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.     

Forensic application of chiral separation of amphetamine-type stimulants to impurity analysis of seized methamphetamine by capillary electrophoresis

The applicability of capillary electrophoresis (CE) with a UV detector using highly sulfated gamma-cyclodextrin as a chiral selector was examined for analysis of impurities in seized methamphetamine. Samples of methamphetamine-hydrochloride dissolved in water at a high concentration (20 mg/mL) were analyzed. Electrokinetic injection has an advantage over hydrodynamic injection for improving the detection of trace impurities. Small peaks of the precursor impurities, such as (1R,2S)-(-)-ephedrine and (1S,2S)-(+)-pseudoephedrine, were detected and quantified without extraction. The seized drugs could be classified into three groups based on the contents of the two impurities.     

Optimization of the separation of organic explosives by capillary electrophoresis with artificial neural networks

The separation of 12 explosives by capillary electrophoresis was optimized with the aid of artificial neural networks (ANNs). The selectivity of the separation was manipulated by varying the concentration of sodium dodecyl sulfate (SDS) and the pH of the electrolyte, while maintaining the buffer concentration at 10 mM borate. The concentration of SDS and the electrolyte pH were used as input variables and the mobility of the explosives were used as output variables for the ANN. In total, eight experiments were performed based on a factorial design to train a variety of artificial neural network architectures. A further three experiments were required to train ANN architectures to adequately model the experimental space. A product resolution response surface was constructed based on the predicted mobilities of the best performing ANN. This response surface pointed to two optima; pH 9.0-9.1 and 60-65 mM SDS, and pH 8.4-8.6 and 50-60 mM SDS. Separation of all 12 explosives was achieved at the second optimum. The separation was further improved by changing the capillary to an extended cell detection window and reducing the diameter of the capillary from 75 microm to 50 microm. This provided a more efficient separation without compromising detection sensitivity.     

Development of a capillary zone electrophoresis method including a factorial design and simplex optimisation for analysis of amphetamine, amphetamine analogues, cocaine, and heroin

A capillary zone electrophoresis (CZE) method was developed for the analysis of amphetamine and 13 amphetamine analogues. A full factorial design was used to screen for important design variables (i.e. carrier electrolyte concentration, pH, and separation temperature), and a modified simplex was employed in a final optimisation step. The resolution values of the target compounds were used as responses in the screening and optimisation phases. This approach made it possible to control the effects of the design variables on the separation of the target compounds. The best results were obtained using a 100mM Tris/phosphate buffer (pH 3.1) at a separation temperature of 10 degrees C, and the analysis time was 23 min under these conditions. After slight modification, the method also enabled baseline resolution of the most commonly encountered amphetamine derivatives, as well as cocaine and heroin, within 7 min. There was a linear relationship between peak area and concentration for all substances, with correlation coefficients in the range of 0.9975-0.9999. Moreover, the technique was repeatable and exhibited relative standard deviation (R.S.D.) values in the ranges of 0.01-0.11% and 0.54-1.60% for relative migration time and corrected peak area, respectively. Lastly, the method was successfully applied to analyse street samples.     

Optimization of a simple method for the chiral separation of methamphetamine and related compounds in clandestine tablets and urine samples by beta-cyclodextrine modified capillary electrophoresis: a complementary method to GC-MS

The chiral separation of (+/-)-methamphetamine, (+/-)-methcathinone, (+/-)-ephedrine and (+/-)-pseudoephedrine by means of beta-cyclodextrine modified capillary electrophoresis is described. The distribution of enantiomers in clandestine tablets and urine samples were identified. Several electrophoretic parameters such as the concentration of beta-cyclodextrin, temperature, the applied voltage and the amount of organic solvent required for successful separation were optimized. The method, as described herein, represents a good complementary method to GC-MS for use in forensic and clinical analysis.     

Enantiomeric separation of amphetamine by high-performance liquid chromatography using chiral crown ether-coated reversed-phase packing: application to forensic analysis

d-Amphetamine and l-amphetamine were clearly separated by HPLC analysis using a chiral crown ether column (CROWNPACK CR(+)). As little as 0.1% d-amphetamine in bulk methamphetamine could be determined. The enantiomeric form of the by-product in a drug sample may be helpful in evaluating the possibility of illicit synthesis. When the isomeric composition of amphetamine present in urine from a methamphetamine abuser was examined, only d-amphetamine was detected as a metabolite. The present method was also applied to the enantiomeric separation of norephedrine.     

Enantiomeric separation of methamphetamine and related analogs by capillary zone electrophoresis: intelligence study in routine methamphetamine seizures

A method for simultaneous enantiomeric separation of ephedrine, pseudoephedrine, and methamphetamine (MA) in a single run by simple capillary zone electrophoresis (CZE) with beta-cyclodextrin as a chiral selector is described. The effects of the buffer pH, phosphate concentration, beta-cyclodextrin concentration, voltage and temperature on the peak resolution were examined. Good enantiomeric resolution was attained for each analyte under our optimized conditions: 15 mM beta-cyclodextrin, 300 mM NaH2PO4 at pH 2.5 with an uncoated capillary (64.5 cm x 50 microm), applied potential at 20 kV and temperature at 30 degrees C. Ultraviolet (UV) detection at a fixed wavelength (200 nm) was employed using a diode array detector. Using phentermine as an internal standard, migration times for all analytes are reproducible within 0.16% for intra-day and 0.6% for inter-day runs. Application of this method to the analysis of confiscated drugs is discussed.     

摇头丸中甲基安非他明和咖啡因含量的检验

目的建立了用毛细管电泳分离和测定摇头丸中的甲基安非他明和咖啡因的方法。方法观察缓冲液的种类以及缓冲液的pH值对分离的影响。结果在10～800mg/L的范围内,用Na2HPO4-Na2B4O7和NaH2PO4-Na2HPO4缓冲液,在pH7.0～9.5时,两种药物成分均有较好的线性(r>0.99),三种摇头丸中的甲基安非他明和咖啡因的含量随浓度的增加其回收率增加。结论该分析方法速度快,重现性好,检测结果准确。     

A comparison of smokeless powders and mixtures by capillary zone electrophoresis

The analysis of inorganic ions present in smokeless and muzzleloading powders has been performed using capillary zone electrophoresis (CZE). Previous publications have examined inorganic low explosives using CZE, but have not looked at the ion profiles from smokeless powders. In this report, seven commercially available smokeless powders were analyzed as unburned powder and burned residue. The results demonstrate that ionic profiles can be used to characterize smokeless powders. Our analysis also included a smokeless powder/ Pyrodex combination to determine if smokeless powder ions are distinguishable in a mixture; however, the high concentration of ions present in Pyrodex RS prevented its detection. In addition, five different smokeless powder samples as well as Pyrodex RS were collected for analysis subsequent to deflagration in fifteen plastic pipe bombs. The relative ion concentrations between these powders can be used to illustrate the differences between open burning and pipe bomb deflagration.     

毛细管电泳技术分析炸药残留物中无机离子

目的　建立炸药残留物中无机离子的毛细管电泳检测方法。方法　运用缓冲溶液使无机离子在毛细管电泳中达到分离 ,采用紫外间接检测法分析。结果　该方法可从爆炸残留物中准确检测出无机阴离子 (Cl- 、SO2 - 4、NO- 2 、NO- 3、ClO- 3和ClO- 4离子 )和无机阳离子 (NH 4、K 、Na 和Mg2 离子 )。结论　操作方法简便快捷 ,检材用量少 ,检测极限可达ng/ml级 ,分析结果满意     

毛细管电泳高频电导法检测饮料中γ-羟基丁酸

目的建立饮料中γ-羟基丁酸（GHB）的毛细管电泳高频电导法检测分析方法。方法样品经稀释后直接进样,采用反向分离模式,以0.5mmol/LNa3PO4＋1.5mmol/LNa2HPO4＋0.1mmol/LCTAB为电泳分离缓冲介质,分离电压为16kV,进样时间为20s进行电泳分离及高频电导检测。结果该实验条件下,6.5min内可实现样品快速分析。GHB在10.0～150μg/mL范围内线性关系良好,r=0.990,检出限为3.0μg/mL（S/N=3）。选择的3种饮料样本不同添加浓度日间和日内RSD均小于6%,回收率均在95%以上。结论采用本文方法,样品处理简单,方法快速、灵敏,操作方便,可作为饮料中GHB的一种快速筛选法在相关检测中选用。     

Separation and quantitation of optical isomers of methylamphetamine samples by capillary electrophoresis

A capillary electrophoretic (CE) procedure using an electrolyte modified with hydroxypropyl-β-cyclodextrin has been used for the separation and quantitation of (S)- and (R)-methylamphetamine in fifteen laboratory prepared samples and ten `street' samples. Excellent separation of (S)- and (R)-methylamphetamine was achieved and the levels of methylamphetamine in the samples were in good agreement with the levels determined by a separate CE procedure previously developed in our laboratory. Both CE systems showed the presence of impurities in some of the samples suggesting that CE could be used in profiling illicit methylamphetamine seizures. Gas chromatography-mass spectrometry (GC-MS) analysis of two of the samples identified one of the impurities as N-formyl methylamphetamine, however, no conclusive proof for the presence of this compound in the electropherograms was available. The chloride and sulphate content of the ten `street' samples was also determined by CE.     

Determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction and capillary electrophoresis with PDA detection

A new method was developed for pre-concentration and determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction (DLLME) and capillary electrophoresis (CE) with photodiode array 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 eight drugs of abuse from the prepared urine solution, was separated by centrifugation. 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 (internal standard) aqueous solution and introduced by electrokinetic injection into CE. Under the optimum conditions, acceptable linear relationship was observed in the range of 3.0–500 ng/mL with the correlation coefficient (r) of 0.9982–0.9994 for spiked urine samples. The limit of detection (LOD) (S/N = 3) was estimated to be 1.0 ng/mL. A recovery of 75.7%–90.6% was obtained for spiked samples. The mean relative error (MRE) was within ±7.0% and the relative standard deviation (RSD) was less than 6.9%. The proposed DLLME-CE procedure offers an alternative analytical approach for the sensitive detection of drugs of abuse in real urine samples.

Key points

• The dispersive liquid-liquid microextraction (DLLME) was involved for the determination of drugs in urine with capillary electrophoresis with photodiode array detection (CE-PDA).
• Good linearity, sensitivity, recovery and precision were achieved.
• The proposed method was eco-friendly with microliter scale solvent consumption.

     

Comparative study of D1S80 typing by capillary electrophoresis and sequencing

The D1S80 locus is very useful for personal identification in Japan. To obtain a correct allele over 45, we examined PCR amplification product of the allele over 45 both by direct sequencing and fragment analysis using capillary electrophoresis. Direct sequencing finally determined the allele as being 57. However, it was calculated to be an allele of 56 by comparison with size markers for capillary electrophoresis. The difference could be attributed to the electrophoretic size markers. This finding indicates that the direct sequencing may be useful to determine the allele over 45 in the D1S80 locus.     

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.     

Use of dynamically coated capillaries for the routine analysis of methamphetamine, amphetamine, MDA, MDMA, MDEA, and cocaine using capillary electrophoresis

A rapid, accurate, precise, reproducible, economical, and environmentally gentle method using capillary electrophoresis (CE) is presented for the routine analysis of methamphetamine, amphetamine, MDA, MDMA, MDEA, and cocaine in seized drugs. The methodology uses a 32 cm by 50 microm capillary (length to detector 23.5 cm) with a commercially available buffer kit and diode array UV detection. Dynamic coating of the capillary surface is accomplished by flushing with base for 1 min, a proprietary polycation for 1 min, and then a proprietary polyanion for 2 min. This approach provides a relatively high and stable electroosmotic flow (EOF), even at low pHs. The background electrolyte (BGE) contains 75 mM phosphate buffer (pH 2.5) with the same polyanion as above. Using this methodology, amphetamine, methamphetamine, MDA, MDMA, MDEA, and an internal standard (n-butylamphetamine) are baseline resolved in less than 5 min. The run-to-run migration time %RSDs and peak area %RSDs are typically <0.3% and <2.1%, respectively. The day-to-day and capillary-to-capillary migration time %RSDs are <1.5% and <2.1%, respectively. The %RSDs of the relative migration times compared with the internal standard on a day-to-day and capillary-to-capillary basis are <0.2% and <0.06%, respectively. The linear dynamic range using peak areas range from 0.003 to 0.10 mg/mL. The correlation coefficients are >0.9998, with all calibration curves passing at or near the origin. Similar data are obtained for cocaine and its internal standard henyltoloxamine. None of the compounds usually encountered in illicit samples interfere with the target compound (e.g., methamphetamine and cocaine) or the internal standard. Quantitative results for synthetic mixtures and seized exhibits are in good agreement with actual values, and also with results obtained from other techniques. The relatively high EOF for the dynamically coated capillary system allows for the screening of basic, acidic, and neutral adulterants in drug seizures; identification is facilitated by the use of automated UV library searches.     

Achiral and chiral quantification of methamphetamine and amphetamine in human urine by semi-micro column high-performance liquid chromatography and fluorescence detection

In this paper, miniaturized achiral and chiral high-performance liquid chromatographic procedures for the determination of methamphetamine and amphetamine in human urine are described. After a simple pretreatment of human urine (i.e., 10 microL of urine or diluted urine were acidified and dried-up under N2 at room temperature) and fluorescence derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride under mild conditions (pH 9.0, 10 min at room temperature), the derivatives were isocratically separated on a semi-micro ODS column with Tris-HCl buffer (0.1 M, pH 7.0): acetonitrile (45 + 55 v/v) at a flow rate of 0.2 mL/min or their enantiomers were separated on a semi-micro OD-RH column with sodium hexafluorophosphate (0.3 M aq.): acetonitrile (44 + 56 v/v) at a flow rate of 0.1 mL/min as the mobile phase. Wide-ranged calibration curves were obtained with detection limits for the achiral and chiral analyses in the atto and femtomol levels, respectively, per injected volume. Satisfactory within- and between-day reproducibility data were obtained with both the methods with the highest relative standard deviation being 9.6%. The methods were applied to the determination of methamphetamine and amphetamine in human urine samples and the concentrations determined by the two methods were well correlated (r = 0.994).     

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.     

Mass spectral and NMR spectral data of two new designer drugs with an alpha-aminophenone structure: 4'-methyl-alpha-pyrrolidinohexanophenone and 4'-methyl-alpha-pyrrolidinobutyrophenone

This study presents and discusses the nuclear magnetic resonance (NMR) spectroscopic and mass spectroscopic data of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) and its homolog 4'-methyl-alpha-pyrrolidinohexanophenone (MPHP) which were seized in 2004 and 2000 in Germany for the first time. The structure elucidation of the aliphatic part of MPBP was carried out by product ion spectroscopy of the immonium ion formed after electron ionization as well as with 1H and 13C NMR. Product ion spectroscopy of immonium ions again proved to be a powerful tool to determine the structure of designer drugs and to distinguish between isobaric structures of the alkyl-amino moiety.