Analyst, May, 1968, Vol. 93, f+. 323-325 323 Efficiency of Extraction of Metabolically Incorporated HEOD (Carbon-14) from Pheasant Tissues, Eggs and Faeces BY YVONNE GREICHUS, DONALD LAMB AND CLIFFORD GARRETT (Experiment Station Biochemistry De#artment, South Dakota State University, Brookings, South Dakota 67006, U.S.A.) Acetonitrile partitioning and Florisil column procedures were examined for efficiency of extraction and purification of HEOD (carbon-14) residues. Fat, liver, eggs and faeces from pheasant hens fed with 1.5mg of HEOD (carbon-14) were used. Gas - liquid chromatographic analysis of HEOD- fortified controls gave results similar to those obtained by liquid scintillation counting when samples were extracted and purified with a Florisil column. Analysis by liquid scintillation counting showed that recoveries of HEOD (carbon- 14) from fortified controls compared favourably with recoveries of HEOD (carbon-14) that had been metabolically incorporated into the tissue for both the partitioning and Florisil column procedures.It was concluded that gas - liquid chromatographic analysis of a fortified control sample gave an accurate measurement of the recovery of HEOD from tissues in which the HEOD was metabolically incorporated within the range of concentrations studied. SAMPLE fortification, or "spiking," is commonly used to determine the efficiency of extraction and isolation of pesticide residues from biological tissues. By using this method, McCully and McKinley1 recovered 73 to 112 per cent. of thirteen pesticides added to mutton and beef fat.Similarly, Parker, Dewey, DeVries and Lau2 obtained 29.4 to 103 per cent. recovery of twelve chlorinated organic insecticides added to human fat. With this method it is assumed that pesticides that have been metabolically incorporated into tissue are extracted with the same efficiency as pesticides added to the tissue just before analysis. The validity of this assumption has been investigated by using liquid scintillation counting and gas - liquid chromatography to analyse various samples from pheasants fed with carbon-14 labelled HEOD (1 ,2,3,4,10,10-hexach1oro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octa- hydro-exo-lJ4-e~do-5 ,8-dimethanonaphthalene). METHODS REAGENTS- Hexane. Light petroleum, boiling range 30" to 60" C . A cetonitrile. Dichloromethane.These reagents used in this work were of Nanograde quality, obtainable from Mallinck- rodt Chemical Works, St. Louis. The maximum interfering gas - liquid chromatographic peaks are no greater than that produced by 10 ng per litre of heptachlor epoxide or 100 ng per litre of parathion: solvents of equal purity can be used. Sodium sulfihate, anhydrous , granular. Florid, 60 to 100 mesh, activated at 650" C-Prepare for use by heating for 12 to 14 hours at 140" C, adding 3 per cent. of distilled water, and storing in an air-tight container. Scintillation jZuid-Dissolve 100 mg of lJ4-bis-2- (5-phenyloxazolyl) benzene , scintillation grade, and 3 g of 2,5-diphenyloxazole. HEOD (carbon-14) , specijc activity 4 millicuries per millimole-This was checked for purity by gas - liquid and thin-layer chromatography and found to be radiochemically more than 99 per cent. pure HEOD.PROCEDURE- Fatty tissue, liver, eggs and faeces of pheasant hens that were fed with 1.6 mg of HEOD (carbon-14) by capsule and sacrificed within 1 week were used in this study. Similar samples 0 SAC and the authors.324 GREICHUS et aZ. : EFFICIENCY OF EXTRACTION OF [Arcalyst, Vol. 93 were also taken from pheasant hens that had received the capsule alone without any HEOD. The extraction and purification by acetonitrile partitioning and by Florisil column treatment were examined. Acetonitrile $artitioning-Samples for acetonitrile partitioning consisted of 2 g of fatty tissue from the pheasants fed with HEOD (carbon-14) and 2-g samples of fatty tissue from the pheasants not fed with HEOD.Similar samples of the latter tissue were used for both the blank and fortification with 0.002 mg (0-0066 microcurie) of HEOD (carbon-14) for testing the extraction procedures. Each sample was placed in 40 ml of hexane and homo- genised in a VirTis “45” homogeniser. One half of this homogenised sample was brought to dryness by using a Rinco rotating vacuum evaporator and reconstituted in 16 ml of scin- tillation fluid. To determine the amount of HEOD (carbon-14) before partitioning, this half was counted in a Packard Tri Carb, Series 3000, Liquid Scintillation Spectrometer. Scintillation samples were adjusted for quenching by comparison of counts, with and without an internal standard. The other half of each sample was partitioned with acetonitrile, as described elsewhere.a Each sample was placed in a 250-ml separating funnel and the HEOD partitioned into four 25-ml portions of hexane-saturated acetonitrile.The acetonitrile phase was combined with 500 ml of distilled water and the HEOD extracted with two 100-ml portions of light petroleum. The combined light petroleum extracts were washed twice with 100ml of distilled water, and the excess of water was removed by pouring the light petroleum through anhydrous sodium sulphate. After evaporating the light petroleum extracts to a small volume, it was analysed by liquid scintillation counting to determine the amount of HEOD (carbon-14) remaining after partitioning. FZorisiZ coZumn treatment-A Florisil column was used for the extraction and purification of fatty tissue, liver, eggs and faeces.The samples were taken from the same sources as those used in the acetonitrile partitioning. The entire content of each egg was homogenised before sampling. The faeces were dried in a forced-draught oven at 60” C to a constant weight and then finally ground with a mortar and pestle before sampling. Two-gram samples of fat, liver, egg and faeces from HEOD-fed pheasants were thoroughly mixed with 10 g of Florisil. These mixtures were then divided into two equal parts by weight. One part was used to determine the carbon-14 activity before it entered the Florisil column. To reduce quenching, this half was placed on 20 g of anhydrous sodium sulphate in a small column and eluted with 125 ml of a mixture of dichloromethane and light petroleum (1 + 1 v/v).The sample was brought to dryness, dissolved in 16 ml of scintillation fluid and counted. An internal standard was then used to determine the amount of any remaining quenching. Internal standards are not reliable if the quenching is too great, and this pro- cedure effectively reduced quenching. Losses of carbon-14 activity on the sodium sulphate column were determined by scintillation counting of the column material and found to range from 1 to 5 per cent. The carbon-14 activity values before entering the Florisil column were adjusted to take these losses into consideration. The second half of each mixture was extracted and purified by using the Florid-column method of Stemp, Liska, Langlois and Stadelman.4 The mixture was placed on top of 40 g of Florisil in a 20 x 400-mm column, and the HEOD eluted with 750 ml of 20 per cent.v/v dichloromethane in light petroleum. The eluates were then reduced to a small volume by using the rotating vacuum evaporator and made up to 10 ml. One 5-ml portion was counted and the other 5-ml portion analysed by gas - liquid chromatography. A 2-g sample of fatty tissue, liver, egg and faeces was also taken from pheasants that had received no HEOD. This was fortified with an amount of HEOD (carbon-14) similar to that metabolically incorporated in the treated pheasants. The fortified samples were treated by the same procedures for the determination of carbon-14 activity, before and after the Florisil column, and for gas - liquid chromatographic analysis as the metabolically incorporated HEOD (carbon-14) samples. The instrument used for gas - liquid chromatography was a Wilkens Aerograph HY-FI, model 600, equipped with an electron-capture detector cell that has a 250-millicurie tritium source.The &inch 0.d. x 5-fOOt Pyrex column was packed with 5 per cent. Dow-11 silicone on 60 to 80-mesh, HMDS-treated Chromosorb W and operated at 185” C, with a nitrogen carrier gas flow-rate of about 44 ml per minute.May, 19681 METABOLICALLY INCORPORATED HEOD (CARBON-14) 325 RESULTS The percentage recovery by acetonitrile partitioning was 89.9 and 79.4 for the meta- bolically incorporated HEOD (carbon-14), and 89.9 and 81.3 for the samples with added HEOD (carbon-14). Samples from pheasants that had received no HEOD (carbon-14) had counts similar to the background count.A comparison of the recoveries of metabolically incorporated and of added HEOD (cabon-14) from samples of fatty tissue, liver, eggs and faeces is shown in Table I for the Florisil column procedure. The samples contained from 0-4 to 40 p.p.m. of HEOD. Un- fortified samples gave the same count as background. The samples were checked for meta- bolites by gas - liquid chromatographic analysis with the Dow-11 column and on a similar column packed with 2 per cent. QF-1 silicone (fluoro) on 60 to 80-mesh, HMDS-treated Chromosorb W. Samples analysed by thin-layer chromatography revealed no radioactivity on the plate, except for the HEOD spot. The presence of undetected carbon-14 metabolites would lower the amount of carbon-14 recovered after extraction and purification of the HEOD.This did not appear to be a serious problem in this study because recovery of carbon-14 from fortified samples was similar to that of the metabolically incorporated HEOD (carbon-14) samples. TABLE I PERCENTAGE RECOVERY OF HEOD (CARBON-14) FROM FLORISIL COLUMN HEOD-fortified tissues Metabolically incorporated HEOD : r A \ scintillation counting Gas - liquid f A \ Scintillation chromatographic Sample Bird 1 Bird 2 Bird 3 Average counting analysis Fatty tissue . . 99 103 94 99 98 94 Liver .. .. 93 92 87 91 92 95 Eggs .. .. 90 91 88 90 91 94 Faeces . . . . 95 80 84 86 84 96 The results show that the recoveries of HEOD from the Florisil column are similar for the metabolically incorporated and the added insecticide and that the results by gas - liquid chromatographic analysis are similar to those obtained by scintillation counting. It is concluded that metabolically incorporated HEOD is efficiently extracted from several pheasant tissues by the techniques used. This paper is published with the approval of the Director of the South Dakota Agricul- tural Experiment Station as publication No. 790 of the Journal series. REFERENCES 1. 2. 3. 4. McCully, K. A., and McKinley, W. P., J . Ass. 08. Agric. Chew., 1964, 47, 652. Parker, K. D., Dewey, M. L., DeVries, D. M., and Lau, S. C., Toxic. Appl. Pharmac., 1965, 7 , 719. U.S. Public Health Service, 1965, “Pesticide Residue Analysis of Foods,” Training Course Manual, Stemp, A. R., Liska, B. J., Langlois, B. E., and Stadelman, W. J., Poult. Sci., 1964, 43, 273. Robert A. Taft Sanitary Engineering Centre, Cincinnati, Ohio L-2-1. Received September 4th, 1967