Robert Wennig

Comparison of MTBSTFA and BSTFA in derivatization reactions of polar compounds prior to GC/MS analysis

In this study, MTBSTFA and BSTFA, which are among the preferred derivatization reagents for silylation were both tested on derivatization of six different groups of polar chemicals to get information about usefulness in terms of sensitivity and specificity of both reagents. Tested compound groups were nitrophenols and methoxyphenols, sterols and sugars, dicarboxylic acids and hydroxylated polycyclic aromatic hydrocarbons. It was found that MTBSTFA-derivates produce characteristic fragmentation patterns presenting mainly the fragments [M](+), [M-57](+) and [M-131](+), of which [M-57](+) is generally dominant on the mass spectrogram. BSTFA-derivates mainly show the fragments [M](+), [M-15](+) and [M-89](+) whereof the molecular ion [M](+) is generally dominant. It was also found that steric hindrance and molecular mass play a very important role in the choice of the best suited derivatization reagent: compounds with sterically hindered sites derivatized with MTBSTFA produce very small analytical responses or no signal at all, and compounds with high molecular mass produce no characteristic fragmentation pattern when derivatization is performed with BSTFA. It was also found that MTBSTFA-derivatization facilitates separation of isomer analytes, suggesting its choice in combination to semi-polar columns, whilst BSTFA seems better for sterically hindered compounds. Findings were confirmed with applications of both reagents to biological and environmental matrices (urine and atmospheric aerosols).

Detection of benzodiazepines and other psychotropic drugs in human hair by GC/MS

For the detection of psychotropic drugs in human hair we collected hair obtained from 21 corpses that died from an overdose of legal or illicit drugs. These persons were known to have taken psychotropic drugs prior to their death as determined by post-mortem toxicological analysis in blood. After washing, cutting hair into segments of 3 cm, pulverization, enzymatic hydrolysis with beta-glucuronidase/arylsulfatase and solid phase extraction, drugs were identified by GC/MS. For quantification of flunitrazepam we used its metabolite amino-flunitrazepam; for oxazepam and lorazepam we used the hydrolysed forms of the corresponding drugs. In the hair of 21 subjects tested we found in 20 cases nordazepam, in 15 cases diazepam, in 15 cases oxazepam and in eight cases flunitrazepam with maximal concentrations of 1.8, 2.2, 3.4 and 9.5 ng/mg hair respectively. In addition to these compounds, in subject 11 to 21 we also analyzed for and detected amitriptyline (seven positive), carbamazepine (eight positive), lormetazepam (three positive) and lorazepam (one positive) and found maximal concentrations of 106.0, 13.5, 29.0 and 4.9 ng/mg hair respectively. The comparison of hair analysis versus post-mortem blood and tissues analysis of all the drugs studied shows that in 40 cases, where a positive result was found in blood, the corresponding drug could also be detected in hair in 37 cases. Our results show that hair testing is complementary to classical post-mortem analysis in forensic toxicology.

Ethyl Glucuronide Determination: Head Hair versus Non-Head Hair

INTRODUCTION In previous studies, hair analysis of ethyl glucuronide (EtG), a non-volatile, water-soluble, direct metabolite of ethanol, was shown to be adequate for the detection of social and chronic excessive alcohol consumption. As in some cases scalp hair is not available, the analysis of hair from alternative anatomical sites becomes of interest. AIMS In this study, hair samples from head, beard, chest, armpit, stomach, pubis, arms and legs from 32 subjects were analyzed when available, in order to compare the EtG concentrations and to study if the cut-offs used for head hair could be used for non-head hair. METHODS EtG was determined by GC/MS in negative chemical ionization mode using EtG-d5 as internal standard, after extraction by solid phase extraction using Oasis MAX columns and pentafluoropropionic anhydride (PFPA) derivatization. RESULTS The results showed that in the cases of negative findings in head hair (EtG < 7 pg/mg), in 7 out of 12 cases negative results could also be found in non-head hair. The five others were positive, due to a positive EtG finding in pubic hair. In 20 cases of positive EtG results for head hair, in all cases positive results could also be found in non-head hair. CONCLUSIONS In conclusion, although preliminary results indicate a clear trend regarding the accordance between EtG results in head hair and non-head hair, interpretation of non-head hair results remains to be carefully done for pubic hair, for which often higher concentrations have been found.

Temporal variations of concentrations of currently used pesticides in the atmosphere of Strasbourg, France

Atmospheric samples have been collected in Strasbourg between April 18 and May 29, 2007 and were analyzed for 71 current-use pesticides, of which 38 were detected. Average concentrations ranged from 0.09 ng m(-3) for Fenarimol to 110.42 ng m(-3) for Dimethachlor, which was slightly higher than the concentrations reported from other, comparable agricultural regions. Significant temporal variations were observed for 30 pesticides, and for most of them it could be shown that these were linked to time, temperature or atmospheric pressure. In several cases this helped to identify pesticide application just before or at the beginning of the sampling period, or ongoing treatment. Humidity, in contrast to previous reports, could not be linked to these variations. For the other 8 pesticides, only very little temporal variations were observed. Generally, these concentrations were low (less than 1 ng m(-3)), and it was assumed that they are not in use in Alsace at present.

Quantitative determination of ethyl glucuronide in sweat.

In this study, ethyl glucuronide (EtG), a specific metabolite of ethanol, was for the first time detected in sweat after alcohol consumption by human volunteers. Sweat was collected using a sweat patch (PharmChek). After collection, chemicals accumulated on the patch were extracted with water and extracts were purified by solid phase extraction. EtG was determined by gas chromatography with mass spectrometric detection in negative chemical ionization mode. In parallel, the amount of sodium deposited on the patch was determined by capillary electrophoresis and used as a correction factor to calculate the volume of sweat accumulated on the patch and, hence, the concentration of EtG in sweat. The EtG sweat concentration observed ranged from 1.7 to 103.0 μg/L for alcohol consumption from 38.0 to 154.6 g equivalent pure ethanol. No EtG was detected in subjects who did not consume alcohol. Our results demonstrate that after ethanol consumption, EtG is detectable in sweat collected using a sweat patch. The simultaneous determination of sodium allows the estimation of the volume of sweat accumulated on the patch and to calculate the concentration of EtG in sweat. This represents the first quantitative determination of a xenobiotic in sweat collected using a sweat patch. This study suggests that EtG determination in sweat could represent an interesting alternative to urine or serum analysis for the control of abstinence of patients included in treatment programs.

Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in human hair by gas chromatography–negative chemical ionization mass spectrometry

The study describes the determination of mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs), metabolites of PAHs, in human hair. Twelve selected OH-PAHs from two to four rings, generally determined in urine analysis, were investigated as markers of human exposure to PAHs. Following hydrolysis of hair specimens of 50-300 mg with 1M NaOH, OH-PAHs were extracted using dichloromethane and submitted to an optimized derivatization with (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride. Compounds were then analyzed using gas chromatography-negative chemical ionization mass spectrometry (GC-NCIMS). The average inter-day and intra-day variability was 12% and 17%, respectively. The average recovery was 52% and the limits of detection and quantification ranged from 20 and 66 pmol/g for 1-OH-phenanthrene (i.e., 3.9 and 12.8 pg/mg) to 311 and 1030 pmol/g for 2-OH-benzo(c)phenanthrene (i.e., 75.9 and 251 pg/mg). The influence of hair washing with water as decontamination step, and enzymatic treatment (beta-glucuronidase) to hydrolyze conjugated derivatives were also tested. The application of the developed method to the analysis of 30 hair specimens (17 from non-smoker and 13 from smoker volunteers) demonstrated inter-individual qualitative and quantitative variations. According to the easiness of hair sampling and based on the extended detection windows provided by hair analysis, this method is proposed as a new promising tool for the assessment of human chronic exposure to PAHs.

Internet suicide guidelines: Report of a life-threatening poisoning using tobacco extract

BACKGROUND Severe nicotine intoxication occurred in a patient after ingestion of a tobacco extract made from a recipe found on a freely available Internet site. OBJECTIVES To determine the levels of nicotine and cotinine in the plasma of a patient who tried to commit suicide by drinking a highly concentrated tobacco extract. CASE REPORT A 67-year-old man tried to commit suicide by following guidelines found on an Internet site. He soaked 300 grams of tobacco for 3 days in water, evaporated most of the extract, and drank the rest of it. He felt sick immediately, with the following signs: respiratory depression, hypothermia, hypersalivation, bradycardia, and myoclonic jerks. Soon after the ingestion he vomited most of the extract. Toxicological analysis revealed potentially life-threatening nicotine and cotinine serum concentrations. Surprisingly, nicotine peak levels (322 microg/L) and cotinine peak levels (9092 microg/L) were reached more than 3 h after ingestion of the extract. Estimated nicotine and cotinine half-lives were 200 min and 1185 min, respectively. Treatment consisted of gastric lavage, ventilation, and monitoring of vital functions. The patient recovered and was discharged from the Emergency Department 4 days later without sequelae. CONCLUSION Nicotinergic intoxication is not always easy to recognize, and without clues from the patient and the toxicologic analysis, might well have been missed in the present case.

Quantification of dextropropoxyphene and its metabolite by HPLC in hair of overdose cases

Hair samples taken from 11 persons suspected to have died from an overdose of legal or illicit drugs were analysed. These hair samples were selected, because classical post-mortem toxicological investigations in blood revealed the presence of dextropropoxyphene (PPX) and its major metabolite norpropoxyphene (NPPX). For the hair analysis, hair strands were cut into segments of about 3 cm, washed with water and acetone, dried and pulverised. An aliquot of about 40 mg of those segments was incubated with acetate buffer pH 4.0 and beta-glucuronidase/arylsulfatase. After liquid-liquid extraction with hexane-3-methylbutanol (99:1), drugs were identified and measured by HPLC using piritramide as an internal standard. Preliminary assays showed that the limit of detection for PPX is below 1.0 ng/mg hair, the limit of detection for the metabolite NPPX being significantly higher (1.5 ng/mg). GC/MS, usually the method of choice for this kind of determination, was not chosen, because of the thermolability of PPX and its unspecific mass spectrum. From the hair of 11 persons, 24 segments were prepared and processed. Our results show that ten persons were found positive for PPX; moreover, when regarding the 24 segments, only three were found negative. PPX and NPPX concentrations were detected at maximal concentrations of 26.4 and 71.0 ng/mg hair respectively. Considering the ratio of PPX to NPPX for each person, we found more PPX than NPPX for three persons, whereas for seven persons we found more of the metabolite than its parent drug.

Time resolved analysis of risperidone and 9-hydroxy-risperidone in hair using LC/MS-MS

Risperidone (RSP) is a second generation anti-psychotic drug used for the treatment of schizophrenia and anxiety disorders. In the last decades, clinical applications of hair analysis have received an increasing attention, because of its wide surveillance window. In this work, we describe a simple and fast method for detection and quantification of RSP and its major metabolite, 9-OH-risperidone (9-OH-RSP), in human hair. The validated method (cv of interday precision, intraday precision and accuracy<15%, r(2) of the calibration curves>0.98, limit of detection (LOD) was 0.90 pg/mg hair (RSP) and 1.52 pg/mg hair (9-OH-RSP), the lower limit of quantification (LLOQ) were 1.8 and 4.56 pg/mg, respectively, extraction yield were 86.9% for RSP and 86.7% for 9-OH-RSP) was successfully applied to quantify both substances in the hair of psychiatric patients treated with RSP. After washing, pulverisation, incubation in an ultrasound bath and liquid/liquid extraction of the hair samples, quantification was performed using LC/MS-MS in selected reaction monitoring mode with methaqualone as internal standard. Concentrations for RSP and its major metabolite ranged from 36 to 4765 pg/mg and from 14 to 57 pg/mg, respectively in the different hair segments. These preliminary results indicate a better relationship between the administered dose and hair concentration for 9-OH-RSP than for the parent drug. Furthermore, the RSP/9-OH-RSP ratio varied from 1 to 83.

Frontline Testing for Drugs of Abuse

Many non-instrument based immunoassays have been developed and commercialized due their ease of use and rapid results. At least 15 different tests are available, such as the Frontline rapid screen for drugs of abuse. Since the early 1990s Boehringer Mannheim (now Roche Diagnostics) has developed and commercialized these single parameter test strips for qualitative and semiquantitative analysis of cannabinoids, cocaine, opiates, benzodiazepines and amphetamines in urine specimens. The test for methadone is currently under development (1999). The test principle and an evaluation of the different Frontline tests will be presented in this chapter.