Identification of furosemide in hair in a post‐mortem case by UHPLC‐MS/MS with guidance on interpretation

Testing for drugs in hair raises several difficulties. Among them is the interpretation of the final concentration(s). In a post‐mortem case, analyses revealed the presence of furosemide (12 ng/mL) in femoral blood, although it was not part of the victim's treatment. The prosecutor requested our laboratory to undertake an additional analysis in hair to obtain information about the use of furosemide. A specific method was therefore developed and validated to identify and quantify furosemide in hair by UHPLC‐MS/MS. After decontamination of 30 mg of hair, incubation in acidic condition, extraction with ethyl acetate, the samples were analyzed by UHPLC‐MS/MS. Furosemide was found in the victim's hair at 225 pg/mg. However, it was not possible to interpret this concentration due to the absence of data in the literature. Therefore, the authors performed a controlled study in two parts. In order to establish the basis of interpretation, several volunteers were tested (four after a single 20 mg administration and twenty‐four under daily treatment). The first part indicated that a single dose is not detectable in hair using our method. The second part demonstrated concentrations ranging from 5 to 1110 pg/mg with no correlation between dosage and hair concentrations. The decedent's hair result was interpreted as repeated exposures. In the case of furosemide analysis, hair can provide information about its presence but cannot give information about dosage or frequency of use.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS: Application to the determination of MDMA after low Ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C₁₈ 5 μm, 2.1 mm × 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A^® (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 °C in NaOH 1 M before liquid–liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1–50 ng/mL in blood and urine; in the range 5–500 pg/mg for MA, MDMA, MDEA and MBDB, and 20–500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T + 12 h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D + 8) and scalp hair at day 60 (D + 60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Nail analysis for the assessment of caffeine abuse in Northwest China: A population-based study

Caffeine is the most widely consumed psychoactive agent worldwide and has the potential for abuse, but studies monitoring caffeine abuse in China are scarce. This study aims to estimate the prevalence of caffeine abuse in northwest China and investigate the correlation between caffeine and other drugs in hair and nails using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Fingernail clippings were collected from 376 participants in northwest China to detect caffeine and 13 other illicit psychoactive drugs and their metabolites. Paired hair and nail samples were collected from 39 participants to investigate the correlation between caffeine and other drugs in hair and nails. The samples were decontaminated, pulverized, and extracted by a high-throughput nail sample preparation method and analyzed by UPLC-MS/MS. The results showed a risk of caffeine abuse in northwest China, with concentrations ranging from 0.43 to 10.6 ng/mg for healthy volunteers, 0.49–246 ng/mg for caffeine abusers, and 0.25–363 ng/mg for drug addicts in community rehabilitation centers. Caffeine was detected together with other illicit psychoactive drugs and their metabolites. Furthermore, positive detection correlations were found between hair and nail samples. This study provides a current perspective on caffeine abuse in northwest China and demonstrates the practical use of UPLC-MS/MS for the simultaneous detection of caffeine and 13 illicit psychoactive drugs and their metabolites in hair and nails. The results highlight the potential of nails as a supplementary matrix when hair samples are unavailable and emphasize the need for handling caffeine carefully given its potential for abuse.     

Analytical Challenge in Postmortem Toxicology Applied to a Human Body Found into a Lake after Three Years Immersion

The body of a 30‐year‐old woman was found in Como lake at a depth of about 120 meters in her own car after 3 years of immersion. The aim of this study was to evaluate psychoactive drugs as well as alcohol biomarkers in biological matrices. The following analyses were initially performed: GC‐MS systematic toxicological analysis on biological fluids and tissues; GC‐MS analysis of drugs of abuse on pubic hair; direct ethanol metabolite determination in pubic hair by LC‐MS/MS. After 7 years, the samples, that had been stored at ?20°C, were re‐analyzed and submitted to an LC‐MS/MS targeted screening method, using multiple reaction monitoring mode. These analyses detected citalopram (150–3000 ng/mL), desmethylcitalopram (50–2300 ng/mL), clotiapine (20–65 ng/mL), and ethyl glucuronide (97 pg/mg). The methods showed an acceptable reproducibility, and the concentrations of citalopram and desmethylcitalopram calculated through the two analytical techniques did not significantly differ in biological fluids.     

Evaluation of the IDS One-Step™ ELISA kits for the detection of illicit drugs in hair

This work presents the validation of a new immunological assay, the One-Step™ enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC–MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 °C. A 100 μL aliquot was collected and evaporated to dryness in presence of 100 μL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 μL of the “sample and standard diluent” solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC–MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC–MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC–MS. Twenty were found positive for cannabis (THC: 0.10–6.50 ng/mg), 21 for cocaine (0.50–55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20–11.60 ng/mg, MOR: 0.20–8.90 ng/mg, codeine (COD): 0.20–5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22–17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step™ ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in workplace drug testing or driving license regranting, especially when many samples have to be tested with a high rate of negative samples, as ELISA is an easy and high-throughput method.     

Alprazolam and Zolpidem in Skeletal Tissue of Decomposed Body Confirms Exposure

In several medico‐legal cases, bone samples analysis may provide the only source of toxicological information. This case study reports the analysis of a human bone specimen, belonging to a 46‐year‐old man, found 3 months after his death due to cervical–thoracic injuries in a motorcycle accident. Bone specimen was the only available material for toxicological analysis, among few skull hair and rotten skin. Analysis was performed by a newly developed and validated ultra‐high‐pressure liquid chromatography–mass spectrometry/mass spectrometry (UHPLC‐MS/MS) method, following simple and efficient sample pretreatment. The results were in accordance with the man's medical record: Alprazolam and zolpidem were found at 2.2 and 5.4 ng/g of bone, respectively. Both these drugs were prescribed to the deceased.     

Quantitative Analysis of Gamma‐Hydroxybutyrate at Endogenous Concentrations in Hair using Liquid Chromatography Tandem Mass Spectrometry

Abstract: A method capable of quantifying endogenous concentrations of gamma‐hydroxybutyrate (GHB) in human head hair was developed and validated using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Hair was digested under alkaline conditions, and GHB was isolated using liquid–liquid extraction. LC/MS/MS was performed using atmospheric pressure chemical ionization in the negative mode, multiple reaction monitoring, and deuterated internal standard (GHB‐D₆). Linearity was observed between 0.1 and 100 ng/mg GHB (R² = 1.000). The limits of detection and quantitation in human hair were 0.2 and 0.4 ng/mg, respectively. Accuracy at 2 ng/mg and 10 ng/mg was determined to be 97% and 94%, and intra‐assay CVs at these concentrations were 5.2% and 7.4% (n = 4). Beta‐hydroxybutyrate (BHB), alpha‐hydroxybutyrate, gamma‐butyrolactone, and 1,4‐butanediol did not produce an interference, and there was negligible ion suppression or enhancement from the matrix.     

Ethyl Glucuronide Concentration in Hair of Detainees: A Preliminary Study

Through the measurement of ethyl glucuronide in hair (hETG), it is possible to assess chronic alcohol abuse over time. In this paper, we present a study on hETG in Italian prison inmates. Analyses were performed by LC-MS according to a previously published method. Results were evaluated using the cut-offs established by the Society of Hair Testing. Positives samples (ETG > 30 pg/mg) accounted for 6% of all subjects, with concentrations ranging from 42 pg/mg up to 270 pg/mg, abstinent subjects (ETG < 7 pg/mg) accounted for 88%, and moderate alcohol consumption (7 < ETG < 30 pg/mg) for 6% of the subjects. No females displayed ETG values above 30 pg/mg. Among positive samples, only two subjects did not declare heavy alcohol consumption and were found strongly positive at 210 and 270 pg/mg. To the best of our knowledge, this represents the first study on ETG hair concentration on prison inmates.     

Deaths Involving Methylenedioxypyrovalerone (MDPV) in Upper East Tennessee

Two deaths involving 3, 4‐methylenedioxypyrovalerone (MDPV) are reported. MDPV is a synthetic cathinone stimulant found in “bath salts” with neurological and cardiovascular toxicity. Biological specimens were analyzed for MDPV by GC/MS and LC/MS. A White man was found dead with signs of nausea and vomiting after repeatedly abusing bath salts during a weekend binge. Femoral venous blood and urine had MDPV concentrations of 39 ng/mL and 760 ng/mL. The second fatality was a White man with a history of drug and bath salt abuse found dead at a scene in total disarray after exhibiting fits of anger and psychotic behavior. Femoral venous blood and urine had MDPV concentrations of 130 ng/mL and 3800 ng/mL. The blood and urine MDPV concentrations are within the reported recreational concentration ranges (blood 24–241 ng/mL and urine 34–3900 ng/mL). Both decedents’ deaths were attributed to relevant natural causes in a setting of MDPV abuse.     

Detection of meperidine and its metabolites in the hair of meperidine addicts

The presence of meperidine and its metabolites in the hair of meperidine addicts was investigated using GC–MS (EI, PCI). Meperidine and its three metabolites – normeperidine, N-methoxy meperidine and acetyl normeperidine, were found in hair samples from addicted subjects. Methods for the simultaneous determination of meperidine and its metabolites by GC–MS-SIM were also established for human hair samples. After the addition of d₄-meperidine as an internal standard, hair samples weighing 5 mg were incubated in 0.1 M HCl at 45°C overnight, and the resulting digests were extracted with ether. The recoveries were greater than 80%, with coefficients of variation (CVs) between 4.48 and 8.31%. The calibration curves for meperidine and normeperidine in hair were linear over a concentration range of 1 to 500 ng per mg of hair, with correlation coefficients of r=0.9990 and r=0.9992, respectively. Values less than 0.25 ng/mg of hair were cut off. Hair samples obtained from 60 drug addicts were analyzed using this method, and the content of meperidine and normeperidine was determined to be 103±130 and 117±143 ng/mg, respectively. Sectional analysis revealed that meperidine was present and stable in hair for at least 20 months, but normeperidine content at the level of the hair root was higher compared to the tip of the hair shaft. The results also revealed that there was a correlation between the subject’s drug abuse history and the distribution of drug along the hair shaft, and between the doses of meperidine and drug content presented in hair.     

GC/MS、GC/NPD法检测毛发中的氯胺酮

目的采用液-液萃取、衍生化和GC/MS、GC/NPD方法,进行毛发中氯胺酮定性定量分析。方法选择4-苯基丁胺为内标,毛发样本用NaOH、HCl及芳基硫酸酯酶/β-葡萄糖醛酸酶等3种方式进行水解,再进行衍生化后,采用GC/MS和GC/NPD方法定性定量分析。对不同水解和衍生化条件以及提取溶剂进行比较优化,并考察方法精密度、稳定性和检出限。结果方法的提取回收率大于95%,精密度和样品稳定性良好,日内和日间标准偏差小于6%;采用GC/NPD和GC/MS直接分析毛发中的氯胺酮,检出限为0.2ng/mg和2.0ng/mg,线性范围为10.0～250.0ng/mg,相关系数均大于0.99;采用酰化衍生化后分析,GC/NPD和GC/MS检出限分别提高至0.1ng/mg和0.2ng/mg。结论该方法回收率高、检测限低,可以用于毛发中氯胺酮的定性定量分析检验。     

Distribution of 6-monoacetylmorphine and morphine in head and pubic hair from heroin-related deaths

There is limited published data to aid interpretation of analytical findings from hair analysis. The aim of the study was to collate 6-monoacetylmorphine (6-AM) and morphine concentrations in head and pubic hair from heroin users and to propose reference ranges and relate these to the amount of heroin used. The ratio of morphine-to-6-AM was also investigated. A total of 82 head hair samples divided into 173 segments of various lengths and 15 pubic hair samples were collected at postmortem from heroin users. The hair was analysed using a previously published method. A statistical evaluation demonstrated that in head hair, the lower, middle and upper concentration ranges of 6-AM were 0.1–0.9, 0.9–12.5 and 12.5–154.1 ng/mg and those of morphine were 0.1–0.8, 0.8–6.0 and 6.0–36.3 ng/mg. In pubic hair, the lower, middle and upper concentration ranges of 6-AM were 0.2–0.5, 0.5–2.3 and 2.3–18.2 ng/mg and those of morphine were 0.2–0.4, 0.4–2.4 and 2.4–13.3 ng/mg. The morphine-to-6-AM ratio showed a large variation. The ratio tended to decrease from proximal to distal segments. The statistical results suggest low, middle and high concentration ranges which we propose can be used for estimating the amount of heroin consumed into corresponding low or occasional, regular or habitual and heavy or excessive drug use. The ratio of morphine-to-6-AM showed great variation and did not support the proposal that a ratio less than 0.77 is needed to prove ingestion of heroin.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS. Application to the determination of MDMA after low ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C(18) 5 microm, 2.1 mm x 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 degrees C in NaOH 1M before liquid-liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1-50 ng/mL in blood and urine; in the range 5-500 pg/mg for MA, MDMA, MDEA and MBDB, and 20-500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T+12h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D+8) and scalp hair at day 60 (D+60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Report of Increasing Overdose Deaths that include Acetyl Fentanyl in Multiple Counties of the Southwestern Region of the Commonwealth of Pennsylvania in 2015–2016

Acetyl fentanyl is a Schedule I controlled synthetic opioid that is becoming an increasingly detected “designer drug.” Routine drug screening procedures in local forensic toxicology laboratories identified a total of 41 overdose deaths associated with acetyl fentanyl within multiple counties of the southwestern region of the state of Pennsylvania. The range, median, mean, and standard deviation of blood acetyl fentanyl concentrations for these 41 cases were 0.13–2100 ng/mL, 11 ng/mL, 169.3 ng/mL, and 405.3 ng/mL, respectively. Thirty‐six individuals (88%) had a confirmed history of substance abuse, and all but one case (96%) were ruled multiple drug toxicities. This report characterizes this localized trend of overdose deaths associated with acetyl fentanyl and provides further evidence supporting an alarmingly concentrated opiate and opioid epidemic of both traditional and novel drugs within this region of the United States.     

HS-SPME-GC/MS法检测尿液及毛发中苯丙胺类毒品

目的采用顶空固相微萃取（HS-SPME）、GC/MS分析方法,对生物样品中苯丙胺（AM）、甲基苯丙胺（MAM）、3,4-亚甲二氧基苯丙胺（MDA）和3,4-亚甲二氧基甲基苯丙胺（MDMA）4种苯丙胺类毒品进行定性定量分析。方法在碱性和饱和盐处理状态下,采用100μm聚二甲基硅氧烷（PDMS）萃取纤维,于顶空瓶中进行生物样品AM、MAM、MDA、MDMA 4种毒品萃取,以2-甲基苯乙胺为内标,经气-质联用选择离子检测（GC/MS/SIM）模式进行定性定量分析。对HS-SPME条件优化,对方法的精密度、准确度和检出限进行测定。结果 AM、MAM、MDA、MDMA 4种毒品尿液中的最低检出限为5ng/mL,毛发中的最低检出限为0.5ng/mg。尿液中线性关系范围为0.05μg/mL~5μg/mL,r〉0.991,回收率为82%~108%,RSD为2.6%~6.1%（n=5）;毛发中线性关系范围为5ng/mg~500ng/mg,r〉0.992,回收率为80%~113%,RSD（%）为1.4%~6.8%（n=5）。结论 HS-SPME-GC/MS各项定量参数符合分析要求。该方法简单、灵活、经济、快速、无溶剂,适用于生物检材中该类毒品的分析。     

A Fatality Following Ingestion of the Designer Drug Meta‐Chlorophenylpiperazine (mCPP) in an Asthmatic—HPLC‐MS/MS Detection in Biofluids and Hair

A 20‐year‐old man, a cocaine addict and regular ecstasy user, with a medical history of allergic asthma died after ingesting half a tablet earlier the same day. The white tablet, stamped with a “smiling sun” logo looked very much like an ecstasy tablet and was sold as such. He experienced a severe asthma attack just after ingesting the half tablet and it evolved over the next few hours into fatal cardiorespiratory arrest. Biological samples, taken after embalming, were analyzed by high‐performance liquid chromatography tandem mass spectrometry (HPLC‐MS/MS). Analysis revealed meta‐chlorophenylpiperazine (mCPP) in concentrations of 45.8 mg in a similar tablet obtained later from the drug dealer, 5.1 ng/mL in the bile, 0.3 ng/g in the liver, 15.0 ng/mL in the urine, and its absence in a hair sample (<0.02 ng/mg), which indicated he was not a regular user (whereas strong concentrations of MDMA and cocaine were found in the hair). Interrogated by the police after his arrest, the dealer said that he had sold the victim and for the very first time two tablets with the same “smiling sun” logo. The tablet used for analysis was from the same brand as the one ingested by the victim. The autopsy excluded other causes of death, while the histological analyses showed a large number of polynuclear eosinophils in the bronchial walls, confirming the asthmatic pathology. None of the other organs examined (larynx, liver, heart, adrenal glands, and kidneys) showed any distinctive signs, and in particular no inflammatory infiltrate. The death was the result of an asthma attack in an asthmatic person, violently decompensated following ingestion of approximately 20 mg of mCPP.     

Separation and Detection of Smokeless Powder Additives by Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry (UPLC/MS/MS),

A reversed phase gradient ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method has been developed for the analysis of smokeless powders. A total of 20 different components were separated by UPLC and detected by MS/MS in multiple reaction monitoring (MRM) mode. These compounds included diphenylamines, centralites, nitrotoluenes, nitroglycerin, and various phthalates. Simultaneous positive and negative electrospray ionization (ESI) was used along with negative atmospheric pressure chemical ionization (APCI) to detect all compounds in a single analysis. Analysis times were under 8 min with a gradient of 10–73% organic at a flow rate of 0.500 mL/min. With this method, ultraviolet and MRM limits of detection ranging from 0.08 to 2.6 ng and 0.4–64 ng injected were achieved. Commercially available smokeless powders were also extracted with methylene chloride and characterized using the developed UPLC/MS/MS method. The procedure permits the determination of compositional differences between different brands as well as lot‐to‐lot variations.