Screening for the presence of drugs in serum and urine using different separation modes of capillary electrophoresis

Capillary electrophoresis (CE) is a modern separation technique that has some distinct advantages for toxicological analysis, such as a high efficiency, fast analysis, flexibility, and complementary separation mechanisms to chromatographic methods. CE can be applied in various modes, which each have a different separation mechanism or selectivity. The most common mode is capillary zone electrophoresis (CZE), in which charged analytes migrate in a buffer under the influence of an electric field. In micellar electrokinetic chromatography (MEKC), micelles are added to the buffer which interact with the analytes. MEKC can also be used for the separation of neutral compounds. In non-aqueous CE (NACE), the aqueous buffer is replaced by a background of electrolytes in organic solvents. A sample that needs to be screened can easily be analyzed subsequently by these CE modes using the same instrumentation.The aim of the study was to develop procedures for the analysis of basic and acidic drugs in serum and urine using CZE, MEKC, and NACE. A test mixture that consisted of six basic and six acidic compounds was used to study the separation behavior of five CE methods. The results showed that three methods (based on CZE, MEKC, and NACE) were suitable for the analysis of basic compounds and three methods (based on CZE and MEKC) for the analysis of acidic compounds.For the extraction of analytes from serum and urine, a solid-phase extraction (SPE) and a liquid-liquid extraction (LLE) method were compared. Both SPE and LLE methods provided clean extracts after extraction of the basic compounds from serum and urine. The extracts of acidic compounds contained more matrix interferences, especially for urine. The SPE method had some advantages compared to LLE, as it lead to cleaner extracts and higher peaks, and as it elutes basic and acidic compounds in one fraction.The potentials and pitfalls of the various methods for screening purposes in analytical toxicology are discussed.     

Application of dispersive liquid-liquid microextraction and CE with UV detection for the chiral separation and determination of the multiple illicit drugs on forensic samples

A new method was developed for pre-concentration, chiral separation and determination of multiple illicit drugs on forensic samples using dispersive liquid-liquid microextraction (DLLME) and capillary electrophoresis (CE) with Ultra Violet (UV) detection. The method was based on the formation of tiny droplets of an organic extractant in the prepared sample solution using water-immiscible organic solvent (chloroform) dissolved in water-miscible organic dispersive solvent (isopropyl alcohol). The organic phase, which extracted heroin, DL-methamphetamine, DL-3, 4-methylenedioxymethamphetamine and dl-ketamine from the prepared sample solution, was separated by centrifuging. The sedimented phase was transferred into a small volume CE auto-sampler vial with 10 μL of 1% HCl methanol solution and evaporated to dryness. The residue was reconstituted in lidocaine hydrochloride aqueous solution (internal standard) and introduced by electrokinetic injection into CE. Parameters affecting extraction efficiency were investigated and optimized. Under optimum conditions, linearity of the method was 0.15-6500 μg/L for all target analytes. The LODs (S/N=3) were 0.05-0.20 μg/L. Excellent repeatability (RSD ≤ 4.4%, n=5) was achieved. The feasibility of this method was demonstrated by analyzing spiked forensic samples. To our knowledge, it is the first time to combine DLLME with CE for chiral separation and determining illicit drugs on forensic samples.     

Analysis of Ecstasy Tablets Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

A method for the identification of 3,4‐methylenedioxymethamphetamine (MDMA) and meta‐chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D). Sample extraction, separation, and detection of “Ecstasy” tablets were performed in <10 min without sample derivatization. The separation electrolyte was 20 mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04 mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE‐C⁴D and compared against routine gas chromatography‐mass spectrometry (GC‐MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90 sec. The advantages of using C⁴D instead of traditional CE‐UV methods for in‐field analysis are also discussed.     

Validation of STR typing by capillary electrophoresis

With the use of capillary electrophoresis (CE), high-resolution electrophoretic separation of short tandem repeat (STR) loci can be achieved in a semiautomated fashion. Laser-induced detection of fluorescently labeled PCR products and multicolor analysis enable the rapid generation of multilocus DNA profiles. In this study, conditions for typing PCR-amplified STR loci by capillary electrophoresis were investigated using the ABI Prism 310 Genetic Analyzer (Applied Biosystems). An internal size standard was used with each run to effectively normalize mobility differences among injections. Alleles were designated by comparison to allelic ladders that were run with each sample set. Multiple runs of allelic ladders and of amplified samples demonstrate that allele sizes were reproducible, with standard deviations typically less than 0.12 bases for fragments up to 317 bases in length (largest allele analyzed) separated in a 47 cm capillary. Therefore, 99.7% of all alleles that are the same length should fall within the measurement error window of +/- 0.36 bases. Microvariants of the tetranucleotide repeats were also accurately typed by the analytical software. Alleles differing in size by one base could be resolved in two-donor DNA mixtures in which the minor component comprised > or = 5% of the total DNA. Furthermore, the quantitative data format (i.e., peak amplitude) can in some instances assist in determining individual STR profiles in mixed samples. DNA samples from previously typed cases (typed for RFLP, AmpliType PM+DQA1, and/or D1S80) were amplified using AmpFlSTR Profiler Plus and COfiler and were evaluated using the ABI Prism 310. Most samples yielded typable results. Compared with previously determined results for other loci, there were no discrepancies as to the inclusion or exclusion of suspects or victims. CE thus provides efficient separation, resolution, sensitivity and precision, and the analytical software provides reliable genotyping of STR loci. The analytical conditions described are suitable for typing samples such as reference and evidentiary samples from forensic casework.     

Simultaneous separation of different types of amphetamine and piperazine designer drugs by capillary electrophoresis with a chiral selector

The recent emergence of a new class of piperazine-type compounds has brought about the need for laboratory screening methods for both seized drugs and toxicological samples. These piperazine compounds, which include 1-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), exhibit comparable physiological effects and can be substituted for the classic amphetamine-type drugs. We have optimized a chiral capillary electrophoresis (CE) separation that detects a set of 6 piperazine and 4 chiral amphetamine compounds in under 23 min using a 200 mM phosphate buffer at a pH = 2.8 with 20 mM hydroxypropyl- beta-cyclodextrin (HPbeta3CD). In addition to the above compounds, a series of "clandestine" BZP diHCl samples were also analyzed using this method to assess the ruggedness of the procedure. The novel CE separation was tailored to simultaneously detect these piperzine compounds in addition to amphetamine-type drugs. Distinct migration time and UV-spectral data were obtained for all compounds of interest.     

Micellar electrokinetic chromatographic screening method for common sexual assault drugs administered in beverages

Recently, much attention has been given to benzodiazepines and gamma-hydroxybutyric acid (GHB) related compounds owing to their alleged widespread use as date-rape drugs. Toxicologists would greatly benefit from a screening method that allows for the simultaneous detection of both groups of substances. A new capillary electrophoresis (CE) method has been developed in the micellar mode to accomplish this separation in under 16 min using a sodium dodecyl sulfate (SDS)/sodium tetraborate/boric acid buffer with an acetonitrile organic modifier. Optimization of SDS and organic modifier concentration, along with pH, were performed on a set of standards containing eight benzodiazepines, GHB, gamma-butyrolactone, and the internal standard, sulfanilic acid. The method was shown to have a detection limit of less than 2 microg/ml for five out of eight benzodiazepines with a linear range of 2.5-100 microg/ml. The detection limit for GHB was 32 mg/ml with a linear range to 2500 microg/ml. This method was applied to the rapid analysis of spiked beverages. GHB spiked beverages were monitored after using a series of simple dilutions to determine the effects of time on the drug analysis. Possible interfering peaks from drugs of abuse and artifacts from a variety of different drink combinations were also studied in detail. A one-step liquid-liquid extraction was the only necessary sample pretreatment.     

Monitoring compliance to therapy during addiction treatments by means of hair analysis for drugs and drug metabolites using capillary zone electrophoresis coupled to time-of-flight mass spectrometry

Capillary electrophoresis coupled to time-of-flight mass spectrometry was used in the present work for the determination of therapeutic and abused drugs and their metabolites in the hair of subjects undergoing addiction treatments, in order to monitor their compliance to therapy. For this purpose a rapid, qualitative drug screening method was adopted based on capillary electrophoresis hyphenated with time-of-flight mass spectrometry, which had earlier been developed and validated for the forensic-toxicological analysis of hair, limitedly to illicit/abused drugs [1]. Sampling of hair was carried out in order to refer to a time window of about two months from the date of sampling (i.e. 2cm ca. from cortex). A single extraction procedure was applied, allowing the determination in the hair matrix of "drugs of abuse" referred to the past abuses, and therapeutic drugs prescribed in the detoxification program as well as their metabolites. Analyte identification was based on accurate mass measurements and comparison of isotope patterns, providing the most likely matching between accurate mass value and elemental formula. Small molecules (<500Da) of forensic and toxicological interest could be identified unambiguously using mass spectrometric conditions tailored to meet a mass accuracy ≤5ppm. In the present study, the proposed approach proved suitable for the rapid broad spectrum screening of hair samples, although needing further confirmation of results by using fragmentation mass spectrometry.     

The affect of tissue depth variation on craniofacial reconstructions

The consumption of synthetic drugs, generally known as designer drugs, has increased drastically in all parts of the world. Typical constituents of designer synthetic drugs are chemical substances derived from amphetamine but significant differences in effects caused and duration may result. In May, 2005, the civil state police of Sao Paulo seized thirty-one gelatinous capsules containing a very small quantity of a white powder inside (approximately 1.5 mg per capsule). This paper describes the analytical assays that were used to identify the seized material. Preliminary assays using colorimetric tests and high performance thin-layer chromatography indicated that the capsules content could be an amphetamine derivative. In the capillary zone electrophoresis assay, it was possible to observe that the analyzed material had basic characteristics. Mass spectrometry analysis revealed that the compound had the same molecular mass as 2,5-dimethoxy-4-bromoamphetamine (DOB) and its identity was confirmed through collision-induced dissociation (CID) experiments. Finally, the comparison of infrared sample spectrum with a spectra library provided further evidence of the DOB presence in the seized material. Although a reference standard material was not available, the information gathered from the different assays allowed the conclusion that the substance was, in fact, DOB, a substance with a powerful hallucinogenic action of proscribed use in the country and which was seized and identified for the first time in Brazil.     

氟硝西泮滥用及检测研究进展

具有镇静催眠作用的氟硝西泮曾在世界范围内被滥用,常被用于自杀、谋杀、迷奸、迷抢等案(事)件。近年其又成为俱乐部滥用药物之一。该药物在体内主要经肝脏代谢为7-氨基氟硝西泮和N-去甲基氟硝西泮,且7-氨基氟硝西泮的血药浓度常常大于血液中的母体药物浓度,大约90%的代谢产物经尿液排出,10%经粪便排出。目前报道的相关分析方法主要是对于血液、尿液、毛发、酒水饮品等检材经LLE、SPE、LPME等净化萃取后,采用毛细管电泳法、色谱法、质谱法及各种技术的联用,检测母体药物及相关代谢产物。本文对氟硝西泮的滥用、体内代谢以及样品的提取净化、仪器分析等进行总结,为相关案(事)件的办理提供参考。     

Rapid Separation of Organophosphate Pesticides using Micellar Electrokinetic Chromatography and Short‐end Injection,

Organophosphate (OP) pesticides are highly toxic substances and are frequently represented as poisons. In order to qualify and quantify the selected OP pesticides (methyl paraoxon, ethyl paraoxon, methyl parathion, fenitrothion, and ethyl parathion), micellar electrokinetic chromatography and short‐end injection were investigated. This is the first time that this combination has been used to separate OP pesticides. A capillary with 8.5 cm effective length was used, and the analytes were separated within 2.1 min. Separation conditions including buffer (type, pH, and concentration), sodium dodecyl sulfate concentration, and separation voltage were optimized. The limit of detection (LOD) was estimated in the range of 10–20 μM. The OP pesticides spiked in artificial saliva and drinking water gave superior peak profiles, and good average recoveries 95.6% and 62.3%, respectively. Overall, a rapid method with excellent resolution and efficiency was developed and successfully applied in the analysis of potential sample matrixes.     

Post-mortem stability and redistribution of carbohydrate-deficient transferrin (CDT)

Post-mortem diagnosis of chronic alcohol abuse is a challenge for forensic experts due to the lack of pathognomonic morphological findings and often also inadequate background information. Objective methods demonstrating chronic excessive alcohol consumption would therefore be a useful tool for forensic pathologists. In clinical practice, several markers of chronic alcohol abuse have recently been introduced, among which carbohydrate-deficient transferrin (CDT) is the most accepted, but the use of these markers in autopsy has not yet been established. We examined post-mortem stability and possible post-mortem redistribution of CDT and compared two analytical methods, capillary zone electrophoresis and high-performance liquid chromatography. According to our results, CDT remains stable for an appreciable time after death. The results further indicate that CDT is not subject to major post-mortem redistribution.     

全血中苯骈二氮杂类药物的胶束电动色谱法分析

目的建立用固相萃取胶束电动毛细管色谱法测定人体全血中苯骈二氮杂艹卓类药物的方法。方法全血以Oasis小柱提取,以克仑特罗为内标,采用未涂层毛细管(75μmID×50.2cm,有效分离长度为40cm),缓冲液为30mmol/LSDS→15mmol/L硼砂→15mmol/L磷酸盐(pH8.2)→18%甲醇。进样条件:压力进样0.5psi×10s,分离电压为25kV,柱温25℃,检测波长为230nm。结果本法分离效率高,9种苯骈二氮杂艹卓类药物的最低检测浓度为5～50ng/ml;血药浓度的线性范围为0.02～1.6μg/ml,日内、日间精密度<12%。结论本法简便、高效、可靠。     

Capillary column pyrolysis-gas chromatography of hair: a short study in personal identification

Human head hairs from 18 individuals were analysed by capillary column pyrolysis-gas chromatography (PyGC). The pyrograms showed several components, three of which were found to differ significantly among individuals. These major components were identified as benzene, toluene and styrene by gas chromatography-mass spectrometry. As the result of capillary column PyGC of 18 amino acids, it was found that differences among the individuals were due to the different amounts of particular amino acids in the hair. The quantities of the components were, further, found not to change in 18 months.     

Application of capillary electrophoresis to examination of color inkjet printing inks for forensic purposes

In the process of questioned document examination, the discrimination of inkjet printing inks is becoming more necessary due to increasingly frequent counterfeiting of documents printed by inkjet printers. Therefore, a method based on micellar electrophoretic capillary chromatography (MECC) has been developed and applied to analysis of such inks extracted from paper. With the use of an optimized and validated analytical procedure, multielectropherograms of inks taken from various models of printers made by various producers (Hewlett-Packard, Epson, Brother, Lexmark and Canon) were created. It was shown that effective differentiation of individual inks was possible in terms of migration time, order and specific shapes of characteristic peaks. By comparison of recorded UV-Vis spectra, the identification of main dyes was also achievable. The usefulness of the method was confirmed by an intralaboratory test of utility, in which several forged printouts were successfully examined. The obtained results proved that the proposed procedure is a useful tool that could be applied to ink discrimination and group identification of dyes originating from inkjet printing inks. Consequently, the developed method can be applied in the forensic field, including investigation of the authenticity of documents.     

Microscopical discrimination of human head hairs sharing a mitochondrial haplogroup

In forensic analyses, determining the level of consensus among examiners for hair comparison conclusions and ancestry identifications is important for assessing the scientific validity of microscopical hair examinations. Here, we present data from an interlaboratory study on the accuracy of microscopical hair comparisons among a subset of experienced hair examiners currently analyzing hair in forensic laboratories across the United States. We examined how well microscopical analysis of hair can reliably be used to differentiate hair samples, many of which were macroscopically similar. Using cut hair samples, many sharing similar macroscopic and microscopic features, collected from individuals who share the same mitochondrial haplogroup as an indication of genetic relatedness, we tested multiple aspects that could impact hair comparisons. This research tested the extent to which morphological features related to ancestry and hair length influence conclusions. Microscopical hair examinations yielded accurate assessments of inclusion/exclusion relative to the reference samples among 85% of the pairwise comparisons. We found shorter hairs had reduced levels of accuracy and hairs from populations examiners were not familiar with may have impacted their ability to resolve features. The reliability of ancestry determinations is not yet clear, but we found indications that the existing categories are only somewhat related to current ethnic and genetic variation. Our results provide support for the continued utility of microscopical comparison of hairs within forensic laboratories and to advocate for a combined analytical approach using both microscopical analysis and mtDNA data on all forensic analyses of hair.     

代谢组学及其在法医学中的应用展望

代谢组学是运用色谱、质谱、核磁共振(NMR)、毛细管电泳(CE)等技术对细胞、体液和器官等样品中的代谢物进行分离、纯化和检测,再用生物信息学的手段对所获得数据进行分析处理,从而得到一个或一组生物标记物信息的一门新兴学科。本文通过分析近年来有关代谢组学研究的文献报道,从历史渊源、概念、优点、分析方法、应用研究、存在问题及发展机遇、专业期刊和网站等方面对其进行综述,并根据现有研究对其在法医学各专业的应用作以展望,相信其能为法医学各专业和法医学鉴定提供一条新的研究思路。     

Analysis of amphetamines by supercritical fluid chromatography,high-performance liquid chromatography,gas chromatography and capillary zone electrophoresis; a preliminary comparison

A supercritical fluid chromatographic method with UV detection (SFC–UV) for the quantitative separation of phenylisothiocyanate (PITC)-derivatised amphetamines is described and compared to high-performance liquid chromatography–diode array detection (HPLC–DAD), gas chromatography–flame ionisation detection (GC–FID) and capillary zone electrophoresis–diode array detection (CZE–DAD) analyses of amphetamine and related compounds. Difficulties in the analysis of common amphetamines by SFC are discussed. Of the methods described in this paper, the SFC method offered the greatest sensitivity at 0.01–0.02 μg of drug on column. Suggestions are made for the use of combinations of these techniques for the identification of amphetamines when gas chromatography–mass spectrometry is not available.     

Developing a quantitative extraction technique for determining the organic additives in smokeless handgun powder.

Compositional analysis of the organic additives in smokeless handgun powder can provide forensic information to associate known and questioned samples. A reliable method for the quantitative extraction of smokeless powder additives would strengthen these measurements. To achieve quantitative recovery, both supercritical fluid extraction (SFE) and ultrasonic solvent extraction (USE) were evaluated as candidate techniques. Following a detailed evaluation of the solvent choice, the recovery of spiked additive compounds, and the effect of the powder matrix, a reliable USE technique was developed. When quantitative USE recovery of the target analytes, nitroglycerin (NG), diphenylamine (DPA), and ethyl centralite (EC), is coupled with additive measurement by micellar capillary electrophoresis (CE), compositional information can be obtained in less than 1 h.     

Analysis of dyes in illicit pills (amphetamine and derivatives)

The determination of dyes present in illicit pills is shown to be useful and easy-to-get information in strategic and tactical drug intelligence. An analytical strategy including solid-phase extraction (SPE) thin-layer chromatography (TLC) and capillary zone electrophoresis equipped with a diode array detector (CZE-DAD) was developed to identify and quantify 14 hydrosoluble, acidic, synthetic food dyes allowed in the European Community. Indeed, these may be the most susceptible dyes to be found in illicit pills through their availability and easiness of use. The results show (1) that this analytical method is well adapted to small samples such as illicit pills, (2) that most dyes actually found belong to hydrosoluble, acidic, synthetic food dyes allowed in the European Community, and (3) that this evidence turns out to be important in drug intelligence and may be assessed into a Bayesian framework.     

The rapid analysis of heroin drug seizures using micellar electrokinetic chromatography with short-end injection

A simple and rapid method for the analysis of heroin seizures by micellar electrokinetic chromatography with short-end injection is described. Separations were performed using an uncoated fused silica capillary, 50 cm x 50 microm I.D. x 360 microm O.D. with an effective separation length of 8 cm. The system was run at 25 degrees C with an applied negative voltage of -25 kilovolts. Injection of each sample was for 2 s at -50 mbar. UV detection was employed with the wavelength set at 210 nm. The background electrolyte consisted of 85:15 (water:acetonitrile, v/v) containing final concentrations of 25 mM SDS and 15 mM sodium borate, pH 9.5. Samples and standards were prepared in 0.1% v/v acetic acid and diluted in the run buffer containing 1 mg/ml of N,N-dimethyl-5-methoxytryptamine as an internal standard. Under these conditions a text mixture containing caffeine, paracetamol, morphine, codeine, heroin, and acetylcodeine was resolved within 1.5 min. The method was used to determine the concentration of heroin in heroin seizure samples, and the results were in good agreement with those obtained by a validated gas chromatographic method.