Jun 7, 1993 - We wish to thank Jos~ Antonio Pascual and Rob Ewin for the design and ... B.K. Logan, D.T. Stafford, I.R. Tebbett, and C.M. Moore. Rapid.
Journal of Analytical Toxicology, Vol. ! 9, March/April 1995
Comprehensive Screening Procedure for Detection of Stimulants, Narcotics, Adrenergic Drugs, and Their Metabolites in Human Urine A. Solans*, M. Carnicero, R. de la Torre, and J. Segura Department of Pharmacology and Toxicology, Institut Municipal d'lnvestigaci6 M~dica, IMIM, Universitat Autbnoma de Barcelona, Doctor Aiguader 80, 08003 Barcelona, Spain
Abstract I An analytical procedure for the detection of stimulants, narcotics, [3-blockers, [3-agonists, and many of their metabolites in urine using a solid-phase extraction procedure and gas chromatography-mass spectrometry (GC-MS) is described. These substances have been specifically banned by the Medical Commission of the International Olympic Committee (IOC) in order to prevent their abuse in sports. Urine samples are submitted to an enzymatic hydrolysis (13glucuronidase arylsulfatase) and extracted by means of Bond-Elut Certify T M columns. The residues are then selectively derivatized with N-methyI-N-trimethylsilyl-trifluoroacetamide (MSTFA), which enables the formation of trimethylsilyl derivatives of hydroxyl, acidic, and phenolic groups, and N-methyl-bis-trifluoroacetamide (MBTFA), which enables the formation of trifluoroacetamide derivatives of primary and secondary amines. A GC-MS system working in scan mode is sensitive and specific enough to detect and identify approximately 100 compounds and metabolites in urine for at least 24 h after the administration of doses typically encountered in therapeutics. Detection in selected ion monitoring mode is needed for the determination of ff-agonist agents. The method was successfully used in doping control of urine samples during the 25th Olympic Games, July 1992, in Barcelona, Spain.
Introduction The development of analytical methods for the detection of stimulants, narcotics, ~-agonists, and [3-blockers is of great interest in toxicology and pharmacology. All of these substances are used in therapeutics, and some stimulants and narcotics are often abused in our society. In addition, these substances have been specifically banned by the Medical Commission of the International Olympic Committee (IOC) in order to prevent their abuse in sports (1). A general analytical screening procedure should take into account the possibility of the simultaneous presence of these kinds of drugs in urine. This group of substances shares several physicochemical characteristics: (a) their pharmacological effects are produced *Author to whom corresponden( e ~hould be addressed.
104
by relatively high doses of drug (clenbuterol, salbutamol, and terbutaline are some exceptions); (b) they can be excreted in urine as free drug or metabolites or as their conjugates with glucuronic acid or sulfate; (c) they are weakly basic compounds (pKa 7-10), as most of them are nitrogen-containing; and (d) almost all of these substances have functional groups that can be derivatized to enhance their gas chromatographic properties. These similarities suggest the possibility of screening all of these substances simultaneously using a single analytical procedure. In addition, when facing massive analysis for different substances, any attempt at unifying analytical screenings is of great value considering the time pressure for releasing results and the number of samples to be analyzed. There have been some efforts to unify the extraction of basic substances in urine using liquid-liquid extraction (LLE) (2,3) or solid-phase extraction (SPE) (4-6) procedures. Methods traditionally used for the extraction of nitrogen-containing basic drugs were based on LLE, and they were designed for the detection of each group of substances separately (7,8). Such methods have automation difficulties, and when analyzing a large number of samples, the high amounts of organic solvent residues generated in the extraction procedure could represent a health biohazard that would then need to be reduced. The development of SPE procedures (9) as an alternative method to LI,E procedures has allowed researchers to obtain cleaner extracts and optimum recoveries. SPE methods save a substantial amount of organic solvent per sample and are more suitable for automation. Some analytical methods for 13blockers (10-13), [3-agonists (13), stimulants (4,5,]4), and narcotic analgesics (15-18) have already been published. Attempts to unify the extraction of these compounds by LLE or SPE methods have revealed different problems, including losses of the most volatile substances in the evaporation step, for example, stimulants like amphetamines (4-6); breaking of some compounds in acidic hydrolysis conditions, for example, some [~-blockers (12,19) and monoacetylmorphine (15); or low extraction efficiency for some compounds (4,7). A general screening method should resolve all of these questions. This paper describes a unified analytical procedure for the simultaneous isolation of stimulants, narcotics, [~-agonists, 13-
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Journal of Analytical Toxicology, Vol. 19, March/April 1995
blockers, and many of their metabolites using an SPE procedure. Gas chromatography-mass spectrometry (GC-MS) was used for separation and detection. For the detection of most compounds, the scanning mode was preferred, except for [~agonists, which were analyzed in selected ion monitoring (SIM) mode. The procedure was successfully used for screening and confirmatory purposes in the analysis of approximately 2000 urine samples during the 25th Olympic Games, July 1992, in Barcelona, Spain.
Human samples Excretion studies involving drug administration and urine collection were performed using healthy male volunteers under the authorization of Hospital del Mar Ethical Committee (Barcelona, Spain) and Spanish Ministry of Health (reference number 88/135). Compounds were administered by the oral route at the doses recommended by the IOC Medical Commission (1). Table I shows the doses given and the main metabolites detected for each compound. Volunteers were under medical supervision throughout the study, and the urine specimens were collected for a period of 24 h.
Experimental
Instrumental analysis
Chemicals and reagents Methanol (HPLCgrade), chloroform, glacial acetic acid, and acetone (all analytical grade) were purchased from Merck (Darmstadt, Germany). lsopropyl alcohol and 25% ammonium hydroxide (both reagent grade) were supplied by Scharlau (Barcelona, Spain). Deionized water was obtained in-house by a Milli-Q system (Millipore, Mulheim, France). Reference standards for all 13blockers, [3-agonists,stimulants, and narcotics were provided by Sigma Chemical (St. Louis, MO). Levallorphan tartrate was donated by Roche (Basel, Switzerland). Stock standard solutions of drugs (1 mg/mL, in free-base form) were prepared in methanol. Working solutions of 100 IJg/mL were prepared by dilution of stock solutions. These solutions were checked by 1N spectrophotometryand stored at -20~ A mixture containing 100 IJg/mL of amphetamine, methoxyphenamine, methylphenidate, ritalinic acid, metoprolol, nadolol, sotalol, codeine, and morphine in methanol was usedas a standard working solution for evaporation and recovery experiments. The 1-mg/mL deuterated solutions of internal standards, codeine-d3 (7,8 didehydro-4,5-epoxy-3-methoxy-17-[methyld3]morphinan-6-ol) and MDMA-d5(d,l-l-[3,4-(methylenedioxy) phenyl]-2-methylaminopropane-l,2,3,3,3-ds), in methanol were obtained from Radian Corporation (Austin, TX). A mixture containing 100 IJg/mL codeine-d3 and 100 1Jg/mL MDMA-d5 in methanol was used as the internal standards working solution. !3-Glucuronidase(containing substantial arylsulfataseactivity) from Helix pomatia type HP-2 was purchased from Sigma Chemical. N-Methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA),Nmethyl-bis-trifluoroacetamide (MBTFA),and trimethylchlorosilane (TMCS) were obtained from Macherey-Nagel (D~ren, Germany). Bond-Elut CertifyTM columns were provided by Analytichem International (Harbor City, CA). This solid phase contains a proprietary bonded-silica sorbent, which exhibits a hydrophobic and cation-exchange extraction mechanism. The vacuum manifold used for the SPE procedure was obtained from Biochemical Diagnostics (Edgewood, NY). Organic phases were evaporated to dryness under a nitrogen stream with a Turbo-Vap LVevaporator from Zymark Corporation (Hopkinton, MA).
Gas chromatography was performed on a Hewlett-Packard (HP) Model 5890 coupled to a 5971A mass selective detector (Hewlett-Packard, Palo Alto, CA). Separation of analytes was carried out using an Ultra-2 HP-5 cross-linked 5% phenylmethyl silicone gum capillary column (12.5 m • 0.2-mm i.d., 0.33-1Jm film thickness). Injector (split mode; ratio, 1:10) and detector temperatures were 280~ The oven temperature was programmed from 100 to 290~ at 20~ (final time, 4 min). Helium flowwas 0.8 mL/min, and the sample injection volume was 2 IJL. The mass spectrometer was operated by electron-impact ionization (EI, 70 eV) in the scan acquisition mode (50-600 amu), and solvent delay was 2 min. Data acquisition was done by an HP 59940 ChemStation (HP UNIX series). Data files were then transferred automatically to a computer server by means of an HP ChemLANproduct. In the server, a laboratory information management system (LIMS) took care of the data management (20). The LIMS, in combination with in-house-developed software (21) specially designed for target analysis, was the tool responsible for local reporting of results (e.g., integration reports and high resolution graphs) after central processing.
Extraction procedure Urine samples (2.5 mL) were added to 25 IJL of the internal standards working solution to obtain a concentration of 1 IJg/mL codeine-d3 and 1 IJg/mL MDMA-ds. Then, 1 mL 1.1M acetate buffer (pH 5.2) and 50 IJL of !3-glucuronidase arylsulfatase (about 2600 units of I~-glucuronidase per milliliter of urine) were added. The samples were vortex mixed, heated to 55~ for 2 h on a water bath, and later cooled to room temperature. Then, sample pH was adjusted to 8-9 with 1M KOH, and the mixture was centrifuged at 2500 rpm for 5 min. Bond Elut Certify columns were inserted into a vacuum manifold and conditioned by washing once with 2 mL methanol and 2 mL deionized water. The columns were prevented from drying before applying specimens. Supernatants of centrifuged samples were poured into each column reservoir and drawn slowlythrough the column. The columns were washed with 2 mL deionized water, 1 mL 0.1M acetate buffer (pH 4), and 2 mL methanol. Elution of analytes was performed with 2 mL of a mixture of chloroform-isopropyl alcohol (80:20, v/v) containing 2% ammonium hydroxide. The eluates were added to 20 IlL MBTFA,vortex mixed, and then evaporated to dryness under a stream of nitrogen in a 50~ water bath. Samples were kept in a desiccator for 30 min.
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Journal of Analytical Toxicology.Vol. 19, March/April 1995
Table I. Stimulants, [3-Blockers, [3-Agonists, and Narcotics Detected in Urine Collected over a Period of 24 Hours Parentcompound Stimulants Amfepramone (Diethylpropion) Amineptine Amphetamine Cathine Chlorphentermine C]obenzorex Dimethamphetamine
Dose (mg) 75 100 10 15 25 30 30
Ephedrine Etafedrine
50 50
Ethylamphetamine Fencamfamine Fenetylline Fenfluramine Fenproporex Heptaminol Mefenorex Methoxyphenamine Methylamphetamine Methylphenidate Phendimetrazine Phenmetrazine Phentermine Phenylpropanolamine Pho[edrine Pseudoephedrine
30 20 25 15 10 50 40 40 I0 30 30 25 30 50 40 60
p-Blockers Acebutolol AlprenoJol Atenolol Bisoprolol Carteolol Celipro]ol Labetalol Metoprolol Nadolol Oxprenolol Penbutolol Pindolo] Propranolol Sotalol Timolol
~-Agonists Clenbuterol Orciprenaline Salbutamol Terbutaline Narcotics Codeine Dextromethorphan Dihydrocodeine Ethylmorphine Methadone Morphine Nalbuphine Pethidine Pholcodine
106
Substances detected N,N-diethylnorephedrine,N-ethylnorephedrine, norephedrine,nordiethylpropion Amineptine, amineptine-Cs-metabolite Amphetamine Cathine (norpseudoephedrine) Chlorphentermine Clobenzorex,amphetamine,hydroxyclobenzorex Dimethylamphetamine,methylamphetamine, pholedrine, amphetamine Ephedrine,norephedrine,hydroxyephedrine Etafedrine,ephedrine,norephedrine, hydroxyetafedrine Ethylamphetamine Fencamfamine Fenetylline,amphetamine Fenfluramine Amphetamine, hydroxyfenproporex Heptaminol Amphetamine, mefenorex,hydroxymefenorex Methoxyphenamine,hydroxymethoxyphenamine Methylamphetamine Methylphenidate,ritalinic acid Phendimetrazine,phenmetrazine Phenmetrazine Phentermine Phenylpropanolamine(norephedrine) Pholedrine Pseudoephedrine,norpseudoephedrine
80 10
Acebutolol, diacetolol Alprenolol, hydroxyalprenolol Atenolol Bisoprolol Carteolol Celiprolol Labetalol, hydroxylabetalol Metoprolol, hydroxymetoprolol Nadolol Oxprenolol, hydroxyoxprenolol Penbutolol, hydroxypenbutolol Pindolol Propranolol,4-hydroxypropranolol, 3-hydroxypropranololmethoxyhydroxypropranolol Sotalol Timolol
0.04 10 2 2.5
Clenbuterol Orciprenaline Salbutamol Terbutaline
200 50 50 10 5 200 200 25 60 40 40 I0 20
200 50 50 I0 I0 200 200 25 60
Codeine, morphine, norcodeine,normorphine Dextrorphan Dihydrocodeine,nordihydrocodeine, dihydromorphine,nordihydromorphine Ethylmorphine,morphine Methadone Morphine, normorphine Nalbuphine Pethidine, norpethidine Pholcodine, morphine
Derivatization procedure The following procedure was used to obtain trimethylsilyl and trifluoroacetyl derivatives. MSTFA (100 laL) was added to the dried residue, vortex mixed, and kept at 60~ for 5 rain to obtain the trimethylsilyl (TMS) derivatives of hydroxyl, acidic, and phenolic groups. After the above mixture was cooled to room temperature, 20 IJL MBTFAwas added, and the mixture was vortex mixed and incubated for 10 min at 60~ to obtain trifluoroacetamide (TFA)derivatives of primary and secondary amines (22). Evaporation experiments Two experiments were designed in order to evaluate the efficiency of two approaches in avoiding losses of some very volatile substances during the evaporation step; the influence of the approaches in the derivatization procedure was also evaluated. Multiple samples of the same urine blank (2.5 mL) spiked with 125 tJL of the standard working solution (equivalent to 5 IJg/mL urine for each compound) and 125 IJL of the internal standards solution (equivalent to 5 IJg/mL urine of each internal standard) were submitted to the same extraction procedure (including the elution step) already described. Subsequently, they were submitted to the following experiments. First experiment. TMCS (20 IJL) was added to three organic eluates, and 20 tJL MBTFA was added to three others. After vortex mixing, samples were evaporated, kept in a desiccator for 30 min, and derivatized as described previously. Second experiment. MBTFA (20 IJL) was added to six organic eluates, vortex mixed, evaporated, and kept in a desiccator for 30 rain. Three tubes were derivatized with 100 IJL MSTFAat 60~ for 5 min; the other three tubes were derivatized as previously described with 100 pL MSTFA at 60~ for 5 rain and 20 laL MBTFAat 60~ for 10 rain. Each sample (2 IJL) was injected into the GC-MS system. Hydrolysis procedure The efficiency of the hydrolysis procedure was tested by means of urine blank samples (2.5 mL) spiked with 1 Iag/mL morphine-3glucuronide (concentration equivalent to morphine free base). Three batches of three aliquots each were combined with 50 IJL 13glucuronidase from Helixpomatia and submitted to hydrolysis at 55~ for 1, 2, and 3 h.
Journal of Analytical Toxicology, Vol. 19, March/April 1995
Samples were extracted by SPE as described previously, together with three control urine samples spiked with 1 IJg/mL morphine free base to evaluate the hydrolysis yield.
Recovery experiments Five urine blank samples (2.5 mL) spiked with 125 IJL of the standard working solution (equivalent to 5 tJg/mL urine for each compound) and 1251JLof the internal standards solution (equivalent to 5 IJg/mL urine for each internal standard) were extracted following the SPE method already described. Simultaneously, an equivalent amount of these solutions was dried and derivatized. Recoveries (n = 5) were calculated by comparing the chromatographic peak areas of the selected ion for each analyte before and after extraction.
Results and Discussion The present analytical method is based on a solid-phase extraction that is said to retain basic, neutral, and acidic drugs under the proper extraction conditions (6). In the procedure presented here, all the extracted substances have an ionizable amine function able to interact with the anionic groups of the extraction column. Working at a pH value near the pKa of these drugs allows hydrophobic interactions and cationic exchange simultaneously between the sorbent and the substances. The evaporation step without the addition of any specific reagent was unsuccessful in avoiding losses of low molecular weight stimulants, even at room temperature. Different authors have described the addition of some reagents that give rise to the formation of salt derivatives (either hydrochlorides or acetates [4,5,6] or acylating reagents [3]), which prevent losses of volatile stimulants. TMCS was used for the formation of hydrochlorides. This reagent succeeded in preventing losses of volatile stimulants. Nevertheless, the overall derivatization was affected, and it was possible to observe broad or double chromatographic peaks or both, especially in those substances belonging to the [~-blockers' group (e.g., atenolol, pindolol, and timolol). The alternative addition of MBTFAto form the trifluoroacetyl derivatives of primary and secondary amines did not introduce any problem in the derivatization procedure, and no losses of substances due to evaporation were observed. The second evaporation experiment evaluated the effect of the addition of MBTFA, prior to the evaporation step, on the subsequent derivatization procedure. It was observed that after the addition of only MSTFA to the dried residue (5 rain at 60~ the formation of O-TMS and N-TFAderivatives was distorted because TMS took the place of TFA in some amino groups. Therefore, the final addition of MBTFA (10 rain at 60~ during the derivatization procedure could not be eliminated. Thus, we recommend adding 20 IJL MBTFAbefore evaporation and derivatizing the dried residues with MSTFA and MBTFA. The experiments on the hydrolysis procedure for morphine3-glucuronide showed that the hydrolysis efficiency was 56% (coefficient of variation [CV], 4.65%) for 1 h of heating, 67%
(CV, 2.52%) for 2 h, and 71% (CV, 1.01%) for 3 h. For qualitative analysis, a hydrolysis time of 2 h at 55~ appears to be enough to detect all the substances of interest. Enzymatic hydrolysis has been shown to be better than chemical hydrolysis (acidic or alkaline) in avoiding degradation of some labile 13-blockers (atenolol, pindolol, timolol) (12,19) and other substances (6-monoacetylmorphine)(15). To establish the recovery of the method, the extraction of several [3-blockers (metoprolol, nadolol, and sotalol), stimulants (amphetamine, methoxyphenamine, methylphenidate, and ritalinic acid), and narcotic analgesics (codeine, levallorphan, and morphine) was investigated. These substances were selected as representative compounds for each pharmacological class studied, and they covered a wide range of volatilities, GC retention times, and MS responses. Table II shows recovery results for the substances described above and for the internal standards. Recoveries are higher than 80% for the majority of compounds except for ritalinic acid. This compound is the main substance found in human urine after the ingestion of methylphenidate; its low recovery, due to its acidic properties, is nevertheless enough to enable detection of the intake of therapeutic doses of methylphenidate for more than 24 h (23). Additionally,a modified sample extraction using more favorable conditions has been designed for ritalinic acid and methylphenidate for confirmatory analysis (23). In the present method, the use of a mixture of two internal standards (MDMA-dsand codeine-d3)to monitor all the steps of the analysis is proposed. MDMA-d5is useful for controlling the amino function derivatization and losses of compounds by volatilization; it also represents the chromatographic properties of the stimulants. Codeine-d3is useful as a control in the derivatization of the hydroxyl functions, is the most appropriate internal standard to prequantitate codeine urinary concentrations (regulated by the IOC until 1993)(1), and represents the chromatographic performance of heavy, volatile compounds like narcotic analgesics and 13-blockers.The relative retention times we report are based on the codeine-d3 retention time.
Table II. Recovery Resultsfor Several Compounds after Extraction from Urine Mean recovery
Drug
(%) (n = 5)
Coefficient of variation (%)
Amphetamine MDMA-d s Methoxyphenamine Methylphenidate Ritalinic acid
91 85 96 86 5
10.8 7.6 5.3 9.5 89.0
Metoprolol Nadolol Sotalol
86 90 56
3.0 5.4 7.4
Codeine Codeine-d3 Levallorphan Nalbuphine
93 91 82 91
4.0 4.5 2.3 6.4
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Journal of Analytical Toxkology, Vol. 19, Mar(h/April 199~
Table III. Relative Retention Time (RRT) to Codeine-d3 and Mass Spectral Data for Some Stimulants, [3-Blockers, ~-Agonists, Narcotics, and Their Metabolites Derivative
RRT
basepeak
Diagnosticions MW*
others
0.42 1.23 1.12 0.28 0.65 0.39 0.45 0.42 0.79 0.26 0.45 0.40 0.38 0.67 1.20 0.37 0.57 0.54 0.34 0.95 0.65 0.66 0.76 0.77 0.56 0.58 0.58 0.58 0.47 0.36 0.53 0.55 0.~6 0.69 (I.47 0.45 0.44 0.40 0.48 0.29 0.39 0.45 0.59 0.35 0.45 0.72 0.51
100 193 193 140 91 179 179 154 125 72 179 86 168 170 346 168 193 81 131 125 267 267 179 179 236 154 ] 58 2] 6 154 154 ] 35 212 72 180 179 100 168 57 167 154 179 179 179 154 179 180 179
205 505 477 231 239 319 391 279 355 163 333 265 259 311 437 327 284 229 313 443 421 353 372 395 ~63 289 294 3[]7 275 245 27~ /47 251 ~2(~ ~47 279 27/ I01 273 245 319 391 ~ ~ 2~1 / ~/ 387 305
190, 105 487, 300 459, 178 118, 91 224, 148 304, 191 376, 212 125, 59 264, 91 118, 91 318, 154 250, 58 140, 91 142, 91 207, 91 308, 140 140, 118 138, 91 186, 140 264, 179 179, 154 338, 86 206, 140 216, 140 209, 154 162, 135 16~, 136 140, 91 140, 91 110, 9] 162, 140 161,135 2 ~6, 16~ 298, 91 //2, 140 I()l, 17!) 140, 105 176, 85 98, 70 230, 91 ~04, 191 376, 212 206, 154 236, 182 H 8, I '~4 /72,118 2fl6, 192
1.25 0.99 0.76 0.90 1.02 1.03 1.02 1.03 0.97 0.91
284 284 284 284 284 284 284 86 235 86
5(]4 576 417 559 417 434 493 364 436 378
242 129 561 129 402 129 544 ] 29 4[]2 ] 29 242 129 478 129 349 163 421,86 363,234
Stimulants and their metabolites Amfepramone (Diethylpropion) Amineptine-N-TFA-O-TMS Amineptine-Cs-N-TFA-O-TMS (metabolite) Amphetamine-N-TFA Benzphetamine Cathine-N-TFA-O-TMS Cathine-bis-N,O-TMS-N-TFAt Chlorphentermine-N-TFA Clobenzorex-N-TFA Dimethamphetamine Ephedrine-N-TFA-O-TMS Etafedrine-O-TMS Ethylamphetamine-N-TFA Fencamfamine-N-TFA Fenetylline-N-TFA Fenfluramine-N-TFA Fenproporex-N-TFA Furfenorex HeptaminoI-N-TFA-O-TMS Hydroxyclobenzorex-N-TFA-O-TMS Hydroxyephedrine-N-TFA-bis-O-TMS Hydroxyetafedrine-N-TFA-bis-O-TMS Hydroxyfenproporex-N-TFA-O-TMS Hydroxymefenorex-N-TFA-O-TMS Hydroxymethoxyphenamine-N-TFA-O-TMS MDMA-N-TFA MDMA-ds-N-TFA Mefenorex-N-TFA Methoxyphenamine-N-TFA Methylamphetamine-N-TFA Methylendioxyamphetamine-N-TFA
Methylendic~xyaml)hetamine-N-TFA-N-TMSt Methylephedrine-O-TMS Methylphenidate N-TFA
N-Ethvln(~rephedrine-N-[FA-O-IMS N, N-Diethyh~orephedr[ne-N-lFA-O-IMS Nordiethylpropion-N-TFA Phendimetrazine Phenmetrazine-N-TFA Phentermine-N-TFA Phenylpropanolamine-N-TFA-O-TMS Phenylt)ropanolamine-bis-N,O-TMS-N-TFAt
Pholedrine-N-TFA-O-TMS Propylhexedrine-N-TFA Pseudoephedrine-C)qMS-N-TFA Ritalinic acid N-TFA-O-TMS Tyramine-N-TFA-O-TMS
/3-Blockers and their metabolites Acebuto[oI-N-TFA-O-TMS AcebutoloI-N-TFA-bis-O-TMSt Alprenok)[-N-TFA-O-TMS AtenoloI-N-TFA-bis-O-TMS* Atenolol-nitriI-N-TFA-O-TMSt,~ AtenoloI-N-TFA-O-TMS* BisoproloI-N-TFA-O-TMS Carteolol-O-TMS Carteolol-bis-O-TMSt CeliproloI-O-TMS * MW = Molecularweight. t Minor derivative. Possiblestructure:an amidegroupof alenolol is supposedto be tost. An amide~un(tion is dehydrated. u S(anmodeacquisitionfrom 50 to 650 amu was used.
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Journalof AnalyticalToxicology,Vol. 19, March/April 1995
Table III (continued). Relative Retention Time (RRT) to Codeine-d3 and Mass Spectral Data for Some Stimulants, [3-Blockers, [3-Agonists, Narcotics, and Their Metabolites Deriva]ive Celiprolol-bis-O-TMS* Diaceto]ol-N-TFA-O-TMS
HydroxyalprenoloI-N-TFA-bis-O-TMS HydroxylabetaloI-N-TFA-tris-O-TMS" HydroxymetoproloI-N-TFA-bis-O-TMS HydroxyoxprenoloI-N-TFA-bis-O-TMS HydroxypenbutoloI-N-TFA-bis-O-TMS 4-Hydroxypropranolol-N-TFA-O-TMS 3-Hydroxypropranolol-N-TFA-O-TMS Labetalol-nitriI-N-TFA-bis-O-TMSisomer 1 Labetalol-nitriI-N-TFA-bis-O-TMSisomer21 LevobunoloI-O-TMS MethoxyhydroxypropranoloI-N-TFA-bis-O-TMS Metoprolol-N-TFA-O-TMS Nadolol-tris-O-TMS Oxprenolo]-N-TFA-O-TMS PenbutoloI-O-TMS PindoloI-N-TFA-bis-N,O-TMS
Pindolol-bis-N,N-TFA-O-TMS~ PractoloI-N-TFA-bis-N,O-TMS PractoloI-N-TFA-O-TMS~ Propranolol-N-TFA-O-TMS SotaloI-N-TFA-bis-O-TMS SotaloI-N-TFA-O-TMSf TimoloI-O-TMS
RRT
basepeak
Diagnosticions MW*
others
0.93 0.95 0.90 1.39 0.96 0.93 0.88 1.05 1.07 1.13 1.14 0.95 1.12 0.88 0.99 0.80 0.82 1.02 0.91 1.03 1.04 0.93 0.96 0.98 0.90
86 284 284 292 284 284 86 284 284 292 292 86 284 284 86 284 86 284 284 284 284 284 344 272 86
450 548 505 638 523 521 451 515 515 550 550 363 545 435 525 433 363 488 512 506 434 427 512 440 388
435,200 533,129 242,129 623,179 478,129 506,129 436,365 242,129 242,129 535, 91 535, 91 348 242,129 420,129 510,409 418 129 348 57 242 129 497 129 491 129 242 129 419 129 497 126 193 126 373 130
0.73 0.80 0.76 0.75 0.70
86 86 355 369 356
349 421 523 455 441
333,262 406,335 508,168 440,86 426,86
0.63 1.24 1.00 1.00 0.84 O.87 0.97 0.98 1.01 0.67 1.02 0.91 1.00 0.95 1.02 0.93 0.87 0.84 1.06 1.03 1.28 1.04 1.02 1.03 1.06 0.72 1.02 1.08 0.90 0.60 1.10 1.47
172 246 371 374 271 59 373 431 112 57 385 395 371 453 429 355 59 72 399 429 573 453 315 373 511 241 459 445 289 71 302 114
261 448 371 374 271 329 373 431 349 261 385 395 371 453 429 355 329 309 399 429 573 453 455 513 511 329 459 445 357 247 393 470
187,84 447,375 234,178 237,181 214,59 272,150 315, 236 236,]46 334,165 188 246 357 327 380 338 356 313 438 381 414 357 272 176 272 150 294 91 340 287 414 401 518 428 313 282 255 225 455 315 496 281 256 143 370,312 430,287 342,245 218,172 378,229 356,100
,6-Agonists ClenbuteroI-O-TMS Clenbuterol-bis-N,O-TMSt Orciprenaline-tris-O-TMS-N-TFA Salbutamol-tris-O-TMS Terbutaline-tris-O-TMS
Narcotics and their metabolites Alphaprodine Anileridine-N-TFA Codeine-O-TMS Codeine-d~-O-TMS Dextromethorphan Dextrorphan-O-TMS Dihydrocodeine-O-TMS Dihydromorphine-bis-O-TMS Dipipanone Ethoheptazine Ethylmorphine-O-TMS Hydrocodone-O-TFA Hydrocodone-O-TMS~ Hydromorphone-O-TFA-O-TMS Hydromorphone-bis-O-TMSt Levallorphan-O-TMS LevorphanoI-O-TMS Methadone Monoacetylmorphine-O-TMS Morphine-bis-O-1MS Nalbuphine-tris-O-TMS Norcodeine-N-TFA-O-TMS Nordihydrocodeine-N-TFA-O-TMS Nordihydromorphine-N-TFA-bis-O-TMS Normorphine-N-TFA-bis-O-TMS Norpethidine-N-TFA Oxycodone-bis-O-TMS Oxymorphone-bis-O-TMS Pentazocine-O-TMS Pethidine Phenazocine-O-TMS Pholcodine-O-TMS
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Journal of Analytical Toxicology, Vol. 19, March/April 1995
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iOr.O0
~2'.o0
t3'.oo
~4.oo
284
1 3 5 7 9
B
0 __?--'.-/!Z-
Alprenolol Metoprolol Propranolol Pindolol Acebutolol
2 4 6 8
Oxprenolol Atenoloi Bisoprolol Practolol
2 4
Timolol Levobunolol Carteolol
OBSERVATIONS: ......
,
12,O3
6.55 86 2~311(
C
1 3 5
2.3
J
PenbutoIol Celiprolol Nadolol
6
OBSERVATIONS:
I
i 9.58
158
371
6~,,0
i t
D
I F
F;I
0 _ _
i~
'~
_
.
tO.83
q.O6
Labetalol (2 isomers) 3aa
o .............. 9 Sotalol
! 9.18
8.19
'=1o
429
G
J:i
9.12
1
T
9.01
10.01
Codeine-d3 (reference) MDMA-d5 (reference) Codeine Morphine
7 ~7
Morphine
9 97
Codeine-d~ (reference)
4 ~1
6 ~)
MDMA-d~ Irefercncc)
Expected
Actual
FACTOR
AREA
ALTITUDE
CONC. (pg/mL)
8.97 5.31 8.98 9.21
8.97 5.31 8.98 9.20
1.01 2.33
164443 310651 163235 70548
109286 204096 121528 45539
1,00 1.00 1.00 1.00
tR (min) SUBSTANCE
I
Codeine
L. . . . .
8.T4
H
RESPONSE
Figure 1. Report format for [3-blockers and some narcotics. (A) Height-scaled total ion chromatogram. (B-E) Four height-scaled selected ion chromatograms (B, m/z 284; C, m/z 86; D, m/z 292; and E, m/z 344) to trace the presence of D-blockers. The updated expected retention times are shown, if-G) Two height-scaled selected ion chromatograpms to trace the presence of codeine (m/z371) and morphine (m/z 429). The integrations and the actual retention times of the identified peaks are shown. (H-I) Two reference substance identification chromatograms showing the integration of the identified peaks and their actual excretion times. Codeine-d3 is used for updating expected retention times and as an internal standard; MDMA-d~ is added as a marker for N-TFA derivatization. A report table shows integrated substance metrics. The spiked urine (control CALl) contains codeine, morphine, and the deuterated internal standards, as well as atenolol, bisoprolol, labetalol, nadolol, oxprenolol, penbuto[ol, practolol, propranolol, and timolol. It also contains the compounds listed in Figure 2.
110
Journal of Analytical Toxicology, Vol. 19, March/April 1995
Excretion studies of doping agents in healthy volunteers were analyzed using the presented method. The mass spectra of all parent drugs and some metabolites were verified with authentic substances. In most cases, confirmation of identity of the main metabolites found in human urine was obtained by combining File:
2A0040201002.d
Computer:
Sample:
CALl L932A004
Lot:
24I
1075I ~
~
mass spectral information and references published elsewhere (2,7,11,19). In instances where drugs were not available for excretion studies, the analytical information corresponds to the analysis of urine specimens spiked with reference substances at concentrations compatible with actual ingestions. The lack of
hpux3 L932A004 271
NORPETHIDINE 4-phenyl-4-piperidineearboxilir
DEXTROMETHORPHAN 3-methoxy- 17-methylmorphinan
acid ethyl
ester N - T F A 0
SAlt
0 l
6.47
6.96
OBSERVATIONS: -
7.01
7.50
It.CO
OBSERVATIONS: Not banned
219
329 1535
PENTAZOCINE (2a,6ec, 11R*)- 1,2,3,4,5,6-hexahydm-6, I Idimethyi-3 -0 -methyl-2-butenyll-2,6-rrmhano-3 -
DEXTRORPHAN ILEVORFANOL | 7- methylmorphinan-3 -ol O - T M S ,A
benzazocin-8-ol O-TMS
0
7.27
7.76
8.26
OBSERVATIONS: Enantiomers
?.$6
1.06
OBSERVATIONS:
-
373
ETHYl,MORPHINE ?,8-didehydro-4,5-epoxy-3 -elhoxyI 7-methylmorphinan-6-ol O - T M S
DIHYDROCODEINE 4,5-epoxy -3-methoxy- 17-methylmorphinan-6..ol O-TMS 8.19
8.68
9.18
OBSERVATIONS:
-
$.~
3520~ ~ [ ~~ ! i
9A3
9.62
OBSERVATIONS: -
5~
399
NALBUPHINE I 7-(c yclobutyl methyl)-4,5-epoxy-motphinan3,6.14-triol tris-O-TMS
MONOACETYLMORPHINE (Sc~,6cr]-7,8-didehydro-4,5 -epoxy- i 7methylmorphinan-6-ol acetate O-TMS
l
0
8.98
9.47
997
OBSERVATIONS:
-
5t.04
53.0I
I3.5t
11.53
52.02
OBSERVATIONS:
-
172
114
53.98
PHOLCODINE t632 7,8-didehydro-4,5.-epoxy- 17-methyl3 -[2 -(4-morpholinyl)ethoxy|-mo~hinan-6-ol O-TMS o OBSERVATIONS: Not banned s,59
ALPHAPRODINE i ,3 -dimethyl-4-phenyl-4-piperidinal propanoate
s.a
e.t7
OBSERVATIONS:
-
112
'
i
0 I 1,62
12.12
I/~i . 1261
DIPIPANONE
ANILERIDINE I -[2-(4-a minophenyl)ethyl]-4-phenyl-4plperidinecarboxyllc acid ethyl ester N - T F A
4,4-diphenyll-piperidinyl)-3-heptanone -6-( 0
OBSERVATIONS: -
8,61
9.10
9..9J
OBSERVATIONS: -
57
HYDROCODONE 4,5-epoxy-3 -methoxy- 17-methylmoq~hinaa-6 -one O-TFA
ETHOHEPTAZINE hexahydro- i -methyl-4-phenyl- I H-azepine-4carboxylic acid ethyl ester 0
5.51
6.OO
6.5O
OBSERVATIONS: -
7.TI
453
8.22
11.72
OXYCODONE
HYDROMORPHONE 4,5-epoxy-3-hydroxyd 7methylmorphinan-6-one O - T M S - O - T F A 802
851
9,01
OBSERVATIONS: Various derivatives
4,5-epoxy- 14-hydroxy-3-n~thoxy-
. 9.65
0 11.68
9A$
9.6?
OBSERVATIONS: Variousderivatives
OBSERVATIONS: Variousderivatives PHENAZOCINE 1.2,3,4.5,6-hexahydro-6, I I-dimethyl-3 (2-phenethyl)-2,6-methano-3-3-be~zocin-8-ol O-TMS
0
L015
t 7-
mcthylmorphina n-6-one BI$-O-TMS
OXYMORPHONE 4..5-epoxy-3.14-dihydroxy- 17me~hytmorph~nan-6-onr b i s - O - T M S 0 915
OBSERVATIONS: Variousderivatives
4~
9.,~
9.89
]0.38
OBSERVATIONS: -
Figure 2. Report format for narcotics analysis. Window ion chromatograms for monitoring several narcotics are included. The spiked urine (control CALl) contains dihydrocodeine, ethylmorphine, nalbuphine, pentazocine, and pholcodine. It also contains the I~-blockers listed in Figure 1 and the deuterated internal standards.
111
Journal of Analytical Toxicology, Vol. 19, March/April 1995
File: Sample:
2A0040301003.d ORP1L932A004
Computer: hpux3 Lot: L932A004
TIC
4711913
A
I 2.O4
,i|
3~.00
6.=
~.00
7'.|
i.=
r
jo'.=
12'.00
l I'.00
13',00
13,99
2 EphedrinelPseudoeph
1 Norpseudoeph/Norephedrine
B OBSERVATIONS: 4.63
!
154
2 Propylhexedrine 4 Chlorphentermine 6 MDMA
l Phentermine 3 Methylamphetamine 5 Methoxyphenamine
C
OBSERVATIONS: i 6.1g
2.04
193
180
140
571328
87aLklg 9
D
E
t35 t1019
t,
F
0 Z~
3,7O
5,31
7.37
9,50
Mcthylphenidate/Ritalinic Acid 168
Amphetamine 167 1991
221849(
10.69
i 3.9~
Amineptinc-C5 Mctabo]itr 17o
f 4.95
MDA 179 215910~
794320
K
H 0 . . . . . .
i 3.83 Phenmetrazine
4.81
3.~ 4.93 Fcnfiure mine/Nordicthylproplon
5.56
Fcncamfumin
6.55
4.~
$.$2
Pholcdrinr
Figure 3. Report format for stimulants analysis. (A) Total ion chromatogram; (B-C) two selected ion chromatograms (m/z 179 and 154) to trace the presence of hydroxyphenylalkylamines and phenylalkylamine analogues; and (D-K) some time window ion chromatograms to monitor several stimulants. The spiked urine (control ORP1) contains amphetamine, chlorphentermine, ephedrine, fencamfamine, fenfluramine, methoxyphenamine, methylphenJdate, phenylpropanolamine, pholedrine, propylhexedrine, and ritalinic acid. It also contains the deuterated internal standards.
112
Journal of Analytical Toxicology, Vol. 19, March/April 1995
metabolic information for these compounds does not mean that they were absent from human urine specimensnor does it mean that there were detection problems related to the analytical method; it simply indicates their unavailability.More than 100 substances, together with their diagnostic ions, were detected with this analytical method and are listed in Table III. In a few cases, the derivatization procedure with MSTFA-MBTFAresulted in the formation of more than one TMS-TFAderivativeof a compound; minor derivatives of these substances are labeled as such in Table III. The amounts detected for each substance were high enough to allowthe use of mass spectrometry with data acquisition in scan mode. The urinary level of each substance is related to the dose given and the collected urine volume. Working in scan mode has some advantages as compared with the SIM mode; one in particular is that a full spectrum can be obtained for the suspicious substance. The ~-agonist agents are the exception to the rule, as they have to be detected in SIM mode because of their low therapeutic doses. A selective derivatization to form cyclic boronates is recommended to obtain more sensitivity and a more characteristic fragmentation pattern (24-26). Although this method can be applied to many different fields, such as pharmacology, toxicology, and pharmacokinetics, it was designed for urinalysis in doping control. In this case, laboratories are confronted with the problem of dealing with a large proportion of negative samples (more than 98%) and at the same time guaranteeing the absence of a list of banned compounds. The printed report of the screening procedure (see Figures 1-3) is a set of mass chromatograms obtained in scan mode with only the most prominent ion shown for each substance in a given window of retention times. Some selected ions (m/z 284, 86, 179, and 154) have been chosen to trace several compound families ([~-blockersand stimulants; see Table III). This report format has several advantages, one of which is the capacity to condense relevant information on monitored substances (approximately70 compounds in a single report) in a few sheets of paper while still maintaining highresolution graphics. In addition to the amount of paper saved as compared with other commercial software packages, the main advantage of this condensed approach is that it enables the analyst to review results concerning many substances in a few seconds. This fact is especially important when dealing with hundreds of samples that need to be analyzed in a short period of time, although it is useful in any situation regardless of the sample workload and the time pressure for releasing results. In conclusion, we have proposed an improved screening method for stimulants, 13-adrenergic drugs (agonists and blockers), narcotic analgesics, and their metabolites in urine. It is simple, fast, and easily lends itself to automation.
Acknowledgments
Wewish to thank Jos~ Antonio Pascual and Rob Ewin for the design and development of the in-house software used to generate analyticalreports, and we also thank Javier Morano for his technical assistance.
References I. Medical Commission, International Olympic Committee. List of doping classes and methods. In International Olympic Charter against Doping in Sport. IOC, Lausanne, Switzerland, 1990, (update May 1992). 2. D.S. Lho, J.K. Hong, H.P. Paek, J.A. Lee, and J. Park. Determination of phenolalkylamines, narcotic analgesics, and beta-blockers by gas chromatography/mass spectrometry. J. Anal. Toxicol. 14: 77-83 (1990). 3. M. Kraft. Approach to combine the present analytical methods for the detection of dope agents to a comprehensive screening procedure with GC/MS detection. In Official Proceedings: lind. I.A.F. World Symposium on Doping in Sport. P. Bellotti, G. Benzi, and A. Ljungqvist, Eds., International Athletic Foundation, Monte Carlo, Monaco, 1990, pp 93-106. 4. P. Lillsunde and T. Korte. Comprehensive drug screening in urine using solid-phase extraction and combined TLC and GC/MS identification. J. Anal. Toxicol. 15' 71-81 (1991). 5. B.K. Logan, D.T. Stafford, I.R. Tebbett, and C.M. Moore. Rapid screening for 100 basic drugs and metabolites in urine using cation exchange solid-phase extraction and high-performance liquid chromatography with diode array detection. J. Anal. Toxicol. 14" 154-59 (1990). 6. K. Chert, J. Wijsbeek, J. Van Veen, J.P. Franke, and R.A. de Zeeuw. Solid-phase extraction for the screening of acidic, neutral and basic drugs in plasma using a single-column procedure on Bond Elut Certify. J. Chromatogr. 529:161-66 (1990). 7. D.S. Lho, H.S. Shin, B.K. Kang, and J. Park. Systematic analysis of stimulants and narcotic analgesics by gas chromatography with nitrogen specific detection and mass spectrometry. J. Anal. Toxicol. 14:73-76 (1990). 8. M. Donike. Overview on present analytical procedures in dope analysis. In Official Proceedings: lind. I.A.F. World Symposium on Doping in Sport. P. Bellotti, G. Benzi, and A. Ljungqvist, Eds., International Athletic Foundation, Monte Carlo, Monaco, 1990, pp 83-92. 9. R.D. McDowall. Sample preparation for biomedical analysis. J. Chromatogr. 492:3-58 (1989). 10. M.S. Leloux, E.G. de Jong, and R.A.A. Maes. Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography-mass spectrometry. J. Chromatogr. 488:357-67 (1989). 11. M.S. Leloux and R.A.A. Maes. The use of electron impact and positive chemical ionization mass spectrometry in the screening of beta blockers and their metabolites in human urine. Biomed. Environ. Mass Spectrom. 19:137-42 (1990). 12. C.L. Davies. Chromatography of 13-adrenergic blocking agents. J. Chromatogr. 531 : 131-80 (1990). 13. M.C. Dumasia and E. Houghton. Screening and confirmatory analysis of [3-agonists, ~-antagonists and their metabolites in horse urine by capillary gas chromatography-mass spectrometry. J. Chromatogr. 564:503-13 (1991 ). 14. R.W. Taylor, S.D. Le, S. Philip, and N.C. Jain. Simultaneous identification of amphetamine and methamphetamine using solidphase extraction and gas chromatography/nitrogen phosphorous detection or gas chromatography/mass spectrometry. J. Anal. Toxicol. 13:293-95 (1989). 15. A. Solans, R. de la Torre, and J. Segura. Determination of morphine and codeine in urine by gas chromatography-mass spectrometry. J. Pharm. Biomed. Anal. 8:905-909 (1990). 16. B.A. Goldberger, W.D. Darwin, 1-.M. Grant, A.C. Allen, Y.H. Caplan, and E.J.Cone. Measurement of heroin and its metabolites by isotope-dilution electron-impact mass spectrometry. Clin. Chem. 39:670-75 (1993). 17. J. Schuberth and J. Schuberth. Gas chromatographic-mass spectrometric determination of morphine, codeine and 6-monoacetylmorphine in blood extracted by solid phase. J. Chromatogr. 490: 444-49 (1989).
113
Journal of Analytical Toxicology,Vol. 19, March/April 1995 18. W. Huang, W. Andollo, and W.L. Hearn. A solid phase extraction technique for the isolation and identification of opiates in urine. J. Anal. Toxicol. 16:307-10 (1992). 19. H.H. Maurer. Systematic toxicology analysis of drugs and their metabolites by gas chromatography-mass spectrometry. J. Chrornatogr. 580:3-41 (1992). 20. J.A. Pascual, R.R. Ewin, and J. Segura. Automated control of doping samples and their analyses preparing for Barcelona '92. Part I. Development of a new laboratory information management system (LIMS) for doping control. In 10th Cologne Workshop on Dope Analysis. M. Donike, H. Geyer, A. Gotzmann, U. MareckEngelke, and S. Rauth, Eds., Sport und Buch Straug, K01n, Germany, 1993, pp 345-67. 21. R.R. Ewin, J.A. Pascual, and J. Segura. Automated control of doping samples and their analyses preparing for Barcelona '92. Part II. Automation, reporting and the local area network. In 10th Cologne Workshop on Dope Analysis. M. Donike, H. Geyer, A. Gotzmann, U. Mareck-Engelke, and S. Rauth, Eds., Sport und Buch Straug, KOIn, Germany, 1993, pp 369-87.
114
22. M. Donike. N-TrifluoracetyI-O-trimethylsilyl-phenolalkylamine: Darstellung und massenspezifischer gas-chromatographischer nachweiss in femtomol-bereich. J. Chromatogr. 103:91-97 (1975). 23. A. Solans, M. Carnicero, R. de la Torre, and J. Segura. Simultaneous detection of methylphenidate and its main metabolite ritalinic acid in doping control. J. Chromatogr. 658:380-84 (1994). 24. J. Zamecnik. Use of cyclic boronates for GC/MS screening and quantitation of beta-adrenergic blockers and some bronchodilators. J. AnaI.Toxicol. 14:132-36 (1990). 25. A. Polettini, M.C. Ricossa, A. Groppi, and M. Montagna. Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography-mass spectrometry. J. Chromatogr. 564: 529-35 (1991 ). 26. A. Polettini, A. Groppi, M.C. Ricossa, and M. Montagna. Gas chromatographic/electron impact mass spectrometric selective confirmatory analysis of clenbuterol in human and bovine urine. Biol. Mass Spectrom. 22:457-61 (1993). Manuscript received January 20, 1994; revision received July 5, 1994.
