Milena M. Madry

Systematic investigation of the incorporation mechanisms of zolpidem in fingernails

Nails are attracting increasing interest in forensic toxicology as an alternative to hair. The goal of this study was to systematically investigate the incorporation of drugs in fingernails after single drug dose, exemplified for zolpidem. Fingernail samples from ring fingers were collected one week before, and then 24 h and weekly after intake for a period of three to five months. Hair samples were taken six weeks after intake. Nail specimens were pulverized and extracted with methanol (internal standard: zolpidem-D6 ) under sonication. Extracts were analyzed by a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method, which was developed and validated for this study. The lower limit of quantification (LLOQ) for a 5-mg sample was 0.1 pg/mg nail. Zolpidem was detected continuously in fingernail clippings. The mean window of detection of zolpidem in fingernail clippings was 3.5 months. Unwashed nail specimens taken 24 h after intake showed the highest zolpidem concentrations indicating external contamination by sweat. External contamination experiments revealed that zolpidem could be incorporated in fingernails by sweat to such an extent that it remained irremovable by daily hygiene. Averagely 3 months after intake a concentration peak was reached, suggesting outgrowth of the nail part which had been formed while the drug circulated in blood. Hair concentrations were higher than the maximum nail concentrations. Pigmented hair contained more zolpidem than non-pigmented hair from the same strand. From all these results it can be concluded, that fingernail clippings may represent a useful alternative and/or complementary matrix in cases of, for example, drug-facilitated sexual assault or monitoring of constant consumption behavior.

Excessive lead burden among golden eagles in the Swiss Alps

Fragments from lead ammunition pose a poisoning risk for predators like golden eagles that scavenge on non-retrieved carcasses or offal left behind by hunters. Three golden eagles were found in the Swiss Alps with an acute lead poisoning. To investigate whether the few cases of lead-poisoned golden eagles are exceptional events or whether a substantial proportion of the Alpine golden eagle population is affected by lead at sublethal levels, we measured body burdens in golden eagles from Switzerland in comparison to eagle owls from the same area and to their respective prey. These two raptor species differ in their food as eagle owls feed on live-caught prey. Lead levels in soft tissues were significantly higher in golden eagles (median 1.14 μg g−1 dry weight in liver, 0.99 μg g−1 in kidney) than in eagle owls (0.14 and 0.23 μg g−1). Bones of golden eagles contained 10 times more lead (median of 12.45 μg g−1 dry weight) than owl bones (1.28 μg g−1), which represent substantially higher levels than previously reported for golden eagles. Bones of prey of both golden eagles and eagle owls had low lead concentrations. In order to investigate whether the sublethal lead of golden eagles originates from ammunition or from generic environmental contamination, we examined lead isotope ratios. Lead isotope signatures of golden eagle bones were very similar to those of ammunition, but differed from the signatures of bones of their prey, eagle owls and soil. Isotope signatures did not change with increasing bone lead concentration in golden eagles or any other group examined. These findings indicate that in the Alps, most golden eagles take up lead from spent ammunition in carcasses or their offal in sublethal quantities throughout their life and a few in lethal quantities leading to acute lead poisoning.

Retrospective monitoring of long-term recreational and dependent cocaine use in toenail clippings/scrapings as an alternative to hair.

BACKGROUND Toenails were assessed as an alternative matrix to hair for retrospective monitoring of cocaine consumption of recreational and dependent users. Results/methodology: Toenail clippings, scrapings and hair samples from recreational and dependent cocaine users were analyzed for cocaine and metabolites. Dependent users displayed significantly higher concentrations in hair and toenail samples compared to recreational users. Cocaine abstinence could be monitored in hair and toenail samples. One postmortem fingernail was analyzed in layers to investigate the cocaine and metabolite concentration profile. Highest concentrations were observed in the dorsal layer, being indicative of contamination. CONCLUSION Having led to comparable results, toenails may be an alternative for retrospective monitoring of cocaine consumption/abstinence. Hair should remain the first choice for assessment of temporal evidence of drug intake.

Hair analysis for opiates: hydromorphone and hydrocodone as indicators of heroin use

BACKGROUND Identification of external contamination is a challenge in hair analysis. This study investigates metabolite ratios of hydromorphone to morphine and hydrocodone to codeine as indicators to distinguish contamination from heroin use provided that hydromorphone/hydrocodone intake is excluded. RESULTS Hair samples after external contamination with street heroin proved to be negative for hydromorphone/hydrocodone. Hair samples from individuals with suspected street heroin use/contamination or opiate medication were analyzed for 6-monoacetylmorphine, morphine, acetylcodeine, codeine, hydromorphone and hydrocodone, and metabolite ratios of hydromorphone to morphine and hydrocodone to codeine were assessed. Hair samples from individuals with medicinal heroin/morphine/codeine use displayed significantly higher metabolite ratios than those with suspected street heroin use/contamination. CONCLUSION Hydromorphone/hydrocodone are solely formed during body passage. Thus, metabolite ratios can be used to distinguish morphine/heroin use from external contamination.

Systematic assessment of different solvents for the extraction of drugs of abuse and pharmaceuticals from an authentic hair pool

Hair analysis has been established as a prevalent tool for retrospective drug monitoring. In this study, different extraction solvents for the determination of drugs of abuse and pharmaceuticals in hair were evaluated for their efficiency. A pool of authentic hair from drug users was used for extraction experiments. Hair was pulverized and extracted in triplicate with seven different solvents in a one- or two-step extraction. Three one- (methanol, acetonitrile, and acetonitrile/water) and four two-step extractions (methanol two-fold, methanol and methanol/acetonitrile/formate buffer, methanol and methanol/formate buffer, and methanol and methanol/hydrochloric acid) were tested under accurately equal experimental conditions. The extracts were directly analyzed by liquid chromatography-tandem mass spectrometry for opiates/opioids, stimulants, ketamine, selected benzodiazepines, antidepressants, antipsychotics, and antihistamines using deuterated internal standards. For most analytes, a two-step extraction with methanol did not significantly improve the yield compared to a one-step extraction with methanol. Extraction with acetonitrile alone was least efficient for most analytes. Extraction yields of acetonitrile/water, methanol and methanol/acetonitrile/formate buffer, and methanol and methanol/formate buffer were significantly higher compared to methanol. Highest efficiencies were obtained by a two-step extraction with methanol and methanol/hydrochloric acid, particularly for morphine, 6-monoacetylmorphine, codeine, 6-acetylcodeine, MDMA, zopiclone, zolpidem, amitriptyline, nortriptyline, citalopram, and doxylamine. For some analytes (e.g., tramadol, fluoxetine, sertraline), all extraction solvents, except for acetonitrile, were comparably efficient. There was no significant correlation between extraction efficiency with an acidic solvent and the pka or log P of the analyte. However, there was a significant trend for the extraction efficiency with acetonitrile to the log P of the analyte. The study demonstrates that the choice of extraction solvent has a strong impact on hair analysis outcomes. Therefore, validation protocols should include the evaluation of extraction efficiency of drugs by using authentic rather than spiked hair. Different extraction procedures may contribute to the scatter of quantitative results in inter-laboratory comparisons. Harmonization of extraction protocols is recommended, when interpretation is based on same cut-off levels.

Acute and Chronic Lead Exposure in Four Avian Scavenger Species in Switzerland

Despite irrefutable evidence of its negative impact on animal behaviour and physiology, lethal and sublethal lead poisoning of wildlife is still persistent and widespread. For scavenging birds, ingestion of ammunition, or fragments thereof, is the major exposure route. In this study, we examined the occurrence of lead in four avian scavengers of Switzerland and how it differs between species, regions, and age of the bird. We measured lead concentration in liver and bone of the two main alpine avian scavengers (golden eagle Aquila chrysaetos and bearded vulture Gypaetus barbatus) over the entire area of the Swiss Alps and two of the main avian scavengers occurring in the lowlands of Switzerland (red kite Milvus milvus and common raven Corvus corax). Of those four species, only the bearded vulture is an obligate scavenger. We found that lead burdens in the two alpine avian scavengers were higher than those found for the same species elsewhere in Europe or North America and reached levels compatible with acute poisoning, whereas lead burdens of the two lowland avian scavengers seemed to be lower. Several golden eagles, but only one red kite with abnormally high bone lead concentrations were found. In all four species, a substantial proportion of birds had elevated levels which presumably represent recent (liver lead levels) or past (bone lead levels) uptake of sublethal doses of lead.