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.     

Nitrate and Nitrite Determination in Gunshot Residue Samples by Capillary Electrophoresis in Acidic Run Buffer,

Simultaneous determination of nitrate and nitrite in gunshot residue has been conducted by capillary electrophoresis using an acidic run buffer (pH 3.5). In previously developed capillary electrophoretic methods, alkaline pH separation buffers were used where nitrite and nitrate possess similar electrophoretic mobility. In this study, the electroosmotic flow has been reversed by using low pH running buffer without any additives. As a result of reversing the electroosmotic flow, very fast analysis has been actualized, well‐defined and separated ion peaks emerge in less than 4 min. Besides, the limit of detection was improved by employing large volume sample stacking. Limit of detection values were 6.7 and 4.3 μM for nitrate and nitrite, respectively. In traditional procedure, mechanical agitation is employed for extraction, while in this work the extraction efficiency of ultrasound mixing for 30 min was found sufficient. The proposed method was successfully applied to authentic gunshot residue samples.     

血尿肝中巴比妥类药物硅藻土提取紫外差示导数光谱测定

目的建立一种操作简单、快速、去除杂质能力强、提取率高的硅藻土提取血、尿、肝中巴比妥类药物的方法。方法尿液不经稀释、血液经稀释过硅藻土柱,乙醚洗脱;肝匀浆用6%高氯酸沉淀蛋白,上清液过硅藻土柱,二氯甲烷洗脱药物。洗脱液挥干用0.45mol/L氢氧化钠溶液溶解,将溶液等分为两份,分别用等量的0.45mol/L氢氧化钠溶液和0.6mol/L硼酸-氯化钾溶液稀释,得到pH10和pH14水溶液,以pH10溶液为参比,测定pH14溶液的紫外二阶导数光谱进行药物检测。结果该法提取率血98.6%~100.3%,尿99.7%~103.2%,肝78.4%~102%,检出限均低于1.0μg/g(m l),变异系数小于2.9%,线性范围0.5~5.0μg/m l。结论硅藻土提取血、尿、肝中巴比妥类药物、紫外差示导数光谱法进行测定,适合作为法医毒物常规检验方法。     

Determination of carbohydrate deficient transferrin (CDT) with capillary electrophoresis: an inter laboratory comparison

Carbohydrate deficient transferrin (CDT) is currently the most specific laboratory marker of chronic or sustained alcohol abuse. CDT is increasingly being used as a diagnostic tool in the areas employment, traffic safety and forensic medicine. In recent times, capillary electrophoresis (CE) has been proposed as a convenient tool for rapid, precise and accurate CDT determination, not only for research but also for routine analyses. Quite recently, commercial kits have been introduced which, reportedly, could simplify and standardize CDT analysis with capillary electrophoresis. The present work was aimed at testing the ruggedness of a capillary electrophoretic method based on a commercial kit (CEofix, Analis), by comparing the results obtained with different instruments in different laboratories, on a panel of sera randomly collected and exchanged. The results showed, notwithstanding few outliers, excellent correlation of the results obtained in the two laboratories (R=0.974). Also high concordance was found when results were classified as positive or negative on the basis of a cut-off (1.25%) established from a control group of teetotalers. In conclusion the present data support the usefulness of capillary electrophoresis for CDT determination for clinical, forensic and administrative diagnosis of chronic alcohol abuse.     

Determination of sex from the clavicle and scapula in a Guatemalan contemporary rural indigenous population

The clavicle has been described as a useful bone for the metric determination of sex of human skeletal remains in a contemporary, predominantly white, North American forensic sample. In this article, measurements of clavicle and scapula are provided for a contemporary Guatemalan rural indigenous sample of forensic origin. Maximum length and circumference at midshaft of the clavicle, and height and width of the glenoid fossa of the scapula, were measured in 35 female and 62 male clavicles, and in 38 female and 65 male scapulae. Discriminant function analysis was used to study sexual dimorphism in this population with a classification purpose. Leave-one-out method (jackknife) matrices produced classification success rates ranging from 85.6% to 94.8%. A comparison with the North American forensic sample showed low percentages of correctly sexed Guatemalan male clavicles, ranging from 29.4% to 54.9%. The choice of an appropriate standard for the metric determination of sex is a crucial step in forensic anthropology.     

A fast ultrasound-assisted extraction procedure for trace elements determination in hair samples by ICP-MS for forensic analysis

An ultrasound-assisted extraction method is proposed for the determination of trace elements in hair samples by inductively coupled plasma-mass spectrometry (ICP-MS) for forensic investigation. Prior to analysis, 25 mg of hair samples were accurately weighed into (15 mL) conical tubes. Then, 2 mL of 20% HNO₃ is added to the samples, sonicated at 2 min (50 W, 100% amplitude), and then further diluted to 10 mL with Milli-Q water. Resulted diluted slurries are centrifuged and the analytes are directly determined in the supernatant. Calibrations against aqueous solutions were carried out with rhodium as internal standard. The method was successfully applied for the extraction of Al, As, Ba, Be, Cd, Co, Cr, Cu, Mn, Pb, Tl, U, V and Zn with a method detection limit (3 s, n = 20) of 0.1, 0.4, 0.2, 0.09, 0.08, 0.04, 0.1, 2.9, 1.0, 0.9, 0.04, 0.05, 0.1 and 4.2 ng/g, respectively. Method accuracy is traceable to Certified Reference Materials (CRMs) 85 and 86 human hair from the International Atomic Energy Agency (IAEA). Additional validation data are provided based on the analysis of hair samples from the trace elements intercomparison program operated by the Institut National de Sante’ Publique du Quebec, Canada. The proposed method is very simple and can be applied for forensic purposes with the elimination of sample digestion step prior to analysis. Then, a considerable improvement in the sample throughput is archived with the use of the proposed method.     

Detection of azide in forensic samples by capillary electrophoresis

Azide salts are highly toxic compounds that have been difficult to detect in forensic samples. Here, anion analysis by capillary electrophoresis with indirect spectrophotometric detection was applied to detect azide in forensic specimens from two suicide victims. Gastric specimens from the victims were shown to have high azide concentrations; azide represented one of the major anionic components and no corresponding component occurred in normal gastric juice. Samples of blood and bile had low concentrations of azide near the limits of detection. The method described for azide analysis used simple steps for sample preparation and analysis time was less than 10 min per sample. It offers a simple and reliable method for detecting azide in biological fluids.     

固相萃取-GC/MS法检测生物检材中的百草枯

目的采用固相萃取-气相色谱/质谱分析方法检测血液、尿液和脏器组织中的百草枯。方法人血液、尿液和猪肺组织样品经三氯乙酸去除蛋白后,取上清用十二烷基三甲基溴化铵和十二烷基硫酸钠处理过的C18小柱提取,提取物用硼氢化钠在碱性条件下还原,产物用气相色谱/质谱法分析,外标法定量。结果生物检材中百草枯回收率为78%～87%,最低检出限为0.1μg/mL,在0.5～1mg/mL范围内线性关系良好,可对实际案例检材进行定量检测。结论本文固相萃取-气相色谱/质谱分析方法能满足中毒生物检材检验及临床毒物检验需要。     

Evaluation and applicability of Alere iCup DX 14 for rapid postmortem urine drug screening at autopsy

Performing point‐of‐care urine drug screen testing at autopsy by a forensic pathologist may provide an early indication of the presence of analytes of interest during autopsy. An evaluation for the screening of 14 classes of common drugs of abuse in postmortem urine by the point‐of‐care screening device, Alere iCup DX 14, is presented. One hundred ninety postmortem urine samples were screened with the iCup occurring at autopsy by the forensic pathologist. Positive and negative results obtained from the screening kit were evaluated against confirmatory test results obtained using routine forensic toxicology analyses that employed LC‐MS/MS and GC‐MS to detect a combination of over 85 common drugs of abuse and medications. Sensitivity for each respective iCup drug class ranged from 66% (buprenorphine) to 100% (methadone, tricyclic antidepressants). Specificity for each respective iCup drug class ranged from 89% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, 3,4‐methylenedioxymethamphetamine, methadone). Positive predictive values ranged from 44% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, methylenedioxymethamphetamine, methadone), while negative predictive values ranged from 96% (methamphetamine) to 100% (barbiturates, methadone, tricyclic antidepressants). A high false‐positive rate was yielded by the benzodiazepine class. The lack of fentanyl screening in the point‐of‐care device is a significant limitation considering its prolific prevalence in forensic casework. The results obtained in the study should be acknowledged when considering the use of the Alere iCup DX 14 in the context of postmortem casework to help indicate potential drug use contemporaneously with autopsy and when requiring such preliminary results prior to the release of a final forensic toxicology report.     

二代测序技术在Y染色体遗传标记分型中的法医学应用

法医遗传学领域常利用Y染色体的父系遗传特点,对非重组区遗传标记进行检测并用于亲缘关系鉴定、混合斑检测、家系排查以及种族推断等研究.目前毛细管电泳仍是应用最为广泛的检测技术,基于该技术的商业化检测试剂盒及数据分析处理系统十分成熟.随着生物信息量的增长,传统检测技术通量低的弊端逐渐显现,推动了法医DNA分型技术的革新.近年...     

毛细管气相色谱法测定酱油中4-甲基咪唑的含量

目的建立毛细管气相色谱法定量分析酱油中有毒成分4-甲基咪唑的方法。方法酱油样品在层析柱中用二氯甲烷洗脱,洗脱液浓缩后加入N,N-二甲基苯胺作为内标,采用DB-FFAP毛细管柱分离样品,氮磷检测器测定4-甲基咪唑含量。结果方法线性范围为4.9~1.5×102μg/L;检测限为0.16μg/L;标准加入0.0102mg和0.0602mg 4-甲基咪唑的平均回收率为97.25%和99.44%。结论本文方法具有操作简便、快速、准确等优点,可用于检验酱油中的4-甲基咪唑。     

Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault

The quali-quantitative determination of two barbiturates, thiopental and its metabolite pentobarbital, in head and pubic hair samples of a woman who had been sexually assaulted during hospitalisation, is reported. Hair was analysed by means of solid-phase microextraction (SPME) and gas chromatography-multiple mass spectrometry (GC-MS-MS), in chemical ionisation conditions. Thiopental and pentobarbital were found in three proximal head hair segments (sample 1A: 0.30 and 0.40 ng/mg; sample 1B: 0.20 and 0.20 ng/mg; sample 3: 0.15 and 0.20 ng/mg) and pubic hair sample. Two distal head hair segments were negative for both barbiturates. Despite the lack of collection and toxicological analysis of blood or urine samples within the hospital setting, analytical findings from hair revealed the use of the anaesthetic agent thiopental to sedate the victim quickly and deeply and commit sexual assault.     

Rapid analysis of 3,4-methylenedioxymethamphetamine: a comparison of nonaqueous capillary electrophoresis/fluorescence detection with GC/MS

Because of the increasing use of 3,4-methylenedioxymethamphetamine (3,4-MDMA), a rapid and sensitive analytical technique is required for its detection and determination. Using nonaqueous capillary electrophoresis/fluorescence spectroscopy (NACE/FS) detection, it is possible to determine this drug at the level 0.5 ppm without any pre-treatment in less than 5 min. After liquid-liquid extraction, the sample can be condensed and a detection limit of 3,4-MDMA in urine of 50 ppb (S/N = 3) can be achieved. The precision of the method was evaluated by measuring the repeatability and intermediate precision of migration time and the corrected peak height by comparison with a 3,4-MDMA-D5 internal standard. With the conventional GC/MS method, it is necessary to derivatize the 3,4-MDMA before injection and the GC migration time also is in excess of 20 min. Therefore, NACE/FS represents a good complementary method to GC/MS for use in forensic analysis.     

Methods for ensuring the highest DNA concentration and yield in future and retrospective trace DNA extracts

In forensic laboratories, increased extraction efficiency of trace evidence is paramount because analytical success is intrinsically dependent on the quantity of DNA recovered. Moreover, highly concentrated nucleic acids are vital for effective downstream analysis and high quality results. This study investigated the efficiency of extraction with the Qiagen® QIAamp® DNA Investigator kit, and explored improvements to the methodology that would maximise the recovery of low concentration forensic samples. Controlled amounts of starting cellular material were used to mimic trace (or low level) DNA deposits prior to DNA extraction with the Investigator kit. Addition of the provided carrier RNA along with conducting two successive elutions of 50 µL improved the net recovery of DNA to 95%. Concentration with centrifugal filters post-extraction were able to concentrate DNA but a large net loss was observed. For the concentration of historic, retrospectively extracted DNA, centrifugal methods are able to concentrate DNA extracts previously too dilute for analysis. These concentrated volumes, however are small, allowing for minimal downstream analysis attempts before the sample is exhausted.     

DNA extraction of burnt bone and teeth casework samples using bead-beating homogenization technique

Sample disruption was a necessary step for DNA isolation. Bone and teeth were useful biological sources particular in human remains and advance decomposed bodies. The compact bone and teeth required several preparation steps prior to analyzing process. However, the methods in standard protocol were laborious and time consuming. An alternating pulverization, bead beating homogenizer, was purposed in its effectiveness for forensic casework. (1) Here, we applied this technique to the burnt cracked bone and tooth that recovered from house fire for forensic DNA analysis. After cleansed an external surface, the eight multidirectional motion tissue homogenizer, Precellys® evolution, was utilized to pulverize bone and tooth followed by a DNA extraction and amplification. For detection with a capillary electrophoresis, full profiles of autosomal STRs and Y-chromosomal STRs were recovered from tooth sample but the partial profile STR was demonstrated in bone sample. The new technique in bone homogenization was less time consuming (around 30 s), less exposure to chemical agents (no need of liquid nitrogen), high efficiency, with high-throughput productivity.     

The relevance of the evolution of experimental studies for the interpretation and evaluation of some trace physical evidence

In order for trace evidence to have a high evidential value, experimental studies which mimic the forensic reality are of fundamental importance. Such primary level experimentation is crucial to establish a coherent body of theory concerning the generation, transfer and persistence of different forms of trace physical evidence. We contend that the forensic context, at whatever scale, will be specific to each individual forensic case and this context in which a crime takes place will influence the properties of trace evidence. It will, therefore, be necessary in many forensic cases to undertake secondary level experimental studies that incorporate specific variables pertinent to a particular case and supplement the established theory presented in the published literature. Such studies enable a better understanding of the specific forensic context and thus allow more accurate collection, analysis and interpretation of the trace physical evidence to be achieved.This paper presents two cases where the findings of secondary level experimental studies undertaken to address specific issues particular to two forensic investigations proved to be important. Specific pre-, syn- and post-forensic event factors were incorporated into the experimental design and proved to be invaluable in the recovery, analysis and in achieving accurate interpretations of both soil evidence from footwear and glass trace evidence from a broken window.These studies demonstrate that a fuller understanding of the specific context within which trace physical evidence is generated and subsequently collected, as well as an understanding of the behaviour of certain forms of trace physical evidence under specific conditions, can add evidentiary weight to the analysis and interpretation of that evidence and thus help a court with greater certainty where resources (time and cost) permit.     

新型微流体液相色谱(micro-LC)串联四极杆质谱检测头发中5种苯二氮卓类药物

目的建立了新型微流体液相色谱(micro-LC)串联四级杆质谱检测头发中5种苯二氮卓类药物(三唑仑,α-羟基咪达唑仑,咪达唑仑,α-羟基阿普唑仑,普拉西泮)。方法采用液-液萃取法(LLE)提取毛发样品,微流体液相色谱串联四级杆质谱检测。结果在回归系数大于0.99的情况下,目标分析物的良好线性。精密度和萃取效率分别在2.7%~17.1%和66.5%~120.5%之间。每个分析物的检出限和定量限分别为0.001~0.08 pg·mg^-1和0.0125~0.25pg·mg^-1。结论本方法与传统的液相色谱串联质谱法(LC/MS-MS)相比灵敏度出高2-10倍。本研究展示了新型微流体分离系统串联质谱方法的实用性,并为法医毒理学领域关于头发样本中痕量药物检测提供了一种新的检测手段。     

Metric determination of sex from the humerus in a Guatemalan forensic sample

The absence of population-specific standards for sex, age and stature estimation for rural Guatemala is problematic for the forensic analysis of skeletal remains recovered from clandestine graves attributed to the recent armed conflict in that country. In order to increase the reliability of the forensic analyses being undertaken in Guatemala, standards for metric determination of sex were developed. Data was collected on several bones; the results for the humerus are presented here. A sample of 118 complete humeri (68 male and 50 female) was studied; maximum length, maximum diameter of the head, circumference at midshaft, maximum diameter at midshaft, minimum diameter at midshaft and epicondylar breadth were measured and subjected to discriminant function analysis. The classification accuracies for the univariate functions range from 76.8% for the maximum diameter at midshaft to 95.5% for the maximum diameter of the head. The classification accuracy for the stepwise procedure was 98.2%.     

GC-MS/MS测定血液中巴比妥类安眠药物

建立GC-MS/MS测定血液中巴比妥类安眠药物的分析方法。方法通过固相萃取提取并富集血液样品中常见巴比妥类安眠药物,采用离子阱二级质谱定性并定量检测其含量,并优化萃取溶液pH值与气相色谱/二级质谱联用分析条件,对巴比妥类安眠药物进行定量分析。结果巴比妥类安眠药物检出限为0.04μg/mL～0.10μg/mL,回收率为80.3%～92.6%。结论该方法高效、简单,灵敏度高,可用于血液中巴比妥类安眠药物同时定性定量检测。     

干生物基质点微样技术及其在法医毒物学中的应用

干生物基质点(Dried Matrix Spot,DMS)微样技术是通过将微量生物液体(如血液、尿液和唾液等)收集于卡纸上并干燥后形成的样本采集和储存形式.干生物基质点微样技术较传统的采样方法有明显的优势:取样量小,样品处理简单,储存运输方便,目前已应用于流行病学调查、毒药物监测、药代动力学研究和毒代动力学研究等领域....