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Anal Bioanal Chem 2007 3881359 1364 DOI 101007s00216 007 1381 1 REVIEW Current role of capillary electrophoreticelectrokinetic techniques in forensic toxicology Franco Tagliaro Federica Bortolotti Jennifer P Pascali Received 20 February 2007 Revised 21 May 2007 Accepted 22 May 2007Published online 16 June 2007 Springer Verlag 2007 Abstract The current application of capillary electropho resis in forensic toxicology has been critically reviewed with special focus on the areas where this technique has shown real advantages over chromatographic methods For example capillary electrophoresis has been most success fully applied to the chiral analysis of some drugs of forensic interest including amphetamines and their congeners Another typical application eld of capillary electrophore sis is represented by protein analysis Recently special interest has been paid to carbohydrate de cient transferrin CDT the most important biological marker of chronic alcohol abuse Other specific applications of capillary electrophoresis of potential forensic toxicological concern are also discussed The review includes 62 references Keywords Capillary electrophoresis Drug analysis Forensic toxicology Introduction About 10000 scienti c articles can be retrieved from the database PubMed using the keyword capillary electropho resis CE On this basis CE commercially introduced in the late 1980s should no longer be defined as new recent or emerging The use of CE in different elds of applied analytical science and research eg molecular biology is nowadays well established whereas in other areas eg chiral analysis CE is receiving great attention F Tagliaro E F Bortolotti J P Pascali Department of Medicine and Public Health Section of Forensic Medicine University of Verona Policlinico GB Rossi Ple LA Scuro 37134 Verona Italy e mail francotagliarounivrit Notwithstanding the many excellent technical reviews of the literature on CE see below in our opinion there is still need for more general critical evaluations on the advantages that CE can realistically offer in each speci c application context where so far chromatography in its different modes has played the leading role Forensic science is typically a eld involving the interface between different analytical technologies among which CEiknown for its versatility and broad eld of applicabilityilooks particularly promising The extent of scienti c interest in using CE in the forensic environment is highlighted by the number of speci c reviews eg see refs 177 just to mention a few which constantly monitor the latest advancements of this technique However despite a wide penetration in forensic STR short tandem repeats analysis and in DNA sequencing where fully automated multicapillary electropherographs are today the standard analytical tools CE is still regarded with suspicion by forensic scientists and particularly by forensic toxicolo gists This apparently unexplainable situation may be justi ed by the lack of sound electrophoretic experience of the majority of the forensic analysts who traditionally use chromatographic and spectroscopic techniques Indeed gas chromatography GC and liquid chromatography HPLC apparently offer solutions to almost all the analytical problems which may be encountered in forensic science so why change The answer to this question is not easy to nd if we stick to tradition Nonetheless a powerful analytical tool such as CE cannot be overlooked if the pace of innovation is to be maintained even in the fairly conservative environment of forensic sciences Hence instead of merely reproducing an updated list of the latest applications of CE in analytical toxicology this review is aimed at highlighting the pros and cons of this technique in selected areas of forensic drug analysis in Q Springer 1360 Anal Bioanal Chem 2007 3881359 1364 which on the basis of a critical review of the recent literature real advantages can be seen from the use of capillary electrophoretic and electrokinetic techniques Analysis of microsamples and online sample enrichment CE methods have been reported for the determination of a variety of analytes including therapeutic and illicit drugs in biological samples and in pharmaceutical and clandestine preparations For these purposes other techniques such as GC and HPLC are available and currently used worldwide In this field in our opinion CE has little advantages to offer in terms of accuracy sensitivity precision and selectivity except for the need for minimal sample and reagent amounts It is worth mentioning the potential of CE to analyse the content of single cells 8711 Also minute samples from microdialysis are very suitable for CE analysis as reported in several publications 12 13 the online coupling of the two techniques is also possible 14 15 Even if to the best of our knowledge these specific applications are rarely of interest for forensic toxicologists microtrace analysis is surely an important approach to forensic investigation for instance in gunshot residue analysis In this specific field as early as in 1992 Northrop and MacCrehan 16 developed a specific sample collection procedure for gunshot residues to be analysed with CE based on the use of masking adhesive tape The film lifts were examined with a binocular stereoscope for gunshot residue particles which were collected with tweezers and analysed by micellar electrokinetic capillary chromatogra phy MECC According to the authors sample collection with adhesive film was superior to the traditional swabbing method Because of the small sample size required by CE it was possible to analyse single particles collected from the skin surface Transferring microsample analysis to the toxicological field could be a new promising application of capillary electrophoretic techniques which so far has never been pursued On the other hand the handling of minute sample amounts limits the loaded mass of analyte and consequently hampers the sensitivity of CE in terms of concentration Typically detection limits of CEUV methods hardly go below micromolar concentrations which is at least 100 times higher than what can be achieved with HPLCUV This limitation can be overcome by sample enrichment methods among which field ampli ed sample stacking FASS and sweeping procedures have been applied to the analysis of forensic samples CE with headcolumn FASS in the presence of a water plug inserted at the capillary tip proved to be a robust approach for drug analysis providing a more than 1000fold sensitivity enhancement when applied to g Springer lowconductivity samples Toxicological examples of this approach were reported by Zhang Thormann and Wey for the determination of opioids in biological uids 17 18 and by Manetto et all for the determination of opiate drugs in hair 19 More recently this online sample preconcentration method proved useful for CEMS analysis as reported by Wey and Thormann 20 for opioids in urine and by Gottardo et all for the determination of 6monoacetylmor phine morphine amphetamine methamphetamine 3 4methylenedioxyamphetamine MDA 34methylenediox ymethampthetamine MDMA benzoylecgonine ephedrine and cocaine in hair 21 Unlike FASS techniques which act only on ionized molecules per se sweeping methods are sample enrich ment techniques which are theoretically also active on neutral compounds Preconcentration occurs due to inter actions chromatographic partitioning complexation be tween analytes and the pseudostationary phase present in the separation buffer but not in the sample matrix 22 Cationselective exhaustive injection CSEI7sweeping MECC combines two online concentration techniques namely sweeping and sample stacking with electrokinetic injection and provides further improvements in sensitivity 23 This technique was applied to determine LSD in mouse blood after experimental administration of LSD 5 Lg g 1 with a reported sensitivity of about 06 ng mL 1 24 More recently ampthetamine metamphetamine and methylenedioxymethamphetamine were determined with picogram per millilitre sensitivity using CSElisweeping MECC 25726 An updated review of the different strategies used to enhance the analytical sensitivity of CE focussed on chiral analysis but easily transferable to achiral compounds was reported by Garcia Ruiz and Marina 27 Other very interesting sample preconcentration methods described in the recent CE literature such as transient isotachophoresis and packed capillary preconcentrators could theoretically be employed to enhance the sensitivity of CE but to the best of our knowledge they have never received adequate attention by forensic analysts Analysis of chiral molecules Therapeutic and illicit drug analysis in biological samples is currently carried out by using liquid and gas chromatogra phy generally coupled to different types of mass spec trometers This analytical strategy has shown excellent reliability and productivity and for this reason it has become a standard in forensic toxicology laboratories In this field we do not believe that CE may offer significant advantages over GC and HPLC excluding potential cases in which the amount of biosample is Anal Bioanal Chem 2007 3881359 1364 1361 particularly limited see Analysis of microsamples and on line sample enrichment or when a new analyte is to be determined which requires the rapid set up of a new method In the latter case the ease and rapidity of method development in CE a technique in which all the separation mechanisms occur in solution may represent a real advantage with respect to chromatographic methods in which stationary phase conditioning always requires time and new columns are fairly expensive Chiral analysis is one of the most important analytical tasks in pharmaceutical and pharmacological analysis not only to verify the enantiomeric purity of products but also to study metabolic transformations pharmacokinetics and pharmacodynamics which are mostly based onto chirally selective interactions erg enzyme reactions protein bind ing and receptor recognition Strangely enough in forensic analysis chiral separation has never gained great popular ity being limited to intelligence studies on clandestine preparations of amphetamine and its analogues and historically of synthetic cocaine However for the above mentioned considerations chiral information could be of the highest interest in the research for the toxicological causes of death and of acute or chronic intoxications In chiral analysis today CE is undoubtedly the most powerful method in comparison to HPLC supercritical uid chro matography and GC 28 In a forensic toxicology laboratory in our opinion the major advantage of CE is represented by the rapid and easy method development which can be done in a few hours by testing different native and derivatized CDs or a few other chiral selectors bile salts crown ethers in proper concentrations To this aim the use of nonaqueous capillary electrophoresis NACE may offer additional advantages These are particularly suitable for compounds poorly soluble in water thus avoiding the use of large amounts of organic solvents as would be necessary in chiral HPLC Chiral NACE for drug analysis has recently been reviewed by Geiser and Veuthey 29 In comparison to HPLC and GC which both require the initial purchase of expensive and delicate columns and or reagents the usefulness of which for the speci c purpose is not known before the test the advantages of CE are patent Once again surprisingly only a few forensic centres adopt CE for chiral analysis particularly in the environment of forensic drugs this issue is discussed in a recent paper by Holzgrabe et all 30 Chiral CE mostly based on separation through com plexation with cyclodextrins CDs classically includes the chiral determination of the stereoisomers of amphetamine and its analogues in biological uids and hair 31735 However chiral CE methods have been reported for methadone and its metabolite 2ethylidene 15dimethyl 33 diphenylpyrrolidine EDDP in urine serum and hair 33 36738 Since it is well known that the activity of the enantiomers of a drug is in many cases different the usefulness of gaining information on the enantiomeric ratios of drugs in biological uids is patent particularly when the blood concentration of a racemic drug andor its metabolites is to be correlated with its pharmacological toxicological activity eg salbutamol see ref 39 In addition to this typical area of chiral analysis other lessknown applications have relevance in forensic toxicol ogy A traditional problem in the forensic analysis of biological samples is the possibility of hair contamination from external sources drugs present in the environment This is particularly critical in hair analysis because of the high surfacetovolume ratio of this biological matrix and its exposure to the environment The determination of metabolites of the parent drug in the hair matrix as indicators of drug intake versus contamination is often of little help since drug degradation may give rise to the same compounds produced by metabolism Moreover metabo lites being more hydrophilic than parent compounds are generally speaking poorly embedded in the hair matrix An important contribution to solving this problem may come from the chiral analysis of the drugs in hair Indeed the enantiomeric discrimination of the drugmetabolizing enzymes produces enantiomeric imbalances of the hair drug content when the compounds enter the hair matrix through blood circulation meaning intake and metabo lism whereas drugs maintain their racemic composition if they enter into the hair directly from the environment ie in cases of hair contamination This was demonstrated by Tagliaro et all who showed a clear enantiomeric disequi librium in the content of 34methylenedioxymethamphet amine MDMA in the hair of an ecstasy user 32 Further applications of chiral analysis may be required when immunoassay results are to be quantitatively correlated with con rmatory instrumental analyses In fact many antibodies have a chiral selectivity whereas chromatographic methods are typically not chirally selective Therefore the quantitative comparison between the two techniques may fail for the irnmunometric underestimation of the drug enantio mer towards which the antibodies have less af nity So far no application of CE nor of other chiral analytical methods to this problem has been described Moreover chiral discrimination is important in deter mining drugs in cases where one isomer is allowed and the other is banned For example the popular antitussive drug dextromethorphan has an illicit enantiomer levomethor phan narcotic analgesic So distinguishing between the two enantiomers is fundamental for studying cases of suspect drug intoxication or abuse For this purpose a chiral CE method using CDs as chiral selectors and MECC was applied to urine samples by Aumatell and Wells 40 Only one of the enantiomers of several other chiral molecules erg propoxifene and norpseudoephedrine are 2 Springer 1362 Anal Bioanal Chem 2007 3881359 1364 controlled drugs but their analysis in biological uids by CE to the best of our knowledge is not currently carried out Special interest has been paid to selegiline a prescribed drug to treat Parkinson s disease Selegiline is known to metabolize to the R enantiomers of methamphetamine and amphetamine and thus chiral analysis is needed to identify if an amphetamine positive urine may be ascribed to previous selegiline use 33 41 Analysis of proteins and peptides of forensic interest Since its introduction in 1930 by Tiselius protein analysis has always been the major field of application of electrophoresis and electrophoretic methods are still at the basis of serum protein analysis in the clinical environment Even if traditionally little attention has been paid to proteins in forensic toxicology in recent years protein molecules have become attractive as biological markers of chronic alcohol abuse Among these carbohydrate de cient trans ferrin CDT has nowadays gained universal acceptance and is determined in many clinical and forensic laboratories worldwide 42 CDT is the collective name of a group of minor isoforms of serum transferrin with a low degree of glycosylation which include asialomonosialoand disialotransferrin Tf CDT concentrations increase after sustained alcohol intake 25L80 g die 1 lasting for at least 7 days and decrease after cessation of drinking with a half life of about 14 days CE was rst applied to CDT analysis in the second half of the 1990s and has since rapidly gained acceptance Together with HPLC CE is today considered the most reliable technique in the international literature 43 After the introduction of commercial reagents and multicapillary instrumentation CE has become the most productive instrumental approach to routine CDT analysis Most CE methods are based on zone electrophoretic separations using borate buffers added with organic amines erg diaminobu tane spermine or diethylenetriarnine to hinder protein interactions with the capillary wall Detection is always based on absorption of UV radiation at 20L214 nm 447 47 An interesting alternative was proposed by Wuyts et all who described a CZE method using proprietary reagents containing polycations and polyanions CEofix CDT buffer system Analis Narnur Belgium which provide a dynamic double coating of the capillary 48 After criticisms for poor peak resolution 49 the method was modi ed to achieve a better separation between disialo and trisialoTf peaks 50 and soon became commercially available It is now distributed by Analis Narnur Belgium as is adopted in many clinical and forensic laboratories as a screening tool for the objective diagnosis of chronic alcohol abuse Further improvements of the results in terms of peak resolution were a Springer obtained by the group of Prof Thormann using the same reagents by fine tuning capillary length temperature and voltage 51753 The penetration of separationbased methods HPLC GC CE in the clinical laboratory is typically hampered by the time required for performing the separation process prior to determination This limitation has been overcome in CE by using multicapillary systems which in recent times have replaced gel electrophoresis for the routine analysis of DNA RNA and serum proteins An automated CE multicapillary system widely adopted in clinical chemistry laboratories for serum protein analysis Capil larys Sebia Evry France has been provided with a reagent kit for CDT analysis CAPILLARYS CDT assay In spite of the wide adoption of this method in many centres surprisingly enough no reports on its application have so far appeared in the international literature Nevertheless we believe that soon multicapillary electro phoresis will play a major role in CDT analysis in most routine laboratories replacing the existing poorly reliable immunoassays Another glycoprotein of potential forensic interest is the hormone erythropoietin EPO which controls erythropoi esis in mammals The biotechnological synthesis of recombinant EPO and EPO analogues such as the novel erythropoiesis stimulating protein NESP has allowed its use as an erythropoiesisstimulating drug Besides its therapeutic use recombinant EPO can be illicitly adminis tered to improve the physical performance of athletes and therefore it is banned as a doping agent Differences in glycosylation speci cally in sialylation characterize the different types of recombinant EPO and therefore can be used to identify different manufacturers andor drug sources Several CE methodologies have been described for the analysis of recombinant human EPO in concentrated solutions but the inherently limited concentration sensitiv ity of CE precludes the detection of EPO at the levels actually found in biological uids In order to achieve the required sensitivity CE has been combined with immu noaf nity with interesting although preliminary results 54 55 Similar considerations could be applied to analysis of recombinant growth hormone rGH 56 Toxins and venoms are products of living organisms which may or may not have a polypeptide nature but which in any case are complex and very unstable molecules For this reason they are hardly susceptible to chromatographic analysis In this eld CE shows very interesting applica tions which have been discussed in a review by Robert Weinberger 57 to which the readers are referred since no determination in human uids or tissues has been published so far The only exception can perhaps be found in an attempt to determine the main toxins of Amam m phallaides alphaamanitin and betaamanitin in body uids reported Anal Bioanal Chem 2007 3881359 1364 1363 by Bruggemann et a1 58 The CE method although successful on toadstool extracts showed a detection limit somewhat too high to cover the clinically relevant range of concentrations Again immunoaf nitymediated CE meth ods may offer a viable solution to the instrumental determination of arnatoxins in bio uids Conclusions Almost twenty years after its introduction CE is still ghting to occupy space in forensic toxicological analysis In our opinion the only two areas in which its superiority over chromatographic techniques is undoubtedly recog nized are chiral drug analysis and protein analysis Thus in these speci c elds its adoption can be highly recommen ded even in laboratories where there is no special expertise in electrophoresis because several companies can provide reagent kits and standardized methods CE shows other attractive features eg minimum sample requirement high mass sensitivity easy and rapid method development which in some instances may be important for the analyst but which require special expertise to be usefully exploited Moreover the lack of a standardized coupling to mass spectrometry MS limits the usefulness of CE in the forensic environment where MS identi cation of compounds has become a 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