LSD - An Overview on Drug Action and Detection

REFERENCE: Paul BD, Smith ML: LSD - An overview on drug action and detection; Forensic Sci Rev 11:157; 1999.

ABSTRACT: LSD is a psychoactive semisynthetic . It is so potent that a small dose (",,25ug) may produce a profound hallucinogenic effect. The compound is a controlled substance under the US code of regulations. One of the major adverse effects of LSD is the "flashback" or spontaneous recurrences of hallucinogenic effects that may occur months to years after cessation of the drug. The major concern of LSD abuse is the long duration of action and fatal accidents and suicides during the state of intoxication. Because LSD metabolizes to a number of compounds and detection methods for these compounds in a large number of samples are not well established, most of the methods are aimed at identifying unchanged LSD in urine. After initial screening by an immunoassay method, the presence of LSD in urine is confirmed by a gas chromatographic-mass spectrometric (GC-MS) method. The immunoassay techniques are simple and cost-effective. In confirmation, selective extraction is preferred because it allows detection of the compound at concentrations as low as 50 pg/mL. Recent methods for detection of an LSD metabolite, 2-oxo-3-hydroxy-LSD, by liquid chromatography-mass spectrometry appeared to be promising in some forensic investigations.

Key Words: Biochemical and pharmacological actions, LSD, metabolism and excretion, methods of analysis, synthesis.

INTRODUCTION LSD was first prepared in 1938 but the pharmacologi- cal studies revealed no unusual effect at that time [33]. It diethylamide (LSD) is a semisynthetic showed an oxytocic action similar to that of ergonovine. compound derived from structural modifications of natu- In 1943, Stoll and Hoffman synthesized the compound rally occurring ergot .Ergot is a rhizomorph of again with the intention of obtaining a compound with , a parasitic that grows on rye analeptic effect because part of the compound showed and wheat, and on other grains in the grass (Gramineae) structural similarities (D-ring with diethylamide) with a family [94]. The alkaloids are also present in the seeds of well-known circulatory stimulant, nikethamide [95]. In morning glory, Rivea corymbosa [32]. Ergot is toxic and the course of these investigations, Hoffman was exposed if appreciable amounts of ergot get into the grain and then to this compound and experienced a remarkable but not into the feed, ergotism may occur. In 994 A.D., ergotism unpleasant state of intoxication that lasted for 2-3 hand killed about 40,000 people in France. Present-day meth- was characterized by intense stimulation of the imagina- ods of ergot production utilize submerged culture fermen- tion and an altered awareness of the world around him tation rather than culti vation on rye or other grains [46,51]. [33]. The ergot alkaloids are hydrolyzed to produce a common An enormous number of articles on chemical, bio- compound, lysergic acid, which is then reacted with chemical, pharmacological, and pharmacokinetic proper- diethylamine to produce the LSD [95]. ties of LSD are available. It is the purpose of this review A resurgence of LSD abuse has been reported among to provide, in short form, an overall background to readers adolescents and college students in the United States [43]. Estimated numbers of LSD use among US children aged Table 1. LSD seizures by the Drug Enforcement Agency a 12-17 years increased from 970,000 in 1996 to 1,174,000 Year Dosage units in 1997, an increase of21 % in one year. According to the

Drug Abuse Warning Network (DAWN), the rate of LSD- 1998 b 701,434 related hospital emergencies increased by 13% from 1989 1997 396,183 1996 68,141 to 1990. Approximately 50% of the patients were younger 1995 100,521 than 20 years [60]. Most of them were high school and 1994 302,446 college students who were male Caucasian. Therefore, (J Source: System to Retrieve Information from Drug Evidence LSD is more prevalent in the suburbs than in the inner (STRIDE).The statistics can fluctuate widely due to the presence or cities. The extent of LSD abuse can be estimated from the absence of large seizures. number of doses seized by the U.S. Drug Enforcement b The 1998 figures are preliminary and are significantly high due to one Agency (DEA) during the last five years (Table 1). large seizure of 621 ,390 dosage units.

Forensic Science Review • Volume Eleven Number Two • Dec.1999 159

whose main interests are in toxicology. Major emphasis appropriate ergot strains in fermenters [5,12,50]. Whereas will be given to the subject that deals with the analysis of C. paspali of New Guinea strain produced a mixture of d- the drug and its metabolites in biological samples. lysergic acid amide and d-isolysergic acid amide - 90% of the total alkaloids of 1.4 g/L of cultured media, C. paspali I. NATURE OF THE DRUG of Portugal strain produced paspalic acid - 95% of the total alkaloids of 0.55 g/L of the cultured media [50]. d- A. Synthesis Lysergic acid was prepared by alkaline hydrolysis of d- lysergic acid amide, or isomerization of paspalic acid Starting materials for LSD synthesis are the ergot under mild conditions (Figure 1).A second method alkaloids. Most of the alkaloids are produced by different involved production of peptide alkaloids by fermentation strains of Claviceps fungus. In classical preparation, the of suitable ergot strains [51]. Most of the alkaloids were rye grown in the field was artificially infected with se- d-lysergic acid alkylamides which on alkaline hydrolysis lected strains of the fungus [97]. The process was time produced d-lysergic acid [5,50,89,90]. consuming and depended on the climatic conditions.Two The biosynthesis of lysergic acid and other ergot other methods for preparation of the compounds are alkaloids was a fascinating subject to many chemists for available. In the first method, lysergic acid amide and nearly forty years. The basic structure is formed from a paspalic acid were prepared by saprophytic cultivation of molecule ofL- and an isoprenoid unit originat-

eOOH

H

HN HN d-Lysergic acid amide Paspalic Acid (natural compound) (natural compound) H. eOOH /

i 7 / Isomerization HYdr~ 11 ('NCH, ~11

H

2 d-Lysergic acid

/CH2CHJ HN'-..CH,CH,

POCI)

12 IJ uv H ) 14 I

I HN HN d-LSD Lumi-LSD

Figure 1. Synthesis of d-LSD and lumi-LSD from naturally occurring ergot alkaloids.

Paul & Smith' LSD - Drug Action and Detection

------160 ing from R-mevaJonic acid (Figure 2). The intermediate skeleton of the ergot alkaloids. Incorporation of R,S- 4-(3,3-dimethylallyl)-L-tryptophan through a number of mevalonate-2-14C,2-3H and 4-3H at rates of 9-23% into steps of decarboxylation, oxidation, and cyclization is the ergot alkaloids strongly suggested a direct precursor transformed into argoclavine, , and paspalic role of mevalonic acid. The rate of incorporation of the R- acid, and finally through isomerization into d-lysergic isomer, however, was demonstrated to be 100 times more acid [21]. TheN-methyl group at 6-position is introduced efficient than the S-isomer. Although d-lysergic acid is a by L-methionine in one of the steps between 4-(3,3- biosynthetic product of ergot, the major source of this dimethylallyl)-L-tryptophan and argoclavine. The pro- compound is the hydrolysis of d-Iysergic acid amides or posed biosynthesis of lysergic acid was supported by the isomerization of paspalic acid. chemical analysis of isotopic biosynthetic products. When Theoretically, four optical isomers are possible from tryptophan-f-v'C was incorporated into the saprophytic the two asymmetric carbons atoms in the lysergic acid cultures of Claviceps, the amount of 14C in elymoclavine molecule (C-5 and C-8 in Figure 1). Many of the ergot was found to be 10-39% of the original radioactivity. alkaloids are isomeric at the C-8 position, but none of Tryptophan deuterated specifically in the indole ring these alkaloids is isomeric at C-5 [52,92]. Therefore, only showed incorporation of the indole moiety and retention two compounds, d-lysergic acid and d-isolysergic acid, of the hydrogens at C-5, C-6, and C-7, but not that at C-4. are formed after alkaline hydrolysis of the ergot alkaloids. These experiments clearly indicated that the entire tryp- In d-lysergic acid, the C-5 hydrogen atom and the C-8 tophan molecule, wi th the exception of the carboxyl group carboxylic acid are in cis configuration, and in d-isolysergic and the hydrogen at C-4, was incorporated into the basic acid, these two functions are in trans configuration. If

J eOOH CH

:O~HC'j H _~~FCH2 CH CH 3 3 3 )j: 3 L'.2-Isopentenol pyrophosphate eOOH HOfr 0 5 00 000 + 6 ~ 4 NH2 R-Mevalonic acid L'.3-Isopelltenol pyrophosphate JI "H

1 HN 2 L-Tryptophan

CH3

)

HN 4-(3,3-Dimethylall)'I)-L-tryptophan Argoclavine

eooH

Elymoclavine Paspalic Acid d-Lysergic acid Figure 2. Biosynthesis of d-lysergic acid.

Forensic Science Review • Volume Eleven Number Two • Dec. 1999

------, ----- 161 these two isomers are not separated before preparation of compound d-iso-LSD is not active. The lethal toxicity LSD, both d-LSD and d-iso-LSD are formed. Moreover, (LDso) of LSD was studied in various species [17]. It has a strong base and long reaction time in the hydrolytic step been suggested that LSD toxicity is related more closely may transform some of the d-Iysergic acid to d-isolysergic to brain than to the body weight. The LDso for mouse, rats, acid [90]. d-LSD could be synthesized from d-lysergic and rabbits were 46, 16, and 0.3 mg/kg. An elephant died acid and diethylamine by refluxing the compounds in after receiving 297 mg of LSD [109]. By extrapolation, chloroform in the presence of phosphorous oxychloride LDso for humans was estimated to be 0.2 mg/kg or 14 mg [42]. average for an adult male [31]. Because the lethal dose is relatively high compared to the minimum effective dose B. Availability and Analysis of Street Samples (-25 ug), the compound exhibited no harmful effects when tested in humans in doses 100 ug/day for several d-LSD is a strong psychoactive compound. It is clas- months. In one instance, an individual swallowed 10 mg sified as a schedule I drug under the Controlled Sub- of LSD without a fatal result [17]. There was a report of stances Act of 1970 and later by the U.S. Code of Federal fatal poisoning with LSD, but the actual dose could not be Regulations, title 21, part 1308.11, 1987. In most clandes- assessed [24]. However, a dose always exists that can tine preparations, the LSD solution is sprayed onto sheets produce death from respiratory failure. The compound is of blotting paper that are dried and perforated in squares. so potent that a small fraction of the toxic dose may Each square represents a dose. The entire square of LSD- produce profound hallucinogenic effect.Overdose of LSD impregnated paper is usually ingested to get the hallucino- may be associated with coma, hyperthermia, and bleeding genic effect. The compound is also available in pills, [49]. The major concern of LSD abuse is the long duration impregnated in sugar cubes, vitamin tablets, or solutions. of action (up to 12 h) and possibility offatal accidents and Typical abuse doses are in the range from 40 to 120 ug and suicides during the state of intoxication [8]. may cost only $3-$5. LSD illicit preparation can be tested by UV-fluores- B. Pharmacological Effects cence and color spot tests with Ehrlich's reagent. These tests produce almost immediate results.Better results are The effects of LSD after oral administration start in obtained by separating the compound on TLC plate and 20-80 min and the time of action depends on the doses and detecting with UV fluorescence and a color test sensitivity of the individual. The intramuscular route [9,19,22,25,69]. GC analysis is unsatisfactory because results in a delay of about 10 min and intravenous admin- underivatized LSD is thermally unstable [39,41,45,48,53, istration decreases the delay to a few minutes. The onset 80]. The GC resolution was improved by derivatizing of activity is almost immediate after the intraspinal admin- LSD to the trimethylsilyl compound [39]. Detection of istration. The intensity of reaction reaches a maximum LSD by LC-UV fluorescence was reported by several within 90 min and may last 3--4 h after the drug adminis- authors [26,39-41,48,107]. Most MS analyses were per- tration.After this period, it begins to decline progres- formed by electron impact using a direct insertion probe sively. Most subjects recover 8-12 h after the ingestion. [106]. LSD was also detected as underivatized compound Plasma concentration of LSD was found to con-elate well or as lumi-LSD transformed by UV irradiation. with human performance [2]. The mean plasma half-life was 2.9 h following intravenous doses of2 ug/kg LSD to II. DRUG EFFECTS five male subjects. The compound stimulates both branches of the autonomic nervous system. Sympathomimetic stimu- Effective doses that produce optimum psychotomi- lation is evidenced in most subjects by a pupillary dilation metic effects range from 0.5 to 2.0 ug/ kg body weight, and moderate increase in heart rate and blood pressure. either given orally [37], intravenously [2], or intramuscu- Parasympathomimetic effects are most common and mani- larly [111]. Mini mum effecti ve dose gi ven orally is about fested on lacrimation and salivation. Objective responses 25 ug [31]. In illicit use of LSD, the oral route is most that can be measured are body temperature, pulse rate, popular. blood pressure, pupillary diameter, and patellar reflex [37,111]. Subjective responses are measured by positive A. Toxicity responses to a questionnaire. LSD induces mental disori- entation characterized by anxiety, alterations in mood d-LSD (hereafter referred as LSD) is a potent halluci- (euphoria), difficulty in thinking and concentration, al- nogen, 10-150 times as potent as and 4,500- tered sensory perception (particularly visual), and halluci- 9,275 times as potent as [37,111]. The isomeric nations. One of the major adverse effects that results from

Paul & Smith> LSD - Drug Action and Detection

~------~- 162 chronic use of LSD is the "flashback" or spontaneous 4 recurrences of hallucinogenic effects that may occur 3 months to years after cessation of the drug [59,62]. \-'.~ 2 ~ ;:;0 C. Biochemical Considerations S .. The LSD-like activity is primarily associated with ~ ~ alterations in the function of the neurotransmitter, 5- ~ .':: hydroxytryptamine (5-HT or ) and its receptors. At least seven major classes of 5-HT receptors ( 5-HT I to ~ 7 5-HT 7) and their subtypes have been characterized [78]. 6 ~ These receptors are widely distributed in the body with 1 increased population in smooth muscle and nervous tis- sue. It is believed that hallucinogenic activity is mediated primarily through activation of 5-HT1A, 5-HTIC, and 5- 4 1 HT2 receptor sites [10,77,102,103]. It has also been pos- 3 tulated that activation of the 5-HT receptors by LSD may ''c.-ASP alter dorsal raphe (DR) neuron electrical properties, in- v , 2 ~ H ,0 hibit DR firing and change the levels of 5-HT in the brain S LSO-N+.··OJ I [3,4,77]. These are the basis of the presynaptic theory CH, which supports the activity of the DR neurons as critical ~ oo~~yr <, for the actions of hallucinogens. Another theory of LSD- ~ like activity is related to increase stimulation of 5-HT2 receptor sites, resulting from blockade of serotonin reuptake ~ 7 and increased concentration of serotonin in the central 6 ~ nervous system [27]. 1 A ligand-receptor approach was used to explain the interaction of LSD with the 5-HT2 receptor sites in which Figure 3. A schematic presentation of helix backbone of 5-HT2 receptor and interaction of LSD with the hydrogen bonding an interconnected seven-helix model of a serotonin recep- between aspartate-86 in helix 3 and tyrosine-216 in helix 7. tor was utilized [29,110]. In this model, an aspartate-86 from helix 3 is linked to a tyrosine-216 in helix 7 by nous administration of is recommended. hydrogen bonding (Figure 3). The position-6 nitrogen in Within a few minutes of an injection of 25-100 mg of LSD dissociates the hydrogen bond and forms a new ionic chlorpromazine, most autonomic and psychic disturbances bond with the anionic aspartate. Disruption in this linkage are completely abolished [30,36,83,85]. Oral may initiate a conformational change which is responsible chlorpromazine may be less active, but 1-3 oral doses of for the receptor activation. Other structural features in 50 mg of chlorpromazine terminated the psychedelic LSD, the essential considerations of structure-activity effects [63]. Because of possible side effects, the drug is relationship, are also important in docking the compound not recommended for adolescents [62]. Diazepam (Valium into the three-dimensional model of the 5-HT receptor 15-30 mg orally) may be given and repeated as necessary sites. until the patient is calm. Another counteractive drug is , 2.0 t04.0 mg intramuscularly, repeated hourly D. Tolerance, Dependence, and Treatment as needed [84].

Tolerance develops after 3 to 6 days of continuous use III. METABOLISM AND ELIMINATION [1,35,111]. However, a period of 4 to 6 days abstinence allows the user to once again experience the effects of the Numerous articles are available describing the phar- drug at normal dose levels. LSD is believed to be not macological effects of LSD on humans. Most of the physically addicting [18], but a psychologic dependence studies were conducted within thirty years after the drug may be encountered [61]. For treatment, the acute panic was reported as hallucinogenic. With increasing restric- reaction can usually be managed at a quite place under tions on administration of LSD to human subjects, current supervision for 8-12 h [68,101]. If the time is not suffi- knowledge on distribution, metabolic profile, and excre- cient to prevent violent actions, intramuscular or intrave- tion of LSD in man is limited. A considerable number of

Forensic Science Review • Volume Eleven Number Two • Dec. 1999

/ ------... __ .. 163 reports are available on the distribution and metabolic guinea pig, and rabbit), N-desethyl-LSD and nor-LSD profile of LSD in animals. In cats and mice, the drug was were detected [70]. N-Desethyl-LSD was also detected as found to be distributed widely in all tissues [7,96]. It is a metabolite when LSD was incubated with human liver extensively bound to plasma proteins and its presence in homogenate [11]. Threecompounds-nor-LSD, lysergic the brain and cerebrospinal fluid indicates that the drug acid ethylvinylamide, and lysergic acid ethyl-2- easily passes through the blood-brain barrier. Plasma half- hydroxyethylamide - were isolated by microbial trans- life in mice, cats, and monkeys were reported as 7, 130, formation of LSD [38]. and 100 min, respectively. Only liver tissue was found to In rats and mice, LSD is oxidized to several com- metabolize the drug when incubated with guinea pig liver, pounds [87]. Two of the compounds were phenolic. Two brain, kidney, spleen, and muscle. other metabolites on glucuronidase hydrolysis produced Metabolic compounds originating from in vitro mi- the same phenolic compounds. Therefore, the conjugated crosomal oxidation, and from animals and human origin, compounds are likely to be the glucuronides of the corre- are schematically presented (Figure 4). LSD was oxi- sponding phenolic metabolites. Three phenolic compounds dized by guinea pig liver microsomal preparations to 2- of LSD are possible (12-,13-, and 14-hydroxy-LSD). The oxo-LSD [6]. No psychological activity was observed mass spectrum and chromatographic characteristics ofthe when the synthetic form of this metabolite was adminis- two phenolic metabolites were compared [88] with a tered orally to humans at a dose of 300 ug. A phenolic synthetic reference of 12-hydroxy-LSD [91]. One of the compound, claimed to be 13-hydroxy-LSD, was isolated metabolites, assumed to be 14-hydroxy-LSD, was un- from rat liver microsomal oxidation of LSD [99]. When stable and cannot be compared directly with the synthetic LSD was incubated with animal liver homogenate (rat, reference. The compound was derivatized to a stable

/~ ~H

Nor-LSD 2-0XO~3-hYdrOXY-LSD ~~

IN~ ~ o ~ .L-~

9 7 ~ 2-0xo-~SD

12 R'~ Ivh, an, hm 13 .o I D ":'", I_n_iC :X« A I •• ? ~ . 1 R2 14 3 H(i B 1 ~~C Jy~CH' R2 Compound N-Ethyl-N-(2- OH H 13·Hydroxy-LSD LSD hydroxyethyl)-LSD H OH 14-Hydroxy-LSD Ogl H LSD-13·hydroxyglucuronide H OgJ LSD-14-hydroxyglucuronide (gl-glucuronide) .;

N-Ethyl-N-vinyl-LSD

N-Desethyl-LSD

Figure4. LSD metabolism in humans (hm) and animals (an), and transformation by liver homogenates (lvh) and microorganism (mic).

Paul & Smith> LSD - Drug Action and Detection 164 methyl ether derivative. The product was then compared specimens in routine analysis showed LSD concentra- with the methyl ether of the l2-hydroxy-LSD. Although tions more than nor-LSD concentrations. In this study, the mass fragmentation patterns of the stable phenolic LSD was detected up to 20-22 h after ingestion. Two other metabolite and the methyl ether of the unstable phenolic compounds, 13- and 14-hydroxy-LSD, were also de- metabolite were similar to that of the l2-hydroxy-LSD tected, but the identities were based on only one mass and its methyl ether, the thin layer chromatographic char- spectral ion with no reference compounds used for com- acteristics of the compounds were distinctly different. parison.Attempts were made to detect N-desethy I-LSD in Therefore, it was concluded that the metabolites were not LSD-positive urine specimens [11]. Preliminary results l2-hydroxy-LSD, but likely to be 13- and 14-hydroxy- indicated the presence of the compound at a very low LSD. To date, the identity of the compounds remains concentration. inconclusive because reference materials are not available A new metabolite, 2-oxo-3-hydroxy-LSD, was de- for comparison. For the glucuronide metabolites, the tected in many human urines submitted for drug testing exact nature of the linkage of the glucuronic acid with the [79]. Identity of this compound was confirmed by com- phenols is not known. Nor-LSD was detectable in pig- paring LC- MS characteristics with a reference compound. mented rat hair following administration of LSD[66]. The average concentration of the metabolite was 20 times In monkeys, LSD is metabolized to 2-oxo-LSD, N- more than that of LSD. However, in some LSD-positive desethyl-LSD, two phenolic LSD (probably 13- and 14- specimens (>200 pg/mL) the compound was found to be hydroxy-LSD), and the glucuronides of the phenolic LSD below the limit of detection (LOD) of the procedure. In [86]. The identity of the 2-oxo-LSD metabolite was deter- routine analysis of human urines, iso-LSD was also de- mined by comparing its physical properties with that of tected [15,67,79]. The compound is suspected to be a the synthetic reference [7], but the compound was found byproduct from the illicit preparation of LSD. to be unstable [91]. It spontaneously transformed to a more stable naphthostyryl compound resulting in a mix- IV. METHODS OF DETECTION ture of the parent compound and the product. LSD was administered 2 ug/kg intravenously to five Due to the very low dose consumed (usually 40-120 human subjects [2]. The plasma half-life was calculated to ug) and to rapid metabolism with less than 1% excreted be 2.9 h. The plasma half-life was found to be 5.1 h when unchanged in urine [7,55,86], identification of LSD in the compound was administered 1 ug/kg orally to a human biological samples is a major challenge to forensic scien- subject [72]. In an experiment, LSD was administered to tists. Furthermore, the instability of LSD in acid [76], heat eight human subjects [100].Each individual was given a [80], and light has made the identification even more single dose of LSD in the range of 200-300 ug. Only two challenging. Because LSD metabolizes to a number of individuals were given a second dose after complete compounds and detection methods for these compounds elimination of the drug from the first dose. Urine speci- in a large number of samples are not well established, most mens were collected up to 24 h after the dose. When tested of the methods are aimed at identifying unchanged LSD in by radioimmunoassay all specimens were found to be biological samples. positive. The results were quantitated as LSD-equivalent. Considerable variations in excretion rate were observed A. Immunoassays among individuals and within the same individual. The peak concentration was observed within 1-3 h after the Immunoassays are the simplest and most time-saving drug administration. screening methods for detection of LSD in urine. A large The metabolism and excretion profile was studied in number of samples can be tested without any sample a human subject (70.5 kg) given LSD orally at a dose of 1 preparation. Radioimmunoassay procedures are based on ug/kg [55]. In urine, nor-LSD was detected as a metabo- competitive binding of 125I-radiolabeled antigen and lite. Both LSD and nor-LSD were analyzed as unlabeled antigen (LSD) with a limited amount of anti- trifluoroacetyl- derivatives. The rates of excretion of LSD body [13,57,81,100]. The specimen is mixed in a test tube and nor-LSD reached maxima in urine at 4-6 and 8-10 h with fixed amounts of antibody and radiolabeled antigen. after the drug administration, respectively. The elimina- Drug and its metabolites present in the specimen compete tion half-life for LSD and nor-LSD were 3.6 and 10.0 h, with the labeled antigen. After a brief incubation, the respectively. The total LSD and nor-LSD excreted in antigen-antibody complex is precipitated with a second urine were only 0.9% and 1.2% of the administered dose, antibody reagent. The supernatant is removed and the respectively. Although the amount of nor-LSD excreted radioactivity is counted in a gamma counter. The radioac- in urine was more than the amount of LSD, many of the tive counts are inversely proportional to the concentration

Forensic Science Review • Volume Eleven Number Two • Dec. 1999 165 of LSD in the specimens. The assay can detect concentra- B. Extraction tions as low as 20 pg/mL of LSD [100]. For urine drug testing, samples are identified as positive or negative by The concentrations of LSD in biological samples are comparing the counts with that of a cutoff standard con- generally low (pg/mL). Extraction oflarge volumes (5-10 taining 500 pg/mL of LSD. The assay at this cutoff mL) of biological fluid is essential to increase detection concentration has been used by the Department of De- sensitivity. In a typical method of solvent extraction, the fense (DoD) for the past 12 years. Under this program, samples are made basic and saturated with salt. The LSD more than ten million specimens were tested for LSD. For is then extracted with a moderately polar solvent (ether, 1- antemortem and postmortem fluids, the drug was initially chlorobutane or methylene chloride). After removing the extracted in methanol and dried, and then tested by the solvent the extract is reconstituted in a suitable sol vent and radioimmunoassay [24]. subjected to chromatographic separation. For tissue At present, the DoD is using a nonisotopic enzyme samples, mixed polar solvents may be used to extract the immunoassay to test urine specimens for LSD. Several compound from the homogenized tissue. These methods nonisotopic homogeneous immunoassays are available of extraction are relatively simple and the recoveries are commercially [56,65,112]. Cloned enzyme donor immu- generally more than 95%. However, impurities that are noassay (CEDIA, Boehringer Mannheim) and enzyme- also extracted with the LSD may seriously interfere with multiplied immunoassay (EMIT, Behring Diagnostics) the detection methods. techniques are based on the principle of enzyme activa- Where interfering impurities are the major problem, tion. The OnLine immunoassay (Roche Diagnostic Sys- selective extraction of LSD from the biological samples is tems) is based on kinetic interaction of microparticles in essential. In the DoD procedure, after the initial solvent solution (KIMS) [58]. These three procedures are spe- extraction, the sample extract was dissolved in a phos- cially designed for large-scale analysis on automated phate buffer of pH 4.5 [76]. The solution was washed with analyzers. Microplate immunoassay (STC Diagnostics) is a solvent to remove the neutral and acidic impurities. The available for small-scale testing [47]. For forensic urine solution was adjusted to pH 9.0-9.5 and saturated with drug testing, a cutoff concentration of 500 pg/mL is salt. The LSD was then extracted with l-chlorobutane. recommended for LSD screening. Comparative studies of Because the extract was free from most acidic and neutral these reagents and radioimmunoassay reagents are also impurities, the interferences in the GC- MS detection were reported [16]. These nonisotopic immunoassays correlate less than the simple solvent extraction method. The over- well with the original radioimmunoassays. In compara- all yield was 92-95%. tive studies, specimens found to be positive by a confirma- Selective extraction was further modified to remove tory procedure (~ 200 pg/mL), were also positive by the some of the basic impurities. Two principles, chromato- immunoassay methods. Although some of the immunoas- say methods are sensitive to LSD at concentration as low Table 2. Cross-reacting compounds of LSD metabolites and as 20 pg/mL, none of the methods are specific for a other structural congeners positi ve identification of LSD . Several specimens showed positive immunoassay results (~ 500 pg/mL) when the Cross-reaction (0/0) a

LSD concentrations were below the 200 pg/mL. It is likely Compound RIA b OnLine C STC d that some of the LSD metabolites in the specimens cross- LSD 100 100 100 react with immunoassay reagents. Major cross-reacting Nor-LSD NR 36 52 metabolites are nor-LSD and 2-oxo-3-hydroxy-LSD 2-0xo-3-hydroxy-LSD NR 40 3.4 (Table 2) [47,54,104]. In many urine specimens, iso-LSD Iso-LSD 0.002' 2.4 0.05 was also detected, but its cross-reaction to LSD-antibody LAMPA 45 14 34 Lysergic acid <0.0025 <0.003 is less than 2.5 %. Other cross-reacting therapeutic agents Lysergic acid-mono ethyl amide 0.7 e _f (haloperidol, fentanyl, sertraline, ) that Lysergic acid-N-hydroxyethylamide 0.056 0.03 are not LSD metabolites were also reported [82]. These 2-0xo-LSD 2e Lumi-LSD 22 e compounds interfere at concentrations of 70-800 ng/mL urine. Several other therapeutic agents cross-react at higher a Compared to signal of LSD at 500 pg/mL. concentrations. Therefore, an alternate method is required b From Roche Abuscreen technical insert. C Data takend from Ref. [54]. to unequivocally confirm the presence of LSD in the d Data taken from Ref. [47]. specimens. The methods for confirmation require extrac- e Results of 50% depression in response compared to 200 pg/ml. tion of the compound from the biological specimens [104]. f Not reported. followed by chromatographic separation and detection.

Paul & Smith> LSD - Drug Action and Detection

------~~ - ~~- 166 graphic fractionation and acid-base separation, were used C. Detection by Color Spot Test in this purification. After the initial solvent extraction, the extract was subjected to a solid-phase chromatographic Color tests were performed on a reaction medium, fractionation using a silica-based propylamine sorbent generally on a filter paper or a porous glass. The extract packed into a polypropylene column [76]. Low polar was dissolved in 5-10 ul, of solvent and adsorbed on the impurities were removed by washing the sorbent with reaction medium using a capillary tube. To test for LSD, either chloroform or methylene chloride containing 0.1 % two drops of 1-2% p-dimethy laminobenzaldehyde in 1M triethylamine (TEA). The LSD was then extracted with ethanolic solution of hydrochloric acid (Ehrlich's re- 3% methanol in chloroform (or methylene chloride) con- agent) was added to the spot. A violet color indicated the taining 0.1 % TEA leaving behind the polar impurities in presence of LSD [44]. The reaction, however, is not LSD- the column. LSD was found to be unstable in the presence specific. Other indole compounds showed similar color of silica. Therefore, during chromatographic fraction- reaction in this test. ation, TEA was included in the mixed solvents to protect D. Fluorescence Detection the compound from breakdown. Although solid-phase extraction removed most of the biological impurities, it Plasma concentrations of LSD were determined by introduced some interfering column materials into the transforming the compound to lumi-LSD. After the initial extracts. These materials and other endogenous interfer- solvent extraction with 2% isoamyl in n-heptane, ing impurities were then removed by the acid-base sepa- the compound was reextracted into a solution of 0.004 M ration. The overall yield was 89-92%. The method was HC!. LSD was then quantitated as lumi-LSD after a brief successfully applied in detecting LSD in a large number of irradiation at 325 nm wavelength and then by measuring urine specimens [76]. the fluorescence at 445 nm wavelength [2,9,98,105] (Fig- An immunoaffinity extraction procedure was described ure 1). The fluorescence detection is not specific for LSD to separate the LSD from urine [108]. An affinity gel was but can detect the compound as low as 1 ng. Many of the prepared from Protein A SepharoseCL-4B and polyclonal ergot alkaloids exhibit fluorescence at that wavelength. LSD-antiserum. Urine sample containing LSD was ap- plied to the gel in a cartridge and allowed to equilibrate for E. TLC and Paper Chromatographic Separation and 30 min. The gel was washed with phosphate buffered Detection saline (pH 7.4), water, and then with a small amount of ethanol. LSD was then eluted with more ethanol and These chromatographic procedures were used to sepa- detected by LC-MS. In this procedure, the' recoveries vary rate LSD from other impurities in the extract [14,20]. The with the LSD concentration. Low recoveries at high drug was then detected by UV fluorescence, color test, or concentrations were reported. It was probably due to mass spectrometric methods. The UV and color tests are saturation of active sites in the gel.Because the availabil- not LSD specific, but in combination with the character- ity of the gel was limited, it was repeatedly used in the istic TLC retention factor (Rr), these methods could be extraction methods. Selection of a suitable internal stan- effectively used for preliminary identification within a dard that shows the same LSD-like affinity appeared to be short period of time and at a reasonable cost. However, it the limiting factor for quantitation of LSD. is only the mass spectral identification that provides A method for extraction of LSD and nor-LSD from rat specific identification of the compound. In one detection hair and human hair was reported [66]. After washing the method,LSD was extracted from biological fluid with hair with O. I % sodium dodecyl sulfate, the compounds solvent, fractionated by HPLC, and then identified on a were extracted with a mixture of methanol and 5M HCl silica gel TLC plate using acetone:chloroform:methanol (20: 1). The extracted solution was filtered, neutralized (15:4: 1) as developing solvents [14]. The LSD was iden- with ammonium hydroxide, evaporated, and partitioned tified as a blue fluorescent spot under irradiation with 360 between methylene chloride and sodium hydroxide (0.1 nm UV light. Following spraying with Ehrlich's reagent M). The compounds in methylene chloride were then and warming, the LSD showed as a colored spot at an Rf evaporated and detected by HPLC with fluorometric de- 0.6. For unequivocal identification, the untreated and UV- tection method or as trimethylsilyl derivatives by GC-MS unexposed spot having the same Rf as the LSD control, method. was scraped from the TLC plate and extracted with 25 ul, of 1 M ammonia and 0.5 mL of methylene chloride. The solvent was removed and the compound was identified by mass spectrum using direct probe. In TLC, the developing solvents used to transport the LSD may vary considerably.

Forensic Science Review • Volume Eleven Number Two • Dec. 1999 167

Ethylacetate with ammonia was used to separate struc- detector was utilized to detect LSD in urine samples [28]. tural analogs of LSD on silica gel TLC plates [71]. The The method used a Cs-reverse phase column. Consider- same solvent system could be used to develop LSD on a able background noise was observed at an LSD concentra- silica gel TLC plate. Better sensiti vity is expected because tion of 500 pg/mL urine. Urine specimens were also tested ammonia may protect LSD from breakdown by silicic by HPLC with a Cl8-reverse phase column and a fluores- acid [76]. cence detector [23]. For quantitation, lysergol was used as In a paper chromatographic method, after the initial an internal standard. Concentrations as low as 400 pg/mL solvent extraction, the sample was spotted on a Whatman were detected by this method.Although good quantitative. No.1 chromatography paper [20]. The developing sol- correlation was observed between HPLC and GC-MS, no vents were n-buranol:acetic acid (10: 1). Instead of detect- chromatogram from HPLC was presented to show the ing LSD directly, an LSD-like compound was detected by background noise. In a similar study on urine and serum UV inactivation ofthe fluorescent compound. Additional specimens, methyllysergol was used as the internal stan- identification of LSD was accomplished by spraying a dard [64]. The LSD was analyzed by LC and UV fluores- separate chromatographic paper strip with Ehrlich's re- cence detector. The serum and urine concentrations were agent and observing a purple color at an Rf of 0.70-0.76. compared with negative and positive responses of LSD by A similar method was applied to detect a phenolic metabo- radioimmunoassay. No LSD was detected in specimens lite (probably 13-hydroxy-LSD) of LSD from a microso- that were negative by radioimmunoassay, indicating good mal media [99]. The detected compound after UV irradia- correlation between the HPLC and radioimmunoassay tion was lumi-LSD [9, 98]. results for negative specimens. In some serum and urine specimens, LSD was detected up to 11 h after multiple F. HPLC Separation and Detection doses of the drug. The limit of detection in this method was considered as 500 pg/mL. Like other chromatographic methods, HPLC was LSD was analyzed by an LC-MS method [108]. After used to separate LSD from interfering compounds. The initial immunoaffinity or solid-phase extraction and evapo- drug was then detected by either UV or MS detector. In ration, the dried extract was dissolved in a minimum some applications, HPLC was used for fractional sample amount of a mixture of 0.1 M ammonium acetate (pH 8) collection. The fraction that corresponded to the retention and acetonitrile (7.5 :2.5) containing 0.25% triethylamine. time of reference LSD was then tested by different detec- The solution was introduced into the HPLC using the tion procedures. In one experiment, after initial solvent same solvent system as the mobile phase. Reverse phase extraction and evaporation, the dried extract was redis- Cls-column was used for the chromatographic separation. solved in a minimum amount of solvent and introduced For detection, an atmospheric ionization MS equipped into an LC column packed with Partisil (silica) [14]. The with an electrospray ionization (ESI) interface was used. flow rate of methanol:0.2% aqueous ammonium nitrate The LSD was initially detected by (M++1) ion (m/z 324) (11 :9) through the column was 1 mUmin. The eluent was by applying an ESI voltage of 4 kV and then by fragment collected in 2 mL fractions. To locate the fraction that ions, m/z 223 and 281 by applying 10-20 V to an octapole contained LSD, reference LSD was injected and the rod assembly between the ESI and quadrupole analyzer. fractions were tested using a UV excitation wavelength of Internal standard, methyl lysergide, was added to the 325 nm and then emission wavelength of 430 nm. For an solutions after the initial immunoaffinity extraction. Me- unknown sample, the fraction that corresponded to the thyllysergide was not retained by the affinity gel and was reference LSD was analyzed on a silica gel TLC plate with unsuitable for immunoaffinity extraction. The drug was acetone-chloroform-methanol (15 :4: 1) as developing sol- then unequivocally confirmed by the relationships be- vents. The compound was then detected by UV fluores- tween the fragment ions. The limit of detection of LSD cence light and finally by Ehrlich's reagent.Verifying was found to be 500 pg/mL. Major background noise was retention times for HPLC and TLC and detection by UV observed with mlz 281 even at LSD concentration of 1,000 and color tests improved confidence in identification of pg/mL. The disadvantage of this procedure was that the LSD in the biological samples. instrument needed to be readjusted after each batch analy- HPLC fractionation was used to analyze human body sis because degree of fragmentation varied between fluids. A reverse phase Cis-column was used. The LSD in batches. the collected fractions was then detected by radioimmu- LSD and nor-LSD were also analyzed by a procedure noassay [104]. The method, however, was not LSD spe- [34] similar to the LC-MS procedure described [108]. cific. Limitofdetectionofthis method was reported as 500 Instead of methyl lysergide, d3-LSD was used as the pg/ml. HPLC coupled with a non-specific fluorescence internal standard. Only m/z 324 (M++1) and a fragment

Paul & Smith> LSD - Drug Action and Detection

------_. ----- 168 ion, mJz 223 were monitored. Common ions in the drug injections.Once the active sites were protected, the sensi- and the internal standard were the major problem for tivity improved substantially. To deactivate the GC col- selecting a third ion for LSD identification. The limit of umn, LSD-TMS was found to be better than the BSTFA unambiguous identification was reported to be 100 pg/mL alone. The limitof detection and quantitation was reported for LSD and 250 pg/mL for nor-LSD. as 50 pg/mL. The method was successfully utilized by the DoD to analyze more than 100,000 urine samples over the G. GC Separation and MS Detection past 12 years. In routine GC-MS batch analysis, a calibra- tor at a cutoff concentration of 200 pg/mL and three A fused silica capillary column (12-15 m long and controls at concentrations of 100, 400, and 800 pg/mL are 0.20-0.25 mm i.d.) coated with either methyl siloxane or used.The DoD requires that the two qualifying ion ratios 5:95 phenyl:methyl siloxane as the stationary phase is of the controls must be within ±20% of that of the generally used. Helium or hydrogen is used as the carrier calibrator and the quantitations of the controls must be gas. Application of capillary GC for chromatographic within ±20% of the theoretical values. separation of LSD in street samples and detection by MS Iso-LSD was detected in many urine specimens that was initially introduced in 1984 [93]. The compound was contained LSD. In some specimens the iso-LSD concen- detected as underi vatized compound using scan mode. In trations were ~200 pg/mL when the LSD concentrations the GC separation, LSD produced poor chromatographic were below the limit of detection of the procedure (50 pg! peak. Poor volatility may have been the reason of unac- mL). In majority of the specimens, the detection of iso- ceptable peak shapes.Volatility and GC characteristics LSD was not conclusive because impurities often inter- were improved by preparing a trimethylsilyl derivative of feredin the chromatogram [15]. Moreover, iso-LSD is not LSD (LSD- TMS) [39]. The advantage of GC-MS over stable.It changes to LSD during standard storage condi- LC-MS is that the capillary column can be directly intro- tions (Table 3). Therefore, a standard solution at a desired duced to the MS source without any interface, allowing concentration cannot be prepared to quantitate iso-LSD in better sensitivity of detection. Loss of compound at the specimens. Iso-LSD is not psychoactive, but the com- ESI interface is the major concern in LC-MS method. The pound, like LSD, is classified as a schedule I drug under method of MS detection of drugs of abuse in urine by the Controlled Substances ACT of 1970.A procedure for monitoring selected ions and by comparing their relative detection of iso-LSD was described [15]. After initial ion abundances with reference compounds was first intro- solvent extraction and evaporation, the compound was duced by the DoD in urine drug testing [74,75]. The isomerized to LSD with sodium ethoxide in ethanol (Fig- concept was successfully utilized to detect LSD in urine ure 5). Within 10 min after the addition of the reagent the [23]. After solvent extraction and evaporation, LSD was reaction was almost complete (98%). The product was derivatized with bis( trimethy lsily l)trifl uoroacetamide purified by solid-phase fractionation and acid-base purifi- (BSTFA). drLSD was used as the internal standard. The cation. Iso-LSD as low as 50 pg/mL was detected by this LSD-TMS was analyzed by monitoring m/; 395 (M+), procedure. When a specimen contained both LSD and iso- 293, and 268.The limit of detection of the procedure was LSD, the amount of LSD determined by this procedure reported as 500 pg/mL. represented the total LSD. The amount of iso-LSD was In a separate procedure, the sensitivity of detection calculated from the difference of total LSD and the LSD was improved lO-fold by solid-phase fractionation and tested without isomerization.A chromatogram of a speci- acid-base purification of the extract [76]. Instead of d3- men analyzed with and without the isomerization step are LSD, LAMPA was used as the internal standard. In the presented in Figure 6. Although two GC-MS procedures acid-base purification, an optimum pH of 4.5 was used to purify the LSD. The compound was found to be unstable when the pH was less than 4.5. The compound was also Table 3.Iso-LSD in methanol isomerized to LSD during storage at 2-6 DC [15] found to be unstable to silica present in glass tubes and

solid-phase extraction columns. During extraction and Storage time (day) Isomerized LSD (%) a purification,LSD was protected by using 0.1 % o 5-7 triethy Iamine. When unextracted LSD as the TMS deri va- 3 16-18 tive was injected repeatedly into a GC column, poor 10 19-22 response was observed at the first few injections. The 132 24-25 response was improved after several injections. 293 33-35

Transsilylation from the LSD-TMS to the silicic acid II Percent based on the total ion chromatogram. active sites may be the reason for poor response in first few

Forensic Science Review • Volume Eleven Number Two • Dec. 1999 169 may be required to determine the iso-LSD concentration chromatogram that showed minimum background inter- in urine, in forensic investigation only one GC- MS proce- ference. Generally, in routine analysis, LSD as low as 80 dure that detects the total LSD (LSD + iso-LSD) is pg/rnl, can be detected by this procedure. recommended because the concentration of each com- pound may fall below the DoD mandated cutoff concen- I. Capillary Electrophoresis and MS-MS Detection tration of 200 pg/mL. A method for separation of LSD and nor-LSD by GC This procedure was utilized to study in vitro meta- and detection by electron capture ionization MS was bolic transformation of LSD by human liver microsomes reported [55]. The compounds were derivatized to 1- [11]. After solid-phase extraction, LSD and its metabo- trifluoroacetyl-LSD and 1,6-bis(trifluoroacetyl)-nor-LSD. lites were separated by capillary electrophoresis (CE) and Only the molecular ions of the compounds were moni- were detected by the MS-MS technique. A capillary of tored. Methods monitoring only one ion are not consid- 100cmlond and 50 urn i.d. at a potential differenceof25.5 ered specific for compound identification and, therefore, kV was used. Samples were introduced electrokinetically have limited applications in forensic investigations of for 10 s with a 5.5 kV drop across the capillary. The CE LSD abuse. However, the method was sensiti ve and cou ld electrolyte was 80 mM ammonium acetate (pH 4.5) con- detect LSD and nor-LSD at concentrations as low as 50 taining 20% methanol. The sheath flow liquid was 5 mM and 30 pg/ml, respectively. It was successfully utilized in ammonium formate in 80% methanol at a flow of 4 ILL! studying excretion profiles of the compounds in human min. Some of the unknown metabolites that could not be urines. separated by a LC-MS-MS method were separated by the CE-MS-MS method. LSD, nor-LSD, and N-desethyl- H. GC Separation and MS-MS Detection LSD were detected at retention times -25 min. The compounds were not quantitated using internal standards. A procedure for GC separation and MS-MS detection of LSD, nor-LSD, and iso-LSD as trimethylsilyl deriva- J. Stability in Standard Solutions tives was reported [67]. Nor-LSD was derivatized to the mono- TMS derivative instead of di-TMS derivative. Hy- LSD in methanol or ethanol containing 0.1 % drogen was used as the carrier gas in the GC column. In the triethylamine is stable for more than two years when MS, the compounds were initially subjected to low energy stored at 2-6 Dc. To verify stability in urine, eleven ionization to form the MH+ ions 396, 382, and 396 for specimens were analyzed, stored frozen at -18 to -20 °c TMS derivatives of LSD, nor-LSD, and iso-LSD, respec- for an average of three years, then analyzed again. The tively. The ions were then selectively separated and again quantitations (111-1051 pg/mL) were within±20%ofthe subjected to collision-induced dissociation (CID) to pro- original values. The effect of freezing on the concentra- duce characteristic fragment ions. Ammonia was used for tion of LSD in urine was also evaluated [73]. The concen- the CID. The compounds were identified by the charac- tration after 45 days at -18 to -20°C was 256.7 ± 6.4 pgl teristic abundance ratios of the selected ions. The initial mL (n = 10) compared to the original concentration of separation of MH+ ion followed by CID produced a 256.8 ± 5.6 pg/mL (n = 15).

12

13\ H

14

B H B HN HN 1 C-8 Carbanion Iso-LSD LSD

Figure 5. Sodium ethoxide isomerization of iso-LSD to LSD.

Paul & Smith s LSD - Drug Action and Detection

----._---- -. ------.------170

CONCLUSION and the effects may last 3-4 hours after administration."Flashbacks", recurrence of effects, may LSD is a semisynthetic compound deri ved from natu- occur for months after cessation of the drug. The com- rally occurring ergot alkaloids. In illicit preparations, iso- pound metabolizes extensively with only 1% excreted in LSD is often present and appears in urine following urine. Because methods for detecting metabolites are not ingestion. Both LSD and iso-LSD are classified as con- well established, most forensic investigations of LSD use trolled substances under the Controlled Substances Act of target detection of the parent compound in urine. 1970. LSD-impregnated blotters, perforated into squares, At present, GC-MS is the most prevalent method for are the most common dosage forms used on the streets. detection of LSD in urine. Because interference is the Each square contains 40-120 ug of LSD, costs only $3-5, major limitation, purification of the compound at the and is taken sublingually or orally. LSD is a powerful extraction step is the most important factor in improving

Abundance Ion 395.00

60000L 7.07 40000 JLill.83

Tim2e~~:06-;=";::::;:::'i9t=;==;=;=;=::;:t=~~"r""T':: ::::;:::::="~~~"" -,r"""T"' .....-,.....,.., -rt~, ~~, ..,.,~~--,..- 6.40 6.60 6.80 7.00 7.20 740 7.60

Ion 293.00 Abundance 7.07

1:::::L~ __ ~~ '::~"~

Time> ~:; ;~'t r= , -- 6.40 6.60 6.80 7.00 7.20 740

Abundance Ion 253.00

100000L 6A2 7A7 -.~ Ttme--> ::::;6t4=0::::::::::::;:6::;:.6:::;0-::::"~;:::::6~.8~0~~~~:;:=r"7;=.0!"7if"'20"""'''''--'-'7""'\-0"""..••.•.•-,-7"'r.-0 i - .,....•...•--,..-,. e o

A

Abundance ion 395.00

2150000000001 JWl6.84 707 100000

50000 6.726.96 Time--> 1-o-..•==;__. ..,,~ ~_~4~=~.,...?~ •...-

4000001 JW6.84 7.07 200000

Tirne-> i===-=o=o-=;,=;===.,.t-=~"'=~~=6.726.96 """"~~=-"""'--"'T'--=~-r-, -..-.---, 6.40 6.60 6.80 7.00 720 7.40 7.60

Abundance Ion253.00

400000\ ~6'84 7.07

200000 ~~~~,-~ __ ~_~6c·7~2~~~6~.9~6~~~-.- --. ~ __ ~~ Time--> 6'.40 6'.60 6.80 7.00 /20 7'.40 7'.60

B Figure 6. Ion chromatogram of a specimen without isomerization (LSD 220 pg/rnL)(A) and the same specimen after sodium ethoxide isomerization (total LSD 416 pg/mL) (B). Retention times of trimethylsilyl derivatives ofiso-LSD, LSD, and LAMPA (internal standard) are 6.71, 6.83, and 7.07 rnin, respectively.

Forensic Science Review • Volume Eleven Number Two • Dec. 1999 171 sensitivity. Despite this limitation, the method has been by a strain of Claviceps paspali Stevens and Hall in sub- successfully used for forensic urine testing for a decade merged culture; Nature 187:238; 1960. and has an LOQ near 50 pg/mL. A GC-MS-MS method 6. Axelrod J, Brady RO, Witkop B, Evarts EV: Metabolism of lysergic acid diethylamide; Nature 178:143; 1956. was reported that has less background interference. The 7. Axelrod J. Brady RO, Witkop B, Evarts EV: The distribu- method requires expensive equipment and a highly trained tion and metabolism of lysergic acid diethy lamide; Ann NY operator. An LC-MS method for simultaneous detection Acad Sci 66:435; 1957. of LSD and 2-oxo-3-hydroxy-LSD metabolite in urine is 8. Bewley TH: Adverse reactions from the illicit use of under investigation. The method does not require Iysergide; Brit Med ] 3:28;1967. 9. BlakeET, CashmanPJ, Thornton IT: Qualitative analysis of derivatization and appeared to be promising for analysis lysergic acid diethylamide by means of the lO-hydroxy of a large number of samples. derivative; Anal Chem 45:394; 1973. In routine analysis, LSD is tested almost at the lowest 10.Burris KD, Breeding M, Sanders-Bush E: Lysergic acid limit of sensitivity of a GC-MS instrument.The following diethy lamide, but not the congeners, is a potent serotonin 5- steps are recommended to improve sensitivity of detec- HT1C receptor agonist; ] Pharmacol Exp Ther 258:891; 1991. tion. 11. Cai J, Henion J:Elucidation of LSD in vitro metabolism by liquid chromatography andcapillary electrophoresis coupled 1. Extensi ve purification at the extraction step is essential with tandem mass spectrometry; ] Anal Toxicol 20:27; to achieve maximum sensitivity during GC-MS analy- 1996. sis. 12. Castagnoli N, Mantle PG:Occurrence of d-lysergic acid 2. A base (triethylamine) is important to protect LSD and 6-methyl-ergol-8-ene-8- carboxylic acid in cultures of from breakdown by the silicic acid in glass tubes or Claviceps purpurea; Nature 211:859; 1966. solid-phase columns. 13.Castro A, Grettie DP, Bartos F, Bartos D: LSD: Radioim- 3. Likemanyotheralkaloids,LSD,iso-LSD,andLAMPA munoassay; Res Commun Chem Pathol Pharmacol6:879; are unstable in halogenated solvents. For storage, 1973. halogenated solvent is removed and the compound is 14. Christie J, White MW, Wiles JM: A chromatographic redissolved in 100 ul, of 0.1% triethylamine in etha- nol. method for the detection of LSD in biological liquids;] Chromatogr 120:496; 1976. 4. Prolonged exposure to acid (pH <5.0) is avoided, 15. Clarkson ED, Lesser D, Paul BD: Effective GC-MS proce- unless it is for a short period of time and part of the dure for detecting iso-LSD in urine after base-catalyzed extraction process. conversion to LSD; Clin Chem 44:287; 1998. 5. Before batch analysis, the GC column needs to be 16. Cody JT, Valtier S: Immunoassay analysis of lysergic acid deactivated by 2-3 injections of unextracted LSD as diethylamide; ] Anal Toxicol2l :459; 1997. TMS derivative. The deactivation improves sensitiv- 17. Cohen S: Psychotomimetic agents; Ann Rev Pharmacol ity of detection. 7:301; 1967. 6. At the injection port, glass wool in the glass insert is 18. Cohen S: The psychotomimetic agents; In Jucker E (Ed): avoided. Columns may be clipped (-20 em) every Progress in Drug Research., Vol XV; pp 68-102; other week. Birkhauser: Basel-Stuttgart, Switzerland; 1971. 7. Baking the column at>280oC may activate the column 19. Dihrberg A, Newman B: Identification and estimation of and reduce the sensitivity. After batch analysis, the lysergic acid diethylamide by thin layer chromatography glass insert is replaced, and the column, injection port, and fluorimetry; Anal Chem 38:1959; 1966. and interface temperatures arc kept at 200 DC. 20.Faed EM, McLeod WR: A urine screening test for lysergide (LSD-25); ] Chromatogr Sci 11:4; 1973. REFERENCES 21. Floss HG: Biosynthesis of ergot alkaloids and related compounds; Tetrahedron 32:873; 1976. 1. Abramson HA, Jarvik ME, Gorin MH. Hirsch MW: Lyser- 22. Fowler R, Gomm PJ, Patterson DA: Thin layer chromatog- gic acid diethylamide (LSD-25): XVII.Tolerance develop- raphy oflysergide and other ergot alkaloids; ] Chromatogr ment and its relationship to a theory of psychosis; ] Psychol 72:351; 1972. 41:81; 1956. 23.Francom P, Andrenyak D, Lim HK, Bridges RR, Foltz RL: 2. 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Markel H, Lee A, Holmes RD, Domino EF: Clinical and sic science casework samples; J Forensic Sci 32:933; 1987. laboratory observations: LSD flashback syndrome exacer- 42. Johnson FN, Ary IE, Teiger DG, Kassel RJ: Emetic activity bated by selective serotonin reuptake inhibitor antidepres- of reduced ; JMed Chem 16:532; 1973. sants in adolescents; JPediatr 125:817; 1994. 43. Johnston LD, O'Malley PM, Bachman JG: National survey 63. Martin AJ: L.S.D. (lysergic acid diethy lamide) treatment of results on drug use from monitoring of the future study, chronic psychoneurotic patients under day-hospital condi- 1975-1992 (Vol I: Secondary School Students; Vol II: tions; Int J Soc Psychiat 3:188; 1957. College Students); National Institute on Drug Abuse: 64. McCarron MM, Walberg CB, Baselt RC: Confirmation of Rockville, MD; 1993. LSD intoxication by analysis of serum and urine; J Anal 44. 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ABOUT THE AUTHORS B.D. Paul, M.L. Smith

Buddha D. Paul received his B.Sc.(1963) and Ph.D. (1970) in chemistry from the University of Calcutta, India. Currently, Dr. Paul is the chief of the Drug Testing Research Branch in the Division of Forensic Toxicology, Office of the Medical Examiner, Armed Forces Institute of Pathology (Rockville, MD). Dr.Paul was a faculty member at the School of Medicine, Johns Hopkins University (Baltimore, MD) during 1974-78 and the Department of Chemistry, University of Wisconsin (Milwaukee, WI) during 1981-83. Dr. Paul worked as a quality assurance supervisor, technical director, and director at the Navy Drug Screening Laboratory, Norfolk, VA for the period of 1983-95.His interests are in development of new methods for drug testing.He developed and introduced new methods for the military drug testing program. Dr. Paul is the author of 47 articles published in professionaljoumals. Dr Paul is a consultant for the Department of Defense and for the Department of Health and Human Services. As an expert witness, he testified in more than 300 trials in the military courts. Dr. Paul also serves as a laboratory inspector for the National Laboratory Certification Program, Department of Health and Human Services. Dr. Paul was honored with the Navy Meritorious Award for sustained outstanding services from March 1983 to June 1995 for the Department of the Navy. He has been a member of several scientific associations, including the American Chemical Society, Society of Forensic Toxicology, Johns Hopkins Medical Surgical Association, and American Association of Clinical Chemistry.

Michael L. Smith received his B.A. from Kansas State Teachers College (Emporia, KS) in 1970 and his Ph.D. in biophysical chemistry from Purdue University (West Lafayette, IN) in 1974. Dr. Smith is currently a chief deputy medical examiner in the Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology (Rockville, MD), and heads the Division of Forensic Toxicology. Dr. Smith's past positions include director of clinical investigations at two different U.S. Army medical centers and commander of the U.S. Army Forensic Toxicology Drug Testing Laboratory that served the European Theater until 1991. He has 62 professional publications and his current research interests are in the development of analytical methods for drug analysis. He is a diplomate (1992) and a Certified Forensic Toxicology Laboratory Inspector (1996) of the American Board of Forensic Toxicology and an inspector (1987) of the National Laboratory Certification Program, Department of Health and Human Services.

Forensic Science Review • Volume Eleven Number Two • Dec. 1999