Anal. Chem. 1992, 64, 1578-1585
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Determination of Lysergic Acid Diethylamide (LSD), Iso-LSD, and NDemethyl-LSD in Body Fluids by Gas Chromatography/ Tandem Mass Spectrometry Chad C. Nelson* and Rodger L. Foltz
Northwest Toxicology, Inc., Salt Lake City, Utah 84124
Procedure8for detection and quantnatlon of ly8ergk acid dC ethylamlde (LSD), b L S D , and MemethyCLSD by caplllary chrmatography/tandemmass8pectrometry(oC/MS/MS) are presented. Several method8 for derlvatlzatkn, sample Introduction, and lonizatlon, in combhation with ma88 8pectrometry/ma88 8pectrometry (MS/MS), have been evaluated for overalllonizatlondfkiency and product-lon wnrltlvtty and 8peMcftv. Fragmentath pathway8derivedfrom low-energy coYdorrlnducedd~tkn(CID)rpectraofprotonaedLSD, and the protonated trknethykllyi derivative8 of LSD (LSDTMS) and deuterlum4abeied a n a m of LSD, have been proposed. Prlnclpaidkrociations prknarlly Involve the amide and plperklh-rlng mdab In which io88e8 of CHs radkal, CHaNHz,CHaNCH2,dlethylamlne,dlethytformamlde,and N,K diethylpropenamlde from MH+ are observed. Podtlvdon ammonla chemkalknizatlon and 8ub8equent MS/MS analydr of the protonatedmolecules (MH+)of the trlmethykllyl (TMS) derlvatlve8 of LSD, b L S D , and MemethyCLSD provide a highdegree of 8peMclty for ldentlfkatlonof these compoundr in urine or bkod at low-pg/mL concentrations. Negatlvalon chemkallonlzatlonand GC/MS/MS analydr of the molecular anlon (I&) of the tritluoroacetyl(TFA) derlvatlve is wdl8uHed for tracdevel ldentkatlon of MemethyCLSD, a metabdlte of LSD.
INTRODUCTION LSD, a popular psychedelic drug of the 19608,continues today to be a significantdrug of abuse.192 There are numerous anecdotal indications of its use, particularly among adolescents. However, documented evidence regarding the extent of LSD use is limited, which may stem partly from the difficulty in detecting this illicit drug and its metabolites in body fluids. LSD is an extremely potent hallucinogenderived from lysergic acid, a member of the ergot alkaloid group of natural p r ~ d u c t s .Of ~ the four possible synthetic diastereomers of LSD only d-LSD has mind-altering proper tie^.^^^ Iso-LSD, a diastereomer of LSD reported to be devoid of psychoadivity,4 is often present in illicit LSD preparations and sometimes constitutes the major component. LSD undergoes such rapid and extensive metabolism that only about 1?6 of the parent drug is excreted in human urine.6 Several LSD metabolites have been identified in laboratory (1)LSD Seizures. Microgram 1990,23,228. (2)New tests for LSD and fentanyl (China White). The Forensic Drug Abuse Advisor 1990,2,61. (3)Eckert, H.; Kiechel, J. R.; Rosenthaler, J.; et al. In Handbook of Experimental Pharmacology, Vol. 49; Ergot Alkaloids and Related Compounds; Berde, B., Schild, H. O., Eds.; Springer: Berlin, 1978;pp 719-803. (4)Isbell, H.; Miner, E. J.; Logan, C. R. Psychopharmacologia 1959, 1, 2. (5)Rossi, V. Am. J. Pharm. 1971,March-April, 38. (6)Lim, H. K.; Andrenyak, D.; Francom, P.; Foltz, R. L. Anal. Chem.
1988,60,1420. 0003-2700/92/0384-1578SO3.0010
animals7 and in humans,6J although the major metabolic producta have not yet been determined. As a result, analytical methods have been aimed primarily toward identification and quantitation of the unchanged parent drug. The N8demethyl metabolite of LSD has been characterized recently in human physiological fluids6 and can be employed as additional evidence for consumption of LSD. Because LSD is ingested in small quantities (typical dosage of 20-80 pg) and its elimination half-life is relatively short (ca. 3.6 h),6 the concentration of LSD in the blood and urine of an LSD user generally drops to the sub-nanogram/milliliter level within a few hours after ingestion. Thus, identification of LSD in body fluids has been a challenging analytical problem for forensiclaboratories and poses a continuing concern for drug enforcement agencies. A number of analytical techniques have been reported for detecting LSD in biological specimens, including radioimmunoassay (RIA),&" high-performance liquid chromatography (HPLC) with fluorescence detection,lP14gas chromatography/mass spectrometry (GUMS),6J6-17andliquid chromatography/mass spectrometry (LC/MS).la However, due to the complex nature of urine, blood, and other biological matrices,most procedures for extraction and isolation of LSD yield samples with varying degrees of purity. As a result, these analytical methods are frequently hindered by coextractants which can severelyinterfere with identification and quantitation of LSD, LSD metabolites, and internal standards. Therefore,without agreater degreeof specificity,some analyses will not provide the degree of confidence in identification of LSD that is required for forensic purposes. An additional problem for laboratories using gas chromatography for the analysis of LSD is the strong tendency for the drug to undergo irreversible adsorption during the chromatographic process, often preventing the detection of the drug at the sub-nanogram/milliliter concentrations normally encountered in body fluids from LSD users. (7)Foltz, R. L.; Foltz, R. B. In Advances in Analytical Toxicology, Vol. ZI; Baeelt, R. C., Ed., Yearbook Medical Publishera, Inc.: Chicago, IL,1989;pp 140-168. (8)Ratcliffe, W.A,; Fletcher, S. M.; Moffat, A. C.; Ratcliffe, J. G.; Harland, W. A.; Levine, T. E. Clin. Chem. 1977,23,169. (9) Tauntan-Rigby, A.;Sher, 5. E.; Kelley, P. R. Science 1973,181, 165. (10)Castro, A.; Grettie, D. P.; Bartos, F.; Bartos, D. Res. Commun. Chem. Pathol. Phnrmacol. 1973,6,879. (11) Loeffler, L. J.; Pierce, J. W. J. Pharm. Sci. 1973,62,1817. (12)Christie, J.; White, M. W.; Wiles, J. R. J. Chromatogr. 1976,120, 496. (13)Twitchett, P. J.;Fletcher, S. M.; Sullivan, A. T.; Moffat, A. C. J . Chromatogr. 1978,150,73. (14)McCarron, M. M.; Walberg, C. B.; Baselt, R. C. J.Anal. Toxicol. 1990,14,165. (15)Paul, B. D.; Mitchell, J. M.; Burbage, R.; Moy, M.; Sroka, R. J. Chromatogr. 1990,529,103. (16)Papac, D. I.; Foltz, R. L. J. Anal. Toxicol. 1990,14,189. (17)Francom, P.; Andrenyak, D.; Lim, H.-K.; Bridges, R. R.; Foltz, R. L.; Jones, R. T. J. Anal. Toxicol. 1988,12,1. (18)Kenyon, C. N.; Melera, A.; Erni, S. J. Chromatogr. Sci. 1980,18, 103. (9 1992 A t "
Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
Tandem mass spectrometry offers several advantages and additional capabilities over conventional mass spectrometry.l9@ For analysis of complex mixtures, a principal strength of MSIMS lies in the high degree of selectivity provided by an additional stage of mass separation (the first mass analyzer). MS/MS can be used to selectively separate and identify components of different molecular weight but of similar chromatographic retention time. An added dimension of specificity is attained with MSIMS through collisioninduced dissociation of a selected precursor ion (parent ion) to generate characteristic product ions (fragment ions), which are then analyzed with the second mass analyzer. Specific dissociations arising from CID can uniquely identify a particular compound and can be used to make distinctions between compounds with the same nominal molecular weight. Also, differentiation of isomers often can be achieved on the basis of relative product-ion abundance8 and unique dissociations.21 Therefore, the combination of gas chromatography (GC) or liquid chromatography (LC) with MS/MS affords both chromatographic separation and retention-time information, as well as mass separation and monitoring of specific collision-induced dissociations. Due to increased specificity, transmission of chemical noise is reduced and sensitivity is often improved correspondingly. Various MS/MS scan functions can serve as valuable tools for determining dissociation pathways based on precursor-to-product ion transitions. This ‘mapping” of related fragment ions is useful for structural determinations and can be a powerful means of identifying similar structures and families of related compounds, such as in metabolite research.22123 The increased specificity gained with tandem mass spectrometry can be particularly important when dealing with complex sample matrices. Liquid chromatography with an electrospray ionization interface, in conjunction with tandem mass spectrometry (LC/ MS/MS), has been employed recently for analysis of underivatized LSD, with promising In addition, an unpublished report submitted to the Naval Research Laboratory describes direct analysis of underivatized LSD by means of chemical ionization (CI) and tandem mass spectrometry and includes a brief evaluation of analysis of LSD in urine.% The tandem mass spectrometry methods described in this paper for the determination of LSD, iso-LSD, and N-demethyl-LSD in urine and blood offer significantly improved sensitivity and specificity compared to published GUMS assays for LSD. As part of the fundamental development of an MSIMS assay, product ions resulting from collisional activation of protonated LSD and LSD-TMS were characterized. Decomposition paths for N-demethyl-LSD closely parallel those of LSD, and dissociation paths analogous to LSD and N-demethyl-LSD can be applied to the identification of other possible metabolic products, as well. In this study, different derivatives (trimethylsilyl and trifluoroacetyl), (19) Buech, K. L.; Glieh,G.L.; McLuckey,S. A. InMSIMS Techniques and Applicatwna of TandemMass Spectrometry; VCH New York, 1989. (20) Tandem MassSpectrometry;McLafferty,F. W., Ed.;Wiley: New York, 1983. (21) For example: (a) Dolnikowski, G. G.;Gross, M. L.; Cavalieri, E. L. J. Am. SOC.MassSpectrom. 1991,2 (3), 256. (b)Proctor, C. J.; McLafferty, F. W. Org. Mass Spectrom. 1983,18,193. (c) Weiez, A.; Iberkleid, E.; Maudelbaum, A.; Blum, W.; Richter, W. J. Org. Mass Spectrom. 1987, 22,3. (d) Unger, S. E. Znt. J. Mass Spectrom. Zon R o c . 1985,66,196. (22) Perchalski, R. J.; Yost, R. A.; Wilder, B. J. AnaZ. Chem. 1982,54, 1466. (23) Straub, K. M. In Mass Spectrometry in Biomedical Research; Gaekell, S., Ed.;Wiley New York, 1986. (24)Hennion, J.; Wachs,T.;Foltz,R.Roceedingsofthe39thAmerican Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, Naehville, TN,May 1991; pp 1653-1654. (26)Yoet, R. A. Final report to the Naval Research Laboratory, Washington, D.C., June 20, 1985, Contract No. “14-84-M-0284.
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methods of sample introduction (GC and direct-exposure probe), and ionization techniques (positive- and negative-ion chemical ionization with ammonia or methane as the reagent gas) in conjunction with MS/MS analysis of protonated LSD, iso-LSD, and N-demethyl-LSD have been compared for overall ionization efficiency and product-ion sensitivity and specificity. EXPERIMENTAL SECTION Materials. d-Lysergicacid diethylamide (LSD)standard was obtained from Alltech-Applied Science (Deerfield, IL). [NBmethyl-2HdLysergicacid diethylamide (LSD-ds),lysergic acid methylpropylamide (LAMPA),and NB-demethyllysergicacid diethylamide (N-demethyl-LSD)standards were purchased from Radian Corp. (Austin, TX). [2HlolLysergicacid diethylamide (LSD-dlo) was kindly provided by David Kidwell of the Naval ResearchLaboratory, Washington, D.C. d-bo-LSD was obtained through the National Institute on Drug Abuse (NIDA). (Trifluoroacety1)imidazole (TFAI) and N,O-bis(trimethylsily1)trifluoroacetamide (BSTFA) derivatizing reagents were purchased from Pierce Chemical Co. (Rockford, IL). Bond-Elute Certify columns (LRC 10 mL, No. 1211-3050)from Analytichem International (Harbor City, CA) were used for solid-phaseextractions. Extraction solvents were HPLC-grade from Fisher Scientific (Pittsburgh,PA). Argon (researchgrade),ammonia,and methane (ultra-high purity) gases were purchased from Air Producte (Allentown, PA). All other chemicals were reagent grade and commercially available. Urine Sample Preparation. A methanolic solution of LAMPA, used as the internal standard, is added to a 4-mL aliquot of urine in a 15-mL screw-capglass tube to give a concentration of 400 pg/mL. The sample is made basic by addition of 300 pL of saturated ammonium carbonate and 200 pL of 2 M sodium hydroxide and briefly vortexed. Five milliliters of toluenelmethylene chloride (7:3 v/v) is added, and the sample and solvent are mixed gently for 30 min. After mixing, the tube is centrifuged. The organic layer is transferred to a clean tube, 5 mL of 0.1 M ammonium hydroxide is added, and the solution is mixed and centrifugedagain. The toluene/methylene chloride organic layer, containing LSD and related compounds,is transferredto a clean, conical glass tube; care must be taken not to transfer any of the aqueous material. To determine the extraction efficiency, three control urines containing 400 pg/mL of LSD were each divided into two equal aliquots; the internal standard (LAMPA) was added to one set of aliquots before extraction and to the other set of aliquots after extraction. By comparing the ratio of LSD to LAMPA in each pair of aliquots, the extraction efficiency for LSD was determined to be approximately 65 % . Blood Sample Preparation. Five milliliters of acetonitrile is added to a 2-5-mL aliquot of whole blood or plasma in a screwcap glass tube, and a sufficient amount of LAMPA is added as the internal standard to give a concentration of 400 pg/mL. The bloodlacetonitrilemixture is vortexed for several minutes and then centrifuged, and the acetonitrilesupernatant is transferred to a clean tube. This initial step permits LSD extraction from either whole blood or hemolyzed blood. The acetonitrileextract is then evaporated to near dryness and reconstituted in 2 mL of 0.1 M HCl. The aqueous solution is washed with 5 mL of hexane, and the hexane layer is discarded. The pH is adjusted to between 9 and 10 by addition of 100 pL of 2 M NaOH and 300 pL of saturated ammonium carbonate. In a manner similar to the urine extraction procedure, LSD is extracted from the basic aqueous solution with 5 mL of toluene/methylene chloride (73 v/v) by rotary mixing for 30 min, followed by centrifugation.The organic layer is transferred to a clean tube and washed with 5 mL of 0.1 M NHdOH. The toluene/methylene chloride layer is then transferred to a clean glass tube and derivatized as described below. Attempts to analyze badly hemolyzed blood samples have resulted in degradation of the chromatographic column, presumably due to coextraction of nonvolatile lipophilic matenals from the blood. Columndegradation can be significantly reduced by additional sample cleanup through a combination of liquid/ liquid and solid-phase extractions. The solid-phase extraction
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Table I. Product Ions from Collision-Induced Dissociation of MH+ and M- of LSD, Iso-LSD, N-Demethyl-LSD, and LAMPA. relative masslcharge (relative intensity) of product ions from CID of the precursor ionb retention time anal* 324 (MH+)(56), 309 (3), 293 (3), 281 (19), 251 (3), 223 (loo), 222 (61,221 (5), 208 (5% 207 (2% LSD (underivatized) 197 (25), 194 (9), 192 (12), 191 (5), 182 (17), 180 (54), 128 (121,100 (W, 74 ( 2 3 , 72 (14) 396 (MH+)(>loo), 381 (3), 353 (38), 323 (6.5), 295 (82), 280 (45), 269 (16), 264 (3), 254 (12), 1.ooO LSD-TMS iso-LSD-TMS
0.979
N-demethyl-LSD-TMSc LAMPA-TMS
1.020 1.030
LSD-TFA
1.000
iso-LSD-TFA
0.996
LAMPA-TFA
1.030
N-demethyl-LSD-TFA
1.050
252 (la), 196 (7), 128 (9), 100 (lo), 73 (100) 396 (MH+)(>loo), 381 (5), 353 (loo), 323 (9,295 (46), 294 (5), 293 (5), 280 (97), 269 (lo), 252 (27)s 240 (7), 100 (15), 73 (12) 382 (MH+)(77), 309 (13), 281 (loo),264 (12), 255 (ll),251 (6), 180 (7), 128 (8), 100 (8), 73 (38) 396 (MH+)(>loo), 353 (17), 323 (8), 295 (loo), 292 (6), 280 (57), 269 (30), 264 (6), 254 (9), 252 (lo), 240 (5),221 (6), 196 (lo), 128 (25), 100 (9),73 (23) 419 (M-) (>loo),399 (3), 371 (15), 356 (lo), 346 (33), 345 (8), 322 (loo), 321 (6), 298 (21), 292 (26), 291 (14), 279 (16), 264 (8), 250 (17), 249 (13), 248 (17),247 (6), 244 (19), 243 (W, 179 (61, 126 (4), 124 (4) 419 (M-) (>loo), 399 ( l l ) , 371 (5), 346 (6), 322 (100), 298 (13), 292 (13h291 (7), 250 (lo), 248 (7), 244 ( l l ) , 243 (7), 166 (5) 419 (M-) (>loo),399 (7), 376 (27), 371 (18),356 (12), 346 (26), 328 (14), 322 (100), 298 (17), 297 (7), 292 (20), 291 (lo), 250 (8), 248 (8), 244 ( 9 , 2 4 3 (7),166 (9) 501 (M-) (
