nium selenite columns (Table I). Titanium selenite is more selective for the separation of cadmium from numerous metals than other exchangers based on titanium (Figure 2). This is the obvious advantage of the synthesis. pH-titration curves for Li, Na, and K of sample 1 show that titanium selenite is also selective for alkali metals. The order of adsorption is K > Na > Li, Li > Na > K and Li = Na > K at pH values 3, 8, and 12, respectively. On stannic arsenate this preference is reversed. Ion-exchange capacity data at different temperatures show that it is a good exchanger and it can be used up to 500 “C successfully. IR spectra are also in support of the above conclusion. In order to explain the effectiveness of titanium selenite for the separation of cadmium from numerous metal ions, a thorough search of the literature (22) was made. It was found that the selenite of Mg, Ca, Ni, Co, Mn, Zn, and AI have been prepared by mixing the carbonate of the respective metal ions (22) J. W. Mellor, “A Comprehensive Treatise on Inorganic and Theoretica 1 Chemistry,” Vol. X, Longmans Green & Co., London, 1961, p 829.
with selenious acid. Except cadmium selenite, all are soluble in water as well as in dilute mineral acids. Therefore, cadmium should be adsorbed more strongly than the remaining cations under study. Hence elution of Mg, Ca, Ni, Co, Mn, Zn, and AI has been made with 2 X 10-4M H N 0 3 while elution of cadmium has been obtained with aqueous 0.40% NH4Cl solution at a low pH value and at a higher ionic strength. The elution of cadmium is sharper than that of the other metal ions. This may be due to the formation of a complex anion (23) (CdCI4*-)with ammonium chloride. ACKNOWLEDGMENT We thank S.M.F. Rahman, B. Rama Rao (R.R. Lab., Hyderabad), M.V. George (I.I.T., Kanpur) for research, X-ray and IR facilities. RECEIVED for review August 4, 1971. Accepted November 9, 1971. One of us (R.K.) thanks the U.G.C. (India) for financial assistance. (23) Reference No. 15, p 606.
Simple Method for Routine Detection of Residues of Diethylstilbestrol (DES) in Meat Contaminated at Levels as Low as One Part per Billion Walter G. Smith1 a n d Edward E. McNeil Animal Pathology Dicision, Health of Animals Branch, Canadian Department o j Agriculture, Animal Diseases Research Institute, Hull, Quebec, Canada DIETHYLSTILBESTROL (DES) may be administered to livestock animals as a growth promoting agent by surgical implantation in pellet form or as an additive in their rations. The detection of residues of DES in animals so treated is difficult. A utilizable, chemical assay must satisfy a number of criteria. It must be suitable for regular and routine use by relatively unskilled personnel. It must permit handling large numbers of specimens in a short space of time for use as a surveillance tool. It must be selective for DES and avoid the simultaneous estimation of natural estrogens. It must unequivocally detect a quantity of DES equivalent to a contamination level in the specimen as low as 1 part per billion. It must permit simultaneous estimation of “free DES” and either “combined DES” (sulfate or glucuronide conjugates) or “total DES”Le., “free DES” together with “combined DES.” Published methods (1-9) satisfy some of these criteria but 1 Present address, Pharmacology Division, Research Laboratories, Food and Drug Directorate, Tunney’s Pasture, Ottawa
(1) “British Pharmacopoeia,” 1968, p 938. (2) “Pharmacopoeia of the United States,” XVI, p 217. (3) “Official Methods of Analysis of the Association of Official Agricultural Chemists,” 10th ed., 1965, Section 32.196 and Section 33.039. (4) R. L. Dryer, Clin. Clzem., 2, 25 (1956). (5) J. M. Goodyear and N. R. Jenkinson, ANAL.CHEM.,33, 853 (1961). 1084
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
none satisfies all. The present communication outlines a new method which in our hands has proved to be rapid, easy to execute, and selective for DES. The present text is restricted to a full description of the method and sufficient discussion to permit its routine use by others with minimum difficulty. The method consists of extraction utilizing acetonitrile :water (9 :l); removal of contaminating lipid from the extracted DES with the aid of immiscible solvent systems; and detection of the extracted DES by electron capture gas chromatography. It seems to us that the detection of nanogram quantities of DES utilizing gas chromatography with an electron capture detector is dependent upon the formation of a derivative of DES on the column (10). This aspect is under further investigation. EXPERIMENTAL Reagents. Ethanol was redistilled in an all-glass apparatus from CaC12 and NaOH. All other reagents used were of (6) E. J. Umberger, D. Banes, F. M. Kunze, and S . H. Colson, J. Ass. Ofjic. Agr. Cliem., 46, 471 (1963). (7) I. E. Smiley and E. D. Schall, J . Ass. Ofic. Ann/. Ckem., 52, 107 (1969). (8) D. L. Simmons, R. J. Ranz, and R. C. Cornell, Cm. J. Pliarm. Sci., 6 , 28 (1971). (9) B. S. Rutherford, J . Ass. Ofic.Ami. Cliem., 53, 1243 (1970). (10) P. Saschenbreker, Animal Pathology Laboratory. Canada Department of Agriculture, Guelph. Ontario, personal communication, January 1971.
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Figure 1. Infrared spectra of standard and extracted diethylstilbestrol in 200 mg of spectral grade potassium bromide A . Blank extract from 20-gram meat sample B. 1 mg of standard diethylstilbestrol C. 1 mg of diethylstilbestrol after extraction from 20 grams of meat
Reagent grade, except benzene (pesticide grade), 2,s-diphenoxyoxazole (PPO), and toluene (both scintillator grade). PPO scintillator reagent consisted of 6 grams of PPO dissolved in 900 ml of toluene. Diethylstilbestrol-l *C was supplied by Amersham Searle, Des Plaines, Ill., with a specific activity of 54 mCi/mmole and diluted with benzene to produce a solution containing 1 pg/ml (440,000 dpm/ml). Glassware. The 500-ml Erlenmeyer flasks and 500-ml separatory funnels were fitted with ?$ 24/40 joints; so also were the cold finger condensers (Supplied by Emerald Glass Co. Ltd., 2 Thorncliffe Park Drive, Unit No. 37, Toronto 17). Insertion of a side arm tube with ?$ 24/40 joints allowed the separatory funnels to be inserted into the Erlenmeyer flasks and used for phase separations without additional support. This was a n essential convenience when processing large numbers of samples. The 2-1n1 microvials in which the
final extracts were taken to dryness were supplied by Regis Chemical Co., Chicago, Ill. Apparatus. The method requires use of a Sorvall Omnimixer or equivalent homogenizer, a vacuum rotary evaporator, a liquid scintillation counter, and an electric hotplate of large surface area to accommodate groups of 500-ml Erlenmeyer flasks. Any commercial gas chromatograph which will accommodate 6-ft columns and which is fitted with a 63Ni electron capture detector would appear to be suitable for the detection of DES. The column configuration, e.g., coiled or U-tube, is unimportant. Glass columns are preferable, but stainless steel columns may be used after a 76-hour conditioning period. Extraction Procedure. Twenty grams of meat were coarsely chopped and transferred to a 400-ml Sorvall Omni-Mixer container. A 100-ml quantity of acetonitiile :water (9 :1) ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
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Micro Tek 220 gas chromatograph. 1.5 OV1 on Anakrom S.D. in. 0.d. glass column. High purity nitrogen 70/80 mesh in 6 ft at 130 ml/min. Column temp = 210 “C. Injection port temp = 240 “C 63Nielectron capture detector at 28 V. Detector temp = 270 T . Purge gas flow = high purity nitrogen at 10 ml/min. in./min. Solvent = Sensitivity = 16 X 102. Chart speed = redistilled ethanol 2 pl. Retention time of DES = 1.5 min under these conditions
was added and the tissue homogenized for approximatel) 90 sec. The resulting slurry was transferred to a 500-ml Erlenmeyer flask. The analysis of a single meat specimen requires the production of 4 such homogenized @quots which were labeled: “cold aliquot,” “cold control,” “heated aliquot,” “heated control.” To both “cold control” and “heated control,” 0.1 ml of a benzene solution of diethylstilbestrol-14C (-44,000 dpm) was added. These controls were used to calculate the percentage of added DES recovered from the specimen. The “cold aliquot” and “cold control” flasks were allowed to stand a t room temperature for 30 min. The “heated control” and “heated aliquot” flasks were transferred t o a heavy duty, even temperature hot plate. One milliliter of 5N hydrochloric acid was added. Into the neck of each was inserted a cold finger condenser 20 cm in length connected to a suitable cooling water supply. These flasks were refluxed gently (82 “C) for 30 minutes to permit estimation of “total DES.” The resulting extracts (2 hot and 2 cold) were then filtered through Whatman No. 1 filter paper (nonacid treated) using a Buchner funnel. Each filtrate was then transferred to a 500-ml separatory funnel using a n additional 10 ml of acetonitri1e:water ( 9 : l ) to wash the Ruchner flask. A 50-ml 1086
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
portion of n-hexane was added to each separatory funnel, and, after shaking, removed as a separated upper phase. This hexane wash, which removes large amounts of material co-extracted with DES, was repeated. A 30-ml portion of benzene was then added to the acetonitri1e:water. The resultant mixture is immiscible with 10% aqueous sodium carbonate, which was then used twice in 30-ml portions to remove acidic co-extractives, especially fatty acids in the “heated aliquot” and “heated control.” The organic phase separated from the sodium carbonate solution was then washed twice with 30-ml portions of distilled water. A 30-ml portion of 1% aqueous sodium hydroxide was added to each separatory funnel and, after separating, drained into a clean 100-ml conical flask. The organic phase was drained from the separatory funnel and rejected. The sodium hydroxide solution was transferred back into the separatory funnel and rinsed from the flask with 20 ml of distilled water. A 2-ml portion of concentrated hydrochloric acid was added followed by 20 ml of benzene. After shaking and separation, the aqueous layer was rejected. The benzene solution of DES was drained into a liquid scintillation vial and taken to dryness in cacuo using a vacuum rotary evaporator. To the “cold control” and “heated control,” 20 ml of PPO scintillator reagent were added. A third vial containing 0.1 ml of the benzene solution of diethylstilbestrol-lF and 20 ml of PPO scintillator reagent was prepared. These three vials were then counted in a Nuclear Chicago Liquid Scintillation Counter and the resultant counts, after corrections for quenching and counting efficiency, used to calculate the percentage recovery of both “free“ and “total” DES. In examining over 100 specimens of meat, the recorded recovery has been within the range 73.2 to 8 3 . 4 x . To the “cold aliquot” and “heated aliquot,” 1 ml of absolute ethanol was added and the resultant solution transferred to a 2-ml microvial. This solution was taken to dryness with the aid of a stream of high purity nitrogen delivered to the surface of the solution through a glass Pasteur pipet. The dry residue was dissolved in 10 pl of absolute ethanol for identification by gas-liquid chromatography. Gas-Liquid Chromatography. The column packing (1.5 OV1 on Anakrom S.D. 70/80 mesh) was prepared with the aid of a fluidiser (Applied Science Labs, P.O. Box 440, State College, Pa.) using a solution of OV1 in toluene. Chromosorb W 60/80 mesh appears to be a suitable substitute for Anakrom S.D. 70j80 mesh. Columns were prepared from 6-ft lengths of 1/6-in. 0.d. stainless steel or 6-ft U-tubes of I/&. 0.d. glass. Coiled stainless steel columns were used in a Victoreen Series 4000 glass chromatograph and U-tubes of glass in a Micro Tek 220 gas chromatograph. Stainless steel columns were conditioned in the Victoreen gas chromatograph after filling with high purity nitrogen according to the following schedule [which is derived from Vandenheuvel and Court ( I I ) ] : (i) 24 hours at 250 “C without gas flow, then (ii) 24 hours a t 300 “C without gas flow, then (iii) 1 hour at 250 “C without gas flow, then (iv) 24 hours at 300 “C with nitrogen at 30 ml/min, then (v) 1 hour at 300 “C with nitrogen a t 60 ml/min, then (vi) 3 hours a t room temperature with nitrogen at 60 mlhnin. Glass columns were conditioned in the Micro Tek gas chromatograph for 48 hours a t 250 “C with nitrogen at 60 ml/min. With either instrument, the identification of DES proved to be sensitive to temperature and gas flow. It is recommended that optimum operating conditions be obtained with a new column by operating at 210 “C isothermal and in(11) F. A. Vandenheuvel and A. S. Court, J . Clirornutogr., 38, 439 (1968).
jecting 250 ng of DES a t gas flow rates of 100 ml/min, increased in steps of 10 ml/min until the gas flow for maximum sensitivity is established. The carrier gas for either instrument was high purity nitrogen. The optimum temperature for maximum sensitivity can be obtained by using repeated injections of the same column load a t temperatures above and below 210 “C after establishing optimum gas Row. When using a Victoreen gas chromatograph, the electron capture detector was operated in the pulse mode and without using a purge gas flow. The detector of the Micro Tek 220 gas chromatograph was operated a t 28 V in the dc mode with a purge gas flow of 10 mllmin of high purity nitrogen. RESULTS AND DISCUSSION
Figure 1 shows the infrared spectra obtained with standard and extracted DES in 200-mg quantities of spectral grade potassium bromide using a Perkin-Elmer Infra Red Spectrophotometer Model 700. These spectra indicate that the extraction procedure recovers DES added to meat a t the mg level in a high state of purity. N o co-extracted material is present in recognizable quantities. Figure 2 shows the gas chromatographic peaks obtained with the Micro Tek 220 gas chromatograph. The 200-ng peak was obtained with a standard solution, the 20-ng peak was obtained with a meat sample to which 20 ng of DES has been added. Similar peaks were obtained with the Victoreen 4000 gas chromatograph. With either instrument, the residue obtained by extraction of 20 grams of meat free of DES produced no peaks at or near the position occupied by DES. Meat contaminated with DES to the extent of 20 nanograms in 20 grams has a contamination level of 1 part per billion. A final extract from such a sample would contain the recovered portion of 20 ng dissolved in 10 pl of ethanol when prepared as described above. If injected in its entirety into a gas chromatograph, such a n extract would produce a signal of the size shown in Figure 26. Since the level of contamination of any given meat sample is not known a t this stage, it is convenient to
use a calibration curve using 20, 40: 80, 160, and 320 ng of standard DES, Heavily contaminated samples, once recognized, may require re-extraction and accurate injection into the gas chromatograph of aliquots of the final ethanol solution smaller than the 10 p1 used on the first run. The method was evohed in a series of steps using diethylstilbestrol-14C to eliminate procedures which adversely affected recovery. Whatman No. 1 (non-acid washed) filter paper is satisfactory. Other grades have not been investigated. The hydrolysis procedure must be performed with cold finger condensers. Experiments in uhich other types were used exhibited low recoveries of added DES (in the 2 0 z range). The extract in 1 ml of absolute ethanol was taken to dryness with the aid of nitrogen to avoid the foaming experienced when some samples were taken to dryness with the rotary vacuum evaporator. The greatest day-to-day difficulty experienced with any extraction procedure which is based upon the separation of immiscible solvents is that of emulsion formation. The procedure described, unlike alternatives in the literature ( 5 , 6 ) and many other systems investigated in this laboratory, does not readily form emulsions a t any stage. We have observed, however, that meat specimens which have been poorly preserved, and those which contain more than 50z fat show greater tendencies to emulsion formation than fresh lean meat. This tendency is more pronounced with the hydrolyzed aliquots, but has not, so far, prevented phase separation unaided by centrifugation. However, any tendency to emulsion formation produces imprecise definition of the boundary between phases and introduces the possibility of poor phase separation. It is in recognition of this ever-present potential source of error that the method specifies that two estimates of “percentage recovery” should be performed in parallel with the extraction of DES residues from each meat specimen under test. RECEIVED for review July 30, 1971. Accepted December 7 , 1971.
Application of Radioactivation to the Sequential Separation of Antimony, Cadmium, Chromium, Cobalt, Iron, Tin, and Zinc from Aluminum and Lead by Ion-ExchangeChromatography Tharwat Z. Bishay Scottish Research Reactor Centre, East Kilbride, Glasgow, Scotland, G.B. VARIOUSMETHODS dealing with the analysis of aluminum and lead have been previously described (1-6). None ofthese has presented a simultaneous determination of seven elements ( I ) F. Girardi and J. Pauly. Nuclear Energy Conference, Milan. 1961 (Conf-140-1 : EUR 306.i). (2) 0. Grossmann and H. G. Dodge. Keriwiiergie. 7, 113 (1964). (3) S. G. Prussin. J. A. Harris. and J. M. Hollander, ANAL.CHEW, 37, 1 127 ( I 965). (4) F. Girardi. G. Guzzi. and J. Pauly. ihid..p 1085. ( 5 ) M. P. Menon. A. P. Rainosek. and R. E. Wainerdi. N~rcl.Appl., 2. 335 (1966). (6) 1’. A. Benson. M’. D. Holland. and R. H. Smith, ;Mod. T r c / d s A r t i ! , .A/i(il,.P K ~ 1111. . C o i i j : . 1961. p 7.
on the same ion-exchange column. Albert et NI. ( 7 ) and Gervis (8) were able to isolate a large number of elemental impurities from aluminum by destructive analysis. However. their techniques are somewhat laborious and time consuming. Analysis of lead through separation schemes is rather scanty. Nondestructive measurements have also been carried out (7) P. Albert and J. Gaittet. International Atomic Energy Agency Conference, Copenhagen. 1960, “Use of Radioisotopes in Physi-
cal Sciences & Industry.” paper RICC/82. (8) R. E. Gervis and W. D. Mackintosh. Proc. 2nd Perrcefirl Uses Ar. Eiiergy. paper 189 (1958). ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
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