Isolation of blood caffeine as a model substance by enzymatic hydrolysis

Organic solvent extraction of toxic compounds from blood either directly or following precipitation of serum proteins yields only free fractions. It is proposed to dissociate toxin-protein complexes in blood samples by enzymatic hydrolysis. Model caffeine complexes in an albumin solution were treated by tripsin, chemotripsin, and papain. The best results were obtained using chemotripsin and papain with which 70.4 +/- 1.6% and 62.2 +/- 1.2% of the caffeine was isolated from blood compared with 45.6% by direct extraction. Validation characteristics of the proposed method are specified.     

尿中MDMA及其代谢物的GC和GC／MS分析

考察MDMA在人体内的代谢以及建立尿中MDMA和体内主要代谢物MDA的分析方法。尿样水解后经液-液提取处理,用GC／MS（EI、PCI）和GC／FID法分析。人摄入MDMA后尿中MDA和原体MDMA比约为0．10～0．14。GC／MS／SIM和GC／FID法的最低检出限为2ng／ml和50ng／ml,回收率大于85％,变异系数小于10％。该法简便快速、灵敏度高、结果可靠,可用于MDMA滥用者的尿样鉴定。MDA／MDMA浓度比可作为评判毒分结果的参考指标。     

气相色谱质谱联用法检测大鼠尿液中2C-B及其代谢物

目的研究4-溴-2,5-二甲氧基苯乙胺（2C—B）在大鼠体内的代谢物以及代谢途径。方法取Wistar大鼠3只,以2C-B灌胃,收集24h内尿液,用B葡萄糖醛酸酶水解,Oasis HLB柱固相提取,DB-35MS柱分析,气相色谱质谱联用检测。结果从大鼠尿液中检出6种2C-B的代谢产物,分别为：4-溴-2-羟基-5-甲氧基苯乙醇、4-溴-2,5-二甲氧基苯乙醇、4-溴-2-羟基-5-甲氧基苯乙酸、4-溴-2,5-二甲氧基苯乙酸、1-乙酰氨基-2-（4-溴-5-羟基-2-甲氧基苯）乙烷和1-乙酰氨基-2-（4-溴-2-羟基-5-甲氧基苯）乙烷。未检出2C—B原药。结论2C-B在大鼠尿液中主要以代谢物形式存在,其在大鼠体内至少仔在两种代谢途径：第一种是2C—B的2位和5位氧上去甲基后氨基被乙酰化;第二种是2C—B去氨基生成醛,接着被还原或氧化生成醇和羧酸。     

Cytokinetics of epidermic cells in skin from human cadavers. II. Dependency on sex, age, and site

Kinetic data on the labeling index (LI), DNA synthesis time (ts), and potential doubling time (tpot) of epidermic cells in relation to sex, age, and site were obtained by in vitro incubation of skin cylinders from 45 human cadavers with DNA precursors 3H- and 14C-thymidine. In a first study on parts of the same material, it was established that LI over a period of more than 70 h and tpot over a period of at least 30 h remained essentially unchanged and are comparable with live humans, when the cadavers were stored at 4 degrees C. The following results were obtained: The female and male cadavers had a LI of 2.6% (+/- 0.8%) or 2.5% (+/- 0.8%), a ts of 3.9 h (+/- 0.2 h) or 5.0 h (+/- 1.6 h), and a tpot of 168.5 h (+/- 34.3 h) or 183.9 h (+/- 27.2 h). The LI for the thigh and knee ranged between 21.3% and 25.8% in different age groups. No statistically relevant differences were established between the sexes or among the age groups. Topographic allocation of the proliferative-kinetic data ultimately showed that, on the average, LI was relatively high at the elbow (3.1% +/- 1.0%) with short tpot (109.3 +/- 72.5 h) and a comparatively large epidermal diameter (47.1 microns); by contrast, LI at the lower abdomen was impressively low (2.1% +/- 0.8%), tpot relatively long (183.0 +/- 138.7 h) and mean epidermal diameter relatively small (23.0 microns). Nevertheless, no statistically relevant differences were established between data for elbow and lower abdomen or between other data for different sites. The proliferative-kinetic data for human cadavers were compared with data reported in the literature for live humans.     

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.     

Rapid isolation of some antiepileptic hydantoins and their analogues with Sep-Pak C18 cartridges and capillary gas chromatography with splitless injection

A simple and rapid method for isolation of seven antiepileptics (2 hydantoin, 2 oxazolidin, and 3 suximide derivatives) from urine and plasma is presented. Urine and plasma (1 ml) samples containing seven antiepileptics were mixed with distilled water (4 ml), and the sample solution was poured into a pretreated Sep-Pak C18 cartridge; this was washed with water and chloroform/methanol was passed through it to elute the antiepileptics. The eluate was mixed with isoamyl acetate and evaporated under a stream of N2. The drugs were detected by gas chromatography with fused silica capillary columns, splitless injection and flame ionization detection. Separation of the seven antiepileptics from each other and from impurities was satisfactory with the use of an SPB-1 capillary column. The detection limit for the seven antiepileptics with the present method was 0.1-1.0 microgram/ml urine or plasma. The recovery of the drugs from urine and plasma was more than 70% and 50%, respectively.     

Expedited, chemically enhanced sperm cell recovery from cotton swabs for rape kit analysis

This report focuses on the development of a method for chemically induced enhancement of cell elution and recovery from cotton swabs. The method exploits the exclusive use of detergents for intact cell removal, and can be utilized in conjunction with, or to circumvent, conventional differential extraction (DE). Samples treated with Sarkosyl (54.4 +/- 1.8%) and sodium dodecyl sulfate (SDS) (78.5 +/- 0.7%) yielded higher sperm cell recoveries than a conventional DE buffer (39.4 +/- 2.1%). The results indicated that the choice of detergent affected sperm cell yield, with anionic detergents having the greatest effect. Storage time of samples affected the concentration of detergent required for optimal sperm cell recovery, longer times requiring increased detergent concentrations. In addition, the extent of sperm cell lysis by proteinase K digestion was evaluated. The results indicate that the exclusive use of SDS enhances the release of sperm and epithelial cells from a cotton swab as compared with DE buffer, providing for a more effective DNA analysis.     

Morphine extraction in necrophagous insects remains for determining ante-mortem opiate intoxication.

To study the potential use of insects remains as toxicological indicators, we measured morphine in desiccated entomological materials using a radioimmunoassay method (Coat-A-Count Serum Morphine, Dade Behring, France) following enzymatic hydrolysis.First, enzymatic cuticle extraction of morphine (pronase digestion preceded by a 2h incubation with a dithiothreitol solution) was performed on various substrates (Calliphoridae puparial cases and desiccated adults, and desiccated pupae of Dermestidae) which were then tested to identify possible interferences with the radioimmunoassay procedure.This same procedure was performed on puparial cases and desiccated adults of Lucilia sericata (Diptera, Calliphoridae) previously reared on minced meat containing various morphine concentrations. Morphine was detected only in cuticle powder from insects reared on meats containing 100 and 1000mg/kg. Higher concentrations were measured in puparial cases. Rearings on psoas from eight heroin overdose victims confirmed previous experimental results.Remains of necrophagous insects, particularly puparial cases, are often preserved for a long time, and consequently can serve as late alternative specimens for toxicological analysis when suitable tissues are not available.     

Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC-MS

Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC-MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E for 12 h. A 100-micron polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d3 and EDDP-d3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0-50 ng/mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng/mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng/mg, and for EDDP from 0.98 to 7.76 ng/mg of hair.     

Determination of cocaine and benzoylecgonine in human amniotic fluid using high flow solid-phase extraction columns and HPLC.

A new solid-phase extraction procedure for the determination of cocaine and benzoylecgonine in amniotic fluid, using high flow co-polymeric sorbents is reported. The recoveries of cocaine and benzoylecgonine within the range 0.1-1 mg/l were 95.7% and 50.3%, respectively. The use of high-flow sorbents allowed the easy extraction of amniotic fluid regardless of sample viscosity or physical nature. The use of these solid-phase columns provided many advantages over the more commonly used solvent extraction, including an increase in extraction speed and efficiency, reduced operator time, reduced solvent use and disposal volumes and exceptional extract quality. Further, the determination of amniotic fluid obtained from pregnant cocaine users may provide important information about handling of cocaine by the fetus at various gestational ages. The procedure was successfully applied to amniotic fluid from suspected cocaine abusers.     

Determination of sex from femora

The determination of sex from bones or bone fragments considerably contributes to identifying unknown bodies or skeletal remains. Due to temporal change and regional differences anthropometric standards have to be constantly renewed. The present study provides measurements of femoral dimensions in a contemporary German population and analyses sexual dimorphism by discriminant analysis. Maximum length (male: 46.4+/-2.4 cm, female: 43.4+/-2.4 cm), maximum midshaft diameter (male: 3.1+/-0.2 cm, female: 2.8+/-0.2 cm), condylar width (male: 8.4+/-1.0 cm, female: 7.7+/-0.5 cm), vertical head diameter (male: 4.9+/-0.3 cm, female: 4.4+/-0.3 cm), head circumference (male: 15.7+/-0.8 cm, female: 13.8+/-1.0 cm) and transverse head diameter (male: 4.9+/-0.3 cm, female: 4.3+/-0.3 cm) were measured in 170 femora, 100 from male (age: 16-92 years, mean: 60.8 years; body height: 153-190 cm, mean: 171 cm) and 70 from female (age: 20-96 years, mean: 72 years; body height: 146-175 cm, mean: 161 cm) individuals. In the discriminant analysis (leave-one-out-method) 67.7% of cases could be grouped correctly with the maximum length alone, 72.4% with the maximum midshaft diameter, 81.4% with the condylar width, 86.8% with the vertical head diameter, 87.7% with the head circumference and 89.6% with the transverse head diameter. The stepwise procedure with all head measurements showed that the results for the transverse head diameter could not be improved. With all measurements subjected to stepwise procedure 91.7% of cases could be classified correctly combining midshaft diameter and head circumference (D=3.012xmidshaft diameter in cm+0.780xhead circumference in cm 20.569).     

The analysis of tiapride in cadaveric material

To optimize conditions of tiapride isolation from cadaveric organs, we compared the results of conventional methods by Stas-Otto, A.A. Vasilyeva and V.F. Kramarenko which provide tiapride isolation up to 50% and a new precise and reproducible method providing 60 +/- 2% tiapride isolation. Identification of tiapride isolated from cadaveric material was made with thin-layer chromatography and high performance liquid chromatography. The latter assay employed the method of external standard. The original techniques proposed identify and measure tiapride in hepatic samples in the presence of unidentified endogenic compounds. The techniques are rapid, selective, sensitive and reproducible.     

Extraction of trifluralin from acetonitrile solutions

Results of extraction of trifluralin from an aqueous acetonitrile solution using various organic solvents are presented. The degree of extraction was shown to depend on the nature of extractant and the water/acetonitrile ratio. An optimal electrolyte and the degree of saturation of the water-acetonitrile layer with this electrolyte were selected. The highest efficiency of extraction was achieved by using water-saturated ethylacetate as an extractant. The indices of extraction were calculated as necessary for the isolation of a given amount of trifluralin from aqueous acetonitrile (4:1) solutions with the solvents considered in the present study.     

Drugs in hair: a new extraction procedure

An enzymatic hydrolysis of hair for drug detection is described. The results obtained with the suggested method are compared with those observed by chemical hydrolysis. Enzymatic digestion provides a high recovery (80%) for cocaine metabolite, while alkaline hydrolysis causes its chemical destruction. The two hydrolytic procedures yielded comparable results for morphine. The outlined procedure is recommended when unstable compounds are to be detected in hair.     

Use of rectal temperature-time of death nomograms at the scene of death

The application of the rectal temperature time of death nomogram at the scene of death by 11 authors from 6 lego-medical institutes resulted in a standard deviation of the differences between nomographic and real death time of +/- 1.3 h in 46 cases (group I) with met requirements and clearly defined points of contact, nevertheless, including 9 cases with a more progressive cooling (0.5 greater than Q greater than or equal to 0.2). In cases with a real death time of more than 4h (N = 26) the standard deviation was +/- 1.0 hours corresponding to permissible variation of 95% of +/- 2.0 hours. Consequently, the permissible variation of 95% was much smaller than that suggested by the nomogram. The nomographic death time interval did not agree with the real one in 5 cases out of additional 30 cases with recognizably unsure points of contact (group II).     

萃取方式对海洛因滥用者毛发中代谢物分析的影晌

目的比较液相萃取和同相萃取对毛发中海洛因毒品代谢物分析的影响。方法对海洛因吸食者毛发和空白添加标准品毛发经甲醇超声后的提取液分别进行液相萃取、同相萃取,然后进行衍生化和GC／MS—SIM检测。结果利用同相萃取法对添加6-单乙酰吗啡的毛发进行萃取和测试,6-单乙酰吗啡的回收率为32．O％,相对标准偏差（RSD）为2．4％;而液相萃取回收率为52．6％,相对标准偏差（RSD）为4．6％。结论固相萃取较之液液萃取,有更好的重复性,更少的杂质干扰和有机溶剂消耗等优势,但甲醇超声液需要挥干后才能进行同相萃取,而且6-单乙酰吗啡的水解率高。     

Application of a radiometric method for evaluation of loss of salicylic acid during isolation from biologic material

A radiometric method for evaluation of loss of salicylic acid in the process of isolation from biologic material is described. According to this study the mean loss during the total process of isolation amounts to 33.59%, the specific values being 19.47% during protein precipitation, 10.68% during extraction, and 3.44% during evaporation of solvent.     

体液中氟乙酰胺SPE-GC/MS检测

目的　利用GC/MS与固相萃取 (SPE)技术相结合 ,开发血和尿样中氟乙酰胺鼠药的GC/MS定量分析新方法 ,并用于实际案例检测。方法　选择乙酰胺为内标 ,通过比较不同固相柱的萃取效率和不同条件对回收率的影响 ,优化用于血和尿样中氟乙酰胺萃取的固相柱和提取条件 ,利用氟乙酰胺与乙酰胺质谱图的分子离子峰面积之比与氟乙酰胺浓度的定量关系 ,建立血和尿样中氟乙酰胺鼠药的GC/MS定量分析新方法。结果　用硅胶柱萃取 ,峰面积之比与氟乙酰胺浓度在 5 0～ 90 μg/ml范围呈线性关系 ,检测限为 1 0 μg/ml。血样中氟乙酰胺检测的平均回收率达 91 6% ,标准偏差小于 7 3 %。结论　此法对实际样品的测定证明可满足氟乙酰胺鼠药中毒的定性定量要求。     

Determination of zimarin in urine

Private technique of extraction isolation and purification, chromatographic detection and photometric determination of zimarin in urine is suggested. Detection limit is 0.01 mg, determination limit is 0.1 mg of glycoside in 100 ml of urine. Method makes it possible to detect 66-80% of zimarin added to 100 ml of urine in quantities 0.5-0.1 mg.     

Fentanyl-related deaths: demographics, circumstances, and toxicology of 112 cases

Since 1979, the potent narcotic analgesic fentanyl and its analogs have been synthesized in clandestine laboratories and sold as heroin substitutes. At least 112 overdose deaths have been associated with their use. In this study, toxicology data, autopsy findings, and coroners' investigative reports were reviewed in order to construct a profile of the typical fentanyl overdose victim and to identify any factors that might heighten the risk of death from fentanyl use. The "typical" fentanyl overdose victim was 32.5 +/- 6.7 years of age (range, 19 to 57 years), male (78%, compared with 22% female), and Caucasian (50%, compared with 29% Hispanic, 20% Black, and 0.9% Asian). With the exception of his or her age, the typical fentanyl overdose victim is quite similar to the typical heroin user. Nearly all the deaths (94%) occurred in California, yet within the state they were widely distributed throughout 17 counties and 44 cities. Pulmonary edema and congestion and needle puncture sites were consistent postmortem findings. No preexisting medical conditions were identified as possible risk factors. Although most of the fentanyl victims had a prior history of intravenous drug use, morphine or codeine were not commonly found, which suggests that the victims had little or no opiate tolerance. Ethanol was present in 38% of the cases and is thought to be a significant risk factor. Mean fentanyl concentrations in the body fluids were quite low: 3.0 +/- 3.1 ng/mL (0.3 +/- 0.31 micrograms/dL) in blood and 3.9 +/- 4.3 ng/mL (0.39 +/- 0.43 micrograms/dL) in urine, measured by radioimmunoassay. Although the potency of the analogs and the purity of street samples varies considerably, it is probably the general availability of the drug rather than the potency of a particular analog that determines the incidence of overdose deaths.