Marie Katrine Klose Nielsen

Simultaneous screening and quantification of 52 common pharmaceuticals and drugs of abuse in hair using UPLC–TOF-MS

An UPLC-TOF-MS method for simultaneous screening and quantification of 52 drugs in hair was developed and validated. The selected drugs represent the most common classes of pharmaceuticals and drugs of abuse such as amphetamines, analgesics, antidepressants, antipsychotics, benzodiazepines, cocaine, ketamine and opioids. Hair samples were extracted with methanol:acetonitrile:ammonium formate (2 mM, 8% acetonitrile, pH 5.3) overnight at 37 degrees C. The target drugs were separated and quantified using a Waters ACQUITY UPLC coupled to a Waters Micromass LCT Premier XE Time-of-Flight mass spectrometer. Total chromatographic run time was 17min. The data were treated with the MassLynx software ChromaLynx XS and QuanLynx for automated identification and quantification, respectively. The limits of detection ranged from 0.01 to 0.10 ng/mg using a 10-mg hair sample and the limit of quantification was 0.05 ng/mg for 87% of the analytes. A good linear behaviour was achieved for most of the analytes in the range from LOQ to 10 or 25 ng/mg except for the amphetamines. The method showed an acceptable precision and trueness, since the obtained CV and BIAS values were <or=25% for 81% of the analytes. The extraction recoveries for 92% of the analytes ranged between 84 and 106% and the extraction recoveries for all analytes were better than 60%. The method was applied to 15 autopsy hair samples from forensic investigations showing a wide abuse pattern of many pharmaceuticals and drugs of abuse within a period of less than three months. The present study demonstrated that the combination of accurate mass and retention time can provide good selectivity, which demonstrates that the TOF instrument is adequate for both screening and quantification purposes. Furthermore, it was shown that screening with the ChromaLynx XS software is less sensitive and selective for some analytes than the QuanLynx software, especially in low concentrations.

Validation of a method for the targeted analysis of 96 drugs in hair by UPLC–MS/MS

The method presented in this study allows the screening and quantification of 96 drugs, from different groups: opiates, amphetamines, hallucinogens, benzodiazepines, antihistamines, antidepressants, antipsychotics, barbiturates and other sedatives, muscle relaxants, etc. in hair. Drugs are extracted from 10mg of washed hair in 18h by a mixture of methanol:acetonitrile:ammonium formate (pH 5.3). Absolute recovery ranged from 70% to 106% for 75% of the analytes. The limits of detection in the low pg/mg range, may allow the detection of single dose drug exposure, with possible application in drug facilitated assaults (DFA); however, chronic use (compliance) can also be examined. The method has been fully validated for the drugs included in the study. The accuracy of the method was demonstrated by the analysis of certified authentic hair samples containing common drugs of abuse. The hair-method has broad potential as the measuring range is wide for the target analytes and new drugs can easily be added to the method due to the versatility of the extraction procedure and chromatographic system.

Simultaneous Determination of 25 Common Pharmaceuticals in Whole Blood Using Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of 25 common pharmaceuticals in whole blood. The selected pharmaceuticals represent the most frequently detected drugs in our forensic laboratory with basic properties such as analgesics, antidepressants, antihistamines, antihypertensives, antipsychotics and β-blockers. Whole blood samples were extracted with butyl acetate after adjusting pH with 2M NaOH. The target analytes were separated on a 100 × 2.1 mm ACQUITY BEH 1.7 µm C18 column by a formic acid/acetonitrile gradient elution using a Waters ACQUITY Ultra-Performance Liquid Chromatography system. Quantification was performed on a Waters tandem quadrupole ACQUITY TQD using multiple reaction monitoring in positive mode. The analytes were eluted within 11 min. The limit of quantification (LOQ) ranged from 0.002 to 0.01 mg/kg depending on the analyte. A good linear behavior was achieved for all analytes in the range from LOQ to 1.0 or 2.0 mg/kg blood. The absolute recoveries were between 55-87% for all compounds except norfluoxetine (44%). The method showed acceptable precision and accuracy for almost all analytes. Only unstable compounds like levomepromazine, methylphenidate, mirtazapine, norfluoxetine and zuclopenthixol deviated more. The method was successfully applied to more than 200 authentic blood samples within a year from forensic investigations.

Pre-analytical and analytical variation of drug determination in segmented hair using ultra-performance liquid chromatography- tandem mass spectrometry

Assessment of total uncertainty of analytical methods for the measurements of drugs in human hair has mainly been derived from the analytical variation. However, in hair analysis several other sources of uncertainty will contribute to the total uncertainty. Particularly, in segmental hair analysis pre-analytical variations associated with the sampling and segmentation may be significant factors in the assessment of the total uncertainty budget. The aim of this study was to develop and validate a method for the analysis of 31 common drugs in hair using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with focus on the assessment of both the analytical and pre-analytical sampling variations. The validated method was specific, accurate (80-120%), and precise (CV≤20%) across a wide linear concentration range from 0.025-25 ng/mg for most compounds. The analytical variation was estimated to be less than 15% for almost all compounds. The method was successfully applied to 25 segmented hair specimens from deceased drug addicts showing a broad pattern of poly-drug use. The pre-analytical sampling variation was estimated from the genuine duplicate measurements of two bundles of hair collected from each subject after subtraction of the analytical component. For the most frequently detected analytes, the pre-analytical variation was estimated to be 26-69%. Thus, the pre-analytical variation was 3-7 folds larger than the analytical variation (7-13%) and hence the dominant component in the total variation (29-70%). The present study demonstrated the importance of including the pre-analytical variation in the assessment of the total uncertainty budget and in the setting of the 95%-uncertainty interval (±2CVT). Excluding the pre-analytical sampling variation could significantly affect the interpretation of results from segmental hair analysis.

Evaluation of metabolite/drug ratios in blood and urine as a tool for confirmation of a reduced tolerance in methadone-related deaths in Denmark

BACKGROUND Methadone blood concentrations in fatal cases are highly variable and there is an appreciable overlap between therapeutic methadone concentrations and the concentrations detected in fatalities. As with other opioids, the background of these methadone-related deaths is unclear. The aim of this study was to investigate if short-time abstinence was contributing to the cause of death in methadone-related deaths by evaluation of the EDDP/methadone ratio in blood and urine. METHODS Samples of blood and urine were collected from 103 autopsy cases and analysed for the concentrations of methadone and its main metabolite EDDP. The cases were divided into three groups according to the cause of death: cases where methadone was the cause of death (N=67), cases where poly-drug poisoning including methadone was the cause of death (N=24) and cases where death were caused by other factors (N=12). Urine samples from 11 living persons receiving methadone were also included. RESULTS In general, a substantial overlap of the methadone concentrations in blood and urine was seen between the groups. There was a tendency of lower median EDDP/methadone urinary ratios in the methadone poisoning group (median: 1.0), poly-drug poisoning group (median: 0.94) and in the fatalities not related to methadone (median: 1.1) compared to the living subjects in methadone treatment (median: 1.6), although the differences were not significant. CONCLUSION It was not possible to reveal a possible abstinence period prior to death by using the EDDP/methadone ratio in blood and urine in methadone-related deaths.

Targeted analysis of 116 drugs in hair by UHPLC-MS/MS and its application to forensic cases

A multi-target method that can detect a broad range of drugs in human hair, such as hypnotics, anxiolytics, analgesics, benzodiazepines, antihistamines, antidepressants, antipsychotics, and anticonvulsants, was developed based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The drugs were extracted from 10 mg of washed hair by incubation for 18 h in a 25:25:50 (v/v/v) mixture of methanol/acetonitrile/2 mM ammonium formate (8% acetonitrile, pH 5.3). For 51% of the basic drugs, the lower limits of quantification (LLOQs) were in the range of 0.05-0.5 pg/mg, and the majority (98%) were ≤ 5 pg/mg. Linearity ranged from LLOQs to 100-500 pg/mg for all the basic drugs. For acid and neutral drugs, the LLOQs ranged from 0.4 to 500 pg/mg, and linearity ranged from LLOQs to 80-40 000 pg/mg. According to published reports on concentrations attained in single dose control studies, the present method is sensitive enough to detect single-dose drug exposure for many of the drugs. The accuracy was within 75-125% for the majority of drugs. Good precision was observed (relative standard deviations [RSD%] < 25%) for most of the compounds, including the prepared quality control (QC) hair samples. The method was applied to forensic cases and concentrations of rarely reported drugs in hair in 25 post-mortem forensic cases were presented. Hair concentrations of amisulpride, gabapentin, mianserin, mepyramine, orphenadrine, and xylometazoline have not been previously reported. Copyright

Evaluation of poly-drug use in methadone-related fatalities using segmental hair analysis

In Denmark, fatal poisoning among drug addicts is often related to methadone. The primary mechanism contributing to fatal methadone overdose is respiratory depression. Concurrent use of other central nervous system (CNS) depressants is suggested to heighten the potential for fatal methadone toxicity. Reduced tolerance due to a short-time abstinence period is also proposed to determine a risk for fatal overdose. The primary aims of this study were to investigate if concurrent use of CNS depressants or reduced tolerance were significant risk factors in methadone-related fatalities using segmental hair analysis. The study included 99 methadone-related fatalities collected in Denmark from 2008 to 2011, where both blood and hair were available. The cases were divided into three subgroups based on the cause of death; methadone poisoning (N=64), poly-drug poisoning (N=28) or methadone poisoning combined with fatal diseases (N=7). No significant differences between methadone concentrations in the subgroups were obtained in both blood and hair. The methadone blood concentrations were highly variable (0.015-5.3, median: 0.52mg/kg) and mainly within the concentration range detected in living methadone users. In hair, methadone was detected in 97 fatalities with concentrations ranging from 0.061 to 211ng/mg (median: 11ng/mg). In the remaining two cases, methadone was detected in blood but absent in hair specimens, suggesting that these two subjects were methadone-naive users. Extensive poly-drug use was observed in all three subgroups, both recently and within the last months prior to death. Especially, concurrent use of multiple benzodiazepines was prevalent among the deceased followed by the abuse of morphine, codeine, amphetamine, cannabis, cocaine and ethanol. By including quantitative segmental hair analysis, additional information on poly-drug use was obtained. Especially, 6-acetylmorphine was detected more frequently in hair specimens, indicating that regular abuse of heroin was common among the deceased. In conclusion, continuous exposure of methadone provide by segmental hair analysis suggested that reduced tolerance of methadone was not a critical factor among methadone-related fatalities. In contrast, a high abundance of co-ingested CNS depressants suggested that adverse effects from drug-drug interactions were more important risk factors for fatal outcome in these deaths.

Post-mortem quetiapine concentrations in hair segments of psychiatric patients — Correlation between hair concentration, dose and concentration in blood

Drug analysis in hair is useful when seeking to establish drug intake over a period of months to years. Segmental hair analysis can also document whether psychiatric patients are receiving a stable intake of antipsychotics. This study describes segmental analysis of the antipsychotic drug quetiapine in post-mortem hair samples from long-term quetiapine users by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The aim was to obtain more knowledge on quetiapine concentrations in hair and to relate the concentration in hair to the administered dose and the post-mortem concentration in femoral blood. We analyzed hair samples from 22 deceased quetiapine-treated individuals, who were divided into two groups: natural hair colour and dyed/bleached hair. Two to six 1cm long segments were analyzed per individual, depending on the length of the hair, with 6cm corresponding to the last six months before death. The average daily quetiapine dose and average concentration in hair for the last six months prior to death were examined for potential correlation. Estimated doses ranged from 45 to 1040mg quetiapine daily over the period, and the average concentration in hair ranged from 0.18 to 13ng/mg. A significant positive correlation was observed between estimated daily dosage of quetiapine and average concentration in hair for individuals with natural hair colour (p=0.00005), but statistical significance was not reached for individuals with dyed/bleached hair (p=0.31). The individual coefficient of variation (CV) of the quetiapine concentrations between segments ranged from 3 to 34% for individuals with natural hair colour and 22-62% for individuals with dyed/bleached hair. Dose-adjusted concentrations in hair were significantly lower in females with dyed/bleached hair than in individuals with natural hair colour. The quetiapine concentrations in post-mortem femoral blood and in the proximal hair segment, segment 1 (S1), representing the last month before death were also investigated for correlation. A significant positive correlation was observed between quetiapine concentrations in blood at the time of death and concentrations in S1 for individuals with natural hair colour (p=0.003) but not for individuals with dyed/bleached hair (p=0.31). The blood concentrations of quetiapine ranged from 0.006 to 1.9mg/kg, and the quetiapine concentrations in S1 ranged from 0.22 to 24ng/mg. The results of this study suggest a positive correlation of quetiapine between both concentrations in hair and doses, and between proximal hair (S1) and blood concentrations, when conditions such as hair treatments are taken into consideration.

Hair analysis in toxicological investigation of drug-facilitated crimes in Denmark over a 8-year period

Hair can serve as a specimen for identifying past drug exposure. Segmental hair analysis may differentiate a single exposure from chronic use. Consequently, segmental hair analysis is useful for disclosing a single drug ingestion, as well as for determining repeated exposures in drug-facilitated crimes (DFCs). This paper presents an overview of toxicological investigations that have used hair analysis in DFC cases from 2009 to 2016 in Denmark. Hair concentrations were determined for 24 DFC-related drugs and metabolites, including benzodiazepines and other hypnotics, antihistamines, opioid analgesics, antipsychotics, barbiturates, and illicit drugs from DFC cases. Drug detection in hair in DFC cases following a single or few intakes of chlorprothixene, codeine, diphenhydramine, oxazepam, oxycodone, promethazine, and phenobarbital is reported for the first time in forensic toxicology. A literature review on concentrations in the published DFC-related hair cases and on concentrations in hair of these substances after single and multiple doses is included. These cases demonstrate the value of segmental hair analysis in DFCs and facilitate future interpretations of results.