Detection and quantitation of methandienone (dianabol®) in urine by isotope dilution—mass fragmentography

Journal of Sreroid Biochemistry. Vol. 13, pp. 169 to 115 Pergarnon Press Ltd 1980. Printed in Great Britain

DETECTION AND QUANTITATION OF METHANDIENONE (DIANABOLB) IN URINE BY ISOTOPE DILUTION-MASS FRAGMENTOGRAPHY INGEMARBJ~RKHEM*, OLLE LANTTO and AGNETA LOF Department of Clinical Chemistry and the Research Unit at Huddinge Hospital, Karolinska Institutet, Stockholm, Sweden (Received 26 April 1979)

SUMMARY A highly accurate method has been developed for detection and quantitation of methandienone (Dianabol) in urine. A suitable internal standard containing a CD,-group in the 17a-position was synthesized. A fixed amount of this internal standard was added to a fixed amount of urine and the mixture treated with Helix pornaria. After extraction, treatment with acetic acid anhydride, and purification by preparative thin-layer chromatography, the purified extract was converted into the methoxime-trimethylsilyl derivative and subjected to combined gas chromatography-mass spectrometry. Unlabeled methandienone could be quantitated from the ratio between the tracings of the molecular ions at m/e 401 and m/e 404. Under the conditions employed, methandienone could be identified and determined in a concentration exceeding 3 ng/ml of urine. The compound could be detected and quantitated for several days after ingestion of a single dose of 10 mg of methandienone. In preliminary experiments the method has been used on urine samples obtained from athletes in connection with a competition. Some of the results are presented. In combination with a positive radioimmunoassay test, the present method should give sufficient evidence that an athlete is guilty of ingesting this steroid.

INTRODUCTION It is well known that there is a wide-spread misuse of synthetic anabolic steroids by athletes. For ethical reasons, and in order to prevent possible irreversible metabolic changes and physical damage, the International Olympic Committee has blacklisted all anabolic drugs. A blacklisting will be effective only if sensitive and specific tests are available for detection of these drugs and their met&olites in urine, In view of the severe legal consequences of a detection of anabolit steroids in the urine from a competing athlete, it should be emphasized that the method used must be highly specific and accurate. Methods based on radioimmunoassay [ 1,2], TLC [3]. HPLC [4], GLC [S-7], and combined gas chromatography-mass spectrometry (GC-MS) [8-l l] have been described. Methods based on radioimmunoassay have sufficient sensitivity, but lack specificity. Thus radioimmunoassay is well suited for screening purposes (cf. ref. [12]) but cannot be used for positive identification. Methods based on TLC, HPLC and GLC give more specific information, but are less sensitive. It may be argued, however, that an * To whom correspondence

should be addressed. Methandienone, 17/?-hydroxy-17a-methylandrosta-l,4-dien-3-one; 17-epimethandienone, 17a-hydroxy-17/3-methylandrosta-1,4-dien3-one; 6/?-hydroxy-methandienone, 6/3,17/%dihydroxy- 17nmethylandrosta-1,4-dien-3-one; HPLC, high performance liquid chromatography. Nomenclature

SB.

13/2--f

and

abbreviations:

169

identification which is based only on the retention time in GLC or HPLC may be insufficient. For identification, GC-MS seems to be the method of choice. This technique has been used in some studies on the metabolism of anabolic steroids [8,9] and is at present in routine use in some laboratories for positive identification of anabolic steroids in urine from athletes [12]. In most of the published studies [8, IO] the anabolic steroids, or their metabolites, have been identified with use of full mass spectra or selected ion chromatograms obtained from repetitive mass spectral data. Such procedures require considerable amounts of material, however, and may thus sometimes be too insensitive. The technique of mass fragmentography (selective ion monitoring), in which some selected present ions are followed through the gas chromatography, should be considerably more sensitive, without loss of essential specificity (cf. ref. [I I]). Use of a deuterium-labeled internal standard should further increase the accuracy of such a technique. In our laboratory we are at present developing a test-program for detection and identification of anabolic steroids in urine. The program starts with radioimmunoassay for detection of 17-methylated steroids and 19-nor steroids [ 11. Positive samples are then analyzed by mass fragmentography, using specific “diagnostic” ions. In the present work we describe a highly accurate method for assay of methandienone (Dianabol) in urine, based on isotope-dilution mass fragmentogra-

INGEMAR BJBRKHEM, OLLE LANTTO and AGNETA L~F

170

phy. Available data seem to suggest that Dianabol is one of the most widely used anabolic steroids for improving athletic performance by illegal means. Some of our experience with the present method on samples obtained in connection with competitions will be presented. MATERIALS 1 7aC2H3-Laheled

METHODS

AND

methandienone

This compound was prepared according to the synthetic route shown in Fig. 1. A solution of dehydroisoandrosterone (I), 2OOmg, in 4 ml dried benzene, was added to a Grignard solution prepared from 146mg of magnesium and 6.9 mmol of C2H3 I and the mixture was refluxed for 3 h. After cooling to 5°C the complex was decomposed by the slow addition of 1 ml of ice water and 2 ml of 50% aqueous acetic acid solution. The mixture was then diluted with water and extracted with ether. The ether phase was washed with water until neutral and dried over anhydrous sodium sulfate. According to TLC (with chloroform-ethyl acetate 4: 1, v/v as solvent) more than 90% of the dehydroisoandrosterone had been converted into the 17a-methylated derivative (II). After evaporation of the solvent, the crude mixture was dissolved in 6 ml of acetone and lOml of anhydrous benzene and treated with 400mg aluminium tert butoxide by reflux for 3 h. After dilution with water and extraction with ether, the mixture was subjected to preparative TLC, using the same solvent system as above. The total yield of C’H,-labeled methyltestosterone (III) was 50 mg. The material was pure as judged by TLC and GLC (as the methoxime trimethylsilyl ether derivative) (methoxime-TMS ether). Part of the material was crystallized from acetone-water. White needles were obtained with m.p. 168°C (reported m.p. for unlabeled methyltestosterone, 164°C) [13-J.

Crystalline C’H,-labeled methyltestosterone, 18 mg, was dissolved in 5 ml of dioxane and refluxed with 40mg of dichloro-dicyano-quinone for 14 h. Water was added and the product was extracted with ether after acidification. The ether phase was washed with water until neutral and the solvent was removed. The residue was subjected to TLC, using the same solvent system as above. The total yield of C’H,-labeled methandienone (IV) was 5.4mg. The material was pure as judged by TLC and GLC as methoxime-TMS. The mass spectrum of the methoxime-trimethylsilyl ether was in complete accord with the proposed structure (Fig. 2) and showed that the material contained less than 3% of unlabeled molecules.

Unlabeled

methandienone

This compound was obtained from CIBA-Geigy Ltd. (Basle, Schwitzerland) and was pure as judged by TLC and GLC of the methoxime-TMS ether.

Urine samples

These were collected from the laboratory staff and from athletes involved in competitions. The samples were stored frozen at -20°C until analyzed.

Radioimmunoassay

A radioimmunoassay kit for analysis of 17-alkylated steroids was obtained from Prof. R. V. Brooks, St. Thomas’s Hospital, London, UK. This kit was used for screening analysis according to the recommendations with only one modification. After treatment with dextran-coated charcoal and centrifugation an aliquot of the supernatant was used for gamma counting.

0

HO

&\

OH

t&l

b

SD3

HOW

I

II

I

Acetone

Al tert butoxide -

DDQ

m

Ip

Fig. 1. Synthetic

route for preparation

of [17a-C2H,]-labeled

methandienone.

-1

Detection and quantitation of methandienone

’

>1

.Z ; OL ; .: ._

171

CHj4-N

so-

z

283

:

Fig. 2. Mass spectrum of the methoxime-trimethylsilyl ether of unlabeled methandienone (upper spec. trum) and [17a-C*H,]-labeled methandienone (lower spectrum).

Preparation of samples for isotope dilution-mass fragmentography In the standard procedure, to lOm1 of urine was standard of lOOng of internal added an 17cc-C2H,-labeled methandienone dissolved in 100 ~1 of acetone. In a few cases (cf. fig. 5C) 5 ml of urine and 400 ng of standard were used. To this mixture, 1 ml of 0.15 M NaAc buffer pH 4.6, was added, together with SO/11of a solution containing Helix pomatia digestive juice. After mixing with a Vortex mixer, the solution was left at 37°C for 24 h. The hydrolysate was then extracted with IOmi of ethyl acetate. The organic phase was washed twice with 8 ml 0.1 M NaOH and once with 8 ml distilled water. After removal of the water with anhydrous Na,SO, the solvent was removed under a stream of nitrogen at 50°C. The residue was treated with 1 ml pyridine and 0.5 ml acetic acid anhydride and left over-night at room temperature. It was confirmed that the 178-hydroxyl group in methandienone was not acetylated under these conditions [ 11. After addition of 1 ml of methanol, the samples were evaporated to dryness. The residue was subjected to TLC (with chloroform-ethyl acetate 1: 1, v/v, as solvent).

In this chromatography, [4-‘4C]-testosterone, 15ng, 6500 d.p.m., was used as marker. The zone containing [4-r4C]-testosterone was detected by radioscanning of the chromatoplate using a Berthold D~nnschicht scanner II (Wildbad, GFR). Under the conditions employed, methandienone had a RF value of 0.43 just below that of testosterone (RF = 0.49). The silica gel from the appropriate chromatographic zone was scraped off and eluted with methanol. After evaporation of the solvent, the methoxime-TMS was prepared as described previously [ 141. The reaction mixture was extracted with 30~1 of hexane and cooled in a freezer in order to obtain-clear phases. Mass fragmentography

About 5~1 of the hexane phase was analyzed by GC-MS using an LKB 2091 instrument. The column was a 1.5% SE-30 (on Chromosorb W, 80-100 mesh, 2 mm x 1.5 m); carrier gas, helium; flow rate lOml/min. The column temperature was 240°C and the temperature of the ion source and flash heater were 275” and 290°C respectively. The electron energy was 20 eV and the trap current 60pA. The electron. multiplier sensitivity was set to 700. The first channel

INGEMAR BJBRKHEM,OLLE LANTTO and AGNETA tin

172

of the MID-unit was focused on the ion at m/e 401 and second at m/e 404. The amplification used for both channels was 200 x In some cases, the identity of methandienone was further confirmed by following other characteristic ions with the multiple ion detector, such as m/e 370 (M-31) and m/e 280 (M-90-31) (Fig. 2). RESULTS

Figure 2 shows the mass spectrum of the methoxime-trimethylsilyl ether derivative of unlabeled and deuterium labeled methandienone. The molecular ion of the derivative of unlabeled methandienone had an intensity about 80% of that of the

peak at m/e 143. This latter ion is derived from the D-ring of the molecule [S]. In the mass fragmentographic analysis of urine extracts, the molecular ion was found to give less interference than the ions at m/e 143, mJe 280 and m/e 370. Thus the molecular ions at m/e 401 and 404 were chosen for the assay. Figure 3 shows multiple ion detector recordings obtained in analysis of derivative of unlabeled and deuterium labeled methandienone. The ratio between peaks at m/e 401 and m/e 404 obtained in mass fragmentographic recordings of different standard mixtures of unlabeled methandienone together with a fixed amount of deuterium labeled methandienone (100 ng) was found to be linear with the amount of unlabeled methandienone (Fig. 4).

base

I_

Ill,.? m e 404 401

0

1

234567

Retention

Fig.

3. Multiple

ion

time

0 (mln)

detector recording of [ 17a-C’HJ-labeled

ng

Fig. 4. Standard

curve

for determination

1

Retention

derivative of methandienone

234567 time

unlabeled (right).

(min)

methandienone

(left)

and

methandienone

of methandienone in the range Materials and Methods).

&20ng/ml

(for details,

see

Detection

and quantitation

In Fig. SA, a typical recording is shown of the derivative of purified extracts of a urine sample to which 17~C2H3 methandienone had been added. Only a very small peak was observed in the tracing at m/e 401, corresponding to unlabeled methandienone. In Fig. 5B, a multiple ion detector recording is shown of purified extract of a urine sample from the same subject as in Fig. 5A, with the exception that a single dose 10mg of methandienone had been ingested. There was a prominent peak in the tracing at m/e 401, corresponding to a concentration of about 60ng methandienone per ml of urine. Under the conditions employed, a significant excretion of methandienone could be detected with the present technique for 4 days after the single oral load of 10mg. The total excretion of unmetabolized methandienone during this time (unconjugated as well as conjugated) was about 140 pg. The small background tracing observed in the recording at m/e 401 (Fig. SA) was obtained in analysis of most urine samples from untreated subjects. This tracing corresponded to an apparent concentration of methandienone of 1.4 k 0.7 ng/ml urine

173

of methandienone

(mean f SD, n = 10). The highest background value obtained in the analysis of urine from an untreated subject was 2.6 ng/ml. This value is however very low as compared to the high concentration obtained after a single dose of 1Omg of methandienone. In connection with doping prior to competition, athletes may take a considerably higher daily dose than 1Omg for several months [ 161. As shown in Fig. 5B, a peak was obtained in the tracing at m/e 401 with a shorter retention time than the derivative of methandienone. This compound was only seen in connection with ingestion of methandienone. The mass spectrum was similar to that of the derivative of methandienone and contained peaks at m/e 401, m/e 370, m/e 280 and m/e 143. This compound was excreted in an amount exceeding the amount of unmetabolized methandienone during the first 2 days after ingestion of 10 mg of methandienone. During the next 2 days, however, the metabolite was excreted in lower amounts than methandienone. The identity of the compound has not so far been established. The coefficient of variation of the method. as calcu-

C

A

\t % L

i

m/e 401

\r

m/e 404

3 4 5 Retention time (mid

6

\i i

m/e 401 m/e 404

..I

0 Retention

-1 I23

time (mid

-1-J

JJ 4

5

6

Retention time tmin)

Fig. 5. (A) Multiple ion detector recording of derivative of a purified extract from a healthy untreated subject. Standard conditions were used with IO ml of urine and IOOng of added [17a-C’H,]-methandienone. (B) Multiple ion detector recording of a purified extract from the same subject as above, with the exception that IOmg of methandienone had been ingested (first 24-hour collection of urine after the ingestion). Assay conditions were the same as in A. (C) Multiple ion detector recording of a purified extract from a urine sample of an athlete taking part in the Swedish championship. In this case only 5 ml of urine was used and the amount of added [ 17x-C’H,]-methandienone was 400 ng.

174

INWMAR BJBRKHEM. OLLE LANTTO and AGNETA LOP Table

I. Recoveryofmethandienone addedtoa urine

Sample

Apparent

concentration

methandienone

Urine

sample

Urine

sample

sample

sample

sample

8

3 . 'i

0

6.2

2

10.9

4

20.1

6

+ methandienone

+ methandienone

20 ng/ml

-t Difference

Error+

+ methandienone

10 ng/ml Urine

(ng/ml)

+ methandienone

5 ng/ml Urine

of

in urine

1.3

2 ng/ml Urine

sample

between

calculated

latedfrom fivereplicate measurements of one urine sample containing about 60 ng per ml, was 6%. The accuracy of the method (in a low concentration range) was tested by addition of known amounts of methandienone to a urine sample (Table 1). The difference between calculated and determined value was always less than 7%. In connection with a competition (Swedish championship), urine samples were obtained from 23 athletes. Radioimmunoassay showed that 2 of these samples contained 17z-alkylated steroids in a concentration significantly above the background value found in untreated subjects (3 ng/ml). Further analysis with the present method showed that one of the samples contained a very high concentration of methandienone (more than I pg/ml) (Fig. 5C). The identity of methandienone was confirmed by tracing also the ions at m/e 370, 280 and rnje 143. A mass spectrum of the material was found to be identical with the mass spectrum of derivative of authentic methandienone. In the mass fragmentographic recording of the ion at m/e 401 a peak was also observed prior to the peak corresponding to methandienone (Fig. 5C). This early peak had the same retention as the metabolite of methandienone (Fig. 5B).

DlSCUSSlON

In accordance with previous studies, it was shown that small amounts of administered methandienone was excreted in urine prior to any metabolism. These small amounts could be detected by radioimmunoassay. In the analysis by CC-MS, however, more reli-

and

found

value.

able results were obtained when more material was available. Thus the conjugates were split by treatment with He1i.r pomatia prior to analysis. It was shown that about l-2% of a single dose of methandienone was excreted as conjugated and unconjugated methandienone. It has been claimed that the major metabelite of methandienone is 6B-hydroxy-methandienone [S]. In our investigation, one metabolite in urine was found, which was not identical with 6B-hydroxymethandienone. Since the mass spectrum of this metabolite was similar to that of unchanged methandienone, it seems possible that it could be an isomer. Very recently, Diirbeck et al. reported that 17-epimethandienone was one of the major urinary metabolites of methandienone [ 151. It seems probable that the metabolite found in our study is identical with that compound. Lack of authentic 17-epi-methandienone prevented however a final identification. Our metabolite was excreted in amounts exceeding the amounts of methandienone during the first and second day after loading with a single dose of methandienone, but was barely detectable during the third and fourth day. Thus it was considered better to develop a specific assay for methandienone itself rather than for its metabolite. Purification was found to be necessary prior to GC-MS. In addition to TLC, HPLC was tried. This latter technique was, however, more time-consuming and did not give better results. 17a-Methylated 17/&hydroxy steroids are less readily acetylated due to steric hindrance [I]. Thus acetylation was included in the procedure prior to thin-layer chromatography. If this step was omitted, sometimes interfering substances were observed during gas chromatography.

Detection and quantitation Different derivatives were tested in connection with GC-MS such as the heptafluorobutyrate, the TMS ether and the methoxime-TMS ether. In order to obtain maximal specificity, it is often advantageous to use a fragment of high molecular weight in the mass fragmentographic analysis. From this point of view, the molecular ion of the methoximeTMS derivative was considered to be the best choice. In order to obtain maximal sensitivity, the fragment should correspond to an ion of high intensity in the mass spectrum. Use of the molecular ion of the methoxime-TMS ether was found to give sufficient sensitivity of the assay under the conditions employed. The heptafluorobutyrate was less suitable, due to occurrence of several isomers of the derivative. In principle, the ion at m/e 143 in the mass spectrum of the TMS-ether or the methoxime TMS-ether may appear attractive due to its high intensity. In addition this ion is common for all 17cc-methylated steroids [S] and can thus be used for detection also of other anabolic steroids than methandienone. As pointed out previously, however [S], this ion is not unique and may arise from different sources in several endogenous steroids or other compounds. In our hands, use of the ion at m/e 143 gave a high background and interfering peaks. As a result the sensitivity in general decreased with at least one order of magnitude when using this ion instead of the ion at mJe 401. In spite of the high specificity of the present method, there was often a small amount of interference when using the molecular ion at m/e 401. Thus an unknown compound gave asmall peak in the mass fragmentographic recording, with the same retention time as the derivative of methandienone. This interference was always small and never exceeded an amount corresponding to 3 ng methandienone per ml. A positive identification of methandienone should thus require an apparent excretion of the compound exceeding 3 ng per ml of urine. Such excretion is very low, however, when compared with the high excretion of methandienone obtained after only a single oral dose of the compound. In view of the very small amounts of interfering material, the nature of the interference could never be established. This background value is analogous to the blank values obtained in the radioimmunoassay. In similarity with other published GC-MS methods for analysis of anabolic steroids, the present method requires 2-3 days for a complete analysis. This time may possibly be shortened considerably by using other methods for deconjugation of the excreted steroid. More rapid methods for deconjugation are tested at present. In combination with a positive radioimmunoassay test, a positive identification with the present method should give sufficient evidence that the athlete has been guilty of use of anabolic steroids. In spite of that, and before more experience has been accumulated, it is advisable to confirm the identity of methandienone

175

of methandienone

by using also other “diagnostic” ions than the molecular ion in the final GC-MS step. Acknowledgements-The skilful assistance of Hgkan Ek in part of this study is gratefully acknowledged. The authors are further grateful to Prof. Arne Ljungqvist for the initiation of the study. This work was supported by a grant from Sveriges Riksidrottsfdrbund. REFERENCES

1. Brooks R. V., Firth R. J. and Summer A. A.: Detection of anabolic steroids by radioimmunoassay. Br. J. Sports Med. 9 (1975) 89-92. 2. Jondorf W. R. and McDougall D. F.: Application of radioimmunoassay method for detecting 19-nortestosterone (nandrolone) in equine and canine plasma. Vet. Rec. 100 (1977) 560-562. 3. Oehrle K.-L., Vogt K. and Hoffman B.: Determination of trenbolone and trenbolone acetate@ by thin-layer chromatography in combination with a fluorescence colour reaction. J. Chromatogr. 114 (1975) 244246. 4. Frischkorn C. G. B. and Frischkorn H. E.: Investigation of anabolic drug abuse in athletics and cattle feed. II. Specific determination of methandienone (Dianabol@) in urine in nanogram amounts. J. Chrotnarogr. 151 (1978) 331-338. 5. Adhikary P. M. and Harkness R. A.: Determination of carbon skeletons of microgram amounts of steroids and sterols by gas chromatography after their high temperature catalytic reduction. Analyt. Chem. 41 (1969) 47&476. 6. Adhikary P. M. and Harkness R. A.: Production of the parent hydrocarbons from steroid drugs and their separation by gas chromatography. J. Chromatogr. 42 (1969) 29-38. 7. Fehtr T. and Sarfy E. H.: A simple gas chromatographic method for the determination of methandienone in human urine. J. Sports Med. 16 (1976) 165-170. 8. Ward R. J., Shackleton C. H. L. and Lawson A. M.: Gas chromatographic-mass spectrometric methods for the detection and identification of anabolic steroid drugs. Br. J. Sports Med. 9 (1975) 93-97. 9. Ward R. J., Lawson A. M. and Shackleton C. H. L.: Metabolism of anabolic steroid drugs in man and the marmoset monkey (Callthrix Jacchus). I. Nilevar and Orabolin. J. steroid. Biochem. 8 (1977) 1057-1063. 10. Brooks C. J. W., Thawley A. R., Rocher P., Middleditch B. S., Anthony G. M. and Stillwell W. G.: Characterization of steroidal drug metabolites by combined gas chromatography-mass spectrometry. J. Chromatogr. Sci. 9 (1971) 3543. 11. Houghton E., Oxley G. A., Moss M. S. and Evans S.: Studies related to the metabolism of anabolic steroids in the horse: A gas chromatographic mass spectrometric method to confirm the administration of 19nortestosterone or its esters to horses. Biomed. Mass Spectr. 5 (1978) 17(rl73. 12. Beckett A. H.: The problems of analysis in dope control in sport. Forensic Sci. 8 (1976) 176177. 13. Fieser L. F. and Fieser M.: Steroids. Reinhold, New York (1959) p. 519. 14. Bjorkhem I., Blomstrand R., Lantto 0.. Lof A. and Svensson L.: Plasma cortisol determination by mass fragmentography. C/in chim. Acta 56 (1974) 241-248. 15. Dtlrbeck H. W., Btlker I.. Scheulen B. and Telin B.: Gas chromatographic and capillary column gas chromatographic-mass spectrometric determination of synthetic anabolic steroids. 1. Methandienone and its metabolites. J. Chromatogr. 167 (1978) 117-l 24. 16 O’Shea J. P.: Anabolic steroids in sport: A biophysiological evaluation. Nurr. Rep. IN. 17 (1978) 607-627.