DIVISION OF NARCOTIC DRUGS Vienna RECOMMENDED METHODS FOR TESTING COCAINE MANUAL FOR USE BY NATIONAL NARCOTICS LABORATORIES Nee UNITED NATIONS 1ST/NAR/7INTRODUCTION, I. ie qr. Iv. contents: DESCRIPTION OF THE PURE COMPOUNDS PRODUCTION OF ILLICIT COCAINE PHYSICAL APPEARANCE AND CHEMICAL CHARACTERISTICS OF COCA LEAF AND ILLICIT MATERIALS CONTAINING COCAINE. . A, Coca leafs... B. Coca paste Cc. Cocaine, THE ANALYSIS OF MATERTALS CONTAINING COCAINE Sampling.. 1. Sompling of single package rceme. ae 2, Sampling of items consisting of more than one package 3. Sampling of materials containing large particles. B. The analysis of coca leaf 1, Physical adentification....sesseeseceee eee : 2. Chemical analysis of coca leaf (whole or poudered). €. The analysis ot coca paste and cocaine. 1, Presumptive tests for cocaine... (a) Colour cest (b) Odour tese...eeeee (c) Nicrocrystal test. 2. Tests for anions associated with cocaine. (a) Solubility tests.. (b) Precipitation tests... 3. Thin layer chromatography of cocaine.. (a) Standard technique... 4. Gas Liquid chromatography of cocaine... (a) Packed column technique.. (>) Capallary columa technique. 5. High performance Liquid chromatography of cocaine......+ 6, Infra rea spectroscopy of cocaine. 7. The analysis of cocaine enantiomers. (a) crocrystal test co differentiate (hb) Alternative methods co aifferentiace 3 13 16 16 16 v7 1s wy 19 y a 2 3INTRODUCTION Background Over the past tew years there has heen a considerable increase in the number of scheduled substances newly included under international control. This increase reflects a rapid diversification of drugs of abuse, and the consequent increase of regulatory efforts results in turn in a larger number of controlled substances and in better buc, at the same time, more stringent national legislation and sentencing provisions. At the same time, the seizes quantities of drugs already under control, such as the opiates, cocaine and coca paste, cannabis products, amphetamine and related compounds have also shown an alarming and unprecedented increase in certain regions. This new situation, involving an increase both 1m the frequency and volume of seizures, presents s challenge not only to national law enforcement authorities, but also co the technical and scientific staff of forensic Laboratories. Owing to the ingenuity of illicit producers anc promoters, unexpected new illicit drugs or combinations of drugs appear on the illicit market, requiring rapid and adequate action as well as ingenuity on the part of forensic chemists, Similarly, the increased number of controlled substances and of related legislative provisions place additional pressure on national forensic and narcotic laboratories and their staft. Analysts have co be able to deal with more substances and preparations ana to use faster, more accurate and more specific methoas of identification and analysis. In addition, the international character of drug trafficking requires che speedy exchange of analytical cata between laboratories and law enforcement authorities both on the national and the international levels. Development of internationally acceptable methods of testing would contribute greatly to the achievement of these objectives, and this possibility has been under consideration tor sone At its eighth special session in February 1984, the Conmission on Narcotic Drugs requested the Secretary-General “to investigate the possibility cr reaching agreement at the regional and interregional levels on recommended methods of analysis of drugs seized from the traffic". The Commission was of the opinion that closer scrutiny and harmonisation of the wide variety of analytical methods in use at the national level would not only ease the tack of the staff of national institutions but would also facilitate the exchange of information at regional ana interregional levels. Purpose of the manual In response to the Commission's request, a group of fifteen experts vas convened in October 1985 by the Division of Narcotic Drugs in Wiesbaden at the invitation of the Federal Republic of GerTany. The Present manual prepared by the United Nations Division of Narcotic Drugs reflects the conclusions of the proup of experts and has been designea to provide practical assistance to national authorities by describing Tecomnendea methods to he used in torensic laboratories for the identification and analysis of illicit coca products. ‘The manual may also serve 3s 3 guide Co national authorities in assessing existing methods used within their own government and university laboratories.This manual is the second in a series of similar publications dealing with the identification and analysis of various groups of drugs under international control; it was preceeded by a manual on heroin analysis (ST/NAR/6) and will be followed by a similar publication covering the analysis of illicit cannabis products. ‘These manuals suggest approaches that may help the forensic analyst to select a technique appropriate to the sample currently being examined. ‘The analyst may then choose to follow any of the methods described in the manual, as each method can be expected to produce reliable analytical information with respect to the samples to which they are applied. Each method has been used for a number of years in reputable forensic laboratories ana has been published in the scientific literature. In identifying these methods, the expert group was aware that many other useful and acceptable methods produce worthwhile analysis and information for the forensic analyst, and that a aumber of other acceptable options are recorded in the torensic scientific literature. Use of the manual Few methoas are perfect, least of all in forensic drug analysis where the materials under examination are very likely to show significant variation both in their physical form and chemical composition. The choice of methodology and approach to analysis remains within the control of the analyst working within his or her own country. The analyst alone has seen the suspect material and can best judge the correct approach to the problem at hand. Furthermore the choice of methods may necessarily depend on the availability of reference materials and of instrumentation. Not all the methods listea need to be applied to all samples suspected to contain cocaine. Requirements may vary, for example, es a result of local trends in samples encountered, focilities available, and the standard of proof acceptable in the prosecution system within which the analyst works. The more complex methods are needed only for certain forensic requirements, such as comparison of samples or the development of typology. In order to establish the identity of any controlled drug, it is suggestea that the criteria should be at Least two independent analytical Parameters. The selection of these parameters in any particular case would take into account the drug involved and the laboratory resources available to the analyst. For example, two uncorrelated TLC systems vould count as tuo parameters, Uncorrelated TLC systems in this context means that either the solvent systems or the coating on the plates are completely different. When possible, three entirely different analytical techniques should be used, tor example: colour test, chromatography (TLC, GLC or HPLC) and spectroscopy (IR or UV). The actual choice of parameters is left to the discretion of the chemist. Attention is also drawn to the vital importance of the availability of textbooks on drugs of abuse and analytical cechoiques. Furthermore, the analyst must continually keep abreast of current trens in analysis, consistently following current analytical and forensic science literature. For this purpose, atcention is drawn co the Nuleilingual Dictionary of Narcocie Drugs and Psychotropic Substances under International Control (ST/NAR/1), a vital tool for Forensic laboratories, and to the Nanval on Staff Skill Requirements and Basic ¥quipment tor-3- Narcotics Laboratories (ST/NAR/2), both published by the Division of Narcotic Drugs. The latter publication lists bibliographic references as well as a selection of well-known journals in the field. Close liaison with national law enforcement and judicral authorities as well as between national narcotic laboratories and those at the regional level can lead to greater awareness of the latest trends in drug presentation, the illicit traffic, smuggling techniques and the preparation of evidence to courts of lav. These in turn will produce @ more meaningful choice of analytical techniques to be applied to the latest submissions. It is equally important that the latest information on changes in drugs available in the illicit traffic be quickly disseminated. This may often neea to be done prior to publication in specialised periodicals dealing with forensic and other chemical analyses, since these publications are available to the forensic community some two to three years after the changes become known. The value of frequently published national reports on the latest information on such changes in drugs and on work being undertaken and analytical results obtained within inaividual laboratories cannot be overemphasized. The Division of Narcotic Drugs would welcome observations on the contents and usefulness of the present manual. Comments and suggestions may be addressed to: Division of Narcotic Drugs United Nations Office at Vienna Vienna International Centre P.O. Box 500 A-1400 Vienna, AustriaOF THE PURE comPOUNDS COCAINE, Melting points (ot) Beta-cocaine Methy lbenzoylecgonine Base Hydroch}oriae Benzoy Imethylecgonine 98 157 (200-202) Solubilities (ig/ml) Base Hydrochloride LCHs Base Hydrochloride ‘OOCH, Water 1300 0.5 Ethanol 7 Diethyl ether 4 almost insoluble ICO— C,H, Chloroform 0.5 18 ©1782 1NOy Mit = 303.4 CINNAMOVLCOCAINE Nelting points (oc) Cinnamoy Imethy lecgonine Cinnany leocaine Methy lbenzoylecgonine Base Hydrochlorige Benzoy Imethy lecgonine han Solubilities (1g/m)) Base Hyerochlorade OCH, water almost insoluble soluble Ethanol soluble soluble Diethyl ether soluble soluble Chloroform solubie slightly CO-CH: Ctl 3804 Mut = 329.4Melting points (oC) Hydrochloriae 195 (anhydrous) (dec. ) 200 86-92 (tetrahydrate) Ecgonine benzoylester “CH; Solubilities (ig/ml) ites) Base Hydrochloride Water, boiling soluble soluble CO-CsHs Ethanol, soluble soluble Cy gt gO, MWe = 289.34 METHYLECGONINE Melting points (ol) Ecgonine methylester Base Hyarochlorige oil 215 Solubilities (ig/ml) cH. Base ydrochtorige yn £oock, Croat NOs Nie = 198.3BoGONTNE Melting poines (0c) Base lyerochlorige 198 (205) 241 (240-275) Solubitieies (1g/al) Uydrochloriae CH ee ‘OOH waver 5 soluble Ethanol 87 Hagnely Methanol 20 soluble Hog thylacetace 75 Cay N03 wie = 185.22 For additional details related to the substances, the reager 1s referred to "The Multilingual Dictionary of Narcotic Drugs ana Psychotropic Substances Under International Control (ST/NAK/1), and to the widely used Merck Index.II. PRODUCTION OF ILLICIT COCAINE The details here outlinea represent one of the approaches to the production of illicit cocaine, which can be achieved in several ways. in the illicit production, variations of technique, reagents ana quanticies are to be expected. However, it would not be unreasonable to assume that the general approach will be similar to that given here. 1, Coca leaves are mixed with water ang with a material such as lime, so 28 to produce an alkaline reaction in the resulting pulp. The mixture 15 crushed, kerosene (or an equivalent hydrocarbon) is added and the mixture stirred. 2, The kerosene is removes and the extracted coca leaf discardea. Acidified water is partitioned with the kerosene, extracting alkaloids into the aqueous layer. The kerosene is removed. If coca paste is required, the water is made alkaline with lime, ammonia or equivalent, which precipitates the more basic alkaloids. The precipitate, vhich eiten contains mixed inorganic salts as well as crude cocaine, is removed and aried. The result 18 coca paste. 3. To produce cocaine hysrochioriae, the coca paste is dissolves in @ilute sulphuric acid. Potassium permanaganate may be addea at this stage until the solution remains pink. The potassium permanganate is adaed to destroy the cinnamoylcocaine isomers present ss an impurity in the cocaine. The solution is allowed to stan, then filtered. made basic with anmonia, precipitating cocaine base and other alkaloids. ‘The precipitate is filterea, washed with water and cried. e filtrate is 4, The crude cocaine base is dissolved in diethyl ether. The solution is filtered, and concentratea hydrochloric acid and acetone are aaded. The cocaine hydrochloride which precipitates is filtered and dried.-38- TIL, PHYSICAL APPEARANCE AND CHEMICAL CHARACTERISTICS OF COCA LEAF AND ILLICIT MATERIALS CONTAINING COCAINE Ie must be stressed that no two samples of coca leaf, coca paste or cocaine hydrochloride will have exactly similar physical appearances. AL Coca leat Coca leaves are somevhat similar in appearance to Laurus nobilis leaves. Different Erythroxylon species produce leaves varying in size and appearance. In all species the upper side of the leaf is darker than the underside which may be grey-green in colour. On the underside of the leaves are founa tuo lines parallel to the mdrib which are considered to be characteristic of coca leaf. The leaves of Erythroxylon coca Lam, are characteristically large and thick, broadly elliptic in shape, more or lese pointed at the apex and aark green in colour. The Leaves of Erythroxylon novogranatense (Morris) Hieron are smaller, narrower, thiner and rounded at the apex. They are bright yellow-green coloured. The leaves of Erythroxylon novograntense var. truxillense (Rusby) Plowman are even smaller and narrower. However they are thicker than the other types and have a rich green colour. B. Coca paste An off-white, creamy or beige coloured powder. Rarely fine, often contains aggregates and generally damp. Unless the aggregates are crystalline (which is rare) they usually break down under slight pressure. It has a characteristic odour. €. Cocaine Although produced from a somewhat variable natural product, by a batch process capable of wide variation, cocaine varies comparatively Litele when compared for example with heroin products. Nevertheless no two illicit samples ot cocaine are exactly identical. For the most part it is a white or off-white powder which is often fine, and rarely dnp. It has a characteristic odour. Adulteration is comparatively rare (but not unknown) within the economically developing countries, the material being internationally trafficked with a purity often of 80 to 90% (es cocaine hydrochloride). Subsequent adulteration and transformation for trafficking purposes within the economically developed countries usually involves the aadition of either an uncontrolled synthetic local-anaesthetic (e.g., lidocaine, procaine or henzocaine) or a carbohydrate (e.g., mannitol, lactose or glucose). In either case the physical appearance is changed only sligntly, tor virtually all adulterants are themselves fine dry white powder:‘The typical purity for trafficking in cocaine within the economically developed countries is about 30%; the internationally trafficked material is subjected to adulteration with about three times its oun weight of the diluent. Occasionally cocaine is encountered as material containing large, sometimes colourless crystals ("rock cocaine"). These crystals can be quite hard. Usually some, if not che major part, of such samples consist of material similar to ordinary "powder" cocaine. If material submitted for forensic examination bears no physical relationship to the description given here, that does not mean, of course, that it is not cocaine or a cocaine containing product.-10- IV. THE ANALYSIS OF MATERIALS CONTAINING COCAINE A. Sampling ‘The principal reason for a sampling proceaure is to produce a correct and meaningful chemical analysie. Because most methods ~ qualitative and quantitative - sed in forensic science laboratories for the examination of drugs require very small aliquots of material, it is vital that these small aliquots be entirely representative of the bulk trom which they have been drawn. Sampling should be undertaken to conform to the principles of analytical chemistry, as laic coun, for example, in national pharmacopoeias or by such organizations as the Association of Official Analytical Chemists. ‘There may be gituations where, for legal reasons, the normal rules of sampling and homogenization cannot be followed if, for example, the analyst wishes to preserve some part of an exhibit as visual evidence. Alternatively, it may be necessary to perform separate assays on two powder items, rather than combining the powders prior to a single assay being performed on the mixture, because each has been separately exhibited by the seizing officer, and the legal system within which the analyst works requires individual results on every exhibit which is to be taken before the courts. To preserve valuable resources and time, forensic analysts should seek, on all possible occasions, to use an approved sampling system and thereby reduce the number of quantitative determinations needea. To facilitate such an approach, the forensic analyst may need to discuss individual situations with both seizing officers and the legal personnel with whom he works. Cocaine is most frequently encountered as a fine powder, although some presentations contain aggregates which may be hard or soft. The aggregates may be of any size. A seizure of cocaine may be ot material within a single container or package, or the material may be inside @ number of packages. Sampling of single package itens ‘The simplest sampling situation is where the submitted item consists of a single package of material ~ in the case of cocaine, the material will most often he a powder. The material should be removea from its container or wrappings, placed in a clean clear plastic bag and the net weight recorded. The material should then be thoroughly homogenised prior to the application of the sequence of chemical tests, although Presumptive testing may be applied at this stage if if is thought that the sampling or homogenization process will be lengthy and there is still some doubt as to the identity of tne material. The simplest way to homogenize a powder is to shake it thoroughly within the clear plastic bag to which it has been transferred. If the powder contains aggregates these may be broken down by passing through successively finer sieves, or by pounding with a mortar and pestle, or by use of an adapted commercial food-mixer or food~processor.-n- Alternatively, the technique of coning-and-quartering can be applied, as follows: the sample is mixed by shaking or stirring. Large fragments are reduced if necessary; the material is then poured on a flat surface to form a cone, The "cone" is flattened and the material is then aivideo at right angles, forming quarters. Opposite quarters are taken for a sample; the remainder of the material is returned to the receptacle from which it was removed. Should further coning-and-quartering be desired to reduce sample size, particle sizes are further reduced, the material mixed thoroughly, poured onto a flat surface, and divided as before. Sampling of items consisting of more than one package ‘The analyst should examine the contents of all packages by eye, and possibly also by simple colour test or TLC to determin 1, If all packages contain suspect cocaine or cocaine-containing material, and/or 2. If one or more packages contain material different to that of the majority of packages. The simplest indicator is the physical appearance of the powder. If one or more packages obviously differ in content, these should be segregated and subjected to separate analysis. ‘The compositing of multiple container items is as tollows: (a) If there are less than 10 packages ~ all packages should be sampled. (b) If there are 10 ~ 100 packages ~ randomly select 10 packages. (c) If there are more than 100 packages - randomly select a number of packages equal to the square root of the total number of packages rounded to the next highest integer. If the powders are founa to be the same then the contents ot a number of packages may be combined; the combined bulk material may then be homogenized in, for example, an adapted commercial food-processor. Alternately, the bulk may be subjected to coning-and-quartering. When different types of material have been identified in the various packages, then each sub-group should be composited in an identical fashion to that previously outline. Sampling errors for quantitative methods are reduced if large aliquots of material are subjectea to sequential dilution with the dissolving solvent. If the total sample size is large, this approach may be adopted. However, when large amounts of material are used for the first dissolution, it may be necessary to add the solvent=by pipette to avoid error due to insoluble materials. It will be rare to find large amounts of insolubles in cocaine samples seizea within the economically developing countries or at importation points into economically developed countries. However, insoluble adulterants are a frequent occurrence in "street" samples seized within economically developed countries.-2- 3. Sompling of materials containing large particles If the particles can be easily reducea to powder, then this approach should be used and sampling procedure followed as outlined previously. Powdering may be achieved by mortar and pestle, commercial food~processor/mixer, or industrial grinder. If the material cannot be easily broken down, then random sized particles should be drawn from at least three different parts of the item. A minimum of 1 granme should be collected, weighed accurately and subjected co assay.Bera B. The analysis of coca leaf Coca leaf, being a vegetable product, requires a different analytical approach to that to be applied co the extracted material, whether it be impure coca paste or the more pure cocaine. The sampling methods may be used on seizures of coca leaf, provided the analyst varies the sampling proceaure to allow tor the aifterent physical makeup of leaf material as opposes to powder. ‘The sampling methodology and the analytical methods described are equally applicable, with minor modification, to coca paste Trafficking in coca leaf is rare (but not unknown) outside chose countries of the developing world where coca is grown. This section has therefore been incluaed in the manual to assist the analyst on those rare occasions he or she may be required to deal with this material. ‘The igentitrcation of both coca leaf and powderea coca leat material should be by @ two part process - botanical and chemical. Ideally the anaivst should be trainea in both botany ana chemistry anc should nave available standard reference materials for both techniques. Le Ph sical identi facati 1, Whole coca leaf This material was described on page 8. Confirmation shoula include microscopy. 2. Powderea coca leat This may he identifies by microscopy. The stanaara textbooks dealing with powdered vegetable drugs contain a section devoted to coca leaf (e.g. "Powdered Vegetable Drugs", Jackson, 8.P. and Snowden, D.W. Churchill, London, 1968, p. 44) 2. Chemical analysis of coca leaf (whole or powdered) Short immersion in boiling ethanol produces eftective extraction of ecgonine-type alkaloids and minimizes the breakdown of cocaine. Extraction with hot methanol has also been found to be effective. A systematic quantitative extraction procedure is to follow the schene illustrated below (taken from Journal of Ethoopharmacolosy, 3 (1981). pp. 293-8).- ue Manicured dried coca leaves (1 Add 40 ml 95% ethanol Reflux 15 minutes Cool Filter Extracted leaves Filtrate discard s Evaporate 20 mi chloroform 10 ml 1.5% (w/v) citric acid (2x) Aqueous layer Chloroform layer JAdjust to pit 8.2 with Nalco, diseara 20 ml chloroform (2x) >, Chloroform layer Aqueous layer discard Dry over Na,S0, Evaporate Residue Add internal standard ec anosisarc If quantitative extraction of the alkaloids is not required, a short extraction at room temperature may be sufficient. The leaves (preferably chopped or powdered) may be pounded with ethanol or methanol in a mortar. The alcoholic extract is subjected to TLC for qualitative analysis of the coca leaf, or (if quantitative extraction has been undertaken) may be subjected to GLC or HPLC to estimate the cocaine content of the leaf.aie C. The analysis of coca paste and cocaine 1. Presumptive tests for cocaine (a) Colour test Ic must be stressed that positive results to colour tests are only presumptive indications of the possible presence of cocaine. The colour tests for cocaine are especially prone to produce talse positives. Many other materials, often harmless and uncontrolled by national legislation or international treaties, may give similar colours with the test reagents. A number of these are either other controlled drugs, often encountered 8s white powders (e.g. methaqualone), or the synthetic local anaesthetics which are often substitutea for cocaine in the illicit traffic. It is mandatory for analysts to confirm such resuits by the use of alternative technique(s). All colour-test reagents should be carefully scrutinizes to ensure that chey have not decomposed. Colour-test reagents that have themselves changed colour may lead to erroneous conclusions shout che nature of the substance under test. The colour test aescribed below is known as the Scoct Test, as tollows: REAGENTS Solution 1. 2% cobalt thiocyanate ~ Co(CNS)z ~ aissolvea in water ana then diluted 1:1 with 96% glycerine (pharmaceutical grade). Solution 2, Hydrochloric acid (concentrated). Solution 3. Chloroform. METHOD The amount of test material should not exceed sutticient to cover the tip of a micro-spatula. STEP 1 The test material is placea in a test tube, 5 crops of solution 1, are added and the mixture is shaken. If cocaine is present a blue colour develops at once. 1f a blue colour is not seen, add more test material. If a blue colour still does not develop, the sample does not contain cocaine. STEP 2. Add one drop of solution 2. and shake. The blue colour wi disappear and a clear pink solution should be seen. Ir all che blue colour does not disappear, add a secon drop (no more) of solution 3 Ado several crops of solution 3. (chloroform) and shake. The chloroform layer will develop an intense blue colour if cocaine as present.-ar- RESULTS Phencyclidine, dibucaine, butacaine and methapyrilene, which give the same colour as cocaine in STEP 1, are all distinguished from cocaine by STEP 3, to which only cocaine gives a blue chloroform layer. Notes 1, The amounts used of solutions 1. and 3. are not critical. However, the ratio of solution I. to solution 2. is critical. 1f excess hydrochloric acid is added to solution 1. after the blue colour has developed with cocaine, a blue rather chan pink solution will result; this blue will not be extracted into che chloroform layer. If excess cocaine is used in STEP 1., then it is sometimes necessary to add 2 drops of hydrochloric acid; no more should be used. 2, The test works on samples containing as little as 1% cocaine. 3. The shelf-life of solution 1, has been shown to be at least 6 months. (>) Odour test ‘The warning given sbout colour tests applies equally co tne odour test for cocaine. REAGENT Methanolic sodium or potassium hydroxice (1 g of potassium or sodium hydroxide dissolved in 20 ml of methanol). METHOD ‘The dried test material is thoroughly moistened witn the reagent ana, after allowing excess alcohol to evaporate, the odour characteristic of the sample is comparea with that of standard cocaine material. Notes 1, Over one hundred drugs were tested for possible interference and only piperocaine (also a benzoate ester) gave a positive result. Certain amines such as amphetamines will produce @ "weak, fishy odour”. 2. The sensitivity is greater than that of existing field tests, e.g. the Scott cest. _ 3. The sample and reagent must be kept free of water which interfers vith the reaction. 4, Ideally the test should be performed concomitantly with a test on standard cocaine material ané the odours cowparea.~ 18 - (c) Microcrystal test The warnings given for colour and odour test apply equally to the microcrystal test. PLATINUM CHLORIDE TEST REAGENT 1 g of platinic chloride is dissolved in 20 ml of water. METHOD Approximately 2 mg of tne sample 18 places on @ microscope slide and dissolved in 1 drap of IN hydrochloric acid. One drop of che reagent is agded. The resulting crystals shoule be viewed with a microscopic magnification of about 10U. Standard cocaine should be analysed concomitantly. The dilution of the test material or the hydrochloric acia may be varied to give the optimum results.~ 19 - 2. Tests for anions associated with cocaine (a) Solubility te METHOD 1. Dissolve @ portion (approximately 1 gramme) of the powder or material in approximately 5 ml of distilled or de-ionised water. For small seizures 0.1 gramme should be used with 0.5 ml of water. 2. Dissolve @ portion (approximately 1 gramme) of the powder or material in approximately 5 ml of ethanol. For small seizures 0.1 gramme should be used with 0.5 ml of ethanol. This will show the presence of any ethanol insolubles such as carbohydrates. The carbohydrates have low solubility in ethanol. Note This test is most useful hen the sample size is large and a considerable quantity of the powder can be used without seriously reducing the total amount of exhibit that can be produced in court. It may be used on small seizures by reducing both the amount of test material ana of solvent. (>) Precipitation tests NTS: 1, Nitric acid: concentrated (contains 70% w/w HNO3) 2. Hydrochloric acid: concentrated (contains 35-38% w/w HCl) 3. Dilute ammonia solution: contains approximately 10% w/w of NH3, ana is made by ailution of concentrated anmonia solution (375 ml to l litre with water). 4. Silver nitrate solution: a 5,0% w/v solution of silver nitrate in water. 5+ Bar1um chloride solution: a 10.0% w/v solution of barium chloride in water. RESULTS Chlorides Solutions of chlorides, when treated with silver nitrate solution, yield a white curdy precipitate which is insoluble in nitric acid, but soluble, after being well washed with water, in dilute ammonia solution, from which it is reprecipitated by the addition of nitric acid.- 20 - SULPHATES Solutions of sulphates, when treated with barium chloride solution, yield a vhite precipitate which is insoluble in hydrochloric acid. Water soluble illicit cocaine products are invariably found to be the chloride salt; the occurrence of any other anion is extremely rare, although sulphate has been encountered. This test should be confirmed, if possible by IR spectroscopy and/or by X-Ray diffraction methods. Complete solubility of the powder or material in ethanol is to be expected for most suspected cocaine samples. Insoluble colourless crystals are a likely indication that the cocaine has been adulterated (eut") by @ carbohydrate such as lactose. The insoluble material may be filtered, dried and subjected to further testing by, for example, 18 spectroscopy. The anount of insoluble material way give a rough guide to the extent to vnich the cocaine has been adulterated, but it must be noted that all the carbohydrates are soluble to various degrees in ethanol.-a- 3. Thin layer chromatography of cocaine (a) Standard technique Coating: activated silica gel G on glass backed plates; the coating contains a fluorescing additive which fluoresces at 254 nm. Layer thickness: 0.25 mm. Plates should be storea in dry conditions, over blue silica gel inside a dessicator and should be protected from chemical vapours. Activation of plates before use should be at 110°C for a minimum of 30 Suze of plate: 20 x 20 cm; 20 x 10 cm; 10 x 5 em; choice depenas on number of samples to be developed simultaneously. Starting point of run = “spotting lane": 1 cm from bottom ot plate. Depth of developing solvent in TLC tank: not more than 0.5 cm, not less than 0.3 em. Distance between applications ("spotting points"): usually 1 cm, never Tess than 0.8 cm. Spots must be positioned at least 1.5 cm from edge of plate to overcome "sige effect". Length of run: optimum is 10 em, for this figure allows easy calculation of Rp values (Method 1. below), However, if Rr values are not required, a simple approach is to allow the solvent to develop to the top of the TLC plate. In such circumstances the plates are arranged so that the maximum development does not exceed 10 cm (Method 2. below). Method 1. For 20 x 20 cm plates a line is drawn 11 cm from the “spotting ena” which gives a 10 cm development for spots applied 1 cm from the bottom. Methoa 2. Plates of 20 x 10 em and 10 x 5 cm dimension are placea in the TLC tank with the 10 cm sides vertical by allowing the solvent to flow co the top of the plete; a 9 cm development is produces. It is important for the analyst to monitor the progress of solvent in both methods; plates must be removed trom the TLC tank as soon as the solvent reaches the “development line” or the top of the TLC plate, otherwise diffuse spots will resule. ~ Size of spot: the solution being applied to the plate spreads outuaras Erom the "spotting pont", This spreading of the solution shoula be restricted as much as possible, otherwise diffuse spots will be produced during development. The ideal size for the application spot is no more than 2 mm in diameter. To achieve, this it may be necessary to apply solutions in aliquots rather than by a single discharge of the spotting equipment. The aliquots may be dried by hot or cold air between discharges. If hot air is usea, care must be taken Co ensure that no component of the mixture under investigation is thermally labile.- 2- The TLC tank and lid: preferably both should be of clear glass; the tank Should be lined with adsorbent paper to assist saturation. The lid should be tight fitting to minimise solvent evaporation. The glass may be ground and/or a smear of petroleum jelly may be applied to the ram. The developing solvent: if a mixture, it should be made as accurately as possible by careful use of measuring cylinaers. If the sane solvent syatens are used daily, it may be convenient to obtain each component via an automatic dispenser. Mixing may be done vithin the TLC tank. The developing solvent, mixture or single component, should be placed vithin the TLC tank in sufficient time to allow saturation to be achieved. With paper-lined tanks this should take approximately 5 minutes. It is important to note that, for certain aeveloping systems, the solvent must be renewed after each development, or at least after 2 to 3 DEVELOPING SOLVENTS SYSTEM A: chloroform 25% by volume Dioxane 60% by volume Ethyl acetate LOR by volume Anmonia (29%) % by volume SYSTEM Bs Methanol, parts by volume Anmonia (29%) pares by volume SYSTEM C: cyclohexane 75% by volume Toluene 15% by volume Daethylamne 10% by volume Preparation of solutions to be applied co the TLC plate Illicit cocaine samples At a concentration of 1 mg per ml in methanol. Standards solutions* All made at a concentration of 1 mg per ml in methanol. 4N.B. The form of standards used , salt or base, is unimportant. Either form will be satisfactory; on the TLC plates che compounas always move as the free base.-23- VISUALIZATION The plates must be dried prior to visualization. This can be done at room temperature or, more quickly, by use of hot air. In the latter case, core must be exercised that no component of interest is thermally labile. It is important for proper colour development that all traces of anmonia or other bases are removed from the plate. Visualization methods 1. UV Light ac 254 am. 2. Aciditied potassium iodoplatinate reagent. 3. Dragendorff's reagent (Munier). Preparation of spray reagents ACIDIFIED POTASSIUM IODOPLATINATE REAGENT ‘ Dissolve 0.25 gramme of platinic chloride and 5 grammes of potassium iodide in water to 100 ml and add 2 mi of concentrated hydrochloric acid is adaed. DRAGENDORFF'S REAGENT Max together 2 g of bismuth subnitrace, 25 ml of concentrated (glacial) acetic acia and 100 ml of water to produce solution (1); dissolve 40 g of potassium iodide in 100 ml of water co produce solution (2). Max 10 mi of solution (1), 10 mi of solution (2), 20 ml of concentrated (glacial) acetic acid and 100 ml of water to produce Dragendorff's reagent. RESULTS Re x 100 values: conrouno DEVELOPING SYSTEM a & cocaine a 39 36 Eegonine 0 a 0 Methy lecgonine a 65 ba Benzoyleczenine 3 25 ° Cinnanoyleocaine 83 3 3 Tetracaine °3 se ~ 8 Benzocaine n 80 n Lidocaine "7 6 40-558) Pethidine a ug °9 Nethaqualone a 8 38 Methadone 75 31-456) 1 Procaine a 35 B16(s) (s) = streak, not spot, produced on the TLC plate.re 4. Gas Liquid chromatography of cocaine (a) Packed column technique Detector FID (Hydrogen at 30 ml per minute, air at 450 mi per minute). colume 6 ft (or 2m), I.D. 2 to 4 mm, Packing SE~30; OV-1; ov-17, carrier gas Nitrogen at 30 ml per minute. Operating conditions Injector temperature: 220°C. Oven temperature: 220°C. Detector temperature: 300°C. Internal standard an-tetracosane or tetraphenylethylene. Derivatizing agent —_N,0 bie-trimethyleilylacetamide (BSA) or N-methy1-N-trimechylsilyltrifluoroacetamide (NSTFA). Conditioning of packed columns Prior to use, all packed columné must be conditioned. Usually the conditioning temperature should be at least 30°C above the temperature at which the analysis is to be performed, unless this would require exceeding the upper operating temperature of the column specified by the manufacturer. In chis case, a smaller temperature differential must be used and the conditioning period substantially extended. Example: for a column to be used at 235°C, which has an upper working temperature of 300°C, an ideal conditioning temperature would be 270°C. A typical conditioning period is usually overnight, or a minimum of 15 hours. 1f, in the example, the upper recommended limit for the column was 280°C, then conditioning could be performed at 260°C for 30 hours. For some columns, conditioning from Friday evening till Monday morning may be required. During conditioning, the carrier gas to be usea experimentally is passed through the column at the same flow rate as in the analysis, e.g., nitrogen at 30 ml per minute measured at conditioning temperature. During conditioning, it is vital that the end of the GC column not be connected to the inlet line of the GC detector. This is because silica bleeds from the solid part of the stationary phase during conditioning and would soon build up within the detector. This would severely affect detector response and ultimately, in the case of an FID, prevent hydrogen combustion by blocking the orifice of the burner. In ordinary operation silica bleeds from the GC column, and the blocking of an FID burner is one of the most common causes of deteFioration of detector response. In such circumstances a simple approach is to raise the operating temperature of the detector by as much as 5U to 100°C (it within the capability of the gas chromatograph). This is to volatilize ("burn-off") the deposited silica. If this is unsuccessful the only solution is removal of the burner from the FID, unit and mechanical removal of the silica deposit. Washing with water, detergent ana abrasives, followed by drying with organic solvents has been found effective.~ 25 - METHOD Standard solution of cocaine hyarochloride is prepared at a concentration of 40 mg per 100 ml in ethanol or methanol. Treat the illicit cocaine similarly, using at least 20 mg sample to give a cocaine concentration approximately equal to that of the standard solution. Derivatization procedure - see capillary column technique. Inject 2 to 5 microlitres as appropriate. ‘The content (%) of any component can be calculated using the general formula: Cr. std. 4x / Aint.std.in sam. chrom, ge = Ty x 100 sam, Ar std./ Aint.std.in std. chrom. Where: ©,% = content of component x in che sample (w/w 2). std, ™ Concentration of substance x in the standard reference solution (w/w). 4, = peak area for substance x during the sample chromatography. Aint. std. in sam. chrom, 7 Peak area of the internal standard obtained curing the sample chromatography Aint. std. in std, chrom. = peak area of the internal standarc. obtained during the standard chromatography Cyam. = concentration of the sample (w/v %). (>) Capillary column technique Column OV-1 = chemically bonded fused silice capillary. Film thickness 0.15 um Length 25 m. by 0.32 mm. ied. Carrier ga Hydrogen. Flow race 2 ml per minute . Split ratio 1:50 Detector FID.Bee) Operating temperatures Injector.....250°C. Detector. ....280°C. Programme....Start at 150°C., immediate increase at 9°C per minute to 280°C, Sample preparation: 3-4 mg of the sample, 1-2 mg of internal standara (n=CgqH59), 1 ml chloroform, 0.2 ml pyridine and 0.15 ml MSTFA (silylating reagent) are mixed and heated at 70°C for 10 minutes. Injection volume: 1 ul-~a- 5. High performance liquid chromatography of cocaine Operating conditions Column 160 mm by 5.0 mm id. Packing material Octadecyl-silica HPLC grade 5 um Mobile phases Eluent A Methanol (300 m1), water (700 ml), 1% (v/v) —— phosphoric acid (1000 ml) ana n-hexylamine (10. 71g5 14 m1) (pH = 2.5). Elueat_B Methanol (1000 mi), 1% (v/v) phosphoric acid (1000 m1) and n=hexylamine (10.712; 14 ml) Apit 2.8). The 1% phosphoric acia is preparea by dissolving concentrated orthophosphoric acid (17 g) in distilled water (1000 ml). Degassing of mobile phase Dissolvea atmospheric gas within the mobile phase must be removed prior to the start of the analysis. If this is not done, the gas comes out ox solution and forms small bubbles either in the tubing between the solvent reservoir and the pump-head(s) or within the cylinder(s) of the punp-head(s). In either case, and especially the latter, pumping will cease and the chromatographic development will be ruined The simplest way of removing dissolve gas is to immerse the eluant mixture in an ultrasonic bath at high power for a minimum of 10 minutes. 4 problem which may arise with this approach is that the water of the ultrasonic bath may become warm during a long degassing period, and the heat may be transferred to the eluant. Addition of ice to the water in the ultrasonic bath will maintain the eluant at ambient temperature. Degassing by this method may be performed in situ by arranging for the dedicated use of an ultrasonic bath which is included as part of the entire HPLC system. The solvent reservoir is placed within the ultrasonic bath. Frequent and relatively short degassing periods shoula be used, possibly between each chromatographic development. The composition of the eluant must be taken into account when degassing. Components which are particularly volatile and/or which constitute only a small proportion of the eluant should not be exposed to frequent degassing. It is vital to keep the eluant reservoir closed to the atmosphere if the problem of dissolved gases is to be avoided. It has been claimed that the wltrasonic technique is agt completely effective, and that the only fully efficient way of cegassing eluants 16 to pass helium or argon gas slowly through the solution. This approach may also be done in situ by having a gas cylinder of helium or argon connected via tubing into the solvent reservoir. Frequent cegassing may be performed between chromatographic developments. It is important to maintain the solvent reservoir beneath an atmosphere of the inert gas chosen for degassing.~ 28 - Flow rate 2.0 mi per minute for both eluents. Detection vv at 230 an Sample preparation all materials are dissolved in the appropriate eluent. Standard solutions dissolve approximately 1 mg in 10 ml eluent of any of the following substances Cocaine Cis-cinnamoyleocaine ‘Trans~cinnamoy cocaine Procaine Lignocaine Any locaine Butacaine Benzocaine Injection volume 20 yl by loop injector. Quantatation by peak areas, internal or external standard methods. RESUL! Elution order and the corresponding capacitance ratios (K')* for eluent A are as follows: Procaine 0.0 Lignocaine 0.79 Cocaine 2.68 Cis-cinnamoyleocaine 6.3 Anylocaine nay Butacaine 8.97 ‘Trans-cinnamoylcocaine 10.65 Benzocaine 20.06 “Capacitance ratio K' = tr - to there ty= time of elution of compound tom time of elution of non-retained material (methanol injection)~ 29 - 6. Infra red spectroscopy of cocaine Sample preparation 1. Halide disc method ‘The finely powdered dry material, about 2 mg, is mixed with an alkali halide, about 200 mg, ground either mechanically in an agate ball mill or by hand in an agate mortar, and pressed into a thin disc. The ideal is to produce a dise as nearly transparent as possible. Originally, potassium bromide was used as the alkali halide, and the technique is often referred to as the "KBr disc method". Potassium chloride has also been widely used and is often considered to be superior to potassium bromide because it is less hygroscopic. Whatever halide is used, it should preferably be of "IR standard", aried at 105°C for a minimum of one hour and stored above a strong dessicant (for example phosphorus pentoxide) in a dessicator. Analytical grade halides, powdered to the same degree as "IR standard" material and dried to tne same degree, are acceptable. ‘The major disadvantage of this method is the need for disc pressing equipment. However, at the present time, a number of commercially available disc pressing systems are very inexpensive relative to the cost of an IR spectrophotometer. Another disadvantage is the possible proguction of an erroneous spectrum if double decomposition occurs during the preparation of the disc: Base.Hcl + KBr =----) Bese.tar + KCL liydrochlorides should, therefore, always be exemined in potassium chloride and not potassium bromide. The anion test using precipitating Feagente should precede IR spectroscopy. The major advantage of the halide disc method 1s that, provided the halide dispersant is free of water, it should contribute no interference to the resulting spectrum. Another minor advantage is that, with care, the discs may be stored indefinitely. This may be important in any subsequent legal proceeding. Aleo, the material under investigation can be recovered from the halide disc for further testing. 2. Micro halide dise method There are commercially available aies which can produce a halide disc only lm in diameter. In this case, the quantity of halide has co be drastically reduced (approximately 1 mg). The use of this technique finds most application in the IR examination of unknown components that have been eluted from TLC plates. It may also be used 1f the analyst cisposes of less chan 1 mg of sample. + Nujol mull method (Liquid paraffin methoa) The finely powdered sample (2-3 me) is mixed with one drop of the liquié paraffin and ground in an agate mortar. Sufticient liquid is then addea so that the final mull is che consistency of a thin cream. The mull is spread on an alkali halide disc, usually NaCl or KBr, and a similar disc placed on top. The film between the halide discs should contain no air bubbles.