Isotope ratio mass spectrometry as a tool for forensic investigation (examples from recent studies)

Scientific and technical

lsotope ratio mass spectrometry as a tool for forensic investigation (examples from recent studies)

James F Carter*, Richard Sleeman, Jenny C Hill, Fay Idoine, and Emma L Titterton Mass Spec Analytical Ltd.. Building 20F, Golf Course Lane, P.O. Box 77, Filton, Bristol, BS99 7AR, UK Science &Justice 2005 45 141 - 149 Received 18 November 2004 accepted 28 April 2005

The versatility of isotope ratio mass spectrometry is demonstrated by reference to diverse case studies. Variations in the natural isotopic composition of non-biological, organic materials are compared as a means by which samples may be associated or discriminated. These techniques may be used to augment or compliment conventional forensic methodologies. 6 13canalysis was used to demonstrate that different masking tape had been recovered in two, apparently similar cases, involving the smuggling of money. Visually similar ecstasy tablets were compared by consideration of the 613c and 615N composition of MDMA extracted from the tablets. Although only a limited number of tablets were analysed, the isotopic similarity between two different seizures was sufficient to induce a guilty plea from a person suspected of possessing both. A combination of h2H, S13c, 615N and S180 together with GC-MS analyses were applied to small samples of seized heroin. Although GC-MS analysis indicated differences between the chemical composition of two of the heroin samples, isotopic analysis suggested similarities, which were confirmed by further a2H, S13c and 6180 isotopic analysis of the clingfilm in which the samples were wrapped. La polyvalence de la spectromCtrie de masse des rapports isotopiques est dCmontrCe en rCfCrence ?I diverses Ctudes de cas. Les variations dans la composition isotopique naturelle de matCriaux non-biologiques organiques sont comparkes comme moyen par lequel les Cchantillons peuvent Ctre associCs ou discriminks. Ces techniques peuvent Ctre utiliskes pour augmenter ou complkter des mCthodologies forensiques conventionnelles. L'analyse du 613c a kt6 utiliske pour dkmontrer que diffkrents adhCsifs de masquage qui avaient kt6 rCcupCr6s dans deux cas apparemment similaires et qui concernaient un trafic d'argent Ctaient diffkrents. Des tablettes d'ecstasy visuellement similaires ont CtC comparCes en tenant compte de la composition en 6 13cet 6I5N extraits des tablettes. Bien que seulement un nombre limit6 de tablettes ait kt6 analysks, les similarit& isotopiques entre deux diffkrentes

saisies ont CtC suffisantes pour obtenir un aveu de culpabilitC d'une personne suspectCe de la possession des deux. Une com, 6 1 5 et ~ 6180 avec des analyses GC-MS binaison de a 2 ~SI3c, ont CtC appliquCes B de petits Cchantillons d'hkroine. Bien que les analyses GC-MS indiquent des diffkrences entre la composition chimique de deux des Cchantillons d'hCroine, I'analyse isotopique suggkrait des similarites qui ont CtC confirmCes par , et S180du film I'analyse complCmentaire isotopique S ~ HS13c plastique dans lequel les Cchantillons Ctaient emballCs. Die Vielseitigkeit der Stabilisotopen-Massenspektrometrie wird anhand verschiedener Fallbeispiele demonstriert. Schwankungen in den naturlichen Stabilisotopenverhaltnissen nicht-biologischer organischer Materialien werden als Grundlage fur die Aufdeckung oder den Ausschluss gemeinsamer Herkunftsbeziige von Proben herangezogen. Diese Techniken konnen zur Erweiterung oder Bestatigung konventioneller forensischer Verfahren eingesetzt werden. Die 6 1 3 ~ - ~ n a l ywurde s e eingesetzt um zu zeigen, dass unterschiedliche Klebebander in zwei zunachst ahnlich erscheinenden Fallen von Geldschmuggel verwendet worden waren. Augenscheinlich ahnliche Ecstasy-Tabletten wurden anhand der Kohlenstoff- und Stickstoff-Stabilisotopenverhaltnisse des aus ihnen extrahierten MDMA verglichen. Obwohl lediglich eine begrenzte Anzahl von Tabletten untersucht wurde, war die Uberein~timmun~ in den Stabilisotopenverhaltnissen von Tabletten zweier unterschiedlicher Sicherstellungen gut genug, um die des Besitzes beider Tablettenchargen verdachtigte Person zu einem Schuldeingestandnis zu bewegen. Eine Kombination von s ~ H - 6I3c-, , 615N-, 6180- und GC/MS-Analysen wurde zur Untersuchung kleiner Proben sichergestellten Heroins eingesetzt. Obwohl die GC/MS-Analyse Unterschiede in den Zusammensetzungen zweier Heroinproben anzeigte, deutete die Stabilisotopenanalyse auf ~hnlichkeitenhin, die durch weitere S2H-, S i 3 c und Sls~-~tabilisotopenanalysen der zum Einwickeln der Heroinproben benutzten Klarsichtfolie bestatigt wurden.

*Author for correspondence O The Forensic Science Society 2005 Key words lsotope ratio, mass spectrometry, drugs, packaging material.

science&justice Volume 45 NO. 3 (2005)141 - I 49

Page 141

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton Isotope ratio mass spectrometry as a tool for forensic investigation

Introduction Many analytical methods, founded on numerous physical and chemical properties or features, are currently applied in support of law enforcement to establish comparisons between one substance and another. Mass spectrometry (MS) is one of the most important techniques used by forensic chemists and has become the method of choice in many areas. MS instruments are now available with a wide range of inlet and ionisation techniques to enable the detection and identification of trace components in diverse complex mixtures with high sensitivity. Although based on the same instrumentation, isotope ratio mass spectrometry (IRMS) finds fundamentally different applications. The technique dates back to Thompson's discovery of the isotopes of neon ( 2 2 ~ e / 2 0 ~ine )the early 20th century and was principally developed in the mid-20th century as a tool for geochemistry. The practice now encompasses virtually every element of the periodic table, although most studies involve the light stable isotopes of the major elements in the biosphere: hydrogen, carbon, nitrogen, oxygen and sulphur. IRMS instruments are highly specialized ~ , l5N/I4N, 180/160 and for the examination of 2 ~ / 1l3c/I2c, 3 4 ~ / 3via 2 ~the analysis of Hz, C02, N2, CO (or 02) and SO;? gases using a high efficiency electron ionisation (EI) source, high transmission magnetic sector, and multiple collectors, delivering relative standard deviations of less than 0.01% [I]. Although numerous other techniques exist for the measurement of isotope ratios [2] the precision attainable by IRMS makes it by far the most popular. The precision to which IRMS can measure isotope ratios enables the determination of very small differences in the isotopic distribution of natural and synthetic materials. The abundance of 13cis frequently quoted as being 1.1% of all naturally occurring earthly carbon, which is correct when quoted to this precision, but high-precision measurements can reveal subtle variations. The thermodynamic and kinetic isotope effects associated with chemical reactions lead to isotopic fractionation, the products of reaction having different isotope ratios to the starting materials depending on the reaction and on the reaction conditions. Studies over 50 years have shown that isotopic fractionation due to physiological processes, specifically C 0 2 transport processes within plants and photosynthesis, leads to variation in isotope ratio in natural compounds [I]. Hence, identical chemicals from different sources may have different isotope ratios, providing a powerful tool for determining their origins. Stable isotope ratios have been compared to fingerprints or DNA evidence for non-biological materials [3]. Indeed, it has been stated that determining the ratios for a number of isotopes can provide a specificity only otherwise attainable from DNA and that the combined specificity obtained from the analysis of four isotope ratios can be as great as 1 in 1.47 billion [3]. Partly because of this potential specificity, recent years have seen an increase in the forensic applications of IRMS. Stable isotope ratio techniques have been used to determine the geographic origins of natural, and modified natural, controlled substances e.g. cocaine [4,5] and heroin [6, 71, in which the stable isotopic signature is controlled by growth conditions such as nutrient and moisture availability [I]. The technique has also been applied to synthetic controlled substances such as amphetamines [8, 93 in which the isotopic

Page 142

signature is dependent on the synthetic conditions employed [lo]. The use of illicit, endogenous drugs, such as testosterone, can now be detected using isotope ratio analysis when referenced against other endogenous steroids [ l 11. Of equal interest to law enforcement authorities are the packaging materials in which drugs are inevitably seized [12]. Because packaging materials are ubiquitous and frequently lack any distinct markings, it is often difficult to provide an unequivocal link between items seized at different times and locations. Conventional optical techniques may be used to compare materials separated by cuts or tears [I31 but are of limited probative value, since characteristic marks change rapidly during the manufacturing process [13]. Packaging materials are also associated with more serious crimes, for example, to bind an individual during a robbery, assault or murder, or to contain a terrorist explosive device. The use of IRMS as a technique to distinguish fragments of pressure-sensitive adhesive (PSA) tape has recently been reported [14]. Importantly, it was demonstrated that use and storage conditions did not significantly effect the isotopic signature of the tape. Recent IRMS instrument developments have employed continuous flow inlets to improve sample throughput and permit a wide range of solid and semi-volatile liquid samples to be analysed by highly automated systems. There are various ways in which isotope ratio analysis may be performed, one technique (referred to as bulk isotope analysis) provides an average isotope ratio for an entire sample which is placed into a tin capsule and combusted in an elemental analyser, converting all organic carbon and nitrogen to C02 and N2, respectively [15, 161. These gases are separated by gas chromatography (GC) and transported to the ion source of an IRMS in a stream of helium. This technique was subsequently extended to the determination ' ~180/'60 ratios through the use of high temperof 2 ~ / and ature pyrolytic decomposition [17, 181. A further refinement of IRMS is the combination of GCxombustion-IRMS (GCC-IRMS) to separate the components of a mixture which are then analysed compound by compound [19]. This article, however, concentrates on the applications of IRMS to bulk drugs and packagings. To simplify reporting, isotope ratios are always reported relative to an international standard (Equation (1)) in parts per thousand using the "delta" notation (permil or %o) [20], in the case of carbon, PeeDee Belemnite (V-PDB, a carbonaceous rock). Similarly, Standard Mean Ocean Water (V-SMOW) is used as the pri1 180/160 ~ whilst atmospheric mary standard for both 2 ~ / and nitrogen (atm N2) is the primary standard for 1 5 ~ / ' 4("V" ~ or "Vienna" is used as a prefix to denote standards recently defined by the International Union of Pure and Applied Chemistry (IUPAC)). Although delta units are neither SI units nor a measure of absolute isotopic content, because all laboratories reference measurements to the same standards, reported values are reproducible globally.

science&justice Volume 4 5 NO. 3 (2005) 142 - 1 4 9

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton Isotope ratio mass spectrometry as a tool for forensic investigation

Figure 1 (A) exhibit 1a and (B) exhibit 1c

Figure 2

Ecstasy tablet seized from suspect

of the second seizure. The police, therefore, required forensic evidence to link the two batches of tablets and IRMS was chosen to investigate the relationship between the two batches.

and Rstandard are the ratios of the signals recorded for the minor and major isotopes corresponding to the sample and standard respectively (e.g. mlz is 45/44 in the case of C02).

Rsample

Case 3 -Heroin wraps Five "wraps" of heroin (small quantities of heroin, typically t IOOmg, bound in clingfilm) were seized by police in close proximity within a short time frame. One of these samples was found to comprise a very small quantity of heroin (approximately 2mg) bound with a large quantity of clingfilm making it appear larger. Both conventional GC-MS and IRMS techniques were used to investigate possible links to a common source of supply.

In an apparently unrelated incident, an individual was apprehended carrying bundles of banknotes wrapped with masking tape (Figure 1B). Although these parcels were different in appearance to the larger parcels, the coincident use of masking tape was obvious and IRMS was employed to try and establish a link between the two types of parcel.

Experimental Case 1-Masking tape Samples were taken from four different regions of the inner layer of the parcels, where the adhesive was in contact with an underlying layer of tape. Before further preparation, the tape was inspected to assure that the adhesive was intact and that no extraneous material had been adhered. Provided that the tape was wound at least twice around the parcel, such that the second layer partially covered the first, there was sufficient unexposed tape available for analysis. These samples were designated la-le. Approximately 1 mm2 samples of tape were analysed, equivalent to between 100-200 p g of material. Samples were prepared on an aluminium surface which was cleaned with acetone between samples and cut with Throwaway knives (Stanley, Sheffield, UK). A total of eight samples were prepared for each exhibit (duplicate samples from four locations) and analysed for 613c composition by combined elemental analyser-IRMS (EA-IRMS).

Case 2 -Ecstasy tablets A suspect was apprehended in possession of 21 ecstasy tablets. A larger quantity of visually similar tablets were subsequently found concealed within the premises at which the suspect was arrested (Figure 2). When questioned, the suspect denied all knowledge

Case 2 -Ecstasy tablets Two tablets selected, at random, from each batch and presented for analysis were designated PH(1) and PH(2). Each tablet was cut into two approximately equal parts, one part being retained for possible subsequent analysis. The active ingredient, together

Case I -Masking tape H. M. Customs and Excise officers investigated four soft backed suitcases which appeared suspicious. Concealed behind the backing material of each suitcase was a large package bound in masking tape (Figure 1A). On investigation, these parcels were each found to contain 16 bundles of sterling banknotes further bound with masking tape. It was estimated that each parcel contained £60,000 which was assumed to be either the proceeds of drug trafficking or intended for the purchase of controlled drugs.

science&justice Volume 45 NO.

3 (2005) 143 - 149

Page 143

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton

Isotope ratio mass spectrometry as a tool for forensic investigation

with other basic compounds, was extracted from the tablets using a simple liquidbiquid extraction procedure already described [8]. The chemical identity and purity of the active ingredient in the extracts was established by GC-MS, according to the method described later. The remaining extracts were evaporated to approximately 0.5 mL under a stream of dry nitrogen and prepared for isotopic analysis. Forty p L aliquots of the concentrated extracts were measured into tin capsules (6 mm x 4 mm, Elemental Microanalysis, Okehampton, UK) containing a few grains of an adsorbent material (Chromosorb G-AW). Two samples were prcpared for each tablet and analysed for 613c and S1 5composition ~ by combined EA-IRMS. Case 3-Heroin wraps A representative sample comprising approximately I 0 mg of powder was taken from each wrap (designated 3a-3d) and accurately weighed into a 10 ml graduated flask. Flasks were then half filled with chloroform (HPLC grade Romil, Cambridge, UK) containing 0.1 mg ml-' of C24 n-alkane which was to serve as an internal standard for quantification. Flasks were subjected to ultrasonic agitation for 2 min to ensure complete dissolution of soluble components and the volume adjusted to 10 ml with the C24 n-alkane solution. The chemical identity of the major components in the solutions and the percentage diacetylmorphine (DAM) content was established by GC-MS, according to the method described later. On opening, sample 3e was found to contain approximately 2 mg of powder, the majority of which was accurately weighed into a 2 ml graduated flask and treated as the other samples. A solution of pure DAM (Sigma-Aldrich Company Ltd., Poole, UK) was prepared in the manner described earlier to enable quantification of the diacetylmorphine content of the illicit heroin samples. A portion of clingfilm was removed from each wrap and cleaned with water (HPLC grade Romil) in an ultrasonic bath for 15 min, followed by drying, in air at ambient temperature. This process removed residual particles of heroin and has been shown not to remove any constituents of the clingfilm and, therefore, not affect the isotopic composition [14]. Approximately 2mm2 samples of clingfilm were analysed, equivalent to between 10&200pg of material. Samples were prepared on an aluminium surface, which was cleaned with acetone between samples and cut with Throwaway knives (Stanley). Three samples of powder (approximately 500 p g and clingfilm from each wrap were prepared and analysed for S i 3 c and S ' ~ N composition by combined EA-IRMS. Five further samples (approximately 150 p g of powder or clingfilm) were prepared and analysed for S 2 and ~ S180 composition by combined EA-IRMS. The first results from these analyses were discounted to account for memory effects associated with the glassy carbon reactor. Instrumentation Gas chromatography-mass spectrometry Combined GC-MS analyses were performed using a Perkin-Elmer Clams 500 GC coupled to a Turbomass Gold MS via a heated transfer line, maintained at 280°C. The GC was equipped with a temperature Pro-

Page 144

Figure 3 Carbon isotopic composition of masking tape samples (A) taken from six exhibits and (B) ten "background" samples summarised as box plots

I -27.5

I

I

8% vs V-PDB

I -24.0

grammable SplitfSplitless (PSS) injector which was programmed from 50 to 300°C at a rate of 200"~min-' and maintained at that temperature for 22 min. One microlitre aliquots of sample were injected by a Perkin-Elmer autosampler. Chromatographic separation was performed using a DB-5MS (J&W Scientific supplied by Thames Restek UK Ltd., Saunderton, UK) column; 30 m in length, 0.25 mm in diameter with a 0.25pm film thickness. The oven was maintained at 40°C for 2min, heated at a rate of 1 0 " min-' ~ to 300°C and maintained at that temperature for 15 min. Helium carrier gas was operated at a constant flow rate of 2 ml min-' . The ion source was maintained at a temperature of 180°C with an electron energy of 70eV and an emission current of 60pA. Data were acquired over a range mlz 45-500 at a rate of two scans per second using a Turbomass 4.5 data system.

Isotope ratio mass spectrometry All isotopic measurements were performed using a ThermoFinnigan DeltaXP IRMS via a ConFlo 111 interface. Carbon and nitrogen isotopic measurements were performed using a ThermoElectron Flash 1112 elemental analyser. Samples were crimped into tin capsules (Elemental Microanalysis) and introduced using an AS200 autosampler. The oxidation reactor, comprising chromic oxide and silver/cobaltous oxide, was maintained at a temperature of 900°C and the reduction reactor, comprising electrolytic copper, was maintained at 680°C. Helium carrier gas was maintained at a flow of 150 ml min-' and a 5 s pulse of oxygen (BOC Research Grade N5.5) was introduced at a flow rate of 175 ml min-' .Water was removed from the evolved gases by anhydrous magnesium perchlorate (Elemental Microanalysis) and the carbon dioxide

Table 1

Isotopic data of masking tape samples from exhibits and combined data.

Sample

6I3cversus V-PDB

1c 1e

-25.73 f 0.20%0 -25.77 f 0.11%O

Difference in means at 95% confidence -0.1 7 to 0.25

science&justice Volume 4 5 NO. 3 (2005) 1 4 4 - 149

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton Isotope ratio mass spectrometry as a tool for forensic investigation

Table 2

Isotopic data of ecstasy tablet extracts.

6 I 3 c VS. V-PDB VS. Air

Tablets PHI11

Tablets PH(2)

Difference in means at 95% confidence

-28.85 f 0.47%0 7.61 f 0.05%0

-28.87 f 0.18%0 6.86 f 0.07%0

-0.22 to 0.26 0.58 to 0.89

and nitrogen formed in the reactor were separated using a GC column containing PorapakQ maintained at a temperature of 35°C. Isotopic compositions were referenced against gaseous carbon dioxide (BOC CP Grade N4.5) and nitrogen (BOC Research Grade N5.5) which were, in turn, calibrated against PEFl polyethylene foil (National Institute for Standards and Technology (NIST) Reference Material (RM8540)), USGS25 ammonium sulphate (NIST RM8550) and IAEA-N3 potassium nitrate (NIST RM8549). Hydrogen and oxygen isotopic measurements were performed using a ThermoFinnigan TC/EA high temperature elemental analyser. Samples were crimped into silver capsules (Elemental Microanalysis) and introduced using an AS200 autosampler. The pyrolysis reactor, comprising an alumina tube lined with glassy carbon, was maintained at a temperature of 1350°C with a helium carrier gas flow of 90mlmin-'. Hydrogen and carbon monoxide, pyrolytically formed in the reactor, were separated using a GC column containing 5 A molecular sieve maintained at a temperature of 90°C. Isotopic compositions were referenced against gaseous hydrogen (BOC Research Grade N5.5) and carbon monoxide (BOC Research Grade N3.7), which were, in turn, calibrated against PEFl and NBS 19 calcium carbonate (NIST RM8544). Data were acquired and processed using ISODAT NT 2.0 software. All statistical manipulations of the acquired data were performed using SYSTAT 11 for Windows. Results If two samples can be shown to be isotopically distinct, considering one or more element, it is reasonable to conclude that they have different compositions, different origins, or have, at some time, been subjected to a different chemical or physical process. If two samples are isotopically indistinct, it is a more difficult task to establish the significance of this similarity, since there must always be a finite probability that this match has arisen solely by chance. In the following examples, case samples were compared to a small number of background samples. The back-

Table 3 Sarn~le

ground materials were not intended as a random, representative, stratified sample but to provide some, limited assessment of the isotopic variability of the population. In these cases, the IRMS data formed only part of the overall investigation and it was sufficient to show either that the isotopic composition was consistent with the samples having a common origin or with the samples having different origins. For IRMS to have the probative value associated with DNA evidence it will be necessary to develop databases of materials which may be the subject of forensic investigations. It is, however recognised that the vast majority of real crime laboratories use databases obtained by convenience sampling from laboratory staff or casework [21]. Case I -Masking tape Masking tape comprises a paper backing and a synthetic or natural rubber adhesive. The carbon isotopic signature obtained from a sample of tape will depend on the origins of both the paper and adhesive and their relative proportions. Batches of tape produced from the same starting materials using the same manufacturing process will have the same isotopic signature. Figure 3 presents the carbon isotopic content of tape removed from each exhibit as a box plot, contrasted with data obtained from 10 background samples collected from laboratory staff. These samples comprised tape with a range of ages, only two being from the same supplier. The length of each box shows the range within which the central 50% of the values fall and the "whiskers" the minimum and maximum values. The overall span of the background data is approximately -27.3 to -24.3%0 versus V-PDB with typical standard deviations for individual samples of 0.15%0. None of the individual measurements corresponding to a particular sample were significantly different from the others. From Figure 3A it is evident that tape taken from the six exhibits form two isotopically distinct populations; a/b/d/f and c/e, the former group corresponding to tape from the four large parcels and the latter to tape from the smaller banknote bundles. This finding may be confirmed by t-tests, some of which are presented in Table 1. These show that the 613c composition of samples from exhibits l c and l e is isotopically indistinct at 95%

Diacetylmorphine (DAM) content and isotopic composition of heroin sample (no standard deviations are reported for sample 3e as no replicate analyses were performed). DAM content

613cVS.V-PDB

science&justice Volume 4 5 NO.

3 (2005) 1 4 5 - 1 4 9

S ' ~ vs. N Air

S*Hvs. V-SMOW

6180

vs. V-SMOW

Page 145

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton Isotope ratio mass spectrometry as a tool for forensic investigation

confidence, whereas the combined data for the two groups identified from the box plot (a/b/d/f and c/e) are isotopically distinct. All of the samples within the a/b/d/f grouping were isotopically indistinct by the same comparison although, for simplicity, these data are not presented.

Figure 4

Isotopic composition of MDMA extracted from tablets (A) PH(I), PH(2) and (B) compared to five batches of seized tablets

The conclusion drawn from these results was that the tape used to bind the small bundles was not the same as that used to bind the larger bundles and the course of the investigation was redirected accordingly. Case 2 -Ecstasy tablets The tablets presented for analysis were described as "visually similar"; being blue in colour, approximately 7 mm in diameter and with a "B" embossed in one face. The two batches of tablets were identified as "PH(1)" and "PH(2)" but no indications were given as to which tablets corresponded to which seizure.

GC-MS analysis showed that all the tablet extracts contained 3,4methylenedioxymethylamphetamine (MDMA) at greater than 98% purity. No other compounds were identified in the extracts and comparison by GC-MS therefore provided very limited discrimination. Table 2 summarises the experimentally determined S13cand S"N composition of the tablet extracts. The S i 3 c compositions of the tablet extracts were consistent, at 95% confidence, whilst the S"N compositions formed two distinct populations, although the sample size (two tablets analysed in duplicate) was extremely small for statistical analysis. The nitrogen isotopic composition of illicit MDMA has been shown to vary between -16 and +19%0 versus atm N2 [9] and so the difference between the exhibits in this case was comparatively small, given this range. When both S i 3 c and S"N are compared, using a bivariate ellipse (Figure 4A), the 95% confidence ellipses for the two batches of tablets overlap, indicating a single population. Furthermore, when these data are compared to background data [8] obtained from 50 other ecstasy tablets (Figure 4B), they can be seen to form a discrete cluster which is distinct from the other batches of tablets. The isotopic difference observed in the S ' ~ Ncomposition may well have resulted from the small sample size or differences in the isotopic composition of the tablet extracts. The latter may reflect the presence of other nitrogen-containing species in the extract such as a small amount of polyvinylpyrrolidone (PVP), used as an adhesive in tablet manufacturer [22], which would not be detected by GCMS but does contain a high proportion of nitrogen. If these data were to have been used in evidence, further analyses would have been deemed necessary, for example, excipient profiling of the existing tablet extracts or isotopic analysis for the retained fragments, or further tablets. However, when presented with these initial findings, together with other evidence, the defendant chose to enter a guilty plea. Case 3 -Heroin and cling film Associations between samples of illicit heroin samples are typically inferred from their physical appearance, the DAM content or from profiling of alkaloids and cutting agents present [6]. Comparisons based on the stable isotope compositions of either the

Page 146

I

I

I

-30 -25 6l3Cvs V-PDB

bulk heroin or its individual components have also been proposed [6,7]. Table 3 presents results for the percentage DAM and isotopic analysis of the five heroin samples from exhibits 3a-3e. The DAM purity for the five samples ranges from 4.5 to 38%, samples 3d and 3e both having DAM purities of 15%. Figure 5 shows the profile of compounds present in the illicit heroin samples determined by GC-MS. Although the percentage DAM content may be taken as an indication that exhibits 3d and 3e were linked, the profiles of these samples differed in the proportions of papaverine (P) and noscopine (N) and in the presence of caffeine and griseofulvin in sample 3e. Different sized and shaped crystals, corresponding to different chemical compounds, may adhere to the wrapping material in different amounts resulting in different chemical profiles of recovered material. This effect would be more evident for small samples such as exhibit 3e. To aid comparison, the isotopic composition of these samples were compared to each other and to powders from the other exhibits,

sciencetkjustice Volume 4 5 No. 3 (2005) 1 4 6 - 1 4 9

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton Isotope ratio mass spectrometry as a tool for forensic investigation

Figure 5 GCMS profiles of heroin powder from exhibits 3a-3e

'"I

a

Figure 6 (A) plot of nitrogen vs. hydrogen vs. carbon isotopic composition and (B) plot of diacetylmorphine content vs. hydrogen vs. carbon isotopic composition for heroin from five exhibits

-33.5

-32.5

-33.0 6°C

-32.0

-31.5

VS V-PDB

Time --+

the isotopic signature of bulk heroin reflecting that of its constituent alkaloids and cutting agents. Unfortunately, there was only sufficient powder remaining in exhibit 3e for two isotopic ~ analyses, one providing 6 I 3 c and S I ~ Ndata the other S 2 and 8180 data. Consideration of Table 3 reveals greater similarity between samples 3d and 3e than between the other samples. These data are summarised in Figure 6A in which the S ~ H6I3c , and 6 1 5 data ~ are combined in a triangular plot, the datum corresponding to sample 3e being closer to data corresponding to sample 3d that the H 6I3c data are combined other samples. In Figure 6B, the S ~ and with the DAM purity presenting a very similar overall pattern. It is possible to present the data in many other combinations, all of which indicate that sample 3e is more closely related to sample 3d than to the other samples. Because the powder within exhibit 3e was ostensibly exhausted, isotopic analysis was applied to the clingfilm to try and confirm a link between samples 3d and 3e. Plastic sheet, including clingfilm, is made by forcing molten plastic through a fine circular die. This forms a large bubble which is supported by a column of hot air and

science&justice Volume 45 No. 3 (2005) 147 - 149

then collapsed to form a solidified plastic tube which is guided through a series of rollers for subsequent processing. "Extrusion features" may appear in the plastic due to fluctuations in the hot air stream or to plastic fragments contaminating the die or rollers, and may be visualised using polarised light. These features vary rapidly during the manufacturing process and may only be considered to determine whether products were manufactured in close proximity [13]. Figure 7A presents the carbon isotopic data derived from the background clingfilm samples as box plots, with the lower region expanded in Figure 7B. This figure demonstrates the large potential variation in the 6 I 3 c content of clingfilm. Data from the clingfilm removed from the heroin wraps is shown in Figure 7C, samples from exhibits 3d and 3e being isotopically indistinct. Table 4 presents all the isotopic data for the clingfilm samples removed from exhibits 3d and 3e which are also isotopically

Page 147

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton Isotope ratio mass spectrometry as a tool for forensic investigation

Figure 7 Carbon isotopic composition of clingfilm samples (A) ten "background" samples, (B) expanded view and (C) samples from five heroin wraps

6°C

drug, the active ingredient and the packaging materials. These analyses may be used in isolation or in combination with more conventional techniques to infer information about the source and supply of illicit materials. There are few analytical techniques that provide this versatility and flexibility.

VS V-PDB

indistinct when considering 6 2 and ~ 613c. These data are also presented as a triangular plot in Figure 8 together with data from the other three clingfilm wraps. It is clear that samples 3a, 3b and 3c form intimate, distinct isotopic groupings whilst samples 3d and 3e are indistinguishable. The combination of DAM purity, chemical profile, heroin isotopic composition, and clingfilm isotopic composition provided compelling evidence that two of the five wraps had been prepared from the same supply of heroin using the same cutting agents and were supplied in the same packaging.

Discussion The case studies presented demonstrate the potential of IRMS as a tool for forensic investigation. An isotopic signature is present in a sample whether it comprises a single, pure chemical compound such as MDMA, or a chemically complex material such as masking tape. As a result, when applied to forensic samples of controlled substances, IRMS has been used to examine the bulk Table 4

The nature of IRMS makes it an ideal tool to discriminate between samples of non-biological material. However, the increased use of IRMS in forensic studies comes with a concomitant need for background databases such that the probability of two samples having the same isotopic signature purely by chance can be fully assessed. The next stage of maturity will be the adoption of common analytical practices and the establishment of databases. This workis currently being undertaken by members of the Forensic Isotope Ratio Mass Spectrometry (FIRMS) network (http://www.forensic-isotopes.rdg.ac.uk),partially funded by the Engineering and Physical Sciences Research Council. Acknowledgements Hugh Grundy and Eve Mason of Avon and Somerset Constabulary, Scientific Investigations are thanked for providing the heroin samples and the ecstasy tablets which produced the background data. Peter Hulmston of Nottingham Constabulary Scientific Support is thanked for the ecstasy tablet case work and Charles Belanger of Iso-Analytical Ltd., for assistance with the analysis of ecstasy tablet extracts.

Isotopic composition of clingfilm removed from exhibits 3d and 3e.

6I3c vs. V-PDB 6180 VS. V-SMOW 6 2 vs. ~ V-SMOW

Page 148

Figure 8 Carbon vs. hydrogen vs, oxygen isotopic composition of clingfilm from exhibits 3a-3e

Clinafilm from exhibit 3d

Clinafilm from exhibit 3e

-31.79 f 0.10%0 20.2 f 0.8%0 -42.8 f 2.0%0

-31.65 f 0.26%0 19.2 f 0.5%0 -37.7 f 1.7%0

Difference in means at 95% confidence

science&justice Volume 45 NO.

3 (2005) 148 - 149

JF Carter, R Sleeman, JC Hill, F Idoine, and EL Titterton lsotope ratio mass spectrometry as a tool for forensic investigation

References 1

nal of Chromatography B. Biomedical Sciences and Applications 1999; 727: 95-

Asche S, Michaud AL, and Brenna JT. Sourcing organic compounds based on natural variations measured by high precision isotope ratio mass spectrometry. Current

105. 12

Organic Chemistry 2003; 7: 1527-1 543. 2

the evidential value of specific techniques. Journal of Forensic Sciences 2001; 46:

Carter JF and Sleeman R, lsotope ratio measurements. In: Worsfold P, Townsend A and Poole C, editors. Encyclopedia of Analytical Sciences, 2nd edition. New York:

28S287. 13

Academic Press. 2004. 3

Meier-Augustein Wand Liu RH. Applications of isotope ratio mass spectrometry. In:

4

14

Ehleringer JR, Cooper DA, Lott MJ, and Cook CS. Geo-locationof heroin and cocaine

Ehleringer JR. Casale JF, Lott MJ, and Ford VL. Trac~ngthe geographical orig~nof

nitrogen-15 analysis. The Analyst 1983; 108: 971-977. 16

7

1985; 12: 510-513. 17

Bescacier F, Guilluy R, Brazier JL, Chaudron-Thorzet H. Girard J, and Lamotte A.

chromatography/mass spectrometry of D/H by high temperature conversion isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry 1999: 13:

Carter JF, Titterton EL. Murray M, and Sleeman R. lsotopic characterisationof 3,4-

12261230. 18

methylenedioxyamphetamineand 3,4-methylenedioxymethamphetamine (ecstasy).

Palhol F, Lamoureux C, and Naulet N. 15N isotopic analysis: A powerful tool to

1999: 13: 1685-1 693. 19

establish links betweenseized 3.4-methylenedioxymethamphetamine (MDMA)tablets. Analytical and Bioanalyt~cal Chemistry 2003; 376: 486490. 10

Aguilera R. Catlin DH, Becchi M. Phillips A. Wang C, Swerdloff RS. Pope HG,

Slater C, Preston T, and Weaver LT. Stable isotopes and the internationalsystem of units. Rapid Communications in Mass Spectrometry 2001; 15: 1270-1273.

21

2591. 11

Meir-Augustein WJ. Applied gas chromatography coupled to isotope ratio mass spectrometry. Journal of ChromatographyA 1999; 842: 351-371.

20

Carter JF, T~tterton EL, Grant H, and Sleeman R. lsotopic changes during the synthesis of amphetamines. Chemical Communlcat~ons2002; 21: 2590-

Kornexl BE, Gehre M. Hofling R, and Werner A. On-line 6180 measurements of organic and inorganic substances. Rapid Communications in Mass Spectrometry

The Analyst 2002; 127: 830-833. 9

Hilkeri AW, Douthiti CB, Sculuter HJ, and Brand WA, lsotope ratio monitoring gas

lsotopic analysis of 13C as a tool for comparison and origin assignment of seized heroin samples. Journal of Forensic Sclences 1997; 42: 42-33, 8

PrestonT and Owen NJP. Preliminary 13cmeasurements using a gas chromatograph interfaced to an isotope ratio mass spectrometer. B~omedicalMass Spectrometry

Besacier F and Chaudron-Thozet H. Chemical profilingof illicit heroin samples. Forensic Science Review 1999; 11: 105-1 19.

Preston T and Owen NJP. Interfacing an automatic elemental analyser with an isotope ratio mass spectrometer: The potential for fully automated total nitrogen and

cocaine. Nature 20M); 408: 31 1-312. 6

Carter JF, Grundy PL, Hill JC, Ronan NC, Titterton EL, and Sleeman R. Forensic isotope ratio mass spectrometryof packagingtapes. The Analyst 2004; 129: 12061210.

15

by stable isotope raios. Forensic Science international1999; 106: 27-35. 5

Pierce DS. Identifiable markings on plastics. Journal of Forensic Identification 1990; 40: 51-59.

Yinon J, editor. Advances in Forensic Applications of Mass Spectrometry. Florida: CRC Press. 2004; pp 14S180.

Maynard PM. Gates K. Roux C, and Lennard C. Adhesive tape analysis: Establishing

Even IW and Weir BS. Interpreting DNA evidence. Sunderland. MA: Sinauer Associates, 1998; pp 44-45.

22

Alderbom G. Tablets and compaction. In: Aulton ME, editors. Pharmaceutics-The

and Hatton CK. Screening urine for exogenous testosterone by isotope ratio

Science of Dosage form Design, 2nd edition. London: Churchill Livingstone. 2002;

mass spectrometric analysis of one pregnanediol and two androstanediols. Jour-

pp 404-41 0.

science&justice Volume 45 NO. 3 (2005) I 49 - I 49

Page 149