Method for confirmation of synthetic corticosteroids in doping urine samples by liquid chromatography-electrospray ionisation mass spectrometry.

In this study, we report on the development of a method to confirm simultaneously nine of the most commonly abused synthetic corticosteroids in urine based on liquid chromatography-electrospray ionisation mass spectrometry. A considerable simplified sample preparation procedure, including liquid-liquid phase extraction with Extrelut-NT3 columns, provided both excellent sample purification and high overall recoveries. Complete HPLC separations were obtained on a reversed-phase column with 1 mM ammonium acetate-acetonitrile (60:40, v/v) as mobile phase. Mass spectral acquisition was done in the negative ion, and selected ion monitoring modes to identify the drugs with at least three characteristic ions. Detection limits were determined at < or =1 ng/ml and the confirmation limits at 1 to 5 ng/ml.

Nutritional supplements as a source for positive doping cases?

We report the findings of the analysis of 75 different nutritional supplements bought through the internet. Seven products (all from the class of prohormones) contained other hormone substances than indicated on the labels, and two further products contained ephedrine and caffeine without a clear indication on the labels.

Techniques for analytical testing of unconventional samples.

Forensic scientists have long detected the presence of drugs and their metabolites in biological materials using body fluids such as urine, blood and/or other biological liquids or tissues. For doping analysis, only urine has so far been collected. In recent years, remarkable advances in sensitive analytical techniques have encouraged the analysis of drugs in unconventional biological samples such as hair, saliva and sweat. These samples are easily collected, although drug levels are often lower than the corresponding levels in urine or blood. This chapter reviews recent studies in the detection of doping agents in hair, saliva and sweat. Sampling, analytical procedures and interpretation of the results are discussed in comparison with those obtained from urine and blood samples.

Salvia divinorum: an hallucinogenic mint which might become a new recreational drug in Switzerland.

Salvia divinorum Epling & Jativa is an hallucinogenic mint traditionally used for curing and divination by the Mazatec Indians of Oaxaca, Mexico. Young people from Mexican cities were reported to smoke dried leaves of S. divinorum as a marijuana substitute. Recently, two S. divinorum specimens were seized in a large-scale illicit in-door and out-door hemp plantation. Salvinorin A also called divinorin A, a trans-neoclerodane diterpene, was identified in several organic solvent extracts by gas chromatography-mass spectrometry. The botanical identity of the plant was confirmed by comparing it to an authentic herbarium specimen. More plants were then discovered in Swiss horticulturists greenhouses. All these data taken together suggest that many attempts exist in Switzerland to use S. divinorum as a recreational drug. This phenomenon may be enhanced because neither the magic mint, nor its active compound are banned substances listed in the Swiss narcotic law.

Is there a place for hair analysis in doping controls?

The actual antidoping control rules applied in sports (as established by the International Olympic Committee and the International Sport Federations) state that a positive case is chemically established by the unequivocal detection of a forbidden parent molecule and/or any of its metabolite(s) in urine, no matter the amounts which were administered and when the drug was taken. Screening is accomplished most of the time by using GC-MS procedures. These have been optimized to detect most if not all of the forbidden compounds which are put on a list. Recently, attempts have been made on scalp hair to demonstrate the value of this matrix as a possible means for differentiating between therapeutic use and doping abuse. In particular, GC-mass selective detector and GC-high resolution MS were successfully applied to treated animals and body-builders for anabolic agents (steroids and beta-2-agonists) at high sensitivity detection (low ng/g level). Naturally occurring molecules, like testosterone and its metabolites, could also be differentiated from their synthetic counterparts. Positive cases are more often challenged in courts and retrospectivity in time of the drug(s) intake is becoming an important issue for evaluating the responsibility of the person. This is can be based on hair analyses if the drugs have been taken at regular intervals. Stimulants and narcotics are often used in sports like drug of abuse in the ordinary social contexts. On the other hand, anabolic agents, when taken to improve the physical performances, follow complex regimens with the mixing of various formulas and dosages. Scalp hair references ranges for these as well as for endogenous substances still wait to be established statistically for competing, well-trained athletes. The incorporation rate into blond or gray hair is poorer than that of dark colored hair raising the question of individuals equality against the controls, a very important matter of concern for the sport's governing bodies. The frequency of hair cutting and short hair cuts necessary to gain speed in specific sports like swimming are other critical factors. On the other hands, irregular hair growth, associated with the washout effect through multiple washing and staining processes over expanded time intervals can cause concentrating or diluting effects. So far, a minority of prohibited substances could be detected in scalp hair with the sensitivity and specificity required in the context of the sport's activities. From the above, clear limitations of the usefulness of hair analysis in doping control analysis are obvious until a lot more data relevant to this particular field have been collected.

Method for quantification of morphine and its 3- and 6- glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine in human blood by liquid chromatography-electrospray mass spectrometry for routine analysis in forensic toxicology.

Simultaneous determination of opiates and their glucuronides in body fluids has a great practical interest in the forensic assessment of heroin intoxication. A selective and sensitive method for quantification of morphine and its 3- and 6-glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine (6-MAM) based on liquid chromatography-electrospray ionisation mass spectrometry is described. The drugs were analysed in human autopsy whole blood after solid-phase extraction on a C8 cartridge. The separation was performed on an ODS column in acetonitrile (analysis time 15 min). For the quantitative analysis, deuterated analogues of each compound were used as internal standards. Selected-ion monitoring was applied where the molecular ion was chosen for quantification. The limits of quantification were 0.5 ng/ml for morphine and 6-MAM and 1 ng/ml for the 6-glucuronide of morphine, codeine-6-glucuronide and codeine and 5 ng/ml for the 3-glucuronide of morphine.

Exit association-mediated suicide: toxicologic and forensic aspects.

The Swiss German chapter of the Exit Association provides conditional assistance to individuals wishing to end their own lives. The Exit Association advocates death with dignity and fights for the right to freely choose the timing of one's own death. According to the Swiss criminal code (articles 114 and 115), altruistic assistance to suicide is not punishable. Active euthanasia is punished by imprisonment. An individual commits active euthanasia if he or she is driven by honorable motives (e.g., pity) and causes the death of another person wishing to die who seriously and insistently requests such action. Based on our information, the preparation for suicide and its completion relies on a well-defined protocol. First, the candidate's eligibility for Exit Association assistance is verified. The candidate then writes a farewell declaration that explicitly confirms the will to terminate his or her own life. A written report describes the events during the suicide procedure. Depending on the circumstances, the investigative judge requests a forensic autopsy and toxicologic analyses. The results of the forensic investigations conducted in the cases presented here are in agreement with the scenario described in the reports of the Exit Association, namely, suicide by massive ingestion of pentobarbital.

[Brief history of recent hemp cultivation in Switzerland and subsequent medico-legal problems resulting from hemp cultivation]. / Bref historique de la culture récente du chanvre en Suisse et problèmes médico-légaux engendrés par cette culture.

In March 1995, a decision about cultivation of cannabis was issued by the Swiss Federal Offices of Public Health, Police and Agriculture in order to satisfy the growing interest of farmers and other people in hemp farming. It pointed out that 1)... each hemp plant contains THC and must be therefore considered a drug, 2)... no permission is required for those who grow hemp without the intention to produce drugs ... meaning that the choice of the plant variety was not restricted to those which are characterized by a low THC concentration and grown in a few countries belonging to the European Union. Claiming that natural hemp must contain significant amounts of THC and thanks to the Swiss legislation, areas dedicated to hemp cultivation develop considerably. Most hemp plants which are submitted to our laboratories by the police for THC quantification belong to the drug-type. Nowadays, a great deal of goods (food and beverages, cosmetics, drugs) made of hemp are marketed in Switzerland. Strong suspicions exist however that several of these products could be used as a screen for the illegal market of cannabis. For instance, despite financial support from the state, fiber hemp cultivation remains unsuccessful. No advantage with regard to seed productivity, edible seed and essential oils qualities and yields have been found for drug hemp over fiber hemp by agricultural research stations up to now. Several clues about the possible illicit use of hemp goods rich in THC, especially hemp tea made of flower tops and "therapeutic" pillows filled with cannabis exist. Recently, two Federal edits were issued in order to restrict the selling of hemp seedlings and of hemp foods and beverages to those containing only low amounts of THC. However, the marketing of hemp plants used for decorating remains free partly explaining the recent success of these "beautiful" plants. Broadly speaking, the Swiss and European legislations about hemp have approached mutually during the last years.

2C-B: a new psychoactive phenylethylamine recently discovered in Ecstasy tablets sold on the Swiss black market.

This study sought to identify, by means of several analytical methods (GC-MS, HPLC-DAD, CE-DAD, FTIR, and NMR), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), which was found in two sets of tablets obtained from the Swiss black market. Unequivocal identification of 2C-B was only achieved by a combination of mass spectrometric and NMR analysis. Quantitation of 2C-B was performed by HPLC-DAD and CE-DAD. The amounts of 2C-B found in the tablets (3-8 mg) were in the range of the minimum quantity required to induce the effects characteristic of this drug.

Car crash after massive ingestion of digoxin and midazolam.

In a case where a 32-year-old man lost control of his vehicle, urine and blood samples were taken 6 h after the crash for toxicological investigations. In the hospital, the driver admitted consumption of some drugs, in particular digoxin and midazolam just before the crash which corresponded to the results of blood analyses. Toxicological findings indicated the presence of digoxin at 12.9 ng/ml and midazolam at 7 ng/ml in the blood. These results suggested that at the moment of the crash digoxin and midazolam blood levels were in the range of toxic and therapeutic concentrations, respectively. Therefore the respective roles of the drugs in the impairment of the ability to drive at the moment of the crash is discussed.

Comparison of different immunoassays and GC-MS screening of benzodiazepines in urine.

A total of 53 urine samples were tested by different immunoassay methods and by gas chromatography/mass spectrometry to determine repeatability of the different methods and to assess whether the immunoassays performed on samples obtained from elderly patients of the emergency section could be considered as reliable enough for identifying a benzodiazepine consumption. Repeatability was excellent for GC/MS and good for immunoassays. The specificity was not different for the three immunoassays (96%). The sensitivity varied from 36, 64 to 75% for OnLine, RIA Immunalysis and RIA DPC, respectively. An other difference between immunoassays and GC/MS was the ability of GC/MS to detect lorazepam and low concentrations of benzodiazepines whereas immunoassays did not.

[Ecstasy--the status in French-speaking Switzerland. Composition of seized drugs, analysis of biological specimens and short review of its pharmacological action and toxicity]. / Ecstasy--la situation en Suisse romande. Composition des saisies, analyse des échantillons biologiques et brève revue de son action pharmacologique et de sa toxicité.

Methylenedioxy-N-methylamphetamine (MDMA, "Ecstasy") and other related phenylethylamines are nowadays used extensively in Western Switzerland at dance clubs and raves. There is a widely held belief among teenagers and misusers that ecstasy is safe. In the last years however, an increasing number of reports of MDMA-related deaths has been reported. Acute clinical toxicity problems following MDMA ingestion include hyperthermia, convulsions and arrhythmias. There is also growing concern that these phenylethylamines are neurotoxic and cause long-term damage to serotonineric nerve terminals in animal brains. Qualitative analyses by GC-MS of street samples of ecstasy showed that only a part of them contain MDMA or related phenylethylamines (MDA, MDEA, MBDB and 2C-B). Most of them were mixed with caffeine and an excipient (sugars or polyols [e.g. mannitol]). Amphetamine cut with caffeine and other drugs (e.g. testosterone), stimulants (e.g. pseudoephedrine) and other drugs unrelated to stimulants and phenylethylamines (e.g. LSD, chloroquine, vasodilators) were also detected. Quantitative determinations performed by HPLC-DAD or EC-DAD reveal huge fluctuations in the amount of active substance(s) per tablet. MDMA and related compounds display unique psychoactive properties, acting as a stimulant and inducing feelings of empathy. The effects of MDMA intake are very likely the results of the large release of serotonin (5-HT) in the synaptic cleft, of the inhibition of the re-uptake inactivation of 5-HT and of the inhibition of a key-enzyme involved in the biosynthesis of 5-HT. Forensic investigations performed at our institute showed significant blood levels of MDMA, MDEA and MDA in samples drawn from people suspected of driving under the influence of psychoactive drugs. Up to now, no death could be attributed to MDMA intoxication only because our analyses always revealed the additional presence of toxic amounts of other psychoactive drugs (e.g. opiates, cocaine). Our study shows that because of the variable composition of ecstasy tablets, unpredictable types and amounts of drugs may be taken by MDMA misusers. Moreover, there is considerable concern that traffic accidents may be caused by MDMA-abusers. MDMA intake could result in severe intoxication and even death, especially when combined with other types of drugs.

Drugs and alcohol among suspected impaired drivers in Canton de Vaud (Switzerland).

Epidemiological and analytical laboratory records concerning living drivers suspected of driving under the influence of drug (DUID) during the 13 years period ranging from 1982 to 1994 were examined. This study included 641 records. 551 men (86%) and 90 women (14%). The average age of the drivers was 27 +/- 7 years (n = 636, minimum 18 and maximum 74) and the 18-30 interval age range was overrepresented (80%) in this population sample. A traffic accident had occurred in 254 (40%) of the records, 273 (43%) drivers were suspected of DUID during police controls and 95 (15%) drivers were suspected of DUID because of their erratic driving. One or more psychoactive drugs were found in 92.8% of the samples. In these records, cannabinoids were found in 57%, opiates in 36%, ethanol in 36%, benzodiazepines in 15%, cocaine in 11%, methadone in 10% and amphetamines in 4%. The majority (58%) of cases presented two or more drugs in biological samples, thus indicating a high incidence of potential interactions between drugs. This observation was specially relevant for methadone and methaqualone. We conclude that police suspicion about drivers under influence highly correlated with positive results for drug analyses in biological samples.

Detection of human growth hormone doping in urine: out of competition tests are necessary.

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2-50 ng hGH/l. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 i.u. every day). The u-hGH excretion after treatment showed dramatic increases of 50-100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and beta 2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.

Intra- and interindividual variation in flank gland secretions of free-ranging shrewsCrocidura russula.

Individual differences in flank gland secretions were examined among males of the monogamous shrewCrocidura russula during the breeding and nonbreeding seasons. Gas chromatography was used to measure intra- and interindividual variation of flank gland secretions of free-ranging shrews from different populations. The number of compounds detected by gas Chromatographic analyses was correlated with body mass, flank gland size, and the presence of blood parasites in individual shrews. Very few compounds were detected from the flank gland area of juvenile males. After they reached sexual maturity, however, the number of compounds detected from the flank gland secretions increased significantly. At the beginning of the reproductive season 48 different compounds were detected from male flank gland secretions. In the middle of the breeding season 70 compounds were detected, while only 11 compounds were detected during the nonbreeding season. Few compounds were common to all males. There were more volatile compounds in the flank gland secretions of males in the beginning of the breeding season than later in the breeding season. Males from the same population had fewer differences in the elution profile of compounds than males from different populations indicating that individuals from a distinct population have similar elution profiles of compounds and that each population has its own type of elution profile. No correlations were found between the number of compounds detected by gas chromatography for each male and the male's body mass or flank gland size. Blood parasites (trypanosomes,Trypanosoma crocidurae) were found in only three of 30 males investigated.

[Cocaine and alcohol: an explosive cocktail!]. / Cocaïne et alcool: un cocktail explosif!

Cocaine is a powerful reinforcer that has become a popular drug of abuse. Cocaine inhibits the reuptake of dopamine into presynaptic terminals, resulting in an overstimulation of the dopaminergic system. When cocaine and alcohol are taken together, cocaine is partially converted by a liver carboxylesterase to its ethyl homolog called ethylcocaine. In 1991, several studies demonstrated that ethylcocaine is a pharmacologically active metabolite of cocaine. Ethylcocaine was shown to be even more potent than cocaine in mediating lethality. In the USA, prevalent studies have indicated that concurrent and simultaneous abuse of cocaine with alcohol were a very common practice. In Switzerland, as far as we know, epidemiologic studies have not been carried out yet. In our laboratory, radioimmunoassays mainly sensitive to benzoylecgonine (an inert metabolite of cocaine and ethylcocaine) are used to detect cocaine abusers. Positive results are always confirmed by combined gas chromatography (GC) and mass spectrometry (MS). Recently, cocaine, ethylcocaine, methyl ecgonine and ethyl ecgonine were detected by GC/MS in urine from three people arrested after erratic driving. Their blood alcohol concentration was 2.89, 0.98 and 1.26 g/kg. In Switzerland, the influence of alcohol, cocaine and ethylcocaine on the incidence of accidents, overdose and death is not known and requires further studies. Because morbidity and mortality are exacerbated by concurrent use of alcohol and cocaine, physicians should discourage their hooked patients from consuming this deadly mixture.

Pharmacognosy of Mimosa tenuiflora (Willd.) Poiret.

A chemical investigation of the bark of Mimosa tenuiflora (Willd.) Poiret, performed in our laboratory, allowed the isolation and identification of three new triterpenoid saponins (mimonosides A, B and C), three steroid saponins (3-O-beta-D-glucopyranosyl campesterol, 3-O-beta-D-glucopyranosyl stigmasterol and 3-O-beta-D-glucopyranosyl beta-sitosterol) together with lupeol, campesterol, stigmasterol and beta-sitosterol. The three new triterpenoid saponins were subjected to in vitro biological tests (immunomodulation and proliferation) using different animal and human cells in culture. The results of these tests contribute to explain the traditional use of this plant material.

Detrimental effect of alcohol intoxication on severity of injuries in male traffic accident victims: a cross-sectional study.

A cross-sectional emergency room study was performed among traffic accident victims aged 16 to 75 years in order to investigate the prevalence of alcohol intoxication (blood alcohol concentration (BAC greater than or equal to 0.8 g/kg) and its potential detrimental effect on injury severity. A BAC greater than or equal to 0.8 g/kg was present in 29% of the male patients. Intoxicated patients had a similar propensity to require inpatient hospital care (65 versus 60%) but had significantly longer hospital stays than the non-intoxicated patients (36 versus 20 days; p less than 0.05), observations which may be explained by the higher prevalence of serious injuries (head and internal injuries) among the intoxicated cohort. This study supports and emphasizes the detrimental effect of alcohol intoxication on the severity of injuries in traffic accident victims, and reinforces the need for further legal and public health measures in preventing alcohol-related traffic accidents.

[Assessment of the intake of opiates (heroin, morphine, codeine and ethylmorphine) by the analysis of intermediate metabolites in the urine: which are the criteria to adopt?]. / Caractérisation de la prise d'opiacés (héroïne, morphine, codéine et éthylmorphine) par l'intermédiaire de l'analyse d'urine: quels critères adopter?

This report presents the different strategies for identifying heroin users. The criteria allowing a clear distinction between an abuse of heroin and a lawful consumption of opiates are deeply discussed. Reliable and sensitive analytical methods are now available for forensic opiate testing. The detection of 6-mono-acetylmorphine (MAM) indicates that heroin was administered within 24 hours or less of specimen collection. In the absence of MAM or after consumption of several opiates, the relative ratios of morphine, codeine and eventually ethylmorphine must be known in order to determine which opiate(s) was (were) taken. A total amount of opiates of less than 0.3 mg/l very often precludes any characterization of the ingested drug(s). Here we have to point out that forensic opiate testing should be done carefully. Interpretation of results requires more than detection of opiates or morphine alone, irrespective of the number of techniques used.