J. Forens. Sci. SOC.(1976), 16, 103

Organic Mass Spectrometry in Forensic Science J. A. ZORO and K. HADLEY Home O&ce Forensic Science Laboratory, Priory House, Gooch Street North, Birmingham, West Midlands, England, B5 GQQ. The principles and methods of modern organic mass spectrometry are brieJy discussed. Applications of the technique to the investigation of cases involving arson, malicious damage, explosizles, toxicology, drugs of abuse and others are illustrated with examples drawn from casework experience. Organic mass spectrometry is a relatively new technique in forensic science and some methods and applications which may be developed in the future are discussed.

Introduction Organic mass spectrometry has been in routine use in forensic science laboratories in England and Wales since 1973 when a mass spectrometer was installed at the Home Office Central Research Establishment (HOCRE). This instrument was used for building up a collection of mass spectra of compounds of forensic science interest (mainly drugs) and also for casework from regional laboratories when particular problems demanded mass spectrometry. Subsequently VG Micromass 12F instruments were installed at the Home Office Forensic Science Laboratories at Birmingham and Chorley and at the Metro. politan Police Laboratory in London. Now all the nine government forensic science laboratories in England and Wales have ready access to the technique for casework by the use of one or other of these instruments. The most recent expansions of instrumentation have been the replacement of the HOCRE instrument by a more sophisticated model and the addition of a sophisticated data system to the Metropolitan Police facility.

Principles Electron impact ( E I ) mass spectrometry depends upon a low pressure reaction between electrons emitted from an incandescent filament and sample molecules. Amongst the products are positive molecular ions and fragment ions usually carrying a unit charge. These positive ions are accelerated by a large potential difference (1 to 10kV) and separated according to their mass-tocharge ratio. Most commonly this separation is achieved by passing the beam of ions through a magnetic field produced by an electromagnet. The separated positive ions are collected, the ion signals are amplified by an electron multiplier and by other electronics and finally the spectrum is recorded at high speed on photosensitive paper. Under ideal conditions complete spectra may be obtained from only tens of nanograms of material.

Chemical Ionisation There are several other ways of generating positive ions and one of these, chemical ionisation ( C I ) , is available on the VG Micromass 12F instrument. Under chemical ionisation conditions the ion source is held at a relatively high pressure (0.5-1.0 Torr) by the introduction of a reactant gas. The reactant gas is ionized by electron impact and ion-molecule reactions then occur giving sample ions of low energy which do not fragment as readily as ions produced under E l conditions. When isobutane is used as the reactant gas proton transfer is the most significant ion-molecule reaction and frequently results in the formation of stable pseudo-molecular ions ( M I)+. Figure 1 illustrates the differences in E I and isobutane C I spectra in the case of the anaesthetic amethocaine. The determination of molecular weights by this method often

+

Figure 1.

Comparison of the

EZ and CZ (isobutane) mass spectra of amethocaine.

leads to the identification of unknown compounds when standard spectra are not available for comparison. Some applications of CI to toxicology and drug analysis have been discussed by Beggs and Day (1974), Saferstein et al., (1974) and Milne et al., (1971).

Mass Fragmentography I n normal operation the field generated by the spectrometer electromagnet is increased to obtain a spectrum covering the mass to charge (m/e) range of 1 to 450. An alternative method of operation is to hold the magnet current steady and to switch the ion accelerating voltage rapidly from one value to another. I n this way a few ions can be specifically monitored and since the spectrometer spends most of the time on the specified ions sensitivity is very much higher than for normal scanning. Figure 2A shows the traces of four ions in a gas chromatographic (GC) analysis of a n aerosol bronchodilator. Traces from a headspace sample of the blood o f a person suspected to have used the aerosol (Figure 2B) show a quite different pattern which was subsequently shown to agree with that of another aerosol in the same case. The ions used are prominent in the spectra of certain freons used as aerosol propellants. This method is rather more specific than the electron capture GC method of Rauws e t al., (1973). Methods of Sample Introduction Probably the commonest method of sample introduction is by means of the direct insertion probe. The sample is placed in a small cup (usually glass or quartz) on a probe and passed through a system of vacuum locks into the ion source, where it is heated to vapourise the sample. This method is ideal for pure materials of appropriate low volatility such as thin layer chromatography (TLC) spot extracts but can also be used to analyse mixtures by a process of

Figure 2.

Mass fiagmentography of aerosol propellants by GC-MS. (A) Traces from a bronchodilator aerosol, (B) traces from a blood sample.

subtracting the spectrum of one component from a mixed spectrum and analysing the remainder or by slow heating to selectively vapourise components of different volatilities. T L C spots are best examined by a method such as that described by Rix et al., (1969). Figure 3 illustrates the analysis of mixed spectra in what was apparently a single T L C spot. Rf measurements and UV spectra indicated this spot to be imipramine, but other drugs were suspected in the case. The mass spectrum (A) is similar to a spectrum of pure imipramine obtained under similar conditions (B) but the process of spectrum subtraction reveals the presence of orphenadrine (C). Sample introduction via a GC offers certain advantages for appropriate

Figure 3.

Analysis of a mixed

EZ spectrum. (A) Mixture. (B) imipramine. (C) orphenadrine.

compounds. Separation of mixtures is achieved before spectra are collected and the sample enters the source already in the vapour state. Carrier gas flow through a conventional packed GC column is commonly in the region of 2&60rnl/min which is far too high to maintain the low pressure necessary in the ion source. Mass spectrometers with large pumps can cope with the carrier gas flow through capillary columns (2-5ml/min) but for packed columns the gas (usually helium) must be separated before the sample enters the source by one of several types of device now in common use (Scaplehorn, 1975). Most spectrometers also have a facility for the direct introduction of gases and volatile liquids, often via a heated reservoir with a capillary leak into the source.

Identification of the Spectra Once a spectrum is obtained the analyst must either search reference collections for a similar spectrum or identify the spectrum by interpretation. One major reference collection is published in book form and on magnetic tape by the Mass Spectrometry Data Centre (MSDC), Aldermaston, and consists

of the most intense eight peaks of 31,000 spectra. MSDC also manages an international conversational data retrieval system accessible to subscribers by telephone. I n addition to such collections most laboratories or organisations concerned with mass spectrometry develop their own specialised data collection for everyday use. In practice it is rarely necessary for a forensic scientist to interpret the spectrum of a completely unknown substance. Usually some useful additional information is available even if it is only smell, appearance and texture and the circumstances in which it came to the laboratory. Often other spectra (UV and IR) are also available.

Applications During the first full year of operation the Birmingham instrument has been used in about 180 cases which have been loosely categorized in Table 1. It TABLE 1 DISTRIBUTION OF CASE TYPES

Type Illegal possession of drugs Suspicious death Explosives Arson Miscellaneous Administration of noxious substances Driving under the influence of drugs Malicious damage Documents Biology

Number 59 47 18 17 10 8

7 7 4 2

will be seen that the technique has been used in a wide range of problems but that applications in biology are so far very few. The examples which follow have been taken from the initial, somewhat experimental, year of operation and include casework from the Birmingham, Cardiff, Nottingham and Bristol laboratories. -Arson -. - ...

In cases of suspected arson headspace samples from fire debris are frequently analysed by gas chromatography. The GC traces may show peaks which could be due to pyrolysis products of building materials, furnishings, etc., or could be due to the presence of accelerant residues. In some cases an "empty" container of the suspected accelerant is available for comparison, but even then peaks due to pyrolysis of furnishings etc., can make correlation of the chromatogram obtained from the debris with that obtained from the container uncertain. The application of combined GC-MS to such a situation can provide much stronger evidence of the use of accelerant than GC alone. A case in which this situation arose is illustrated in Figure 4 and Table 2. Figure 4 shows chromatograms of headspace samples of a fire residue and of a suspected accelerant. Table 2 gives the identity of each peak as deduced from the mass spectra. Some identifications (prefixed by question marks) are uncertain but this is of little consequence since it is the similarity or otherwise of the spectra which is most important and not the absolute accuracy of identification. Further work could often establish the precise chemical identity of peaks beyond doubt but would not be justified since it would be of no evidential value. I n this case it will be seen (Table 2) that all five major components of the suspected accelerant are present in the fire debris but that three other major debris peaks, acetone, carbon disulphide and an octene are not derived from this source.

Malicious Damage Malicious damage to automobiles is a frequent occurrence in urban areas;

Figure 4. GC-MS analysis of hcadspace samples. (A) Suspected accelerant (B) fire debris. The chromatograms shown are freehand copies which eliminate discontinuities in the integrated ion trace produced by the spectrometer. See Table 2 for identification of the peaks.

one common method is the use of some solvent to strip paint from vehicles. The forensic scientist is likely to receive a sample of the damaged paint from which headspace samples can be taken for analysis. One such case is illustrated 108

TABLE 2 ANALYSIS OF HEADSPACE SAMPLES* Peak

Accelerant IdentiJication

1

? cyclopentadiene

2

benzene toluene

3

4 5 6

7 8

Peak

1 2

xylene xylene and styrene ? methylethylbenzene ? propyl and propenyl benzene indene

Debris Identification carbon disulphide

3 4 5 6 7

? acetone benzene toluene ? octene xylene styrene

8

? propyl and propenyl benzene

9

unknown

* See Figure 4 for chromatograms. in Figure 5. The solvent was a complex mixture and identification of the components could certainly be of use in determining the source of the mixture and hence in tracing the culprit. - r - - -- ~ -

Mass spectrometry is one of several analytical methods which have been advocated for the detection and identification of explosives. Many explosives however give only weak molecular ions or indeed no measurable molecular ions at all by EI and so several workers have examined the CZ spectra of explosives using various reactant gases (Gillis et al., 1974; Zitrin and Yinon, 1974; Yinon, 1974 and Saferstein et al., 1975) and in many cases large pseudomolecular ions have been obtained. One common explosive problem in the United Kingdom is the identification of traces of nitroglycerine. Mass fragmentography would be a n ideal method but all the intense ions in the EI spectrum are small (Figure 6A) and confusion with other materials is possible. The closely related compound pentaerythritol tetranitrate (PETN) has been reported (Yinon, 1974) to give an intense pseudo-molecular ion with water as reactant gas but the authors of this paper have only been able to produce similar spectra (Figures 6B and 6C) when large quantities (tens of micrograms or more) of PETN were introduced into the source. No water adduct ion is observed in these spectra and it is presumed that the effective reactant gas is one or more thermal decomposition products of PETN. The authors have also been unable to produce intense pseudo-molecular ions of nitroglycerine with either hydrogen or water as reactant gas. EZ mass fragmentography of nitroglycerine using the ions m/e 30, m/e 46 and m/e 76, though not a n ideal method is in use in this laboratory in conjunction with other tests and good results have been obtained with samples of only a few nanograms.

Polymers I n some cases it is possible to discriminate between polymeric materials by the detection of trace additives such as anti-oxidants. Following the theft of some polyethylene coated copper cable, small fragments of polyethylene were recovered from a vehicle and from a hacksaw. Polyethylene from the stolen cable was fbund to contain the anti-oxidant 1,1,3 -tris-(5-tert-butyl-4 hydroxy-2-methyl-pheny1)-butanewhich was absent from the fragments taken from the vehicle but present in fragments from the saw. The spectrum obtained by direct probe insertion of 100pg of the polyethylene containing this antioxidant is shown in Figure 7. Fragments of polyethylene without the antioxidant produced similar spectra except for the absence of ions m/e 339 and m/e 544, but were not readily distinguishable by pyrolysis gas chromatography.

Figure 5. GC analysis of a headspace sample from damaged paintwork. The main peaks were identified by GC-MS. (1) Air (2) methylene chloride (3) iso-butanol (4) n-butanol (5) methyl butyl ketone (6) toluene (7) butyl acetate (8) xylene.

Even greater sensitivity could be achieved by the use of mass fragmentography on such samples.

Toxicology and Drugs of Abuse A general discussion of mass spectrometry and its applications in drug identification is given by Scaplehorn (1975). More detailed accounts of the use of elaborate GC-MS-computer systems in routine toxicology have been given by Costello et al., (1974) and by Finkle et al., (1974). About 70°/, of cases involving mass spectrometry in the Birmingham laboratory are concerned with toxicology or drugs (Table 1) but just a few examples will serve to illustrate the methods used. Heroin preparations are often complex mixtures and the identity of the minor

Figure 6. Mass spectra of explosives. (A) EZ spectrum of nitroglycerine (B) CZ type spectrum of PETN without reactant gas (C) CZ spectrum of PETN using water as reactant gas.

components is valuable information which can be useful for linking samples of heroin in different cases. I n one case TLC and MS were used to identify heroin, morphine, acetyl morphine, codeine, acetyl codeine, strychnine, quinine and caffeine in an illicit heroin preparation. Occasionally speed of analysis is important in forensic science and mass spectrometry can then be very useful, for example fragements of tablets can be analysed directly using the probe and in most cases give good spectra of the active constituents. Sometimes these spectra are contaminated with ions due to excipients such as palmitic and stearic acid but these ions do not usually hinder the experienced analyst. Mass spectrometry has proved useful in many cases where unusual drugs are encountered. I n such cases identification by conventional techniques could be tedious or impossible, particularly if only small quantities are involved. In recent casework a number of unusual pharmaceuticals including sulphonal, millophylline, pyrovalerone, cinchocaine, maprotiline, oxprenolol, prazepam and methyl phenidate have been identified. O n a number of occasions metabolites of common drugs such as aspirin, the benzodiazepines and pethidine have been identified.

Miscellaneous unknown substances Occasionally substances are encountered in forensic science which are difficult to identify either because they are unfamiliar, or because they have been found in unusual surroundings. A mass spectrum obtained quickly by the use of the direct insertion probe can often provide a n answer or at least a clue which can save many hours of a n analyst's time. I n one such case it was alleged

1SU

Figure 7.

Direct probe

LUU

EZ spectrum of a polyethylene sample.

that in the course of a dispute between neighbours a white powder had been used to damage certain garden plants. Conventional analysis ruled out common herbicides after which the material was submitted for mass spectrometric analysis. The spectrum enabled the major component of the powder to be identified as flavone-a naturally occurring compound found as a white powder on the leaves of certain plants. Several substances bcar a superficial resemblance to cannabis resin and though chemical tests can easily eliminate cannabis it can be useful to identify these materials. By inserting a tiny sample of the material directly into the spectrometer using the probe slabs of brown material have been rapidly examined and gave spectra which showed the main constituent to be abietic acid. This acid is the main component of rosin. A fragment of a tablet, suspected to contain controlled drugs was found not to contain anv constituents of si~nificantUV absorbance. Direct mass snectrometric analysis gave a spectrum of metaldehyde. Occasionally it is desirable to learn the chemical nature of polymeric materials and pyrolysis GC-MS can be useful here. For example the identification of butadiene, isoprene, styrene and dipentene among the major peaks of a pyrolysis gas chromatogram allowed a foam material to be identified as a styrene-butadiene co-polymer. U

Future Trends Mass spectrometry is a new technique in forensic science and will certainly find many new applications in the years to come. The mass fragmentography technique for the quantitative analysis of sub-nanogram levels of drugs in body fluids is now being widely used in clinical chemistry, for example for barbiturates (Lee and Millard, 1975), for heroin and STP (Holmstedt and Lindgren, 1972), and for Ag-tetrahydrocannabinol (Rosenfeld et al., 1974).

I t has been established (Lee and Millard, 1975) that the use of stable isotope labelled carrier material greatly increases sensitivity by reducing column losses and this method could undoubtedly permit the detection and quantification of many drugs in blood samples submitted under the Road Traffic Act (1972) of the United Kingdom and reduce still further the detection limit of other compounds of forensic science interest. Still on the subject of drugs, derivatisation methods such as methylation and silylation used prior to gas chromatography can improve chromatographic behaviour, provide cleaner mass spectra and permit the use of lower column temperatures which result in less column "bleed" and lower detection limits. Gas chromatography columns of the capillary or SCOT type can also result in cleaner spectra by reducing peak overlap in complex chromatograms. Pyrolysis GC-MS has already been mentioned and direct pyrolysis MS is another promising technique which has shown potential use for the identification of bacterial cultures (Meuzelaar pt al., 1974) and could be applied in forensic science to discriminatc between organic materials of many types such as cosmetics, wood, soil, fibres, residues on knife or saw blades and perhaps even dirty marks left by tools or fingers. For each such application it will be desirable to obtain considerable background information to strengthen the evidential value of the results. For example, in the case of the polyethylene fragments containing a particular anti-oxidant it is desirable to know how often this compound is used and for what purposes. This type of information should be fairly readily available, but much work will be involved when experimentation is the only way of obtaining the information, as it would be for example for the direct pyrolysis mass spectral "fingerprints" of soils. The use of mass fragmentography could of course make direct pyrolysis MS and pyrolysis GC-MS even more sensitive while useful discriminating power could be retained in some instances at least. Computers, particularly small laboratory computers, are being used more and more for the analysis of mass spcctrometric data (Zoro, 1976) and forensic science will surely benefit just as bio-medicine and environmental science (e.g., Eglinton et al., 1975) have done. Simple data systems can perform the laborious processes of counting and normalising spectra and subtracting background ions, and more complex systems can carry out automatic file searching and spectrum classification and permit the collection of hundreds of spectra during a single GC-MS run. Important spectra may then be selected and examined, or the change in intensity of any ions during the run may bc plotted (the technique known as mass chromatography). Mass chromatograms can indicate the presence of minor constituents of complex mixtures and could be used to "fingerprint" many kinds of materials which could be introduced into the source by heating on the probe, by direct pyrolysis, by GC or by pyrolysis-GC. References BEGGS, D. P. and DAY,A. G., 1974,J. For. Sci., 19, 891 COSTELLO, C. E., HERTZ,H. S., SAKAI,T. and BIEMANN, K., 1974, Clin. Chem., 20, 255. EGLINTON, G., SIMONEIT, B. R. and ZORO,J. A., 1975, Proc. Roy. Soc. Lond. B., 189.> 41.5. FINKLE,B. S., FOLTZ,R. L. and TAYLOR, D. M., 1974, J. Chrom. Sci., 12, 304. GILLIS,R. G., LACEY,M. J. and SHANNON, J. S., 1974, Org. Mass Spectrom. 9 , 359. HOLMSTEDT, B. and LINDGREN, J.-E., 1972, Anal. Chem., 261, 291. LEE,M. G. and MILLARD, B. J., 1975, Biomed. Mass Spectrom., 2, 78. M. A., 1974, MEUZELAAR, H. L. C., KISTEMAKER, P. G. and POSTHUMUS, Biomed. Mass Spectrom., 1, 3 12. MILNE,G. W. A., FALES,H. M. and AXENROD, T., 1971, Anal. Chem., 43, 1815. R ~ u w s ,A. G., OLLING,M. and WIBOWO,A. E., 1973, J. Pharm. Pharmac., 25, 718. - - -

Organic mass spectrometry in forensic science.

J. Forens. Sci. SOC.(1976), 16, 103 Organic Mass Spectrometry in Forensic Science J. A. ZORO and K. HADLEY Home O&ce Forensic Science Laboratory, Pri...
5MB Sizes 0 Downloads 0 Views