Contribution of thermal desorption and liquid-liquid extraction for identification and profiling of impurities in methamphetamine by gas chromatography-mass spectrometry

Impurity profiling of methamphetamine (MA) using thermal desorption (TD) and gas chromatography-mass spectrometry (GC-MS) was examined. Using TD/GC-MS, impurities were extracted and separated under various conditions. Optimal chromatograms were obtained when a 20 mg MA sample was extracted at 120 degrees C for 3 min using a TD instrument, followed by separation of the extracts using a non-polar capillary column coated with (5%phenyl)-methylpolysiloxane. MA samples from nine different batches were analyzed under optimized conditions. Compounds related to the structure of MA, such as benzaldehyde, benzyl alcohol, amphetamine, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, and N-acetylephedrine, were detected in the chromatograms without any laborious extraction procedure. Compounds such as ethanol, diethyl ether, and acetic acid, which are considered reagents and solvents for MA synthesis, were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different among the samples. Impurity profiling of MA using TD was compared with that using liquid-liquid extraction (LLE). Better reproducibility of peak areas was obtained using LLE, whereas higher intensities and numbers of peaks were detected using TD. Solvents were extracted more effectively using TD. The nine batches of MA were classified using both extraction procedures. The nine batches were divided roughly into two groups using data from LLE. Subsequently, the groups were classified in detail using data from TD. TD can be used to provide supplemental information for LLE, and the combination of these extraction methods can be helpful for impurity profiling of MA.     

生物检材中甲基苯丙胺及苯丙胺的分析研究进展

对近几年国内外22篇有关生物检材中甲基苯丙胺及苯丙胺测定的文献进行了综述。介绍了血、尿、毛发等生物检材的收集与预处理方法,比较了生物检材中甲基苯丙胺及苯丙胺的液-液萃取(LLE)、固相萃取(SPE)、固相微萃取(SPME)和顶空固相微萃取(HS-SPME)等提取方法,以及内标的选取、不同的衍生化方法和包括免疫、GC/MS、GC/NPD、GC/ECD、GC/FID、HPLC、HPCE在内的各种检测方法。最后,对分析结果的评定进行了讨论。     

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.     

Cross-examination of liquid-liquid extraction (LLE) and solid-phase microextraction (SPME) methods for impurity profiling of methamphetamine

Impurities in 48 methamphetamine (MA) samples were analyzed by liquid-liquid extraction (LLE) and headspace solid-phase microextraction (HS-SPME) methods. MPS-2 autosampler was used to improve reproducibility of SPME method, and nonadecane (C(19)) diluted with potassium bromide (KBr) powder was used as an internal standard for standardizing retention time. Impurities identified by SPME method showed different patterns compared with LLE method. Non-volatile impurities like methamphetamine dimer were not identified by SPME method, but some volatile impurities like diphenylketone, caprolactam and lots of unknowns were identified only by SPME method. 1-Phenyl-2-propanone (P2P), 1-phenyl-2-propanol and benzylcyanide peaks could be discriminated clearly by SPME method without interference of amphetamine, an artifact originates from MA degradation. Differences in the impurity patterns resulted in different clustering results. When 48 MA samples were classified into 5 LLE and 5 SPME clusters, cross-matching of the clusters resulted in 8 sub-clusters. It shows that combination of the different extraction methods can distinguish the differences which cannot be distinguished by LLE or SPME method alone, and can improve reliability of the profiling results.     

Headspace solid-phase microextraction (HS-SPME) and liquid-liquid extraction (LLE): comparison of the performance in classification of ecstasy tablets. Part 2

Headspace solid-phase microextraction (HS-SPME) is assessed as an alternative to liquid-liquid extraction (LLE) currently used for 3,4-methylenedioxymethampethamine (MDMA) profiling. Both methods were compared evaluating their performance in discriminating and classifying samples. For this purpose 62 different seizures were analysed using both extraction techniques followed by gas chromatography-mass spectroscopy (GC-MS). A previously validated method provided data for HS-SPME, whereas LLE data were collected applying a harmonized methodology developed and used in the European project CHAMP. After suitable pre-treatment, similarities between sample pairs were studied using the Pearson correlation. Both methods enable to distinguish between samples coming from the same pre-tabletting batches and samples coming from different pre-tabletting batches. This finding emphasizes the use of HS-SPME as an effective alternative to LLE, with additional advantages such as sample preparation and a solvent-free process.     

体液中常见滥用药物的系统筛选分析

本文建立了体液中常见滥用药物的筛选分析体系.尿液或血液经固相萃取(SPE)或液提取(LLE)后,直接用GC/NPD分析或经TFA、BSTFA衍生化后用GC/MS分析.方法适用于同时分析甲基苯丙胺、MDMA、度冷丁、去甲度冷丁、曲马多、美沙酮、EDDP、可卡因、苯甲酰芽子碱、可待因、安定、氯丙嗪、吗啡、单乙酰吗啡等十四种常见滥用药物及代谢物.SPE法和LLE法回收率分别为66～102%和50～86%,最低检出限为2-5ng/ml尿.涉毒案件的鉴定应用表明该分析方法简便、快速、可靠.     

全血中扎来普隆两种萃取方法的比较

目的建立血样中扎来普隆的液液萃取和固相萃取方法。方法液液萃取方法是在碱性条件下用二氯甲烷提取。固相萃取是采用OasisHLB固相萃取柱提取,萃取液均用GC/NPD检验,并对其进行比较。结果血样中扎来普隆固相萃取平均回收率达89%以上,液液萃取平均回收率为84%,GC/NPD最低检出限为2×10-2μg.mL-1。结论两种方法均能满足实际检案的需求,而固相萃取方法在减少仪器损耗和内源性杂质干扰方面具有较大的优势。     

Development of microwave-assisted extraction procedure for organic impurity profiling of seized 3,4-methylenedioxymethamphetamine (MDMA)

Abstract: Organic impurity profiling of seized 3,4‐methylenedioxymethamphetamine (MDMA) tablets aims to link tablets to common production sources. Conventionally, organic impurities are extracted from tablets using a liquid–liquid extraction (LLE) procedure prior to analysis by gas chromatography–mass spectrometry (GC‐MS). In this research, the development of an alternative microwave‐assisted extraction/headspace solid‐phase microextraction (MAE/HS‐SPME) procedure is described. The optimal procedure used phosphate buffer (1 M, pH 8), with an HS‐SPME extraction temperature of 70°C for 40 min, using a divinylbenzene/Carboxen?/polydimethylsiloxane (DVB/CAR/PDMS) fiber. Impurities were extracted from seized MDMA exhibits using the MAE/HS‐SPME procedure, as well as HS‐SPME alone, and a conventional LLE procedure. The HS‐SPME procedure was deemed to be the most practical because of the affordability and need for less analyst involvement. Although the LLE was limited in the number of impurities extracted, the procedure is still useful for the extraction of less volatile impurities that are not extracted by HS‐SPME.     

Solid-phase extraction for profiling of ecstasy tablets

A solid-phase extraction (SPE) procedure has been developed for impurity profiling of illicit tablets containing 3,4-methylenedioxy-N-methyl-amphetamine (MDMA, ecstasy). Following initial comparison of liquid-liquid extraction and solid-phase extraction, SPE was found to be preferable because it afforded higher extraction efficiencies and shorter extraction times. Procedure blank samples were also analyzed to identify constituents of the extracts which did not originate in the ecstasy tablets. The developed procedure was subsequently applied to 12 samples of seized ecstasy tablets and a comparison was made of these samples to determine similarities and obtain inferences with respect to commonality of origin.     

Comparative Analysis of the Chemical Profiles of 3,4-Methylenedioxymethamphetamine Based on Comprehensive Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry (GC × GC-TOFMS)*

Abstract: The chemical profiling of illicit drugs is an important analytical tool to support the work of investigating and law enforcement authorities. In our work, comprehensive two‐dimensional gas chromatography–time‐of‐flight mass spectrometry (GC × GC‐TOFMS) combined with nontargeted, pixel‐based data analysis was adapted for the chemical profiling of 3,4‐methylenedioxymethamphetamine (MDMA). The validity and benefit of this approach was evaluated by analyzing a well‐investigated set of MDMA samples. Samples were prepared according to a harmonized extraction protocol to ensure the comparability of the chemical signatures. The nontargeted approach comprises preprocessing followed by analysis of variances as a fast filter algorithm for selection of a variable subset followed by partial least squares discriminant analysis for reduction to promising marker compounds for discrimination of the samples according to their chemical profile. Forty‐seven potential marker compounds were determined, covering most of the target impurities known from the harmonized one‐dimensional profiling as well as other compounds not previously elucidated.     

Comparison and classification of methamphetamine seized in Japan and Thailand using gas chromatography with liquid-liquid extraction and solid-phase microextraction

Methamphetamine (MA) is one of the most frequently abused drugs worldwide. The aim of this study is to improve the analytical method for profiling MA impurity in order to compare and classify MA crystals seized in different countries and to investigate the relationships between seizures. To compare MA samples seized in Japan and Thailand, the following analytical method was adopted. A 50mg sample of MA.HCl was dissolved in 1ml of buffer solution (four parts 0.1M phosphate buffer, pH 7.0, and one part 10% Na(2)CO(3)), impurities were extracted with 0.5ml of ethyl acetate containing four internal standards (n-decane, n-pentadecane, n-eicosane and n-octacosane) and analyzed by gas chromatography with a flame ionization detector on a DB-5 capillary column (0.32mm i.d.x30m, film thickness 1.0mum). Fourteen characteristic peaks on chromatograms were selected for the comparison and classification of samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. Sixty-nine samples seized in Japan and 42 seized in Thailand were analyzed. The samples were classified into four groups roughly by cluster analysis. In addition, when it was difficult to compare samples that had fewer impurities on chromatograms obtained from liquid-liquid extraction (LLE), solid-phase microextraction (SPME) was effective. Because many characteristic peaks were detected using SPME, SPME made it easy to compare samples of high purity. The combination of LLE and SPME was useful for impurity profiling of MA samples seized in different countries.     

Development of a harmonised method for the profiling of amphetamines VI: Evaluation of methods for comparison of amphetamine

Amphetamine samples were analysed by gas chromatography-mass spectrometry (GC-MS), and the peak areas of 33 target compounds were transformed by applying various pretreatment techniques. The objective was to optimise the ability of a number of distance metrics to establish links between samples of amphetamine originating from the same batch (henceforth referred to as linked distances). Furthermore, partial least squares discriminant analysis (PLS-DA) was used to evaluate the effects of various pretreatment methods on separation of amphetamine batches synthesised by the Leuckart reaction, reductive amination of benzyl methyl ketone, and the nitrostyrene route. The most efficient way to pretreat GC-MS data varied for the different distance metrics, although best results were obtained when data were normalised to the sum of peak areas, and either the fourth root or a logarithm was applied to the normalised data. When pretreating normalised data by fourth root transformation, Pearson correlation was the distance metric that was most successful at finding linked samples. Normalisation and the use of fourth root also represented the best method of pretreating data when employing PLS-DA to separate samples synthesised by different routes. To achieve a faster and more user-friendly procedure for evaluating chromatograms, experiments were performed in which the number of target compounds used to compare samples was reduced. The effect of each compound that was removed was studied by applying PLS-DA and by using Pearson correlation to calculate linked distances as well as unlinked distances (between samples from different batches of amphetamine). Considering both links between samples from the same batch and separation of samples synthesised by different routes, the best results were obtained with the data set comprising 26 compounds. Finally, it was found that the profiling method developed in this work was superior to an existing technique with respect to separating linked and unlinked distances.     

Development of a harmonized method for the profiling of amphetamines. II. Stability of impurities in organic solvents

The present study focused on the stability of 22 amphetamine impurities dissolved in six organic solvents: isooctane, toluene, ethanol, dichloromethane, ethyl acetate, and diethyl ether. The aim was to find the most inert, and thereby most suitable, solvent for amphetamine profiling. Mixtures of the impurities were prepared in the different solvents, and changes in the concentrations of the individual compounds over-time were monitored by gas chromatographic analysis after 0, 4, 12, 24, 48, and 96 h. Isooctane and toluene provided the most inert conditions, although, a few of the impurities were insufficiently stable in these two solvents. The present experiments were performed as a part of the development of a harmonized method for profiling of amphetamine. The results can be used to support the choice of organic solvents for sample preparation. They also provide information about the stability of the impurities that are found in profiles of illicit amphetamine. This is essential due to the fact, that unstable compounds can have a negative influence on the comparison of profiles.     

固相萃取技术（SPE）在苯丙胺类毒品分析中的应用

固相萃取（SPE）技术是利用固体吸附剂将目标化合物吸附,使之与样品的基体及干扰化合物分离,从而达到分离和富集的目的。SPE是20世纪70年代后期发展起来的一种样品前处理技术,本文主要综述了近10年来SPE技术在苯丙胺类毒品分析中的应用现状及其趋势。     

GC法检测血液和尿液中甲基苯丙胺和咖啡因

目的建立同时测定血、尿中甲基苯丙胺和咖啡因含量的方法。方法应用GC／NPD技术,以4-苯基丁胺为内标,直接碱化,用氯仿提取,三氟乙酸酐衍生化,8CB熔融石英毛细管柱（30m×0．25mm×0．25μm）分析。结果生物样品中甲基苯丙胺与咖啡因在0．012—7．5μg/mL浓度范围内线性关系良好,检测限（S／N=3）依次为1．2ng／mL,0．6ng／mL（血）;1．6ng／mL,0．8ng／mL（尿）。苯丙胺在0．017—10．0μg／mL浓度范围内线性关系良好,检测限为1．6mg／mL（血）,3．2ng／mL（尿）。所有样本回收率均大于85％。结论本方法准确、灵敏,适用于血、尿中甲基苯丙胺及其代谢物苯丙胺的三氟乙酸酐衍生化物和咖啡因的同时检测,为判定滥用毒品种类、追查毒品来源以及研究生物体内甲基苯丙胺和咖啡因的交互影响提供了检测手段。     

LC-MS/MS-MRM筛选血液中22种有毒生物碱成分

目的建立液相色谱-串联质谱法对血液中的22种常见有毒生物碱成分进行筛选分析。方法血液以丁丙诺菲为内标经液液提取后,用液相色谱-串联质谱仪以电喷雾电离(ESI )、多反应监测(MRM)方式进行分析。结果以化合物的保留时间、两对母离子/子离子对定性,最低检出限为0.1～20ng/mL。结论该方法选择性佳、灵敏度高,适用于法医毒物分析和临床毒物分析中有毒生物碱的分析。     

Application of solid-phase microextraction to the profiling of an illicit drug: manufacturing impurities in illicit 4-methoxyamphetamine

This article describes the application of solid-phase microextraction (SPME) to the recovery of manufacturing by-products and impurities from an illicit drug seizure. The preparation chosen for examination using this technique contained 4-methoxyamphetamine, an hallucinogenic amphetamine that has been encountered frequently in South Australia. Compounds found in the PMA preparation included 4-methoxyphenol, 4-methoxybenzaldehyde, 4-methoxyphenyl-2-propanone, 4-methoxyphenyl-2-propanol, 4-methoxyphenyl-propene, and (tentatively) 4-methyl-5-(4'-methoxyphenyl) pyrimidine. The presence of these compounds suggests that the active drug was prepared from 4-methoxybenzaldehyde via 4-methoxyphenyl-2-propanone using a Leuckardt reductive amination. In this instance, SPME was found to be a simple, rapid, and non-destructive recovery technique that gave results complementary to those provided by conventional liquid-liquid extraction. There is an indication that SPME might find application in profiling of illicit drugs.     

超高效液相色谱-串联质谱法同时测定尿液中16种抗生素

目的 建立同时分析尿液中16种抗生素的超高效液相色谱-串联质谱(UPLC-MS、MS)方法.方法 以哌拉西林为内标.尿样中的目标化合物经Oasis HLB固相萃取柱富集、纯化,利用ZORBAX SB-C18色谱柱,以0.1%的甲酸溶液-乙腈为流动相经梯度洗脱分离,采用多反应监测(MRM)模式进行分析.结果各化合物的最低...     

Effect of Extraction Procedure and Gas Chromatography Temperature Program on Discrimination of MDMA Exhibits

Analysis of impurities in seized MDMA tablets can be used to determine the synthesis method used and to identify links among exhibits. However, no standardized method exists to generate impurity profiles, limiting comparisons among different laboratories. This research investigated the effect of extraction procedure and gas chromatography temperature program on the resulting impurity profiles. Five exhibits were extracted using liquid–liquid extraction (LLE) and headspace solid‐phase microextraction (HS‐SPME), then analyzed using two different temperature programs. Profiles were statistically assessed using principal components analysis. While LLE was more reproducible, more compounds were extracted using HS‐SPME, thus providing more informative chemical profiles. The longer temperature program (53 min vs. 36 min) allowed greater discrimination of exhibits, due to improved precision as a result of an extended hold time (12 min). This research further highlights the need for standardized extraction and analysis procedures to allow comparison of chemical profiles generated in different laboratories.     

Development of a harmonised method for the profiling of amphetamines V: Determination of the variability of the optimised method

This paper is the fifth in a series of six in relation to the development of a harmonised method for the profiling of amphetamine [L. Aalberg, K. Andersson, C. Bertler, H. Borén, M.D. Cole, J. Dahlén, Y. Finnon, H. Huizer, K. Jalava, E. Kaa, E. Lock, A. Lopes, A. Poortman-van der Meer, E. Sippola, Development of a harmonised method for the profiling of amphetamines I. Synthesis of standards and compilation of analytical data, Forensic Sci. Int. 149 (2005) 219-229; L. Aalberg, K. Andersson, C. Bertler, M.D. Cole, Y. Finnon, H. Huizer, K. Jalava, E. Kaa, E. Lock, A. Lopes, A. Poortman-van der Meer, E. Sippola, J. Dahlén, Development of a harmonised method for the profiling of amphetamines II. Stability of impurities in organic solvents, Forensic Sci. Int. 149 (2005) 231-241]. The third paper [K. Andersson, K. Jalava, E. Lock, L. Aalberg, Y. Finnon, H. Huizer, E. Kaa, A. Lopes, A. Poortman-van der Meer, M.D. Cole, J. Dahlén, E. Sippola, Development of a harmonised method for the profiling of amphetamines III. Development of the gas chromatographic method, Forensic Sci. Int., in press] dealt with the optimisation of the gas chromatographic and detection methods whereas the fourth paper [K. Andersson, K. Jalava, E. Lock, Y. Finnon, S. Stevenson, L. Aalberg, H. Huizer, E. Kaa, A. Lopes, A. Poortman-van der Meer, M.D. Cole, J. Dahlén, E. Sippola, Development of a harmonised method for the profiling of amphetamines IV. Optimisation of sample preparation, Forensic Sci. Int., in press] concerned the optimisation of the extraction method prior to GC analysis. This paper is a study of the optimised method in order to determine its stability. Investigations of within and between day variations were carried out in four laboratories. Moreover, variations between laboratories were also determined. Both flame ionisation detector (FID) and MS detection were used. One laboratory studied nitrogen-phosphorous detector (NPD) detection as well. For this task, 12 batches of amphetamine were prepared. Six of them were synthesised via the Leuckart route, three via the nitrostyrene route and three via the reductive amination route [A.M.A. Verweij, Impurities in illicit drug preparations: amphetamine and methamphetamine, Forensic Sci. Rev. 1 (1989) 2-11]. Taking into account all studied target compounds and the average results from four laboratories, the within day variation was around 6% for FID and 5% for MS, the between days variation was around 10% for FID and 8% for MS. For NPD detection, within day variation was 5% and between days variation 9% (only one laboratory). Finally, the inter-laboratory variation was about 12% for FID (four laboratories) and 10% for MS (three laboratories).