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[61 Royal Society of Chemistry, Analytical Methods Committee, Analyst, 112 (1987) 679. E.A. Maier, Ph. Quevauviller and B. Griepink, [71 Anal. Chim. Acta, 283 (1994) 590. [8l K. Vercoutere, U. Fortunati, H. Muntau, B. Griepink and E.A. Maier, Fresenius’ J. Anal. Chem., 352 (1995) 197. 191 Ph. Quevauviller, H. Muntau, U. Fortunati and K. Vercoutere, The certification of the total and aquaregia trace element contents in calcareous loam soil (CRM 141R), sewage sludge from domestic origin (CRM 144R) and sewage sludge from industrial origin (CRM 146R), EUR Report, European Commission, Brussels, in press. and [ 101 Certification of Reference Materials-General Statistical Principles, IS0 Guide 35, 1985, International Organization for Standardization, Geneva, Switzerland. II111 E.A. Maier, B. Griepink, H. Muntau and K. Vercoutere, EUR Report, 1994,15282 EN, 15283 EN and 15284 EN, European Commission, Brussels, Belgium. I121 G. Rauret and Ph. Quevauviller (Editors), Proc. Workshop on the Sequential Extraction of Trace Metals in Soils and Sediments, Int. J. Environ. Anal. Chem., 5 1 ( l-4) ( 1993). A. Ure, Ph. Quevauviller, H. Muntau and B. GrieI131 pink, EUR Report, 1993, 14763 EN, European Commission, Brussels, Belgium. 1141 Ph. Quevauviller, G. Rauret, A. Ure and H. Muntau, The certification of the EDTA- and acetic acid-extractable contents (mass fractions) of Cd, Cr, Cu, Ni, Pb and Zn in terra rossa and sewage

sludge amended soils, EUR Report, European Commission, Brussels, Belgium, in press. [ 151 Ph. Quevauviller, M. Lachica, E. Barahona, G. Rauret, A. Ure, A. Gomez and H. Muntau, Sci. Total Environ., 178 ( 1996) 127. [ 16 ] E. Cortes Toro, R.M. Parr and S.A. Clement, Biological and environmental reference materials for trace elements, nuclides, and organic microcontaminants - A survey. IAEA/RL/ 128 (Rev 1 ), International Atomic Energy Agency, Vienna, Directory of Certified Reference Materials, Secretary for REMCO, ISO, Case Postale 56, 12 1I Geneva, Switzerland, 1990. [ 17 ] COMAR Data Bank, Laboratoire National d’Essais, 1 rue Gaston Boissier, 75015 Paris.

Philippe Quevauviller and Eddie Maier are at the European Commission, Standards, Measurements and Testing programme (formerly BCR), 200 Rue de la Loi, 1040 Brussels (Belgium). Bernard Griepink was also formely at the BCR but recently moved to the PHARE programme of the European Commission. Umberto Fortunati is at the Studio di lngegneria Ambientale, Via Monti29,20123 Milan0 (Italy); he was the coordinator of the certification of the CRMs 14 lR, 144R and 146R. Kristien Vercoutere is at the University of Ghent, Department of Nuclear Chemistry, Proeftuinstraat 86,900O Ghent (Belgium). Herbert Mun tau is at the European Commission, Environment Institute of the Joint Research Centre, 2 1020 lspra (Italy).

Forensic capillary electrophoresis technique, then some applications to forensic problems are discussed.

F. Tagliaro Verona,

Italy

F.P. Smith* Birmingham,

AL, USA

Capillary electrophoresis (high-performance electrophoresis) has recently become established as a versatile and powerful separation technique. It has a broad applicability and can be used with a variety of detection systems. An introduction is given to this analytical *Corresponding

author.

0165-9936/96/$15.00 P/IS01

65.9936

I96)00055-6

1. Introduction Capillary electrophoresis (CE), known also as high-performance capillary electrophoresis (HPCE), has established itself as an analytical technique with an extremely wide field of application. It originated by optimization of the basic electrophoretic principles largely applied in slab gels for use in an instrumental configuration. Soon, CE extended to include separation mechanisms typical ,f 1996 Elsevier Science R.V. All rights reserved.

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Detector

+

(FBI), as witnessed by several papers in analytical chemistry and forensic science journals (for a review, see Ref. [ 5 I), and a specific course on CE has been held in the Graduate Program in Forensic Science at The University of Alabama at Birmingham, Birmingham, AL. The purpose of this article is to give a short but up-to-date overview of the applications of CE in the main analytical fields of forensic interest, including analytical toxicology, DNA fingerprinting, and explosive- and gunshot-residue-analysis. A short introduction to the basic concepts of the technique is given to help forensic scientists who are not familiar with CE.

Power supply Fig. 1. Scheme of a capillary eletropherograph. Polarity is set according to the normal mode, with a uncoated fused-silica capillary having an inner surface negatively charged by the ionization of silanols.

of chromatography, thus becoming a versatile and flexible tool in separation science. Consequently, CE rapidly found widespread application in analytical chemistry, biochemistry, and biotechnology as well as in various areas of pharmaceutical and biomedical analysis, as witnessed by the increasing number of papers, books and symposia dedicated to this technique [ l-4 1. The most attractive features of CE are its broad analytical spectrum (from inorganic ions to large DNA fragments), variety of separation modes (electrophoretic and chromatography-like) and detection systems (including mass-spectrometry ), high separation efficiency (up to millions of theoretical plates) and mass-sensitivity (from femtomoles - lo-l5 moles - down to yoctomoles lo-*i moles, with the most sensitive detectors), its negligible consumption of samples (nl) and solvents (few ml per day), easy and inexpensive operation and instrumental ruggedness. All these characteristics fit exceptionally well the needs of forensic science laboratories, which cover extremely diverse analytical problems such as gunshot residues, low- and high-explosives, inks, dusts, soils, illicit drugs and toxicants, and DNA fingerprinting. The samples are often limited in quantity, heavily contaminated, and must be preserved as far as possible in order to allow further investigations. In recent years, great attention has been paid to CE by leading forensic science laboratories at the DEA and the Federal Bureau of Investigation

2. Analytical principles - separation,

injection and detection modes 2.7. Capillary zone electrophoresis CE is substantially a high voltage ( lo-30 kV) electrophoresis carried out in tiny fused-silica (or Teflon@) capillaries (20-100 pm I.D., 20-100 cm in length) in order to minimize analyte diffusion and zone broadening. The simplest application of this technique is known as capillary zone electrophoresis (CZE). Silica capillaries have excellent heat dissipation, because of the high surface-tovolume ratio, good antidiffusive-anticonvective action, high transparency to UV light (allowing in-column optical detection) and good physical resistance. These features permit one to accomplish high voltage electrophoresis with almost negligible band broadening, thus making CE by far the most efficient separation technique in the liquid phase. The main separation mechanism taking place in CZE is based on the different electrophoretic mobilities, p, of the charged analytes, which are described (although with some approximation) by the well-known equation: pi = qi/hnqri where ui is the ion mobility, 9; is the ion charge, q is the solution viscosity and riis the ion’s effective radius. When an electric field (E) is applied, the ion’s migration velocity will equal the product of Pi-E, causing the physical separation of the components of a mixture with different pi. As in most electrophoretic separations, also in CZE the voltage generates a so-called ‘electroendo-osmotic flow’ (EOF). It originates from the ions of the buffer, which are electrostatically

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attracted by the charges present on the capillary wall (SiO- in the case of fused-silica). These ions (cations in the case of silica capillaries) in part are fixed, in part mobile. When the voltage is applied, the latter migrate towards the respective electrode: this migration of osmotically active species drags water in the same direction, resulting in a measurable flow of liquid, which actually is the EOF. Since the capillary electropherograph system (Fig. 1) usually has with the detector close to the end of the capillary towards which the EOF is directed, this liquid flow will drag towards the detector all the substances contained in the injected sample, excluding those with an electrophoretic countermigration higher than the EOF itself (small ions with high charge-to-mass ratio). Thus, ionized species will migrate with a velocity, v, resulting from the sum of their intrinsic mobility (pi) and the mobility of the EOF (pEoF ):

Neutral compounds will all migrate together at the velocity of the EOF. In short. with the usual instrumental arrangement having the injector at the anodic end and the detector close to the cathodic end of an uncoated fused-silica capillary (negatively charged at the inner wall), the cations with the highest mobility will arrive at the detector first, followed by the cations with progressively lower mobility; then the bulk of neutrals will appear, followed by the anions in reversed order of mobility (slow anions first, fast anions last). The EOF, being driven by a force generated close to the capillary wall, has a peculiar plug-like flow profile (differing from the parabolic profile of pressure-generated laminar flow), which is ideal for limiting zone diffusion. 2.2. Micellar electrokinetic

capillary

chromatography

Micellar electrokinetic capillary chromatography (MECC or MEKC) was introduced by S. Terabe in order to separate uncharged (neutral) analytes, which in CZE migrate all together at the same velocity as the EOF. A micellar phase (formed by a surfactant at a concentration above its critical micelle concentration) with electrophoretic mobility opposite to the EOF is added to the running buffer. In this system, resembling reversed-phase liquid chromatography, the micelles (made of anionic or cationic surfactants) act as a ‘pseudostationary phase’ (see the end of this Section)

exerting a retarding effect on the compounds they interact with, while the EOF acts as a chromatographic mobile phase, driving the analytes towards the detector. Thus, the uncharged polar analytes are excluded from the lipophilic core of the micelles and consequently migrate first, at a velocity matching the EOF. On the other hand, the most lipophilic compounds are highly retained by the micelles and move at a velocity matching the ‘apparent’ migration of the micelles towards the detector (i.e., the velocity of EOF minus the velocity of the counter migration of the micelles ). The compounds with an intermediate polarity interact selectively with the micelles and are differently retarded, according to their individual degrees of lipophilicity. As in reversed-phase chromatography, the addition of organic modifiers (methanol, acetonitrile etc.) to the running buffer can be used to modify selectively the ‘elution’ of the analytes. The term ‘pseudo-stationary phase’ is used above because the micelles are not actually stationary, but counter-migrate in the running buffer. For situations where the EOF is higher than micelle counter-migration velocity, they actually move towards the detector with a lower velocity than EOF. 2.3. Other separation

modes

The addition to the running buffer of an additive (in solution or in micellar form), which can interact with the analytes with stereospecific selectivity (e.g., crown ethers, bile salts, cyclodextrins), produces highly efficient chiral separations. Cyclodextrins, the most popular chiral selectors in CE, are cyclic oligosaccharides (with six, seven, or eight glucose units for a-, p- and y-cyclodextrin, respectively) with an external hydrophilic surface and a hydrophobic cavity, which can form stereoselective (and mass-selective) inclusion complexes with the analytes. Native cyclodextrins are neutral and move with the EOF. Recently, charged cyclodextrins have been introduced in order to better modulate the mobility of the complexes they form with the analytes. Other separation modes. rarely applied in forensic CE, but potentially important in the near future, are capillary electrochromatography (CEC) and capillary isotachophoresis (CITP). In CEC, a real stationary phase is packed or wallimmobilized in the capillary and a mobile phase (the running buffer) is driven through the capillary by electro-osmosis. In brief, CEC seems to offer an alternative approach to capillary liquid chroma-

516

tography with better mass transfer kinetics and efficiency. In CITP, the ionic analytes are separated and migrate in discrete zones at the same velocity between two ionic solutions, one with the highest mobility (leading electrolyte) and the other with the lowest mobility (terminating electrolyte) of all the analytes in the sample. Because of its band focusing ability, CITP can be used not only for direct separation, but also for sample pre-treatment (with concentrating ability up to 100-1000 times), followed by CZE. Gel-electrophoresis in capillaries (CGE), as in agarose or polyacrylamide slab gels, produces separations based on molecular sieving. It is mainly applied for separation of bio-polymers in which, because of the similarity of the constituent monomeric units, the charge and mass change proportionally, giving almost constant charge-to-mass ratios. In these cases, the potential of plain electrophoretic separations is extremely poor and, consequently, an additional mechanism, i.e., molecular sieving, must be introduced in the separation process. CGE is typically used for DNA fragment analysis and DNA sequencing (both fundamental for forensic DNA fingerprinting) and for the characterization of proteins (resembling slab gel SDSPAGE). A difference from slab gels is that, in CE, entangled polymer solutions (linear polyacrylamide, hydroxyalkylcellulose, polyvinylalcohol, etc.) are often preferred to cross-linked gels. Isoelectric focusing, popular in protein analysis, has also been converted for use in CE technology, and is known by the acronym CIEF. 2.4. Injection modes In order to maintain a high efficiency of separation, with standard capillaries the volume of the sample injected should not exceed a few tens of nanolitres. The accurate and reproducible injection of such volumes is usually carried out by applying a pressure difference between the ends of the capillary, while the injection end is dipped in the sample vial. Then the injection end is moved into the running buffer vial, the voltage is applied and the separation started. An alternative, electrokinetic mode of injection can be accomplished by applying a voltage, while the inlet end of the capillary is dipped into the sample vial; the sample components will be driven into the capillary by electrophoresis and by the EOF. In hydrodynamic injection, the injected plug is representative of the sample com-

trends in analytical chemistry, vol. 15, no. IO, 7996

position, whereas the electrokinetic mode is ‘selective’, because sample components enter the capillary according to their mobility and with dependence of mobility and concentration of the total ions in the sample. 2.5. Detection modes Usually, detection is accomplished ‘in-column’ by UV(-visible) absorption. Modern electropherographs can be fitted with diode array or fast-scanning absorption detectors, which can collect on-line UV(-visible) spectra of the separated zones, although with a moderate loss in sensitivity. Fluorescence (mostly, laser induced) and conductimetric detection have also become available in some commercial instruments. Mass spectrometry (mainly with electrospray ionization) has been successfully coupled to CE and some interfaces have already become commercially available. Amperometric detection, although feasible and highly selective and sensitive, needs special instrumental arrangements and, so far, is limited to a few specialized laboratories. A detection mode which is typical in, although not exclusive to, CE is the so-called ‘indirect detection’ , which allows the determination of ionic molecules that, because of their structure, are not susceptible to ‘direct detection’ (e.g., inorganic ions, amino acids, organic acids). It is based on the local variations of concentration of an ionic additive, detectable at trace levels by the detector used, induced by the presence of the analytes. This compound, added on purpose to the running buffer, is displaced from the zones where the ionic analytes migrate to preserve electroneutrality, thus giving rise to ‘reversed peaks’.

3. Applications sciences

of CE in the forensic

3.7. Forensic drug analysis One of the major tasks for forensic science laboratories is the analysis of preparations of illicit or controlled drug substances, in order to identify the major components, and also trace components, in order to infer the sources and pathways of production and to compare different seizures with a possible common origin. For reviews of the applications of CE for the analysis of seized drugs see

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[ 6,7 1. Other important tasks are the determination of controlled drugs, toxicants, and their metabolites in biological fluids and tissues, for investigation of causes and circumstances of death or of conditions of acute or chronic intoxication. In a forensic science environment, it is obvious that all the analytical results must be defensible in court, but different countries have different standards to achieve legal defensibility. However, in practice they have some fundamental common points. The analytical methods must rely on sound, widely accepted analytical principles and must be thoroughly validated in the laboratory and at an international level. The results must be confirmed by using at least two independent methodologies, based on different physico-chemical principles, but with comparable sensitivity. Other requirements concern intra- and inter-laboratory proficiency testing, laboratory certification or accreditation, etc. On the grounds listed above, CE appears to be particularly interesting, because the electrophoretic separation principles it relies on are relatively novel in drug analysis, which is traditionally based on chromatographic and /or spectrometric techniques. Thus, being based on specific separation principles, CE is highly complementary to the traditional analytical approaches and is suitable for cross-validation of methodologies and/or confirmation of results. This view was experimentally verified by Lurie et al. [ 8 1, who compared the MECC separation patterns of more than twenty anabolic steroids of forensic interest with those from gas-chromatography (GC) and high-performance liquid chromatography ( HPLC ). Good agreement was obtained between all three techniques and, notably, a statistical evaluation using the Principal Component Analysis (on 17 steroids) proved that CE, HPLC, and GC were highly orthogonal (i.e., not correlated). This supports the complementarity of CE which, in combination with chromatographic methods, may increase the overall discriminatory power of the analytical strategy. Substantially different separation patterns from CE and HPLC had already been reported, for example in the separation of bulk heroin, heroin impurities, degradation products, and adulterants by Weinberger and Lurie [ 9 ] (Fig. 2). These authors first applied CE to the screening of illicit drug substances, including psilocybin, morphine, phenobarbital, codeine, methaqualone, LSD, heroin, amphetamine, cocaine, methamphetamine, benzodiazepines, phencyclidine, cannabinoids, heroin impurities, degradation products and adulterants, cocaine, and cocaine-

517

m,$

Fig. 2. Separation of bulk heroin, heroin impurities, degradation products and adulterants. (Top) HPLC chromatogram. Conditions: column, 11 .O cm x 4.7 mm I.D., Partisil 5ODS-3; mobile phase, phosphate buffer containing hexylamine 23 mM, pH 2.2-methanol, gradient elution; detector wavelength, 210 nm. (Bottom) MECC electropherogram. Conditions: capillary, 25 cm x 50 pm I.D.; voltage, 20 kV; temperature, 40°C; buffer, 85 mMSDS-8.5 mMphosphate-8.5 mM borate-l 5% acetonitrile, pH 8.5; detector wavelength, 210 nm. Peaks (a) acetic acid, (b) morphine, (c) 3monoacetylmorphine, (d) 6-monoacetylmorphine, (e) acetylcodeine, (f) heroin, (g) phenobarbital, (h) noscapine, (i) papaverine, (j) methaqualone. Reprinted with permission from Ref. [ 9 1. Copyright 1991, American Chemical Society.

related compounds. For this purpose, the authors employed MECC in bare fused-silica capillaries using a pH 8.5 phosphate-borate buffer containing 85 mM SDS as the micellar agent with an organic modifier ( 15% acetonitrile). Detection was ‘in column’ by UV absorption at 2 10 nm. The method also achieved a high-efficiency separation of the acidic and neutral impurities in illicit heroin seizures. The migration pattern of heroin-related substances obtained with MECC was, again, clearly different from the elution order in reversed-phase HPLC.

518

Moreover, MECC allowed the resolution of about twice as many peaks as HPLC, but HPLC-UV was far more sensitive than MECC-UV. In order to overcome this limitation of UV, the authors tested lampbased fluorescence detection (excitation wavelength 257 nm, emission 400 nm) which increased the sensitivity by a factor of twenty for the fluorescent impurities in illicit heroin with a remarkable improvement in selectivity, also. Pursuing a further increase in sensitivity, Lurie et al. [ lo] have recently reported the successful use of laser-induced fluorescence detection with a krypton-fluoride laser source at 248 nm. Unfortunately, the fluorescence of the trace compounds present as impurities in heroin preparations depends on simple fluorophores (e.g. phenanthrene rings) which require excitation at relatively low wavelengths. This imposes the use of often-expensive laser sources, a limitation which could be overcome using the development of derivatization procedures with highly fluorescent tags or reagents which would give rise to fluorescent adducts by reaction with the analytes of interest. This approach, which would allow the use of much higher excitation wavelengths, has not yet received enough attention, particularly in drug analysis. The excellent improvements in sensitivity which can be attained by laser-induced fluorescence in comparison with UV ( 1000 times and more) and the reduction in cost of laser sources, along with the increase in emitted wavelength allows us to hypothesize that, in the near future, greater attention will be paid to fluorescent derivatization in the field of drug analysis. As far as reproducibility is concerned, Weinberger and Lurie’ s, pioneering paper [ 9 1, reported relative standard deviations (RSD) of about 0.5% for migration times and 4-8% for areas and peak heights. However, poorer reproducibility was observed for the peaks with migration times > 40 min, which was ascribed essentially to inconsistent evaporation of the organic modifier from the open buffer reservoirs, and to capillary-wall fouling. On the detection side, another improvement in the CE of illicit drug preparations (namely, heroin and cocaine) was reported by Staub and Plaut [ 111, who employed an ‘in column’ fast-scanning UV detector to record the absorption spectra of the peaks, and provide a further tool for the identification of the sample components. Krogh et al. [ 121 published the validation of a quantitative method for the analysis of heroin and amphetamine-related substances using MECC with 25 mM SDS. Within-day and between-day RSDs

trends in analytical chemistry, vol. 15, no. 70, 1996

for migration times (relative to an internal standard) were in the ranges 0.5-l .9% and 0.89-2.23%, respectively. Standard curves were linear in the range 0.02-0.5 mg/ml, with correlation coefficients of 0.997-0.999: in quantitative analyses the RSDs ranged from 2.0 to 4.3%. Samples were injected every 13 min and the same fused-silica capillary lasted as many as 500 injections. The authors concluded that CE can be a valuable complement to HPLC and GC. MECC with 40 mM SDS was also adopted by Walker et al. [ 13 ] for the quantitation of heroin illicit samples. They reported, with minor exceptions, complete separation within 5 min of numerous compounds, including substances which are difficult to analyze by GC - such as morphine, 6monoacetylmorphine, aspirin, and salicylic acid. Comparisons with a commonly used GC method showed excellent correlation. The cationic surfactant cetyltrimethylammonium bromide (CTAB ) was proposed by Trenerry et al. [ 14-161 as a micelle former as an alternative to SDS for the MECC separation of heroin and cocaine samples and for the analysis of morphine related alkaloids in crude morphine, poppy straw, and opium preparations. Quantitative results from MECC correlated well with HPLC, showing a greater resolving power for CE, but with a slightly lower precision. In cocaine analysis, MECC was also compared with GC, showing good quantitative correlation and comparable precision. Chiral separation is one of the most challenging tasks in pharmaceutical and pharmacological analysis. In this field, CE has rapidly become a ‘first choice’ technique because of its high efficiency, excellent chiral resolution power, and economy. The enantiomeric composition can provide investigators with precious information on the method of production of some chiral drugs. At the DEA laboratories Lurie [ 17 ] optimized the MECC separation of the enantiomers of amphetamine, methamphetamine, ephedrine, pseudoephedrine, norephedrine, and norpseudoephedrine after reaction with a chiral derivatizing reagent (2,3,4,6-tetra-G-acetyl-b-bglucopyranosyl isothiocyanate) to form the corresponding diastereomers which were separable using a non-chirally-selective MECC method. Notably, a ‘direct’ approach, avoiding any derivatization, was later reported by Lurie et al. [ 18 ] for amphetamine, methamphetamine, cathinone, methcathinone, cathine, cocaine, propoxyphene, etc., by using as chiral selectors neutral and anionic cyclodextrins added to the buffer. As mentioned above,

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195 nm

3 $

o-010* 0

1

2

5

-.;I:IJ, 195

nm

J

-0.001

30.0

Ii.0

0

0.060

195

g B

iii > -0.002

P-cyclodextrins, has been reported by Aumatell et al. [ 19,201, Cladrowa-Runge et al. [ 211 and by Varesio and Veuthey [ 221, while Flurer et al. [ 23 ] resolved ten stereoisomers of ephedrine compounds using hydroxypropyl-/3-cyclodextrin. A third way, resembling chromatography, to achieve chiral separation is by using a chiral stationary phase and CEC. Li and Lloyd [ 241, used at -acid glycoprotein as stationary phase packed in fused-silica capillaries, and reported the enantioselective analysis of benzoin, hexobarbital, pentobarbital, ifosfamide, cyclophosphamide, disopyramide, metoprolol, oxprenolol, alprenolol. and propranolol. In comparison to HPLC, the CE mode of separation, by miniaturizing the volumes of the separation compartments, provided higher efficiency and reduced the costs of consumables. MECC, which couples electrophoretic and chromechanisms, has matography-like separation proved highly compatible with biological matrices, and is thus suitable for the analysis of drugs in biological fluids [ 25 1. To the best of our knowledge, Wernly and Thormann [ 26 ] were the first to use a borate-phosphate buffer containing 75 mM SDS to achieve the qualitative analysis of benzoylecgonine, morphine, heroin, 6-monoacetylmorphine, methamphetamine, codeine, amphetamine, cocaine, methadone, methaqualone and benzodiazepines in urine (Fig. 3). A sample pre-treatment with solid-phase extraction and concentration allowed the authors to achieve a sensitivity of about 100 ngl ml. Further confirmation of the peak identity was accomplished by means of a fast-scanning UV detector; the on-line recorded peak spectra matched the reference spectra of standard compounds stored in the computer data base. According to the authors, MECC showed a sensitivity comparable to most non-isotopic immunoassays and was proposed for confirmation testing. After this paper, MECC with UV detection has been reported in the recent literature for the determination of several drugs of abuse and toxic compounds in biological fluids, even if often only qualitatively. Some notable examples include: 0 barbiturates [27-291 l THC metabolite [30] l morphine-3-glucuronide [ 3 1] l benzodiazepines [32] l tricyclic antidepressants [33] As an alternative to MECC, highly efficient separations of drugs of forensic interest were obtained by Chee and Wan [ 341 by using plain CZE in phosphate buffer pH 2.35. The authors

320 14.0

20.0 TIME

5

26.0 (mid

Fig. 3. Determination of cocaine metabolites and opioids in urine with MECC. Conditions: capillary, 90 cm x 75 ym I.D.; voltage, 20 kV; buffer, 75 mill SDS-10.0 mM phosphate-6.0 mM borate, pH 9.1. Electropherograms: (A) directly injected urine blank, (B) extracted urine blank, (C) extracted urine spiked with (1 ) benzoylecgonine, (2) morphine and (5) codeine (10 pg/ml each), and (D) a three-dimensional data plot of the extracted spiked urine. Reprinted with permission from Ref. [26]. Copyright 1991, American Chemical Society.

cyclodextrins form chirally selective inclusion complexes with analytes, thus selectively retarding one of the two stereoisomers of a racemate ( because of a different charge-to-mass ratio of the complex, in comparison with the free analyte). An optimization of the enantiomeric separation of amphetamine, methamphetamine and other ring-substituted analogues based on the use of native and substituted

520

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achieved the separation of seventeen basic drugs, including amphetamine, methamphetamine, medazepam, lidocaine, diazepam, and methaqualone in only 11 min. Under these conditions, analytes were separated according to a typical electrophoretic separation, depending on mass and charge: thus, drugs having lower pKa values, and consequently less positive charge, showed higher migration times. The reported migration time RSDs were less than 1% and the peak-area RSDs were between 1.5 and 4.3%. The sensitivity of this method, according to our experience, is better than that of MECC, because of a higher efficiency of CZE with consequent lower peak dilution. On the other hand, a limitation of CZE is its inability to analyze charged and neutral drugs together, which is possible with MECC. According to Taylor et al. [ 35 ] CZE in 100 mM phosphate buffer of pH 6 allowed the complete resolution of pholcodine, 6-monoacetylmorphine, morphine, heroin, codeine and dihydrocodeine in urine, with a sensitivity of about 10 ng / ml (electrokinetic injection). Hair is a skin annex without metabolism in its stalk and has recently become an important sample to be analyzed when past, chronic exposure to illicit drugs is to be investigated. In this field, GC, GC (-MS ) and HPLC are the usual analytical tools, but CE could play an important role, as reported by

Tagliaro et al., who used both CZE [ 36,371 and MECC [ 38 ] with UV detection for the determination of cocaine and morphine in hair samples. Fairly good resolution, efficiency and repeatability (intra-day migration time RSDs: < 1% in CZE and < 2% in MECC, intra-day peak-area RSDs in the range 3-5%) were achieved but, particularly in MECC, the sensitivity was substantially lower than that of HPLC. Notwithstanding this limitation, CE could meet the sensitivity limits generally required in hair analysis (0.5-l .O ng of analyte/ mg of hair). In conclusion, CE seems to be an excellent complement to existing chromatographic techanalysis (screening niques for toxicological or confirmation), with advantages over GC in terms of its ability to deal with nonvolatile, polar or thermally labile compounds, and over HPLC in terms of efficiency, resolving power and consumption of expensive and often toxic solvents. 3.2. Analysis of explosives

and gunshot

residues

The analysis of gunshot and explosives residues is aimed at the identification of persons involved in firearm use or in terroristic blasting, but also at the

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0.0093

38 CALIBER 45 CALIBER SWAB BLANK

0.0074 2 0.0055 0.0036 0.0017

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Fig. 4. MECC analysis of extracts from ammunition shell casings. Conditions: capillary, 67 cm x 100 pm I.D.; voltage, 20 kV; buffer, 25 mM SDS-2.5 mM borate, pH 8.9; detector wavelength, 220 nm. Bottom trace: blank. Middle trace: 45 calibre. Upper trace: 38 calibre. Peaks: EtOH, ethanol; EGDN, ethylene glycol dinitrate; NG, nitroglycerine; EC, ethylcentralite; DBP, dibutyl phthalate. Reprinted with permission from Ref. [ 391. Copyright 1991, American Chemical Society.

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investigation of the sources and supplying pathways of ammunitions and explosives. Highly sophisticated and expensive instrumentation is used worldwide, such as neutron activation analysis, mass spectrometry, X-ray, and infrared techniques, and all types of chromatography to analyze either organic or inorganic constituents of ex- plosive mixtures or post-blast residues. In this field, CE is suitable for the determination of both organic and inorganic compounds, showing greater versatility and economy than the traditional methods. Northrop et al. [ 391 optimized a MECC method for the analysis of 26 organic constituents of explosives and gunshot residues. Experimental conditions were very simple, as the authors used uncoated silica capillaries ( 100 pm I.D.), 2.5 mA4 borate buffer and 25 mM SDS; the potential applied was 20 kV. Detection was by UV absorption at 250 nm (200 nm for nitroglycerine). Baseline separation of gunshot residue constituents, namely nitronitroglycerin, guanidine, 2,4-dinitrotoluene (DNT), 2,6-DNT, 3,4-DNT, 2,3-DNT, diphenylamine (DPA), N-nitrosoDPA, 2-nitro( DPA), ethylcentralite, and dibutyl phthalate, was achieved in 10 min, with efficiencies between 200000 and 400 000 theoretical plates and mass detection limits of fractions of a nanogram. The same MECC method allowed the separation of fifteen components of high-explosives, including nitroguanidine, ethyleneglycol dinitrate, diethyleneglycol dinitrate, 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), nitroglycerine, 2,4,6-trinitrotoluene (TNT), pentaerythritol tetranitrate, and picric acid. Application of the method to forensic cases included the analysis of spent ammunition casings (Fig. 4), reloading powders and plastic explosives. Differences in composition were found between reloading powders from different manufacturers and also between powders from the same producer. In a further paper, a method was reported for sample collection of gunshot residues, optimized for CE, which took advantage of the minimal sample needs of this technique 1401 . Instead of the usual swabbing of the area of interest (for users of revolvers and pistols, the back of the hands along the thumb and forefinger and the webbing in-between), they used masking adhesive tape ( 1 inch square sections) to collect residues. The film lifts were examined with a binocular stereoscope and gunshot residue particles were collected with tweezers, placed in glass micro vials and dissolved in 50 pl of ethanol; the solvent was then evaporated

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and the residue reconstituted with 25 pl of MECC buffer and injected. Using this sampling method, it was possible to analyze a single particle collected from the skin surface, conceivably allowing a more careful picture of the spatial distribution of the gunshot residue particles than with swabbing techniques. Firing range experiments with subjects who fired different weapons demonstrated that characteristic gunshot residue constituents were found on the person who discharged the firearm and on the weapon itself. CE was also applied to low-explosive investigations and compared to ion chromatography, the leading technique in this field. Low explosives are based on the reaction of strong inorganic oxidants (e.g. nitrate, chlorate) with fuels (e.g. carbon, sulphur, sugars): anions left behind after the blast are the most important pieces of evidence for inferring the type of the explosive material used. Although ion chromatography is suitable for this purpose, it reportedly suffers from lack of adequate complementary techniques and could find a complement in CE, which is based on different separation and detection principles. The most important ions present in the blast residue are chloride, nitrite, nitrate, sulphate, sulphide, chlorate, carbonate, hydrogencarbonate, cyanate, thiocyanate and perchlorate, which can be separated by CZE in a single run. Hargadon and McCord [ 4 11 at the FBI Laboratories used 65 cm X75 pm I.D. bare silica capillaries with a borate buffer (2 mM borate, 40 mM boric acid) containing 1 mM diethylenetriamine as an EOF modifier, at the final pH of 7.8. The applied potential was 20 kV, with reversed polarity, to obtain the migration of anions with high mobility towards the detector. Indirect UV detection at 280 nm was used, by adding to the running buffer a dichromate chromophore ( 1.8 mail). In a comparison with ion chromatography, CZE showed clear differences in the separation patterns, with a superior efficiency. The CZE reproducibility of the migration times was evaluated over two months with the same capillary, with RSDs better than 1%. Also, peak identity could be confirmed by comparing the ‘negative’ electropherograms recorded at 280 nm (by indirect detection) with the profiles recorded at 205 nm, a spectral region where nitrite, nitrate and thiocyanate absorb, generating ‘positive’ peaks. The complementary nature of CZE and ion chromatography was tested with pipe-bomb fragments prepared with different explosive mixtures (potassium chlorate-Vaseline, black powder, smokeless

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powder) and experimentally detonated. Ion chromatography and CZE were carried out with concordant results. In short, CE offers powerful novel tools suitable for both high- and low-explosive and gunshot residue analysis. Today, this is particularly important because of the trend of the producers towards the elimination of heavy metals from the ammunition primers, which could soon make useless the traditional investigation tools based on the application of atomic absorption spectrometry and scanning electron microscopy. 3.3. Analysis of forensic DNA As is well known, DNA polymorphism analysis is the modern approach to personal identification (in criminal cases or in mass disasters) and paternity testing, while the traditional strategies based on the analysis of phenotype variants have almost been abandoned worldwide. The forensic analysis of DNA polymorphism is based on exponential amplification by polymerase chain reaction (PCR) of individual loci, followed by analysis of differences in length or sequence. Length polymorphism are most often studied by forensic genetists, who focus their attention onto specific regions of DNA known as variable number tandem repeats (VNTRs) showing a high variability in the population: the length of a repeat unit can be as short as two base pairs and, at a given locus, each individual has a pair of such sequences (alleles). Short ( < 1000 base pairs long) or very short (up to about 200 base pairs) fragments, having length differences as little as l-2%, are particularly interesting for forensic genotyping. While PAGE and agarose slab gel electrophoresis have long been established as the standard separation methods for DNA fragments, CE using molecular sieving separation modes has proved to be a rapid and highly efficient alternative. In fact, especially with laser-induced fluorescence detection, CE achieves sensitivity limits that cannot be met by traditional techniques. Also, because of its instrumental configuration, CE is more suitable for GLP and interlaboratory quality controls than a ‘manual’ technique such as slab gel electrophoresis. Recently, a fully automated instrument based on CE technology (with laser-induced fluorescence detection) has become commercially available for DNA sizing, quantitation, screening and sequencing. For separation of DNA fragments of different size, CGE with cross-linked polyacrylamide, or

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agarose, gel-filled capillaries has been used. Even if separations in these capillaries have proved exceptionally efficient, some pitfalls of gel-filled columns have soon become evident, especially in routine applications, because of localized overheating of the gel, irreversible binding of DNA strands to the gel network, and clogging at the injection end by particulate material present in the samples. As an alternative to gel filled columns, ‘non-gel’ sieving media have been introduced, using the sieving effect of entangled solutions of water-soluble linear polymers, such as hydroxyalkylcellulose or linear polyacrylamide. The main advantage of this approach is that the non cross-linked sieving media can easily be renewed in the capillary, getting rid of the old buffer. In an early stage of development, non-gel sieving buffers showed a worse resolution than gel-filled capillaries, but polymeric hydroxyethylcellulose has recently been reported to have a separation power close to, or even better than, cross-linked gels [ 421. Also, linear polyacrylamide at concentrations from 4-10% has shown excellent resolution of DNA fragments in the 5 l-23 130 base pair range [ 43 1. In the field of forensic DNA analysis, non-gel sieving media (i.e., OS-0.75% hydroxyethylcellulose in 100 n-&Zn-is-borate buffer, pH adjusted to 8.7 with CsOH, containing 0.635-1.27 pM ethidium bromide) with a 100 pm I.D. capillary coated with a moderately hydrophobic phase (DB-17), have been used by McCord et al. [ 441 at the FBI Laboratories, Quantico, VA. The authors applied this method with UV detection and, for the analysis, two genetic markers, namely DlS80 and SE 33. D lS80 has a tandem repeat unit of sixteen base pairs: a mixture of standard D 1S80 alleles, spanning from 403 to 1069 base pairs, was resolved. The marker SE 33 has alleles differing by only four base pairs, in a molecular range of 230-350 base pairs, with possible two-base-pair repeats (half alleles). Notably, CE under the conditions mentioned above was capable of resolving also the SE 33 allelic test mixtures. More recently, McCord et al. [ 45 ] and Srinivasan et al. [ 42 ] reported the application of laserinduced fluorescence detection with an argon-ion source. PCR products were stained with fluorescent intercalating dyes, namely YO-PRO- 1 and TOTO1, achieving the excellent sensitivity of about 500 pg/ ml of DNA. Using 1% hydroxyethyl or 0.5% methylcellulose, respectively, as the sieving media, PCR-amplified DNA fragments from 120 to 400 base pairs were separated, with resolution of four

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Fig. 5. Separation of HUMTHOI allelic ladder (between 150-bp and 300-bp markers) with non-gel sieving buffer. Conditions: capillary, 37 cmx50 t_rrn I.D., DB-17 coated; buffer, 100 mM M EDTA, 1% hydroxyethylcellulose pH 8.1, 50 ng/ml YO-PRO-l; voltage, O-5.2 min -15 kV, 5.2-10 min -5 kV; temperature, 25/C; laser fluorescence: excitation 488 nm, detection at 520 nm. From Ref. [ 51, with permission.

base pairs or less. Application to analysis of genetic markers of forensic interest, such as apolipoprotein B, DISSO, mitochondrial DNA, MBP and HUMTHOI ( Fig. 5), was reported by the two groups. The same authors also showed applications of CE for restriction-fragment length polymorphism (RLFP) analysis. In a recent paper Butler et al. [ 461 reported the use of dual internal standards for typing PCR products for the locus HUMTHOI ( 179-203 bp) with high reproducibility and achieving precise fragment sizing with an inter-assay standard deviation of 0.3 bp. DNA sequencing is another promising field of application for CE, especially using laser-induced fluorescence detection [ 47 1. However, sample throughput is a limitation with the current instrumentation fitted with a single capillary. CE instrumentation with multiple ( 100 and more) capillaries in parallel and automatic sample-handling systems promises to increase dramatically the productivity in DNA sequencing. 3.4. Other applica Cons A typical area of forensic science investigation is ink analysis. which is currently carried out by using

thin-layer chromatography, liquid chromatography, and slab-gel electrophoresis. Recently, and still tentatively, the application of CZE has been reported by Fanali and Schudel [ 48 ]. Clear differences between the electrophoretic patterns from different fibre-tip pen inks were found, using a mixture of 0.1 A4 ammonium acetate pH 4.5 with methanol (3:l ) as running buffer, and a coated capillary (20 cm X 25 pm I.D. ) with UV detection at 206 nm. Excellent peak shape and resolution of the ink components were observed. Dye spots separated by thin-layer chromatography were extracted and added to the same ink sample to check by analyte addition the identity of the well resolved, but individually unknown CZE peaks. Also, CE has been applied for the identification of acetaldehyde protein adducts, which could be considered as potential markers of chronic alcoholism. Distinguishable changes have been found in the CZE electropherograms from the hair of rats chronically administered with ethanol [ 491. This was tentatively explained by charge changes in the protein structure induced by acetaldehyde, the main endogenous metabolite of ethanol, reacting by Schiff’s base formation with the protein-free amino groups. Other applications to the analysis of proteins of potential forensic interest include the separation and determination of globins [ SO] and saliva and semen proteins [ 5 1,521.

4. Conclusion Probably, the most impressive characteristic of CE is its great versatility. As we have shown, an incredibly wide spectrum of applications and separation modes can be covered with the same instrumental hardware. This makes CE unique in modem analytical technology and fits very well the diverse needs of forensic sciences. The diffusion of this emerging technique into the forensic science environment is still limited. This is understandable, given the limited electrophoretic background of many analysts working in forensic chemical analysis. In forensic biology, where electrophoresis has a long tradition, there is little familiarity with an instrumental technique such as CE. Despite these problems of ‘mutual comprehension’ between CE and forensic scientists, this technique is already established as a sound, mature technology, ready for use in the analytical disciplines of major forensic interest. One might also note recent preliminary

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reports of the application of the hyphenated CEelectrospray MS to the analysis of a panel of forensic drugs [ 53 ] and the demonstration of a high degree of orthogonality between CZE and MECC in the analysis of illicit drugs, which allows, to a certain extent, the mutual confirmation of results without changes in the CE instrumental hardware [ 541. Thus, we believe that in the near future CE will not be overlooked by the forensic science community. As stated by Kuffner et al. in a recent paper [ 55 ] on the admissibility of new technologies in the USA courts, “given the economical and efficiency advantages of CE for forensic science and the judicial process, expert testimony based on CE analysis can be anticipated. The legal criteria of Daubert, as long as they are met by the scientific community, will allow CE and other legitimate scientific processes into evidence as acceptable expert testimony”.

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Franc0 Tagliaro, MD, is forensic pathologist, toxicologist at the institute of forensic Medicine, University of Verona, Verona, Italy, where he is deputy director and lectures in Forensic Medicine. His major research field is analytical toxicology, and particularly the analysis of hair for drugs of abuse for which he pioneered the use of liquid chromatography and capillary electrophoresis. He is Visiting Professor in the Graduate Program in Forensic Science, Department of Justice Sciences, The University of Alabama at Birmingham, Birmingham, AL, USA. He is a member of the editorial board of Forensic Science International and of The International Journal of Drug Testing, an Internet based journal. Frederick Paul ‘Smith, PhD, is Associate Professor of Forensic Science at the Department of Justice Sciences, The University of Alabama at Birmingham, Birmingham, AL, USA. The majorpati of his research work is devoted to the application of immunochemical and chromatographic analytical techniques in forensic toxicology, particularly directed to the pathways of incorporation of xenobiotics the in hair matrix. Since 1994, Drs. Smith and Tagliaro are cooperating in a joint research project on the applications of capillary electrophoresis in forensic science.

Evolution and potential of the BiAS procedure for the determination of non-ionic surfactants Zenon Lukaszewski*, Andrzej Szymanski, Bogdan Wyrwas Poznan,

carbons or adsorbed on particles) and polyethylene glycols were discussed.

Poland

The evolution of the BiAS procedure is reviewed and its standard recommended version was compared with the modified version combined with the indirect tensammetric method (BIAS-ITM). New applications of the use of BIAS-ITM for the determination of nonionic surfactants (in the presence of hydro-

*Corresponding author. ‘A glossary of terms and abbreviations the end of the paper.

is given at

1. Introduction’ The BiAS procedure is routinely used for the determination of non-ionic surfactants (NS). The name of the procedure is an abbreviation of its full name, ‘bismuth active substances’. The procedure is also known as the Wickbold method or as the method for the determination of NS with the modified Dragendorff reagent. This last name for the method is the oldest one. Nowadays, the Dragendorff reagent is known as the anionic complex species of bismuth( III) and iodides. Originally it was