Microcrystalline Tests in Forensic Drug Analysis

Microcrystalline Tests inForensic Drug Analysis

Mathieu P. Elie and Leonie E. ElieSchool of Natural and Applied Sciences, Universityof Lincoln, Lincoln, UK

1 Introduction 11.1 History 21.2 Modern Use 2

2 Chemical and Forensic Context 22.1 Microchemical Identification 22.2 The Value of Microcrystalline Tests for

Forensic Drug Analysis 22.3 Type of Evidence Provided by

Microcrystalline Tests 33 Technical AspecT 4

3.1 General Principle of MicrocrystallineTests 4

3.2 Sample and Reagent Preparation 43.3 Crystal Growth 53.4 Crystal Classification and Description 63.5 Storage, Shelf Life, and Sample

Recovery 84 Application Examples 8

4.1 Cocaine 84.2 Diamorphine (Heroin) 94.3 Methadone 94.4 γ -Hydroxybutyrate 94.5 Ketamine 104.6 Phencyclidine 104.7 Amphetamines and Methamphetamine 10Acknowledgments 12Abbreviations and Acronyms 12Related Articles 12References 12

Drug detection in the forensic context requires numerousanalytical techniques. Depending on locally adoptedstandard procedures, different techniques are used forscreening solid samples for potential illicit substances(e.g. Fourier transform infrared spectroscopy (FTIR)). Asample suspected of containing prohibited material passesthrough another layer of techniques to confirm that theillegal substance is present and to identify it (e.g. liquid/gas

chromatography). Finally, the drug is systematicallyquantified (e.g. mass spectrometry). Microcrystalline testsfall within the second step of this analytical process. Theyare low cost because of the minute amount of reagents usedand the simplicity of the instrumentation and consumablesrequired to perform the analysis. They offer all the featuresrequired by a good confirmation technique while beingvery fast to administer and interpret, although they do notoffer quantification capabilities.

Microcrystalline tests are chemical tests resulting in theformation of unique microcrystals for a given substancewhen combined with a specific reagent. Microcrystalsare observed under a microscope and micrographs ormicrovideos constitute the results of the test.

Thanks to the chemical mechanism by which micro-crystals develop, microcrystalline tests can be appliedto molecules of various sizes, shapes, charges, and withdifferent functional groups, and can naturally distinguishbetween enantiomers.

1 INTRODUCTION

Forensic drug analysis is a vast field that involves theapplication of a range of analytical techniques to identifyand quantify drugs of abuse, to support authorities duringinvestigation proceedings. Abuse of drugs is a worldwidehealth problem touching all layers of the populationirrespective of age and socioeconomic background.

In 1961, an international treaty was signed: the SingleConvention on Narcotic Drugs (SCND), which prohibitsthe production and supply of specific drugs (or relateddrugs) except those under license for medical or researchpurposes. The substances addressed by the SCND wereopiates, cocaine, and cannabis. In 1971, the Conventionon Psychotropic Substances (CPS) added lysergic aciddiethylamide (LSD), Ecstasy, and other psychotropicdrugs. Finally, the United Nations Convention AgainstIllicit Traffic in Narcotic Drugs and PsychotropicSubstances introduced regulations on precursors in 1988.

The SCND offered a base to develop nationalregulations across the globe, for example, the SubstanceControl Act in the United States (1970) and the Misuseof Drugs Act in the United Kingdom (1971).

Commonly abused substances regulated in theseacts include opioids (morphine, heroin, codeine, andmethadone), cocaine, amphetamine, methamphetamine,3,4-methylenedioxymethamphetamine (MDMA), canna-bis, benzodiazepines, γ -hydroxybutyrate (GHB),flunitrazepam, phencyclidine (PCP), and barbiturates.

Numerous analytical techniques exist to screen seizedmaterial for the presence of drugs and to quantifyany amount detected. In this analytical framework, the

Encyclopedia of Analytical Chemistry, Online © 2006–2009 John Wiley & Sons, Ltd.This article is © 2009 John Wiley & Sons, Ltd.This article was published in the Encyclopedia of Analytical Chemistry in 2009 by John Wiley & Sons, Ltd. DOI: 10.1002/9780470027318.a9102

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purpose of microcrystalline tests is to make a definite andfinal identification when a drug is present in a sample.

1.1 History

In the early 1800’s chemists started to apply reagentson drugs, pharmaceuticals and food stuffs and observedreactions such as color changes through a microscope.With the development of the polarized light microscope,the observation of crystals evolved rapidly because itreveals local differences in specimen structures, althoughit was mostly utilized for mineralogy.(1,2)

In 1827, Raspail introduced the term ‘‘chemicalmicroscopy’’.(3) By then he had already discoveredmicrochemical tests for the detection of sugars, oils,and albumin within cell material. In the middle of thenineteenth century, toxicologists started to study crystalformations of alkaloids when combined with reagents,and the first crystal tests emerged. From then on,microcrystalline tests developed more systematically upto the publication of ‘‘Microchemistry of Poisons’’ byWormley in 1867, which included microcrystal illustra-tions engraved upon steel.(4)

The technique evolved across inorganic and organicchemistry from the simple use of a microscope to aidchemical identification to the development of reactionsthat led to the formation of characteristic crystals.

Fulton published the only textbook on microcrystallinetests, which covers numerous microcrystalline tests for theidentification of narcotics and drugs of abuse from simpletest setup to result in the interpretation and developmentof new tests.(5) This provided a detailed classification forcrystal characterization including numerous micropho-tographs showing various shapes and appearances.

Clarke extended the range of microcrystalline testsmostly focusing on toxicology applications.(6) At a timewhen analytical techniques such as chromatography wereevolving fast but could not yet routinely be used toidentify unknown drugs, reliable microchemical testssuch as microcrystalline and color tests were neededand developed.

After 1969, publications on microcrystalline tests fordrugs were based either on Fulton’s or on Clarke’s workand only rarely contained new tests.

However, when modern separation and detectiontechniques, especially gas chromatography/mass spec-trometry (GC/MS) and liquid chromatography/massspectrometry (LC/MS) became widely available andreliable, techniques that did not provide any molec-ular, structural, and/or compositional information weregradually neglected and eventually used as presump-tive/screening tests. Microcrystalline tests were entirelyremoved from Clarke’s Isolation and Identification ofDrugs, second edition (1986).(7)

1.2 Modern Use

It is difficult to assess how widely microcrystalline testsare now used for forensic drug analysis. On the basis of thesmall number of recent publications, research activity inthe development of microcrystalline tests seems limited,and existing guides issued by the American Society forTesting and Materials (ASTM) describing such tests areoutdated.

However, the recent emergence of GHB on thedrug facilitated sexual assault (DFSA) scene has seenthe sudden publication of three papers proposingmicrocrystalline tests for this drug.(8 – 10)

Indeed, GHB being a very small (104.1 g mol−1)and simple molecule, finding a specific test for itwas challenging. Nevertheless, GHB can be detectedeffortlessly using a microcrystalline test, thanks to thetechnique mechanism.

2 CHEMICAL AND FORENSIC CONTEXT

2.1 Microchemical Identification

A microcrystalline test is a chemical test and the result is amicrocrystal observed under a microscope. After allowingthe sample to meet with a carefully chosen reagent, testingwhether a drug is present using this technique relies oncomparing the shape, size, color, and spatial arrangement(also called habit)(6) of the microcrystals formed with thethose formed in a control with the same reagent. Thecontrol is an authentic sample of the drug tested for. Ifthe crystal’s shape, size, color, and spatial arrangementsmatch, the analyst can conclude a positive identificationof the drug.

2.2 The Value of Microcrystalline Tests for ForensicDrug Analysis

The microcrystalline test technique is often wronglyclassified as a screening technique and/or a presumptivetechnique. A microcrystalline test should be classified andconsidered as a confirmation technique with screeningcapability.

A good confirmation technique must have threemain qualities. First, the technique needs to presenta certain predictive aspect where the results obtainedare expected. The confirmation technique is chosenbecause of its specificity and sensitivity for the suspectedsubstance. Second, the technique should consider thewhole compound and not just its reactive groups.Finally, a confirmation technique must be different, eitherin its nature or its results, from any test previouslyused to screen and identify the substance. Differentanalytical techniques base substance identification on


MICROCRYSTALLINE TESTS IN FORENSIC DRUG ANALYSIS 3

different principles: e.g. retention times/Kovacs indicesin liquid or gas chromatography, fragmentation patternsin mass spectrometry, and transmitted energy at specificwavelengths in FTIR spectroscopy. Similarly, the typeof data generated ranges from the intensity dischargedat a particular emission wavelength, measured witha fluorescence detector, to the relative abundances offragment species in a mass spectrum, depending onthe detection technique employed. A good confirm-ation technique is carefully chosen considering theidentification technique employed, because it needs toadd a new layer of evidence to the entire analyticalprocess.

GC/MS is the main confirmation technique employedfor forensic drug analysis because it fulfills all criteria of agood confirmation technique by combining two differentidentification techniques (GC and MS), and presents theadvantage of coupling confirmation and quantificationwhen necessary. However, before injecting into a GC/MS,it is often required to derivatize substances to reduce thepolarity and enhance the volatility of high molecularweight polar drugs, to stabilize a thermally unstable drug(e.g. GHB dehydrates to GBL in the injector’s liners),(11)

or to increase the molecular weight of very volatile drugsto complex the fragmentation profile, thus increasing theconfidence in identification/confirmation. In such cases,the substance is not directly analyzed: the derivative ofthe drug is considered. Therefore, while considering thewhole molecule, the GC/MS technique is often indirectwhen the derivatization of the compound is requiredbefore analysis.

Microcrystalline tests are direct tests: the testedsubstance is directly the cause for an observable effect.These tests also fulfill all the criteria of a good confirma-tion technique. First, the test reagent is specifically chosento induce the development of specific microcrystals withthe analyte and the test is invariably compared with acontrol test. This demonstrates the predictive mannerin which microcrystalline tests are used. Secondly, thetested drug molecule is an integral part of the micro-crystal formed (Section 3.3) unlike other tests in whichthe analyte simply induces a change in the reagent (likemost color tests). The shape and growth directions of thecrystals are dictated by the physical and chemical proper-ties of the molecule. Finally, the microcrystalline testingtechnique principle and the type of results produced(micrograph/microvideo) are unique compared to anyother analytical technique in forensic drug analysis, there-fore adding new evidence to the analytical case.

Moreover, to be carried out microcrystalline tests donot require the highest purity of substance. Some purifi-cation procedures can sometimes be used before testing(Section 3.2). However, provided that other substancesdo not react in a similar way, if at all, with the reagent

used to carry out the test, and provided that the impu-rities, diluents, and adulterants reactions of the with thereagent do not prevent or mask the formation of charac-teristic microcrystals for the drug tested, the sample canbe analyzed directly without predetection separation ofthe target compound from its matrix.

An analyst could be tempted to try to relate theamount or the size of the microcrystal formed with theabundance of the tested drug in the sample. However,the number of crystals formed and their sizes can greatlyvary with the temperature, hygrometry, and pressureunder which the experiment takes place as well as with thepresence of diluents/adulterants. No published attemptto quantify a substance has been reported using themicrocrystalline testing technique.

Finally, microcrystalline tests need to be specific asopposed to indicative or characteristic. Sometimes, areagent induces resemblance between the microcrystalsformed with two or just a small number of drugs whilebeing very different from any other substances. In caseswhere differences between the two microcrystals formedare too minute, the microcrystalline test is consideredhighly characteristic for the singled out compoundsbut not specific enough to be used as a confirmatorytest. However, this nonspecific but highly characteristicreagent can be employed as a powerful screening test.

In absolute terms, a test can only be called specificafter having tested every single existing substance witha reagent and witnessing that the specific microcrys-tals obtained occur only for one particular targetedcompound. However, because of the predictive contextin which microcrystalline tests are utilized, this is not anissue.

2.3 Type of Evidence Provided by MicrocrystallineTests

Recording the results of a microcrystalline test is verystraightforward. In addition recording the shape, size,color, spatial arrangement of the microcrystals formed,and the magnification used in writing, the analysttakes a micrograph of both sample and control. Themicrophotographs must show a scale for comparisonpurpose between sample and control. The scale used hasto be introduced using a graticule slide. Nowadays, digitalmicroscope software (such as GE5 Digital MicroscopeR1.0.0.1 from View Solutions, Inc.) are capable ofconverting pixels into micrometers instantaneously,taking into account the magnification used. They alsoallow very precise direct measurements of the crystalsdimensions, angles, areas, and cluster radius, instead ofjust showing a scale of the micrograph.

Moreover, the digital microscope is capable ofrecording videos of a microcrystalline test. This is a


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considerable advantage over a classic microscope becauseit addresses various problems intrinsic to the techniquewhen it comes to microcrystal observation. First, severalmicrocrystalline tests see the formation of reagent crystals(crystals formed by the reagent on its own) on top of thecrystals of interest, sometimes leading to difficulties inspotting them. Fortunately, reagent crystals and drugcrystals do not form at the same time: the reagentcrystals generally appear later. Without the capacityto film the test, the analyst has to keep monitoringthe development of the microcrystals because thereis no definite time when the crystal of interest willhave grown enough to be interpreted. The developmenttime is dependent on temperature, hygrometry, andanalyte concentration. Moreover, reagent crystals canform very suddenly and rapidly all over the monitoredarea; sometimes, even while the analyst is trying to takethe microphotograph. Secondly, some microcrystallinetests see the development of transient microcrystals ofvariable lifetime, which may be missed by the observer.This is particularly true for very low concentrations of thedrug. Possessing a full record of the test allows the analystto go back in time on suspicion that transient crystals mayhave occurred.

Therefore, the analysts can record the full length of thetest and study the video afterward and easily snapshot thetest at any point in time.

Micrographs and microvideos constitute permanentrecords that are very straightforward to present in court.Microcrystals are specific and a simple rapid comparisonbetween the sample and the control will undeniably provethe drug present if the test is positive.

3 TECHNICAL ASPECT

3.1 General Principle of Microcrystalline Tests

Performing a microcrystalline test is straightforward andis described in Figure 1.

Step 1 is discussed in detail in Section 3.2. Step 2consists of applying a microdrop of the sample in thesolution on a glass slide and depositing a microdrop of thereagent solution on top of it. Microdrops volume usuallyranges from 1 to 50 µL depending on the available samplesize and should not be generalized. Typically, the reagentmicrodrop will have the same volume as the sample. Usinga plastic pipette tip, some gentle stirring of the combinedliquid droplets can be applied to encourage the reagent todiffuse in the sample when their respective solvents are ofvery different viscosity (e.g. when glycerol is used in thereagent solution). The stirring should not exceed a fewseconds because this will result in the distortion of thecrystals that are forming. Alternatively, it is possible tomix the sample and the reagent solutions before applyinga microdrop of the mixture on the slide. However, thisshould not make any significant difference because of thelow concentrations generally utilized in microcrystallinetests. In Step 3, the analyst constantly monitors the testlooking for the formation of microcrystals specific to thesubstance tested for. Alternatively, a digital microscopecan be set to record the length of the test (Section 2.3).

Chemically, microcrystalline tests are simple additionreactions where the targeted drug complexes with areagent; the addition products formed crystallize astheir concentration increases. The chosen solvent shouldfavor the reaction by ensuring optimum solubility of thedrug and the reagent. Typically, the reagents are heavymetals complexed with halogens, but any complex acidsand anions are potential reagents when testing for newsubstances (Table 1).

3.2 Sample and Reagent Preparation

Before the test, solid samples may need to undergosome purification procedure. It is always advisableto observe powders under a microscope to evaluatetheir homogeneity. If they are found heterogeneous, amechanical separation of the different grains is performedunder a microscope when possible.

SampleMicroscope slide

Dissolve 10 µL 10 µL

Reagent

Microcrystals

Microscope

Monitor the formation ofcrystals while the solventevaporates

Step 1 Step 2 Step 3

Figure 1 General microcrystalline test principle.



Table 1 Examples of reagents

Complex metal halides Organic acids Simple oxygen acids

HAuCl4 Picric acid Cr2O72−

HAuBr4 Picrolonic acid MnO4−

H2PtCl6 Trinitrobenzoic acid ClO4−

H2PdCl4 5-nitrobarbituric acid (dilituric acid)

Basic reagents Complex oxygen acids Neutral or basic reagents

Na2CO3 Phosphorimolybdic acid Iodine-KINa3PO4 Arsentimolybdic acid HgCl2NH4OH NH4Cr(NH3)2(SCN)4 (Reinecke salt)

Most good microcrystalline tests do not require furtherpreparation; however, in cases where the diluents oradulterants interfere strongly with the formation of theexpected characteristic microcrystals obtained for thetarget compound, the analyst may choose to use thinlayer chromatography (TLC), liquid–liquid extraction(LLE), or solid phase extraction (SPE) to purify thesample before microcrystalline testing it.

In the case of liquid samples (e.g. freebase cocaine indiethyl ether, GHB syrup), the samples are evaporated todryness at room temperature under a stream of inert gasbefore testing, although applying heat could induce loss ofmaterial and/or denature the sample. This has the addedadvantage to facilitate the preparation of an adequateconcentration of the sample in an adequate solvent forthe test.

Most microcrystalline tests require the drug to beinitially put into aqueous solution. The solvent ofchoice was water for a long time until Fulton startedto modify aqueous tests with HCl, then extended therange to phosphoric, acetic, and sulfuric acids of variousconcentrations.(5) Indeed, if acid drugs readily unite withcations in water, an acidic environment helps basic drugsand amphoteric substances to connect with the reagents.

Up to 50% ethanol is sometimes incorporated in sometests to help dissolving certain substances; however, ahigher proportion is to be avoided because it tendsto precipitate reagents. Glycerol is often used as anadditive to the reagent because it slows down thecrystallization process and allows more time for theion–drug complexes to pack harmoniously.(5) It alsooften lengthens the time necessary for reagent crystalsto appear, which is desirable. It is unwise to try to slowdown the evaporation rate using a cover slip becausecrystals are three-dimensional entities and would end upbeing distorted and uncharacteristic.

Typically, reagents range between 0.1% and 2%in concentration.(5,6) Few tests necessitate a higherconcentration and numerous tests show more crystalformation with far less reagent. A forensic analystalways tries to minimize the concentration of the

reagent used because it lowers the formation of reagentcrystal, potentially interfering with the observation of themicrocrystals of interest. It also has the benefit of loweringthe cost of the tests.

Ideally, the drug concentration should be approxi-mately the same as that of the reagent. This is oftendifficult to achieve because of the unknown quantityof diluents, impurities, and adulterants present in thesample; however, an experienced analyst knows whatkind of levels are to be approximately expected andinvariably prepares a series of dilutions of the sampleready to be tested.

Given a drug and a reagent, the composition andchemical structure of the microcrystal formed is extremelyuseful to know when it comes to choose the bestconcentration to be used for the test. Indeed, knowing themolecular/atomic ratio between drug and reagent allowsevaluating the order of magnitude in which the reagentis incorporated into the crystal, hence avoiding too highreagent concentrations that would lead to the formationof a high density of reagent crystals.

Unfortunately, information on crystal stoichiometryis sparse, although recent work using X-ray diffractionshowed that the GHB : Ag+ molar ratio was 2 : 2 in themicrocrystal formed between silver (I) and GHB.(9)

3.3 Crystal Growth

The formation of a crystal starts with an event callednucleation, which is the beginning of the transition fromliquid phase to solid phase. Nucleation begins when theconcentrations of the drug and the reagent are sufficientfor the molecules to form enough addition products,which will then result in a number large enough to forma protocrystal that grows until it reaches a critical size. Itthen falls out of solution, becoming a microcrystal, whichcontinues to grow.

In the context of microcrystalline testing, nucleationcan be unassisted: the nucleation sites such as surface ofthe liquid, suspended impurities, and minute gas bubblesoccur naturally. In the case of assisted nucleation, crystalnucleation is encouraged by introducing a solid into the


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solution or scoring the microscope slides used for the testwith a diamond point.

It is tempting to try to increase the concentrationof the molecules in solutions to speed up the nucleationprocess by applying heat to the test to increase the solventevaporation rate. However, molecules naturally try toposition themselves to achieve the closest packed spacebecause it is the configuration that requires the lowestenergy to maintain inducing the most stable state. Formicrocrystalline tests relying on shape comparison, it isessential to allow time for the molecules to completethis proper packing so that the properly developed

100 µm

100 µm

100 µm

(a)

(b)

(c)

Figure 2 Drying speed effect on crystal shape. (a) Typicalelongated right-angled GHB + lanthanum/silver nitrate micro-crystals allowed to grow at room temperature. (b) The samemicrocrystals after gently blowing hot air on the test area duringthe analysis are distorted, extremely thin, and are all mergedtogether. (c) The same microcrystals after positioning the slideon a hot plate throughout the experiment are malformed anddo not show any right angles.

100 µm

100 µm

(a)

(b)

Figure 3 Support surface tension effect on microcrystalspatial configuration. (a) GHB + lanthanum/silver nitratemicrocrystals developed on a SEM carbon stub; (b) the samemicrocrystals grown on a generic glass slide.

microcrystals can be observed. This is why speedingup the drying process is not advised because it willinduce distortions and malformations of the crystals(Figure 2).

The different surface tensions of support surfaces usedto develop the crystals, impact on the spatial arrangementof the crystals. Typically, microcrystalline tests are carriedout on generic microscope glass slides that are slightlyhydrophilic. This type of surface tends to favor thehorizontal spreading of microcrystals clusters. On thecontrary, a hydrophobic surface such as a scanningelectron microscope (SEM) carbon stub encourages themicrocrystal clusters to bloom vertically (Figure 3).

3.4 Crystal Classification and Description

Crystals can be classified according to their chem-ical/physical properties. Within this classification, crys-tals fall within four categories: covalent crystals (e.g.diamond), metallic crystals (e.g. metallic titanium crystal),



(a) (b) (c) (d)

(e) (f) (g) (h)

(l) (m) (n) (o)

(p) (q) (r) (s)

(i) (j) (k)

Figure 4 Examples of microcrystalline structures. (a) Dendrites; (b) tufts; (c) stairs; (d) rods; (e) bundles; (f) prisms; (g) brushes;(h) X’s; (i) clusters of prisms; (j) wires/feathers; (k) spindles; (l) skeletons of blades; (m) Blades; (n) spirals of feathers; (o) fans; (p)hairs; (q) rosettes of needles; (r) disks/globes; (s) skeletons of needles.

ionic crystals (e.g. sodium chloride), and molecular crys-tals (e.g. sugar). Usually, microcrystals created during amicrocrystalline test are ionic and molecular crystals: theunits composing the crystal are drug molecules and metalions. The internal order of the crystal is dictated by howthe drug molecules arrange themselves around the metalions and how these addition products further positionthemselves relative to each other.

A forensic analyst records the crystalline structure todescribe the crystal obtained with a test. Both Fultonand Clarke suggested descriptive terms to depict micro-crystals; however, they both agree that the descriptions

cannot be exact as different forms overlap. For example,a needle becomes a blade when it is broad enough, anda blade transforms into a plate when length and widthare similar. The spatial arrangement of the microcrystalsrelated to each other is also recorded if characteristic, suchas rosettes, clusters, and fans. Because the shape, size,color, and spatial arrangement of microcrystals obtainedfor the sample need to perfectly match the control micro-crystals for a positive identification, micrographs are a farsuperior mean to record microcrystalline test results thana written description. Examples of crystalline structurecan be seen in Figure 4.


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NH2

CH3

H

NH2

HCH3

L-Amphetamine

D-Amphetamine

D-Methamphetamine Phencyclidine Methadone

Cocaine Diamorphine

Ketamine Dihydrofuran-2(3H)-one(GBL)

g -Hydroxybutyrate(GHB)

NH

CH3

H

NHClO

N

O

O

O

O

O

O

N

O

OH

OH

N

O

O

O

O

O

O

HN

Figure 5 Chemical structures.

3.5 Storage, Shelf Life, and Sample Recovery

The interpretation of the test is generally performedbefore complete drying because as the solvent leveldecreases, more and more reagent crystals form, inter-fering with the analyst’, observations. However, specificmicrocrystals can often still be observed at this stage, espe-cially when using low concentrations of reagents. Oncethe test drop is entirely dry, the shelf life of the resultingcrystals varies depending on the reagent and the sampleitself. Over time, microcrystals degrade and their shapeschange, making it impossible to interpret them correctly.For the preservation of the crystals for a prologed periodof time, if required, Clarke describes a procedure using thehanging microdrop technique to perform the microcrys-talline test (which consists in depositing the microdropon a glass cover slip and positioning it upside down on acavity slide to minimize the evaporation rate) followed bythe application of a sealing agent, bitumen in toluene, toenclose the cavity and prevent evaporation.(6) He claimsthat the slide may be preserved for several months withthis technique; however, bitumen contains heavy metalsand could interfere with the test. Alternative substances

with low gas permeability, such as ceramic-based sealant,could be used.

At first glance, the microcrystalline test techniqueseems destructive, which is always a problem whendealing with minute amounts of sensitive, potentiallyincriminating material. This is a misconception becausethe chemical reactions occurring during a microcrystallinetest are addition reactions involving only the creation ofionic bonds between the drug and the reagent (Section3.1). It is possible to recover the sample by redissolving thecrystal formed using an adequate solvent and using thisextract for further analytical investigations because themolecule was unaltered during the microcrystalline test.

4 APPLICATION EXAMPLES

The chemical structures of the drugs discussed in thissection can be found in Figure 5.

4.1 Cocaine

Three main microcrystalline tests exist for cocaine. Thefirst two use 5% gold chloride or 5% platinic chloride as



reagent and require the sample to be dissolved in 10%acetic acid or HCl.(12 – 15) The characteristic microcrystalsexpected to be seen are serrated needles with goldchloride and thin needlelike branched skeletons withplatinic chloride. Both show immediate precipitationthroughout the test on application of the reagent onthe sample droplet, resulting in difficulties in finding thecrystals of interest.

Alternatively, 1% gold bromide reagent can be usedto microcrystalline test for cocaine.(5) The sample isdissolved in concentrated sulfuric acid and 10% aceticacid (1 : 1). On application of the reagent, instantaneousprecipitation occurs and very distinct characteristic X’swith ragged blade arms and pale orange tint microcrystalsstart to grow on the periphery of the test area (Figure 6).

4.2 Diamorphine (Heroin)

Diamorphine is dissolved in 10% hydrochloric acid andmicrocrystalline tested using 1% mercuric chloride,(5,6)

or 1% mercuric iodide,(5) both resulting in dendriticcrystal formations. However, the mercuric chloride showssmall dark dendrites with thin branches, whereas mercuriciodide induces dendritic stems (Figure 7).

This example demonstrates that different halogens canprovoke divergences in the microcrystal shape, althoughretaining an identical spatial configuration.

4.3 Methadone

The sample is dissolved in water and 1% mercuric chlorideis applied.(6) Initially, the test area shows a uniform cloudof dark particles in suspension. Over time, characteristicrosettes of branching rods appear and grow, consumingthe cloud (Figure 8).

300 µm

Figure 6 Cocaine + gold bromide microcrystals.

300 µm

300 µm

(a)

(b)

Figure 7 (a) Diamorphine + mercuric chloride microcrystalsand (b) diamorphine + mercuric iodide microcrystals.

300 µm

Figure 8 Methadone + mercuric chloride microcrystals.

4.4 γ -Hydroxybutyrate

γ -Hydroxybutyrate readily dissolves in water and a 1%lanthanum/silver nitrate reagent is applied on the sampledroplet.(9,10) Specific right-angle microcrystals appearafter a few minutes, initially on the edge of the testarea (Figure 9).


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300 µm

Figure 9 GHB + lanthanum/silver nitrate microcrystals.

4.5 Ketamine

The sample is dissolved in water and 5% platiniciodide reagent is applied.(6) Instantaneous precipitationis observed and characteristic rhomboidal plates can beobserved fixed on the slide below a layer of reagentcrystals suspended in the liquid (Figure 10).

4.6 Phencyclidine

The reagent of choice for phencyclidine microcrystallinetest is potassium permanganate.(6,14,16) When the sample

300 µm

Figure 10 Ketamine + platinic iodide microcrystals.

300 µm

300 µm

(a)

(b)

Figure 11 PCP + 0.2% potassium permanganate microcrys-tals. (a) Solvent: 10% HCl and (b) solvent: 10% acetic acid.

is dissolved in 10% HCl and tested with 2% potassiumpermanganate in 0.5% phosphoric acid, instantaneousprecipitation occurs and the test area is too highlypopulated by reagent crystals to be able to distinguish thecharacteristic purple razor-blade-shaped microcrystalsexpected. Lowering the reagent concentration to 0.2%helps obtain a readable test, although the small crystalsobtained appear pale and often misshaped (Figure 11).

Dissolving the sample in 10% acetic acid using the0.2% potassium permanganate reagent greatly improvesthe test and very distinctive deep purple razor blademicrocrystals form (Figure 11).

4.7 Amphetamines and Methamphetamine

Amphetamine and methamphetamine are chiral mole-cules. Being able to distinguish between single enan-tiomers and racemic mixtures is often important forpolice investigation because it permits the discrimi-nation between clandestine and genuine productionproducts. Typically, clandestine manufacture results inracemates, whereas asymmetric synthesis techniquesused by pharmaceutical companies produce mostlydextro compounds.

The sample is dissolved in concentrated phosphoricacid and tested with 5% gold chloride reagent.(17) TheD-amphetamine produces yellow rods and blades after10 min, whereas the DL-amphetamine shows growingirregular blades with serrated edges instantaneously.



300 µm

300 µm

300 µm

(a)

(b)

(c)

Figure 12 Gold chloride reagent. (a) Microcrystals formedwith D-amphetamine; (b) microcrystals formed with DL-amphe-tamine; (c) microcrystals formed with D-methamphetamine.

The D-methamphetamine produces long blades of joinedcrystals within 1 min (Figure 12).

Using 5% platinic chloride as the reagent, D-methamphetamine instantaneously generates grains with

300 µm

Figure 13 D-methamphetamine + platinic chloride micro-crystals.

100 µm

300 µm

300 µm

(a)

(b)

(c)

Figure 14 Gold bromide reagent. (a) Microcrystals formedwith D-amphetamine; (b) microcrystals formed with Dl-amphet-amine; (c) microcrystals formed with D-methamphetamine.

sharp edges, which aggregate into skeletal ferns over time(Figure 13).(17)

Alternatively, the sample is dissolved in a mixtureof concentrated phosphoric and sulfuric acid and 1%gold bromide reagent is applied.(5) D-amphetamineproduces characteristic small red trapezoidal blades,DL-amphetamine forms small red cigars, and D-methamphetamine shows pale orange, segmented,squared-cut, elongated crystals (Figure 14).

These tests demonstrate the high discrimination powerof microcrystalline tests when trying to differentiatebetween enantiomers and racemic mixtures. This isbecause of the mechanism by which the microcrystalsare formed. The addition complexes pack in a specificpreferential manner depending on their physical andchemical properties, ending up in a unique characteristicform. This is corroborated by the differences betweenD-amphetamine and D-methamphetamine results.


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ACKNOWLEDGMENTS

The authors thank Dr. Mark Baron for his valuable adviceand suggestions. They also want to express their gratitudeto Dr. Julian Bartrup for his understanding and kindnessduring the writing of this article.

ABBREVIATIONS AND ACRONYMS

ASTM American Society for Testing and MaterialsCPS Convention on Psychotropic SubstancesDFSA Drug Facilitated Sexual AssaultFTIR Fourier Transform Infrared SpectroscopyGBL γ -ButyrolactoneGC/MS Gas Chromatography/Mass SpectrometryGHB γ -HydroxybutyrateLC/MS Liquid Chromatography/Mass SpectrometryLLE Liquid–liquid ExtractionLSD Lysergic Acid DiethylamideMDMA 3,4-MethylenedioxymethamphetaminePCP PhencyclidineSCND Single Convention on Narcotic DrugsSEM Scanning Electron MicroscopeSPE Solid Phase ExtractionTLC Thin Layer Chromatography

RELATED ARTICLES

Drugs of Abuse, Analysis ofStructure Determination, X-ray Diffraction forLiquid Chromatography/Mass SpectrometryMass Spectrometry in Pharmaceutical AnalysisChiroptical Spectroscopy in Drug Analysis

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Microcrystalline Tests in Forensic Drug Analysis