The detection of group-specific component from urine samples

The detection of group-specific component (Gc) from serum and bloodstains has been widely used in the forensic laboratory. A recent increase in substituted or adulterated urine samples in various drug screening programs has necessitated methods to determine the donor. This paper discusses the detection of Gc from urine samples. The samples were concentrated and applied to ultrathin polyacrylamide gel and focused for 150 min. This method separates the samples into the three common phenotypes found in all human populations. A nitrocellulose membrane blotting technique was used to detect the Gc bands. Serum and urine samples were collected from each individual and were typed for Gc. Urine samples tested after 6 months of storage (4 degrees C) were still readable. This method provides the forensic laboratory with an additional test from a body fluid which, until recently, provided little information.     

Completely automated interpretation of reference samples

Inspired by fully continuous interpretation a completely automated interpretation workflow for reference samples was implemented in our lab. It's based on a quite simple model, which includes DNA amount and degradation as well as backward and forward stutters. The current implementation can handle CE results of any number of replicates, even from different autosomal or Y-chromosomal STR kits. Results, lessons learned in a first set of test series and potential future extensions are discussed.     

Concentration of urine samples by three different procedures: ABO typing from concentrated urine samples

Urine samples from 28 donors with known blood group and secretor status were concentrated by three different procedures, and ABO typing on the concentrated samples was successfully performed after 12 weeks of storage. The effects of storage with or without sodium azide on ABO typing and on the pH values at several different temperatures were also studied.     

血液和尿液样品中海洛因代谢物稳定性研究

目的对尿液和血液中海洛因代谢物3-β-D-葡萄糖醛酸吗啡（M3G）,吗啡,O6-单乙酰吗啡（O6）在180d内的稳定性进行研究。方法准备空白添加血液、尿液、染毒动物（大白兔）血液、尿液和吸食海洛因者血液、尿液样本,分别置于20℃、4℃、-20℃下,分别于0、1、2、4、7、14、28、56、112、156、180d时间点测定样品中M3G、吗啡,O6相对含量。结果在3种不同温度下,随保存时间的延长,血液、尿液中的O6含量均逐渐下降至零;血液中吗啡含量升高（空白血液添加组）或下降（染毒动物组）,在尿液则均升高;血液样中M3G含量均升高,尿样中则略有下降。下降和升高的幅度均随保存温度的下降而缩小。结论海洛因代谢物在-20℃时保存稳定性最佳。     

The detection of psilocin in human urine

Pharmacokinetic studies of psilocybin in humans have shown the rapid dephosphorylation of psilocybin to psilocin with further conversion to 4-hydroxy-tryptophole (4HT) and 4-hydroxyindole-3-acetic acid (4HIAA) in plasma. Our study shows that psilocin also undergoes conjugation and can be found in the urine as the psilocin-glucuronide conjugate. Recoveries after enzymatic hydrolysis of the urine with beta-glucuronidase (Helix Pomatia or E. Coli) when compared to non-hydrolyzed urine confirmed the presence of the glucuronide. Detection of psilocin from hydrolyzed and extracted samples was optimized for GC/MS by derivatization with MSTFA. The method developed allows for the detection of psilocin in urine with a limit of quantitation of 10 ng/mL, based on 5 mL of spiked urine. Using this method, our laboratory has confirmed the presence of psilocin in 6 out of 8 urine samples, with concentrations ranging from 10 ng/mL to greater than 200 ng/mL. Before implementation of the hydrolysis and derivatization steps, our limit of detection was 200 ng/mL, based on spiked urine standards. No case samples were positive without hydrolysis and derivatization.     

Capillary gas-liquid chromatography separation of phenethylamines in amphetamine-positive urine samples

Good gas chromatography (GC) separation of molecules is essential for clean gas chromatography/mass spectrometry (GC/MS) confirmation of compounds. The trifluoro derivatives of ephedrine (E) and methamphetamine (MA) coelute on dimethyl silicone capillary columns, such as DB-1, which are most commonly used by chromatographers. Methods are described to separate E and MA to aid GC/MS confirmations of methamphetamine, ephedrine, or both E and MA together, whichever may be present in Enzyme Immunoassay (EIA)-analyzed amphetamine-positive urine samples. The use of the heptafluoro derivatives of E and MA on a DB-1 column, or the trifluoro derivatives of E and MA on a DB-17 column, is suggested for good gas chromatographic separation.     

Analysis of nitrite in adulterated urine samples by capillary electrophoresis

A simple method for analyzing nitrite in urine has been developed to confirm and quantify the amount of nitrite in potentially adulterated urine samples. The method involved separation of nitrite by capillary electrophoresis and direct UV detection at 214 nm. Separation was performed using a bare fused silica capillary and a 25 mM phosphate run buffer at a pH of 7.5. Sample preparation consisted of diluting the urine samples 1:20 with run buffer and internal standard, and centrifuging for 5 min at 2500 rpm. The sample was hydrodynamically injected, then separated using -25 kV with the column maintained at 35 degrees C. The method had upper and lower limits of linearity of 1500 and 80 microg/mL nitrite, respectively, and a limit of detection of 20 microg/mL. The method was evaluated using the National Committee for Clinical Laboratory Standards (NCCLS) protocol (Document EP10-A2), and validated using controls, standards, and authentic urine samples. Ten anions, ClO-, CrO4(-2), NO3-, HCO3-, I-, CH3COO-, F-, SO4-, S2O8(-2), and Cl-, were tested for potential interference with the assay. Interferences with quantitation were noted for only CrO4(-2) and S2O8(-2). High concentrations of Cl- interfered with the chromatography. The method had acceptable accuracy, precision, and specificity.     

Radioreceptor assay and radioimmunoassay of triazolam in urine samples from racing greyhounds

Two complimentary assay techniques were used to determine triazolam levels in greyhound urine samples following a single oral dose. The results from the trials were statistically compared. The relative non-specificity of the benzodiazepine antibody used in radioimmunoassay caused a significant difference in teh two sets of results. This was independent of hydrolysis.     

Poppy seed consumption and toxicological analysis of blood and urine samples

Poppy seeds contain morphine in different amounts. Reported concentrations are up to 294 mg morphine/kg poppy seeds. Since penalties based on Street Traffic Law (parapgraph 24a StVG) in Germany (administrative offence) require definitive proof of morphine in blood samples, and the "Grenzwertkommission" in consultation with the Ministry of Transportation recommended a threshold of free morphine of 10 ng/mL, the question arose whether the consumption of poppy seeds can lead to a blood concentrations equal or higher than 10 ng/mL of free morphine. Therefore, five volunteers ate poppy seed products (50 mg morphine/kg poppy seeds). In urine, all on-site tests were enzyme immunologically positive for opiates and were positive to morphine by GC/MS. All the blood samples were negative to morphine by EIA and to free morphine by GC/MS. However, after hydrolysis, morphine was detected by GC/MS in all cases. Accordingly, in Germany, penalties based on parapgraph 24a StVG are not likely to cause road users any concerns should they have consumed poppy seeds. Driver Licensing Authorities, however, should be advised of this problem to avoid unjustified legal measures.     

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。     

SPME-GC-MS法快速检测尿液中的甲基苯丙胺

目的建立SPME-GC-MS快速检测吸毒人员尿液中的甲基苯丙胺的方法。方法以SPME法提取尿液中的甲基苯丙胺,以1-萘胺作内标,用GC-MS法检测。结果在2～2000ng/mL的范围内呈线性关系(r=0.9985,n=7),甲基苯丙胺的检测限为0.5ng/mL(信噪比3),在低、中、高(200、500、1000ng/mL)浓度的平均相对回收率为102.6%、98.5%、93.2%,日内及日间RSD分别小于8.1%、7.2%。结论用此方法检测尿液中的甲基苯丙胺,灵敏度高,简单快速,易操作,适用于吸毒人员的快速定性定量检测。     

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－STD分型技术对尿样DNA进行FIBRA和D18S535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℃及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。     

尿蛋白酶抑制剂对尿样STR分型的影响

目的研究尿蛋白酶抑制剂(urinary trypsin inhibitor,UTI)对提高尿样STR分型成功率的作用。方法收集男女各5名健康志愿者中段尿,分装后分别添加不同质量浓度的UTI,-80℃保存,于8个不同的时间点进行DNA提取与Identifiler系统STR分型。比较15个STR基因座在不同组别尿样及来自同一个体对照血样的分型结果,计算不同组别尿样STR分型成功率。结果对照血样及保存期限为1 d的尿样15个STR基因座均分型成功,未经UTI处理的女性尿样3 d即发生基因座的丢失,9 d时检测不到任何基因座,而经UTI处理的女性尿样在9d之内均可以检测到15个STR基因座;未经UTI处理的男性尿样7d时检测不到任何基因座,而经UTI处理的男性尿样9d时STR基因座平均检出数约为9个。当尿液保存时间为30d时,经0.2、0.4和0.6μg/mL UTI处理的女性尿样的STR基因座检出率差异无统计学意义,平均为0.840 0±0.042 3,显著高于男性尿样的0.160 0±0.042 3。结论 UTI处理可显著提高尿样STR分型成功率,一定程度上延长尿样用于个体识别的保存期限。     

Immunohistochemical antigen detection in dried tissue samples

Investigations were carried out on dried tissue samples for the identification of ABH antigens and human hemoglobin using the indirect immunoperoxidase technique (PAP). Samples from various organs were stored at room temperature over a period of 1 year and periodically examined immunohistochemically. By means of a rehydration medium, blood group and species identification were successfully demonstrated in the complete experimental series.     

Screening for drugs in forensic blood samples using EMIT urine assays

A screening method for the detection of drugs in haemolysed whole blood has been evaluated. Methanolic extracts of 300 forensic blood samples known to be positive or negative for drugs were analysed with EMIT d.a.u. assay kits for amphetamine, cannabinoids, opiates and benzodiazepines (the latter to analyse for diazepam and the main metabolite N-desmethyldiazepam). There were very few false positive results, except for the amphetamine assay in postmortem blood samples, where 9% were false positive. For amphetamine and cannabinoids a few false negatives were found, these were from samples with very low drug concentrations. No false negatives were found for opiates and diazepam. The present modification of the EMIT d.a.u. method seems to be a good method for screening of drugs in forensic blood samples, except for amphetamine in postmortem samples. The method is simple and requires only 0.5 ml blood.     

Detection of haptoglobin from concentrated urine samples by enzyme immunoassay

The detection of haptoglobin (Hp) from serum and bloodstains is utilized extensively in forensic science laboratories in order to include or exclude possible donors. There is an increasing need to make the same discriminations utilizing genetic markers from urine samples. This paper describes the use of enzyme immunoassay and Western blotting (electrophoretic) techniques to determine Hp phenotypes from concentrated urine samples. Serum and urine specimens were collected from volunteer donors. The serum sample from each donor was typed for Hp. The urine specimens were concentrated 3000-fold from the starting volume of 15 mL to a final volume of 5 microL and applied to the gradient polyacrylamide gels. This procedure allows the separation of Hp samples into the three common phenotypes as well as the other rare variants found in humans. The Western blotting electrophoretic technique was used to achieve the transfer of Hp bands from the gels to the nitrocellulose membranes. Enzyme immunoassay with goat anti-Hp antiserum and rabbit anti-goat immunoglobulin alkaline phosphatase conjugate were used to identify the Hp bands from the concentrated samples. Specimens stored for six months at -22 degrees C were also concentrated and typed successfully. Recent implementation of drug-screening policies has resulted in an increase in the submission of substituted urine specimens. The above procedure can be used to detect an additional genetic marker from urine samples and thus facilitate the identity of the donor.     

Passive exposure in detection of low blood and urine cannabinoid concentrations

Whenever small amounts of drugs are present in blood or urine samples, especially of substances that are preferentially smoked such as cannabinoids, the discrimination between active and passive inhalation may cause severe problems. The statement of a passive exposure by marijuana smoke has been scrutinized reviewing the literature. The pharmacokinetics of smoked marijuana as well as experimental data on cannabinoid concentrations in plasma and urine samples following passive exposure are summarized. As a conclusion it seems urgent to enlarge the existing data base.