Forensic Science International, 14 (1979) 221 - 227 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
A ROUTINE OF METHYL
SCREENING PHENIDATE
221
METHOD FOR THE MAJOR METABOLITE IN THE URINE OF GREYHOUNDS
JOHN H. LEWIS Department
of Pharmacology,
University
of Sydney,
N.S. W. 2006 (Australia)
(Received February 7, 1979; in revised form May 14, 1979; accepted June 1, 1979)
Summary A qualitative urine screening procedure for the determination of the major urinary metabolite of methyl phenidate, a-phenyl-2-piperidine acetic acid (ritalinic acid), has been devised. The method involves a salting-out extraction, conversion of the metabolite to the parent drug using methanol-hydrogen chloride, and simultaneous two-column gaschromatographic detection. Final confirmation of the presence of the drug is by twosystem thin-layer chromatography. The method is suitable for the detection of illicitly administered methyl phenidate in greyhounds.
Introduction The determination of methyl phenidate and its urinary metabolite, (Yphenyl-2-piperidine acetic acid (ritalinic acid), has been reported previously by several authors [ 1 - 51. Earlier work by the author [l] proposed a method utilising thin-layer chromatography (TLC) of the urinary metabolite, following an ion pair solvent extraction using bromthymol blue. More recent investigation has shown TLC to be insufficiently sensitive for the purpose required. The general inertness of the piperidine nucleus of the ritalinic acid molecule to chromatographic sprays precludes TLC as a primary screening system. Schubert [2] isolated ritalinic acid from human urine by evaporation of the urine in uucuo and conversion, using diazomethane, of the metabolite to the parent drug, methyl phenidate. Wells et al. [3], also with human urine, u tilised freeze-drying coupled with diazome thane me thylation. The pharmacokinetics of methyl phenidate in man, dog, rate and mouse have been extensively discussed by Faraj et al. [4] . Using butanol as the extracting solvent, followed by ion-exchange chromatography, “C-labelled metabolites were identified by TLC and derivative formation. Milberg et al. [ 51 isolated ritalinic acid from human urine by a salting-out technique, diazomethane reaction and gas chromatography-mass spectrometry. Delbeke and Debackere [6] used gas chromatography for the detection of unchanged methyl phenidate and other doping agents in sporting events. No metabolites were detected.
222
A method was required for the detection of ritalinic acid in the urine of racing greyhounds. None of the published methods could be applied routinely in a screening programme for racing greyhounds. Firstly, a method was required that could be performed by technical personnel without access to mass spectrometry and without having to resort to the toxic and unstable diazomethane. Secondly, greyhound urine, being heavily pigmented in comparison to human urine, requires extensive clean-up procedures prior to chromatography. Thirdly, the method should utilise simple extraction equipment and reagents and be reproducible. Experimental
Reagents.
All reagents were AR grade and were used without
further
purification.
TLC plates. The plates were prepared by mixing a slurry of 20 g silica gel G (Merck) with 40 ml distilled water, and spreading a layer 0.25 mm thick. The plates (20 cm X 20 cm) were air dried for 5 min and then baked at 100 “C for 30 min. TLC solvents. System a: methanol-ammonia (100:0.3). ethyl acetate-acetone-chloroform-ammonia (100:10:5:5).
System
b:
Potassium triiodide spray. 10 g of potassium iodide and 10 g of iodine were dissolved in 250 ml of ethanol. 60 ml of concentrated hydrochloric acid were added and the solution made up to 500 ml with distilled water. Methyl phenidate (Ritalin@) and a-phenyl-2-piperidine acetic acid were generously donated by CIBA-Geigy (Australia and Switzerland). Sodium acetate buffer. 2 g of sodium acetate were dissolved in 200 ml of distilled water and the pH adjusted to 4.5 by the dropwise addition of glacial acetic acid. Extraction with Teflon-lined
Vortex-type
These were 50-ml glass tubes (Corex, screw caps.
tubes.
mixer.
Bench centrifuge.
Rota-Line
U.S.A.)
fitted
(Australia).
Hettich Universal 11.
Methanol-hydrogen chloride. Dry hydrogen chloride gas was prepared by the dropwise addition of concentrated hydrochloric acid onto concentrated sulphuric acid. The evolved gas was passed through a sulphuric acid drying trap and bubbled into anhydrous methanol. 1 ml aliquots of the methanol solution were transferred to 2 ml glass ampoules and sealed. Ampoules were stored at 4 “C until required.
223
Acid-methanol.
1 ml of concentrated hydrochloric acid was added to
60 ml of methanol.
Method Extraction of ritalinic acid 20 ml of urine which had been extracted for both acidic and basic drugs
[7] was transferred to a 50 ml glass centrifuge tube. A salting-out extraction procedure [ 51 was used as follows. 2 ml of propan-2-01 were added followed by 15 g of anhydrous potassium carbonate. The sample was shaken on a vortex-type mixer for 2 min and then centrifuged for 5 min. The upper alcohol phase was transferred to a 15 ml centrifuge tube. A second extraction was performed on the urine using 1 ml of propan-2-01. The combined organic fractions were centrifuged to remove any residual aqueous phase and then evaporated to dryness on a water bath at 45 “C under a stream of air. Purification
of crude
extract
The crude dry extract containing ritalinic acid was purified as follows. The extract was dissolved in 2 ml of 1 M sodium hydroxide; 1 ml of n-butanol was added and the sample extracted on the vortex mixer for 1 min. After brief centrifugation the upper phase was transferred to a centrifuge tube. The extraction process was repeated twice using 1 ml and then 0.5 ml of butanol. To the combined butanol phases was then added 1 ml of sodium acetate buffer (pH 4.5). The sample was extracted on the vortex mixer for 1 min. After brief centrifugation the lower aqueous phase was transferred via a Pasteur pipette to another centrifuge tube. The extraction process was repeated twice using 1 ml and then 0.5 ml of sodium acetate buffer. The combined aqueous phases were adjusted to pH 1 - 2 by the addition of 5 drops of concentrated hydrochloric acid and the ritalinic acid taken up in butanol using three extractions of 1 ml, 1 ml and 0.5 ml of solvent. The combined butanol fractions were evaporated to dryness in a 15 ml Quickfit centrifuge tube at 45 “C under a stream of air.
Methylation
The purified extract was then methylated by the addition of 1 ml of methanol-hydrogen chloride reagent. The tube was stoppered and the contents allowed to react for at least 1 h. Recovery
of methyl phenidate
The sample containing methylated ritalinic acid was gently evaporated to dryness under a stream of air. The extract was dissolved in 0.5 ml of 0.1 M hydrochloric acid and washed with 2 ml of ether, which was discarded. The extract was adjusted to pH 8 by the slow addition of solid sodium bicarbonate. 2 ml of chloroform were added and the sample extracted on the vortex
224
for 2 min. The extraction was repeated using a second ahquot of 1 ml of chloroform. The combined chloroform phases were then extracted into 1 ml of 0.1 M sulphuric acid. The chloroform was discarded. The acid phase was washed in 1 ml of fresh chloroform which was then discarded. The acid was diluted to 2 ml with distilled water, adjusted to pH 8 by the slow addition of solid sodium bicarbonate and extracted with 2 ml of chloroform as before. A second extraction was performed using 1 ml of solvent. The combined organic layers were transferred to a tapered tube, 2 drops of acid-methanol were added and the whole evaporated to dryness at 30 “C under a stream of air.
Gas-liquid chromatography The purified extracts were subjected to gas-liquid chromatography (GLC) using two columns simultaneously. Glass columns of length 1.5 m, internal diameter 4 mm, were used as follows: (a) 3% OV-1 on Gas Chrom Q loo-120 mesh (Applied Science Labs.). (b) 5% Free fatty acid phase (Varian Pty., Ltd.) coated on Chromasorb W AW DMCS 80-100 mesh (Applied Science Labs.). Conditions. Instrument: Bendix 2500 Series Gas Chromatograph fitted with flame ionisation detectors. Carrier gas: nitrogen 40 ml min-‘. Air: 300 ml min ‘. Hydrogen: 40 ml min-‘. Injector and detector: 245 “C. Oven: 170 “C. Recorders: Twin Perkin Elmer Model 56, chart speed 5 mm min-‘. The extracts were dissolved in 50 ~1 of chloroform containing n-eicosane as internal marker, and 2 ~1 injected into each column.
Thin-layer chromatography Samples that gave matching peaks to methyl phenidate on both systems were then subjected to TLC. Two solvent systems were employed to confirm the presence of the (see above). 15 ~1 of the remaining extracts were spotted onto each of two plates, along with standard methyl phenidate hydrochloride. The plates developed to a distance of 15 cm, air dried and finally lightly sprayed the potassium triiodide reagent.
GLC drug TLC were with
In vivo experiments Methyl phenidate hydrochloride was administered orally to a greyhound on three occasions, in doses of lo,15 and 20 mg. In each experiment control urines were obtained prior to dosing and thereafter at 1 h intervals up to 7 h after administration. In vitro experiments Ritalinic acid was added to a series of drug-free urine specimens and the detection limits determined.
225
Results From the in uivo experiments good separation of methyl phenidate from other urinary constituents was achieved on both GLC systems (Figs. 1 and 2). In both TLC systems methyl phenidate was resolved from other compounds in the extracts. Ritalinic acid could be detected as methyl phenidate in the urine of greyhounds up to 7 h after the administration of 10 mg of methyl phenidate. The excretion of the metabolite was detected from 2 h after administration at all dose levels. No unchanged methyl phenidate was detected at any dose level administered. Following the addition of ritalinic acid to a series of blank urine samples, positive recordings were obtained on the gas chromatograph from as little as 1 pg of drug per ml of urine. Retention data [8] for methyl phenidate are given in Table 1.
i
iii min.
Fig. 1. GLC on Methyl phenidate;
i
i
i
min.
OV-1 of urine 2, n-eicosane.
extract
following
methyl
phenidate
administration.
1,
Fig. 2. GLC on FFAP of urine Methyl phenidate; 2, n-eicosane.
extract
following
methyl
phenidate
administration.
1,
226 TABLE Retention
1 data for methyl
phenidate Column
Retention index Relative retention
time (neicosane
= 1.0)
ov-1
FFAP
1718 0.31
1818 0.49
Discussion The method described here for the determination of ritalinic acid in the urine of greyhounds has the distinct advantage over other published methods by utilising flame-ionisation GLC with confirmation by TLC. The use of methanol-hydrogen chloride as the methylating reagent enables technical personnel to perform the entire analysis without having to resort to the unstable, toxic and potentially hazardous diazomethane. Positive results with all four chromatographic systems were considered to be satisfactory proof of the presence of the metabolite and hence of the ingestion of the parent drug. As methyl phenidate is almost completely metabolised in the greyhound, the analytical method is dependent on the maximum yield of metabolite. Since greyhound urine is highly pigmented, extensive clean-up procedures are necessary in order to obtain satisfactory chromatograms. In spite of substantial losses due to these parameters ritalinic acid was easily detected for many hours following the administration of 10, 15 and 20 mg of pure drug.
Acknowledgements The author is grateful to Dr. D. Jackson of this department for his advice and comments, and to Miss S. Shadbolt for technical assistance. The author is funded by the New South Wales Greyhound Racing Control Board.
References 1 J. H. Lewis and R. H. Thor-p, Methyl phenidate, coping with doping. Forensic Sci., 5 (1975) 147. 2 B. Schubert, Detection and identification of methyl phenidate in human urine and blood samples. Acta Chem. Stand., 24 (1970) 433 - 438. 3 R. Wells, K. B. Hammond and D. 0. Rodgerson, Gas-liquid chromatographic procedure for measurement of methyl phenidate hydrochloride and its metabolite, ritalinic acid, in urine. Clin. Chem., 2014 (1974) 440 - 443.
227 4 B. A. Faraj, Z. H. Israili, J. M. Perel, M. L. Jenkins, S. G. Holtzman, S. A. Cucinell and P. G. Dayton, Metabolism and disposition of methyl phenidate-14C: studies in man and animals. J. Pharm. Exp. Ther., 191 (1974) 535 - 547. 5 R. M. Milberg, K. L. Rinehart, R. L. Sprague and E. K. Sleator, A reproducible gas chromatographic mass spectrometric assay for low levels of methyl phenidate and ritalinic acid in blood and urine. Biomed. Mass Spectrom., 2 (1975) 2 - 8. 6 F. T. Delbeke and M. Debackere, Isolation and detection of methyl phenidate, phacetoperane and some other sympathomimetic central nervous stimulants with special reference to doping. J. Chromatogr., 106 (1975) 412 - 417. 7 J. H. Lewis, unpublished data. 8 L. Kazyak and R. Permisohn, Retention indices for compound identification by gas chromatography. J. Forensic Sci., 15 (1970) 346 - 356.
A ROUTINE OF METHYL
SCREENING PHENIDATE
221
METHOD FOR THE MAJOR METABOLITE IN THE URINE OF GREYHOUNDS
JOHN H. LEWIS Department
of Pharmacology,
University
of Sydney,
N.S. W. 2006 (Australia)
(Received February 7, 1979; in revised form May 14, 1979; accepted June 1, 1979)
Summary A qualitative urine screening procedure for the determination of the major urinary metabolite of methyl phenidate, a-phenyl-2-piperidine acetic acid (ritalinic acid), has been devised. The method involves a salting-out extraction, conversion of the metabolite to the parent drug using methanol-hydrogen chloride, and simultaneous two-column gaschromatographic detection. Final confirmation of the presence of the drug is by twosystem thin-layer chromatography. The method is suitable for the detection of illicitly administered methyl phenidate in greyhounds.
Introduction The determination of methyl phenidate and its urinary metabolite, (Yphenyl-2-piperidine acetic acid (ritalinic acid), has been reported previously by several authors [ 1 - 51. Earlier work by the author [l] proposed a method utilising thin-layer chromatography (TLC) of the urinary metabolite, following an ion pair solvent extraction using bromthymol blue. More recent investigation has shown TLC to be insufficiently sensitive for the purpose required. The general inertness of the piperidine nucleus of the ritalinic acid molecule to chromatographic sprays precludes TLC as a primary screening system. Schubert [2] isolated ritalinic acid from human urine by evaporation of the urine in uucuo and conversion, using diazomethane, of the metabolite to the parent drug, methyl phenidate. Wells et al. [3], also with human urine, u tilised freeze-drying coupled with diazome thane me thylation. The pharmacokinetics of methyl phenidate in man, dog, rate and mouse have been extensively discussed by Faraj et al. [4] . Using butanol as the extracting solvent, followed by ion-exchange chromatography, “C-labelled metabolites were identified by TLC and derivative formation. Milberg et al. [ 51 isolated ritalinic acid from human urine by a salting-out technique, diazomethane reaction and gas chromatography-mass spectrometry. Delbeke and Debackere [6] used gas chromatography for the detection of unchanged methyl phenidate and other doping agents in sporting events. No metabolites were detected.
222
A method was required for the detection of ritalinic acid in the urine of racing greyhounds. None of the published methods could be applied routinely in a screening programme for racing greyhounds. Firstly, a method was required that could be performed by technical personnel without access to mass spectrometry and without having to resort to the toxic and unstable diazomethane. Secondly, greyhound urine, being heavily pigmented in comparison to human urine, requires extensive clean-up procedures prior to chromatography. Thirdly, the method should utilise simple extraction equipment and reagents and be reproducible. Experimental
Reagents.
All reagents were AR grade and were used without
further
purification.
TLC plates. The plates were prepared by mixing a slurry of 20 g silica gel G (Merck) with 40 ml distilled water, and spreading a layer 0.25 mm thick. The plates (20 cm X 20 cm) were air dried for 5 min and then baked at 100 “C for 30 min. TLC solvents. System a: methanol-ammonia (100:0.3). ethyl acetate-acetone-chloroform-ammonia (100:10:5:5).
System
b:
Potassium triiodide spray. 10 g of potassium iodide and 10 g of iodine were dissolved in 250 ml of ethanol. 60 ml of concentrated hydrochloric acid were added and the solution made up to 500 ml with distilled water. Methyl phenidate (Ritalin@) and a-phenyl-2-piperidine acetic acid were generously donated by CIBA-Geigy (Australia and Switzerland). Sodium acetate buffer. 2 g of sodium acetate were dissolved in 200 ml of distilled water and the pH adjusted to 4.5 by the dropwise addition of glacial acetic acid. Extraction with Teflon-lined
Vortex-type
These were 50-ml glass tubes (Corex, screw caps.
tubes.
mixer.
Bench centrifuge.
Rota-Line
U.S.A.)
fitted
(Australia).
Hettich Universal 11.
Methanol-hydrogen chloride. Dry hydrogen chloride gas was prepared by the dropwise addition of concentrated hydrochloric acid onto concentrated sulphuric acid. The evolved gas was passed through a sulphuric acid drying trap and bubbled into anhydrous methanol. 1 ml aliquots of the methanol solution were transferred to 2 ml glass ampoules and sealed. Ampoules were stored at 4 “C until required.
223
Acid-methanol.
1 ml of concentrated hydrochloric acid was added to
60 ml of methanol.
Method Extraction of ritalinic acid 20 ml of urine which had been extracted for both acidic and basic drugs
[7] was transferred to a 50 ml glass centrifuge tube. A salting-out extraction procedure [ 51 was used as follows. 2 ml of propan-2-01 were added followed by 15 g of anhydrous potassium carbonate. The sample was shaken on a vortex-type mixer for 2 min and then centrifuged for 5 min. The upper alcohol phase was transferred to a 15 ml centrifuge tube. A second extraction was performed on the urine using 1 ml of propan-2-01. The combined organic fractions were centrifuged to remove any residual aqueous phase and then evaporated to dryness on a water bath at 45 “C under a stream of air. Purification
of crude
extract
The crude dry extract containing ritalinic acid was purified as follows. The extract was dissolved in 2 ml of 1 M sodium hydroxide; 1 ml of n-butanol was added and the sample extracted on the vortex mixer for 1 min. After brief centrifugation the upper phase was transferred to a centrifuge tube. The extraction process was repeated twice using 1 ml and then 0.5 ml of butanol. To the combined butanol phases was then added 1 ml of sodium acetate buffer (pH 4.5). The sample was extracted on the vortex mixer for 1 min. After brief centrifugation the lower aqueous phase was transferred via a Pasteur pipette to another centrifuge tube. The extraction process was repeated twice using 1 ml and then 0.5 ml of sodium acetate buffer. The combined aqueous phases were adjusted to pH 1 - 2 by the addition of 5 drops of concentrated hydrochloric acid and the ritalinic acid taken up in butanol using three extractions of 1 ml, 1 ml and 0.5 ml of solvent. The combined butanol fractions were evaporated to dryness in a 15 ml Quickfit centrifuge tube at 45 “C under a stream of air.
Methylation
The purified extract was then methylated by the addition of 1 ml of methanol-hydrogen chloride reagent. The tube was stoppered and the contents allowed to react for at least 1 h. Recovery
of methyl phenidate
The sample containing methylated ritalinic acid was gently evaporated to dryness under a stream of air. The extract was dissolved in 0.5 ml of 0.1 M hydrochloric acid and washed with 2 ml of ether, which was discarded. The extract was adjusted to pH 8 by the slow addition of solid sodium bicarbonate. 2 ml of chloroform were added and the sample extracted on the vortex
224
for 2 min. The extraction was repeated using a second ahquot of 1 ml of chloroform. The combined chloroform phases were then extracted into 1 ml of 0.1 M sulphuric acid. The chloroform was discarded. The acid phase was washed in 1 ml of fresh chloroform which was then discarded. The acid was diluted to 2 ml with distilled water, adjusted to pH 8 by the slow addition of solid sodium bicarbonate and extracted with 2 ml of chloroform as before. A second extraction was performed using 1 ml of solvent. The combined organic layers were transferred to a tapered tube, 2 drops of acid-methanol were added and the whole evaporated to dryness at 30 “C under a stream of air.
Gas-liquid chromatography The purified extracts were subjected to gas-liquid chromatography (GLC) using two columns simultaneously. Glass columns of length 1.5 m, internal diameter 4 mm, were used as follows: (a) 3% OV-1 on Gas Chrom Q loo-120 mesh (Applied Science Labs.). (b) 5% Free fatty acid phase (Varian Pty., Ltd.) coated on Chromasorb W AW DMCS 80-100 mesh (Applied Science Labs.). Conditions. Instrument: Bendix 2500 Series Gas Chromatograph fitted with flame ionisation detectors. Carrier gas: nitrogen 40 ml min-‘. Air: 300 ml min ‘. Hydrogen: 40 ml min-‘. Injector and detector: 245 “C. Oven: 170 “C. Recorders: Twin Perkin Elmer Model 56, chart speed 5 mm min-‘. The extracts were dissolved in 50 ~1 of chloroform containing n-eicosane as internal marker, and 2 ~1 injected into each column.
Thin-layer chromatography Samples that gave matching peaks to methyl phenidate on both systems were then subjected to TLC. Two solvent systems were employed to confirm the presence of the (see above). 15 ~1 of the remaining extracts were spotted onto each of two plates, along with standard methyl phenidate hydrochloride. The plates developed to a distance of 15 cm, air dried and finally lightly sprayed the potassium triiodide reagent.
GLC drug TLC were with
In vivo experiments Methyl phenidate hydrochloride was administered orally to a greyhound on three occasions, in doses of lo,15 and 20 mg. In each experiment control urines were obtained prior to dosing and thereafter at 1 h intervals up to 7 h after administration. In vitro experiments Ritalinic acid was added to a series of drug-free urine specimens and the detection limits determined.
225
Results From the in uivo experiments good separation of methyl phenidate from other urinary constituents was achieved on both GLC systems (Figs. 1 and 2). In both TLC systems methyl phenidate was resolved from other compounds in the extracts. Ritalinic acid could be detected as methyl phenidate in the urine of greyhounds up to 7 h after the administration of 10 mg of methyl phenidate. The excretion of the metabolite was detected from 2 h after administration at all dose levels. No unchanged methyl phenidate was detected at any dose level administered. Following the addition of ritalinic acid to a series of blank urine samples, positive recordings were obtained on the gas chromatograph from as little as 1 pg of drug per ml of urine. Retention data [8] for methyl phenidate are given in Table 1.
i
iii min.
Fig. 1. GLC on Methyl phenidate;
i
i
i
min.
OV-1 of urine 2, n-eicosane.
extract
following
methyl
phenidate
administration.
1,
Fig. 2. GLC on FFAP of urine Methyl phenidate; 2, n-eicosane.
extract
following
methyl
phenidate
administration.
1,
226 TABLE Retention
1 data for methyl
phenidate Column
Retention index Relative retention
time (neicosane
= 1.0)
ov-1
FFAP
1718 0.31
1818 0.49
Discussion The method described here for the determination of ritalinic acid in the urine of greyhounds has the distinct advantage over other published methods by utilising flame-ionisation GLC with confirmation by TLC. The use of methanol-hydrogen chloride as the methylating reagent enables technical personnel to perform the entire analysis without having to resort to the unstable, toxic and potentially hazardous diazomethane. Positive results with all four chromatographic systems were considered to be satisfactory proof of the presence of the metabolite and hence of the ingestion of the parent drug. As methyl phenidate is almost completely metabolised in the greyhound, the analytical method is dependent on the maximum yield of metabolite. Since greyhound urine is highly pigmented, extensive clean-up procedures are necessary in order to obtain satisfactory chromatograms. In spite of substantial losses due to these parameters ritalinic acid was easily detected for many hours following the administration of 10, 15 and 20 mg of pure drug.
Acknowledgements The author is grateful to Dr. D. Jackson of this department for his advice and comments, and to Miss S. Shadbolt for technical assistance. The author is funded by the New South Wales Greyhound Racing Control Board.
References 1 J. H. Lewis and R. H. Thor-p, Methyl phenidate, coping with doping. Forensic Sci., 5 (1975) 147. 2 B. Schubert, Detection and identification of methyl phenidate in human urine and blood samples. Acta Chem. Stand., 24 (1970) 433 - 438. 3 R. Wells, K. B. Hammond and D. 0. Rodgerson, Gas-liquid chromatographic procedure for measurement of methyl phenidate hydrochloride and its metabolite, ritalinic acid, in urine. Clin. Chem., 2014 (1974) 440 - 443.
227 4 B. A. Faraj, Z. H. Israili, J. M. Perel, M. L. Jenkins, S. G. Holtzman, S. A. Cucinell and P. G. Dayton, Metabolism and disposition of methyl phenidate-14C: studies in man and animals. J. Pharm. Exp. Ther., 191 (1974) 535 - 547. 5 R. M. Milberg, K. L. Rinehart, R. L. Sprague and E. K. Sleator, A reproducible gas chromatographic mass spectrometric assay for low levels of methyl phenidate and ritalinic acid in blood and urine. Biomed. Mass Spectrom., 2 (1975) 2 - 8. 6 F. T. Delbeke and M. Debackere, Isolation and detection of methyl phenidate, phacetoperane and some other sympathomimetic central nervous stimulants with special reference to doping. J. Chromatogr., 106 (1975) 412 - 417. 7 J. H. Lewis, unpublished data. 8 L. Kazyak and R. Permisohn, Retention indices for compound identification by gas chromatography. J. Forensic Sci., 15 (1970) 346 - 356.
