CI-mass fragmentographic analysis of methamphetamine and amphetamine in human autopsy tissues after acute methamphetamine poisoning

A 22-year-old male methamphetamine abuser was put under police protection owing to his abnormal state of excitation, but died 1 h later. Distribution of methamphetamine and amphetamine in the body was analyzed by the chemical ionization mass fragmentographic method. Amphetamine/methamphetamine concentrations (μmol/100 g) were $\text{0.24}\text{5.59}$ in blood, $\text{0.41}\text{9.43}$ in liver, $\text{0.41}\text{10.02}$ in brain, $\text{0.37}\text{9.80}$ in kidney, $\text{0.18}\text{4.57}$ in muscle, $\text{0.02}\text{0.63}$ in subcutaneous fat and $\text{1.87}\text{1464}$ in gastric contents. Total amount of methamphetamine hydrochloride in stomach contents was about 54 mg. Amphetamine concentrations in tissues ranged from 3.2% to 4.3% of methamphetamine, and was 0.1% in stomach contents. Amphetamine in tissues seems to be a metabolite of methamphetamine, and amphetamine in gastric contents is presumed to result from gastric mucous excretion. The blood concentration of methamphetamine was at a fatal level, and the total amount of the drug in gastric contents indicates that fatal poisoning occurred by ingestion.     

Detection and diagnostic interpretation of amphetamines in hair

A review with 22 references on detection and incorporation of amphetamines in hair is presented. This review deals with the detection, incorporation into hair, behavior in the hair shaft, confirmation of past drug use and diagnosis of dependence mainly regarding amphetamine and methamphetamine, along with methoxyphenamine, methylenedioxymethamphetamine, bromomethamphetamine, deprenyl, benzphetamine, fenproporex and mefenorex. First, pretreatment, extraction and analytical methods for amphetamines in hair using immunoassay, HPLC and GC/MS are discussed. This is followed by sections describing the animal experiments, incorporation rates of amphetamines from blood to hair and relationship between drug history and drug distribution in hair. Finally, the diagnosis of amphetamine dependence and confirmation of methamphetamine baby by hair analysis is discussed. The paper concludes with a brief outlook.     

Nails as useful materials for detection of methamphetamine or amphetamine abuse

Methamphetamine and amphetamine could be demonstrated in nail clippings obtained from methamphetamine users by sensitive gas chromatography/chemical ionization mass spectrometry with N-methylbenzylamine as an internal standard. The methamphetamine levels in fingernails were comparable to those in hair. Both stimulants were more concentrated in toenails than in fingernails. The detection of methamphetamine and amphetamine in nails provides an alternative informatian to that in hair on their past abuse.     

甲基苯丙胺在家兔体内的毒物代谢动力学研究

目的研究甲基苯丙胺及其代谢物苯丙胺在家兔体内的毒物代谢动力学行为。方法GC/MS法测定家兔灌胃甲基苯丙胺后不同时间点血、尿中甲基苯丙胺和代谢物苯丙胺浓度,采用3P97程序进行房室模型拟合以及毒物代谢动力学参数计算。结果甲基苯丙胺和苯丙胺在家兔体内的毒物代谢动力学过程均呈一级动力学特征,符合二室开放模型。甲基苯丙胺在家兔体内Cm ax为1.457 mg/L±0.094 mg/L,Tm ax为1.557h±0.078h,t1/2 ka、t1/2α和t1/2β分别为0.384h±0.052h、1.614h±0.036h和3.007h±0.430h,CL为1.769 L/h/kg±0.114 L/h/kg。甲基苯丙胺的毒物代谢动力学方程为:C t=2.767 e-0.746 t+1.454 e-0.234 t+4.119 e-1.746 t。结论甲基苯丙胺在家兔体内吸收、消除和代谢都较快。建立的甲基苯丙胺毒物代谢动力学方程和参数可为甲基苯丙胺分析的合理取样、从血药浓度推断服毒时间以及甲基苯丙胺滥用的法医学鉴定提供理论依据。     

应用一滴溶剂萃取（SDE）技术提取尿样中苯丙胺类毒品

目的建立一滴溶剂萃取技术（SDE）在尿样中苯丙胺类毒品检验的提取优化方法。方法通过GC／NPD分析，系统考察了溶剂体积，萃取溶剂，搅拌速度，萃取时间等参数对苯丙胺类毒品的SDE萃取效率的影响。结果经实验研究，建立了SDE最优化方法。结论SDE技术是一种新型样品前处理技术，具有操作简单快速，成本低廉等特点，在毒物毒品检验中具有广泛的应用前景。     

毛发中甲基苯丙胺及代谢产物苯丙胺的分析研究

介绍了毛发中甲基苯丙胺（ MAMP）及代谢产物苯丙胺（ AMP）的 GC/NPD、 GC/MS的定性定量分析方法。毛发用 0.1 mol/L HCl水解, 4－苯基丁胺（ 4－ PBA）为内标,液－液提取,三氟乙酸酐（ TFA）衍生化。毛发用量为 10mg,检出限为 GC/NPD 0.5ng/mg, GC/MS 0.1ng/mg,回收率大于 78％。该方法成功应用于染毒豚鼠毛发中 MAMP及其代谢产物 AMP浓度变化过程的测定。     

Phenobarbital in hair and drug monitoring

Phenobarbital analysis was performed in vertex hair of patients by gas chromatography mass spectrometry (GC/MS). After washing with dichloromethane, about 250 mg were ground to dust in a ball mill. A 50-mg sample was stirred mechanically for 10 min with 3 ml of NH₄Cl/HCl buffer (pH 2.0) containing phenobarbital D₅. A solid phase extraction was performed (extrelut Merck) and elution was achieved with chloroform/isopropanol/n-heptane (50:17:33; v/v). A full scan (40–240 uma) acquisition was realized by GC/MS with an ion trap (ITD 700 Finnigan) using a DB5-MS chromatographic column. Quantification was achieved by integrating dominants ions (phenobarbital, 204; phenobarbital D₅, 209). Compared to serum, hair concentrates phenobarbital during anti-epileptic therapy (average value 36.4 ng/mg, n = 40 vs. 18.7 mg/l, n = 23). A group correlation exists between phenobarbital in hair and phenobarbital in serum, and between phenobarbital in hair and clinic observation in some typical cases. Phenobarbital in hair yields good information over a long period, especially when blood collection has not been made, when clinical disorders are observed on long-term therapeutic observance.     

Ethyl glucuronide: unusual distribution between head hair and pubic hair

Ethyl glucuronide (EtG) is a minor metabolite of ethanol that can be detected in hair. In some specific situations, head hair can be missing, and therefore, alternative anatomical locations of hair are of interest. In this study, paired hair specimens (head hair and pubic hair) from eight social drinkers were analyzed for EtG. Each sample was decontaminated by two dichloromethane bathes (5 ml) for 2 min. After cutting into small pieces, about 50 mg of hair was incubated in 2 ml water in the presence of 10 ng of EtG-d5, used as internal standard and submitted to ultra-sonication for 2 h. The aqueous phase was extracted by SPE using Oasis MAX columns. The hair extract was separated on an ACQUITY BEH HILIC column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 221-85 and 75 and m/z 226-75 for EtG and the IS, respectively. This laboratory is using a positive cut-off at 50 pg/mg. All eight head hair specimens were negative for EtG at a limit of quantitation fixed at 10 pg/mg. Surprisingly, EtG was identified at high concentrations in pubic hair, in the range 12-1370 pg/mg. It appears, therefore, that it is not possible to document the drinking status of a subject by simply switching from head hair to pubic hair.     

唾液、尿液中甲基苯丙胺浓度分布及初筛情况分析

目的获得吸毒者唾液和尿液检材中甲基苯丙胺浓度分布及胶体金试剂条初筛情况。方法液相色谱串联质谱法获得吸毒者唾液和尿液检材中甲基苯丙胺浓度,通过胶体金试剂条检测获得初筛情况。对两者结果进行比对分析。结果采用直接沉淀蛋白法和液质MRM扫描法检测,唾液线性范围是1~100ng/m L,线性相关系数0.9987,检出限是0.1ng/m L,定量限是1ng/m L;尿液线性范围是1~100ng/m L,线性相关系数0.9943,检出限是0.5ng/m L,定量限是1ng/m L。唾液和尿液检材按一定比例稀释,使浓度在线性范围内。采用唾液和尿液四种型号甲基苯丙胺胶体金试剂条初筛,直接点样,目测判断结果。结论胶体金试纸条初筛尿液检出率为79%左右;唾液检出率大概为81%,两种试剂条结合使用,检出率可以提高到93%以上。结合此次初筛结果和仪器确认浓度可以发现:灰区设置和灵敏度的设置对检出率有一定影响,建议提高灵敏度以满足筛查工作需要。     

Theoretical limits of the evaluation of drug concentrations in hair due to irregular hair growth

When examining concentration relationships of doses it must be taken into account that hair growth is irregular. Hair growing from the shaved skin after a single dose of a certain drug cannot possibly contain the same concentration as hair after the same dose that has not been cut over a long period. Concentrations can even change during the hair growth in cases where the hair had been cut a couple of months before the hair sample was taken. The variations in the expected concentrations can exceed 20%. On the other hand, the evaluation of a hair tuft which has grown after the last drug consumption may be important in forensic cases where the hair which has grown earlier is not available. This may lead to misinterpretations at low concentrations. Expected concentrations are calculated assuming a telogen part of 10%.     

Interlaboratory comparison of quantitative determinations of drug in hair samples

Testing human hair for drugs of abuse is a relatively new technique which requires control before being fully accepted in Justice applications. A consensus procedure was recently proposed to the four French Laboratories performing hair analysis for opiates and cocaine. Results of two independent controls have shown that the laboratories have performed very well quantitatively, using the recommended method. In order to compare these results with those obtained by other procedures, one sample was sent to 15 laboratories concerned with the analysis of human hair for drugs of abuse in Germany, Italy, Spain, and United States. Results from this study have indicated that the French recommended method is in accordance with the general procedures.     

The study of metabolite-to-parent drug ratios of methamphetamine and methylenedioxymethamphetamine in hair

The metabolite-to-parent drug ratios were determined in the hair of 2444 methamphetamine (MA) abusers who had produced MA-positive hair results from 2001 to May 2005 and in the hair of 53 ecstasy abusers who had produced positive methylenedioxymethamphetamine (MDMA) hair results from 2002 to May 2005. For the hair analyses, hair strands were washed, cut into small pieces and extracted for 20 h in 1 mL methanol containing 1% HCl. Drugs in the extract were determined by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring after derivatization with trifluoroacetic anhydride. The six range groups were divided as follows on the basis of MA concentrations in hair (n = 2389): 0.5-5 ng/mg (n = 950), 5-10 ng/mg (n = 582), 10-20 ng/mg (n = 503), 20-30 ng/mg (n = 160), 30-40 ng/mg (n = 80), more than 40 ng/mg (n = 114) to assess the correlations between MA concentrations and metabolite-to-parent drug ratios. In groups of higher MA concentrations, lower ratios of AP/MA were found, and there was a statistically significant difference among six range groups. Comparisons of age groups (tens, twenties, thirties, forties, fifties, and sixties) and male and female subjects for the ratios of AP/MA showed a statistically significant difference. The detection of metabolites and the parent drug with reasonable ratios was found to be a useful indicator for distinguishing internal drug incorporation from external contamination. In our study, MA users can produce 0.4-116% (mean = 9%) of amphetamine (AP) concentrations in hair, and ecstasy users 1-110% (mean = 12%) of methylenedioxyamphetamine (MDA) in appropriately washed hair samples.     

Preparation and application of a fortified hair reference material for the determination of methamphetamine and amphetamine

Quality assurance is one of the major issues in forensic analytical laboratories, where the need for a reference material (RM) has rapidly increased. RMs are very useful for method development and validation, internal quality control or proficiency tests. In the present study, we prepared a RM using drug-free hair for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) according to the recommendations of ISO Guide 35. The concentrations of MA and AP were determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC/MS) after derivatization with trifluoroacetic anhydride (TFAA). The assignment of values was conducted through the homogeneity study and characterization of the material. Furthermore, an internal proficiency test was performed with the prepared RM, of which the results were compared with those of the authentic hair RM prepared in our previous study. As a result, a hair RM containing MA and AP was prepared at the level of 4.86+/-0.69 ng/mg and 4.63+/-0.44 ng/mg, respectively. Most participants showed satisfactory performances in the internal proficiency test with the both RMs. The hair RM prepared in this study demonstrated its suitability for quality assurance in forensic laboratories.     

Segmental analysis for cocaine and metabolites by HPLC in hair of suspected drug overdose cases

Hair samples of eight postmortem cases were analyzed in segments of 1 to 3 cm for cocaine, benzoylecgonine and cocaethylene. Samples were prepared for analysis by digestion in 0.1 M HCl and subsequent extraction with mixed-mode solid-phase extraction columns. Measurement was made by reversed-phase, narrow-bore HPLC and fluorescence detection using two laboratory-made internal standards. The concentrations were in the region of 0.29–316 ng/mg of hair for cocaine, 0.43–141 ng/mg of hair for benzoylecgonine and 0.93–1.83 ng/mg of hair for cocaethylene. All eight investigated cases had cocaine-positive segments. In six of the cases, all segments were positive, suggesting regular cocaine use and two showed in-between negative segments indicating an interruption or a change of the abuse intensity. The results showed a second, remarkable observation, i.e. enormous concentration differences (factor >150) for both cocaine and benzoylecgonine between the different subjects. Furthermore, interindividual cocaine/benzoylecgonine ratios ranged from 0.02 to 8.43. We believe these observations could in part be attributed to both some of the still existing limitations in the analytical approach(es), especially the mandatory hair washing steps, and in our still too limited knowledge of the hair incorporation processes. Nevertheless, in some cases, segmental analysis proved to be an important tool to distinguish, together with postmortem examination, deadly chronic abuse from single acute drug overdosage.     

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。     

Fast LC-MS/MS method for the determination of amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB and PMA in urine

A fast method was designed for the simultaneous determination of amphetamine (A), methamphetamine (MA), PMA, MDA, MDMA, MDEA and MBDB in urine. The drugs were analysed by LC (ESI)-MS/MS, after a simple liquid-liquid extraction in the presence of the deuterated analogues. Reverse phase separation on an Atlantis dC18 Intelligent Speed column was achieved in less than 4 min under gradient conditions, and the total run time was 8 min. The method was fully validated, including linearity (1-1000 ng/mL for A, MDMA, MDEA and MBDB; 2-1000 ng/mL for MDA and PMA; 1-200 ng/mL for MA; r2>0.99 for all compounds), recovery (>80%), within-day and between-day precision and accuracy (CV and MRE<12.7% for intermediate level and ULOQ, and <17.2% for LLOQ), limit of detection (0.2 ng/mL for MDMA, MDEA and MBDB; 0.5 ng/mL for A, MA and PMA; 1 ng/mL for MDA) and quantitation (1 ng/mL for A, MA, MDMA, MDEA and MBDB; 2 ng/mL for MDA and PMA) and relative ion intensities. No matrix effect was observed. The procedure proved to be sensitive, specific and rapid, and was applied to real forensic cases.     

Hair analysis for drug abuse: I. Determination of methamphetamine and amphetamine in hair by stable isotope dilution gas chromatography/mass spectrometry method

Determination of methamphetamine and amphetamine in hair was performed by gas chromatography/mass spectrometry using stable isotope-labeled internal standards, 2-methylamino-1-phenylpropane-2,3,3,3-d4 and 2-amino-1-phenylpropane-2,3,3,3-d4. Extraction of hair with methanol/5M hydrochloric acid (20:1) using ultrasonication was chosen as the standard method. The calibration curves for amphetamines in the hair were linear from 1 to 100 ng/mg (r greater than 0.99). The detection limit was 0.5 ng/mg at the 95% confidence level. The coefficients of variation (CV) (n = 8) of analysis using the spiked hair with methamphetamine were from 0.7 to 6%. The CV (n = 8) of analysis of the methamphetamine abuser's hair was 17.5%. Sectional analysis of monkey and human hair after methamphetamine ingestion suggested a good correlation between the duration of drug use and drug distribution in the hair.     

Differentiation between drug use and environmental contamination when testing for drugs in hair

The differentiation between systemic exposure and external contamination for certain drug groups has been frequently referred to as one of the limitations of in drug testing in hair. When hair samples are used, three steps are usually employed in order to minimise the possibility of external contamination causing a misinterpretation. The first consists of decontaminating hair samples by washing the hair before analysis, the second is the detection of the relevant metabolites in the hair samples and the third is the use of cut-off levels. Difficulty in the interpretation arises when metabolites are not detected either due to external contamination of the hair or low doses of the drugs used. A wash protocol needs to be practical and ideally remove any drug deposited on the external portion of the hair.     

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.     

Correlation of methamphetamine results and concentrations between head, axillary, and pubic hair

This study was designed to compare the qualitative results and concentrations of methamphetamine (MA) and its metabolite amphetamine (AP) in head hair and hair collected from different parts of the body (axillae and pubis). Hair from subjects (N = 14) suspected MA users was simultaneously collected. Hair preparation involved washing step, fine cutting, overnight extraction, derivatization by the trifluoroacetic anhydride, and gas chromatography/mass spectrometry (GC/MS) using selective ion monitoring. In this study, we found a good correlation of the qualitative results for MA between head hair and hair on other parts of the body, but there were some differences in concentrations of MA and AP. Namely, the concentrations of MA and AP were higher in axillary and pubic hair than in head hair.