Metabolites of ephedrines in human urine after administration of a single therapeutic dose

Ephedrine (EPH), pseudoephedrine (PEPH), phenylpropanolamine (PPA), methylephedrine (MEPH) and cathine are sympathomimetic amines. These drugs are commonly found in over-the-counter (OTC) cold medicines and some dietary supplements. In Taiwan, the misuse of these drugs has resulted in an increase in athletic violations. Excretion studies of the ephedrine-related drugs have been performed to better understand the metabolic yields of ephedrines in urine. After consuming a single clinical dose of each of these drugs, urine samples from volunteers (n=3 for each drug) were subjected to tert-butyl-methyl-ether (TBME) extraction and trifluoroacetic acid (TFAA) derivatization before gas chromatography-mass spectrometry (GC-MS) analysis. Most ephedrines were excreted unchanged in urine, including EPH (40.9%), PEPH (72.2%), and PPA (59.3%). However, only a relatively small amount of MEPH (15.5%) was excreted unchanged in urine. In addition, a trace amount of PPA (1.6%) and cathine (0.7%) was found to be the metabolites of EPH and PEPH, respectively. Urinary EPH, PEPH, and PPA reached peaks at 2-6h and disappeared in urine at approximately 24-48 h post-administration. For MEPH, the peaks of excretion extended from 4 to 12h post-administration and were undetectable at approximately 48 h. A single clinical dose of EPH (25 mg) may exceed threshold level (10 microg/mL) in sport drug testing if the urine samples are tested within approximately 8h post-administration. However, a single dose of MEPH (20 mg) never reached the threshold value (10 microg/mL).     

Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a single dose of Rohypnol

The hypnotic benzodiazepine flunitrazepam (Rohypnol) has been identified as the drug of choice for the purposes of "drugging" unsuspecting victims and raping them while they are under the influence of this substance. The objective of this paper was to study elimination of flunitrazepam and 7-aminoflunitrazepam in urine collected from ten healthy volunteers who received a single 2 mg oral dose of Rohypnol, to determine how long after drug administration 7-aminoflunitrazepam can be detected. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of flunitrazepam (LOQ 100 pg/mL) and 7-aminoflunitrazepam (LOQ 10 pg/mL) in urine was developed. All samples were screened for benzodiazepines using optimized micro-plate enzyme immunoassay. The highest concentrations of 7-aminoflunitrazepam (70-518 ng/mL) and flunitrazepam (0.7-2.8 ng/mL) in urine were observed 6 h after drug administration in nine subjects and after 24 h in one subject. In six subjects 7-aminoflunitrazepam was detected up to 14 days after flunitrazepam administration, in one subject up to 21 days and in three subjects up to 28 days. In urine samples collected from six volunteers, flunitrazepam was detected three days after Rohypnol intake, in three subjects 24 h, and in one subject 5 days later. Benzodiazepine micro-plate enzyme immunoassay kit allowed the detection of flunitrazepam and metabolities 5 to 21 days after drug administration.     

Deposition of 7-aminoflunitrazepam and flunitrazepam in hair after a single dose of Rohypnol

In recent years, there has been a notable increase in the number of reports on drug-facilitated sexual assault. Benzodiazepines are the most common so-called "date-rape" drugs, with flunitrazepam (Rohypnol) being one of the most frequently mentioned. The aim of this study was to determine whether flunitrazepam and its major metabolite 7-aminoflunitrazepam could be detected in hair collected from ten healthy volunteers after receiving a single 2 mg dose of Rohypnol using solid phase extraction and NCI-GC-MS. Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault in order to reveal drug use. Ten healthy volunteers (eight women and two men, 21 to 49 years old) participated in the study. The following hair samples were collected from each volunteer: one before flunitrazepam administration, and 1, 3, 5, 14, 21, and 28 days after. In five volunteers, 7-aminoflunitrazepam was detected 24 h after flunitrazepam administration and remained in hair throughout the entire 28-day study period (0.6-8.0 pg/mg). In two cases, 7-aminoflunitrazepam appeared in hair 21 days after drug intake (0.5-2.7 pg/mg), and in two subjects 14 days later (0.5-5.4 pg/mg). In one volunteer, 7-aminoflunitrazepam was detected on day 14 and 21 but concentrations were below the quantitation limit. Flunitrazepam was detected in some samples but all concentrations were below the quantitation limit (0.5-2.3 pg/mg).     

Determination of paraquat from formalin-fixed tissue

In this paper, a sulfuric acid digestion method and a clean-up technique by using cation exchange resin followed by XAD-2 resin has been developed for the determination of paraquat from formalin-fixed tissue at the submicrograms per gram level. Formalin-fixed tissue is dissolved by hot sulfuric acid, then paraquat is isolated and purified with cation exchange chromatography. The eluted paraquat forms an ion-pair with sodium dodecyl sulfate, it is then adsorbed on XAD-2 resin. Paraquat is eluted, extracted and reduced with solvent mixtures, NaCl solution and dithionite reagent, respectively. The calibration graphs of zero-order and second-derivative spectroscopy are linear in the range of 0.01-5.0 mg/kg. The relative standard deviation was less than 5% and the detection limit was 0.02 mg/kg based on 0.5-g samples. The sensitivity of the proposed method could be increased by using larger sample sizes. The method was precise and gave a quantitative recovery of paraquat spiked into formalin-fixed liver homogenates (78%). The proposed method has been satisfactorily applied to the determination of paraquat in the formalin-fixed tissues of suspected poisoned cases. It has been shown to be of great value in the field of forensic toxicology especially when formalin-fixed tissue only is available.     

Headspace solid phase microextraction for the gas chromatographic analysis of methyl-parathion in post-mortem human samples. Application in a suicide case by intravenous injection

A simple and rapid procedure for the determination of methyl-parathion (m-p) in post-mortem biological samples was developed using headspace solid phase microextraction (SPME) and gas chromatography (GC) with nitrogen-phosphorous detection (NPD). Methyl-parathion was extracted on 85 microm polyacrylate SPME fiber. Salt addition, extraction temperature, and extraction time were optimized to enhance the sensitivity of the method. The linearity (y = 0.0473x - 0.0113, R2 = 0.9992) and the dynamic range (0.1-40 microg/ml) were found very satisfactory. The recoveries of methyl-parathion were found to be 46% in spiked human whole blood, 53% in spiked homogenized liver tissue, and 54% in spiked homogenized kidney tissue compared with samples prepared in water. The coefficients of variations for 2, 4, and 20 microg/ml of methyl-parathion in blood ranged from 0.9 to 5.1%, whereas the detection limit of the method was satisfactory (1 ng/ml in aqueous samples, 50 ng/ml in whole blood). The developed procedure was applied to post-mortem biological samples from a 21-year-old woman fatally poisoned (suicide) by intravenous injection of methyl-parathion. The intact insecticide was found in the post-mortem blood at a concentration of 24 microg/ml. No methyl-parathion was detected in the liver, kidneys, and gastric contents.     

Determination of blood/serum ratios of different forensically relevant analytes in authentic samples

For forensic toxicological investigations only whole blood, but no serum is often available. Pharmacokinetic data are helpful for interpreting the results, but most of these studies indicate serum or plasma concentrations. In order to obtain reliable conversion factors which also take intersubject variability into account, the blood/serum ratios (B/S) of oxycodone, morphine, fentanyl, hydromorphone, zopiclone, MDMA, dexamphetamine, alprazolam, risperidone and 9-hydroxyrisperidone were determined by LC-MS/MS using authentic samples. Blood and corresponding serum samples were obtained from driving studies performed with controlled or known dosages of the above drugs. The analytes were analysed in blood and serum and the following mean B/S ratios (relative standard deviations) were determined: oxycodone 1.48 (8.19 %); morphine 1.03 (3.59 %); fentanyl 0.87 (13.9 %); hydromorphone 1.04 (8.11 %); zopiclone 0.89 (16.1 %); MDMA 1.19 (8.04 %); dexamphetamine 0.89 (10.9 %); alprazolam 0.81 (5.84 %); risperidone 0.65 (7.52 %); 9-hydroxyrisperidone 0.73 (12.3 %). These mean values are largely in line with those reported in the literature. The B/S ratios did not appear to depend on partition coefficients, whereas there was strong evidence that B/S ratios decreased with increasing plasma protein binding.     

过敏性休克豚鼠血浆咽喉肺组织中P物质的观测

目的探讨过敏性休克法医学鉴定的诊断指标。方法制备豚鼠过敏性休克的动物模型,应用放射免疫法测定其血浆中P物质浓度;免疫组化SABC法和BI-2000图像分析系统对咽喉和肺组织的P物质免疫反应进行染色观测,计算阳性指数(PI)。结果与对照组含量(87．70pg±7．60pg／μl)相比,过敏性休克豚鼠血浆中P物质含量 (131．01pg±18．93pg／μl)增加,其差异具有极显著意义(P<0．01);呼吸道内P物质免疫阳性反应增强,实验组咽喉和肺组织PI值分别为63．59±14．51和55．98±14．8,对照组咽喉和肺组织PI值分别为33．32±8．04和20．51±6．76, 两组差异具有极显著意义(P<0．01)。结论 P物质在过敏性休克豚鼠血浆浓度增加和呼吸系统组织中免疫阳性反应增强,可能有助于过敏性休克的法医病理学诊断。     

Fatal intravenous injection of potassium in hospitalized patients

We describe 4 cases of fatal intravenous injection of potassium in the hospital setting. These cases illustrate the subtlety of findings in such deaths and remind the forensic pathologist to consider this type of event in sudden, unexpected death of hospitalized patients.     

单个细胞DNA样本的制备及其在灵敏度分析中的应用研究

目的建立一种用于高精确灵敏度分析的微量DNA样本制备方法。方法用显微捕获技术制备以细胞为单位的DNA样本,并用于ProfilerPlus试剂盒及ABI310遗传分析仪的灵敏度测试。结果建立了单细胞捕获操作方案,成功制备了依次含1～11个细胞的一组DNA分析样本,并可准确用于ProfilerPlus誖试剂盒及ABI310遗传分析仪的灵敏度研究。结论基于显微操作技术的高精确微量DNA分析样本制备方法可准确用于分析技术方法的灵敏度研究     

Determination and distribution of clotiapine (Entumine) in human plasma, post-mortem blood and tissue samples from clotiapine-treated patients and from autopsy cases

The antipsychotic drug clotiapine (Entumine^®) has been marketed for more than 35 years, however there is little published data on the therapeutic and toxic concentrations of this drug. To fill this gap, two rapid and sensitive methods were developed for the determination of clotiapine (2-chloro-11-(4-methyl-1-piperazinyl)dibenzo-[b,f][1,4]-thiazepine), in human plasma and post-mortem blood and tissue samples. After simple liquid–liquid extraction at pH 9.5 with n-hexane/dichloromethane (85/15, v/v), clotiapine was quantitated by HPLC-DAD and by GC-NPD. The calibration curve was linear between 10 and 1000 μg/L. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 2 and 6 μg/L for the GC-NPD method and 5 and 15 μg/L for the HPLC-method, respectively. These methods were applied to 12 plasma samples from patients treated with clotiapine, to seven autopsy cases and to one case of driving under the influence of drugs (DUID). Concentrations ranged for the clotiapine-treated patients between 6 and 155 μg/L (mean 46 μg/L), and for the autopsy cases between 22 and 341 μg/L (mean 123 μg/L).     

Haemostatic plugs as a histological vital reaction in the skin wounds of guinea pigs.

Applying a histological staining method (Carstairs' modification of picro-Mallory staining), haemostatic plugs could be demonstrated at points of vascular lesion in one-minute vital, or older, skin wounds of the guinea-pig on the average in 33% if the specimen was taken immediately after the reaction period, and in about 10% of wounds examined if the specimen was taken five days after death. No haemostatic plugs could be observed in postmortally inflicted skin wounds.     

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.     

Typeability of AmpFlSTR SGM Plus loci in brain and thyroid gland tissue samples incubated in different environments

Autolysis and putrefaction are crucial factors responsible for degradation of cells, tissues, and organs. Postmortem changes may assume different course depending on extrinsic and intrinsic conditions. The aim of the study was assessment of environmental effect on typeability of AmpFlSTR SGM Plus loci: D3S1358, VWA, D16S539, D2S1338, D81179, D21S11, D18S51, D19S433, TH01, FGA, and gender marker amelogenin. Brain and thyroid gland tissue specimens collected during autopsies of five persons aged 20-30 years were incubated at 21 degrees C and 4 degrees C in different environmental conditions. DNA was extracted by organic method from tissue samples collected in 7-day intervals and subsequently typed using AmpFlSTR SGM Plus kit and ABI 310. A fast decrease in typeability rate was seen in specimens incubated in peat soil and in sand. Brain tissue samples were typeable in all AmpFlSTR SGM Plus loci within 126 days of incubation at 4 degrees C. Faster DNA degradation was recorded in thyroid gland specimens. In samples with negative genotyping results, no DNA was found by fluorometric quantitiation.     

Determination of the hand from single digit fingerprint: a study of whorls

In this investigation 400 bilateral rolled fingerprints of Punjabi Jat males have been examined for the determination of the hand from single digit fingerprint from whorl patterns. Direction of the flow of the apex ridges, rotation of centrally circular ridges, angles between the core and the delta, ridge tracing, ridge counting and the position of the perpendiculars drawn between the delta and the core are useful for establishing in most of the cases the hand and finger involved from the fingers having whorl pattern.     

Determination of manganese in human brain samples

A method is presented for the determination of manganese (Mn) in human tissue samples (especially brain) by graphite furnace atomic absorption spectrophotometry (GFAAS). After complete digestion by a mixture of concentrated nitric acid (HNO₃)/concentrated perchloric acid (HClO₄) (50:50, v/v), the samples are assayed on a Perkin-Elmer 5100 PC apparatus, equipped with transversal graphite tubes and a Mn-specific hollow cathode lamp. The furnace conditions are as follows (for each step: temperature (°C)/ramp (s)/duration (s)) dry 120/1/40; char 1200/5/10; atomization 2250/0/4; pyrolysis 2400/1/1. Zeeman correction is employed. The method is linear over the range 0.05 to 5.00 μg/g wet tissue, and the limit of detection for Mn is about 0.01 μg/g wet tissue. This simple and rapid method may be of value for the post-mortem assessment of Mn accumulation in brain structures due to occupational or iatrogenic exposure. An application is presented in which elevated levels of Mn were determined in the brain samples of a 63-year-old female deceased after long-term total parenteral nutrition involving Mn supplementation.     

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。     

利多卡因及MEGX在蛛网膜下腔麻醉致死犬体内死后再分布

目的研究利多卡因及其代谢产物单乙基甘氨酰二甲苯胺(MEGX)在蛛网膜下腔麻醉致死犬体内的死后再分布规律。方法犬6只随机分为A、B两组,分别经蛛网膜下腔注射0.5倍(6.34mg/kg)和5倍(63.35mg/kg)硬膜外麻醉极量的盐酸利多卡因,于死后0h、12h、24h、36h、72h取心血、外周血、肝、脑等,采用高效液相色谱法(HPLC)检测其中利多卡因及MEGX的含量。结果 A组犬死后72h,心血、外周血和脑中利多卡因含量与死亡当时的比值(Ct/C0)分别为4.74,14.87,7.67,均呈上升趋势(P0.05),MEGX含量与死亡当时含量差异无统计学意义(P0.05);B组犬死后72h,心血中利多卡因含量Ct/C0值为0.36,呈下降趋势(P0.05),脑中为3.48(P0.05)呈升高的趋势,肝中MEGX含量与死亡当时相比差异无统计学意义(P0.05)。结论蛛网膜下腔不同剂量麻醉致死犬体内利多卡因均会发生死后再分布,MEGX未发生死后再分布。     

Multiple interchanging of tissue samples in cases of breast cancer

Due to the suspicion of a gynaecologist, a pathologist was suspected of incorrect diagnoses in cases of breast cancer and the interchanging of tissue samples. Many women applied to the attorney's bureau to clarify the reproaches. The privately owned laboratory for pathology was searched and 926 histological slides, roughly the same number of paraffin blocks and about 20 formalin fixed tissue samples were confiscated. Together with other confiscated material, at least 1236 histological slides and additional 249 paraffin blocks had to be sorted. Histological slides and paraffin blocks were matched with patients as far as possible following the laboratory book. Many of the warranted samples which were diagnosed as containing the carcinoma by the pathologist were missing. A total of 160 samples were chosen and rediagnosed by two independent pathologists. The formalin fixed tissue was negative for DNA most likely due to storage in formalin for years. Most of the histological slides were positive for DNA. On the whole, 18 expertises about histological findings and the DNA results were given. In some cases only DNA results could be presented, as previous experts had only performed DNA examinations without controlling the histological diagnosis. In six cases a carcinoma could be confirmed and the DNA profile matched with patient's DNA; in seven cases a carcinoma was confirmed without match with the patient; in two cases the carcinoma could not be confirmed in the presented samples. A jurisdictional solution was impossible because the accused pathologist died during the investigation. In conclusion, it must be stated that a DNA examination of histological slides should never be performed without a rediagnosis of an independent pathologist and photographic documentation of the findings. Whenever possible, material should be left on the slide.