Detection of orosomucoid 1 phenotypes in semen and semen stains

Orosomucoid 1 phenotypes were detected in seminal plasma by isoelectric focusing and immunoprinting. The orosomucoid 1 phenotypes in seminal plasma correlated with the types found in the corresponding serum specimens. Semen stains stored for ten days could be typed for orosomucoid 1. The present work revealed that orosomucoid 1 is a useful genetic marker for the medicolegal grouping of semen stains.     

人体不同体液内PSA含量及其法医学意义

目的检测人体不同体液内前列腺特异抗原(PSA)含量,探讨其法医学价值。方法收集成年人(19～63岁)晨尿40份(男28份、女12份)、血液58份(男45份、女13份)、唾液25份(男14份、女11份);青少年(10～15岁)男性晨尿205份;哺乳期(25～31岁)女性乳汁9份;使用Cobas e411型全自动电化学发光免疫分析系统及T-PSA定量测定试剂盒,检测各样本T-PSA含量;分析不同体液及不同年龄青少年男性尿液PSA含量差异。结果除男、女性唾液外,其它样本均可检测到PSA,其中成年男性尿液含量最高,与其它体液比较具有显著性差异(P<0.000 1);青少年男性各年龄组尿液PSA含量随年龄逐年增高,11岁及以下年龄组含量不足1ng/mL,14岁及以上年龄组可超过1 000ng/mL。结论前列腺发育成熟的男性尿液PSA含量较高,在进行精液斑的法医学检验时应给予充分注意。     

尿中苯基脲类除草剂代谢物气相色谱分析

目的建立尿中苯基脲类除草剂代谢物的气相色谱分析方法。方法在检材中加入环己烷、固体氯化钠等试剂,分取有机相,在60℃水浴中加入衍生化试剂反应0．5h,冷却后洗去衍生化试剂,分取有机相进样进行气相色谱分析。结果尿中苯基脲类代谢物4一氯苯胺和3,4-二氯苯胺的检出限在5ng／mL以下,回收率良好,相对标准偏差均低于5％。在0．1μg～5μg／mL间线性关系良好。结论该法高效、灵敏、简便,适合对尿等生物检材中脲类除草剂的代谢物的定性和定量测定。     

Analysis of dimethylamphetamine and its metabolites in human urine by liquid chromatography-electrospray ionization-mass spectrometry with direct sample injection

An analytical method to identify and determine dimethylamphetamine (DMA) and its metabolites in human urine was developed with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) involving the direct injection of a urine sample. The urine samples were directly injected by using a gel permeation column, whose stationary phase was polyvinyl alcohol with a small amount of a carboxyl group, so DMA and its metabolites were analyzed rapidly and simply without pretreatment such as extraction, concentration and derivatization. DMA and its metabolites were identified in drug-free human urine spiked with 1 microg of DMA, dimethylamphetamine N-oxide (DMANO) and methamphetamine (MA), and 3 microg of amphetamine (AM) in 1 ml of urine under the full-scan mode. Under the selected ion monitoring (SIM) mode, the limits of detection (signal-to-noise ratio=5) for DMA, DMANO, MA and AM were 20, 20, 20 and 60 ng in 1 ml of urine, respectively. This method was applied to the identification and determination of DMA and its metabolites in urine samples of 10 DMA abusers. The concentrations of DMANO were higher than those of unchanged DMA in all urine samples; thus, DMANO is considered to be a useful metabolite as an indicator to prove DMA intake.     

PCR-STR分型技术在尿样DNA分型中的应用

目的　对尿样的DNA分型进行研究。　方法　 10份尿样随机收集于无关个体 ,同时采集血液样本做DNA分型对照 ,用PCR -STR分型技术对尿样DNA进行FIBRA和D18S5 35基因座分型。　结果　 10份尿样 10ml、1ml及 0 .2ml体积均获得准确的分型结果 ,且与同一个体的血样DNA分型结果完全相同 ;室温储存 4天及 4℃保存 4周的尿样均分型成功。　结论PCR -STR分型技术对尿样DNA分型是一种有效的方法 ,在尿样的个人识别中具有极高的实用价值。     

Orosomucoid (ORM) typing by isoelectric focusing: description of two new alleles in a German population and thermostability in bloodstains

The genetic polymorphism of serum orosomucoid (ORM) was studied in 168 unrelated German individuals using isoelectric focusing followed by immunoprinting. Two new alleles, tentatively designated ORM1*14 and ORM2*13, were identified. The method was successfully applied to demonstrate ORM1 types in dried bloodstains. Each type of ORM1 was also correctly determined in bloodstains heated at 130 degrees C for 30 min. The results indicated that ORM1 is a new powerful genetic marker system for the grouping of bloodstains.     

Transferrin polymorphism detected in human urine using isoelectric focusing followed by immunoblotting

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF*C1 = 0.7265, TF*C2 = 0.2624, TF*D = 0.0083 and TF*B = 0.0028.     

Rapid confirmation of enzyme multiplied immunoassay technique (EMIT) cocaine positive urine samples by capillary gas-liquid chromatography/nitrogen phosphorus detection (GLC/NPD)

A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 microL of pentafluoropropionic anhydride and 25 microL of pentafluoropropropanol at 90 degrees C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 micron of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency.     

Gamma-hydroxybutyric acid stability and formation in blood and urine

Gamma-hydroxybutyric acid (GHB) can cause problems in interpretation of toxicological findings due to its endogenous nature, significant production in tissues after death and potential formation in stored samples. Our study was designed to determine the influence of storage conditions on GHB levels and its possible in vitro formation in blood and urine in cases where no exogenous use of GHB or its precursors was suspected. The samples were prepared by validated method based on liquid-liquid reextraction with adipic acid internal standard and MSTFA derivatization and assayed on a GC-MS operating in EI SIM mode. The first part of the study was performed with pooled blood and urine samples obtained from living and deceased subjects stored with and without NaF (1% w/v) at 4 and -20 degrees C over 8 months. In ante-mortem samples (both blood and urine) no significant GHB production was found. After 4 months of storage, the substantial GHB rise up to 100 mg/Lwas observed in post-mortem blood stored at 4 degrees C without NaF with subsequent gradual decrease in following months. The inhibition of GHB production was apparent during storage in NaF treated frozen blood samples. In post-mortem urine only slight temporary GHB levels were ascertained (up to 8 mg/L). The second part of our study was aimed to analyse 20 individual post-mortem blood samples stored at 4 degrees C for 16-27 days between autopsy and analysis without preservation followed by storage at 4 degrees C with NaF for 4 months. The temporary GHB production with maximum of 28 mg/Lwas detected in some samples.     

Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine

The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm×25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio=25. The linearity studied on spiked urine was satisfactory (r=0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10–300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n=10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.     

Genotyping for DQA1 and PM loci in urine using PCR-based amplification: effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing.

Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for individualization of biological samples have reached a new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investigated the influence of storage conditions, sample volume, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based genotyping for DQA1 and PM loci in urine. Urine specimens from male and female volunteers were divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by dialfiltration, using 3000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin method gave comparable typing results. Larger sample volume yielded a higher amount of DNA, but the typing rates were not affected for sample volumes between 1 and 5 ml. The quantifiable amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage samples produced significantly higher success rates than that of frozen samples, while there was only marginal difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of DQA1 + PM genotype was achieved for all samples of fresh urine, independent of gender, starting sample volume, or concentration method. Preservation by 0.25% sodium azide was acceptable for sample storage at 4 degrees C during a period of 30 days. For longer storage duration, freezing at -70 degrees C may be more appropriate. Thus, the applicability of the DQA1 + PM typing was clearly demonstrated for individualization of urine samples.     

Simultaneous determination of THC-COOH and THC-COOH-glucuronide in urine samples by LC/MS/MS

A fast method using liquid-liquid extraction and HPLC/tandem-mass spectrometry (LC/MS/MS) was developed for the simultaneous detection of 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid beta-glucuronide (THC-COOH-glucuronide) and 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. This highly specific method, which combines chromatographic separation and MS/MS analysis, can be used for the confirmation of positive immunoassay results even without hydrolysis of the sample or derivatisation of extracts. Liquid-liquid extraction was optimised: with ethylacetate/diethylether (1:1, v/v) THC-COOH-glucuronide and THC-COOH could be extracted in one step. Molecular ions of the glucuronide (MH(+), m/z 521) and THC-COOH (MH(+), m/z 345) were generated using a PE/SCIEX turboionspray source in positive ionisation mode; specific fragmentation was performed in the collision cell of an API 365 triple-quadrupole mass spectrometer and yielded major fragments at m/z 345 (for THC-COOH-glucuronide) and m/z 327 as well as m/z 299 for both cannabinoids. Chromatographic separation was performed using a reversed-phase C8 column and gradient elution with 0.1% formic acid/1 mM ammonium formate and acetonitrile/0.1% formic acid. Retention times were 22.2 min for the glucuronide and 26.8 min for THC-COOH. After enzymatic hydrolysis of urine samples with beta-glucuronidase/arylsulfatase (37 degrees C, 5 h), THC-COOH-glucuronide was no longer detectable by LC/MS/MS in urine samples. However, the THC-COOH concentration was increased. For quantitation of THC-COOH, THC-COOH-D(3) was added to the urine samples as internal standard prior to analysis. From the difference of THC-COOH in the native urine and urine after enzymatic hydrolysis, molar concentration ratios of THC-COOH-glucuronide/THC-COOH in urine samples of cannabis users were determined and found to be between 1.3 and 4.5.     

Morphine to codeine concentration ratio in blood and urine as a marker of illicit heroin use in forensic autopsy samples

A morphine to codeine ratio greater than unity (M/C>1) has been suggested as an indicator of heroin use in living individuals. The aim of this study was to examine the morphine to codeine ratio in a large population (N=2438) of forensically examined autopsy cases positive for 6-monoacetylmorphine (6-MAM) and/or morphine in blood and/or urine. Blood and urine concentrations of 6-MAM, morphine and codeine were examined using GC-MS and LC-MS/MS methods. In 6-MAM positive samples, the M/C ratio was greater than unity in 98% (N=917) of the blood samples and 96% (N=665) of the urine samples. Stratification of 6-MAM negative cases by M/C above or below unity revealed similarities in morphine and codeine concentrations in cases where M/C>1 and 6-MAM positive cases. Median blood and urine morphine concentrations were 8-10 times greater than codeine for both groups. Similarly to 6-MAM positive cases, 25-44 year-old men prevailed in the M/C>1 group. In comparison to cases where M/C ≤ 1, the M/C ratio was a hundred times higher in both 6-MAM positive and M/C>1 cases. The range of morphine concentration between the lowest and the highest quintile of codeine in M/C>1 cases was similar to that in 6-MAM positive cases. This range was much higher than for M/C ≤ 1 cases. Moreover, linear regression analyses, adjusted for age and gender, revealed a strong positive association between morphine and codeine in 6-MAM positive and M/C>1 cases. The M/C ratio appeared to be a good marker of heroin use in post-mortem cases. Both blood and urine M/C>1 can be used to separate heroin users from other cases positive for morphine and codeine.     

A research note: the outcome of GC/MS/MS confirmation of hair assays on 93 cannabinoid (+) cases

This article reports the outcome of gas chromatography/tandem mass spectrometry confirmations for THC and carboxy-THC on 93 hair samples screened by RIA for cannabinoids. The samples were taken from probationers in Pinellas County, FL, who voluntarily provided the research staff with six hair and six urine specimens, collected at 1-month intervals. There were 40 samples that were RIA (+), urinalysis (−). Samples were selected which had cannabinoid (+) outcomes for hair, urine, or both. The THC and/or the carboxy-THC was (+) on confirmation. Of these 40 samples, 22 were (+) for both THC and carboxy-THC, 15 were (+) for THC but not carboxy-THC, and three were carboxy THC (+), but THC (−). Only one sample had a (+) RIA, but was (−) for both THC and carboxy-THC on confirmation. RIA detection of cannabinoids was confirmed in nearly all cases. Most cases that were RIA (−) but urine (+) were cannabinoid (+) when analyzed by GC/MS/MS.     

大鼠血液、尿液中阿米替林的气相色谱快速分析

目的建专：m液及尿液中阿米替林（AMTL）的气相色谱分析方法,、方法以正常大鼠m液及尿液为空F1样奉,分别添加AMTI-标准品和内标SKF525A。实验大鼠以AMTL2倍LD50灌胃,致大鼠急性中毒后提取血液及尿液。用乙醚提取样本中AMTL,采用GC／FID法进行定量分析,并考察实验条件,结果采用该方法,血液及尿液中AMTL线性池用分别为5～150μg／mL（r=0．993）和5～150μg／mL（r=0．998）;最低检测限（S／N／〉3）均为1．0陆g／mL;口内、口间精密度均小于6％,同收率存95.5％～105．6％之间。结论该方法方操作便捷、捧确度高,适用=fAMTL临床治疗中血药浓度快速监测和法医毒物分析鉴定。     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.     

HS-SPME-GC/MS联用法测定生物检材中的百草枯

目的建立检测生物检材中百草枯的顶空固相微萃取-气相色谱-质谱联用（HS-SPME-GC/MS）的分析方法。方法尿样中加乙基百草枯作为内标,在氯化镍作催化剂的条件下,用硼氢化钠在碱性条件下进行还原,HS-SPME萃取,提取物经GC/MS分析。全血需先离心,沉淀血细胞提取上清液,再用甲醇沉淀蛋白。最终得到的上清液加内标乙基百草枯,以下操作同尿样。结果尿样和血样中的百草枯的还原产物在1.0μg/mL~100μg/mL范围内线性关系良好,回归方程分别为y=0.0957x-0.0163,r=0.9974（n=6）;y=0.1096x＋0.0871,r=0.9964（n=6）。尿样、血样低、中、高三个质量浓度,RSD值均小于7%。回收率分别为尿样85.49%~100.83%,血样94.72%~99.68%。结论本法操作简便易行、灵敏度高、快速准确。为检测生物检材中的百草枯提供了有效的方法。     

血液和尿液中草甘膦的净化富集及检测

目的建立血液、尿液中草甘膦的净化富集及定性定量方法。方法取适量血、尿样品,用80%甲醇水溶液稀释,经Agilent Accu Bond NH2（弱阴离子交换柱）净化富集,用3m L 5%氨水溶液洗脱后进行ESI/LC/MRM对草甘膦的定性及阴离子色谱法（IC）的定量检验分析。结果血液、尿液中草甘膦的IC法检验回收率均大于90%,方法的线性范围均在1-100μg/m L,血检测限为0.1μg/m L,尿液为0.08μg/m L。血液、尿液中草甘膦的LC/MRM的方法检测限均在5ng/m L左右。结论建立的血、尿中草甘膦的前处理方法和LC/MRM定性分析及IC定量分析方法,可满足司法检验要求。     

Detection of drugs of abuse in simultaneously collected oral fluid, urine and blood from Norwegian drug drivers

Blood and urine samples are collected when the Norwegian police apprehend a person suspected of driving under the influence of drugs other than alcohol. Impairment is judged from the findings in blood. In our routine samples, urine is analysed if morphine is detected in blood to differentiate between ingestion of heroin, morphine or codeine and also in cases where the amount of blood is too low to perform both screening and quantification analysis. In several cases, the collection of urine might be time consuming and challenging. The aim of this study was to investigate if drugs detected in blood were found in oral fluid and if interpretation of opiate findings in oral fluid is as conclusive as in urine. Blood, urine and oral fluid samples were collected from 100 drivers suspected of drugged driving. Oral fluid and blood were screened using LC-MS/MS methods and urine by immunological methods. Positive findings in blood and urine were confirmed with chromatographic methods. The analytical method for oral fluid included 25 of the most commonly abused drugs in Norway and some metabolites. The analysis showed a good correlation between the findings in urine and oral fluid for amphetamines, cocaine/benzoylecgonine, methadone, opiates, zopiclone and benzodiazepines including the 7-amino-benzodiazepines. Cocaine and the heroin marker 6-monoacetylmorphine (6-MAM) were more frequently detected in oral fluid than in urine. Drug concentrations above the cut-off values were found in both samples of oral fluid and urine in 15 of 22 cases positive for morphine, in 18 of 20 cases positive for codeine and in 19 of 26 cases positive for 6-MAM. The use of cannabis was confirmed by detecting THC in oral fluid and THC-COOH in urine. In 34 of 46 cases the use of cannabis was confirmed both in oral fluid and urine. The use of cannabis was confirmed by a positive finding in only urine in 11 cases and in only oral fluid in one case. All the drug groups detected in blood were also found in oral fluid. Since all relevant drugs detected in blood were possible to find in oral fluid and the interpretation of the opiate findings in oral fluid was more conclusive than in urine, oral fluid might replace urine in driving under the influence cases. The fast and easy sampling is time saving and less intrusive for the drivers.     

Comparison of results for quantitative determination of morphine by radioimmunoassay, enzyme immunoassay, and spectrofluorometry.

The quantitative results (accuracy and precision) for determination of opiates by radioimmunoassay (RIA), enzyme immunoassay (EMIT), and spectrofluorometry on split samples are compared. A variety of physiological samples were studied, including random urine from a methadone maintenance clinic and postmortem urine, blood, bile, brain, and lung tissue from heroin-induced or heroin-related deaths. The opiate concentrations detected by the two immunoassay methods were in good agreement with each other in the absence of interfering substances which are believed to react with the antimorphine antibodies. The immunoassay results were in agreement within the relative standard deviation with the fluorometry results in 55% of the urine samples and 80% of the blood samples. The immunological methods are superior to fluorometry for quantitation of morphine in urine samples due to quenching interferences in fluorometry from urine. They were comparable to fluorometry for quantitation of morphine in blood samples.