Toxicologic analysis of 35 drugs in post mortem human blood samples with focus on antihypertensive and antiarrhythmic drugs.

Antiarrhythmic and antihypertensive drugs are frequently encountered in post mortem analysis, and the question may arise as to whether they were administered in therapeutic doses, and if they were taken in accidental, intentional, or suicidal overdose scenarios. Therefore, a novel analytical method was developed and validated for the quantification of 35 drugs with toxicological relevance, including antihypertensive and antiarrhythmic drugs (ajmaline, amlodipine, amiodarone, atenolol, bisoprolol, carvedilol, clonidine, desethylamiodarone, diltiazem, donepezil, doxazosin, dronedarone, esmolol, flecainide, lercanidipine, lidocaine, metoprolol, nebivolol, nimodipine, pindolol, prajmaline, propafenone, propranolol, sotalol, urapidil, and verapamil), as well as other medications commonly found in combination (sildenafil, tadalafil, atorvastatin, clopidogrel, dapoxetine, memantine, pentoxifylline, rivastigmine, and ivabradine). The method enables simultaneous identification and quantification in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation exhibited excellent linearity across the concentration range for all analytes. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 9 % and 10 %, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated very high sensitivity, with limits of detection (LOD) as low as 0.01 ng/ml and limits of quantification (LOQ) as low as 0.04 ng/ml. Extraction recovery exceeded 57.5 % for all analytes except atenolol, and matrix effects were <17 % for all analytes except pindolol. Processed sample stability evaluations revealed consistent results with acceptable deviations for all analytes. In addition, the method was specifically tested for the use in post mortem analysis. The applicability of our method was demonstrated by the analysis of two authentic human autopsy blood samples.

Chiral analysis of amphetamine, methamphetamine, MDMA and MDA enantiomers in human hair samples.

A novel analytical method was developed for the simultaneous quantification of the R/S-enantiomers of amphetamine, methamphetamine, MDA and MDMA in hair samples using liquid chromatography-tandem mass spectrometry (LC-MS-MS). This method involved a straightforward derivatization step with dansyl chloride and the use of a chiral column, enabling the separation and quantification of all eight enantiomers in a single analysis. The method exhibited excellent linearity across a concentration range of 0.03-3.00 ng/mg for each enantiomer. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 6% and 9%, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated high sensitivity, with limits of detection (LOD) below 8 pg/mg and limits of quantification (LOQ) below 19 pg/mg for all analytes. Extraction recovery exceeded 79%, and matrix effects were minimal for all analytes. Processed sample stability evaluations revealed consistent results with deviations below 11% for all analytes. Application of the method to 32 authentic human hair samples provided valuable insights into amphetamine use patterns, allowing differentiation between medical amphetamine consumption and illicit use based on enantiomeric composition. Additionally, the method detected co-use of methamphetamine, MDA or MDMA in some samples, highlighting its applicability in drug monitoring and real-life case scenarios within a forensic institute. This innovative analytical approach offers a sensitive and selective method for enantiomeric differentiation of amphetamine, methamphetamine, MDA and MDMA in human hair samples, providing a valuable tool for forensic and clinical investigations.

Morphine concentrations in fatalities after palliative treatment of acute burn injury.

The evaluation of a morphine concentration in postmortem blood is routine for a forensic toxicologist. We here report three fatal cases where we found high morphine concentrations with 7.96, 4.30, and 5.82 mg/l in femoral blood that have to be estimated as unusually high. All these individuals died due to severe burn injuries and obtained morphine in the context of their palliative care in the last hours of their lives. According to the autopsy results, the cause of death in case 1 was burn disease with burns of about 90% of the body surface area (BSA), case 2 burn trauma, and case 3 burn shock. Besides morphine, propofol, fentanyl, sufentanil, midazolam, diazepam, lorazepam, cefazolin, and rocuronium were detected in femoral blood. The findings fitted well with the detailed clinical documentation. Further evidence of therapeutic concentrations of quetiapine, duloxetine, and melperone could be matched to preexisting medication of the individuals. Physiologically based pharmacokinetic modelling (PBPK) was applied, developed for the intravenous administration of morphine, to find an explanation for the high morphine concentrations in femoral blood. Quantification of morphine in body fluids and tissue was performed to calculate morphine tissue concentration ratios to the morphine concentration in femoral blood. The presented cases show that pharmacokinetic simulations can reflect decreased renal clearance and decreased hepatic metabolism in general. However, this prediction is not sufficient to explain the high morphine concentrations in femoral blood measured here. It can be assumed that burn shock in particular leads to altered pharmacokinetics, namely decreased distribution of morphine.

Stability of PEth 16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2, and 18:1/18:1 in authentic whole blood samples (at room temperature).

Phosphatidylethanol (PEth) is a direct alcohol biomarker to monitor individuals' drinking behavior that has gained recognition in clinical and forensic settings. The increasing application of the marker makes investigation of the preanalytical handling necessary, and analyte stability deserves major attention. This study was conducted to investigate the change of six PEth homologues' concentration, stored in authentic samples of EDTA blood over a course of 30 days at room temperature (n = 62). The stability criterion of concentration being ±15% of the original concentration was fulfilled at mean for 10, 3, 2, 5, 2, and 7 days for PEth 16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2, and 18:1/18:1, respectively. Regarding all homologues, there were samples in which concentration had declined by >15% or by more than the critical difference on day 1. Overall, calculated concentration declines were very inhomogeneous, with inter-sample differences of 43%-73% after 30 days. PEth 16:0/18:2, 16:0/20:4, and 18:0/18:2 declined to a greater extent than PEth 16:0/18:1. Blood alcohol concentration was measured >0.1‰ in 25 samples. Three of the six samples that exceeded 115% of initial concentrations were positive for blood alcohol. The study results add to the previously reported information on PEth stability and firstly look at six homologues in comparison. Due to the high scatter of stability among the samples and the observed poor stabilities in some, it can be concluded that transportation and storage times, especially if cooling cannot be provided, must be kept short. If analyzing from dried blood, spotting should preferably be conducted at the site of sampling.

Beta-Glucuronidase Activity: Another Source of Ethyl Glucuronide.

Numerous classes of endogenous and xenobiotic compounds are conjugated to uridine-5'-diphospho (UDP)-alpha-D-glucuronic acid which is catalyzed by human UDP-Glucuronosyltransferases (UGTs). The resulting beta-D-glucuronides can be hydrolyzed to ß-D-glucuronic acid and the corresponding aglycone in a configuration retaining manner by beta-glucuronidases (GUSBs), which are widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants. This study investigates GUSBs' activity in the presence of ethanol (0-70% by volume) using different ß-D-glucuronides (phenolphthalein-ß-D-glucuronide, 4-nitrophenol-ß-D-glucuronide, morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide) as substrates. It was found that ß-D-ethyl glucuronide (EtG), which is a minor UGT-derived metabolite of ethanol in man and one of the most frequently used biomarkers of alcohol consumption today, builds up from all investigated ß-D-glucuronides by means of GUSBs in the presence of ethanol. The glucuronyl transfer reaction, which was neither detected in the absence of ethanol nor in absence of GUSBs, is minor at ethanol concentrations which are commonly observed in blood and tißues after consumption of alcoholic beverages, but predominant at higher concentrations of ethanol. In spite of in vitro characteristics, our observations point to an additional biochemical path and another source of EtG, which should be further evaluated in the context of alcohol biomarker applications. The detection of EtG in several settings independent from of human UGT-metabolism (e.g. EtG post post-collection synthesis in E.coli coli-contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the described mechanism.

PEth 16:0/18:1 and 16:0/18:2 after consumption of low doses of alcohol-A contribution to cutoff discussion.

Phosphatidylethanol in blood has gained recognition as a direct alcohol biomarker. Although different cutoffs have been suggested, there is no consensus for differentiating abstinence from alcohol consumption. In this study, 75 participants (72% female) consumed 20 g of ethanol on three consecutive evenings. Blood was sampled on each following day and PEth 16:0/18:1 and 16:0/18:2 were determined. PEth 16:0/18:1 ranged from 8.9-21.5, 8.7-19.3, and 8.8-42.3 ng/ml and PEth 16:0/18:2 from 8.7-31.7, 9.0-39.3, and 9.4-43.0 ng/ml after the respective days of ethanol consumption. PEth 16:0/18:1 yielded a sensitivity of 25%, 45%, and 49% and PEth 16:0/18:2 of 40%, 61%, and 68% for the consumption days, respectively (cutoff 10 ng/ml). PEth 16:0/18:1 reached >20 ng/ml in five samples overall. Sensitivity of PEth 16:0/18:2 > 20 ng/ml was better with 35% after the three drinking days. Overall, PEth 16:0/18:1 was >35 ng/ml in one sample and PEth 16:0/18:2 in three samples. Significantly, more women had PEth 16:0/18:1 > 10 ng/ml after the third day of consuming 20 g of alcohol (p = 0.02) and PEth 16:0/18:2 > 10 ng/ml after the second (p = 0.023) and the third (p = 0.002) consumption, which can be led back to the higher blood alcohol concentration women reach after consuming the same alcohol amount as men. Although the response rates of PEth to alcohol uptake are subject to strong interindividual differences, results suggest that PEth cutoff should be lowered for better detection of consumption of low to medium amounts of alcohol. Furthermore, it is advantageous to analyze both PEth 16:0/18:2 and 16:0/18:1.

Phosphatidylethanol in patients with liver diseases of different etiologies: Analysis of six homologues and comparison with other alcohol markers.

BACKGROUND AND AIMS: Phosphatidylethanol (PEth) is a direct alcohol biomarker. Aim of the study was to evaluate the performance of six homologues of PEth in comparison to other alcohol markers in patients with liver diseases. METHODS: The study included 234 patients with liver disease, who gave statements about alcohol consumption during the three months prior to the doctor's appointment. Ethylglucuronide in urine (uEtG) and in hair (hEtG) and carbohydrate-deficient transferrin (CDT) were analyzed in addition to PEth. RESULTS: Of all patients 47% stated to have drunk alcohol during the past three months. UEtG, hEtG and CDT showed a sensitivity of 29% and a specificity of 92% together for ingestion of at least two standard drinks (24 g) per week. With PEth 16:0/18:1 in addition, sensitivity increased to 59%. For consumption in the last week uEtG's sensitivity and specificity was 28% and 100%, respectively. PEth's was 75% and 93%. When looking at patients who consumed at least two standard drinks per week during the past three months and of which a hair sample could be obtained, hEtG's sensitivity was 37% and specificity 90%. PEth had a sensitivity of 53% and specificity of 100%. Quotients of PEth 16:0/18:1 with 16:0/18:2, 16:0/20:4 and 18:0/18:2 were smaller when alcohol had been consumed more recently. CONCLUSION: Despite the rather poor overall sensitivity of alcohol biomarkers in this study, PEth showed best sensitivity for all time periods of alcohol consumption.

Investigating the use of PEth, CDT and MCV to evaluate alcohol consumption in a cohort of homeless individuals- A comparison of different alcohol biomarkers.

In a cohort including individuals with suspected high alcohol consumption, the concentrations of the indirect alcohol biomarkers carbohydrate-deficient transferrin (CDT) and mean corpuscular volume (MCV) and the direct alcohol biomarker phosphatidylethanol (PEth) were investigated. Blood alcohol concentration (BAC) was analysed as a marker for acute alcohol ingestion. In addition to questions about subjective alcohol consumption behaviour, 147 homeless persons underwent a physical examination with blood sampling. BAC, PEth, CDT and MCV were determined in the blood samples. Special focus was on the comparison of PEth and CDT for indicating excessive alcohol consumption. BAC was measured above 0.1‰ in 39 blood samples (0.1-2.5‰, median 0.75‰). PEth was detected in all of them. Overall, PEth was positive (≥10 ng/ml) in 104 samples (71%) (11-5687 ng/ml, median 650 ng/ml) with 68 (46%) being above the cut-off for excessive alcohol consumption (210 ng/ml). In 26 subjects PEth was the only positive alcohol biomarker. CDT was ≥ 1.7% in 66 cases (47%) (1.8-22.2%, median 4.4%) and ≥ 2.5% in 52 (35%) cases. MCV was elevated (≥95 fl) in 58 subjects (39%). CDT and PEth concentrations showed a significant positive correlation (spearman's correlation coefficient ρ = 0.77, p < 0.001). PEth concentrations were significantly higher in samples that were also CDT positive than solely PEth positive (p = 0.004). PEth did not indicate excessive alcohol consumption (< 210 ng/ml) in eight and two cases in which CDT was ≥ 1.7% and ≥ 2.5%, respectively. On the other hand, CDT was< 1.7% and< 2.5% in ten and 18 cases, respectively, in which PEth was above cut-off for excessive alcohol consumption. Taking the self-reports of the participants into consideration, PEth's sensitivity for detecting excessive alcohol consumption was 100% (10 ng/ml) and 94% (210 ng/ml) and CDT's was 88% (1.7%) and 75% (2.5%). In individuals of the investigated cohort unusually high concentrations of the alcohol consumption markers PEth and CDT were quantified, which proves the assumption of chronic excessive alcohol consumption in parts of the cohort. PEth was the marker that was positive most often and was more sensitive for excessive alcohol consumption than CDT.

Analysis of six different homologues of phosphatidylethanol from dried blood spots using liquid chromatography-tandem mass spectrometry.

Phosphatidylethanol (PEth) is a direct biomarker for alcohol consumption consisting of a fraction of different ethanol-modified, homologue phospholipids. The aim of this study was to validate an ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantitate six different homologues of PEth (16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2, and 18:1/18:1) from dried blood spots (DBSs). DBSs were prepared volumetrically (20 µL of whole blood) and extracted with 1 mL of methanol (0.02 ng/µL internal standards). PEth homologues were separated on a BEH C18 column (2.1 × 150 mm, 1.7 µm) using methanol and ammonium acetate buffer (25 mM) in a 7 min isocratic run. Multiple reaction monitoring mode was used for the detection of PEth and the internal standards. Calibrators (10-1000 ng/mL) and quality controls (40, 400, and 700 ng/mL) were prepared from spiked whole blood; external control samples were obtained from proficiency testing schemes. After a comprehensive validation of the method, quantitative patterns of the different homologues were investigated in PEth positive samples (n = 57) from patients in a transplant setting. Satisfactory chromatographic separation, sensitive detection, and reliable quantification of the PEth homologues in DBSs can be achieved using the liquid chromatography-tandem mass spectrometry (LC/MS/MS) procedure. Validation results, including accuracy, linearity, recovery, matrix effects, and in-process stability, complied with international standards, and the analytical performance of the procedure was not affected by the hematocrit of the blood samples. Different quantitative patterns of the investigated PEth homologues were observed in authentic samples from liver transplant patients. This method will enable the study of the kinetics of six PEth homologues simultaneously and investigate the meaning of the homologues' distribution in individuals.