to pulverize large pieces of frozen sample, up to ~5 cm, into a fine free- flowing matrix. Seal frozen composite in jars as described above. A more uniform composite is usually obtained by this method. Also the sample is kept completely frozen throughout the grinding and compositing cycles. Samples such as grains, nuts, seeds, coffee beans, and other similar dry solid samples may be ground in a grinder to pass a 20-mesh screen. ‘Use a Wiley mill or a grinder that has metal blades in a metal container. ce. General Extraction Procedure First add 5 gm of Celite to the extraction blender. Then select the sample aliquot as designated in Table I, and add to the blender. Add an appropriate amount of ZR-2791 internal standard dissolved in isooctane. Ensure that the total amount of isooctane added to the blender does not exceed about Sml. The amount of internal standard to use is about 3 to 10 times the expec- ted level of fluvalinate in the samples. Thus, for a 10-gm sample expected toTABLE I EXTRACTION, CLEANUP, AND Recovery Data FoR FLUVALINATE Column Fluvalinate — Fluvalinate Grams* Gram cleanup! Comments on fortification’ recovery* ‘Commodity (,) equivalents" Defat* (lengths) chromatogram* (ppm) CA Alfalfa Forage 10 7 2 GPC c 0.021 94 Seed 50 1 2 GPC GL 0.013 0 Almonds Meats 20 2 3 F 20) c 0.019 4 Meats 20 7 2 GPC c 0.021 7 Hulls 20 2 2 F (10) c 0.020 86 Hulls 20 2 2 ‘GPC QL 0.020 4 Apples Red Delicious 20 2 2 F (10) Gs 0.024 86 Red Delicious 20 4 2 GPC c 0.024 96 Golden Delicious 20 2 2 F (10) c 0.021 93 Golden Delicious 20 7 2 GPC c 0.021 96 Granny Smith 20 2 2 F (10) Cs 0.020 ai Granny Smith 20 7 2 GPC c 0.020 38 Beets, red table Roots 50 2 2 F (10) Gs 0.013 a Roots 50 7 2 GPC c 0.013 3 Leafy tops 50 2 2 Flo) P 0,022 4 Leafty tops 50 7 2 GPC c 0.022 98 Broccoli 50 2 2 F (ly c 0,025 84 50 7 2 GPC c 0.025 83 ‘Cabbage 30 2 2 F (10) c 0,009 110 50 2 2 F (10) c 0.108 100 50 7 2 GPC Ck 0.044 106 (Continued)88 TABLE I (Continued) Column Fluvalinate Grams* Gram eleanup* Comments on recovery? ‘Commodity cr) equivalents” Defat® (lengths) chromatogram* (%) Cauliflower 0 2 2 F (10) c 82 x0 1 2 GPC GL a Celery 50 2 2 F (10) c 93 50 7 2 GPC c 106 Charas Grapefruit, pink 20 2 3 F 20) c 0.028 9 Lemons 20 2 2 F (10) c 0.017 84 Limes 20 2 3 F Qo) c 0.018 83 Oranges ‘Valencia 50 2 2 at) c 0.023 94 Valencia so 7 2 GPC c 0,023 99 Navel 50 2 2 F (10) c 0.023 95 ‘Navel 50 7 2 GPc c 0.023 7 Coffee beans, green (with shells) 10 2 3 GPC c 0.021 104 Corn, sweet Kernel 50 2 2 F (0) c 0.020 83 Kernel 50 7 2 GPC GL 0.020 92 Ear husks 20 2 2 F (10) 8c 0.022 87 Ear husks 2» 7 2 Pc S,P 0.020 8 Collards 50 2 2 F (10) Cc 0.013, 3 30 7 2 GPC Cc o.013 90 ‘Grain kernel Wheat 0 7 2 Pc cs o.o12 92 Barley 50 2 2 F (10) c e010 7 Grapes Zinfandel juice 2» 2 2 F (10) c 0.026 n63 Zinfandel juice Raisins (Thompson seedless) Raisins (Thompson seedless) Thompson seedless ‘Thompson seedless Red Emperor Red Emperor Lettuce Head Head Head Leaf Onions Green Yellow Yellow Peamats Nuts oil Shells Shells: Pears D’Anjou D'Anjou Bartlett Bartlett Comice Comice 20 20 20 20 20 50 50 50 10 10 20 20 20 20 aaa ae a a awa w Be REN hip wis Ne RRR crc F (10) GPC F (10) Fo) GPC F (10) GPC GPC F (10) F (20) GPC F (10) F (10) F (10) GPC F (io) GPC F (10) F (10) GPC a a nanan oa o mon anan an rm 2 qagaanaangn annn 0.026 0.022, 0.022 0.019 0.019 0.020 0.020 0.011 0.010 0.110 0.017 0.024 0.027 0.027 0.020 0.020 0.018 0.018 0.026 0.026 0.019 0.019 0.021 0.021 n w 88 87 0 1” 90 116 88 no 13 94 95 39 76 88 97 8 a me nD 103 91 91 (Continued)TABLE I (Continued) Column Fluvalinate — Fluvalinate Grams* Gram cleanup’ Comments on —_ fortification’ —_—_recovery® Commodity F)) equivalents’ Defat’ (lengths) chromatogram‘ (ppm) (he) Potatoes Russet 50 2 2 F (Id) c 0.027 86 Russet 30 7 2 GPC c 0.027 7 Red 0 2 2 F (10) c 0.021 83 Red 30 7 2 GPC c 0.021 82 Radish root 50 7 2 GPC GL 0.019 92 Soybeans 0 2 3 F (20) c 0.020 4 Tomatoes Whole tomatoes so 1 2 GPC c 0.013 a Whole tomatoes so 2 2 F (10) c 0.011 7 Whole tomatoes 50 2 2 F (10) c 0.011 108 Tomato juice so 2 2 F (10) c 0.010 mn Tomato puree 50 2 2 F (10) c 0.012 102 ‘Wet pomace 20 2 3 F (10) c 0.011 9 Wet pomace 20 2 3 F (10) c 0.010 106 Dry pomace 20 2 3 Foy c 0.010 106 Dry pomace 20 2 3 F (10) c O01 toi Cottonseed Oil 10 1 3 F (20) c 0.020 34 ‘Whole 10 2 3 F (10) c 0.010 85 Bovine matrices 10 2 3 F (10) c 0.010 89 8 T 2 GPC c 0.010 102 25 7 2 GPC c 0.010 2B 25 2 2 F (10) c 0.020 93 2 7 2 GPC c 0.020 83 10 2 3 F (10) c ool »16 Poultry matrices ‘Muscle (breasts) 10 2 2 F (ia) c 0.019 a ‘Muscle (breasts) 10 2 2 GPC Cc 0.020 90 Fat 25 2 2 F (10) C 0.018, 94 Fat 25 2 3 GPC Cc 0.018, 100 Skin 25 2 2 Flo) Cc 0.019 92 Skin 5 2 2 GPC c 0.019 104 Liver 25 2 2 F (10) c 0.018 80 Liver 28 2 3 GPC c 0.018 93 Whole eges (white, fresh) 20 2 2 F (10) Cc 0.017 94 Whole eggs (white, frozen} 20 2 2 GPC GL 0.020 101 Whole eggs (brown, fresh) 20 2 2 FO) c 0.017 95 Waters Pond 1000 1000 a F (10) Cc 0.01 ppb 92 Pond 1000 1000 o F (10) c 0.10 ppb 1 Pond 1000 1000 o None Cc 1.00 ppb m2 Pond 1000 1000 a None Cc 2.00 ppb 109 Runoff (sandy loam soil) 20 2 a None c 0.89 ppb 100 Soils ‘Sandy loam 50 1 1 GPC c 0.012 87 ‘Sandy loam 50 1 a GPC c 0.100 89 Silty clay 50 4 0 F (10) c oon 86 Pebbly sandy loam 50 4 0 F (10) c 0.020 ” “Grams of sample (H’,) used for extraction. ® Gram equivalent selected from extraction volume V, for further analysis, “Number of defatting steps when using the acetonitrile partitioning method for each step. + Method of cleanup to be used: GPC or F (Florisil), with the Florisil column length (cm) shown in parentheses. * Chromatograms: C, clean chromatograms in region of ZR-2791 and fluvalinate ; P, some significant peaks eluting closely to ZR-2791 or fluvalinate; S, large solvent front; L, some late-eluting compounds (negative peaks) with the ECD, / Amount of fluvalinate used to fortify the sample prior to analysis (in ppm unless otherwise indicated). Statistical results are shown for samples fortified at or near the established limit of detection for each commodity. For most analyses, “C-labeled fluvalinate was used to fortify samples and confirm recoveries at various steps throughout the method. * Percentage recovery of fluvalinate.92 ‘W. L. FITCH ET AL. contain 0.1 ppm residues, fortify with 3.0-10.0 yg of internal standard. At lower levels, fortify with 10 times the expected fluvalinate level. The larger amount of ZR-2791 climinates any effect of potential interferences during GLC analysis. For solvent extraction, add to the blender 200 ml of hexane—acetone (1:1 v/v), Blend at high speed for 3-5 minutes, Filter the hexane—acetone extract through a Biichner funnel. Rinse the blender and lid with 25 ml of hexane-acetone (1:1 v/¥), and pour over the cake with slow filtration. Re- peat with another 25 ml hexane-acetone. Samples extracted with polar solvents will require the following parti- tion to leave the extract in hexane ready for any required cleanup steps. Add filtrate to a 1-liter separatory funnel. Add 750 ml of distilled water (0.03 N in HCl, with about 3 gm NaCl). Extract. Recover the upper hexane layer in a glass-stoppered flask, avoiding any droplets of water or emulsion from the lower aqueous layer. The extract may be stored at 0-5°C prior to further analysis. f. Cleanup Procedures The specific cleanup procedures to be utilized for each commodity type are listed in the following section. This section describes the three general procedures used for cleanup: defatting, Florisil chromatography, and gel permeation chromatography. Either GPC or Florisil column cleanup can be used in most cases. However, for a few commodities only one or the other cleanup method can be used, as designated in Table I. i, Defatting Steps Measure total volume (V’,) of hexane extract, Accurately measure an aliquot (V3) of the hexane extract for analysis. This volume (V’,) represents an appropriate gram equivalent of sample. The amount to select is desig- nated in Table 1. Adjust volume to 50 ml by evaporating or adding more hexane. Add to 100 ml acetonitrile in a 0.5-liter separatory funnel. Extract, and discard the upper hexane layer containing the fat. After recovering the acetonitrile, extract again with 50 ml of isooctane. Discard isooctane layer. During the partitionings, avoid collecting any of the upper isooctane or hexane layers along with the lower acetonitrile layer. In certain cases a third defatting step with an additional 50 ml of isooctane is required. These cases are in- dicated in Table I. Extract the fluvalinate~ZR-2791 into pentane by adding 750 ml of distilled water (0.03 in HCI, with about 3 gm NaCl) to the acetonitrile layer in a 1-liter separatory funnel, Add 100 ml pentane, extract, and recover the upper pentane layer in a glass-stoppered flask, avoiding any5. FLUVALINATE 93 droplets of water from the lower aqueous layer. The extract may be stored at 0-5°C prior to chromatography. ii, Florisit Chromatography (a) Calibration of Florisil, Each batch of Florisil is calibrated before use to assure adequate recovery of fluvalinate and internal standard. This is best accomplished by conducting the chromatography as described below with a reference standard containing 5.0 yg of fluvalinate and 15.0 vg of ZR-2791 internal standard. The two compounds should be recovered to an extent greater than 95% in the 35- to 235-ml fraction, If less than 95% of the fluvalinate~ZR-2791 is found in this fraction, adjust the height of future columns, Column heights that have been required to obtain greater than 95% of the fluvalinate-ZR-2791 in the 35- to 235-ml fraction have varied only about 1-3. cm from the normal 10-cm height. (b) Column Chromatography. Evaporate the hexane or pentane extract from extraction or defatting steps to 9 ml. Add 1 ml ether to obtain 10 ml of extract in 10%, ether-pentane. Prepare column as follows: Place | cm glass wool in the bottom of the column, and top with 1 em anhydrous sodium sulfate. Slurry the Florisil with the ether-pentane (10:90, v/v), and pour required column height. Top with 1 cm anhydrous sodium sulfate, and elute to surface. Add the 10 ml sample extract, and collect 35 ml of eluate, Change to a new receiver, and collect the 35- to 235-ml eluate for fluvalinate-ZR-2791. Evaporate the eluate, using a warm-water bath (40 + 5°C) and a stream of nitrogen into an appropriate volume of isooctane for GLC. Do not allow the extract to go to dryness at any time, iii, Gel Permeation Chromatography (GPC) (a) Calibrationof GPC. Standardize the GPC with reference standards containing 0.3 yg fluvalinate and 1.0 ug ZR-2791 dissolved in cyclohexane- methylene chloride (85:15 v/¥). Inject 7.0 ml of the reference standard to completely fill the 5.0-ml injection loop. Collect fifteen 20-ml fractions. at a flow rate of 5 ml/minute. Evaporate into an appropriate volume of isooctane, and analyze each fraction by capillary GLC-ECD. The majority (> 95%) of the fluvalinate— ZR-2791 should be in samples No. 6-13, 120-260 ml (140 mil). If not, adjust collection to reflect new or shifting profiles. The selected fraction should completely span the elution profile of the fluvalinate-ZR-2791 to obtain complete recoveries. Fig. 2 shows a typical separation of fluvalinate and ZR-2791 from lipid.94 W. L, FITCH ET AL. Units @.00 27.50) 55.02 B2.SD NP. 197.S065.OR 192.Se 220.88 Volume (mt) F16, 2, GPC elution profile of corn oil lipids (2), fluvalinate (I), and ZR-2791 (©) in eyelohexane-methylene chloride (85:15 v/v) {b) Sample Chromatography. Add the appropriate gram equivalent of extract to 1-2 ml cyclohexane-methylene chloride (85:15 v/v), and evaporate to a volume of 0.5-1 ml with a stream of nitrogen and a 40°C water bath. Dilute with cyclohexane—methylene chloride (85:15 v/¥) to 7.0 ml of final volume, and inject into the GPC so the 5.0-ml injection loop is over- filled, Set up the chromatography to collect the appropriate yolume as determined under calibration. To remove traces of ¢lectron-capturing advantageous to filter the GPC eluate through a short 2-gm plug of deactivated Florisil (deactivated with 30% water) held in a filter funnel. Evaporate eluate into an appropriate volume of isooctane for GLC- ECD analysis. As an example, if a 5-g equivalent/5 ml is injected, evaporate the extract after GPC cleanup into a final volume of 5.0 ml isooctane. The extract will contain | g equivalent/ml, which provides an adequate response by GLC-ECD for a 0.01-ppm limit of detection. g. Extraction and Cleanup Steps for Specific Commodities i. Vegetables, Grains, Beans, Nuts, Fruits and Ferage Grasses For these commodities, use the general extraction procedure. with the sample amounts as indicated in Table |. Table | also indicates the aliquot size and the number of defatting steps required, and it contains specific recovery data for experiments utilizing the cleanup steps listed in the table. In general, either Florisil or GPC can be utilized as the final sample cleanup5. FLUVALINATE 95 method, Table I indicates which methods have been verified for specific commodities. ii, Cottonseed Select at least 25 gm of a composite cottonseed sample for analysis. Grind seed in a grinder that has metal blades in a metal container. The blades should rotate closely to the walls and base of the container to effect adequate grinding of the entire seed, including the seed kernel, shell, and outer cotton- cellulose layers. An electric coffee grinder meets the requirements. Grind cottonseed sample for 30 seconds. Most of the cotton—cellulose on the outside of the seeds will separate from the seeds during grinding. Weigh cellulose (x) and seed (y) separately, and record weights. To select a representative ground composite of 10 gm of the cottonseed (cellulose plus seed) for analysis, calculate the ratio that x and y are of the whole weight, and select the appropriate aliquots to total 10 gm. Extract as described in the general procedure except extract with 5 gm of Celite and 200 ml hexane-ethanol (97:3 v/v), and rinse filter cake with 2 x 25 ml of hexane-ethanol. Select an aliquot, and perform three defatting steps prior to Florisil column cleanup. lil, Coitonseed Oil Analyze 10 gm with the following changes: Extract with 200 ml hexane only and 5 gm of Celite. Rinse filter cake with 2 x 25 ml of hexane. Select an aliquot, and perform three defatting steps prior to chromatography on a Florisil column, prestandardized to collect the 85- to 410-ml fraction, not a 35- to 235-ml fraction. In order to obtain the fluvalinate and ZR-2791 in the 85-410 ml, a 20-cm column height is usually required. Usually column heights do not vary more than a few centimeters above or below 20 cm to obtain the fluvalinate~ZR-2791 in the 85- to 410-ml fraction iv. Poultry and Bovine Samples For muscle, liver, or kidney, grind sample thoroughly in a food proces- sor to obtain a suitable composite. For fat and skin alone, cut and chop sample into 1-cm pieces. Place 10 gm of subsample of muscle or 25 gm of fat, skin, kidney, or liver over ~1 cm glass wool in a Whatman 35 = 94 mm cellulose thimble in a 45/50 Soxhlet chamber. Pipette 2 ml of 0.1 V HCl over the surface of the sample, and add an appropriate amount of ZR-2791. Top thimble with glass wool. Add a Teflon stir bar, 2 ml of 0.1 NHCI, and 200 ml of anhydrous ethyl ether to a 250-ml round-bottomed receiver flask. Stir and soxhlet extract vigorously for 5-6 hours. Filter the extract through 25 gm anhydrous sodium sulfate saturated with ethyl ether, and rinse the extract through with 25 ml ether. Perform defatting steps and either Florisil or GPC cleanup as summarized in Table I.96 W. L. FITCH ET AL. In the analysis of whole eggs, follow the procedure as described above with certain exceptions as follows; Beat yolks and whites together from a representative composite sample until well mixed. Select aliquot and place in a beaker. Adjust to pH 0 (4.0 ml of 1.0 N HC! per 20 gm, in most cases) while stirring with a glass rod. Stir in 7-10 gm of Celite, enough to make a paste. Add and mix sufficient glass wool to make all the paste adhere and become suspended by the glass wool. Transfer to a Soxhlet thimble, and place in Soxhlet chamber. Add the appropriate amount of ZR-2791, and Soxhlet-extract as above. For bovine milk samples, follow the general extraction procedure, with these changes: Blend 25 ml of stirred, homogeneous milk with 200 ml of acetonitrile and 10 gm of Celite. Filter, and take the appropriate aliquot of the acetonitrile extract directly to the two defatting steps, prior to either Florisil or GPC cleanup. v. Waters The method of extraction and cleanup will be dictated by the sensitivity of the GLC-ECD used, the limit of detection desired, and the types of im- purities and degree of turbidity of the water. (a) Clear, Transparent, Water (Free of Turbidity). Place a 1-liter sample of the water in a 2-liter separatory funnel, and add ZR-2791 internal standard, Add 3 gm NaCl and 25 ml 0.25 N HCl to prevent emulsion. Extract 1-2 minutes with 2 x 200 ml of pentane, and combine the pentane fractions. Evaporate into 1.0 ml isooctane for GLC. (b) Turbid Pond Waters, For waters that contain a large amount of suspended organic material, extract sample by adding 5 ml of 1.0 NHCI and 6gm of NaClto | liter of water sample in a 2-liter separatory funnel. Extract gently with 2 x 200 ml ethyl ether—pentane (1:10 v/v). Combine the ether— pentane fractions, and filter through 25 gm anhydrous sodium sulfate saturated with ethyl ether. Rinse extract through with 25 ml ether—pentane (1:10 v/v). Evaporate to ~5 ml, add to 50 ml isooctane, and perform two defatting steps prior to Florisil column cleanup. (c) Soil Runoff Water (High Turbidity from Suspended Soil). Into a 50-ml glass-stoppered centrifuge tube add 20 ml sample water, 25 ml aceto- nitrile-acetone (2:1 v/v), and an appropriate amount of ZR-2791 internal standard. Stopper and shake vigorously 2 minutes. Centrifuge 10 minutes at high speed, until soil has settled and supernatant is clear. Decant the uppet clear liquid, and add to a 0.5-liter separatory funnel. Add 200 ml distilled water (0.03 N in HCl with 1.5 gm NaCl), and extract with 100 ml pentane for 1-2 minutes. Collect the pentane extract, and evaporate into an appropriate volume of isooctane for GLC.5. FLUVALINATE 97 Most soil runoff water from agricultural soils has required no additional column cleanup for a I-ppb limit of detection. To obtain less than a 1-ppb limit, Florisil or GPC cleanup is usually required. If column cleanup is required, use the entire pentane extract for chromatography rather than selecting an aliquot. vi. Soils (Al Types of Sand, Silt, Clay, and Organic Soils) Select a composite of soil for analysis, crush and mix thoroughly, and spread thinly over aluminum foil. If the water content of the soil is below the saturation level, pipette water over the surface of the soil until the soil saturation level is just reached. Add 50 gm of the soil to a Whatman 35 x 94 mm cellulose thimble in a standard taper 45/50 Soxhlet-extraction chamber. Pipette 10 ml 0.1 N HCL over the soil surface, and add an appropriate amount of ZR-2791. Top the thimble with glass wool. To a 250-ml round-bottomed receiver flask, add 10 ml of 0.1 NW HCI and 175 ml hexane-acetone (1:1 v/v). Stir and Soxhlet- extract vigorously for 5-6 hours, Partition as described for the general extraction method. Recover the upper hexane layer, and go directly to column cleanup by Florisil or GPC. Some soils may provide chromatograms with significant interferences at the desired limit of detection. The hexane extracts will then require at least one defatting step prior to the column cleanup step. h. Gas Chromatographic Analysis Fluvalinate levels in sample extracts are determined using capillary GLC-ECD. The capillary chromatography is conducted in the split- injection mode at the relatively low split ratio of 5:1. This low split ratio is possible only with the use of a packed injector insert. The use of a packed insert also protects the front end of the column from buildup of nonvolatile contaminants. Alternate injection techniques such as splitless or on-column injection are not practical for isothermal capillary GLC-ECD. i, Conditions Split flow rate: 5 ml/minute helium. Column flow rate: | ml/minute (measured at detector outlet), Septum purge rate: 0.5 ml/minute. Makeup flow rate: 30 ml/minute nitrogen. Oven temperature: 210°C. Detector iemperaiure : 270°C. Injector temperature: 240°C. Injector insert: Use a packed 2 mm id. glass injection port insert for split injection.98 W, L, FITCH ET AL, ii, Procedure (a) Concentrate or dilute samples so that 10-50 pg/yl of fluvalinate are: present. Thus, an injection of a few microliters of the sample extract will produce an adequate response. In this case the ECD will be working near maximum sensitivity. These conditions of analysis prolong the life of the precolumn, capillary column, and detector. Variations in detector response are also kept to a minimum. (b) Inject 1-2 yl of extract into the chromatograph. Record the peak area for the internal standard and for fluvalinate. Fig. 3 is a representative ] A 6 Fic. 3. **Ni electron capture gas chromatogram fram the analysis of fluvalinate, Peak 1, ZR-2791; peaks 2 and 3, fluvalinate, (A) Standard: (B} Thompson seedless taisin extract (0.022 ppm fluvalinate).