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。     

分子印迹固相萃取法在血中苯丙胺类毒品分析中的应用

目的建立分子印迹固相萃取（MISPE）、GC/MS分析方法,用于血液中苯丙胺类毒品检测。方法 10mmol/L醋酸铵缓冲液（pH8.0）4倍稀释空白添加血液,1mL甲醇,1mL10mmol/L醋酸铵缓冲液（pH8.0）活化苯丙胺类分子印迹固相萃取柱;2×1mL去离子水、1mL60%的乙腈去离子水、1mL1%醋酸乙腈洗涤杂质;2×1mL1%甲酸/甲醇洗脱,洗脱液挥干定容,经GC/NPD、GC/MS分析检测。结果各种苯丙胺类毒品回收率均在90%以上,在20～5 000ng/mL浓度范围内线性关系良好,r2为0.995 7～0.998 9,LOQ在16～30ng/mL之间,LOD在8～15ng/mL之间。结论本方法回收率高,净化效果显著,稳定性好,杂质干扰少,可用于血液中低浓度苯丙胺类毒品的分析检测。     

Drug intelligence based on organic impurities in illicit MA samples

One major objective of the European project "Collaborative Harmonisation of Methods for Profiling of Amphetamine Type Stimulants" (CHAMP), funded by the sixth framework programme of the European Commission, consisted of the harmonisation of a gas chromatography/mass spectrometry (GC/MS) method for the analysis of organic impurities found in illicit methamphetamine (MA) samples in a drug intelligence perspective. Statistical analysis provided a selection of pertinent variables among the 43 organic impurities identified in the chromatograms. As for the 3,4-MethyleneDioxyMethAmphetamine (MDMA) study, correlation coefficients were used as a discrimination tool between populations of linked samples (from the same seizure) and unlinked samples (from different seizures). It was also shown that correlation measurements based on Pearson and cosine functions applied to the data pre-treated by normalisation to the sum of peak responses followed by the square root provided excellent discrimination between the two populations. The organic impurities profiling method was proved to be relevant for the characterization of samples from different seizures and their synthesis route patterns.     

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.     

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.     

The impurity characteristics of methamphetamine synthesized by Emde and Nagai method

Impurity profiling and classification of seized methamphetamine may play an important role in the interpretation of analytical results, the determination of the synthetic method employed, and the criminal investigations of drug traffic routes. Our study is focused on classifying seized methamphetamine samples according to the groups sorted by the types and quantities of impurities present in illicit methamphetamine samples. The samples (100 mg) were dissolved in 2 mL of potassium phosphate buffer (pH 7.0), extracted with 200 μL of ethyl acetate under basic condition, and then analyzed by gas chromatography-mass spectrometry (GC–MS) with a DB-1 capillary column (30 m × 0.25 mm i.d., 0.25 μm). Five impurities are used as criteria for the classification of seized methamphetamine samples by Emde and Nagai method. A total of fifty-two samples of seized methamphetamine were analyzed by GC–MS and classified by five organic impurities, and then sorted into four groups, which are Nagai type, Emde Type, Undetermined I type, and Undetermined II type.     

Analysis of the impurities in the methamphetamine synthesized by three different methods from ephedrine and pseudoephedrine

Organic impurities of methamphetamine may show different patterns by synthesis. In the present study, we tried to find the impurities reflecting the conditions of synthesis by comparing impurity patterns of the methamphetamine samples synthesized by different methods. Sixteen methamphetamine samples were synthesized from ephedrine and pseudoephedrine by the three different manufacturing methods of Emde, Nagai and Moscow. The synthesized samples were extracted with ethyl acetate containing four internal standards, and the patterns of the organic impurities were investigated by GC-MS and GC-FID . Through the investigation, we found 10 peaks appearing in the latter part of GC chromatograms are characteristic to synthesis. The areas of the selected peaks were converted to the variables suitable for the statistical calculation, and the synthesized samples could be classified into four groups through the resultant cluster analysis.     

Determination of gamma-hydroxybutyrate in water and human urine by solid phase microextraction-gas chromatography/quadrupole ion trap spectrometry

A simple method of detection was developed for gamma-hydroxybutyrate (GHB). The method involves the derivatization of GHB using a hexyl-chloroformate procedure in aqueous media (such as water or urine), extraction of the derivatization product directly from the sample using solid-phase microextraction, and subsequent separation and detection with gas chromatography quadrupole ion trap mass spectrometry. The deuterated form of GHB (GHB-D6) is used as an internal standard for quantitation. The method was linear for GHB-spiked pure water samples from 2 to 150 microg/mL GHB with a detection limit of 0.2 microg/mL. Spiked urine samples showed linearity from 5 to 500 microg/mL GHB with a detection limit of 2 microg/mL. The SPME-GC/MS method is applied to actual case samples, and the results are compared to those values obtained using a conventional GC/MS method. Sensitivity and linearity are comparable to those seen using traditional methods of separation, yet the SPME method is superior due to the simplicity, speed of analysis, reduction in solvent waste, and ability to differentiate between GHB and gamma-butyrolactone (GBL).     

Effective injection in pulsed splitless mode for impurity profiling of methamphetamine crystal by GC or GC/MS

Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) are commonly used for the impurity profiling of illegal drugs. For the impurity profiling of methamphetamine, it is very important to obtain information about impurities related to the manufacturing route and the precursor chemicals [B. Remberg, A.H. Stead, Drug characterization/impurity profiling, with special focus on methamphetamine: recent work of the United Nations International Drug Control Programme, Bull. Narcotics LI (1999) 97-117 ]. There are many artifact impurities arising from the preparation of samples and conditions of GC. Moreover, some impurities pose a barrier to the statistical processing of methamphetamine profiling. We investigated capillary GC analysis using pulsed splitless (PS/L) injection to minimize the thermal decomposition of impurities at the injection port and improve the transfer of samples into the column. We confirmed that the optimal conditions of PS/L-mode are 230 degrees C (injection temperature), 50 psi (pulsed pressure) and 1.1 min (pulsed time) for the methamphetamine profiling. Based on the impurity profiles of 48 methamphetamine crystals in PS/L-mode, we can achieve very easy handling and obtained, good results.     

Are there possibilities for the detection of chronically elevated alcohol consumption by hair analysis? A report about the state of investigation

The analysis of suitable ethanol markers in hair would be an advantageous tool for chronic alcohol abuse control because of the wide diagnostic window allowed by this specimen and the possibility of segmental investigation. Between the markers practically used or thoroughly investigated in blood or urine, ethylglucuronide, fatty acid ethylesters, phosphatidylethanol, acetaldehyde adducts to protein and 5-hydroxytryptophol can be regarded as possible candidates also in hair, but preliminary data were found in the literature only for ethylglucuronide and acetaldehyde modified proteins. By using headspace gas chromatography and headspace solid phase microextraction in combination with gas chromatography-mass spectrometry (SPME-GC/MS), in alkaline hydrolysates of hair it was possible to determine between 17 and 135 ng/mg of ethanol beside acetone and several other volatile compounds with slightly higher ethanol values for alcoholics than for social drinkers and teetotalers. A part of this is ethanol only absorbed in the hair matrix from the surrounding environment and consequently is not applicable as a diagnostic criterion. By extraction with aqueous buffer, methanol or a methanol/chloroform mixture and subsequent alkaline hydrolysis it was found that another part is generated from ethylesters, which are preferentially deposited in the lipid fraction of hair. In a specific search for ethylesters of 17 carboxylic acids by GC/MS-SIM in most cases ethyl 4-hydroxybenzoate (0.1 to 5.9 ng/mg, a preservative in hair cosmetics) and in four cases traces of indolylacetic acid ethylester were found. Furthermore, diethyl phthalate (a softening agent, present also in many cosmetic products) was identified in the hair of alcoholics as well as of children. As potential markers of alcohol intake, ethyl palmitate, ethyl stearate and ethyl oleate were detected in hair samples of alcoholics by headspace SPME-GC/MS of the chloroform/methanol extracts.     

Detection of methamphetamine and amphetamine in a single human hair by gas chromatography/chemical ionization mass spectrometry

A detailed procedure of an extremely sensitive method for quantitation of methamphetamine and amphetamine in human hair by gas chromatography (GC)/chemical ionization (CI) mass spectrometry (MS) is presented. N-methylbenzylamine was used as an internal standard. The samples, after extraction with an organic solvent, were derivatized with trifluoroacetic anhydride before the GC/MS analysis. Quantitation was made with quasi-molecular ions of the derivatives by selected ion monitoring in the CI mode. The detection limit was about 10 pg in an injected volume. The high sensitivity enabled us to measure both stimulants in a single human hair in actual cases.     

尿中苯丙胺、甲基苯丙胺、MDA和MDMA的固相微萃取和GC/MS/SIM测定

目的研究固相微萃取(SPME)用于尿中苯丙胺(AMP)、甲基苯丙胺(MET)、3,4-亚甲二氧基苯丙胺(MDA)和3,4-亚甲二氧基甲基苯丙胺(MDMA)的提取。方法样品调节至碱性和用盐饱和后用顶空SPME,内标为MET-d5。萃取纤维为100μm聚二甲基硅氧烷(PDMS)。用气质联用选择离子检测(GC/MS/SIM)。结果0.2μg/ml加标尿样,AMP、MET、MDA和MDMA的富集倍数分别为22,60,13和47。检出限(S/N=3)为0.4～9.5ng/ml。线性范围为0.05～1μg/ml。0.2、0.5和1.0μg/ml加标尿样,相对回收率77.9%～112.4%,变异系数2.7%～18.0%(n=5)。用该方法分析5个案件样品,和常规液液萃取结果接近。结论顶空SPME法用于尿中AMP、MET、MDA和MDMA等化合物的分析,无需有机溶剂,富集效率高,提取-富集-进样一体化,简单方便实用。     

Quantitative determination of amphetamines, cocaine, and opiates in human hair by gas chromatography/mass spectrometry

Hair of young subjects (N = 36) suspected for drug abuse was analysed for morphine, codeine, heroin, 6-acetylmorphine, cocaine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA). The analysis of morphine, codeine, heroin, 6-acetylmorphine, cocaine, and methadone in hair included incubation in methanol, solid-phase extraction, derivatisation by the mixture of propionic acid anhydride and pyridine, and gas chromatography/mass spectrometry (GC/MS). For amphetamine, methamphetamine, MDA, MDMA, and MDEA analysis, hair samples were incubated in 1M sodium hydroxide, extracted with ethyl acetate, derivatised with heptafluorobutyric acid anhydride (HFBA), and assayed by GC/MS. The methods were reproducible (R.S.D. = 5.0-16.1%), accurate (85.1-100.6%), and sensitive (LoD = 0.05-0.30ng/mg). The applied methods confirmed consumption of heroin in 18 subjects based on positive 6-acetylmorphine. Among these 18 heroin consumers, methadone was found in four, MDMA in two, and cocaine in two subjects. Cocaine only was present in two, methadone only in two, methamphetamine only in two, and MDMA only in seven of the 36 subjects. In two out of nine coloured and bleached hair samples, no drug was found. Despite the small number of subjects, this study has been able to indicate the trend in drug abuse among young people in Croatia.     

Identification of impurities and the statistical classification of methamphetamine using headspace solid phase microextraction and gas chromatography-mass spectrometry

The profiling of impurities in methamphetamine (MA) using headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) is described. The extraction of the impurities with an SPME fiber was examined under varying conditions. Optimal chromatograms were obtained when a 50 mg MA sample at 85 degrees C for 30 min was extracted using a fiber coated with divinylbenzene/carboxen/polydimethylsiloxane. MA samples from nine different origins were analyzed under optimized extraction conditions. Compounds related to MA such as benzaldehyde, benzyl alcohol, amphetamine, benzyl methyl ketone, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, N-acetylamphetamine, N-acetylmethamphetamine and N-formylmethamphetamine were detected in the chromatograms. Trace amounts of ethanol, diethyl ether and acetic acid were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different, depending on the sample. After the areas of the eight principal peaks were converted to their square root and logarithm, similarities among the samples were evaluated by Euclidian distance, cosine distance and correlation coefficient. The results showed that a combination of logarithmic conversion and cosine distance was the most suitable for discriminating and classifying the samples. HS-SPME/GC-MS is a simple and effective method for the extraction and identification of impurities. The present method, in combination with an appropriate statistical analysis, would be useful for developing a profile of impurities in MA.     

气相色谱-串联质谱法分析尿和血中除草剂百草枯

目的建立尿和血中百草枯的离子交换固相萃取-气相色谱-串联质谱分析方法。方法尿样加内标乙基百草枯,用732阳离子交换树脂提取;血样加内标乙基百草枯,用三氯乙酸凝聚蛋白质后取上清液用732阳离子交换树脂提取。提取物用硼氢化钠在水溶液中碱性条件下还原,还原物用有机溶剂提取进行气相色谱-串联质谱法分析。结果尿和血中百草枯的提取率分别为76％和74％,检测限分别为2ng／mL和10ng／mL,尿添加百草枯100ng／mL和血添加百草枯500ng／mL水平的回收率分别为99．6±5．6％和99．3±7．6％（Mean±CV）。结论本文建立的分析方法灵敏度高,能够满足中毒致死案件检验及临床毒物检验的需要。     

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.     

Illegal Route Estimation of the Seized Illicit Drug, Methamphetamine, by the Comparison of Striation Marks on Plastic Packaging Films

Abstract:  In Japan, the most common illicit drug is methamphetamine. It is possible to trace the origin of this drug by analyzing its organic and inorganic impurities and/or byproducts using several methods, such as GC, GC/MS, and inductively coupled plasma-mass spectrometry (ICP-MS). As reported here, one other method includes comparison of the striation lines of polymer sheet layers from packaging using a polarized light method. Other alternative methods include analyzing the heat sealer pattern, layer thickness surface characteristics, and/or components of polymer sheet layers using infrared spectroscopy. Several of these alternative methods were used to analyze the origins of 29 packages confiscated from three regions over a 1000 km distance in Japan. Results indicated that packages seized from different regions had some polymer sheet layers which contained striation lines and heat sealer patterns that were similar.     

Basic and neutral route specific impurities in MDMA prepared by different synthesis methods. Comparison of impurity profiles

In this work, the neutral and basic impurities found in the precipitate of MDMA(*)HCl are presented. MDMA.HCl was prepared by the most popular synthesis methods used in clandestine manufacture, i.e. safrole bromination, Leuckart method and reductive amination with various reducing agents: Al/Hg, NaBH(4), NaBH(3)CN. 3,4-Methylenedioxyphenyl-2-propanone (MDP-2-P), the starting material in Leuckart reaction and reductive amination, was prepared by two different synthesis methods, i.e. by isosafrole oxidation and MDP-2-nitropropene reduction. The extraction of impurities was performed under alkaline and neutral conditions. Impurity profiles were obtained using GC/MS. Each synthesis method is characterised by its own route specific impurities. The influence of pH on the extraction of synthesis markers from 3,4-methylenedioxymethamphetamine (MDMA) samples is discussed and comparison of the profiles of basic and neutral impurities is presented.     

Simple analysis of arylamide herbicides in serum using headspace-solid phase microextraction and GC/MS

A simple and sensitive method for analysis of four arylamide herbicides (butachlor, propanil, diphenamide and propyzamide) in serum was developed using a headspace–solid phase microextraction (SPME) and a gas chromatograph–mass spectrometer (GC–MS). A vial containing a serum sample and sodium chloride was heated at 90°C. The extraction fiber of the SPME was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of the GC–MS. The calibration curves, using an internal standard method, demonstrated good linearity throughout the concentration range from 0.25 to 10.0 μg/ml. Propyzamide was used for an internal standard. The limit of detection was 0.10, 0.05, and 0.25 μg/ml for butachlor, diphenamide, and propanil, respectively. No interferences were found, and the time for analysis was 60 min for one sample. In addition, this proposed method was applied to a suicide case in which the patient ingested Kusanon A^®, a herbicide. Propanil, which was the main ingredient in the herbicide, was detected in the eight serum samples collected from the patient during the hospitalization at the concentration range from 26.7 to 1.1 μg/ml.     

芬氟拉明和苯丙胺类兴奋剂的固相微萃取

用固相微萃取技术从血中提取芬氟拉明、苯丙胺和甲基苯丙胺。在 70℃条件下用 10 0 μm聚二甲基硅氧烷萃取头吸附 15min。重氢甲基苯丙胺作内标 ,采用柱前衍生化的进样方式 ,气质联用仪测定。选择离子m /z2 6 8(芬氟拉明 )、m/z2 40 (苯丙胺 )、m /z2 5 4(甲基苯丙胺 )和m/z2 5 8(重氢甲基苯丙胺 ,内标 )的峰面积比定量。血中检测浓度可达 0 0 1～ 0 0 3μg/g。通过解剖例中芬氟拉明的实际测定 ,证明这是一个从血液中提取分析苯丙胺类衍生物的快速准确的方法