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Thin-layer chromatographic method for the detection of anabolics in fatty tissues
213
Journal of Chromatography, 489 (1989) 213-218 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
CHROMBIO. 4598
THIN-LAYER CHROMATOGRAPHIC OF ANABOLICS IN FATTY TISSUES
L. VAN LOOK, Ph. DESCHUYTBRE
METHOD FOR THE DETECTION
and C. VAN PETBGHEM*
Lxdmratory of Food Analysis, Faculty of Pharmaceutical Sciences, State University of Ghent, Harelbekestraat 72,900O Ghent (Belgium)
SUMMARY A modification of the method of Verbeke [J. Chromatogr., 177 (1979) 691 is presented. The fatty tissue is dissolved in hexane, partitioned against methanol-sodium acetate buffer (pH 5.2) and extracted with dichloromethane. The crude extract is then purified on a disposable Cl8 column. The final extract together with a set of reference compounds is spotted on two opposite sides of a highperformance thin-layer chromatographic plate (10X 10 cm), which is developed with two different eluents in two opposite directions. This mode of operation has the advantage that more reference compounds can be spotted and that the identification is based on two independent chromatographic runs. Also the total analysis time is decreased.
INTRODUCTION
In the control of the illegal use of anabolics in fatting stock there are two analytical approaches. The first directs all efforts to the detection of one specific compound, which usually is selected by its presumed or sometimes proved intensive use. Immunochemical methods (radioimmunoassay, chemjluminescence immunoassay, enzyme-linked immunosorbent assay, etc. ) make use of antibodies which are specifically directed against the analyte or against a group of structurally very related substances. These methods are characterized, from the analytical viewpoint, by high sensitivity, limited selectivity and relative ease of operation. The second approach consists in multi-residue methodology. These methods try to detect as many compounds as possible in one analytical run. It is obvious that chromatographic procedures are most appropriate for this purpose. If sufficient sensitivity can be attained, thin-layer chromatography (TLC) offers the considerable advantage of visible colour differentiation. In order to increase the resolving power of the chromatographic system often two-dimensional development with different eluting solvents is carried out [ 1,2]. Various purifi0378-4347/89/$03&O
0 1989 Elsevier Science Publishers B.V.
214
cation methods prior to the final chromatographic step have been described [l-
71.
One of the most extensively used methods is that of Verbeke [ 21. It is intended for the detection of various anabolic residues in tissues-or urine at levels as low as 0.5-10 ppb. Starting from 50-g samples, residues are recovered with an efficiency ranging between 60 and 80%. The detection limit of most anabolics is of the order of l-10 ng. As a particularly interesting feature, a group separation into an estrogen and a non-estrogen fraction is performed. Mainly because of this fractionation, which at the same time is a clean-up step, a serious drawback of the method is that is very laborious; one analyst is supposed to complete ten analyses per week. In this paper a method is presented which is based on that of Verbeke [ 21 but which contains a reversal in the extraction procedure, permitting a better release of the fat matrix and an alternative clean-up procedure, yielding in a shorter time a final extract that permits the detection of l-10 ppb of anabolic in fatty tissue. EXPERIMENTAL
Instrumentation The equipment consisted of a laboratory blender (Stomacher Type 80, Colworth, London, U.K.) with disposable polythene bags, a water-bath at 4O”C, centrifuge tubes (70 ml), a mechanical shaker, a rotary vacuum evaporator, Pasteur pipettes, chromatographic tanks (Camag, Muttenz, Switzerland) and a sample applicator (Merck, Darmstadt, F.R.G., Art. No, 10226) with microcapillaries (Merck, Art. No. 10289). Disposable 3-ml Bond Elut C,, columns (Analytichem International, Harbor City, CA, U.S.A.) were used with a Baker-10 extraction system (J.T. Baker, Phillipsburg, NJ, U.S.A). Reagents and reference solutions Silica gel 60 TLC plates (10 x 10 cm) from Merck (Art. No. 5631) were used. All reagents were of analytical-reagent grade and used as received. Lipidex5000 was obtained from Packard Instrument (Brussels, Belgium). Reference solutions for TLC were prepared in methanol from commercially available bulk products. The concentration of each anabolic was 0.1 mg/ml. Extraction Bovine fatty tissue (50 g ) spiked in the l-10 ppb concentration range with 50 ~1 of a methanolic solution is minced with a scalpel and suspended in 60 ml of hexane. The mixture is homogenized for 2 min in a blender and then heated in a water-bath a 40°C until a yellowish liquid is obtained. This liquid is centrifuged at 470 g for 1 min. A clear solution of the lipids in hexane is obtained. The hexane phase is homogenized with 180 ml of methanol by mechanical shaking for 10 min, transferred to a separating funnel and mixed with 80 ml of 0.04 M sodium acetate buffer (pH 5.2). On gently swirling phase separation occurs and the hexane layer is discarded. The turbid aqueous layer is then partitioned three times against 50 ml of hexane by mechanical shaking for 10 min.
215
Next, the aqueous layer, which has become clear, is extracted with 150 ml of dichloromethane, followed by another two extractions with 90 ml of dichloromethane. The combined organic layers are evaporated to dryness at 40°C in a rotary vacuum evaporator. The crude extract is transferred into a conical tube by means of two 3-ml portions of methanol and.evaporated to dryness in a stream of nitrogen. Clean-up The Bond Elut Cl8 (3 ml) columns are pretreated by rinsing with two 6-ml portions of methanol, followed by two 6-ml portions of doubly distilled water. The extract is taken up in 0.5 ml of methanol and 4 ml of doubly distilled water and transferred on to the Cl8 column. Washing is carried out with 3 ml of doubly distilled water, 3 ml of methanol-water (55 : 45 ) and 1.5ml of hexane. Elution is performed with 3 ml of methanol. The eluate is evaporated to dryness under a stream of nitrogen and finally taken up in 50 ,ul of methanol. Thin-layer chromatography Two chromatographic runs are performed in two opposite directions on one TLC plate. On a starting line 1 cm from one edge, aliquots (1.5 and 3.75 ~1, corresponding to 1.5 and 3.75 g of fatty tissue, respectively) are spotted with a microcapillary. On the opposite side of the plate, again 1 cm from the edge, the spotting is repeated. The reference standards are spotted in groups of six in five different lanes also on both starting-lines. The plate is eluted in one direction as far as the middle of the plate with chloroform-acetone (9O:lO). After drying, the opposite half of the plate is eluted with cyclohexane-ethyl acetate-ethanol (77.5:20:2.5 ). The chromatogram is then sprayed with 10% sulphuric acid in methanol and heated for 10 min at 90°C. The spots are examined under UV light at 366 nm. Identification is achieved by comparing the RF values and the colours of the spots in the extract with those of the reference mixtures on both chromatograms. A residue is not present when it is not unambiguously identified in both runs. RESULTS AND DISCUSSION
The main advantage of the alternative procedure described here is a more complete extraction of the fatty tissue. Mechanical homogenization using an Ultraturrax or a similar device always leaves a considerable amount of solid material. In the proposed modification, the fat is dissolved with slight heating in hexane. After centrifugation a clear yellow organic phase is obtained with mostly only a minute amount of precipitate. Concerning the efficiency of both extraction procedures, very little information can be deduced from spiked fat samples, as the conditions of the injected analyte are in no way comparable to those of endogenous residues of the analyte. Anyway, from a purely theoretical point of view a higher recovery can be expected from an almost completely dissolved tissue than from a minced but actually undissolved pulp. In most instances the bulk amount of lipids is removed by the solid-phase ex-
216
traction on C,, columns. The extract is sufficiently pure to allow direct identification by comparison of the qualitative data (RF and spot colour) of both chromatograms. The recovery from the C,, columns is reasonable to very good for most of the compounds; by visual comparison of the intensity of the spots with that of standards of known concentration, it may be estimated to be at least 80%. This figure is only of limited importance, however, as spiked samples can never replace real samples. The addition of a certain amount of a substance in the form of a methanolic solution does not imitate the conditions under which the same compound is present as a residue in the fatty tissue. It is worth noting that the recovery is poor for several compounds (mestranol, nortestosterone decanoate, testosterone cypionate, estradiol cypionate, testosterone isocaproate, testosterone enanthate, estradiol phenylpropionate, testosterone phenylpropionate and estradiol valerate). This is of limited diagnostic value, however, as the anabolic esters in general are deposited as the free form in the fatty tissue. The double chromatogram has the advantage over the normal two-dimensional chromatogram that considerably more reference compounds can be applied on one plate. Indeed, several lanes are available and the composition of the standard mixtures can be freely chosen as a function of the resolution between certain constituents. The double chromatogram also yields a much quicker and simpler overall picture of the composition of the final extract. The results on both parts of the double chromatogram must correspond for a reliable identification. It certainly matches the possibilities offered by the overspotting technique applied by Verbeke [ 2 1. On some double chromatograms the identification can be hampered in one of the chromatograms, owing to interferences which disguise some zones of the chromatographic lane. If there is evidence from the other chromatogram that the probable analyte might be covered, the identification becomes questionable. In such instances a more thorough clean-up of the extract must be applied. Before the solid-phase extraction on Cl8 a delipidation is performed on Lipidex-5000. The crude extract is taken up in 0.6 ml of hexane-dichloromethane (85:15, v/v) and applied on top of a small glass column ( 145 mm x 6 mm I.D. ), plugged at the bottom with glass-wool and filled with 6 cm of Lipidex-5000, swollen and conditioned with the same solvent. The column is then eluted with the same solvent. The first 1 ml is discarded and the next 6 ml are collected and evaporated to dryness at 40°C in a stream of nitrogen. The procedure is then continued as described under Clean-up. Of the commonly encountered unesterified anabolics, estradiol and progesterone are not eluted from the column. As these are endogenous hormones, their detection in fatty tissues does not indicate any demonstrative illegal use of xenobiotic anabolics. The chromatographic data are summarized in Table I. They are related not only to the free alcohols but also to derivatives which are commonly encountered in pharmaceutical and/or illegal preparations. Quoting the detection limits as absolute values makes little sense for two reasons. First, there are variations in the efficiency of the detection process. The time and temperature of the heating and certainly the amount of reagent, which is difficult to control, play an important role and lead to varying sensitivity. Sec-
217 TABLE I
RF VALUES OBTAINED AND COLOURS AFTER VISUALIZATION Eluent 1, chloroform-acetone
(9:l); eluent 2, cyclohexane-ethyl acetate-ethanol (77.5:20:2.5).
Substance
Colour UV (366 nm )
RF value
Eluent 1 Eluent 2 Eluent 1
Eluent 2
Chlormadinone acetate E&radio1 phenylpropionate Testosterone propionate Testosterone enanthate Testosterone
0.35 0.45 0.72 0.99 0.75 0.78 0.81 0.87 0.39
0.17 0.29 0.43 0.63 0.14 0.40 0.37 0.40 0.10
Ethinylestradiol
0.44
0.23
19-Norethisterone
0.49
0.14
E&radio1 valerate l’i’-Hydroxyprogesterone Testosterone phenylpropionate
0.73 0.77 0.83
0.42 0.25 0.35
Dienestrol Norethisterone Estradiol bensoate Mestranol Trenbolone acetate
0.44 0.53 0.73 0.82 0.85
0.29 0.22 0.34 0.49 0.32
Nortestosterone decanoate Estradiol
0.95 0.46
0.51 0.18
Diethylstilhestrol Medroxyprogesterone acetate Estradiol cypionate Norethisterone acetate Testosterone cypionate
0.59 0.87 0.89 0.91 0.95
0.28 0.20 0.47 0.31 0.46
19-Nortestosterone
0.49
0.12
Methyltestosterone Megestrol acetate Testosterone isocaproate Nortestosterone laurate Hexestrol
0.58 0.86 0.94 0.96 0.49
0.18 0.22 0.40 0.46 0.28
Yellow Purple Purple Purple Orange-brown Yellow-green Yellow-green Green Yellow (brown centre) Yellow (orange centre ) Yellow (orange dots) Yellow-orange Yellow-green Orange (red centre) Purple Yellow Orange Orange Green (fluorescent) Brown Yellow (orange centre ) Brown Orange-brown Yellow Yellow Green (fluorescent ) Yellow (orange centre) Green Yellow-brown Yellow-green Brown Purple
Progesterone Diethylstilbestrol dipropionate
Yellow Purple Purple Purple Yellow-brown Yellow-green Green Green Yellow (brown centre ) Orange (red centre ) Yellow (orangedots ) Yellow-orange Yellow Orange {red centre) Purple Yellow Orange Orange Green (fluorescent ) Brown Yellow (orange centre) Brown Orange-brown Yellow Yellow Green (fluorescent) Yellow (orange centre) Green Yellow-brown Yellow-green Brown Purple
ond, there is the subjectivity of the plate reading: some eyes are more sensitive and/or more trained to detect spots whose intensities in some instances hardly differ from the background.
218
The detection limit can be estimated to be in the range l-10ppb, which is of the same order as obtained by Verbeke [ 21 and which is the concentration range in which residues in fatty tissues occur. The analytical capacity, however, is enhanced; one can expect a skilled analyst to complete fifteen analyses in five days. ACKNOWLEDGEMENTS
This work was supported by the Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Grant No. 3.0068.86). The technical contribution of M. Monteyne is acknowledged. REFERENCES B. Wortberg, R. Woller and T. Chulamorakot, J. Chromatogr., 156 (1978) 205. R. Verbeke, J. Chromatogr., 177 (1979) 69. H.-J. Stan and F.W. Hohls, Z. Lebensm,-Unters.-Forsch., 166(1978) 287. F.W. Hohls and H.-J. Stan, Z. Lebensm.-Unters.-Forh. 167 (1978) 252. F. Smets and M. Vandewalle, Z. Lebensm.-Unters.-Forth., 175 (1982) 29. F. Smets and M. Vandewalle, Z. Lebensm.-Unters.-Forsch., 178 (1984) 38. W.G. De Ruig, J.M. Weseman, G.M. Binnendijk and H. Hooijerink, Ware(n) Chemicus, 14 (1984) 89.
Journal of Chromatography, 489 (1989) 213-218 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
CHROMBIO. 4598
THIN-LAYER CHROMATOGRAPHIC OF ANABOLICS IN FATTY TISSUES
L. VAN LOOK, Ph. DESCHUYTBRE
METHOD FOR THE DETECTION
and C. VAN PETBGHEM*
Lxdmratory of Food Analysis, Faculty of Pharmaceutical Sciences, State University of Ghent, Harelbekestraat 72,900O Ghent (Belgium)
SUMMARY A modification of the method of Verbeke [J. Chromatogr., 177 (1979) 691 is presented. The fatty tissue is dissolved in hexane, partitioned against methanol-sodium acetate buffer (pH 5.2) and extracted with dichloromethane. The crude extract is then purified on a disposable Cl8 column. The final extract together with a set of reference compounds is spotted on two opposite sides of a highperformance thin-layer chromatographic plate (10X 10 cm), which is developed with two different eluents in two opposite directions. This mode of operation has the advantage that more reference compounds can be spotted and that the identification is based on two independent chromatographic runs. Also the total analysis time is decreased.
INTRODUCTION
In the control of the illegal use of anabolics in fatting stock there are two analytical approaches. The first directs all efforts to the detection of one specific compound, which usually is selected by its presumed or sometimes proved intensive use. Immunochemical methods (radioimmunoassay, chemjluminescence immunoassay, enzyme-linked immunosorbent assay, etc. ) make use of antibodies which are specifically directed against the analyte or against a group of structurally very related substances. These methods are characterized, from the analytical viewpoint, by high sensitivity, limited selectivity and relative ease of operation. The second approach consists in multi-residue methodology. These methods try to detect as many compounds as possible in one analytical run. It is obvious that chromatographic procedures are most appropriate for this purpose. If sufficient sensitivity can be attained, thin-layer chromatography (TLC) offers the considerable advantage of visible colour differentiation. In order to increase the resolving power of the chromatographic system often two-dimensional development with different eluting solvents is carried out [ 1,2]. Various purifi0378-4347/89/$03&O
0 1989 Elsevier Science Publishers B.V.
214
cation methods prior to the final chromatographic step have been described [l-
71.
One of the most extensively used methods is that of Verbeke [ 21. It is intended for the detection of various anabolic residues in tissues-or urine at levels as low as 0.5-10 ppb. Starting from 50-g samples, residues are recovered with an efficiency ranging between 60 and 80%. The detection limit of most anabolics is of the order of l-10 ng. As a particularly interesting feature, a group separation into an estrogen and a non-estrogen fraction is performed. Mainly because of this fractionation, which at the same time is a clean-up step, a serious drawback of the method is that is very laborious; one analyst is supposed to complete ten analyses per week. In this paper a method is presented which is based on that of Verbeke [ 21 but which contains a reversal in the extraction procedure, permitting a better release of the fat matrix and an alternative clean-up procedure, yielding in a shorter time a final extract that permits the detection of l-10 ppb of anabolic in fatty tissue. EXPERIMENTAL
Instrumentation The equipment consisted of a laboratory blender (Stomacher Type 80, Colworth, London, U.K.) with disposable polythene bags, a water-bath at 4O”C, centrifuge tubes (70 ml), a mechanical shaker, a rotary vacuum evaporator, Pasteur pipettes, chromatographic tanks (Camag, Muttenz, Switzerland) and a sample applicator (Merck, Darmstadt, F.R.G., Art. No, 10226) with microcapillaries (Merck, Art. No. 10289). Disposable 3-ml Bond Elut C,, columns (Analytichem International, Harbor City, CA, U.S.A.) were used with a Baker-10 extraction system (J.T. Baker, Phillipsburg, NJ, U.S.A). Reagents and reference solutions Silica gel 60 TLC plates (10 x 10 cm) from Merck (Art. No. 5631) were used. All reagents were of analytical-reagent grade and used as received. Lipidex5000 was obtained from Packard Instrument (Brussels, Belgium). Reference solutions for TLC were prepared in methanol from commercially available bulk products. The concentration of each anabolic was 0.1 mg/ml. Extraction Bovine fatty tissue (50 g ) spiked in the l-10 ppb concentration range with 50 ~1 of a methanolic solution is minced with a scalpel and suspended in 60 ml of hexane. The mixture is homogenized for 2 min in a blender and then heated in a water-bath a 40°C until a yellowish liquid is obtained. This liquid is centrifuged at 470 g for 1 min. A clear solution of the lipids in hexane is obtained. The hexane phase is homogenized with 180 ml of methanol by mechanical shaking for 10 min, transferred to a separating funnel and mixed with 80 ml of 0.04 M sodium acetate buffer (pH 5.2). On gently swirling phase separation occurs and the hexane layer is discarded. The turbid aqueous layer is then partitioned three times against 50 ml of hexane by mechanical shaking for 10 min.
215
Next, the aqueous layer, which has become clear, is extracted with 150 ml of dichloromethane, followed by another two extractions with 90 ml of dichloromethane. The combined organic layers are evaporated to dryness at 40°C in a rotary vacuum evaporator. The crude extract is transferred into a conical tube by means of two 3-ml portions of methanol and.evaporated to dryness in a stream of nitrogen. Clean-up The Bond Elut Cl8 (3 ml) columns are pretreated by rinsing with two 6-ml portions of methanol, followed by two 6-ml portions of doubly distilled water. The extract is taken up in 0.5 ml of methanol and 4 ml of doubly distilled water and transferred on to the Cl8 column. Washing is carried out with 3 ml of doubly distilled water, 3 ml of methanol-water (55 : 45 ) and 1.5ml of hexane. Elution is performed with 3 ml of methanol. The eluate is evaporated to dryness under a stream of nitrogen and finally taken up in 50 ,ul of methanol. Thin-layer chromatography Two chromatographic runs are performed in two opposite directions on one TLC plate. On a starting line 1 cm from one edge, aliquots (1.5 and 3.75 ~1, corresponding to 1.5 and 3.75 g of fatty tissue, respectively) are spotted with a microcapillary. On the opposite side of the plate, again 1 cm from the edge, the spotting is repeated. The reference standards are spotted in groups of six in five different lanes also on both starting-lines. The plate is eluted in one direction as far as the middle of the plate with chloroform-acetone (9O:lO). After drying, the opposite half of the plate is eluted with cyclohexane-ethyl acetate-ethanol (77.5:20:2.5 ). The chromatogram is then sprayed with 10% sulphuric acid in methanol and heated for 10 min at 90°C. The spots are examined under UV light at 366 nm. Identification is achieved by comparing the RF values and the colours of the spots in the extract with those of the reference mixtures on both chromatograms. A residue is not present when it is not unambiguously identified in both runs. RESULTS AND DISCUSSION
The main advantage of the alternative procedure described here is a more complete extraction of the fatty tissue. Mechanical homogenization using an Ultraturrax or a similar device always leaves a considerable amount of solid material. In the proposed modification, the fat is dissolved with slight heating in hexane. After centrifugation a clear yellow organic phase is obtained with mostly only a minute amount of precipitate. Concerning the efficiency of both extraction procedures, very little information can be deduced from spiked fat samples, as the conditions of the injected analyte are in no way comparable to those of endogenous residues of the analyte. Anyway, from a purely theoretical point of view a higher recovery can be expected from an almost completely dissolved tissue than from a minced but actually undissolved pulp. In most instances the bulk amount of lipids is removed by the solid-phase ex-
216
traction on C,, columns. The extract is sufficiently pure to allow direct identification by comparison of the qualitative data (RF and spot colour) of both chromatograms. The recovery from the C,, columns is reasonable to very good for most of the compounds; by visual comparison of the intensity of the spots with that of standards of known concentration, it may be estimated to be at least 80%. This figure is only of limited importance, however, as spiked samples can never replace real samples. The addition of a certain amount of a substance in the form of a methanolic solution does not imitate the conditions under which the same compound is present as a residue in the fatty tissue. It is worth noting that the recovery is poor for several compounds (mestranol, nortestosterone decanoate, testosterone cypionate, estradiol cypionate, testosterone isocaproate, testosterone enanthate, estradiol phenylpropionate, testosterone phenylpropionate and estradiol valerate). This is of limited diagnostic value, however, as the anabolic esters in general are deposited as the free form in the fatty tissue. The double chromatogram has the advantage over the normal two-dimensional chromatogram that considerably more reference compounds can be applied on one plate. Indeed, several lanes are available and the composition of the standard mixtures can be freely chosen as a function of the resolution between certain constituents. The double chromatogram also yields a much quicker and simpler overall picture of the composition of the final extract. The results on both parts of the double chromatogram must correspond for a reliable identification. It certainly matches the possibilities offered by the overspotting technique applied by Verbeke [ 2 1. On some double chromatograms the identification can be hampered in one of the chromatograms, owing to interferences which disguise some zones of the chromatographic lane. If there is evidence from the other chromatogram that the probable analyte might be covered, the identification becomes questionable. In such instances a more thorough clean-up of the extract must be applied. Before the solid-phase extraction on Cl8 a delipidation is performed on Lipidex-5000. The crude extract is taken up in 0.6 ml of hexane-dichloromethane (85:15, v/v) and applied on top of a small glass column ( 145 mm x 6 mm I.D. ), plugged at the bottom with glass-wool and filled with 6 cm of Lipidex-5000, swollen and conditioned with the same solvent. The column is then eluted with the same solvent. The first 1 ml is discarded and the next 6 ml are collected and evaporated to dryness at 40°C in a stream of nitrogen. The procedure is then continued as described under Clean-up. Of the commonly encountered unesterified anabolics, estradiol and progesterone are not eluted from the column. As these are endogenous hormones, their detection in fatty tissues does not indicate any demonstrative illegal use of xenobiotic anabolics. The chromatographic data are summarized in Table I. They are related not only to the free alcohols but also to derivatives which are commonly encountered in pharmaceutical and/or illegal preparations. Quoting the detection limits as absolute values makes little sense for two reasons. First, there are variations in the efficiency of the detection process. The time and temperature of the heating and certainly the amount of reagent, which is difficult to control, play an important role and lead to varying sensitivity. Sec-
217 TABLE I
RF VALUES OBTAINED AND COLOURS AFTER VISUALIZATION Eluent 1, chloroform-acetone
(9:l); eluent 2, cyclohexane-ethyl acetate-ethanol (77.5:20:2.5).
Substance
Colour UV (366 nm )
RF value
Eluent 1 Eluent 2 Eluent 1
Eluent 2
Chlormadinone acetate E&radio1 phenylpropionate Testosterone propionate Testosterone enanthate Testosterone
0.35 0.45 0.72 0.99 0.75 0.78 0.81 0.87 0.39
0.17 0.29 0.43 0.63 0.14 0.40 0.37 0.40 0.10
Ethinylestradiol
0.44
0.23
19-Norethisterone
0.49
0.14
E&radio1 valerate l’i’-Hydroxyprogesterone Testosterone phenylpropionate
0.73 0.77 0.83
0.42 0.25 0.35
Dienestrol Norethisterone Estradiol bensoate Mestranol Trenbolone acetate
0.44 0.53 0.73 0.82 0.85
0.29 0.22 0.34 0.49 0.32
Nortestosterone decanoate Estradiol
0.95 0.46
0.51 0.18
Diethylstilhestrol Medroxyprogesterone acetate Estradiol cypionate Norethisterone acetate Testosterone cypionate
0.59 0.87 0.89 0.91 0.95
0.28 0.20 0.47 0.31 0.46
19-Nortestosterone
0.49
0.12
Methyltestosterone Megestrol acetate Testosterone isocaproate Nortestosterone laurate Hexestrol
0.58 0.86 0.94 0.96 0.49
0.18 0.22 0.40 0.46 0.28
Yellow Purple Purple Purple Orange-brown Yellow-green Yellow-green Green Yellow (brown centre) Yellow (orange centre ) Yellow (orange dots) Yellow-orange Yellow-green Orange (red centre) Purple Yellow Orange Orange Green (fluorescent) Brown Yellow (orange centre ) Brown Orange-brown Yellow Yellow Green (fluorescent ) Yellow (orange centre) Green Yellow-brown Yellow-green Brown Purple
Progesterone Diethylstilbestrol dipropionate
Yellow Purple Purple Purple Yellow-brown Yellow-green Green Green Yellow (brown centre ) Orange (red centre ) Yellow (orangedots ) Yellow-orange Yellow Orange {red centre) Purple Yellow Orange Orange Green (fluorescent ) Brown Yellow (orange centre) Brown Orange-brown Yellow Yellow Green (fluorescent) Yellow (orange centre) Green Yellow-brown Yellow-green Brown Purple
ond, there is the subjectivity of the plate reading: some eyes are more sensitive and/or more trained to detect spots whose intensities in some instances hardly differ from the background.
218
The detection limit can be estimated to be in the range l-10ppb, which is of the same order as obtained by Verbeke [ 21 and which is the concentration range in which residues in fatty tissues occur. The analytical capacity, however, is enhanced; one can expect a skilled analyst to complete fifteen analyses in five days. ACKNOWLEDGEMENTS
This work was supported by the Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Grant No. 3.0068.86). The technical contribution of M. Monteyne is acknowledged. REFERENCES B. Wortberg, R. Woller and T. Chulamorakot, J. Chromatogr., 156 (1978) 205. R. Verbeke, J. Chromatogr., 177 (1979) 69. H.-J. Stan and F.W. Hohls, Z. Lebensm,-Unters.-Forsch., 166(1978) 287. F.W. Hohls and H.-J. Stan, Z. Lebensm.-Unters.-Forh. 167 (1978) 252. F. Smets and M. Vandewalle, Z. Lebensm.-Unters.-Forth., 175 (1982) 29. F. Smets and M. Vandewalle, Z. Lebensm.-Unters.-Forsch., 178 (1984) 38. W.G. De Ruig, J.M. Weseman, G.M. Binnendijk and H. Hooijerink, Ware(n) Chemicus, 14 (1984) 89.