Journal of Chromatographic Science, Vol. 20, July, 1982

IdentiUsing ficationChromatographyof Major Drugs of Abuse ; T,A. Gough* and P.B. Baker _: Laboratory of the Government Chemist, Stamford Street, London, United Kingdom i

: Introduction chromatography (GLC), and MS reference data are available (1,2). The methods of choice will depend on the particular pro- _, This paper reviews the use of chromatography for the separa- blem at hand, the use to which the data will be put, and the _. .... |ion and subsequent identification of drugs that are commonly time and facilities available, it is hoped that this review will abused in Western society. Analytical data on drugs of abuse enable the analyst to select the chromatographic method most _' are or value in various fields, notably law enforcement, rex- appropriate to the situation. _ icology, pharmacy, pharmacology, and chemobotany. In the past few years there have been a number of papers However, the analytical approach will of necessity depend upon in which a particular chromatographic technique has been us- which of these fields is involved. In law enforcement there is ed for the separation of a wide range of drugs of abuse. Apart usually no shortage of the drug, and one problem facing the from a short list of very commonly encountered drugs, including analyst is thai of obtaining representative samples from perhaps heroin, cannabis, and cocaine, many drugs are seen only occa- several kilograms of material, in contrast, the toxicologist may sionally and some are almost never encountered, even at "street be required to detec! nanogram quantities in blood or urine, level". A breakdown of illicit importations into the United l! is the intention of the reviewers to consider the role of Kingdom (UK)of drugs controlled by the Misuse of Drugs Act ii' chromatography in relation only to the analysis of the raw or (1971) showed that in 1979, 80o/0 of all cases examined by this prepared materials, and not [o their detection after administra- Laboratory on behalf of the customs authorities related to can- lion to animals or humans. The reviewers do not claim to cover nabis products. Heroin and other narcotics represented 16¢/0, all papers falling within this category, but have deliberately cocaine 3o/0, and all other controlled drugs made up the remain- : selected for discussion papers typical era particular procedure, ing 1°70.Over [he same period, the proportions seized within i There have been major advances in instrumentation over the London and Southeast England by the civil police were 660/0 _ last few years, particularly in liquid chromatography, and this for cannabis products, 9O7ofor narcotics, 2°7ofor cocaine, and i: has had a major impact on drug analysis. The reviewers 23°7ofor all olher drugs (whether or not controlled by the Misme r_'cognise, and indeed emphasise, that there are many instances of Drugs Act). In the United States of America (USA), the pro- where a simple and cheap lechnique is adequate, and it is not portions of seizures, estimated from numbers of arrests during always necessary to resort to sophisticated instrumentation. This 1979 reported by the Drug Enforcement Administration, were review covers all aspects of chromatography from thin layer cannabis 24070, narcotics 20°7o, cocaine 32°70, and other drugs syslems to combined gas chromatography and mass spec- 24°70. trometry (although mass spectra are not discussed), and covers the main publications over the past decade. There are references to earlier papers, particularly on thin layer chromatography (TLC), but only where the work is of special interest or where I no significant advances have since been made. There is, in the Heroin* and Related Narcotics _! reviewers' opinion, no "best method" of analysis; methods i!; other than chromatography are indeed valuable in many cases. Thin Layer Chromatography l[ is, however, uncommon to rely solely on non-separatory Proof of trafficking of illicit heroin necessitates its rapid iden- : techniques simply because drugs are usually encountered as mix- tificalion on seizure by law enforcement agencies. The purity ._;_ lures, either with other relaled materials, cutting agents, or ex- of illicit heroin, when intercepted, ranges from grossly

and infra-red (IR) spectroscopy (as appropriate) are used for at importation. More typically the purity of importations, at • scrcipientseening. Inorcoconnjuncfirmationtionw. ithMasschromaspecttogrrometryaphy, ul(MS)tra-violeis alsot (UusV)- ladulleraledeast into thematerialUK, is inatth"estreeranget levof el"30 toto80almost°70, andpurethe matmaterialerial

= i e...... d for confirmation, often on-line with gas/liquid "In this review, "heroin" means pure diacetylmorphine. "Illicit heroin" means impure {_ "Author to whom correspondence should be addressed diacetylmorphine, which contains related narcotics and olher materials. i [: Reproduction(photocopying)ofeditorialcontentofthisjournalis prohibitedwithoutpublisher'spermission. 289 Journalof ChromatographicScience,Vol.20, July,1982

may contain several related narcotics including codeine, tech||iques. In a manner analogous to paper chromatography, acetylcodeine, the acetylmorphines, morphine, noscapine, and cellulose plates, treated with 5% sodium dihydrogen citrate and papaverine. A variety of cutting agents are found in "street then dried, have been used to separate morphine from level" heroin, from sucrose to strychnine, barbiturates to brick 6-acetylmorphine. Heroin and acetylcodeine were unresolved. dust. Detection of the major narcotics is the basis of identifica- The plates were developed in a solution of citric acid, water, tion of illicit heroin, l-butanol (4.8 g/i 30 ml/870 ml) (10). The first reported satisfac- Paper chromatography was originally used to separate heroin, tory separation of heroin from acetylcodeine was achieved us- codeine, acetylcodeine, morphine, the acetyhnorphines, and ing activated silica gel plates with an alkaline solvent mixture procaine, using mixtures of l-butanol (or 3-methyl-l-butanol), (I-butanol, butyl ether, ammonium hydroxide, 25/70/5) (I I). water and acetic acid (3). Insufficient resolution was obtained Alumina plates, heated at 100°C in the presence of water, between several of the narcotics, and acetylcodeine was not well separated heroin, morphine, 3-acetylmorphine and resolved from heroin. It has, however, been separated from 6-acetylmorphine using benzene, methanol, ammonium hydrox- heroin after its hydrolysis to codeine (4). Detection was by ex- ide (90/10/0.2) (12). amination under UV light and by spraying with potassium As part of a procedure for the assay of morphine for phar- iodoplatinate. These methods of detection are used by the vast maceutical use, Holcomb et al. (13) used silica gel plates (Merck majority of workers in present-day TLC systems. Paper chro- GF) with chloroform, methanol, diethylamine (80/15/5) as sol- matography can be used to separate morphine from codeine vent. This system separated morphine, pseudomorphine, (5-7), but improvements in TLC and the availability of com- morphine-N-oxide and parabens. The use of TLC for the separa- mercially coated plates in recent years has rendered paper tion of morphine and its metabolites was studied by Yell (14), chromatography obsolete. It is, however, still occasionally who used silica gel plates with a variety of basic solvent mix- reported, for example, in the preparative scale separation of tures. A satisfactory separation of codeine and morphine-N- 6-acetylmorphine from/_-chloromorphide in a report of the lat- oxide was obtained. Morphine and norcodeine were not resolv- ter compound in an illicit opium-like preparation (8). ed. Pre-coated silica gel plates were developed with ethyl acetate, .... TLC is widely used for the separation of narcotics in view methanol, ammonium hydroxide (85/10/5), the solvent system of its simplicity. It thus offers a screening method prior to ex- de'scribed by Davidow (15). Sherma et al. (16) developed a TLC amination by other chromatographic techniques. No figures are system for the quantitation of morphine, using a densitometer. available for the UK, but in the USA, 93 out of 125 crime Silica gel G and GF pre-coated plates were used without pre- laboratories involved in heroin analysis reported the use of TLC treatment, with chloroform, acetone, methanol, t-butylamine (9). These laboratories did, of course, also useother analytical (30/40/10/20). Morphine was detected by UV and the

Table I. References to the TLC of Narcotics

Reference 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Acetylcodeine X X X X X X 3-Acelylmorphine X X 6-Acetylmorphine X X X X X X X X Codeine X X X X X X X X X X X X X X X X Ethylmorphine X X X X Heroin X X X X X X X X X X X X X X Methadone X X X X X X X Methaqualone X Morphine X X X X X X X X X X X X X X X X X X X . X I_orphine-N-oxide X X X Nalorphine X X X Norcodeine X Normorphine X --- Noscapine X X X X Papaverine X X X X X X Thebaine X X X X

Reference 44 45 50 92 93 94 95 ,, 96 97 122 132 162 163 170 191 211 215 217 250 Acetylcodeine 3-Acetylmorphine 6-Acetylmorphine X X Codeine X X X X X X X X X X X X Ethylmorphine X X Heroin X X X X X X X Methadone X X X X X X X X X X X X X Melhaquatone X X X X Morphine X X X X X X X X X X X X X X X X Morphine-N-oxide Nalorphine X Norcodeine X Normorphine X Noscapine X Papaverine X X Thebaine X X

290 _" Journal of Chromatographic Science, Vol. 20, July, 1982

[[ fluorescence measured by scanning either in the transmission 6-acctylmorphine using activated silica gel plates. The solvent

- ed after spraying with an aqueous 1/1 mixture of 20/0 ferric monium hydroxide (75/25/5/1). chloride and 2°70potassium ferricyanide. While the method was Moffat and Clare (26) examined 11 published papers con- sensitive (limit 25 ng) and a linear response with respect to con- raining 26 TLC systems that have been used for the analysis i or reflectance mode. Alternatively, quantitation was perform- system was chloroform, diethyl ether, methanol, 25% am- !_i centration was obtained, the authors gave no information on of basic drugs. They considered each system in terms of: i) speed potentially interfering compounds. A valuable report by Manura of analysis, it) sensitivity, iii) repeatability, iv) distribution of et al. (9) examined four TLC solvent systems for the separa- Rf values, and v) inter-system correlation. They selected 100 _ tion of heroin from 56 structurally related compounds. Silica drugs in common use and on the basis of the above criteria, gel G plates were used with: two systemswereselected.These may be combined into a single,

(5/50/40/5); sprayed with 0.1N sodium hydroxide were used. Solvents were A) ammonium hydroxide, benzene, dioxan, ethanol two-dimensionalsystem, since the same plates (silica ge160) pre- ,. B) ethyl acetate, methanol, ammonium hydroxide (85/10/5), cyclohexane, toluene, diethylamine (75/15/10) and chloroform, from reference 15; methanol (90/10). Development time for each solvent was I to i' C) methanol, ammonium hydroxide (100/I.5), from reference ! V2 hr for a 10-cm plate. 17; Using iodoplatinate reagent, most narcotics give a similar col- D) ethanol, acetic acid, water (60/30/10). our, which therefore has little diagnostic value. Vinson et al i None of these systems, individually or collectively, were able (27) recommended N,2,6-trichloro-p-benzoquinoneimine to distinguish between all of the compounds, although in prac- (TCBI) with tentative identification based on colour as well as tice it is most unlikely that more than a few would be en- Rf. A 16-fold better detection limit was claimed for morphine :: countered in illicit preparations. The study was valuable in draw- using this reagent. Dutt and Poh (28) also drew attention to ing attention to the possibilityoferroneous conclusions when the value of using a spray reagent, which gives colours using TLC alone. Of the narcotics commonly encountered in characteristic of particular groups of drugs, and examined illicit heroin and examined by Manura et al. (i.e., heroin, co- ninhydrin for the detection of 56 basic drugs. Activated silica deine, acetylcodeine, morphine, and 6-acetylmorphine), only gel plates were used with Davidow's system (15). After spray- solvent systems A and B gave adequate separation. Using either ing, the colours of the spots at room temperature, 80°C, 120_U, system A or B, 16 compounds were insufficiently resolved from and 160°C were noted. At room temperature and 80°C, heroin heroin. System A had the drawback that it used benzene. System and codeine were not detected; at 120°(:: both gave blue spots, B is used in many laboratories, including that of the reviewers, and at 160°C they were both brown. Masoud (29) examined It does not resolve methadone (a synthetic narcotic) from co- various TLC systems for the separation of 43 drugs (including caine (18). Several TLC solvents were examined by Clark, for 19alkaloids) encountered at "street level", using silica gel GF2,, use with silica gel plates (19). Again there was insufficient resolu- plates activated for I hr at 110°C. A systematic investigation tion between heroin and several other narcotics. Chloroform, of five solvents and five spray reagents enabled a tentative iden- cyclohexane, diethylamine (8/10/3) separated heroin, codeine, tity of all these compounds to be made. morphine, and 6-acetyhnorphine, but caffeine, which is From this summary of published methods, it is evident that sometimes present in illicit heroin, was not separated from despite the variety of solvent systems which have been used, 6-acetylmorphine. However, these two compounds can be no one system will resolve all the narcotics. For law enforce- distinguished after spraying with iodoplatinate reagent, as cat- merit purposes the system of choice should be one that at least, feine does not react (20). in the shortest possible time, resolves heroin from the other ma- Marumo et al. (21) have separated heroin, codeine, acetylco- jor narcotics and from major interferants commonly en- deine, morphine, and 6-acetylmorphine on Merck silica gel G countered. Such a system is described by Davidow (15), although plates using diethyl ether saturated with water, acetone, this does result in hydrolysis of heroin to acetylmorphine. This diethylamine (85/8/7). Quantitation of the heroin present was can be avoided by using the solvent proposed by Marumo (21). based on UV absorption at 280 nm and 360 nm. The detection Any TLC system should be regarded only as an aid to screen- limit was 1l*g. Similar detection limits were reported by Engelbe ing prior to further examination. (22) for morphine (I .5 #g), codeine (2 #g), and thebaine (2 #g). These workers also used silica gel plates for I1 other Gas/Liquid Chromatography papaveraceous alkaloids. Three solvent systems were used, the most satisfactory being toluene, acetone, ethanol, ammonium Methods for the estimation of heroin based on GLC can be hydroxide (20/20/3/1). Quantitative analysis of morphine and divided broadly into two categories. Heroin, and some of the its degradation products has been reported by Ebel (23) using related narcotics, can be detected directly using a flame ionisa- UV reflectance at 285 nm, with Kieselgel 60 (Merck) plates. Co- tion detector (FID) after their separation. However, the polar deine, morphine, pseudomorphine, morphine-N-oxide, and the- nature of these compounds makes chromatography difficult, baine were resolved with chloroform, ethanol, acetone, am- and several procedures in which the narcotics are derivatised monium hydroxide (20/20/5/I) as solvent. The precision* of prior to chromatographic analysis have been described. With quantitation was 2 to 4°/o. Vincent et al. (24) reported that the- appropriate choice of derivative this can have the added ad- baine appeared to be unstable above 60°C, as an additional spot vantage that by using an electron capture detector (ECD) there was observed on TLC. The procedure, for the identification is an improvement in the lower limit of detection compared with of drugs encountered at "street level," used by van Welsum the FID. Curry and Patterson (10) separated heroin, codeine, (25) separated the opium alkaloids, heroin, and acetylcodeine, and 6-acetylmorphine using a column contain- ing cyclohexane dimethanol succinate (CDMS) at 250°C. Caf-

:-Througnoui-th,sre-vie-w, procision and accuracy are expressedinlermsofcoefficients feine (a common adulterant of illicit heroin from the Far East) : ofvaria[,on, was eluted with the solvent dimethylformamide (DMF) under

_z 291 Journal of Chromatographic Science, Vol. 20, July, 1982

these conditions, and morphine could not be successfully phine, 6-acetylmorphine, and caffeine. The most Satisfactory chromatographed. Most workers have used methyl- (e.g. OV-I) was silanised OV-210 (trifluoropropyl-, methyl-silicone), and - or methylphenyl- (e.g. OV-17) silicone stationary phases. While this was used to analyse a ratio of mixtures prepared in the resulting in less loss of compounds by adsorption, these laboratory to determine the validity of the work. Reproducibility materials fail to adequately resolve acetylcodeine from of retention times over a 7-week period were typically 0.407o, 6-acetyimorphine. Morphine has been found by several workers and variation of detector response over the same period was (30-32) to be difficult to chromatograph, most probably because 4 to 8°7odepending on the compound. This paper also gave data of its high polarity, which favours adsorption. Dezan and on relative retentions, on-column losses, and peak asymmetry Fasanello (33) used 3°70OV-I stationary phase at 235°C in a for the five narcotics on all the columns studied. Two groups method for the quantitation of heroin and quinine. Morphine (39,40) have observed transacetylation when heroin and mor- was not included in their study and although samples were ex- phine are both present in samples, although no systematic study amined for the presence of acetylcodeine and 6-acetylmorphine, of this phenomenon has been reported. High injection these were not resolved from one another, temperatures were used in both studies. Later work (38) has An extensive study to measure the heroin and quinine con- shown that because some of the narcotics are thermally labile, tents of samples was later described by Moore and Bena (34), the lowest possible injection and column temperature should using the same stationary phase at 275°C. Heroin was present be used, which should also minimise the risk of transacetylation. over the range 2 to 20070, with a typical precision of replicate All of the above methods suffer from the disadvantage that determinations of 3°7o. Accuracy was within the range 2 to 607o. the narcotics, and morphine in particular, suffer adsorption Codeine, morphine, the acetylmorphines, papaverine, losses within the GLC column, although such losses are methadone, and some local anaesthetics were included reproducible (38) under some conditions. Zoccolillo (41) used qualitatively in the study. Vincent et al. (24) analysed thebaine glass beads treated with a surfactant (sodium dodecylbenzene- and obtained a precision of 1070on replicate analyses, despite sulphonate) prior to coating with stationary phase for the small adsorption losses on new columns and their own obser- separation of drugs, including narcotics. Morphine gave an ex- vation (see above) that the compound was thermally unstable, cellent elution profile with little tailing when using beads with With the objective of correlating the composition of illicit heroin a very low percentage of FFAP and SP-525, respectively. with its origin, Van der Slooten and Van der Helm (35) measured Although no data were reported on the use of this system for the proportions of heroin, morphine, and 6-acetylmorphine in quantitative analysis, it is unlikely to be of much routine value 32 samples and achieved a 5% precision of these ratios on because of serious column overloading by the sample. Fontan replicate analyses of a given sample. Two stationary phases were and Hill (42) used a specially prepared Carbowax 20M used, SE-30 (methyl silicone) and QF-! (trifluoropropyl, methyl- (polyethylene glycol) coated column. Excess stationary phase silicone), both at 235°C. The paper did not consider the effect was deliberately removed from the support by Soxhlet extrac- on the results of interference by acetylcodeine. The same two tion after prolonged heating in nitrogen. This modified phase columns were used to examine illicit heroin for adulterants, in- was compared with OV-17 and SE-30 for the separation of cluding caffeine, quinine, methadone, cocaine, and strychnine, several polar drugs, including heroin, codeine, and morphine. all of which were resolved from each other and from the nar- Columns were also prepared of OV-17 and SE-30, each on a cotics. This information was used to deduce whether different support treated with modified Carbowax 20M. No chromato- seizures were originally part of the same lot. The authors pointed grams were shown of morphine, and it is therefore difficult to out that different conditions of storage could affect the rate judge the success of the technique in reducing adsorption losses. of hydrolysis of the narcotics. However, combined with other Further, none of the specially treated columns resolved heroin information pertaining to the seizure, chemical information may from morphine, and retention times were substantially longer give evidence to support or disprove a connection, than on conventionally prepared silicone phases. Gough and Nakamura et al. (36) used combined GLC and MS to separate Baker (38) prepared six modified phases by in situ oxidation heroin from procaine, which were identified on the basis of their of silicone gums. Results were expressed in terms of adsorp- low resolution spectra. Total ion monitoring was used to tion losses and peak asymmetry. Using modified OV-61 generate chromatograms. No quantitative data were given. An (phenyl-, methyl-silicone) excellent peaks were obtained for all OV-17 column was used and therefore, resort to MS did not the narcotics, and adsorption losses were small, even for mot- solve the problem of lack of resolution between acetylcodeine phine. This phase also resulted in the partial separation of and 6-acetylmorphine, at least from the point of view of their acetylcodeine from 6-acetylmorphine. quantitation. Clark (19) used on-line GLC/MS to identify 18 Lim and Chow (43), as part of their method for analysing morphine derivatives, several of which were co-eluted. Although illicit heroin, described a novel method for the estimation of each compound is recognisible on the basis of its spectrum, in- acetylcodeine and 6-acetylmorphine. Using OV- !7 as stationary terpretation of overlapping spectra of related substances is dif- phase, the heroin, caffeine, and codeine (as internal standard) ficult. Manura et al. (9) examined 3 silicone phases and a contents were determined. The sample was then acetylated, carborane-silicone phase (Dexsil 400) for the separation of whereby any 6-acetyimorphine present was converted to heroin. heroin and structurally related compounds. The use of any two The sample was re-analysed under the same GLC conditions of these columns enabled a distinction to be made between most and the acetylcodeine content, not now subjected to interference of 57 compounds, simply on the basis of retention times. Hueger from 6-acetylmorphine, was measured. The 6-acetylmorphine et al. (20) used OV-I 7 to examine 50 seizures of illicit heroin content was calculated from the increase in the amount of heroin for the presence of heroin and caffeine. Shaler and Jerpe (37) resulting from the acetylation. Morphine, which would also be quantified heroin by FID and confirmed its identity by IR spec- converted into heroin, was not included in this study, as it was troscopy after effluent trapping. Baker and Gough (38) studied usually absent from the samples encountered by these workers. the suitability of several stationary phases for use in the separa- The GLC analysis time per run was 12 min, and precision of tion and quantitation of heroin, codeine, acetylcodeine, mot- results was 1.4070for heroin and 3070 for caffeine.

292 Journal of Chromatographic Science, Vol. 20, July, 1982

Morphine has also been detected as heroin, its diacetyl samples after preparation of the trimethylsilyl (TMS) ethers of derivative, by Wallace eta/. (44). Although useful in the analysis some of the diluents, which were then analysed on SE-]0. The of biological fluids, because heroin is converted in vivo to mot- method could, in principle, be used to determine the presence phine, it is less valuable in illicit heroin analysis. On-column of sugars, although only partial separation of heroin and the

'_ methodacetylation does,does nohowevet giver,quanteliminaitativete convethersiondiffi(45,culty46). Theof heroin,lactose danerivd athetivederivativeswas achievofed.morphineThe otheranddra6-awcetbackylmorphinwas thate _ chromatographing morphine, were incompletely resolved. However, quantitative results for !; Derivatisation, commonly used to obtain an enhanced and heroin in illicit samples were in reasonable agreement with UV selective response in toxicological analysis (47-52), is also rele- absorption measurements. Sobol and Sperling (54) described ::_": vant in the present context, as it may enable satisfactory a quantitative method for the comparison of exhibits contain-

choice of derivative also achieves separation of acetylcodeine morphine, using OV-25 (methylphenyl-, diphenyl-silicone). from 6-acetyimorphine. Grooms (53) studied illicit heroin Derivatisation with bis-trimethyisilyl-2,2,2-trifluoroaeetamide i chromatography to be performed on morphine. Appropriate ing heroin, codeine, acetylcodeine, morphine, and 6-acetyl- I i '_ Table II, References to the GLC of Narcotics Reference 9 10 14 18 19 20 24 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Acetylcodeine X X X X X X X X ii_. 3-Acetylmorphine X X X _ 6-Acetylmorphine X X X X X X X X X X X j! Codeine X X X X X X X X X X X X X _ Ethylmorphine X X X Heroin X X X X X X X X X X X X X X X X X Methadone X X X X X X Melhaqualone X Morphine X X X X X X X X X X X X X X Morphine-N-oxide X Natorphine X Norcodeine X Normorphine X Noscapine X X X Papaverine X X X X X X Thebaine X X X X X

Reference 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 68 Acetylcodeine X X 3-Acetylmorpnine X X X 6-Acetyimorphine X ' X X X X X X X X Codeine X X X X X X X X X X X X X Ethylmorphine X X X Heroin X X X X X X X X X Methadone X X X Methaqualone Morphine X X X X X X X X X X • X X X X X X X X X Morphine-N-oxide Nalorphine X X X X X X Norcodeine X Normorphine X X Noscapine X X Papavedne X X Thebaine X X X

Reference 92 106 179 180 185 191 218 219 220 221 222 250 309 333 ,_ Acetylcodeine ,_ 3-Acetylrnorphine !!, 6-Acetylmorphine X !_ Codeine X X Ethylmorphine X i Heroin X X X Methadone X X X X X X X _ Methaqualone X X X X X X X X Morphine X X X X i_. Morphine-N-oxide _ Nalorphine X l_ Norcodeine _:_ Norrnorphine

• Papaverine X X Thebaine X i Noscapine X

i 293 Journal of Chromatographic Science, Vol. 20, July, 1982

(BSTFA) was used to separate, and hence quantify, the acetylco- significance can be attached to its quantitative determination deine and 6-acetylmorphine, although it was noted that it is not in illicit heroin. An inter-laboratory comparison has been made always possible to use a silanisation procedure on illicitly between direct and derivative methods for the quantitative prepared samples. This is due to competing reactions from cut- analysis of codeine and morphine in opium (63). Direct analysis ling agents, particularly sugars, which could result in erroneous of morphine was regarded as unsatisfactory and attention was quantitation. Despite incomplete resolution between acetylco- therefore concentrated on the use of TMS-derivatives for which deine and 6-acetylmorphine, analysis rnust then be carried out a mixed OV-225 (phenyl-, cyanopropyl-, methyl-silicone) and on the underivatised sample. Precision of the quantitative results OV-25 stationary phase was found to be the most suitable. Preci- using both methods varied from 0.20/o to 17% depending, in- sion was between 207o and 707o for the data provided by 8 ter alia, on the concentrations of the constituents. The detec- laboratories, each analysing 5 different opium samples in tion limit for both codeineand morphinewasapproximately replicate. i, ! 5 ng. Morphine has been determined in opium after treatment In summany the GLC analysis of opiates suffers from the I with N,O-bis-trimethylsilylacetamide (BSA) or BSTFA (52,55). following problems: Other compounds are, of course, also derivatised during this i) losses of compounds, particularly morphine, can be reaction. Thebaine was only partially resolved using UCW-98 substantial; (vinyl-methyl-silicone), although the authors (52)claimed that ii) derivatisation is subject to interference from cutting other constituents did not interfere quantitatively with the agents; analysis. OV-17 was unsatisfactory because codeine and mor- iii) transacetylation can result in erroneous analysis; i phine were incompletely resolved. Rasmussen (56) showed that, iv) it is difficult to separate acetyicodeine from i in contrast to simple acetylation, on-column silylation gave 6-acetylmorphine. satisfactory results using N-trimethylsilyl-imidazole (TSi). Us- No one method described above overcomes all these pro- ing combined GLC/MS, no underivatised codeine or morphine blems, and the method of choice will therefore depend on the was detected in the GLC el'fluent, from which it was conclud- type of sample under analysis and the reasons for carrying out ed that the reaction was complete. However, any remaining the analysis. For illicit heroin, direct analysis on silanised traces of these cotnpounds would probably be lost on the col- OV-210 will result in minimal and reproducible losses, with suf- umn by adsorption. Dexsil-300 (carborane-silicone) stationary ficient resolution of the major narcotics for quantitation. phase was used in this work, because SE-30 did not separate Analysis of opium is most satisfactorily carried out after silanisa- : TMS-morphine from thebaine and OV-17 did not separate the tion. The use of the lowest possible injection and column morphine and codeine derivatives. In a subsequent paper, Brug- temperature should minimise the possibility of transacetylation. gard and Rasmussen (57) studied on-column acylation using In view of these limitations, HPLC may offer an alternative N-trifluoroacetylimidazole (TFAI) and N-heptafluorobutyl- and more attractive method of analysing narcotics. The extent rylimidazole (HFBi). The use of TFAI occasionally resulted in to which this is realised in practice can be judged by consider- the formation of both mono- and bis- (TFA) morphine. Neglec- ing the following papers. ting these data, precision of quantitative results was between 1 and 3%, and in general, was better using HFBI. The detec- High Performance Liquid Chromatography tion limit for morphine by electron capture was 20 pg and for codeine 100 pg, provided heart cutting was used to prevent detec- Cashman and Thornton (64) were among the first workers tor contamination by excess derivatising reagent (58). The same to use HPLC for the analysis of heroin, morphine, and group used a wail-coated open tubular (WCOT) SE-30 capillary 6-acetylmorphine, which were completely resolved in 25 rain. column to exanaine an illicit heroin sample, after treatment with Detection was by UV absorption, in which the effluent was BSA, BSTFA or TSI. GLC analysis time was 40 rain despite scanned over the range 210 to 400 nm: identification was bas- temperature programming to 260°C, and this method appeared ed on the resulting spectrum. The column was l-m x 4.5-ram to have no advantage over packed column analysis (59). Prager i.d., containing silica (Porasil T), with a mobile phase of et al. (60) used a I/1 mixture of SE-30 and OV-17 to separate chloroform and methanol. Despite varying the proportions of heroin (and cocaine) from the TMS derivative of morphine, solvent, it was not possible to separate heroin from procaine. Other narcotics encountered in illicit heroin were not considered. Using an alumina column, 6-acetyimorphine and procaine were The quantitative results had a precision of 3°7oand an accuracy co-eluted, Wittwer (65) used ion e_chang¢ chromatography to of 5%. t4eptafluorobutyric anhydride (HFBA) has been used separate 22 compounds, including the major opium alkaloids. as a derivatising reagent to determine trace amounts of codeine, Knox and Jurand (66) also used ion exchange, which partially morphine, and 6-acetylmorphine. Detection limits were 20 pg, separated codeine, morphine, papaverine, thebaine, cryptopine, 80 pg, and 100 pg respectively. Various silicone (OV) phases and noscapine, with an analysis time of 50 min. Detection was were studied, the most suitable being OV-17. Precision of the by UV absorption at various fixed wavelengths. Methadone was quantitatitve results fell in the range 2 to 6% (61). The use of separated from morphine and its derivatives using an anion ex- fluorine-containing silicone phases was ruled out, as detection change column. A subsequent paper by the same authors (67) of the derivative was by electron capture, although it is only examined the use of this system for quantitative analysis, bas- necessary to use this detector in the determination of trace quart- ed on peak area measurements at particular wavelengths. titles. These authors also conceded that derivatisation is un- Calibration curves were presented, but no quantitative analyses suitable for the analysis of adulterated illicit heroin, particularly were reported. The identification of the compounds was based when procaine is present. Moore (62) has described a method on complete UV and electron impact mass spectra after effluent of distinguishing between 3-acetylmorphine, 6-acetyhnorphine, collection. Hays et al. (68) used an ion exchange column with and the other constituents in illicitly prepared heroin, using gradient elution and separated morphine from (i) HFBA derivatisation. 3-acetyhnorphine is, however, unstable, 3-acetylmorphine, 6-acetylmorphine, and codeine, which were both as the isolated compound and in solution, and little themselves only partially resolved; and (ii)acetylcodeine and

294 7:

_- Journal of Chromatographic Science, Vol. 20, July, 1982

_! Table III. Referencesto the HPLCof Narcotics

f' Relerence 20 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 Acetylcodeine X X X X X X X X _i 3-Acetylmorphine X X 6-Acetylrnorphine X X X X X X X X X X X X X Codeine X X X X X X X X X X X X X X X X _! Ethylmorphine X X X _i Heroin X X X X X X X X X X X X X X X X X X _'.'i Methadone X X X X X X X X ,; Methaquatone X X X X Morphine X X X X X X X X X X X X X X X X X X X X _', Morphine-N-oxide

I ' NoNalorphinercodeine X , Normorphine

, __, NosPapcaaveprineine X X X X X X X X X X X X X .... Thebaine X X X X X X X X X X X

Reference 85 86 87 88 89 90 91 205 207

Acetylcodeine X Most of the above procedures gave inherently poor resolu- 63--AceAcetylylm0lmorphirpllinnee X X X tion and broad peaks, but the introduction of small particle size Codeine X X X X X X ( < 10 #m) packings resulted in very significant improvements. Ethylm0rphine X For example, Jane (73) publishedsomeexcellentchromatograms Heroin X X X X showing the separation of major opium componentsusing 6-#m MethMethadoaqual0nene X silica (Partisil) in l0 min. This material was fined to reducethe Morphine X X X X X X X X particle size range, prior to packing in 25-cm x 4.6-mm i.d. col- M0rphine-N-oxide umns. Detection was at a singlewavelength,selectedon the basis Nat0rphine X X X of the UV absorption maximum of eachcompound. The detec- N0r0rc0rn0rdeiphinene X tion limit for heroin and morphine was 50 ng. Replicate injec- N0scapine X X X lions gave peak height variations better than 2°70. Replicate Papaverine X X quantitative results for morphine in opium had a precision of Thehaine X X X 5%. Twitchett (74) usedion exchangechromatography with gra- dient elution to examine thecomponents of illicit heroin, which were eluted in 7 minutes. The effluent was monitored at 270 nm, which was found to give less baseline drift than the use heroin, which were incompletely resolved. Despite the authors' of shorter wavelengths. The heroin, morphine, and 6-acetyl- claims, this is no advanlage over GLC unless only morphine morphine contents of 20 samples were based on peak height is to be determined. Wu e/el. (69) compared the performance measurements.Caffeine, strychnine, and quinine were frequent- of Corasil (silica) columns, either statically or dynamically ly detected. No mixtures of known composition were examin- coated with polyethylene glycol (Poly G 300). Separation of ed in order to validate the system for quantitative analysis, morphine, codeine, papaverine,and thebainewas achieved,with although repetitive analyses of illicit mixtures had a precision a shorter analysistime when using dynamiccoating. An accuracy of 2.307ofor heroin and 4.5070for 6-acetylmorphine. The coef- of 10% was obtained for the quantitative analysis of the last ficient of variation of retention times for heroin was 1.2070.The three compounds, although insufficient information was given same laboratory (75) also used a 10-#m octadecylsilane (ODS) to conclude that the system was satisfactory over a wide con- bonded silica column 0t-Bondapak C,,) for the analysis of 30 centration range. The analysis of many drugs encountered at drugs, including narcotics. Various aqueous methanol mixtures "street level" was accomplished by Chanet al. (70) using gra- covering pH 3 to 9, were used as the mobile phase. Although dient elution with a Corasil II column. Codeine was eluted in the system gave satisfactory chromatograms for acidic and 17 rain and heroin in 20 min. Detection was at 254 nm with neutral drugs, the performance for basic drugs was unaccep- confirmation by complete UV spectra on collected effluent. The lably poor. Wheals (76) investigated various laboratory-prepared problem of peak identity resulting from irreproducible reten- chemically bonded phases, based on silica, in an attempt to im- tion times, common in gradient elution, was reduced by using prove their use for basic drugs. several internal standards. Opium alkaloids were studied by Ver- Huizer et al. (20) achieved excellent separation of heroin, co- poorte and Svendsen (71) using a Merckosorb Si 60 column with deine, acetylcodeine, 6-acetylmorphine, and caffeine using a either chloroform, methanol, or diethyl ether, methanol as silica column (Lichrosorb Si 60 5 #m), with a mobile phase of mobile phase. Detection was at 254 nm or 280 rim. No quan- diethyl ether, 2,2,4-trimethylpentane, methanol, diethylamine _ titative analyses were reported, and no chromatograms were (52.8/35/12/0.2). A number of illicit heroin samples were ex- _ shown. Heroin, morphine, and methadone have been examin- amined, and the distribution of each, compared throughout the i ed by reverse-phase chromatography (72) using Bondapak C,,. samples, was used as a basis upon which to group the seizures The mobile phase was acetonitrile, water containing ammonium for intelligence purposes. AIbanbauer et al. (77), using #- . carbonate to generate the free bases of the narcotics. No infor- Bondapak C,, with water, acetonitrile, ammonium carbonate, marion on repeatability or interference from other narcotics was separated heroin, acetyicodeine, morphine, 6-acetylmorphine, given, noscapine, and papaverine. Rather broad peaks were obtained

ii 295 1 Journal of Chromatographic Science, Vol, 20, July, 1982 and that of acetylcodeinc, it_ particular, was poor. Reuland and a propylsulphonic acid silica, and untreated silica. On each of Trinler (781 used reverse-phase chromalography to separate these columns, 161 drugs were examined, of which 93 were heroin from other narcotics, contaminants, and culling agents, found to have unique retention characteristics. Fifteen opium including procaine, quinine, and lactose, in "street" samples, alkaloids were included, and the pairs papaverine-noscapine and The mobile phase was acetonitrile, water in varying proportions, dihydrocodeine-hydromorphone were not resolved on any col- and ammonium carbonate. Despite using 10-_m ODS-siliea umu. rwo columns were required to distinguish between the (Partisil), broad peaks were obtained with incomplete resolu- remaining alkaloids and commonly encountered cutting agents. tion between several of the narcotics. Rasmussen et aL (79) us- Acetylcodeine was not examined. Poochikian and Cradock (86) ed similar conditions to determine the morphine content of used reverse-phase chromatography with aqueous methanol and opium, h_ addition, they used 5-#.m silica (Spherisorb) with 10-tim ODS-silica (p.-Bondapak C_) packing. This separated methanol, chloroform, ammonium hydroxide. Quantitative heroin, morphine, 3-acetylmorphine, and 6-acetylmorphine. results for morphine were compared with data obtained spec- Detection was at 235 nm with a limit of 10 to 15 rig. Precision troscopically and were, in general, in good agreement. Preci- of the qt, antitation of heroin was 1.6%. Using the same pack- sion of the determinations were 2.3% and 0.8% respectively ing, Baker etal. (87) compared observed and calculated reten- for the two different column systems. Feher etal. (80) also corn- tion indices for several narcotics, but agreement was found only ,_ pared silica (7-/_m) normal-phase chromatography with 10-#m in some instances. Vincent and Engelke (88) used 5-#m silica ! reversed-phase ODS silica for the separation of codeine and (/_-Porasil) and hexane, methylene chloride, ethanol, morphine in pharmaceutical preparations, diethylamine (300/30/40/0.5) to separate and quantify the five Lurie (81)used reverse-phase ion pair chromatography to major opium alkaloids, which were detected at 285 nm. separate 5 major opium alkaloids (codeine, morphine, thebaine, Replicate quantitative analyses of morphine had a precision of noscapine, and papaverine) in 20 rain. A/_-Bondapak C,, pack- 4%. Nine other alkaloids were also examined, but not all were ing was used with a mobile phase of methanol, acetic acid, resolved; heroin and its hydrolysis products were not included. water, and a counter io,1of I-heptanesulphonate. Unfortunate- A reverse-phase system designed specifically for the analysis of ly, codeine was unresolved from 6-acetylmorphine, and heroin illicit heroin was described by. Love and Pannell (89), using was unresolved from aeetylcodeine. Ion pair reverse-phase acetonilrile and aqueous ammonium acetate with a #-Bondapak chromatography was used by Soni and Dugar (82) with a similar C,, packing. Satisfactory chromatography was achieved and the mobile phase, and they reported the separation of several nar- separation of eight narcotics, caffeine and strychnine, was cotics on 10-_ttm#.-Bondapak C,, packing. Counter ions were demonstrated. A linear response, based on peak height tetrabutylammonium phosphate or I-heptanesulphonate. lden- measurements, was found for all the compounds, except mor- tification of the opiates was based on the ratio of the responses phine, for which it was necessary to base quantitation on area at 254 nm and 280 nm. While the authors claimed that this gave measurements. Although these authors did not expect that ion much enhanced reliability compared with the use of retention pairing methods would be suitable for illicit heroin analysis on times alone, several related narcotics have similar absorbance the basis of preceding publications, Baker and Gough (83) subse- ratios, including soine which are not resolved on this system, quently separated the same narcotics using tetrabutylammonium Baker and Gough (83) also used one of these ion pair systems phosphate. The packing was aminopropyl bonded silica and the and reproduced the results of Lurie (81), in particular the lack solvent acetonitrile, water. Quantitative analysis of mixtures of of resolution between heroin and acetylcodeine. No explana- known composition, representing illicit heroin, was carried out, tion can be offered for the fact that this this does not agree and satisfactory recoveries and percentage compositions for the with the data reported by Soni and Dugar (82), who obtained constituents were achieved. a relative retention of 6.2 for heroin and acetylcodeine. Absor- All the above papers use UV absorption as the basis for detec- bance ratios have also been used by Baker et aL (84), who ex- tion, and al!hough this has adequate sensitivity for most pur- amined 101 drugs using three solvent-colunm systems. On the poses, a more sensitive detector may be desirable for the detec- basis of retention times alone, only 9% of the drugs could be tion of trace quantities of narcotics, for example in clothing. distinguished on any one system. But by considering all the Wallace etal. (90) used an electrochemical detector for the detec- systems and absorbance ratios at 254 nm and 280 nm, 95% of tion of morphine in urine extracts. The column was ODS-silica the drugs could be distinguished although, of course, many were with an aqueous phosphate buffered methanol mixture as structurally unrelated. Nevertheless, this is far higher than eluant. Precision of the quantitation of morphine was 6.5% for distinction based only on retention times on two dissimilar col- 250 pg and 12% at the detection limit (50 pg). Peterson et al. ureas. Long term precision of the ratios was 21%. Codeine and (91) used a U V and an electrochemical detector in parallel to morphi,le, which were not resolved on #-Porasil with methanol, detect opium alkaloids after reverse-phase separation on #- ammonium hydroxide, had similar absorbance ratios and it Bondapak C,,, using aqueous methanol as solvent. The detec- would therefore be difficult to distinguish between them on this tion limit for morphine was 100 pg. The electrochemical detec- basis, even assuming only one was present. The study did not for was selective towards compounds having a catechol struc- include the impurities usually present in illicit heroin, ture and did not therefore respond, for example, to codeine. Wheals (85) examined 22 packing materials for use in the Further investigation is necessary to determine the parameters separation of basic drugs using aqueous methanol (methanol, affecting the response of electrochemical detectors. 2N ammonium hydroxide, IN ammonium nitrate, 27/2/1) as In the opinion of the reviewers, TLC still has a role to play mobile phase. All the packings were laboratory-prepared from in the analysis of narcotics, particularly as a screening method. a single batch of silica of particle size range 3 to 7 #m. Eleven For many applications this is a more economical approach than test compou,lds (including heroin, codeine, and morphine) were submitting samples directly to a multi-column GLC or HPLC run on each column and the retention data compared with that system. Although morphine can be chromatographed on most obtained on untreated silica. The three most suitable packings of the HPLC systems, its behaviour is far from ideal. For ex- selected for further study were a mercaptopropyl modified silica, ample, it is necessary to use peak area measurements rather than

296 Journal of Chromatographic Science, Vol. 20, July, 1982

heights, to achieve linearity of response with respect to con- distinguished after spraying, the former giving a colour only centration (83,89). Several HPLC systems will separate most with DAB. Choulis (94) examined 17 drugs of abuse, including of the narcotics, but there is no one system which will resolve cocaine, on silica gel using chloroform, dioxan, ethyl acetate, all the narcotics and common cutting agents. In the absence ammonium hydroxide (25/60/10/5). Detection was with _'_ of mass spectrometry, the reliability of identification, and in- potassium permanganate or iodine. Although complete separa- deed quantitation, is substantially enhanced by the careful selec- tion of all compounds was claimed, the Rfs of amobarbitai and ;_ tion of one GLC method and one HPLC method, phenobarbital were very close to that of cocaine. The com- Ii pounds were quantified using UV absorption, but no data were _. givenfor cocaine.Other localanaestheticswerenot included !_ Cocaine and Other Local Anaesthetics in the study. Van Welsum (25) included cocaine and procaine I _ ina generalTLCsystemforillicit"street"drugs,usingactivated i _ Thin Layer Chromatography silica gel G (Merck), and chloroform, diethyl ether, methanol, ammonium hydroxide (75/25/5/!). Moffat and Clare (26) in- _,_ Illicit cocaine frequently contains other local anaesthetics not eluded cocaine in their examination of TLC systems for screen- ,/: subject to abuse control, and occasionally suspected cocaine is ing a wide range of drugs. Cocaine was separated from the vast • found to consist exclusively of the latter. The chromatographic majority of drugs including benzocaine, butocaine, lignocaine, process must be capable of separating cocaine from these com- mepivacaine, piperocaine and procaine, using either of the two !;_ pounds. The percentage purity of cocaine in the seizure is also solvents which the authors recommended for screening (see required. Local anaesthetics are also encountered as additives above). Conine and Paul (95) reported the separation of co- : in illicit heroin, although cocaine itself is less commonly found caine from major narcoticst but other local anaesthetics were than the non-controlled compounds. Nevertheless, it is desirable not examined. Vinson et al. (27) examined four solvent systems to have a system that will resolve heroin from cocaine. In a for use with common "street" drugs and used TCBI for review of methods of detection for cocaine, Bastes and Hoff- visualisation. Cocaine was separated from three other local man (92) state that "the separation of cocaine from other drugs .anaesthetics and from narcotics using ethyl acetate, methanol, of abuse is not a simple matter." They showed that even using ammonium hydroxide (100/18/!.5). The spray reagent gave four different TLC solvent systems, it was not possible to characteristic colours for various major drug classes, although distinguish between cocaine and phencyclidine, in the case of the local anaesthetics this is not particularly The TLC system described by Davidow et al. (I 5) (see above) helpful. distinguished between cocaine and heroin. Unfortunately co- There are several publications which consider the determina- caine, procaine, noscapine, and papaverine all had the same tion of cocaine metabolites in urine. The principal metabolite Rf. Brown et al. (93) examined laboratory-prepared mixtures is benzoylecgonine, which is also a major product of the acid of seven local anaesthetics with illicit heroin and obtained hydrolysis of cocaine and may be present in illicit cocaine separation of cocaine from heroin; not all the other anaesthetics samples. It can be separated from cocaine by TLC using silica were separated from each other. The TLC plates were silica gel- gel G plates with chloroform, methanol, ammonium hydrox- developed with ethyl acetate, propanoi, ammonium hydroxide ide (100/20/1) (96). Various spray reagents were investigated (40/30/3). Detection was by acidic iodoplatinate and p- with a view to obtaining the best possible detection limit. Us- dimethylaminobenzaldehyde (DAB). Cocaine gave a dark blue ing Dragendorff's reagent, followed by 20% sulphuric acid, a colour with iodoplatinate, but no colour with the other reagent, limit of 0.5 #g was obtained for cocaine, in contrast to 1 to 10 Benzocaine and butacaine, which were not resolved, were /_g obtained without sulphuric acid, or with iodoplatinate. Us-

Table IV. References to the TLC of Local Anaesthetics References 10 15 17 18 25 26 27 28 29 92 93 94 95 96 97 99 100 101 102 132 163 191 211 215 217 Amylocaine X Benzocaine X X X X X Benzoylecgonine X X Bupwacaine X Butacaine X X X X Cinchocaine X Cinnamoylcocaine • Cinnamoylecgonine Cocaine X X X X X X X X X X X X X X X X X X X X X X X X Cyclomethycaine X Ecgonine Holocaine X Lignocaine X X X X X X X X X Mepivacaine X X X X Methylecgonine Piperocaine X X Prilocaine X ,' Procaine X X X X X X X X X X ' Proxymetacaine X ; Tetracaine X X Tropacocaine X ,_ Otherlocalanaesthetics X

297 Journal of Chromatographic Science, Vol, 20, July, 1982 ing high performance TLC plates, Gubitz and Wintersteiger (97) caine and codeine on OV- 17 and SE-30, although Fontan and obtained a limit of 50 #g with Dragendorff's reagent. Masoud Hill (42) give relativeretentiontimes, with respectto cocaine, (29,98) described a four-solvent screening system for a variety of 1.71 and 1.99 on these two phases. The reviewers have also of drugs, with detectionofcocaine byiodoplatinate. Several clearly separated cocaine from codeine using OV-17. other local anaesthetics were co-eluted on at least one of the Christophersen and Rasmussen (59) separated cocaine from co- systems. Gough and Baker (99) used silica gel 60 F_, plates with deine on a WCOT SE-30 capillary column after making the methanol, 2N ammonium hydroxide, IN ammonium nitrate TMS- derivative of the latter and other narcotics. (27/2/1) to examine 15 local anaesthetics. Detection was by ex- Jain et al. (106) proposed the use of a mixed phase of 2% amination under UV light (254 nm and 366 nm), followed by UCW-98 and i% OV-17 to separate and quantify cocaine, i spraying with iodoplatinate reagent. Cocaine was separated methadone, methaqualone, phencyclidine, and propoxyphene, i from methaqualone. Proxymetacaine and butacaine were co- Resolution between cocaine and methaquaione was poor, with eluted with cocaine, but the first mentioned could be distinguish- a relative retention of i.06. These authors claimed no in- _ ed because it was visible at 366 nm. The only other local terference from codeine, barbiturates, and amphetamines, but ! anaesthetic detected in this wavelength was cinchocaine. Buta- retention data were not quoted. Baker and Gough (99) used car- caine, but not proxymetacaine or cocaine, gives an orange col- rier gas flow programming to reduce the analysis time inherent our on spraying with DAB. All 15 local anaesthetics studied in the isothermal separation of local anaesthetics. For were visible at 254 nm and gave a colour with iodoplatinate, laboratories where the daily sample throughput is high, flow The colour was of no diagnostic value, but Dutt and Poh (28), programming has the advantage over temperature programm- using ninhydrin, found that cocaine gave a brown colour when ing in that inter-run equilibration time is significantly reduced. the TLC plate was heated above 120°C. Lignocaine changed Using OV-17 under isothermal conditions to separate all but from purple to brown over the range 80 to 160°(? and procaine two of 15 local anaesthetics, analysis time was 93 min isobaricai- from pink Io brown over the same range. Jukofsky et al. (100) ly and 37 min by flow programming. Methaqualone was not distinguished between lignocaine (lidocaine) and cocaine on the resolved from cocaine, and codeine was eluted with butacaine. basis of minor differences in their reactions with iodoplatinate, Ji0dal et al. (107)examined illicit cocaine and separated two although they were unresolved on the systems used. it is not major components using OV-17. On-line MS was used to iden- clear why the several published systems that resolve these corn- tify these as cocaine and lidocaine. Prager et al. (60), using a pounds were not used. Despite the at, thors' claim that both corn- I/1 mixture of SE-30 and OV-17, quantified" cocaine with a pounds could be detected and even semi-quantitated in mixtures, precision and accuracy of 3%. it would seem to be an unnecessarily risky basis on which to Aynilian et al. (108) examined nine species of the genus make such determinations. Erythroxylon, including E. coca, for the presence of cocaine. Eskes (101) separated the optical isomers of cocaine indirectly All except one contained cocaine in amounts ranging from traces after hydrolyses to the corresponding ecgonines and esterifica- to 0.53%0, the latter being in freshly harvested E. coca. A 5% tion with enantiomeric 2-octanols. Separation was achieved on OV-101 (methyl-silicone) column was used and quantitation was silica gel plates with methanol as solvent. Siegel and Cormier by area measurement of the FID response. Turner et al. (109) (102) were able to separate/-cocaine and d-cocaine (pseudoco- used 6% OV-I in the determination the cocaine content of E. caine) directly on silica gel plates using chloroform, methanol coca from Peru, which varied between 0.57070 and 0.60% for (9/I ), with visualisation by iodoplatinate reagent, three different samples. Each was determined in triplicate with a precision of 4%. Gas/Liquid Chromatography A collaborative study on the quantitation of cocaine in powders and tablets (I 10) was carried out by eight laboratories, One of the earliest papers on the application of GLC to local using 3%0 OV-I. The inter-laboratory precision ranged from anaesthetics was published by Koehler and Hefferren in 1964 0.9% to 3.2% for five different samples, covering the concen- (103). Cocaine was not included in the study, but 18 other local tralion range 6 to 100%0 cocaine. Roberson (111) studied the anaesthetics were examined. The most satisfactory stationary repeatability of quantitation of cocaine using a high boiling n- phase was SE-30, but even this only partially resolved the ma- alkane as internal standard. Both OV-I and OV-17 columns jority of the cornpounds. Detection in this, and all subsequent were used, with injection volumes between 0.2 and i #1. Many papers unless otherwise noted, was by flame ionisation. This parameters affecting quantitation are, of course, not unique to was followed by some Japanese work (104) in which 10 local cocaine and from Ihis point of view the study has general ap- anaesthetics, including cocaine, were examined on QF-I and plicability. The possibility of incomplete or slow conversion of Thermol-3 (believed to be a phenetidine derivative). Again many cocaine hydrochloride to the free base by thermal decomposi- of the compounds were only partially resolved and tailed bad- tion on injection was considered. It was found that the pro- ly. Suzuki et al. (105) examined cocaine and 13 other local blem of irreproducibility was the result of preferential loss of anaesthetics on four different silicone stationary phases and on solvent on injection causing deposition of the hydrochloride "Fhermol-3. The bes! separation was obtained on OV-17. Detec- within the needle. This is simply solved by suitable choice of tion limits were in the range 0.1 to 0.8 #g depending on the tom- solvent and good injection technique. pound. Using XE-60 (cyanoethyl-, methyl-silicone), cocaine was Illicit cocaine contains several minor coca alkaloids, and only partially resolved from mepivacaine and procaine and with Moore (112) has shown cinnamoylcocaine to be present in over the other phases, quatacaine and propitocaine (prilocaine) were hall" of the samples examined, usually at concentrations less than unresolved. No chromatograms were shown, so it is not possi- 1%0of cocaine itself. The cis- and trans-isomers of this alkaloid ble to comment on the quality of the separations. Unfortunately were separated on 3% OV-I, and confirmation of identity was OV-I, OV-17, and SE-30 did not resolve cocaine from several obtained by several spectroscopic techniques. The same author other commonly encountered drugs, in particular methaqualone (I 13) examined cocaine for the presence of benzoylecgonine and (92). The same paper quoted identical Kovats Indices for co- ecgonine after silylation with BSA. The derivatives were

298 i Journal of Chromatographic Science, Vol. 20, July, 1982 i Table V. References to the GLC of Local Anaesthetics ! i Reference 10 30 31 34 35 36 39 40 41 42 53 54 59 60 92 99 103 104 105 106 107 108

Amylocaine X +:: Benzocaine X × X X : Benzoylecgonine Bupivacaine X ; Butacaine X X X + Cinchocaine X Cinnam0ylc0caine Cinnarnoylecgonine + Cocaine X X X X X X X X X X X X X X X X X X Cyclomethycaine • X _" Ecgonine Holocaine X _, Lignocaine X X X X X X ' Mepivacaine X X X Methylecgonine Piperocaine X X Prilocaine X X Procaine X X X X X X X X X X X X Proxymetacaine X Tetracaine X X X X X Tropacocaine X X Otherlocal anaesthetics X X X

Reference 109 110 11'1'112 113 114 115 116 179 180

BenzoArnylocainecaine X X X formed by the acid hydrolysis of cocaine. Siegel and Cormier Benzoylecgonine X X X (i02) found that pscudococaine and cocaine had a relative reten- BupivacaJne X lion of 1.05 on 3°7oOV-17 although they weredistinguished by Butacaine X X combined GLC/MS. It is, however, preferable and easier to Cinchocaine X use TLC for preliminary separation. Levin el al. (116) found Cinnamoylcocaine X X X Cinnamoylecgonine X a relative retention of 1.03on 3% SP-2250-BD (phenyl-, methyl- Cocaine X X X X X X X X X silicone) phase for the same two compounds. Under the same Cycl0methycaine conditions allococaine and ailopseudococaineeach gave riseto Ecg0nine X X two peaks of identical retention times, one sharp peak prior Holocaine to elution of cocaine and the other much broader and later. All Lignocaine X X Mepivacaine X the ecgonine methyl estersappeared to be resolved from each Methylecgonine X other, ahhough alloecgonine methyl estergave riseto two peaks. Piperocaine " X Examination by chemical ionisation MS and combined PPrrocail0cainanee X X X GLC/MS showed that for all the isomers giving two GLC peaks, Pr0xyrnetacaine both peaks were due to decomposition products. For each com- Tetracaine X pound, the decomposition peak of longer retention time was Tr0pac0caine benzoic acid. All the ecgonine methyl esters dehydrate on in- Otherlocal jection into the GLC. anaesthetics High Performance Liquid Chromatography

separated on 10% OV-101 with temperature programming. Since cocaine and most of the other local anaesthetics have Levels down to at least 0.3°70 benzoylecgonine and 0.1% excellent GLC properties, there is little need to seek HPLC ecgonine, with respect to cocaine, could be detected. Other methods, unless there is a particular separation problem, for silylating reagents and silicone pha_s were considered, but gave instance, cocaine from methaqualone. Cocaine has been includ- less satisfactory results. On OV-17, for example, cocaine and ed in a number of HPLC methods for drugs in general. For ,t TMS-benzoyiecgonine were unresolved. Javaid et al. (I 14) example Chan et al. (70) separated 16 common "street" drugs detected the same compounds by electron capture after forma- by gradient elution on Corasil II. Cocaine was at least partially lion of fluorinated derivatives. Detection limits were I ng for resolved from phencyclidine, propoxyphene, and methadone, benzoylecgonine and 50 pg for ecgonine. The derivatising but not from methaqualone. Win et al. (69) used dynamic reagent was HFBA comaining l,l,l,3,3,3-hexafluoropro- coating HPLC (Poly G300 on Corasil 11), but found that co- pan-2-ol (2:1). Cocaine was also detected as the fluorinated caine was eluled with caffeine. The Metropolitan Police ! derivative after its reduction with lithium aluminium hydride, Laboratory (73,85) measured the retention times of cocaine, ahhoagh such methods have advantages over direct detection other local anaesthetics, and many other drugs on various silica only in the case of trace quantities. Lukaszewski e/al. (1 15) columns with methanol, 2N ammonium hydroxide, IN am- considered hydrolysis products and artefacts in illicit cocaine monium nitrate (27/2/!). On untreated silica several local analysis, using on-line MS after GLC separation on 3070OV-I. anaesthetics and methaqualone had similar retention times. Co- l'hey frequently lom_d methylecgonidine, although this was also caine, procaine and tetracaine were poorly retained and fo| reed from cocaine on i,ajcction into the GLC. It can also be tmresolved on both a mercapto-propyl-silica bonded phase and

+_ 299 Journal of Chromatographic Science, Vol. 20, July, 1982 on a cation exchange packing and hence, none of these columns adsorption losses, and quantitation is therefore straightforward. is suitable. Baker et al. (84) found that the UV absorption ratios The HPLC of local anaesthetics is in general rather poor. HPLC (254:280 nm) were 0.86 for cocaine, 0.44 for procaine, and can has little to recommend it as an alternative to GLC, except in thus aid distinction between these compounds. Sugden et al. the analysis of cocaine isomers where GLC causes decomposi- (117) examined the behaviour of amylocaine, benzocaine, buta- tion and could therefore give rise to erroneous conclusions. caine, and cocaine on silica and ODS- silica using an alkaline 70% aqueous methanol solution. Using reverse-phase ion pair chromatography, Lurie (81) found that benzocaine, cocaine, lignocaine, procaine, and tetraeaine were completely resolved, LSD and Other Hallucinogens although the retention time of methaqualone relative to cocaine was only I. 1! ; phencyclidine was also well resolved. Crommeu The hallucinogen most frequently encountered in illicit use 1118) also used reverse-phase ion pair chromato!,,raphy and is lysergide (LSD, lysergic acid diethylamide), usually as the tar- separated amylocaine, benzocaine, lignocaine, and procaine us- trate. The compound can exist in four stereoisomeric forms, ing 10 #m silica. Trinler and Reuland (72), using tt-Bondapak but it is only D--lysergic acid diethylamide, which does not oc- C,, with aqueous acetonitrile, showed that cocaine was not pro- cur naturally, that has significant hallucinogenic activity (121). perly resolved from heroin and morphine. In a later paper the same authors (119) examined simulated "street" drugs for five Thin Layer Chromatography local anaesthetics. A phenyl-silica bonded phase (Bondapak A study of LSD analysis was undertaken by Genest and Far- phenyl/Porasil B) was used, with UV detection at 254 nm. Elu- mile (122), with particular reference to its TLC separation from tion was with acetonitrile, water (85/15). Cocaine was resolv- narcotics in illicit drugs. The ergot alkaloids and narcotics were ed from other compounds, and identity was confirmed by frac- run on silica gel G plates using a solvent mixture of chloroform, tion collection and subsequent IR spectroscopy, methanol (90/10). Detection was by UV absorption and by Cocaine and three isomers were separated by reverse-phase spraying with DAB. Most subsequent workers have used the ion pair HPLC on ODS-silica, using tetrahydrofuran, water same reagent. The detection limit was 0.05 /zg. In order to (20/80) as solvent (120). The detection limit was 3 ng at 235 distinguish between LSD and alkaloids of similar Rf, the pro- nm. Shortly afterward Levin et al. (116) reported excellent ducts of irradiation were also subjected to TLC. Irradiation separation and quantitation of the same four compounds and results in the formation of hydroxy-derivatives (the lumi- the ecgonine methyl esters. The column was Partisil 10-PXS compounds), which reveal a different TLC pattern. In addi- (cation exchange) and the mobile phase 2-propanol, heptane, tion, hydrolysis of ergot alkaloids generates amino acids, which diethylamine (25/70/0.1), which was flow programmed to 4 are detected by spraying with ninhydrin. The hydrolysis pro- mi/min/min from 0.48 ml/min. The absorbance maxima for duct of LSD is diethylamine, which gives no reaction with the compounds were between 226 nm and 228 nm, and the detec- ninhydrin. Quinine, heroin, morphine, codeine, and related tot was operated at 230 nm. alkaloids were all separated from LSD. Some years later Blake TLC is valuable in screening for local anaesthetics and will et aL (123) pointed out that identification of LSD in illicit separate cocaine from methaqualone, in contrast to most GLC samples could be subject to error by the presence of fluoresc- and HPLC systems. A combination of TLC and GLC will ing excipients or degradation products. The presence of both enable a distinction to be made between all the local anaesthetics 10-hydroxy-LSD and residual LSD in samples after controlled and many other drugs of abuse. GLC elution profiles of the irradiation was taken as supportive evidence for the presence local anaesthetics are excellent; the compounds do not suffer of LSD in the original sample.

Table VI. References to the HPLC of Local Anaesthetics

Reference 65 69 70 • 71 72 73 74 75 76 78 81 84 85 92 116 117 118 119 120 150 205 206 Arnylocaine X X 8enzocaJne X X X X X X X Benzoylecgonine Bupivacaine Butacaine X X Cinchocaine Cinnarnoylcocaine Cinnamoylecgonine Cocaine X X X X X X X X X X X X X X X X X Cyclomethycaine Ecgonine X Holocaine Lignocaine X X X X X Mepivacaine Methylecgonine Piperocaine X Procaine X X X X X X X X X X X Proxymetacaine Tetracaine X X X Tropacocaine Other local anaesthetics

300 1 &t:_ Journal of Chromatographic Science, Vol. 20, July, 1982

Several workers have examined morning glory (Ipomoea anti Fowler et al. (130) studied the resolution of LSD and 14 Convolvulus) seeds and plants for the presence of ergot related ergot alkaloids using 18 different TLC systems, with alkaloids. The major hallucinogenic compound is lysergamide various silica, alumina, and cellulose plates. None of the systems (lysergic acid amide, ergine). Taber and Vining (124) used unequivocally resolved LSD from all of the other compounds; :i chloroform, methanol (85/15), and the same detection methods in particular, ergotamine and ergosine frequently interfered _ as used by Genest and Farmilo (122) for LSD. Quantitation was when using neutral solvents. Dihydroergotamine and : by removing the TLC spots and measuring the optical density dihydroergocristine were also close to LSD, but did not fluoresce of the acidic solutions after treatment with DAB. Genesl (125) at 350 nm and gave distinctive colours with DAB. Using basic carried out direct densitometry on TLC plates to determine the solvents with silica gel, or a trichloroethane, methanol, mix- amount of ergine and isoergine (8_-Iysergamidc) in morning ture with alumina, LSD was resolved from all compounds in glory seeds. After examining five different solvent systems, the study except iso--LSD, ergocristine, ergocryptine, and Genest showed that chloroform, ethanol (96/4) with alumina ergocornine. The authors concluded that for LSD analysis, it : plates gave the best separation of the alkaloids. Detection was was simplest and most satisfactory to use acetone as mobile by examination at 254 nm and 366 nm, followed by treatment phase with either silica or alumina plates. Reichelt and Kudr- with DAB. nac (131)reported the completeseparationof the L- and D- " Chad and der Marderosian (126) measured the indole alkaloid alkaloids of the ergotoxine and ergotamine groups. LSD was content of seeds of Argyreia nervosa using TLC and IR spec- not included and D-lysergic acid, together with D-isolysergic troscopy, after fractionation of the alkaloids using column acid, remained at the origin. Their amides (i.e., ergine and chromatography. Ergine and isoergine represented 2307oand isoergine) had the same Rf values as ergometrine and 31%, respectively, of the total alkaloid content of the seeds, ergomelrinine respectively. Dried silica gel plates were treated LSD has been quantified by fluorimetry at an excitation with formamide and developed with di-isopropyl ether, wavelength of 360 nm and fluorescence at 436 nm (127). Using tetrahydrofuran, toluene, diethylamine (70/15/15/0. i). Ardrey alumina plates with l,i,l-trichloroethane, methanol(98/2), LSD and Moffat (132) have compiled a comprehensive list of was well separated from related compounds and quinine. Delec- analytical data on 19 ergot alkaloids. For TLC, four solvents lion was by UV absorption, after which spots were removed "were examined; the conclusion of Fowler et al. (130), that the for quantitation, most useful solvent is acetone, was confirmed. Detection was LSD has also been quantified by direct UV absorption by DAB, sodium 1,2--naphthaquinone-4-suiphonate or measurements on TLC plates (128). Precision over the range 2-nitroso-l-naphthol. 0.1 to 2 #g was between 1 and 5°70. Using silica gel coated Phillips and Gardiner (133) applied TLC to the separation polyester sheets, Alliston et al. (129) recommended morpholine, of 20 tryptamines and tabulated the Rf values of these corn- toluene (i/9) as solvent for ergot alkaloids. This separated a pounds on four systems. Most satisfactory was methanol, am- number of the alkaloids but LSD, N,N-dimethyltryptamine monium hydroxide (100/1.5) (17), using either silica gel plates (DMT) and psilocin (4--hydroxy-N,N-dimethyltryptamine) had or polyester sheets. However, none of the systems adequately similar Rf values. However, the two latter compounds do not resolved DMT from N,N-diethyltryptamine (DET) and in all fluoresce at 360 rim. The detection limit for LSD was 4 rig. cases, psilocybin remained on the baseline. After development, Neither this system nor the more commonly used chloroform, the tertiary amines were distinguished from primary and secon- methanol mixture was satisfactory for lysergic acid and dary amines on the basis of the colour reaction with iodine. The psilocybin (4-phosphoroyloxy-N,N-dimethyitryptamine) as former gave orange fading to yellow, and the latter both gave neither exhibited any significant mobility. The advantage of the pale yellow fading to fawn. Hydroxylated tryptamines gave a proposed system lies in the fact that LSD is not subject to in- pink colour when examined under UV light at 254 nm, whereas terference from ergotamine, which has a virtually identical Rf other tryptamines gave a dark purple colour. The same group value using chloroform, methanol. (129,1341 subsequently used morpholine, toluene (see above)

Table VII. References to the TLC of Hallucinogens Reference 25 26 27 29 92 94 97 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 143 158 161 163 216 Bufotenine X X X • DET" X X X X X DMI X X X X X X X X X X Ergocristine X X X X X X Ergocristinine X X • Ergometrine X X X X X X X X X Ergotamine X X X X X X X X Ergotaminine X X X X LSD X X X X X X X X X X X X X X X X X X X X X Lysergamide X X X X X X X X X Lysergicacid X X X X X X X Methyl ergometrine X X X X Methysergide X X X X X X Psilocin X X X Psttocybin X X X X X X Otherergot alkaloids X X X X X X X X X X X

"See List ot Abbreviations

301 Journal of Chromatographic Science, Vol. 20, July, 1982

and achieved resolution between DMT and DET. Methanol, structure of tile eluted compounds by combined GLC/MS. The chloroform did not resolve this pail. Shaw and Peel (135) resolv- MS of many ergot alkaloids has been reported by Ardrey and ed I)MT from DET on activated silica gel using chloroform, Moffat (132). Positive and negative mobility spectra of LSD cyclohexane, diethylamine (50/40/10). havc been reported by Karasek e/al. (141). The plasma detec- Several TLC screening methods for drugs in general have in- tor has been proposed for the detection of trace amounts of cluded the hallucinogens, t_Jsing chloroform, dioxan, ethyl- organics, but when used in conjunction with GLC does of course acetate, ammonium hydroxide (25/60/10/5), LSD was resolv- demand the elution of the compound unchanged, as with any ed from some narcotics, barbiturates, and cannabinoids (94). detector. Methadone had a similar Rf value but unlike LSD, has only weak UV absorption. With chloroform, diethyl ether, methanol, ammonium hydroxide (25) several of the hallucinogens had Table VIII. References to the GLC of Hallucinogens retention times similar Io some barbiturates and amphetamines. References 136137138139140141142143144158179180183 * They were, however, distinguished by using DAB for visualisa- tion. The narcotics and most of the local anaesthetics did not Buf0teine X X X DET X interfere. Psilocin and DET had the same Rf, but only the laI- DMT* X X X X tar strongly absorbs at 366 nm. DMT and LSD were included Erg0cristine X in tile screening system of Viuson et al. (27) and although several Ergocristinine other drugs had the same Rf values, of the major drugs includ- Erg0metrine X ed in the study only codeine inter fared. Masoud (29) examined Erg0tamine X Erg0taminine four systems in which LSD, psilocybin, and psilocin were in- LSD X X X X X X X X X eluded. On the basis of the retention data and visualisation pro- Lysergarnide X cedures given in this paper, a preliminary identification of these Lysergicacid hallucinogens can be made. Methyl ergometrine Melhysergide Gas/Liquid Chromatography Psilocin X Psilocybin X X Radecka and Nigam (136) found thai LSD could not be chro- Otherergot matographed due to thermal instability. In an attempt to im- alkaloids X X

part stability, the authors hydrogenated LSD, after which it was "See List of Abbrevialions. examined on a 0.2070SE-30 column at 270°C. The resulting chromatograms, although giving a major peak, gave rise to two minor peaks indicating the presence of impurities or decom- Other hallucinogens have also been studied by GLC with lit- position products. The method was less sensitive than TLC and tie success. DMT, 5-methoxy-DMT, and 5-hydroxy-DMT even at the 50 _g level, an unconvincing response was obtain- (bufotenine) were separated, as TMS-derivatives, on 5% SE-30 ed. Katz et al. (137) chromatographed LSD directly, using 0.307e with temperature programming from 140°C (142). Confirma- SE-30 on glass beads at 280°C. Symmetrical peaks were obtain- tion of structure was by on-line GLC/MS. N-Methyitryptamine ed, and response was linear with respect to LSD concentration and N-methylserotonin were also studied, and each gave rise over the range 1 to 4 #g. Lerner (138) achieved similar results to two peaks by GLC. GLC/MS showed these to be the mono- with underivatised LSD and attributed this to the use of an all- and di-TMS derivatives of N-methyltryptamine and the di- and glass column system. He also used a lower column temperature, tri-TMS derivatives of the other tryptamine. A third major pro- but even so there was some evidence of decomposition. Tile duct was also sometimes encountered after silanisation, but the atnhor examined the stability of derivatives and found that bet- authors were unable to establish its identity. Tryptamine itself tar chromatography was obtained using tile TMS rather than gave only the tri-TMS derivative. Psilocin has been the I'FA derivative. Agurell and Ohlsson (139) used several chromatographed on SE-30, but the injection of methanolic silicone phases for the separation of ergot alkaloids. LSD and solutions of psilocybin results in its conversion to psilocin (139). lysergamide were the only lysergic acid-related compounds By reacting psilocybin (143) and psilocin (144) with BSA, the which could be chromatographed. Lysergic acid methyl car- resulting TMS derivatives were separated on silicone stationary ,, binolamide decomposed on injection to form lysergic acid phases (SE-30 and SE-52 phenyl-methyl-silicone). Combined amide. Jaue and Wheals (140) confirmed that glass must be used GLC/MS confirmed that the tri-TMS and bis-TMS derivatives, to minimise decomposition, even when chromatographing the respectively, had been formed. silyl derivatives. Derivatisation was carried out in the injection

port, and BSA was found to give higher yields than N-methyl- High Performance Liquid Chromatography N-trimethylsilyl-trifluoroacetamide (MSTFA). The method was not found satisfactory for all I.SD samples examined. Several Wittwer et al. (145) examined two HPLC systems for 15 ergot unidentified peaks were frequently observed, some of which in- alkaloids (including LSD). Phencyclidine, 2,5,-dimethoxy-4-me- terfered with the peak attributed to TMS-LSD. The use of thylamphetamine (STP) and strychnine were also included, as silylating reagents also gave rise to substamial deposits of silica they are sometimes encountered in combination with LSD. Us- in the detector, a common problem when using silanised ing a silica (SiI-X) column with acetonitrile, isopropyl ether derivatives. (40/60), I.SD was separated from all these compounds except It is apparent that GLC is not, in general, satisfactory for ergotamine and 8tx-lysergamide. Using Corasil il and the same LSD and related compounds. It does not resolve stereoisomers eluting solvents (25/75), LSD was unresolved from and even after silanisation, some compounds are thermally dihydroergocristine and 8u-lysergamide. Quantitation of LSD unstablc. If GLC is use, it is clearly necessary to confirm tbe was based on absorption at 254 nm and in most cases, gave

302

=..----.-= Journal of Chromatographic Science, Vol. 20, July, 1982 !+

results in agreement with direcl UV and Ihtorescence of IOOpg LSD, using a fluorimeter with excitation at 320nm i[ measurements. Repeatabil{ty of quantitation by HPLC was and emission at 400 llll'l. The dihydro-derivativcs were detected ;i 3.4%. Heacock e/ al. (146) used fluorimetric detection after by UL absorption at 280 nm. The mass spectra of the same tom- ! HPLC separation of ergot alkaloids on Zipax and Corasil (silica) pounds have also been published (132). Wurst et al. (149) under- : columns with various solvents. Fluorescence maxima for the took the quantitative analysis of ergot alkaloid mixtures using 16 ergot alkaloids studied were between 390 nm and 410 nm. a Micropak-NH. column with gradient elulion by a diethyl ether, Little variation in fluorescence intensily was observed using dif- ethanol mixture. Detection was by UV absorption at various ferent solvents, except that substantial quenching occurred at wavelengths. For example, agroclavine, lysergine, and erginine 350 nm when chloroform was included in the solvent mixtures, all absorb at 225 nm, but only the latter two compounds ab- It is well known thai even trace amounts of some compounds sorb at 310 tam. Satisfactory quantitation of mixtures of known can result in major quenching, and it may be that the quen- composition were obtained with a precision of 2 to 8%. Meg- ching observed with chloroform was the resuh of impurities in ges (150) partially separated lysergic acid methylpropylamide this solvent. This is unfortunate, because chloroform is a par- and L SD (relative retention 1.07), on a #-Bondapak-C,, col- ticularly valuable solvent in TLC (and hence potentially in umn using acetonitrile, water, I% ammonium carbonate HPLC) for the separation of ergot alkaloids. (400/572/28). Detection was by UV absorption at 313 nm. An Indeed, several authors (122,133,146) separated the HP[.C system devised for "street" drug analysis (70) at least stereoisomers ergotaminc and ergotaminine using methanol, partially separated I.SD and DMT from other common drugs, chloroform mixtures. Using a reverse-phase system eliminates using methanol, cyclohexane, cyclohexylamine, and Corasil I1 the necessity to use chloroform. Jane and Wheals (140), using packing. Corasil C,, with methanol, 0. I% aqueous ammonium carbonate HPLC has been applied to the separation of psilocin and (6/4), separated 20 ergot alkaloids from LSD. The pairs psilocybin using Partisil 5 with methanol, water, IN ammonium lysergamide and ergometrine, and lysergic acid and nitrate (240/50/10) at pH 9.7 (151). Extracts of Psilocybe methylergometrine, were unresolved. Detection was by UV and semilanceata mushrooms contained a third compound, only par- fluorescence spectrometry in series. Sensitivities of the two detec- tially resolved from psilocybin. The extract was subjected to tors were similar with a detection limil of 2 ng for LSD. TLC, using silica gel plates with n-butanol, acetic acid, water Ahhough fluorescence detection is much more selective than (2/1/1), which gave three well-resolved spots. MS of extracts UV detection, this is of little value in distinguishing between of these spots provisionally identified the unknown compound ergot alkaloids themselves, other than the dihydro-compounds, as 4-phosphoryloxy--N-methyltryptamine (baeocystin). Ion ex- which do not fluoresce. Lurie (81) used reverse-phase ion pair change chromatography has been used to separate psilocybin chromatography to separate LSD from other ergot alkaloids, from psilocin and DMT (152). A Partisil SCX-10 column was Hartmarm el al. (147) separated the four components of used with aqueous methanol at pH 4.5 and S0°C. Detection was dihydroergotoxine, which includes two isomers, using a reverse- based on fluorescence. Psilocybin fluoresces strongly at 335 nm phase system of water, acetonitrile, triethylamine (32/81/1) with with excitation at 267 nm, giving a detection limit of 250 pg. ODS-silica (5-/,tm Lichrosorb RP-18). Detection and quantita- Psilocin has a weak fluorescence at 312 nm with excitation at tion was by UV absorption at 280 nm. Precision of the deter- 260 nm, resulting in a detection limit of 30 ng. Maximum UV minations in various pharmaceutical preparations varied from absorbance for both compounds is 267 nm, giving detection 1.2 to 2.7%. limits of 7 and 150 ng, respectively. Repeatability of quantita- Twitchett et al. (148) published retention data on ergot tion was 3.9% for psilocybin and 2.8% for psiiocin. No specific alkaloids on silica (Spherisorb S5W) and reverse-phase ODS- mention was made of possible interference from baeocystin, silica (Spherisorb S5ODS) systems. Although LSD was resolv- although unidentified compounds were observed. Recently ;, ed from the other alkaloids on both systems, the bonded-phase Thomson (153) reported the separation of psilocybin from cohtmn separated many more ergot alkaloids from each other psilocin using reverse-phase chromatography with a number of than did the silica colunm. The detection limit was on the order different ion-pair reagents. :,

Table IX. References to the HPLC of Hallucinogens Relerence 70 76 81 84 132 140 145 146 147 148 149 150 151 152 153 208 359 Bufotenine ! DMT X X X i I Ergocristine X X X X X i Ergocristinine ! Ergo_iline X X X i Ergotamine X X X X X X i Ergotaminine i LSD X X X X X X X X X. X Lysergamide X X X X Lysergicacid X X X t Methylergometrine !i, Methysergide X X X X _

Psitocin X X X r}, Psilocybin X X X ; Otherergotalkaloids X X X X X X X X

"See Lis! of Abbreviations i '

303 __ Journal of Chromatographic Science, Vol. 20, July, 1982

Amphetamines and Related Stimulants The TI.C system developed by Davidow et al. (15) has been widely used for drug screening (see above). Afler development, The principal stimulants to be misused have been am- the silicagel plates were sprayed with ninhydrin, which detected phetamine [(RS)-t_-ntelhylphenethylamine], dexamphetamine amphetamine. Few other stimulants were used in this original [(S)-o_-methylphenethylaminel, and methylamphetamine (N- study, and detection limits were not given. Cimbura (159) methyl-c_-methylphenethylamine). Many other structurally chromatographed 3,4-methylenedioxyamphetamine (MDA) on related stimulants have been encountered. In addition a great silica gel G developed in chloroform, ethanol, benzene, am- variety of ring-substituted phenethylamines and cz- monium hydroxide (80/40/175/5): Visualisation was with I_0 methylphenethylamines, which are potent hallucinogens, have potassium permanganate solution, and i #g could be detected. been synthesised. The former drugs are either lawfully manufac- The authors stated that amphetamine, methylamphetamine, and tured material obtained by theft, fraud, or over-prescription, STP had similar Rf values to MDA, but of these three only or are of illicit manufacture. The commercial presentations are MDA reacted with permanganate. Marquis reagent will often of high quality and, provided a manufacturer's marking distinguish between MDA and STP, the former giving a blue or olher recognisable product characteristic can be identified, colour, the latter yellow-green. confirmation of identity is by comparison with an authentic sam- Phillips and Gardiner (133) examined many phenethylamines pie. TLC or UV spectroscopy is frequently used. However, the in two TLC systems: silica gel developed in methanol, am- " widespread forgery of licit preparations meansthat the analyst monium hydroxide (100/1.5) (17) and alkaline silica gel must exercise special care with some preparations purportint developed in chloroform, methanol (9/1) (122). The authors to be of licit origin (albeit containing controlled drugs). Illicit found that considerable discrimination between phene- preparations are of variable quality and may contain substan- thylamines was possible, using these two systems visualised with tial quantities of both impurities and other drugs. They may an iodine in methanol spray. Differences between Rf values ob- be encountered as powders, liquids, home-made tablets and cap- rained by Clarke (157) and by these authors were attributed to sules, and an indication of their identity canno often be obtained the different solvents used to dissolve the samples. Cantoni et by a simple visual examination, it may also be necessary to al. (160) presented TLC data on stimulant drugs on four dif- undertake quantitative analysis or to identify a particular isomer t'er_nt absorbent layers (cellulose, silica gel, polyamide, and of the substance being examined. This is of particular impor- alumina) developed in butanol, formic acid, water (20/1/2). tahoe where distinction is made in national legislation between Good differentiation between methylamphetamine, am- two isomers: for example in the UK, between amphetamine and phetamine, phentermine, methyl phenidate, phenmetrazine, dexamphetamine, phendimetrazine, mephentermine, and diethyipropion was Unlike Cannabis products, chemical spot tests are unable to achieved. A different visualising reagent was used for each of identify drugs within this group with any certainty, although the absorbents employed, but details of detection limits and careful use of several tests may identify class or eliminate other spray specificity were not given. A two-dimensional TLC system classes (154, 155). Similar considerations apply to the use of was also described: The first solvent was ethanol, 5N ammonium microcrystalline tests (156). TLC, GLC, and HPLC have all hydroxide (9/!) and the second l-pentanol, 5N ammonium been applied to identification and quantitative analysis of the amphetamines, hydroxide (1/1). Eleven of fourteen amphetamine-like com- pounds were separated, but amphetamine was not separated Thin Layer Chromatography from fencamfamin and tranylcypromine. Several TLC systems have been devised for rapid screening An early and valuable paper by Clarke (157) examined many of "street" drugs. Brown et al. (161) used silica gel plates amphetalnine-type drugs by TLC on silica gel plates using developed in ethyl acetate, l-propanol, ammonium hydroxide methanol, ammonium hydroxide (100/l.5) as developing sol- (40/30/3), visualised with iodoplatinate. 20 #g of mescaline, i manganate,vent (17). Visuandalisation10% waformalins with ioindoplatinate,sulphuric potaacidssium(Marquisper- authorsmethylamphetamineconsidered thatandthisamphetaminesystem providedwere usefuldetected.evidenceThe 1 reagent): These reagents were used on separate plates. Good of drug class but little evidence of identity. Van Welsum (25) [ separation between amphetamine, methylamphetamine, and suggested preliminary sorting by colour tests to establish class many stimulants was obtained: 1"he author considered that with of drug prior to TLC screening. Silica gel plates were developed careful use of the different spray reagents, most of the corn- in chloroform, diethyl ether, methanol, ammonium hydroxide pounds examined could be identified with some certainly, down (75/25/5/I) and visualised with iodine in methanol, which was _, to about 10 _g. Genest and Hughes (158) examined the TI.C more sensitive than iodoplatinate. The author noted that great of STP and although separation of the compound from rues- care should be taken in making up the solvent mixture if caline (3,4,5-trimethoxyphenethylamine), amphetamine, and reproducible results are to be obtained. Choulis (94) used silica * methylamphctamine was claimed in all of these systems, the gel plates developed in chloroform, dioxan, ethyl acetate, am- published data do not bear this out, and only one system, silica monium hydroxide (25/60/10/5). The author examined a very gel G, with methanol, chloroform (I/I), was wholly successful, limited range of compounds and suggested that quantitative These authors used four visualising reagents: TCBI followed analysis was possible by elution of the compound from the ab- by iodine vapour, DAB, the modified Prochazka reagent, and sorbent and quantification by UV absorption. Vinson et al. (27) potassium iodoplatinate. They considered the general alkaloid used four different solvent systems and silica gel sheets. TCBI reagents, such as potassium iodoplatinate, to be rather insen- was used for visualisation, as the detection limit for am- strive towards amphetamines (detection limit I #g for STP); phetamine and methylamphetamine (0.25/zg) was lower than however, the TCBI-iodine reagent detected 30 ng of STP. This that using ninhydrin (1 #g). Many stimulants were reagent also gave a violet colour with amphetamine and a yellow distinguishable on colour developed alone, and this spray colour with methylamphetamine, but detection limits for these reagent appears more valuable than the non-selective reagents. two compounds were not given. Niwaguchi and lnoue (128) described a method for the quan- :304 i/

_: - Journal of Chromatographic Science, Vol. 20, July, 1982

tilalive UV densitomelry of STP on TLC plates. Sherma et al. useful approach to the screening of sample for low levels of

_: (16,162) quantitatively determined amphetamine on TLC plates, these drugs. , after spraying the plate with fluorescamine, and detected a Although a number of physical methods (171) have been ! minimum of 100 ng. Many other stimulants are primary amines developed to distinguish between optical isomers of am- _. and therefore respond, phetamines, few of these are likely to be applicable to impure _: The reviewers consider that quantitative measurements such samples or illicitly produced material. Eskes (172) described a :_ as those described above are so non-specific as to be almost simple TLC system for the separation of the optical isomers valueless, in view of the impure nature of many stimulants. Dutt of amphetamine and methylamphetamine. Samples on silica gel '.. and Poh (28) used ninhydrin as a spray reagent for basic drugs, plates were overspotted with either N-trifluoroacetyI-L-prolyl _ Considerable discrimination between drugs was possible on the chloride (TPC) or N-benzyloxycarbonyI-L-prolyl chloride so'lu- # basis of colour at different temperatures, but few stimulants tions (ZPC): Optical isomers of both compounds were separated were included in the study; 5 /.tg of amphetamine could be using the latter reagent. Visualisation was with Marquis reagent i _ detected. Masoud (29) suggested ninhydrin-phenylacetaldehyde with a detection limit of 20 tog. TLC is of use for screening on- as an improved reagent for the detection of stimulants, but no ly (i 33), because many of the stimulants are structurally related " detection limits were given. Following a study of published TLC and samples often contain many impurities. A system such as ii_: methods, Moffat (26,122,163,164)recommended alkaline silica that of Davidow (15), Sunshine (17), or Moffat (26) may be _ gel plates developed in chloroform, methanol, which can be used used with appropriate standard compounds to provide tentative _ for the tentative identification of stimulants (see above). In a evidence of identification. TLC is unsuitable for any quantitative series of papers, Bailey and co-workers examined a number of analysis of these drugs by scanning densitometry or similar substituted amphetamines by Tt.C, some of which have been techniques, as specificity is lacking. Trace quantities of some encountered illicitly. The authors studied the ring-substituted stimulants may be tentatively identified using the over-spotting series dimethoxyamphetamines (! 65), mono-methyl and mono- techniques of Gubitz and Wintersteiger (97) and optical isomers methoxy amphetamines (166), dimethylamphetamines (167), and by the technique of Eskes (172). _r their N-methylated ring-substituted analogues (166). in general, r even using pure compounds, individual isomers could not be Gas/Liquid Chromatography _ distinguished, although separation into different groups was i possible. These authors found chromotropic acid Genest and Hughes (158) studied STP and related (4,5-dihydroxy-2,7-naphthalenedisulphonic acid) was a specific phenethylamines: good elution profiles were obtained on both reagent for the visualisation of amphetamines with a 3% XE-60 and 5%0SE-30. The former column did not separate methylenedioxy substituent in the ring. Shaw and Peel (135) ex- amphetamine from methylamphetamine, but the retention times amined MDA, MDMA (3,4-methylenedioxy-N-methylamphtha- of STP and mescaline were rather long on the latter column. mine) but were unable to separate them. However, spraying with The authors suggested that compound identities could be con- gallic acid gave a green colour with MDA and a blue colour firmed by preparation of isothiocyanates with carbon with MDMA, with a detection limit of I #g. disulphide. Shaler and Padden (173) chromatographed Several methods have been developed for the detection of low 2,5-dimethoxyamphetamine on 3% OV-I 7 and 3% OV-I col- levels of amphetamines using TLC. Although these find limited umns. Beckett and Rowland (174) used 10% Carbowax 20M application apart from toxicological studies, the preliminary mixed with 5% potassium hydroxide (KOH) in a successful screening of drug traces from clothing or vehicles may be car- separation of amphetamine from methylamphetamine. ried out by TLC if appropriate visualisation reagents are used. Caddy et al. (175) found neither steel nor glass WCOT coi- Dickes and Ellis (168) used hexane-2, 5-dione followed by DAB umns satisfactory for amphetamines analysis; but support- but could only detect ! t_gof amphetamine, which is inadequate coated open tubular (SCOT) columns with either Apiezon in screening for traces of drugs. Loh et aL (169) reported a rapid L/KOH or Carbowax 20M/KOH as coating separated a large and sensitive method for the estimation of amphetamine and number of stimulants, and low levels (10 rig) could be detected. methylamphetamine: the compounds were dansylated and In a second paper (176), these authors established that retch- subsequent TLC was performed on 3 x 3-cm polyamide plates, tion indices were sufficiently reproducible for the routine iden- Good separation of the two derivatives was reported with several tification of these compounds. Using dicyclohexylamine as an developing solvents, and a detection limit of 0.3 ng was achiev- internal standard, they found that quantitative analysis was ed. Cantoni et al. (160) found that 30 ng of the dansyl derivatives possible on SCOT columns (177). Even greater sensitivity was of phenmetrazine, amphetamine and methylamphetamine could reported when a nitrogen-selective detector was used. While the be detected on normal-sized TLC plates. The dansylation pro- use of capillary columns is attractive, the presence of many im- cess was long (overnight), compared to that of Lob et al. (2 purities in illicit samples is likely to result in fairly rapid hr), and therefore impractical lor routine application. Gupta deterioration of the column. et al. (170) found that 4-chloro-7-nitrobenzofurazan (NBD Bailey and co-workers have examined ring-substituted am- chloride) rapidly formed highly fluorescent derivatives with am- phetamines by GLC.(see above), using 5% OV-7 (phenyl- phetamine and methylamphetamine. Gubitz and Wintersteiger methyl-silicone); 2.5% OV-225 (165-167,178); 5% OV-I; and (97) used high performance TLC plates (Merck) for the separa- 5% OV- i 7 ( 166,178). Although separation of many of these tion of amphetamines: Dragendorff's reagent (detection limit compounds was achieved, the authors conclude that other (spec- 200 ng), fluorescamine (25 ng), NBD chloride (25 ng), and dan- troscopic) techniques are necessary to identify even the pure syl chloride (25 ng) were used as visualising reagents. There was compounds. some increase in sensitivity using these special plates. Derivatives Moffat et al. (I 79) compared eight different stationary phases were made by overspotting with derivatising reagent. There used for the GLC of basic drugs and concluded that a low polari- seems to be no reason why this technique cannot be applied to ty phase such as SE-30 or OV-17 was preferred. In a later study normal I'LC plates or sheets, and it would appear to be a very IVloffat (180) compiled retention data for 480 drugs, which

305 Journal of Chromatographic Science, Vol. 20, July, 1982

TableX, Referencesto theTLCof AmphetaminesandRelatedStimulants Reference 10 15 16 20 25 26 27 28 29 92 94 95 97 121 122 128 132 133 135 143

Amphetamine X X X X X X X X X X X X Benzphetamine Bromo-STP" Caffeine X X X X X X X X Oiefhylpropion X X Ephedrine X X X X X X X X MDA X MDMA X Mephentermlne X X Mescaline X X X X X X X X X Methylamphefamine X X X X X X X X X Methylphenidate X X X X X X Norephednne Norpseudoephedrine _, Phendimefrazine X Phenmetrazine X X X X Phentermine X X Pseudoephedrine X STP X X X X X X Otherstimulants

Reference 157 158 159 160 161 162 163 165 166 167 168 169 170 172 178 191 215 217 250 333

Amphetamine X X X X X X X X X X X X X Benzphetamine Bromo-STP Caffeine X X X X X Deithy_propion X X X Ephedrine X X X X MDA X MDMA X Mephentermine X X X Mescaline X X Methy_aml_etamine X X X X X X X X X X X X Methylphenidate X X X Norephedrine florpseudoephedrine Phendirnetrazine X Phenmetrazine X X X Phentermine X X X Pseudoephedrine X X STP X Otherstimulants X X X X X X X

"S(Je List Of Abbreviat0ons.

showed that the majority of common stimulants were separated FID). For both compounds, intra-batch variations were found on SE-30. Donike and Stratmann (181) measured retention in- to be far less than inter-batch variations, and the authors con- dices for stimulant drugs on two columns, 12.5070Apiezon L sidered this a valuable technique for the comparison of illicit + 2.5% lgepal CO-880 (nonyl phenoxy poly (ethyleneoxy) drug samples. Barron e/al. (186) used 10070OV-101 and 3070 ethanol) and 10tr/0Carbowax 20M, the support for both these OV-25 for the separation of impurities found in illicit methylam- , phases being alkali-washed prior to coating. Separations on Car- phetamine samples. Good separation was obtained on both col- bowax 20M were less good and added little useful information, umns of methylamphetamine, N-formyl-methylamphetamine, Huber et al. (182) chromatographed 42 stimulant drugs on u-benzyI-N-methylphenethylamine, and N,tx,a'-trimethyldi- , OV-101, OV-225, Apiezon L, and Carbowax 20M coated alkali- phenethylamine. Other studies on impurities include those by washed supports. The authors compared the correlation bet- Lomonte el al. (I 87) on amphetamine, Kram and Kreugel (! 88) ween retention indices on these four columns and the mass slx=c- on methylamphetamine, and Lukaszewski (189) on MDA. tra of the drugs. They concluded that the information derived Bruce and Maynard (190) prepared heptafluorobutyryl (HFB) from GLC on two columns (OV-101 and Carbowax 20M) was derivatives of amphetamine, methylamphetamine, and chlor- equivalent to that derived from electron impact low resolution phentermine and chromatographed them on a 5°70OV-101 col- mass spectra for the identification of specific stimulants. Can- umn with electron capture detection. Good separation of each field e/al. (183) separated 12 stimulants on 10070Apiezon L/2% derivative was obtained, and 1 ng could be detected. Jain el KOH and 10% Carbowax 20M/207o KOH, although ampheta- al. (191,192) and Brugaard and Rasmussen (57) prepared HFB mine and methylamphetamine were not included, and TFA derivatives of amphetamine "on-column." Although Stromberg 084) and Stromberg and Maehly (185) examined tile use of fluorinated derivatives with electron capture is an trace impurities i'n samples of amphetamine sulphate and extremely sensitive method, many other drugs and impurities phenmetrazine hydrochloride using two detectors (ECD and in illicit samples also react with HFB and TFA.

306 Journal of Chromatographic Science, Vol. 20, July, 1982

Two approaches have been made to the problem of separating ing !07ocarbowax 20M, which gave retention times of 12.5 min and quantifying enantiomers by GLC (193): A racemate (or an and 15.5 rain. The method was applied to the quantitative unknown single optical isomer) is reacted with a chiral reagent analysis of the pure compounds and proprietary preparations. to yield a diastereoisomeric mixture that may be resolved on in a collaborative study 098) of this method, acceptable i a normal GLC phase, or the racemale itself may be resolved recoveries were obtained and precision of quantitation was in on a chiral phase. This latter approach has not proved fruitful the range 0.6 to 3070.The method was found to be applicable

successfor the sheasparationbeen acofhievedisomesusinofgampchiralhetaminereagents.(194),Gordisbut (19some5) variablto singlee resultstablet wh(5enmg)analysinassays.g ampNichholsetaminet eal. enantiomer(199) obtaineds us-

TrePCsolvederivativd dexames,phetwahmicineh werefromchrolevamatompgrhetapaminehed onby 507preopSE-30aring rineagg TPentC durinderivatives,g preparation.probably because of racemisation of the

poor and retention times were rather long, quantitative results phenylacetic acid (MTPA), which does not undergo racemisa- with a detection limit of 5 #g. Although elution profiles were These same authors used a-methoxy-a-trifluoromethyl compared well with polarimetry measurements. On-column tion, to prepare diastereoisomeric derivatives. Retention times i !_ derivitisation was found to be as effective as external derivitisa- were 20 to 30 min on Carbowax 20M with 4 to 6 rain between i_ tion. Beckett and Testa (196) used this technique and found only diastereoisomers. Sourer (200) prepared diastereoisomeric +! partial resolution of methylamphetamine enantiomers; again, derivatives from a number of amphetamines and four optical, _: retention times were rather long (50 rain) for routine work. Wells ly active acylated amino-acid chlorides (derived from proline, _: (197) improved the chromatography of TPC derivatives by us- valine, leucine, and alanin¢). Best separation was obtained on

Table Xl. References to the GLC of Amphetamines and Related Stimulants Reference 10 20 30 31 32 35 38 39 41 43 57 59 92 110 143 158 166 167 173 174 175 176 177 178 179 Amphetamine X X X X X X X X X X Benzphetamine ._ Bromo-STP" i Caffeine X X X X X X X X X X X X X X ' . Diethylpropion X X X Ephedrine X X X X X X X X MDA MDMA X Mephentermine X X Mescaline X X X X Methyl- amphetamine X X X X X X X X Methylphenidate X X ! Norephedrine Norpseudo- ephedrine Phendimetrazine X X Phenmetrazine X X X X Phentermine X X X Pseudoephedrine X X STP X Otherstimulants X X X X X X X

Reference 180 181 182 183 184 185 186 187 188 189 190 191 192 194 195 196 197 198 199 200 201 202 211 218 222 Amphetamine X X X X X X X X X X X X X X X X X X X X X Benzphetamine Bromo-STP Caffeine X X X X Diethylpropion X Ephedrine X X X X X MDA X X X X MDMA ,* Mephentermine X :!, Mescaline X X Methyl- amphetamine X X X X X X X X X X X X MethylphenidateX Norephedrine Norpseudo- ephedrine PhendimetrazineX X Phenmetrazine X X X X X X X Phentermine X X PseudoephedrineX X STP X X X X + Otherstimulants X X X X X X X X X X

"See List of Abbreviations. T

307 Journal of Chromatographic Science, Vol. 20, July, 1982

a 5°70 DEGS (Diethyleneglycol succinate) column using TPC (70,204) chromatography, resulting in poor efficiencies by derivatives with a relative retention of 1.34 for dex- and levam- present-day standards. Using solvents at different pH, Twit- phetamine. Gal (201) made further studies on MTPA derivatives chert and Moffat (75) evaluated #-Bondapak C,, as a packing and obtained partial resolution of the enantiomers of am- for the analysis of basic drugs, including amphetamine and phetamine and complete resolution of the enantiomers of STP methylamphetamine, but even this exhibited poor efficiency for and 4-methoxyamphetamine. The column was 3% OV-17 and such drugs. In an important publication, Jane (73) examined detection limit 5 ng using ECD. Reaction of MTPA with a wide range of drugs of abuse by HPLC using 6-p.m silica and primary amines was complete in 30 rain, a more rapid and con- a mobile phase of methanol, 2N ammonium hydroxide, IN am- venient method than that recommended by Nichols (199). Secon- monium nitrate (27/2/1). Detection was by UV absorption at dary amines were much slower to react and were essentially 254 nm. Good chromatography was achieved, but by no means underivatised under the conditions recommended for primary were all the major stimulants resolved. Quantitation was by peak b amines. An advantage of this reagent is the commercial height measurement, and the column was reported to have a availability of the optically pure free acid. Matin et al. (202) long life. Twitchett et al. (205) evaluated a microparticulate ca- ! prepared l-(l-pentafluorobenzoyI-L-prolyl)imidazolidine tion exchange column, but found that the column life was rather derivatives of amphetamine and dexamphetamine and several short. Waiters and Waiters (206) examined diethyipropion and 'P ring-substituted oL-methyl phenethylamine enantiomeric pairs, its decomposition products on an ODS-silica column using two GLC was performed on a 3% OV-17 column and, using an solvents: acetonitrile, 0.1% ammonium carbonate solution, ECD, the detection limit was i ng. Although the authors claimed methanol (70/20/10) and 0. i % ammonium carbonate solution, resolution of lev- and dex-amphetamine derivatives, the reten- acetonitrile, methanol (60/35/5). Good separation between lion times were rather close (relative retention 1.07). The corn- diethylpropion and l-phenyl-l,2-propanedione was achieved in plex preparative procedure also limits the use of this reagent, both systems (the second decomposition product, diethylamine, Non-polar or semi-polar stationary phases have been prefer- was not observed). Detection was by UV absorption at 255 nm. red by most workers for the analysis of stimulant drugs. Con- Achari and Theimer (207) chromatographed amphetamine and siderable use has also been made of alkali-washed solid sup- other drugs on Partisil 10 using methanol, dichloromethane, ports and alkaline-treated stationary phases. Although good ammonium hydroxide (75/25/!) as mobile phase. Lurie and separation is achieved between many of the common stimulants, Weber (208) examined "street" samples of methylamphetamine retention data are only of value in providing supportive evidence using ion pair HPLC: good chromatography of this drug was of identification because of the many structurally similar corn- obtained on a _-Bondapak C,, column using water, methanol, pounds that have been encountered, acetic acid (79/20/I) with 0.005M pentane sulphonic acid at Where the identity of an amphetamine has been determined pH 3.5 as the mobile phase. Baker et al. (84) used three solvent unequivocally, preparation of an appropriate diastereoisomer systems on a #-Bondapak C,, column to characterise drugs on by reaction with an optically active derivatising agent, follow- the basis of retention times and UV absorbances at two different ed by GLC, will provide identification and quantitation of the wavelengths (see above). Several stimulants were distinguished isomers present, by this system. Wheals (85), in a similar study, recommended three different packings (silica, a mercaptopropyl modified High Performance Liquid Chromatography silica, and an n-propylsulphonic acid modified silica) with a The forensic applications of HPLC to phenethylamine single mobile phase, that of Jane (73), for basic drug : analysis have been briefly reviewed by Wheals (76,203). Early characterisation. In the case of the stimulant drugs, this triple systems used ion exchange (203) or large-particle adsorption column system has little to offer over the single system (73).

Table XII. References to the HPLC of Amphetamines and Related Stimulants Reterence 20 66 69 70 73 74 75 76 77 81 82 83 84 85 89 118 145 204 205 206 207 208 260 Amphetamine X X X X X X X X X X X X Benzphetarnine X X X X X Bromo-STP" X • Caffeine X X X X X X X X X X X X X X X X X Oiethylpropion X Ephedrine X X X X X X X X X X X X MDA X X X MDMA Mephentermine X X X X Mescaline X X X Methylamphetamine X X X X X X X X X X X Methylphenidate X X X X Norephedrine X X Norpseudoephedrine X X Phendimetrazine X X X X Phenmetrazine X X X X Phentermine X X Pseudoephedrine X X STP X X X X Otherstimulants X X X X X

"See List ol Abbreviahons.

308 _ i Journal of Chromatographic Science, Vol. 20, July, 1982

t_ althoughHPLC thhaes snotystembeendevisexteednsivelbyyJaneappli(73)ed tiso tsthimule mostant usdreugsful., (separ> 1 atehr din bally usingcases).allDtheetesysctiontems.limitDesvelopmentof 0.5 to 1.0times/tg werewere lonob-g The main value of HPLC and GLC in this field is for providing tained by using alkaline fluorescein as visualisingagent.In ad.

ili solventluther eorvidmulti-columnence of identityscreeningand forsystemsquantthatitationhave. Thebeen multi-devis- detecteddition, aliobarbital,with iodine vacyclobarbital,pour. Sunshineand(17secobarbital) used silica gelcouldplatesbe t ed are very time-consuming and expensive compared with TLC. developed in chloroform, acetone (9/1) to examine 23 bar- ': It is emphasised that chromatographic techniques alone are not biturates, and a good spread of Rf values was obtained. usually able to provide unequivocal identification of a particular Visualisation was by spraying with mercuric sulphate followed '_ member of this class of drugs, and confirmation by specific by diphenylcarbazone (DPC), or with potassium permanganate. _ techniques, such as IR or mass spectrometry (combined with The author recommended that standards should be run _, GLC where necessary), is often mandatory, alongside samples, as Rf values alone could not be relied upon #_' for tentative identification. Pentobarbital was not separated from amobarbital, but other major barbiturates were Separated Barbiturates from each other. Berry and Grove (211) used this system and recommended that acetone should be carefully dried before use. Visualisation was with mercuric chloride/DPC, which these Although barbiturates are not yet controlled in the UK under authors found more sensitive than mercurous nitrate/DPC. In the Misuse of Drugs Act (1971), many are subject to control in North America. In addition, some are listed in Schedules 3 a lengthy method, Rosenthal et al. (212) studied five barbiturates and 4 to the United Nations Convention on Psychotropic and glutethimide on activated silica gel plates. After spotting, Substances (1971), and it is therefore appropriate to include the plates were placed for 30 min in a TLC tank containing two them in this review. Large numbers of barbiturates have been beakers of ammonium hydroxide. Developing solvent of synthesised, and many have been widely used in commercial chloroform, n-butanol, formic acid (70/40/3.5) was then add- ed to the tank (the ammonium hydroxide remaining), and the preparations. Illegally obtained, but licitly manufactured pro- ducts make up the majority of seizures in the UK. Barbiturates plates were developed in the normal way. Good separation bet- •ween glutethimide, pentobarbital, secobarbital, amobarbital, combined with amphetamines have also been used in certain licit preparations, but the availability of such products is now barbital, and phenobarbital was obtained. Visualisation was with DPC followed by mercuric nitrate. Abu-Eittah et al. (213) limited. Small quantities are found mixed with other drugs such studied four barbiturates and an hydroxybarbiturate on several as heroin. In contrast to amphetamines, there is little indica- tion that barbiturates are being illicitly manufactured, different silica gel-based layers in eleven different solvent systems. No one system completely resolved all five compounds: Thin Layer Chromatography Visualisation was by spraying with Cobalt (I1) or Mercury (II) salts, with detection limits of 10 t_gand 0.5 pg respectively. Bress Many samples of barbiturate-containing preparations are of et al. (214) examined the TLC separation of barbiturates using commercial origin and may be recognised by product identifica- reverse-phase TLC. Silica gel plates, after spotting, were dip- tion codes and other manufacturers' markings. The identity of ped briefly into a tank of mineral oil, light petroleum (1/10), the ingredients may be made by spectroscopic techniques or by developed in water, methanol, ammonium hydroxide (80/20/2), simple TLC alongside authentic samples. Many paper dried and sprayed with DPC/mercuric sulphate. Separation of chromatographic and TLC systems for the separation of bar- phenobarbital, butobarbital, amobarbital, pentobarbital, biturates have been devised, and these have been summarised secobarbital, diphenylhydantoin, ethchlorvynol, and by Jain and Cravey (209). Most systems use silica gel plates, glutethimide was achieved. The authors suggested that use of but Huang and Wang (210) used polyamide plates developed this system in conjunction with normal phase TLC would pro- in seven different solvent mixtures to study seven barbiturates, vide further evidence of identity of a particular barbiturate. No one system separated all the compounds, but all may be Many authors have included barbiturates in general drug

Table XIII. References to the TLC of Barbiturates

Reference 15 17 20 25 27 29 94 122 170 209 210 211 212 213 214 215 216 217 322 AIIobarbital X X X X X Amobarbital X X X X X X X X X X X X X X Aprobarbital X X X X Barbital X X X X X X X X X X X Butobarbital X X X X X X X X Butalbital X Cyclobarbital X X X X X X Heptabarbital X X X Hexobarbital X X X X X Mephobarbital X X Metharbital X Pentobarbital X X X X X X X X X X X Phenobarbital X X X X X X X X X X X X X X X X X X Ouinalbarbital X X X Secobarbital X X X X X X X X X X X Talbutal X X Otherbarbiturates X X X X

309 Journal of Chromatographic Science, Vol. 20, July, 1982 screening TLC systems. Davidow (15) used silica gel developed ammonium hydroxide (47.5/45/5/2.5) and visualised bar- in ethyl acetate, methanol, ammonium hydroxide (85/10/5) biturates with mercuric sulphate/DPC. Amobarbital was not sprayed with DPC/mercuric sulphate. In general, rather poor separated from secobarbital, nor meprobamate from phenobar- separation of barbiturates was achieved by this system, although bital. Vinson et aL (27) used silica gel sheets developed in four phenobarbital was well separated. The spray reagents did not different solvents in a screening system for "street" drugs: TCBI visualise any other commonly occurring drugs of abuse except was used as visualisation reagent. In general, poor separation meprobamate, which had a similar Rf to the barbiturates. Broich of barbiturates was obtained, but discrimination from other et aL (215) developed silica gel sheets in hexane, ethanol (93/7) drugs could be made on the basis of high (>0.9) Rf values for visualised with mercurous nitrate. Little separation was achieved the barbiturates. Blue colours were given by barbiturates with between the barbiturates in this system, but the authors sug- TCBI, which also gave some discrimination from other drugs. gested that this should be used as a screening method, with iden- TLC has been widely used for the detection of barbiturates, tification by other techniques. Owen et al. (216) studied 15 dif- particularly in the toxicological field. Identification is usually ferent TLC systems and recommended that the best systems for tentative, as separation between even the major barbiturates is the identification of common barbiturates (amobarbital, bar- poor in most solvent systems. Combination of a reverse-phase bital, butobarbital, cyclobarbital, hexobarbital, methohexital, TLC system (214) with normal-phase TLC is a useful aid to iden- pentobarbital, phenobarbital, and secobarbital) were Davidow's tity. Visualisation is almost universally by mercury (il)/DPC (15), combined with either ethyl acetate or chloroform, sprays, which have a high specificity towards barbiturates and methanol (9/1), these latter two also on silica gel plates. Mer- related drugs and can therefore provide considerable evidence curie chloride/DPC was used as visualising agent, giving white of the presence of a barbiturate. spots on a purple background. A number of diuretic drugs (ben- drofluazide, frusemide, hydroflumethiazide) also gave a similar Gas/Liquid Chromatography response: however, these three drugs were easily distinguished GLC has been widely used for the analysis of barbiturates: on the basis of their Rf values in chloroform, methanol (9/I). The free acid barbiturates exhibit considerable adsorption and Masoud (29) examined the TLC characteristics of three bar- consequent poor elmion profiles on the majority of biturates (amobarbital, phenobarbital, and secobarbital) in five chromatographic packings. Many workers have therefore form- solvent systems on silica gel plates visualised with mercuric ed alkylated derivatives prior to GLC analysis. Jain and Cravey chloride/DPC. Separation was poor in all systems. The author (209) list and briefly discuss much of the literature up to 1974. reported that acetylsalicylic acid (aspirin) may also respond to Alber (218) proposed 3%00V-17 on Gas Chrom Q as a general this reagent. GLC column for pharmaceuticals. Barbital, amobarbital, Single TLC systems have been suggested for the identifica- secobarbital, mephobarbital, and phenobarbital were successful- lion of more common drugs of abuse. Choulis (94) used silica ly separated on this column, but tailing of phenobarbital was gel plates developed in chloroform, dioxan, ethyl acetate, am- evident in the published chromatogram. Taylor (219) tabulated monium hydroxide (25/60/10/5) and separated a number of retention data for many barbiturates and related compounds drugs including amobarbital, phenobarbital, pentobarbital, and on this column, although many were not completely resolved. secobarbital, although Rf values were very close. Van Welsum Temperature programming and an AFID were used. Berry (220) (25) used silica gel plates developed in chloroform, diethyl ether, used 12 different stationary phases (including 5% OV-17) coated methanol, ammonium hydroxide (75/25/5/I) sprayed with on Chromosorb W(HP) in a critical examination of the many DPC/mercuric chloride. Preliminary screening prior to TLC phases recommended for barbiturate analysis. Several were was carried out using the Marquis reagent, to which barbiturates reported to give useful separations, and the author concluded do not respond, and alkaline cobalt nitrate. Although separa- that a 4% CDMS column was best. Butobarbitai and amobar- tion was poor, preliminary screening and the spray reagent af- bital were not completely separated, and methaqualone and forded good evidence for the presence of a barbiturate. Speaker meprobamate had Rts similar to the barbiturates on this col- (217) developed silica gel plates in dioxan, cl'doroform, acetone, umn. Considerable tailing of cyclobarbital, heptabarbital, and

Table XlV. References to the GLC of Barbiturates

Reference 30 32 42 180 209 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 . Atlobarbital X X X X X X X X X X Amobarbital X X X X X X X X X X X X X X X X X Aprobarbital X X X X X X X X X X X , Barbital X X X X X X X X X X X X X X X Butobarbital X X X X X X X X X X X X X X Butalbital X X X Cyclobarbital X X X X X X X X X Heptabarbital X X X X X X Hexobarbital X X X X X X X X X X X MephobarbitalX X X X X X X X Metharbital X X X X X Pentobarbital X X X X X X X X X X X X X X X X X Phenobarbital X X X X X X X X X X X X X X X X X X X X X Quinalbarbital X X X X Secobarbital X X X X X X X X X X X X X l-atbutal X X X Otherbarbiturates X X X X X X X X X X X X X X X X X X

310 Journal of Chromatographic Science, Vol. 20, July, 1982

phenobarbital is apparent from the published chromatograms, methanol before chromatography on 5% XE-60 or 10% NPGS The author considered that high performance supports were (neopentyl glycol succinate). Kurata et al. (229) identified satisfactory for the analysis of sub-microgram amounts of bar- N-methyl-2-phenylbutyrimide as the principal by-product of the biturates, but noted that the system, which should be all glass, flash heater methylation of phenobarbital with trimethylanilium must be silanised. The author considered that additional precau- hydroxide. tions (not specified) would be necessary to chromatograph very Giovaniello and Pecci (230) suggested on-column hexylation small quantities successfully. The same column was recommend- and heptylation of barbiturates, and Budd (231,232) prepared ed for the analysis of methaqualone (221). A second column numerous dialkyl derivatives for study by GLC. Although many (4% OV-225) was recommended to confirm identities. Moffat useful separations were achieved by using alkyl derivatives (other (180) chose SE-30 as the "preferred stationary phase" for the than methyl), the reviewers consider that methylation provides GLC of drugs, although separation was poor between a number adequate information for the routine identification and quan- !, of barbiturates. Methaqualone was, however, separated from titative analysis of the majority of the barbiturates. Dilli and the barbiturates. Chromatograms were not shown, and adsorp- Piilai (233) prepared the chioroethyl derivatives of ten bar- tion problems were not discussed, biturates for GLC. Minimum detectable amounts by ECD were Fontan and Hill (42) found considerably less tailing by drugs <0.5 ng in all cases, a considerable increase over the FID. chromatographed on a specially prepared Carbowax 20M col- Garle and Petters (234) used an SE-30 SCOT column to give umn whether or not it was coated with a second stationary phase good separation of the ethyl derivatives of 14 barbiturates and (SE-30 or OV-17) (see above). Separation of amobarbital and glutethimide. Quatitation was based on dipropylbarbituric acid pentobarbital was achieved on this column. Cyclobarbital was as internal standard for compounds of short retention time and chosen by Street et al. (222) to study the reduction in adsorp- allylphenylbarbituric acid for the remainder of the compounds. tion of drugs on specially prepared packings: the solid sup- Precision of quantitation varied a great deal depending on the port was acylated prior to coating with stationary phase, the particular barbiturate, the best being 6% for phenobarbital and glass column was similarly treated, and the packed column was heptabarbital, and the worst, 40% for metharbital. Dunges et then heat treated in nitrogen. On a conventional OV-I column, al. (235) prepared methyl, allyi, and benzyl derivatives of bar- it was not possible to detect less than I #g of cyclobarbital due biturates prior to WCOT chromatography using SF-96 (methyl- to complete adsorption; whereas, using the modified support, silicone) stationary phase. By preparing all three derivatives, nanogram amounts were detected. Methaqualone and most of the 29 compounds could be identified on the basis of meprobamate were also successfully chromatographed, but no retention times; however, butethal and butalbitai, secobarbital details were given. Riedmann (223) considered that adsorption and vinbarbital, cyclopentobarbital and heptabarbital pairs were of barbiturates by the support could be eliminated if Haloport not distinguished. Rasmussen and Christopherson (236) F (polytetrafluoroethylene)were used instead of diatomaceous prepared derivatives, by flash heater methylation with DMF earths. Nanogram quantities were detected using an AFID. dimethylacetal. Good separation between seven barbiturate Breimer and van Rossum (224) chromatographed hexobarbital derivatives was achieved using OV-I and SE-30 WCOT columns. with methohexital as internal standard on a 3% OV-17 column, Three principal stationary phases have been used for the 0.5 ng of both compounds could be detected, using an AFID; analysis of underivatised barbiturates: OV-17 (218), CDMS adsorption of these compounds on this column was not discuss- (220), and SE-30 (180). Berry (220) found CDMS the most ed. Dvorchik (225) chromatographed barbital, pentobarbital, suitable stationary phase, but the chromatograms showed eon- secobarbital, and hexobarbital on 3% SP-2250. Response of siderable tailing of the longer retained components. Good the AFID was linear over the range 10 to 100 ng, but losses separation and minimal adsorption losses by using the methyl by adsorption were noted below 10 ng. O, ly partial separation derivatives have been achieved by several groups: considerable between pentobarbital and secobarbital was achieved, evidence of the identity of a particular barbiturate may be ob- To overcome losses of barbiturates by adsorption, rained by comparing retention times ofunderivatisedanddif- Brockmann-Hanssen and Oke (226) prepared the methylated ferent derivatives of the same barbiturate. The AFID is useful derivatives using trimethylanilinium iodide. A 3% SE-30GLC when barbiturates are mixed with other volatile organic colunm was used, and 12 barbiturate derivatives were separated materials. Although capillary GLC overcomes most of the pro- within 25 minutes. Complete derivitisation on injection was blems of separation, quantitative analysis is insufficiently precise reported, and response of the FID with respect to the amount in many cases. Whichever GLC procedure is adopted, it is still of barbiturate injected was linear over the range 0. I to 1.0/zg usually necessary to confirm the presence of a particular bar- and passed through theorigin, indicating that adsorption losses biturate by other techniques, including combined GLC/MS were insignificant. The authors suggested that barbiturates that (237,238). Plasma spectra have also been proposed for the gave the same derivative on methylation (e.g., barbital and qualitative analysis of barbiturates (239), but this system is not metharbital) may be differentiated by chromatographic analysis widely available. of the sample before and after methylation. Ehrsson (227) prepared methylated barbiturates by performing the derivitisa- High Performance Liquid Chromatography lion step in carbon disulphide with methyl iodide and tetrahex- ylammonium iodide: carbon disulphide gives a very low response Despite the fact that GLC alone is unable to resolve all bar- with the FID. Interference from trihexylamine formed by ther- biturates, there have been relatively few papers using HPLC mal decomposition of the counter-ion was eliminated by main- with this objective. Wheals (76,203) listed papers up to 1976, raining the injection temperature below 180°C. The column was and there have been few publications since then. 3% SE-30 and detection limit 20 ng of dimethyl phenobarbital. Roos (240) used a strong anion exchange column (SAX, Mraz and Sedivec (228) used trimethylphenyl ammonium acetate quaternary ammonium substituted methacrylate polymer) to or tetramethylammonium acetate in order to eliminate reactions study 17 barbiturates using either an alkaline (borate/nitrate) leading to side products. Methylation was carried out in or acidic (citric acid) mobile phase. Although elution profiles

311 Journal of Chromatographic Science, Vol. 20, July, 1982

were rather broad, separation of a number of these compounds 5,5-dialkylbarbituric acids. The mobile phase was made alkaline was achieved. Detection, down to nanogram levels, was by UV after elution by the post-column addition of a pH 10 borate absorption at 254 nm. Twitchett et al. (205) evaluated a buffer solution. The barbiturates were therefore detected by UV microparticulate cation exchange column, but found that it ex- absorption, as their anionic chromophores, giving a 20-fold in- hibited little ability to separate barbiturates even over a wide crease in sensitivity over detection of the un-ionised compounds. pH range. Tjaden el al. (241) used a methyl-silica column and Enhanced detection limits were obtained by Hulshoff et al. (247) eluted barbiturates with methanol, water mixtures. Good separa- after preparing 2-naphthacyl derivatives of barbiturates, which tion of the majority of the 15 barbiturates studied was achier- were separated by reverse-phase chromatography. At 249 nm, ed, with a detection limit at 220 nm of 2.4 ng. A linear calibra- 1 ng of the phenobarbital derivative was detected. The struc- tion curve was obtained for hexobarbital over the range 20 ng tures of the derivatives were confirmed by MS and "C-NMR to 5 #g, but precision of quantitation varied substantially with spectroscopy. Lurie (81) and Lurie and Weber (208) used amount injected. Baker et al. (84) found that the determina- reverse-phase ion pair chromatography (see above) to separate tion of many barbiturates could be made on ODS-silica _- several barbiturates, although amobarbital and pentobarbitai Bondapak C,,) at pH 7 with 0.025 M sodium hydrogen were unresolved. phosphate in methanol, water (2/3). Out of 17 barbiturates only There have been no reports of irreversible adsorption of bar- .¢ the phenobarbital and aprobarbitai pair was unresolved, biturates on HPLC columns, and this technique therefore ap- although each bad different absorbance ratios (see above). Pen- pears to be more suitable for the separation and determination tobarbital was not completely resolved from glutethimide, but of barbiturates, without resort to derivitisation, than GLC. With methaqualone was separated from all the barbiturates examin- so many different barbiturates available, no one system is like- ed. Baker (242) has also published observed and predicted reten- ly to provide unequivocal evidence of identity. However, the tion indices of barbiturates, single column, dual detection concept of Baker (84) and White Twitchett and Moffat (75) chromatographed phenobarbital, (244) may provide useful additional evidence in particular cases. phenytoin, glutethimide, and secobarbital on ODS-silica (#- Alternatively, analyses using both normal and reverse phase Bondapak C,,) using a pH 7 methanol, water (11/9) eluent: Elu- HPLC with aqueous methanol as solvent may be used (241,246). tion profiles in the published chromatogram were rather broad There is considerable scope for further development in the and baseline separation was not achieved. Court el aL (243) ob- HPLC separation of barbiturates. rained good chromatography on ODS-silica _-Bondapak C,,) using methanol, aqueous pH 6, 2.6 mM ammonium carbonate buffer (60/40) as eluent. Phenobarbital was separated from butobarbital, hexobarbitai, amobarbital, pentobarbital, and Phencyclldine and Analogues secobarbital; however, amobarbital was not resolved from pen- tobarbital. White (244) used a similar system to that of Court Phencyclidine [PCP, l-(l-phenylcyciohexyl)piperidine] has el al., combined with dual-wavelength UV detection, in a study been the subject of increasing abuse in the USA and is controlled of 29 barbiturates. By combining retention data and absorbance both there and in Canada. It is made in clandestine laboratories, ratios, 27 barbiturates could be positively identified. Butalbital together with its analogues and is often contaminated with in- and butobarbital could not be distinguished using this system, termediates and by-products. In 1979 it came under the con- The author recommended a guard column between the pump trol of the Misuse of Drugs Act (1971) in the UK, although there and the injection port to ensure reproducible retention data over is little evidence of its illicit use and availability is very limited. i extended periods. Reverse-phase chromatography on ODS-silica (10 #m) was used with gradient elution (methanol, water by Thin Layer Chromatography Jandera el al. (245). Detection of ten separated barbiturates was by UV absorption at 254 nm. Clark and Chan (246) used a Phencyclidine is included in several general TLC screening similar system isocratically and separated several methods. Van Welsum (25) separated phencyclidine from corn-

Table XV. References to the HPLC of Barbiturates i Reference 70 74 75 81 82 84 205 208 240 241 242 243 244 245 246 247 322 . Atlobarbital X X X X X X Amobarbital X X X X X X X X X X X Aprobarbital X X X X X X Barbital X X X X X X X X X X X Butobarbital X X X X X X X X X X : Butalbital X X X Cyclobarbital X X X X Heptabarbital X X X X X He_obarbital X X X X X X X X Mephobarbital X X X X X Metharbital X X X X Pentobarbital X X X X X X X X X Phenobarl_ital X X X X X X X X X X X X X Quinalbarbital X X X Secobarbital X X X X X X X X X i _ Talbutal X X X X Otherbarbiturates X X X X X X

312 Journal of Chromatographic Science, Vol. 20, July, 1982

mealy encountered narcotics, hallucinogens, barbiturates, and were methylene chloride, I-butanol, ammonium hydroxide amphetamines using activated silica gel G plates with (88/15/0.2) on silica gel (Quanta Gram) and ethyl acetate, chloroform, diethyl ether, methanol, ammonium hydroxide methanol, diethylamine (90/10/1.6) on silica gel (Merck), (75/25/5/!). Cocaine was not resolved and gave the same col- although not all compounds were completely resolved. our with iodoplatinate. Noscapine, which was also not resolv- ed, was distinguished by its reaction with the Marquis reagent. Gall/Liquid Chromatography Masoud (29) used five different solvent systems (see above). None unequivocally distinguished phencyclidine on the basis Illicit phencyclidine was first identified by Lindgren etaL (253) of Rf alone, and heroin, caffeine, LSD, methadone, methylam- using combined GLC/MS, when it was successfully chromato- phetamine, some canttabinoids, and some local anaesthetics in- graphed on OV-17 at 160°C. A mixed stationary phase of 20/0 UCW-98 + 1%00V-17 has been used to separate phencyclidine terfered. All except the cannabinoids, caffeine, and LSD, gave from cocaine, methaqualone, methadone, and propoxyphene ; a reaction with iodoplatinate. Brown el al. (161) examined some hallucinogens and amphetamines, from which phencyclidine was (106). The reliability of quantitation of phencyclidine has been assessed in a collaborative trial (254) using 3% OV-I. Preci- well resolved, using activated silica gel plates with ethyl acetate, sign of the results of three determinations for each of six samples °*j l-propanol, ammonium hydroxide (40/30/3). Detection was by in six laboratories varied between 1.1 and 3.4%. iodoplatinate. Phencyclidin¢ is not visible under UV light at 254 Using 5% OV-7 (phenyi-methyl-silicone) at 150"C, Bailey am. (249) found that phencyclidine was unresolved from TCP, although the latter also gave rise to a degradation product. Table XVI. References to the TLC of Phencyclidine Under the sameconditions phencyclidine, PHP, and PCE were and Analogues well resolved. Combined GLC/MS confirmed that PCE pass- ed through the column unchanged. Bailey and Legault (251) Reference 25 29 92 135161170191248 249250251252258 extended the study to sixother related compounds, all of which PCC" X X were resolved, using the same conditions as in the earlier study. PCE X X X X Electron impact mass spectra were also published. GLC/MS _x has been used by Cone et at. (252). Four different silicone gum Phencyclidine X X X X X X X X X X X X PHP X stationary phases of varying polarity were used, the best separa- TCP X X tigris being obtained on SE-30. Phencyclidine was not resolved Other from TCP on OV-17. Identification of the compounds was bas- phencyclidine ed on chemical ionisation mass spectra. Phencyclidine, PCE, analogues X X methadone, methaqualone, and severalother basic drugs have •so= t.,mo_Atmf=v+at_o,s been separated on OV-17 and SE-30 (250). Trace amounts of phencyclidine have been detected using an AFID, both with packed OV-17 (255,256) and capillary OV-101 columns (256), A common impurity in phencyclidine is the intermediate with a precision of quantitation of 0.3 and 6°/0 respectively. l-piperidinocyciohexanecarbonitrile (PCC). With conventional PCC is unstable, although some unchanged PCC, as well as activated plates this compound decomposes, first to the major degradation product, was observed by Helisten and l-piperedinocyclohexene (PCX) and ultimately to cyclohex- Shulgin (248) using GLC. This was assumed to be PCX and anone and piperidine. Helisten and Shulgin (248) have describ- was later confirmed by Seine et aL (257) using GLC/MS. Ball- ed a method to separate phencyclidine, PCC, and the thiophene inger et al. (258) examined 12 "street" sample for the presence analogue, l-[l-(2-thienyi)cyclohexyl]piperidine (TCP). The sol- of phencyclidine and PCC using 3% OV-7 at 125°C. Although vent was benzene, acetone, pyridine (16/8/1) with deactivated decomposition of PCC to PCX occurred, the authors claimed silica gel plates. By pre-wetting the plate at the site of applica- that this was sufficiently reproducible to allow quantitation of lion, decomposition of PCC was minimised, provided no heat PCC. The quantitation of PCC in "street" samples of phen- was applied until development was complete. The plate was then cyclidine has also been made by Cone et al. (259). Quantita- heated to 110°C and sprayed with ninhydrin, after which it was lion was based on the assumption that breakdown to PCX was cooled and sprayed with iodoplatinate. Phencyclidine gave no both rapid and complete on injection into the chromatograph. • colour with ninhydrin, the other compounds, including Detection was by AF1D with a limit of 4 ng. Confirmation of piperidine, giving purple spots. Bailey (249) synthesised N-ethyl-l-phenyl-cyclohexylamine (PCE), which has been encountered in "street" samples, and Table XVII. References to the GLC of Phencyclidine and Analogues separated it from phencyclidine, TCP, and l-(l-phenylcyclo- hexyl) pyrrolidine (PHP), usingsilicagel with acetone, 12N am- Reference 106180248249250251252253254255256257258259 monium hydroxide (99/1). Phencyclidinewasnot resolved from PCC* X X X X TCP. Smialek et al. (250) separated PCE from morphine, co- PCE X X X X X deine, quinine, caffeine, methaqualone, and methadone using PCX X X X X X two different solvent systems.Bailey and Legault (251) alsoex- PhencyclidinXe X X X X X X X X X X X X amined the N-aikyl derivatives of l-phenylcyclobexylamine. Rf PHP X TCP X X values were quoted using nine different solvent systems. Most Other of the compounds were resolved, although none of the systems phencyclidine separated the N-n-propyl and N-iso-propyl isomers, analogues X X X X Cone el al. (252) examined 15 phencyclidine precursors, metabolites, and analogues. The most satisfactory TLC systems "sooCislofADbrov+afions

313 Journal of Chromatographic Science, Vol. 20, July, 1982

the presence of PCC in the sample was by direct chemical ionisa- material, and in most cases, no quantitative analysis (other than tion MS. PCC (as PCX) was also analysed by Soine et aL (257) simple weighing} is necessary. Unambiguous identification of on a 3% OV-17 column coupled to MS. It was noted that phen- Cannabis plant material may be made at all stages of the plant's cyclidine itself partially decomposed to give l-phenylcyclohex- growth by microscopic or macroscopic recognition of its ene, which was not completely separated from PCX. It is thus botanical characteristics (261). It is usually desirable to corn- possible to assign this peak incorrectly and erroneou_,ly con- plemem this with chemical tests, particularly when evidence of elude that the sample originally contained PCC. identity may he presented in a court of law. Furthermore, many samples have no microscopically identifiable feature, and High Performance Liquid Chromatography chemical tests are then mandatory. Cannabis extracts and smok- In view of the instability of some of the phencyclidine ing residues often fall within this category. The presence of a analogues, it is surprising that lhere are so few studies using Cannabis product (which may comprise all or part of a sam- HPLC. Wittwer (145) included phencyclidine in a study of LSD pie) may be confirmed by any analytical technique that iden- , rifles the presence of one or more of the cannabinoids, which and other ergot alkaloids (see above). Phencyclidine was resolv- Mechoulam (262) reports as unique to Cannabis sativa. Since ed from all other compounds in the study, using a Corasil !1 column with acetonitrile, isopropyl ether (40/60). Pheucyclidine that report, no record of the natural occurrence of cannabinoids ,_ in any other platn or animal species has appeared in the scion- was included in the drugs studied by Chanet el. (70), which covered hallucinogens, amphetamines, barbiturates, THC, co- tific literature. Fenselau et el. (263) demonstrated, using com- caine, heroin, and methadone. Phencyclidine was unresolved bined GLC/MS, that cannabinoids were absent from genus from methadone in all four solvent/column combinations. Humulus (hops), the only other member of the family Can- nabaceae. A number of colour tests with various degrees of However, partial resolution was obtained using gradient elu- tion with a solvent mixture of ethanol, dioxan, cyciohexylamine, specificity have been developed; this topic has been reviewed hexane on Corasil !1. Phencyclidine was detected by UV ab- by Crombie (264). Certain of these tests, when performed by sorption at 254 nm, and response of the detector was linear at an experienced analyst, can identify many samples with little least over the range 2 to 20 p.g phencyclidine, uncertainty. Confirmation of the presence of Cannabis products Trinler et al. (260) also used reverse phase HPLC can thus be achieved by some combination of visual examina- (Phenyl/Corasil) with acetonilrile, water (50/50) and separated tion, colour tests, and chromatography. Useful discussions on phencyclidine from several amphetamines and caffeine. Lurie the identity of Cannabis products have been presented by Courts (8 i ) included phencyclidine and TCP in a system for separating and Jones (265), McBay (266), and Hughes and Warner (267). hallucinogens by ion pair reverse-phase HPLC (see above); TCP The major psychoactive constituent in Cannabis is was unresolved from LSD. Phencyclidine was included in the (-)-A+-tetrahydrocannabinol. The A'-compound has also been found, but many papers on the determination of cannabinoids comprehensive list of drugs studied by Baker et el. (84) (see above). Using aqueous metimnol at pH 7 and a #-Bondapak do not specifically discuss one or both compounds, referring C,, column, phencyclidine was resolved from all compounds to them collectively as THC. The reviewers are therefore obliged studied except pentobarbital. On #-Porasil, with methanol, 2N to follow this procedure and have only been able to draw a ammonium hydroxide, IN ammonium nitrate (27/2/I), phen- distinction between the isomers in a few cases. There is clearly cyclidine was unresolved from methadone, The absorbance a need for authors to carefully define the cannabinoids includ- !+ ratios of phencyclidine were different from the co-eluted ed in the study, particularly when the use of trivial abbrevia- :i compounds, tions is widespread. It is not within the remit of this review to discuss nomenclature, but attention is drawn to a recent discus- The reviewers have been unable to locate any publications describing separations of the many analogues of phencyclidine, sion on this topic in which the name "hempan" was offered for the basic cannabinoid structure (268). Using IUPAC rules, the major psychoactive tetrahydrocannabinol would become 1,3,4a(10b),9-hempantetraen-l.ol. Table XVIII. References to the HPLC of Phencyclidine and Analogues Thin Layer Chromatography

Reference 70 73 81 84 145 260 TLC extends the colour test concept by separating and PCC* visualising the components as different coloured spots using an t PCE appropriate spray reagent to give a characteristic pattern, it is PCX important to differentiate between the use of TLC for identi- i Phencyclidine X X X X X X + PHP fying a sample of a Cannabis product and the application of TCP X TI.C in further studies, such as comparison with other samples. Otherphencyclidine For the former application, almost any system which separates analogues the principal cannabinoids may be used. For further study more "see Llstof Abbtev+ation.s specific systems must be applied. Extraction of samples, usually with light petroleum or chloroform prior to Tt,C, is performed at room temperature to minimise decomposition of the cannabinoid acids that are thermally labile (269,270). Many different TLC systems have Cannabis Products been devised, and brief reviews have been presented t (264,271,272). The systems fall into three main categories in The majority of forensic examinations of Cannabis products which: (i) the absorbent is modified by the addition of a base, i!!! are carried out in order to establish the identity of a suspect either to the plate prior to development or to the developing q. I , 314 Journal of Chromatographic Science, Vol. 20, July, 1982

solvenl; (it) the absorbent is modified by addition of a sah or sulphanilic acid, and Fast Blue B gave similar limits of detec- t a buffer during preparation of the plate; (iii) normal absorp- lion (0.1/,,g for THC). These authors prepared pure standards l lion TLC is employed. Early workers tended to use one or the by column chromatography and purity was checkedby GLC. :i other of the first two categories, but the advent of commer- De Faubert Maunder (286) used toluene, diethylamine (98/2) cially prepared TLC plates and sheets with minimal batch-to- as the second solvent for a two-dimensional TLC system on silica batch variations meant that time-consuming processes of plate gel sheets and concluded that solvent systems that contained preparation and pre-washing could be eliminated. Consequently, diethylamine were unsuitable for confirming the presence of the most workers now use normal absorption TLC. Visualisation major cannabinoids, because the spots tended to streak and of cannabinoids after development of the plates is by a variety overlap. Grlic (287) dipped silica gel sheets in diethylamine, of spray reagents, most of which have been adapted from the which were then air-dried and developed in toluene. The system colour test reagents. Korte and Sieper (273) impregnated silica separated CBD, THC, and CBN well, and the sheets were gel plates with DMF and used hexane for development. Separa- reported to be usable for several weeks after the initial amine lion of several cannabinoids, which were assigned as cannabidiol treatment. 50 ng of THC was detected with Fast Blue B. (CBD), cannbinol (CBN), and three isomers of THC was The most common absorbent used for normal TLC of can- reported. The system, with minor modifications, has been widely nabinoids has been silica gel: development solvents have, in the used and studied. However, in a later publication (274), this main, been aromatic or aliphatic hydrocarbons with a small same author identified tile three "isomers" as A_-THC and two amount of a polar solvent such as ethyl acetate or diethyl ether. other cannabinoids. Merkus (275) compared this system with Parker and Fiske (272) have reviewed many of these systems. several others and considered that it provided good separations Machata (288) developed commercially-prepared Kiesegel plates and reliable results. In a second paper (276), this author also in light petroleum, diethyl ether (4/I) and separated CBD, THC, indicated that the two "isomers" were other cannabinoids. CBN, and cannabidiolic acid (CBDA). Fast Blue B and TCBI Several authors have identified problems with this system: were used as visualising agents, but no sensitivity figures were Aramaki et aL (277) found that the cannbinoid Rf values were given. De Faubert Maunder (289) considered that modified ab- affected by the moisture content of the DMF, and Nielson (278) sorbents and multi-component solvent systems were inconve- found that Rf values were affected by ambient humidity. Verwey n[ent for rapid routine use such as is required in a forensic and Witte (279) found that although the system was useful for laboratory. He devised a system using toluene and silica gel the separation of neutral cannabinoids, some information was sheets and resolved CBD, THC, CBN, cannabigerol (CBG), and lost because of the poor separation of the acidic cannabinoids, cannabichromene (CBCh). (The last two were, at that time, Korteand Sieper (273) used several visualising reagents including unidentified). The system did not resolve THC from Ihe Beam, Duquenois, and Ghamrawy reagents, which gave tetrahydrocannabivarin (THV) or CBN from cannabivarin detection limits between 0.5 and I/_g of THC. They found that (CBV). The cannabinoid acids ran as a streak near the baseline, a basic sohtlion of Fast Blue B (3,3'-dimethoxybiphe- This author emphasized the importance of small spot volume nyl-4,4 '-bisdiazonium chloride) was the most sensitive, detec- (< 0.5 /LI) and diameter (< 0.2 cm) and pointed out that loss ting 10 ng of CBD, THC, and CBN. In view of the possibility of resolution occurred if the plates were overloaded. Fast Blue of erroneous assignment of spots using this solvent system, these B was used for visualisation, but no detection limits were figures may be in error. Betts& Holloway (280) using a similar quoted. In a further discussion of TLC systems (290), the same TLC system, compared a number of spray reagents and found author cited this simple system as the only one suitable for con- Fast Blue B to be the most sensitive. Miras et ai. (281) obtain- firming the absence of CBD in cannabis samples. It is unfor- ed good separation of CBD, THC, and CBN. They were tunate that these TLC sheets (Kodak-Chromagram) can no visualised with TCBI, but the sensitivity of this reagent was not longer be used for this application, since a change in the binder reported. Caddy and Fish (282) prepared TLC plates modified has resulted in considerable loss of resolution. Verwey and Witte with (i) citric acid, disodium hydrogen phosphate, and (it) (279) using the published hexane, diethyl ether (4/1) system (291) i_ phthalate buffer, silver nitrate. The former plates were studied the variations in the amounts of the cannabinoid acids _:!i developed in chlorotorm, 0.5_0 ethanol in light petroleum (b.pt. in samples of different geographical origins. Good resolution i_ 40 to 60°C) (I/I), but this did not resolve THC from CBN. The of CBDA, tetrahydrocannabinolic acid (THCA), and can- ' latter plates were developed in chloroform, !.5_70ethanol in light nabinolic acid (CBNA), and separation of CBD, Ag-THC and :i petroleum (b.pt. 40 to 60°C) (I/!), and good resolution was CBN were achieved; however, THV was not resolved from obtained between CBD, THC, and CBN; Fast Blue B was used A_-THC and CBV was not resolved from CBN. These authors for visualisation. Other silver nitrate impregnated plate systems noted the decarboxylation of TI-ICA after heating at 150°C on have been described by Turk et al. (283), Hively et al. (284), a TLC plate. Although differences in cannabinoid patterns in and Patterson and Stevens (285). Silver nitrate impregnated samples from different sources were discernible, the reviewers i_ plates suffer from deterioration in light and require special consider that the lack of resolution of the cannabivarins from !i handling. In addition, it is a feature of this and many other the C-5 cannabinoids is a serious limitation when TLC patterns early TLC systems that little mention was made of either the are used to correlate country of origin with cannabinoid con- cannabinoid acids or the propyl analogues of the cannabinoids tent. As an alternative approach, Parker and Fiske (272) pro- (the cannabivarins). Examination of the published posed the use of wedge-shaped TLC plates to resolve com- chromatograms casts doubt as to the purity, and in many cases ponents of low Rf, but in the reviewers' opinion, a return to the identity, of the cannabinoid standards, laboratory-prepared plates would be a retrograde step for Early solvent systems developed for use with unmodified TLC routine analysis. These authors also suggested two simple plates often incorporated a base. Aramaki et al. (277) separated systems, both using silica gel plates for the rapid identification CBD, THC, and CBN on silica gel developed in benzene, hex- of Cannabis products, the first developed in chloroform, the ane, diethylamine (25/10/i). These authors examined a number second in pentane, dioxan (9/1). Resolution of CBD, A'-THC of spray reagents and found that diazotised benzidine, diazotised and CBN was acceptable, but no mention was made of the can- Journal of Chromatographic Science, Vol. 20, July, 1982

Table XlX. References to the TLC of Cannabinoids Reference 27 29 94 121265 267 270 272 273 274 275 276 277 278 279 280 281 282 283 284 286 287 288289 290291 293294 296 CBCh" X X X X X CBChA CBCy CBD X X X X X X X X X X X X X X X X X X X X X X X X X X X X CBDA X X X X X X X X X X CBG X X X X X CBN X X X X X X X X X X X X X X X X X X X X X X X X X X X X CBNA X X X CBV X X X X THe X X X X X X X X X X X X X X X X X X X X X X X X X X X X THCA X X THV X X X X THVA Othercannabinoids X X X I

Relerence 297 298 300 301 303 304 313 322 331 343 zoylamino-2,5-diethoxybenzcnediazoniumchloride)was found CBCh X X X X X to be superior to Fast Blue B, both in sensitivity and improved CBChA colour, although it wasslightly slower in response. De Faubert CBCy X X X X Maunder recommended that Fast Blue BB should replace Fast CBDA X X X X X X X X X Blue B in Cannabis analysis, for thesereasons, and alsobecause CBG X X X X X X of the potential hazard of unreacted o-dianisidine (3,3 '-dime- CBN X X X X X X X X thoxybenzidine) in Fast Blue B. It wasalso recommended that CBNA X the plate should be sprayed with diethylamine both before and THCCBV X X X X X X X X XX after chromogen spraying for best preservation of THCA X chromatograms. Hughes and Kessler (296) demonstrated that THV X X X X Fast Blue BB is also more specific toward cannabinoids than THVA X Fast Blue B. Othercannabin0ids X X X Krejci and Hanus (297) studied the TLC of cannabinoid acids •soe L,mofAbbrevations after their extraction into basic solution and methylation. The resulting esters were separated on silica gel in hexane, dioxan, methanol (7/2/1). Visualisation was with diazotised benzidine. nabivarins or the cannabinoid acids. De Zeeuw et al. (270) com- This is now regarded as a hazardous reagent and should not pared the systems of Macha/a (288); Korte and Sieper's DMF be employed. These authors identified CBDA, THCA, and impregnated plates (273) developed in hexane, dioxan (3/1) in CBNA and made tentative identification of other components.

ter (276). In all systems, considerable overlap between can- nabinoids in smoke. Two TLC systems were employed: neutral nabivarins and the C-5 cannabinoids occured. De Zeeuw et al. silica gel sheets developed in toluene, diethylamine (19/1), for lili andidanolkalint reproduce atmospe hMerkus'sere; and sepaMerkrautions's mofodifiTHcVatioandn ofCBNthe andiat- cannabisDahiya andsmokeJain products,(298) usedandTLCalkalinefor thesilicaidentifigelcationplates ofin chan-ex- did not mention the cannabinoid acids. It is not clear whether ane, ethyl acetate (9/I) for cannabis resin smoke products. Many a single development of DMF-impregnated plates in cyclohex- compounds were separated but only three (CBD, THC, CBN) ane was used; bul if so, this may account for the lack of resolu- were identified. No reason was given for the use of two dif- tion obtained in comparison to that of Merkus, who developed ferent solvent mixtures. Rao (299) proposed the use of his plates three times in cyclohexane. De Zeeuw et al. (292) l-nitroso-2-naphthol as a chromogen for Cannabis products. reported the presence of alkanes in Cannabis, but pointed out From a cannabis sample, five components were separated; three their presence did not affect TLC, as they were non-polar and gave a blue colour (changing to violet after 2 hr), and two gave did not react with Fast Blue B. In two very similar papers a grey-blue colour when sprayed with this reagent. The com-

b(2e9n3,2t 9(alu4), mTewarina, i calciuet alm. descrsulpibedhate,a sys22tem/3) inwaswhicdevelopedh the absorin- Mobarakponents weelreaLnot(300)idenusedtifiedt,wo-dimensionaland no detectionTLClimforits thwere eanalysisgiven. 1 chloroform, benzene (1/1). The THC standard was prepared of a single sample of cannabis resin. Silica gel plates were us- ] according to Turk (283) and gave three spots identified by ed, the developing solvents being (i) pentane, diethyl ether (9/I), f Tewari et al. as "THC 1," "THC li," and "THC Ill." Turk and (it) pentane, acetone (9/1). Two spray reagents were used: records his sample as being a single major component con- 2-hydrazono-2,3-dihydro-3-methylbenzothiazole hydrochloride taminated with CBD and CBN. The reviewers consider that the (HMBT), followed by ammonium eerie sulphate in dilute colours developed by the cannabinoids after Fast Blue B treat- sulphuric acid and Fast Blue B. Good separation of many com- ment were rather unusual, and these authors appear to be rely- ponents was achieved. The authors noted that HMBT was less ing upon identifications similar to those of Korte and Sieper sensitive than Fast Blue B. The use of HMBT did enable the (273). De Faubert Maunder (295) discussed the preservation of diols (CBD and cannabidivarin, CBDV) to be distinguished, thin layer chromatograms, an important consideration when although no information was given on any visual differences. these may have to be produced in evidence long after analysis. Baggi (301) suggested the use of HMBT as a new reagent for This author identified the main parameters that affected col- the visualisation of cannabinoids in view of the possible hazard our stability as chromogen basicity, the actual dyestuff and of Fast Blue B (302). This author suggested that no progress residual solvent at the time of spraying. Fast Blue BB (4-ben- had been made in seeking a replacement for Fast Blue B, but

316 Journal of Chromatographic Science, Vol. 20, July, 1982

appeared to have overlooked the work of de Faubert Maunder marion is lost during the GLC analysis of Cannabis extracts, (295) on Fast Blue BB and of Mobarak et al. on HMBT itself as the cannabinoid acids are decarboxylated, upon injection, (300). Their procedure for colour development is long and to the corresponding cannabinoids (267,270). Various derivatisa- tedious compared to Mobarak et al. and in the reviewer's opt- lion techniques have been described to overcome this problem, nion. is not suitable for routine rapid use. Furthermore, the but none are completely satisfactory. Detection of cannabinoids detection limit (5 #g THC) and the developed colours (varia- after GLC is almost invariably by FID. GLC/MS has been us- lions on brown) render it of limited valne in Cannabis product ed to identify many components present in Cannabis products analysis. (305,306) including non-cannabinoids, but this technique finds Fowler et al. (303) had observed the problems associated with few applications in the routine forensic analysis of these the system of de Faubert Maunder (289): these authors used materials. GLC/MS is not usually available for routine drug silica gel plates developed in alcohol-free chloroform, analysis, and simpler techniques should be sought. It does, of 1,l-dichlorethane (15/I 0) and separated CBD, A_-THC, CBN, course, have considerable application to toxicology because of _. THV, CBV, CBG, and CBCh from the cannabinoid acids, its selectivity. ,which ran as a streak near the baseline. The relative amounts The efficiency of extraction of cannabinoids from a Cannabis of CBD, Ag-THC and THV vary widely with the geographical product and the stability of the resulting extract are two mat- origin of the sample and consequently this system has been us- ters of concern to the forensic analyst: Both these problems have ed in a study which related the cannabinoid TLC patternstothe been studied in considerable detail by GLC. Fairbairn and Lieb- geographical origin of the sample (304). Fowler et al. (303) mann (307) reported that 88 to 94070of THC was recovered from also devised a two-dimensional TLC system for preliminary a sample of cannabis in a single extraction by shaking and 98 comparison of Cannabis products prior to more detailed ex- to 99070 with a double extraction using light petroleum. With amination by HPLC. Following the first development, the plate chloroform, almost quantitative extraction of THC from both was sprayed with diethylaminc and then developed in xylene, cannabis and cannbis resin was obtained. Ethanol was regard- dioxan (19/I). After visualisation with Fast Blue BB, some 15 ed as unsuitable because co-extracted non-cannabinoid com- to 2(1 components were observed. Modification of this sytem pounds may interfere with the chromatography. Turner et al. by heating for 5 rain at 150°C between developments allowed ('308), in a survey of different solvents, also found chloroform the acids to be studied qualitatively as their decarboxylated pro- to be the most satisfactory for extracting THC from plant ducts. As the acids are decarboxylated during smoking, an in- material. Both these reports cover in some detail the problems dication of the "quality" of the Cannabis product being ex- associated with previously reported extraction systems. Turner amined can be obtained, et al. (308) used a modification of Lerner's (309) extraction pro- It is clear that there is no single TLC system that will resolve cedure: After chloroform extraction, the extract was dried in all the major cannabinoids and their corresponding acids, it vacuo, dissolved in ethanol and filtered prior to GLC analysis. is therefore necessary to use a TLC system that achieves the The authors stated that the chloroform extracts were stable for aim of the analyst with the minimal use of materials and time. at least 6 days. Parker et al. (310) found that CBD was unstable The reviewers consider that simple absorption TLC using corn- in chloroform even in the dark. After one day there was ap- mercially prepared plates provides confirmation of the presence proximately 10070loss, while, solutions in ethanol were essen- of cannabinoids within a sample, provided the following con- tiaUy stable over 8 days. Turner and Henry (311) attributed pro- ditions are met: (i) that the system separates at least the major blems of instability to impurities in chloroform and conceded cannabinoids, CBD, Ag-THC and CBN; (ii) that an authentic that cannabinoids should not be stored in chloroform for ex- reference sample, such as Tincture of Cannabis BPC 1949, is tended periods. Fairbairn et aL (312) studied solutions of pure used during each analysis, a view expressed by Parker and Fiske cannabinoids, cannabis, and cannabis resin extracts. Exposure (272) and Coults and Jones (265); and (iii) that a selective to light was the greatest single factor affecting their stability, visualising agent is used. and they should be protected from light at all times. These There are many TLC systems that fulfill the first condition, authors confirmed the results of Parker et aL (310) on the stabili- and it is largely a matter of personal choice and experience which ty of ethanolic solutions, provided these were kept in the dark. system is chosen. Fast Blue B has been the most widely used Kubena et al. (313) examined an old cannabis extract, from chromogen, but in view of the potential hazard and the possibili- which he concluded that A'-THC was stable in ethanol. Fair- ty of its manufacture being banned in the USA, Fast Blue BB bairn et al, (312), in an examination of this evidence considered should be used as a visualising agent. More detailed knowledge it unlikely that more than 10070of the original A'-THC remain- of cannabinoid acid or cannabivarins content may also be ob- ed unchanged. rained using an appropriate TLC or two-dimensional TLC Narayanaswami et al. (314) also found that considerable system. Two-dimensional TLC is a valuable screening system degradation occurred on storage of alcoholic extracts over ex- for samples prior to detailed comparative analysis, and the tended periods. Many other extraction systems have been used systems of Fowler et al. (303) or Mobarak et al. (300) are useful prior to GLC: Coutts and Jones (265) and Mobarak (300) used for this purpose, hexane, Novotny et aL (315) used cyclohexane and soxhlet ¢x, GulLiquid Chromatography ' .. traction, and Harvey (305) used ethyl acetate. Bailey (316) and Repetto et al. (317) used hexane with soxhlet extraction. Baker In contrast to TLC, GLC has been little used in the identifica- et ai. (318) used ethanol and soxhlet extraction for l hr for GLC lion of Cannabis products. Applications of GLC to the foren- analysis or methanol, chloroform (4/1) with ultrasonic vibra- sic examination of Cannabis" products include (i) accurate quan- tion for 10 min if analysis of the cannabinoid acids by HPLC titation of a particular cannabinoid (usually A'-THC), and (ii) was required (319). No problem with ballast substances was en- determination of the relative amounts of all the separated can- countered using either of the solvents: GLC column life was nabinoids. This information has been used to compare samples not noticeably reduced, and interfering peaks were not observ- and tentatively assign origins. However, much valuable infor- ed in the chromatogram. Both these extractions were found to

317 Journal ol Chromatographic Science, Vol. 20, July, 1982

be quanlilative and repl'odt,cible. These anthors found it nabjvarins. File reviewers consider thai these compounds are necessary to gri_td cannabis resins, parlicularly those I'ronl tile unsttilable Ior GI.L' o1 _tnnabis products and that an internal Indian sub-continent, as non-quantitative extraction was other- standard is not norrnally necessary, provided an injection wise obtained; this result was in contrasl 1o Fairbairn & Lieb- volume of a reasonable size (5 _1) is used. mann (307), who found it unnecessary to grind resins prior to Turner et al. (327) recommended glass columns for GLC extraction. In terms of extraction efficiency, the reviewers con- analysis of cannabinoids: Steel columns were found to cause sider thai there is liltlc It) choose belween tile many procedures losses of up to 30%. Silanisation of steel columns partially for exlraction prior to GI.C, especially those where chloroform reduced this, bul losses of 15%0were still recorded (308). or alcohols (or mixtures) are used. Chloroform has been shown 'The wide range of stationary phases and supports used for to be an excellent solvent, but may have deleterious effects on the (;LC analysis of Cannabis products Ires been listed (264,328) the FID. The safety of chloroform for general use is also now and reviewed briefly (328,329). Although many early workers open to question (320). The reviewers therefore consider that used non-polar stationary phases such as SE-30, more recent alcoholic extraction should be used wherever possible prior to work has been carried out on semi-polar phases, the most com- GI.C and that all solutions should be stored in the dark forthe mon being OV-17. This is usually at a 2% loading on minimum time possible prior to analysis. Chromosorb W AW-DMCS (279,307,309,324,330-334) or 3% Rasmussen et al. (321)used direct micro-solid injection to on Gas Chrom Q (318,321,325,329,335-340). OV-17 will minintise contamination with compounds normally co-extracted separate CBDV, THV, CBV, CBD, Ag-THC and CBN, and has with cannabinoids. These authors claimed that Img of plant been recommended as a stationary phase for general drug material could be identified in this way. If a A'-THC content analysis (218). Fish (328) preferred to use a column of in- of 20/0 is assumed for a cannabis sample, this corresponds to termediate polarity such as XE-60 (4W0 on Chromosorb W or a detection limit of 20 t,tg A_-THC, far inferior to TLC using 1%oon Chromosorb G) and obtained good and rapid separa- Fast Blue B as visualising agent. 1"his method of detection has lion with sharp peaks. This author also obtained good results no advantage over a simple -I'LC; it is less specific and )nay not with 1% CDMS, a phase used successfully by several groups give results representative of the whole sample. In an extension (285,328,341). Hoffman and Yang (342) demonstrated that, of the original system, Rasmussen and Aaro (322) removed wlien supports coated with 3% or less of several silicone phases samples from TL.C plates and transferred them to the heated were used for the Gt+C analysis of small quantities of A+-THC, injection port of the chromatograptt. Volatiles were trapped on substantial and variable losses were observed. These authors the top of the cold column which was subsequently heated to recommended that a minimum of 5% of either OV-i or OV-210 release the canuabinoids. Confirmation of identity was made be used for the trace analysis of A'-THC. Although these losses by on-column silanisation. The detection limit for THC was are not significant when larger amount of ,_'-THC are I p.g. Again confirmation at this level would be better done by chromatographed, confirmation of the presence of trace TI.C (on more than one system if required) and at lower levels amounts of cannabinoids is often made by GLC/MS, and ap- by GLC/MS. Fairlie and Fox (323) used a similar process to propriately loaded columns should be used for that technique. determine the A'-THC content of small samples; they found Although there are no reports in the literature of other corn- that when I to 4 mg of cannabis was analysed, THC conteut pounds co-eluting with z_9-THC, and therefore giving erroneous could be determined with an accuracy of better than 5%. results, de Zeeuw et al. (292) observed two long chain n-alkanes Although this appears to be a successful system, it is of limited (C,, and C:,) in cannabis: Other n-alkanes, from C,_ to C,, ii forensic application in view of the problems of representative were also detected in smaller quantities. The authors staled that sampling at this level, interference by alkanes was likely to be minimal, but recom- il There has been considerable discussion in the literature on mended that they should be removed by column the use of internal or external standards when quantitation of chromatography. This procedure has not been used by other a cannabinoid is required. Lerner (309) considered the external workers and is more likely to introduce quantitative errors by standard technique to be inherently imprecise, as it requires corn- loss of cannabinoids than is the presence of low levels of n- plete reproducibility of injection volumes and detector response, alkanes. Eskes et al. (333) did not report any interference Lerner and Zeffert (309,324) obtained a precision of 13% for resulting from the presence of opium in Cannabis products. A_-TttC using an external standard. With an internal standard Bailey (316) observed the formation of small quantities of (methadone), a precision of !.1_0 was obtained. Fairbairn et olivetol from CBD at 150°C, and Turner and Hadley (343) al. (307) used androst-4-ene-3,17-dione, which was recommend- observed the cyclisation of CBCh to cannabicyclol (CBCI), ed by Fetterman el al. (325) as an internal standard and ob- Although such heat-induced changes may be important when rained a precision of 1.4%. This compound is eluted much later minor components or trace quantities of cannabinoids are be- than the principal cannabinoids on silicone stationary phases, ing analysed, they are unlikely to be significant in the routine ' Chiesa et al. (326) used '._-THC as an external standard and forensic context. Of major significance is the decarboxylation obtained a precision of 2.9%. it was pointed out that the of cannabinoid acids on injection into the GLC (267,270). As presence of numerous peaks in the chromatograms, resulting a result, the THC content is the sum of the original THC plus from the GLC analysis of some samples of cannabis, may make THC resulting from the decarboxylation (3 i 8). It is sometimes the use of internal standards an inherently unsafe process, necessary to differentiate between "total" THC and original Crombie (264) considered that peak area integration relative to THC in a sample. Methods have been discussed by Baker et internal standards was best for quantitative GLC and listed the al. (344) and are further described under HPLC of Cannabis. large number of compounds used for this purpose in Cannabis To prevent decarboxylation of the cannabinoid acids and product analysis. A number of these internal standards, par- therefore retain information for comparison purposes, ticutarly those used by early workers, e.g., cocaine hydrochloride derivatives which are stable and may be chromatographed have (277) and methadone (309), are eluted prior to the principal can- been prepared: Crombie (264) has listed many of these. nabinoids and may have similar retention values to the can- Particular problems arise when silyl derivatives are used (345), i 318 Journal of Chromatographic Science, Vol. 20, July, 1982

and these have been discussedby "lurner and Hadley (346), Con- with a high sugar content. Extraction of the cannabinoids with lamination of FIDs, when highly silylated extracts are routine- a non-polar solvent is necessary to prevent contamination of ly analysed over long periods, is a serious drawback. Other the GI.C column. derivatives suffer many of the problems associated with silyl 11is often necessary to confirm the presence of traces of Can- derivatives, and the varied nature of Cannabisproducls makes nabis, for example in personal effects. One approach is to [ reliable formation of derivatives difficult. Consequently, most prepare electron-capturing derivatives (330,353), although recent work on comparalive Cannabis analysis has been car- (;L.C/MS of underivatised cannabinoids is preferable for unam- ried out by HPLC. Apart from GLC of Camrabis extracts biguous detection (provided the work of Hoffman & Yang (342) J (directly or after derivatisation), several other procedures have is noted, i been suggested for comparative analysis: These include capillary in a comparative study of different methods of Cannabis i GL(? (315), analysis of minor components (347), and headspace analysis, Courts and Jones (265) concluded that GLC was not :i , analysis of volatile constituents (348); but these have not found necessary for identification purposes and added nothing to [L wide application. Attempts have been made to correlate can- microscopy, colour tests, and TLC. GLC finds limited foren- I

, 'Ihis has beet] discussed by Baker el al. (304,318,344), although in comparative analysis. Most cannabinoids, apart from the in the main these melhods have had little success, itood et al. acids, do, however, possess satisfactory GLC properties and nabinoid patterns derived from GLC with geographical origin, sic application in quantitative cannbinoid analysis and few uses i!l! (348,349) observed uo correlation between tile preseqce of can be separated on phenyl-methyl- or dimethyl-silicone phases. volatile constituents and sample origin. Comparison of the can- GLC is particularly useful in obtaining total THC contents of !' nabinoid patterns of a Cannabis product with that of the samples. [ distillateresultingfrom smokingindicatedthat only limited t changes take place during smoking (350,352). In this case, decar- High Performance Liquid Chromatography :_i boxylation on-coh, mn is an advantage, as the distillate would There have been few papers published of the application of i1, be ilself decarboxylated on smoking, in a similar manner, the HPLC to the analysis of Cannabis products. HPLC has little !! acids in hash-oil samples are decarboxylated during tile prepara- or nothing to add to their identification, but has found use in ! tion of" this product. When comparing hash-oil samples with Ihe comparison of these materials and in the study of the can- either cannabis resins (from which they may have been prepared) nabinoid acids as naturally present in a sample. Wheals and or with other "hash-oil" samples, GLC can be of greater value Smith (354) prepared their own reverse-phase column pack- than HPLC. GLC has also been used to determine the total ing by reacting Partisil 5 (5-v.m silica) with oc- THC content in illicit rums from the West Indies, which con- tadecyltrichlorosilane: The mobile phase was methanol, 0.02N rain small amounts of cannabinoids in aqueous ethanol, often sutphuric acid (4/1). Detection was by ultraviolet absorption

Table XX. References to the GLC of Cannabinoids

Reference 121 141 180265 269 270 277 280 285 292 300 305 306 307 308 309 310311 312313 314 315 316 317 318 319 321 322 323 CBCh" X X X X X X X CBChA CBCy X X X X X CBD X X X X × X X X X X X X X X X X X X X X X X CBDA X CBG X X X X X X CBN X X X X X X X X X X X X X X X X X X X X CBNA CBV X X X X X X THC X X X X X X X X X X X X X X X X X X X X X X X X X X ' THCA X X THV X X X X X THVA X Other cannabinoids X X X X X X X

Reference 324325 326 327 330331 332333 334 336 337 338 339 340 341 342 343 344 345 346 347 348 350 351 352 353 355 358 362 CBCh X X X X X X X CBChA . X CBCy X X X X X X X CBD X X X X X × X X X X X X X X X X X X X X X X CBDA X X X X X CBG X X X X X X X CBN X X X × X X X X X X X X X X X X X X X X X CBNA × X CBV X X X X X THC X X X X X X X X X X X X X X X X X X X X X X X X X X X X X THCA X X X X THV X X X X X X THVA Other cannabinoids X X X X X X X X X X X X X "Soe LISI OJ Abbrewallons. ii ] Journal of Chromatographic Science, Vol. 20, July, 1982

at 254 nm. Eight major peaks were detected, but the compounds only ,_V-THC, A'-THC, and CBD were identified. The detec- were not, at thai time, identified. In a study of the use of this tion lintit for THC was 2 ng. The authors examined a _%THC system to distinguish different samples of Cannabis products, standard, two "street" samples of cannabis and one of can- 34 different samples were examined by TLC, GLC, and itPLC, nabis resin, and a ten-year-old sample of Mexican cannabis. HPi_.C was able to classify these into 30 separate batches: By The "street" samples of cannabis were reported to be devoid i contrast, GLC classified them in 25 batches and TLC, only l I of ,..k"-Tl-t(..?and the Mexican sample to contain 10°/0 A%THC. batches. None of the samples indistinguishable by HPLC could This is surprising, because cannabis sample that are several years be distinguished by GLC or "EL('. Aging of the extract, ,,vcr old normally contain little or no A%THC and considerable a few days did not affect the I-tPI.C results, amounts of CBN as the major breakdown product. Smith (355) identified many of the constituents separated by Although sensitivity is an important attribute when HPLC i the above system; the principal peaks were attributed to CBD, is being used for toxicological analysis, the reviewers do not CBDA, CBN, A%THC, CBCh, CBNA, A*-THCA, and can- consider that there is any need for a more sensitive technique nabichromenic acid (CBChA). Identification was by preparative than UV absorption in the routine HPLC analysis of Cannabis HPLC (from the analytical column) followed by GLC/MS, or products. Kanter et al. (361) determined A_oTHC and A_-THCA by retention coincidence with authentic compounds. The acidic contents as part of a study to define optimum conditions of cannabinoids were separated by fractionation into alkaline solu- A%THC production from A%THCA by thermal degradation. tion and identified by GLC/MS. Detection was at 254 nm, Extracts were subjected to HPLC before and after heating. A although the authors noted that a lower wavelength (210 to 220 Sperisorb $5 (5-/_m silica) column and a mobile phase of hep- i nm) is preferable for THC itself. Smith and Vaughan (356) utilis- tane, 1°7o2-propanol in heptane (70/30), with UV detection at ed this HPLC system for the quantitative analysis of acidic and 215 nm, was used. The authors recommended a pre-column to : neutral cannabinoids. Quantitation of neutral cannbinoids was prevent contamination of the analytical column, but observed by comparison with authentic samples, while cannabinoid acids that tiffs may result in broad elution profiles. This is a useful were quantified indirectly by a separative step followed by technique for determining A%THCA and A%THC contents when HPLC and GLC. Partially resolved and unresolved components GLC is not available (as was the case for Kanter et aLL but were quantified either by varying the column temperature and sin'auhaneous GLC and HPLC analyses of the same extract is hence increasing column resolution, or by using two detectors a preferable approach. Baker et al. (344) used HPLC and GLC in series, operating at different wavelengths (220 nm, 254 rim). to study the amounts of A'-THC and A'oTHCA in Cannabis Precision was measured on 23 replicate injections of a single products of different geographical origin. A wide range of extract from one resin, by I0 extracts from 10different samples relative and absolute amounts of these two components was of a similar resin, and in both series of analyses was between found in samples of both cannabis and cannabis resin. The 2 to 3_0 (except for CBN in the former series, which was bet- authors considered that combination of this information with ween 4 to 6%). These results also indicated that the resin was other data such as TLC patterns is useful in assigning of a uniform nature. These authors suggested that, as A'-THC, geographical origin. Ag-THCA, CBD. and CBDA were of prime interest, 220 nm Baker et al. (319) used a Spherisorb S5ODS (ODS-silica) col- " was the most appropriate detector wavelength. No long-term umn and 0.02N sulphuric acid, methanol, acetonitrile (7/8/9) stability problems associated with the HPLC itself were as eluant to separate the major cannabinoids, which were iden- reported, tiffed either by comparisonwith authenticsamplesor by MS In a second study (357), the same authors measured the ex- after preparative HPLC. Extraction, by ultrasonic agitation into traction efficiencies and the stabilities of eannabinoids in methanol, chloroform (4/1), was shown to be essentially quan- methanol, chloroform, ligh! petroleum (b.pt. 40 to 60°C) and titative. Detection was by UV absorption at 220 nm in order '. methanol, chloroform (9/1). Methanol was the more effective, that the cannabinoids of major importance could be detected except for the extraction of CBCh and CBChA: This is in con- with adequate sensitivity. CBD, CBV, CBG, THV, CBDA, trast to the results of Turner et al. (308) (see above), and it was CBN, A'-THC, A_-THC, THVA, CBCy, CBCh, and THCA suggested that the alcohol used by Turner et al. may have con- were identified, although not all these components were fully rained water. Double extraction with chloroform, as suggested resolved. This system was applied to the comparison of can- by Fairbairn & Liebmann (307), was found satisfactory. Knaus nabis resin samples. Cannabis resins from Pakistan and el al. (358) separated CBD, A%THC, and CBN on a #-Bondapak Lebanon were shown to be homogeneous products with respect C,, packing using methanol, water (3/1) as mobile phase and to the distribution of cannabinoids within a single slab of resin, UV detection at 254 ran. The detection limit for THC was about although there were minor differences between the surface and 150 ng. Silyl and phosphate derivatives were also prepared for the interior of Pakistani slabs. In general, inter-slab coefficients HPLC, in order to minimise the risk of decarboxylation, of variation, in a given batch, were no higher than the correspon- isomerisation, and oxidation. However, there is no evidence that ding intra-slab values, indicating that resins, at least from these these effects are significant at room temperature. Although these two countries, are relatively uniform products. No two slabs, authors recognised the existence of the cannabinoids acids, they from 94 unrelated seizures from Pakistan, were found to have were not included in the study, coincident cannabinoid profiles: No correlation was found bet- Although most workers in this field have used UV absorp- ween the "identification" marks on some resins from Pakistan tion for detection of cannabinoids, other methods of detection and their cannabinoid content. The authors consider that, while I have been studied. Twitchett et al. (359) used post-column on- it would be preferable to fully resolve all the components of 1' line photochemical detection and detected 1ng of CBN. Masoud Cannabis products, the conditions described provide sufficient and Wingard (360) separated cannabinoids on a 10 #m information to distinguish resins from different batches, even i' Lichrosorb RP-18 (ODS-silica) with methanol, 0.05M when these are from the same geographical area. phosphoric acid (3/I) as eluant, and used an electrochemical An earlier study, by Smith and Vaughan (357), of the can- _ detector. Eleven constituents were separated and detected, but nabinoid prolile across a block of Middle-Eastern cannabis resin

: 320 Journal of Chromatographic Science, VoI. 20, July, 1982 i

showed that, in general, acidic cannabinoid concentrations in- List of Abbreviations _: creased towards the middle of the block, but that variations Abbreviation Full Name ',_ in the neutral cannabinoids were random, it is difficutl to com- pare results of these studies with that of Fairbairn (312), because AFID Alkali-flame ionisation detector (Thermionic I the latter used only GLC. detector) _i The primary application of HPLC to Cannabis analysis is Bromo-STP 4-bromo-2,5-dimethoxyamphetamine ._!i in comparison of samples. Two principal systems have been BSA N,O-bistrimethylsilylacetamide _i described (319,354), and both have been used to study varia- BSTFA bis-trimethylsilyl-2,2,2-trifluoroacetamid¢ _; tions in cannabinoid content of many different samples. Ex- CBCh cannabichromene traction with methanol, chloroform mixtures has been found CBChA cannabichromenic acid to be most suitable, as high extraction efficiencies are achieved CBCy cannbicyclol and good elution profiles result. The columns and eluting CBD cannabidiol solvents used by these two groups are similar. Wheals and Smith CBDA cannabidiolic acid (354) preferred detection at 254 nm, as more peaks were detected CBDV cannabidivarin in their extracts; while Baker et al. (319) found 220 nm to be CBG cannabigerol more sensitive for the detection of CBD, A'-THC, and THV. CBN cannabinol Smith (355) did not identify the cannabivarins in his CBNA cannabinolic acid chromatogram but, in a later publication with Vaughan (356), CBV cannabivarin he considered these to be only minor components of Cannabis DAB p-dimethylaminobenzaidehyde products. Baker et al. (304) and Field and Arndt (362) found DET N,N-diethyltryptamine significant amounts of cannabivarins in some samples of Can- DMF dimethylformamide nabis products. Apart from choice of wavelength, there seems DMT N,N-dimethyltryptamine .... to be little to choose between these two methods for comparison DPC diphenyicarbazone of samples. There appears to be no advantage in using other ECD electron capture detector detection systems for routine forensic purposes. Fast Blue B 3,3 '-dimethoxybiphenyi-4,4'-bisdiazonium chloride Fast Blue BB 4-benzoylamino-2,5-diethoxybenzene Table XXl. Reference to the HPLC of Cannabinoida diazonium chloride FID flame ionisation detector Ref_.earence 70 76 319344353355356357356359360361 GLC gas/liquid chromatography CSCh X X X X HFB- heptafluorobutyryl- CBChA X X HFBA heptafluorobutyricanhydride CCBCyBD X X X X X X X X HFB! N-hcptafluorobut yrylimidazol¢ CBDA X X X X HMBT 2-hydrazono-2,3..dihydro-3-methylbenzothiaz. CBG X X o1¢ hydrochloride CSN X X X X X X X X IR infra-red CBVCBNA X X X X LSD lysergide THC X X X X X X X X X X X lysergic acid diethylamide THCA X X X X X X MBTH see HMBT THV X MDA 3,4-methylenedioxyamphetamine THVA X M DM A 3,4-methylenedioxy-N-methylam phetamine Othercannabinoids X X X MS mass spectrometry MSTFA N-methyl-N-trimethylsilyi-t rifluoroacetamide MTPA oe-methoxy-ot-trifluoromethylphenylacetic acid The reviewers do not consider that an internal standard is NBD chloride 4-chloro-7-nitrobenzofurazan essential for the HPLC of extracts of cannabis products. With NMR nuclear magnetic resonance good chromatographic techniques, reproducible results can be ODS octadecylsilyl- obtained using external standards. Furthermore, comparisons PCC l-piperidinocyciohexanecarbonitrile between samples are normally based on relative, rather than PCE cyciohexamine absolute, amounts of cannabinoids. N-ethyl-l-phenylcyclohexylamine PCP phencyclidine L l-(l-phenylcyclohexyl)piperidine PCX l-piperidinocyclohexene PHP 1-(l-phenyicyclohexyl)pyrrolidine Acknowledgments SCOT support coated open tubular STP 2,5-dimethoxy-4-methylamphetamine We thank the Director of the Metropolitan Police Forensic TCBI N,2,6-trichloro-p-benzoquinone imine Science Laboratory, London, for providing the data on civil TCP thiophencyclidine police seizures quoted, in the Introduction and the Drug En- l-[l-(2-thienyl)cyclohexyllpiperidine forcement Administration for the US data quoted there. We TFA- trifluoroacetyi- also thank the Government Chemist for permission to publish TFAI N-trifluoroacetylimidazole this review. THC tetrahydrocannabinol

321

__ J Journal of Chromatographic Science, Vol. 20, July, 1982

THCA tetrahydrocannabinolic acid Table Xll. References to the HPLC of Amphetamines and THV tetrahydrocannabivarin Related Stimulants I! THVA tetrahydrocannabivarinic acid 'Fable XIII. References to the TLC of Barbiturates I TLC thin layer chromatography Table XIV. References to the GLC of Barbiturates ! TMS- trimethylsilyl- Table XV. References to the HPLC of Barbiturates TPC N-tritluoroacetyI-L-prolylchloride Table XVI. References to the TLC of Phencyclidine and TSI N-trimet hylsilylimidazole Analogues ii UK United Kingdom Table XVII. References to the GLC of Phencyclidine and I USA UnitedStatesof America Analogues UV ultra-violet Table XVIII. References to the HPLC of Phencyclidine and WCOT wall-coated open tubular Analogues ZPC N-benzyloxycarbonyl-k-prolyl chloride Table XIX. References to the TLC of Cannabinoids Table XX. References to the GLC of Cannabinoids Table XXI. References to the HPLC of Cannabinoids

List of GC StationaryPhases

Abbreviation Identification References

Carbowax 20M polyethylene glycol 1. B.S, Finkle and DM. Taylor. A GC/MS reference data system for the iden- CDMS cyclohexane dimethanol succinate tification of drugs of abuse. J. Chromatog. Sci. 10:312-33 (1972). DEGS diethylene glycol succinate 2. B.S. Finkle, R.L. Foltz, and D.M. Taylor. A comprehensive GC-MS Dexsil-300 carboraue-silicone reference data system for toxicological and biomedical purposes. J. Dexsil-400 carborane-silicone Chromatogr. Sci. 12:304-28 (1974). FFAP Free fatty acid phase • .3 .G,R. Nakamura. Chromatography of monoacetylmorphine and acelylco- deine. J. Forens. ScL 5:259.65 (1960). i i Igepal CO-880 nonylphenoxypoly(ethyleneoxy)ethanol 4. G.R. Nakamura and T. Ukita. A study of hydrolysis of heroin by paper , NPGS neopentylglycol succinate chromatography. J. Forens. Sci. 7:465-73 (1962). i i OV- I methyl silicone 5. H.V. Street. The paper chromatographic separation of codeine, morphine OV-7 20070phenyl, methyl silicone and nalorphine. J. Pharm. Pharmacol. 14:56-57 (1962). 6. e.G. Farmilo and K. Genest. In Toxicology. Vol. 2, C.P. Stewart and A. OV-17 50_'0 phenyl, methyl silicone Stolman, ads. Academic Press (London) 1961. OV-25 75 % phenyl, methyl silicone 7. T.W.M. Davis, C.G. Farmilo, and K. Ganest. Analysis of an impure heroin OV-61 33% phenyl, methyl silicone seizure. Bull. Narc, XlV(3): 41-57 (1962). OV-101 methyl silicone a.G.J. Herman and N-N. N. Ken. Identification of an atypical constituent OV-210 50070 trilluoropropyl, methyl silicone of a clandestine opiate. Biomed. Mass Spat. 1:350-51 (1974). 9, J,J. Manure, J-M Chad, and R. Saferstein, The forensic identification of OV-225 25070 phenyl, 25070cyanopropyl methyl heroin. J. Forens. Sci. 23:44-56 (fg78). Silicone 10. A.S. Curry and D.A. Patterson. A procedure for the analysis of illicit QF-I 50% trifluoropropyl, methyl silicone diamorphine samples. J. Pharm PharmacoL 22:198-201 (1970). SE-]0 methyl silicone 11. G.K Nakamura. Separation of acetylcodeine from illicit heroin by thin layer SE-52 5070phenyl, methyl silicone chromatography. J.A.O.A.C. 49:1086-090 (1966). 12. E.A. Davey, J.B. Murray, and AR. Rogers. The determination of diamor- SF-96 methyl silicone phine by thin layer chromatography and spectrophotometry. J. Pharm. SP-525 aromatic polymer (?) Pharmacol, 20: 51s-53s (supplement) (1968). SP-2250-BD 50% phenyl, methyl silicone 13. I.J. Holcomb, R.B. Luers and S.A. FusarL Chromatographic separation Thermol-3 phenetidine deriwttive (?) and assay of morphine in injoctables. J. Pharm. Sci. 62:1504-509 (1973). 14. S.Y. Yeh. Separation and identification of morphine, its metabolites and UCW-98 vinyl, methyl silicone congenersJ.. Pharm.Sci. 62:1827-829(1973). XE-60 25070cyanoethyl, methyl silicone 15 B, Davidow, NL. Petri, and B. Quame, A thin layer chromatographic screening procedure for detecting drug abuse. Amer. J. Chem. Path. 50:714-19 (1968). 16. J. Sherma, M.F. Dobbins, and J.C. Touchstone. Quantitative spectroden- sitometry of morphine and amphetamine on thin layer chromatograms. List of Tables J. Chromatog. ScL 12:300-03 (1974). 17. I. Sunshine. Use of thin layer chromatography in the diagnosis of poison- Table I. References to the TLC of Narcotics ing.Amer. J. C/in.Path. 40:576-82(1963). Table I1. References to the GLC of Narcotics 18. S.J. Mule. Methods for the analysis of morphine and related surrogates: current status, d. Chromatog. ScL 12:245-53 (1974). "Fable Ill. References to the HPLC of Narcotics 19. C.C. Clark. A study of procedures for the identification of heroin. J. Table IV. References to the TLC of Local Anaesthetics Forens.Sci. 22:418-28(1977). Table V. References to the GLC of Local Anaesthetics 20. H. Huizer, H. Logtenberg and A.J. Steenstra. Heroin in the Netherlands. Table VI. References to the HPLC of Local Anaesthetics Bull Narc. XXlX:65-74(1977). "Fable Vii. References to the TLC of Hallucinogens 21. Y. Marumo, T. Indue, T. Niwase, and T. Niwaguchi. Studies on direct quantitative analysis of diacelylmorphine (heroin) on thin layer Table VIII. References to the GLC of Hallucinogens chromatogramKiagakuKeis. atsuKenkyushoHokoku31:60.63(1978). Fable IX. References to the HPLC of Hallucinogens 22. B.F. Engelke and P.G, Vincent. Thin layer chromatography combined with ]able X. References to the TLC of Amphetamines and colourspottestreactionfsorpreliminaryidentificationofpapaveraceous alkaloids, J.A.O.A.C. 62:538-44 (1979). Related Stimulants 23. S. Ebel and D. Rest. Determination of morphine and its degradation pro- tt,i_l ]'able XI. References to the GLC of Amphetamines and ducts in drugs by TLC Arch Pharm. 313:337-43 (1980). Related Stimulants Gentnerl l:_:' 24. PG. Vincent, W.A.F.J.E.M Kuppers, and C.A. Salemink.

Iiii 322 : Journal of Chromatographic Science, Vol. 20, July, 1982

Pepaver bracteatum Lmdl: quantitative extraction and determination 53. J,O. Grooms. Quantitative gas chromatographic determination of heroin ot Ihebaine, J. Pharm, ScL 60:87-89 (1979), in illicit samples. J.A.O,A.C. 51:1010-013 (1968). 25. RA. Van Welsum. A simphlied procedure for the identification of drugs 54. SP. Sobol and A.R Sperling. A comparison ol heroin samples. In Foren- trom the illicit street market by thin layer chromatography. J. Chromatog. sic Science, G. Davies, ed. American Chemical Society Symposium 76:-237-40 (1973). Series 13, (Washington) 1975, pp. 170-82, 26. A.C. Moffat and B. Clare. The choice of paper and thin layer 55. K.D. Parker, J.A. Wright, A.F. Halpern, and C.H. Hine. Pre_iminaryreport i chromatographic systems for the analysis of basic drugs. J. Pharm. Phar. on the detection and quantitation of opiates and certain other drugs of macol. 26:665-70 (1974). abuse as tremethysilyl derivatives by gas-liquid chromatography. J. 27. J,A. Vinson, JE Hooyman, and C.E. Ward. Identification of street drugs Forens. Sci. Soc. 10:17-22 (1970). _ by thin layer chromatography and a single visualization reagent. J. 56, K.E. Rasmussen. Quantitative morphine assay by means of gas liquid Forens. Sci. 20:552-56 (1975). chromatography and on-column silylation, J. Chromatog. 120:491-95 28. M.C. Dutt and T.T. Poh. Use of ninhydrin as a spray reagent for the detec- (1976). tion of some basic drugs on thin layer chromatograms. J. Chromatog. 57. G. Brugaard and KE. Rasmussen, Quantitative gas liquid 195:133-38 (1980). chromatography of amphetamine, ephedrine, codeine and morphine after 29. A.N. Masoud. Systematic identification of drugs of abuse I1: TLC. J. on-column ecylation. J. Chromatog. 147:476-80 (1978). Pharm. ScL 65:1585-589 (1976). 58. A.S. Christophersen and K.E. Rasmussen, On-column acylation, heart- 30. K.D. Parker, C.R. Fontan, and P.L. Kirk. Rapid gas chromatographic cutting and electron-capture detection of opiates. J. Chromatog.lU: method for screening ol toxicological extracts for alkaloids, barbiturates, 216-20 (1979). sympathomimefic amines and tranquilizers. Anal. Chem, 35:356-59 59. A.S. Christophersen and K.E. Rasmussen. Glass capillary column gas (1963). chromatography of narcotic drugs after flash-heater trimethylsilylation. 31. E. Brockmann-Hanssen and C.R. Fontan. Gas chromatography ot J. Chromatog. 174:454-60 (1979). alkaloids with polar stationary liquids. J. Chrornatog. 19:296-99(1965). 60. M.J. Prager, S.M. Harrington and T.F. Governo. Gas liquid 32. C. McMartin and H.V. Street. Gas liquid chromatography of sub-micro chromatographic determination of morphine, heroin and cocaine.

gram amounts ol drugs, I. J. Chromatog. 22:274-85 (1966). J.A.O.A.C. 62:304-07 (1979). i 33. P. DeZan and J. Fasanello. The quantitative determination of heroin in 61, J.M. Moore. Rapid and sensitive gas chromatographic quantitation of mor- illicit preparations by gas chromatography. J. Chtomatog. ScL 10:333-35 phine, codeine and 0e-acetylmorphine in illicit heroin using an electron (1972). capture detector. J. Chromatog. 147:327-36 (1976). 34. J.M, Moore and F.E. Bena. Rapid gas chromatographic assay for heroin 62. J.M. Moore and M. Klein, Identification of 0:Lmonoacetylmorphine in il- in illicit preparations. Anal. Chem. 44:385-87 (1972), licit heroin using gas chromatography electron capture detection and 35. E.P.J, Van der S|ooten and H.J. Van der Helm, Analysis of heroin in relao mass spectrometry. J. Ct_romalog154:76-83. (1976). • tion to illicit drug traffic. Forens. Sci. 6:83-88 (1975). 63. Committee Report. Determination of morphine in raw opium, opium tinc- 36. 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Use of glass beads in gas chromatographic analysis of drugs• chona alkaloids, opium alkaloids, heroin and related narcotics by J. Chromatog. 178:311-13 (1979). dynamic-coating high speed liquid chromatography. Anal. Chim. Acre, 42, C.R. Fontan and H H, Hill, Gas chromatography of drugs on a Carbowax 63:393-402 (1973). 20M deactivated support. J. Chromatog. 170:249-53 (1979). 70. M.L. Chan, C. Whetsoll and J,D. McChesney. Using high pressure liquid 43. H-Y Lim and S-T Chow. Heroin abuse and a gas chromatographic method chromatography for the separation of drug ot abuse. J. Chromatog. Sci. for determining illicit heroin samples in Singapore. J. Forens. Sci. 23: 12:512-16 (1974). 319-28 (1978). 71. R. Verpoorte and A.B. Svendson, High speed liquid chromatography of 44. ,J.E. Wallace, J.D, Biggs, and K. Blum. Gas liquid and thin layer alkaloids, I. J. Chromatog. 100:227-30 (1974), chromatographic determination of morphine in biologic specimens. C/in. 72. W,A. Trinler and D.J. Reuland. Rapid screening of street drugs by high Acte Chirn. 36:85-91 (1972). pressure liquid chromatography: The screening of cocaine, heroin, 45. S.J. Mule. Determination of narcotic analgesics in human biological methadone and morphine by reverse phase HPLC. J. Forens. Sci. Soc. materials: application of ultraviolet spectrophotometry, thin layer and gas 15:153-58 (1975). liquid chromatography. Anal. Chem. 36:1907-914 (1964). 73. I. Jane, The separation of a wide range of drug abuse by high pressure 46. G.R, Nakamura and T.T. Noguchi. A simple GLC assay of opium. J. liquid chromatography, J. Chromatog. 111:227-33 (1975). Forens. Sci. Soc. 14:347-53 (1974). 74. P.J. Twitchett. Analysis of illicit diamorphine preparations by high pressure 47. N. Ikekawa, K. Takanyama, E. Hosaya, and T. Oka. Determination of mor- liquid chromatography. J. Chromatog. 104:205-10 (1975). phine in urine by gas chromatography. Anal. Biochem. 28:156-63 (1969). 75. P.J. Twitchett and A.C. Mortar. High pressure liquid chromatography of 48. G.R. Wilkinson and E.L. Way. Submicrogram estimation of morphine in drugs: and evaluation of an octadecylsilane stationary phase, J. biological fluids by gas liquid chromatography. Bioc,_em. Pharm. 18: Chromatogr. 111:149-57 (1975). 1435-439 (1969). 76. B.B. Wheals. Forensic aspects of high pressure liquid chromatography. 49. JE. Wallace, H.E. Hamilton, K. Blum, and C. Petty. Determination of mor- J. Chromatog. 122:85-105 (1976). phine in biologic fluids I:)y electron capture gas liquid chromatography. 77. J. Albanbauer, J. Fehn, W. Further and G. Megges. Quantitative high Anal Chem. 46:2107-111 (1974). performance liquid chromatography of narcotic drugs, I. Heroin and its 50, G.J, Digregorio and C. O'Brien. Chromatographic detection of narcotic formulations. Arch. Krim. 162:103-07 (1978). antagonists in human urine. J. Chromatog. 101:424-27 (1974), 78. D.J. Reuland and W.A. Trinler. An unequivocal determination of heroin 51. B. 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325 Journal of Chromatographic Science, Vol, 20, July, 1982

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