Techniques for the detection and identification of amphetamines and amphetamine-like substances
Infra-red and Raman spectrophotometry
Operation of TLC screening data
Formation of derivatives
High performance liquid chromatography
Column solvent systems
Muiti-detection and multi-column systems
Author: K. E. RASMUSSEN, P. KNUTSEN
Pages: 95 to 112
Creation Date: 1985/01/01
P. KNUTSEN Department of Forensic Science, National Bureau of Crime Investigation, Oslo, Norway
This paper reviews analytical techniques for the detection and identification of amphetamines and amphetamine-like substances in non-biological samples. It shows the wide range of methods available, from simple testing procedures to the use of the most powerful instruments available in analytical chemistry. The following techniques are discussed: colour tests, microcrystal tests, ultraviolet spectrophotometry, fluorescence spectrometry, infra-red and Raman spectrophotometry, thin-layer chromatography, gas chromatography and high performance liquid chromatography.
Amphetamines are stimulants of the central nervous system and have become a major class of drugs of abuse. The many closely related amphetamines present a challenge to the forensic chemist who must design analytical procedures to distinguish and identify the individual drugs. "Street drugs'' are seldom pure ; they are usually encountered as mixtures with cutting agents, excipients and other related drugs. In some situations it is necessary to identify optical isomers of amphetamines. By using multiple analyses and combining different methods, large amounts of analytical data are gathered. However, there will not be much improvement in analytical information unless discriminative power is improved through new or better methods of analysis.
This article reviews common methods used in the analysis of amphetamines from simple inexpensive methods to advanced instrumental analysis. The methods used to identify the individual drugs in street drug samples are discussed briefly. Articles on the forensic analysis of drugs of abuse [ [ l] , [ 2] , [ 3] ] and a textbook on amphetamines and related stimulants [ 4] have been published elsewhere.
Colour tests of different types are the most common form of preliminary drug screening. Commercial drug detection kits for field use are based on colour tests. The law enforcement agents who use the kits may not always understand the forensic chemistry involved ; some may be under the misapprehension that the kit is a drug identification kit instead of a drug screening kit. Colour tests do not identify a drug but serve to narrow down the list of drugs possibly present in a sample. Clarke [ 5] has drawn up a comprehensive list of colour tests and has also developed a technique using microdrops.
Colour tests are not specific for a single drug but may give useful hints about chemical groups in a molecule. Amphetamine gives an orange colour with Liebermann's reagent (sulphuric acid and nitrous acid) and so will many compounds containing a monosubstituted benzene ring. With Marquis' reagent (sulphuric acid and formaldehyde), amphetamine gives an orange-brown colour as do phenformin (an antidiabetic) and other drugs. If no colour is observed with Marquis' reagent, it is fairly reasonable to assume that amphetamine is not present or is present in quantities below the detection limit.
The colour formation may be influenced by many factors, such as the reaction time, temperature, the stability and concentrations of the reactants and the presence of dyes or other substances giving colour reactions.
The assignment of colour to the result of a colour test is subjective; standardization of the colour assignment procedure would decrease the ambiguities associated with the interpretation of the colour. Velapoldi and Wicks [ 6] carried out a study of colour tests. The colours produced were assigned numbers and descriptions corresponding to colours in the ISCC-NBS central colour charts using colour chips and evaluating the colour lightness, saturation and hue. They concluded that a positive identification of a pure drug according to the colour produced with a single reagent is difficult and probably incorrect, even if the interpretation is by a trained investigator. They suggested that seven reagents would be necessary for a reasonable, multi-reagent testing scheme that would decrease the number of false positives and increase specificity. The use of multiple reagents in a logical fashion will help in sorting out the possible drug classes and in eliminating others.
The use of multiple colour tests generates large amounts of data that have to be interpreted. Johns and others [ 7] presented the colour test results in tabular form, which facilitated manual or computer searches. The colour test results were assigned numerical codes. Given reliable and reproducible colour test results, the numerical code system can be time-efficient for handling large amounts of colour test data.
Colour tests work well with pure drugs. In spite of the improvements discussed above, however, the lack of specificity and ability to discriminate between chemically closely related compounds limit the colour tests to screening, which gives only preliminary and presumptive evidence. Law enforcement agencies that contemplate using colour test kits as field tests for amphetamines and other drugs would be well advised to plan with the forensic chemistry laboratory that is going to follow up the field screening tests with definite drug identification. It would be wise for law enforcement agencies to be very cautious about taking legal action on the basis of results obtained by colour test kits before a drug has been definitively identified.
Microcrystal tests are not suited to systematic drug screening but are an excellent means of confirming results. They are performed under the microscope, mixing solutions of the sample with test reagent and observing the crystals formed. The tests vary in specificity. There is no systematic identification system, and it takes both time and skill to learn the tests. Closely related crystals may be reported as false positives and problems in getting the crystals to form at all may give false negative results.
Clarke [ 5] has listed many microcrystal tests. One example is a test to differentiate between related compounds such as amphetamines, phenmetrazine and phendimetrazine that give different crystal forms with picrolonic acid. Fulton [ 8] has written an extensive book on microcrystal tests. One of their particular uses is to distinguish optical isomers of a compound. Differentiation between d-, l- and the racemic dl-form of amphetamine can be made on the basis of microcrystal tests. 1
Related to microcrystal tests are the crystallographic tests to determine such crystal optic properties as the refractive index and the extinction of the crystal. A simple but useful test to detect small amounts of amphetamine in a mixture is to put the mixture in alkaline solution. The amphetamine base is volatile, and it can be trapped in a hanging drop of diluted hydrochloric acid. The amphetamine hydrochloride is allowed to crystallize, and the crystals are observed between crossed polars under the microscope. The test can also be scaled up to a preparative isolation procedure, and the amphetamine hydrochloride can be analysed using spectrophotometry or chromato-graphy. When doing the colour or microcrystal tests, it may also be convenient to do traditional wet-chemistry ion tests for sulphate, phosphate and chloride.
The ultraviolet (UV) absorption spectrum contains information about the chemical functional groups involved in electronic transition. It is not, however, specific enough to provide proof of identity for single drugs such as amphetamine. On the other hand, if a sample does not show any absorption in the 200 to 400 nm range, it is reasonable to conclude that the sample contains no amphetamine or that the quantity of amphetamine is too small to be detected.
More data may sometimes be obtained using multiple solvents with different polarities. Differences in solute-solvent interactions may give recognizable shifts in the ultraviolet absorption spectra and help to distinguish between compounds. Clarke [ 5] has listed ultraviolet absorption data for many amphetamines, most recorded in 0. l N sulphuric acid. Recording ultraviolet absorption spectra at different pH may provide more information about compounds containing ionisable groups. The Drug Identification Atlas [ 9] contains ultraviolet absorption spectra recorded at pH 2 and pH 12 for many amphetamines. Ultraviolet spectrophotometry may be used to determine the quantity of amphetamines, provided that there are no other compounds in the sample that would interfere on the absorption band used for quantitation.
Amphetamines have rather low specific absorptivities in the ultraviolet region, however, and it may be better to use chromatographic methods to determine the quantity of amphetamine in street drug samples. Gill and others [ 10] used a diode array rapid-scanning spectrophotometer and found that the differential spectra made it easier to recognize benzenoid spectra which would otherwise be masked by a broad background absorption in a mixture. They also reported differential spectra to be of value in distinguishing amphetamine from benzphetamine and ephedrine from phenelzine.
Fluorescence spectrometry may be much more sensitive than ultraviolet spectrophotometry, and the characterization of a compound by spectra for excitation and for emission may give high specificity [ 11] , although strongly fluorescent impurities may interfere. It is possible to make strongly fluorescent derivatives of amphetamines. Nix and Hume [ 12] reported on a procedure for the determination of amphetamine as a fluorescing lutidine derivative. The reported detection limit for the amphetamine derivative was 250 ng/ml in the cuvette. The method was claimed to be highly specific for amphetamine. However, phenethylamine and methamphetamine also formed fluorescent derivatives and might interfere if present. A fluorescamine derivative of amphetamine was studied by de Silva and Strojny using a xenon arc energy source [ 13] and later a dye laser as energy source [ 14] for the determination of amphetamine as the fluorescamine derivative.
Organic molecules such as amphetamines absorb infra-red radiation under transitions between rotational/vibrational energy levels in the electronic ground state of the molecule. The fundamental, overtone and combination infra-red absorption bands produced give very characteristic spectra, ensuring good selectivity and capable of identifying a pure compound in almost all cases. Amphetamines in mixtures may have to be isolated to obtain good infra-red spectra. For screening purposes, infra-red spectrophotometry may yield good results provided that not too many interfering compounds are present and that the amphetamine concentration is reasonably high. Polymorphism may occur in some compounds and give slightly different spectra. There are collections of infra-red spectra of manyamphetamines in Clarke [ 5] and the Drug Identification Atlas [ 9] . Optical isomers of amphetamines may be distinguished by infra-red spectrophotometry as isomeric mandelate derivatives [ 1] [ 15] .
Computer-aided chemistry with digital storage of the spectra and rational programmes for search, identification and quantitation may be of great help to the forensic chemist handling and documenting large amounts of spectrophotometric data. Moss and others [ 16] used numerical taxonomy techniques in an attempt to classify the infra-red spectra of amphetamines and other drugs of abuse.
Raman and infra-red spectrometry are complementary techniques. Antisymmetric vibrations and polar groups are most easily studied by the infra-red technique, while symmetric vibrations and nonpolar groups are best studied by Raman, and the sample preparation is very simple. At the empirical level, both techniques provide excellent "fingerprint'' spectra for qualitative identification of molecules [ 17] .
Thin-layer chromatography (TLC) is a low-cost and very versatile technique, and the choice of plates, stationary or mobile phases and visualization means is large. TLC may be run qualitatively with many samples in parallel, two-dimensionally, quantitatively or as a preparative technique. The chromatographic data may be documented as Rf values, by comparison with standard drugs, or by preserving the plates with varnish, photocopying the plate or photographing it in colour. An improved version of TLC is high performance thin-layer chromatography (HPTLC). The favourable cost and effectiveness ratio for TLC makes it well suited for forensic chemistry laboratories that cannot afford expensive analytical instruments. To get the maximum amount of analytical information from the TLC plate, the chromatographic data should be interpreted by a forensic chemist.
Stead and others [ 18] noted that the use of a single TLC system was of little help in identifying an unknown compound ; more information could be gained using multiple TLC systems. However, if the systems had correlated chromatographic properties, multiple systems would not give much further information. They stressed the evaluation of the discriminating power of a TLC system, the discriminating power being defined as the probability that two drugs selected at random can be separated by a TLC system. Twentynine TLC systems were evaluated and 4 systems were selected for basic drugs:
Methanol-ammonia (100 : l.5)
Cyclohexane-toluene-diethylamine (75 : l5 : 10)
Chloroform-methanol (9 : l)
Silica gel was dipped in 0.1 M KOH and dried. For screening for basic drugs using TLC the chloroform-methanol system was best. The increase in the discriminating power for the combination of TLC systems for basic drugs was shown to be 0.860 for system l alone, 0.962 for systems l and 2, 0.988 for systems 1, 2 and 3 and 0.993 for all four systems. The spray reagents for basic drugs were put on to a plate; the reagents were, sequentially: ninhydrin solution, FPN solution (iron-III-chloride, perchloride acid, nitric acid in water), Dragendorff's reagent and acidified iodoplatinate. Marquis' reagent was put on another plate. Stead and others have pointed out that by standardizing TLC systems, chromatographic data can easily be transferred from one laboratory to another. They list Rf values for 794 drugs on eight TLC systems.
O'Brien and others [ 19] have used TLC to differentiate amphetamine and its major hallucinogenic derivatives with two solvent systems:
ethylacetate-methanol-water-ammonia (95 : 3.5 : 1.5 : 0.75); and
acetone-ammonia (100 : 0.5). The developed silica gel plates were sequentially exposed to formaldehyde vapour, dipped into Mandelin's reagent, viewed under 366 nm UV radiation and dipped into a modified Dragendorff's reagent. Gallic acid or chromotropic acid were used as confirmatory reagents for methylenedioxy groups.
Bailey and others [ 20] reported the identification of the N-methylated analogs of hallucinogenic amphetamines using TLC, UV, infra-red, mass spectrometry (MS), nuclear magnetic resonance (NMR) and melting points. Six TLC systems were used to separate N-methylamphetamines. The developed plates were examined under 254 nm UV radiation and sprayed with ninhydrin or chromatropic acid.
Vinson and others [ 21] proposed the TLC system ethylacetate-methanol-ammonia (100 : 18 : 1.5) for the general screening of street drugs and the TLC system methanol-ammonia (100 : l .5) as a confirmatory system for basic drugs. As a single visualization reagent they propose TCBI (N-2,6-trichloro-p-benzoquinoneimine).
Loh and others [ 22] chromatographed in less than five minutes the dansyl derivatives of amphetamine and methamphetamine in several TLC systems using 3 x 3 cm polyamide plates.
Cartoni and others [ 23] presented chromatographic data for amphetamines on silica, cellulose, polyamide and alumina developed in butanol-formic acid-water (20 : l : 2). A two-dimensional method was also reported on silica with the first TLC solvent system as before, the second system being n-amylalcohol-5 N ammonia ( 1 : l ). Four visualization reagents were used.
Brown and others [ 24] proposed a TLC screening system for street drugs using silica plates and ethylacetate-n-propanol-28 per cent ammonium hydroxide (40 : 30 : 30) as solvent system. The developing time was 70 minutes. The chromatograms were visualized at 254 nm UV radiation, using iodoplatinate spray for the amphetamines.
Genest and Hughes [ 25] analysed 2-amino-l-(2,5-dimethoxy-4-methyl)phenylpropane (STP) and other amphetamines in three different TLC systems. Sundholm [ 26] described a rapid TLC method for the separation of amphetamines and other drugs using a horizontal developing chamber with HPTLC plates, silica gel G 60 F254, 10 x 10 cm. One half of the plate was immersed in 0.1 M KHSO4 and dried. The developing solvent was methanol containing 0.0125 M KBr to reduce tailing, run simultaneously from both sides. Visualization was performed by UV, ninhydrin and iodoplatinate. The effect of various KBr concentrations on acidic silica and untreated silica was studied. The correlation factor was 0.63, and the discrimination power was 0.94 for Rf with untreated silica versus Rf with acidified silica at 0.01 M KBr. The compounds studied were divided into four groups according to their retention characteristics in the different systems. The first group contained amines with high basicity such as amphetamine with higher Rf value on the acidic silica than on the untreated silica. The studied compounds could be nicely arranged into four groups. However, compounds such as amphetamine and ephedrine, with similar basicities, were not well resolved.
Eskes [ 27] reported a TLC procedure for the differentiation of the optical isomers of amphetamine and methamphetamine. The samples spotted on a silica plate were overspotted with an optically active reagent, and the plate was developed as usual. The optically active reagents used were N-trifluoroacetyl-L-prolylchloride (TPC) or N-benzyloxycarbonyl-L-prolylchloride (ZPC). Three developing solvents were used : chloroform- methanol (197 : 3), hexane-chloroform-methanol (10 : 9 : 1) and hexane-ethylacetate-acetonitrile-diisopropylether (2 : 2 : 2 : l). Marquis' reagent was used for visualization.
TLC may be used as the primary screening procedure to determine the Rf values. Searching lists of Rf values, and matching other chromatographic and spectrophotometric data in order to identify the unknown compound may be greatly facilitated by a rational computer system. McLinden and Stenhouse [ 28] reported a chromatography system for drug identification based on three types of TLC data from different solvents ; two were gas chromatography (GC) data and one a UV parameter. A drug on a TLC plate was classified with reference to a series of standard drugs with defined zones that were coded alphabetically.
Since the introduction of GC as a method of separating and identifying sympatomimetic amines in 1962 [ 29, 30] , numerous reports on the qualitative and quantitative determination of amphetamines and related compounds have been published. GC is today one of the most widely used methods of analyzing these drugs. The technique is reliable, highly selective and very sensitive. The flame ionization detector (FID) is used as a universal detector. Selective detection of compounds containing nitrogen is offered by the nitrogen-phosphorus selective detector (NPD), and the electron capture detector (ECD) can be used after the formation of a suitable derivative.
Many stationary phases have been used for gas-liquid chromatography (GLC) of amphetamines [ 31 , 32] . Moffat and others [ 33] compared eight different stationary phases (SE-30, Apiezon L/KOH, OV-17, Carbowax 20 M/KOH, Carbowax 20 M, CDMS, DEGS/KOH, DEGS) and concluded that a low polarity phase such as SE-30 should be chosen as the preferred liquid phase for GLC of basic drugs. The retention index values of 62 basic drugs were reported. In a later study Moffat [ 34] compiled retention index values of 480 drugs on SE-30, which showed that the majority of amphetamines and amphetamine-like substances were separated. Moffat's paper reported retention indices arranged in alphabetical order of drug name and in ascending order of retention index. These data provide valuable information for drug identification. Huber and others [ 35] reported on retention index values for 43 stimulant drugs on four stationary phases (OV-101 , OV-225, Apiezon L, PEG 20 M) and the correlation between retention index values on these four phases and the mass spectra of the drugs. The use of retention index values and temperature-programmed gas chromatography, which is very useful for drug screening, was discussed by Perrigo and Peel [ 36] . They reported retention index values of 289 compounds, including amphetamines, for SE-30, as well as retention index values for common compounds on OV-7 and OV-17.
These papers demonstrate that a non-polar phase such as SE-30 is the most popular liquid phase for the analysis of basic drugs. A large amount of data in the form of retention index values is available from the literature. However, some peak tailing of amphetamines occurs on SE-30, and there is also considerable use of KOH-treated stationary phases 2 such as Apiezon/KOH and Carbowax 20 M/KOH.
Derivative formation is often used as an aid to identification. The derivative formed with a particular reagent indicates the functional groups that an unknown drug may contain, and the retention characteristics of the derivatives provide additional data. Another important reason for the formation of derivatives is to increase the sensitivity of the analysis, either by improving the chromatographic behaviour of a drug or by forming derivatives which makes it possible to use a high sensitivity detector. Derivatives can be formed in a tube or on-column by injecting the sample solution along with the reagent onto the column.
Primary amines may be converted to Schiff's bases by aldehydes or ketones. Brochmann-Hansen and Baerheim Svendsen- [ 29] chromatographed the acetone and butanone derivatives of sympathomimetic amines. Schiff's bases were later studied by several groups.
Beckett and others [ 32] chromatographed a number of Schiff's bases and acyl derivatives on both SE-30 and Carbowax 20 M/KOH in order to identify stimulant drugs. O'Brien and others [ 37] formed trifluoroacetamide derivatives with trifluoroacetic anhydride to provide symmetrical peaks for the GLC analysis of amphetamine and phentermine on a OV-1 column.
2 The KOH coating of the support is done in order to reduce adsorptive effects.
In a screening of amphetamines Jain and others [ 38] chromatographed free bases on an Apiezon L/KOH column and trifluoroacetamide derivatives on a OV-17 column. On-column trifluoroacetylation was employed.
On-column derivatization was also used by Brettell [ 39] to form trifluoroacetyl derivatives of amphetamine analogues. MBTFA was used as the reagent and Carbowax 2() M/KOH as a stationary phase. Sharp symmetrical peaks were obtained.
On-column acylation with N-acylimidazoles as reagents has also been reported [ 40] . To eliminate interferences with amphetamine and methamphetamine from ephedrine and phentermine Budd and Leung [ 41] chromatographed the trifluoroacetamide derivatives on a SP-25 10-DA Column and on a SP-1240-DA column. These columns eliminated interferences with amphetamine, methamphetamine and n-propylamphetamine from ephedrine and beta-phenetylamine and provided satisfactory separation between amphetamine and methamphetamine.
Optical isomers can be separated by GLC by reacting a racemate or an unknown optical isomer with a chiral reagent to yield a diastereoisomeric mixture that may be separated on a normal GC column. Another approach is to resolve the racemate on a chiral phase. Several papers have been published on the separation of optical isomers of amphetamines by GLC [ [ 42] , [ 43] , [ 44] , [ 45] ].
Recent years have been a period of innovation with regard to capillary gas chromatography. A new generation of glass capillary columns and fused silica columns has been created that combines high separation power with good column stability, flexibility and load capacity. The chemical bonding of stationary phases has been shown to increase the stability of the stationary phases compared with conventionally coated films. Modern gas chromatographs are designed for capillary columns and different injectors permit on-column injection, split injection and splitless injection of the sample. Important contributions to the analysis of amphetamines by capillary gas chromatography have been published in recent years.
Kinberger and others [ 46] used a 25-m fused silica capillary column deactivated with Carbowax 20 M and with SP 2100 as a stationary phase to analyse underivatized stimulants. The gas chromatograph was equipped with a flame ionization detector and a split injector.
Schepers and others [ 47] compared retention index values for various drugs on three capillary columns with the corresponding values on packed columns.
Plotczyk [ 48] discussed considerations for optimizing on-column and splitless injection as part of a study of system discrimination and reproducibility.
A nitrogen detector and a 10-m glass capillary column coated with SP-2250 were used by Pettitt [ 49] for the rapid screening of drugs of abuse.
Alm and others [ 50] installed two differently coated columns in a common split-splitless injector and connected the column ends to an NPD and to an FID. Fused silica columns were used with immobilized stationary phases. One 11-m-long column with SE-54 as stationary phase was connected to an FID and the other, a 10-m-long column with OV-215 as stationary phase, was connected to an NPD. The relative retention times from both detector signals were calculated from two internal standards by use of a Basic program. The values obtained were then compared to empirically determined values of individual substances, and the names of compounds identified were printed on the chromatogram. This screening method was combined with a quantitative determination of the drugs. Amphetamine was quantitatively determined on the SE-54 column connected to the FID. With this dual-column system, considerable and accurate chromatographic information could be obtained from a single run. By using an auto-sampler in combination with the Basic program, both qualitative and quantitative analysis could be performed routinely.
Mass spectrometry in combination with gas chromatography (GC-MS) is a very powerful analytical tool, but few laboratories have the resources to use GC-MS as a screening method. The principal use of this technique is in the confirmation of the identity of a drug tentatively identified by other techniques. MS of amphetamines has been described in several articles [ 32] , [ 35] , [ 51] as well as MS of their N-trimethylsilyl derivatives [ 52] and their N-mono-trifluoroacetyl derivatives [ 39] .
High performance liquid chromatography (HPLC) is a technique of major importance for the analysis of drugs of forensic interest. It is particularly suitable for drugs that can be troublesome to analyse by gas chromatography because they are thermally degradeable, non-volatile or polar. The technique is non-destructive, and compounds can be isolated for identification by other methods. Several detectors are available such as the UV detector, the fluorimetric detector and the electrochemical detector ; UV detection is most commonly employed in amphetamine analysis- The forensic applications of HPLC to the analysis of amphetamines have been briefly reviewed [ [ 53] , [ 54] ], and a textbook on HPLC in forensic chemistry has been published [ 55] .
Several HPLC systems have been reported for the analysis of amphetamines.
An important contribution was published by Jane [ 56] . The author used a 25 cm x 4.6 mm ID column packed with 6 μm silica (Partisil) and methanol-2 N ammonia-l N ammonium nitrate (27 : 2 : l) as a mobile phase. A wide range of drugs of abuse were examined, and relative retention times of 27 amphetamine-type stimulants were tabulated. The majority of the stimulants were separated using this system. A variation in the retention of the compounds was affected by changes in either the methanol-water ratio, the concentration of ammonia or the concentration of ammonium nitrate. Detection was made by UV absorption at 254 nm and quantitation by measurements of peak heights. Good column stability was obtained.
Reversed phase chromatography on μ-Bondapak C 18 was reported by Twitchett and Moffat [ 57] ; 30 compounds including amphetamine and methylamphetamine were studied, but the column efficiency for basic drugs was poor. Later Twitchett and others [ 58] chromatographed the same 30 compounds on a microparticulate cation-exchange column. Tolerable efficiency for most drugs was obtained but the authors observed that the column life was rather short.
To reduce peak tailing in reversed-phase HPLC, Gill and others [ 59] examined a series of eluents containing amines as part of the buffer system. Large improvements in peak shape were demonstrated, and practical guidelines for the selection of suitable amine additives were given.
Lurie [ 60] used reversed-phase ion-pair chromatography for the analysis of drugs of forensic interest. The optimum resolution of amphetamines was with μ-Bondapak C 18and methanol-water-acetic acid (20 : 79 : l) with 0.02 M methane-sulphonic acid as counter-ion at pH 3-5. Detection was made by UV absorption at 254 nm.
Flanagan and others [ 61] chromatographed basic drugs on a silica column with non-aqueous ionic eluents and reported retention data of a variety of compounds. A Spherisorb 5 silica column was used, and the eluent was methanol-hexane (85 : 15) containing perchloric acid. The non-aqueous ionic systems showed high efficiency, stability and reproducibility and gave long column-life.
The identification of drugs using HPLC with dual wavelength UV detection was reported by Baker and others [ 62] . The column was connected to a 254-nm detector then in series to a 280-nm detector enabling the absorbance ratio of the drugs at 254 nm to be determined. Three column solvent systems were used : a μ-Bondapak C18 with methanol-water (2 : 3) pH 7 with phosphate buffer, a μ-Porasil column with methanol-2 N ammonia-l N ammonium nitrate (27 : 2 : l) and a μ-Porasil column with dichloromethane and ammonia ; 101 drugs were tested and only 9 per cent of the drugs could be distinguished using relative retention times alone, while when both the retention times and absorbance ratios were used, 95 per cent of the drugs could be distinguished.
Isocratic multi-column HPLC was reported by Wheals [ 63] as a technique for the qualitative analysis of basic drugs. Three columns were studied, a column packed with silica (Si), a mercaptopropyl bonded phase (SH) and a column packed with an aliphatic strong cation exchanger (SCX). The eluent was methanol-2 N ammonia-1 N ammonium nitrate (27 : 2 : l) ; 161 drugs, including amphetamines, were studied. The retention sequence was found to parallel closely the order of increasing basicity of the drugs.
Although HPLC has not been extensively applied to stimulant drugs, these reports show that several systems can be used to separate and identify them. The system devised by Jane [ 56] is particularly well documented in the literature. Retention data used in combination with data obtained from either stopped-flow UV scanning or multiple wavelength monitoring can provide a very effective method of characterizing compounds. Both column technology and instrumentation have been greatly improved during the last years. The new generation of diode-array spectrophotometers used as HPLC detectors represent a significant improvement. These detectors provide simultaneously chromatographic and spectral data on drugs.
A number of methods are available for the detection and identification of amphetamines, from simple testing procedures to the use of the most powerful instruments available in analytical chemistry. Simple and low-cost screening techniques can be used in many cases, however, and it is not always necessary to depend on sophisticated instruments. The strategy for solving a particular problem will depend on the expertise and experience built up in the laboratory, as well as on the time and the facilities available. It is, however, not advisable to rely only upon non-separatory methods, because street drugs are seldom pure.
At present IR and UV spectroscopy are the most frequently used spectroscopic methods, and TLC and GLC are the work-horses of the separation methods. HPLC has also much to offer in terms of separation power, selectivity and reproducibility. The information derived from chromatographic and spectroscopic analyses confirms the identity of a drug.
Forensic drug identification and quantitation has progressed rapidly during the last years. Large improvements both in instrumentation and separation technology have been seen. This progress will certainly continue, and analysts must keep themselves up-to-date not only with scientific literature but also with developments in analytical instrumentation.
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