Quantification of Antibiotics in Patient Samples: State of the Art in Standardization and Proficiency Testing.

BACKGROUND: For many antibiotics, the convenient one-fits-all dosing regimen had to be abandoned. Owing to highly variable pharmacokinetics, therapeutic drug monitoring has become an indispensable prerequisite. It is based on a suitable measuring method, sample materials, and standardization. Appropriate quality control including external quality assessment (EQA) is essential. For many antibiotics, EQAs have been established for many decades, whereas others have only lately been introduced. This article gives an insight into the state of the art regarding the therapeutic drug monitoring of antibiotics regarding standardization, EQAs, and reference measurement procedures (RMPs). METHODS: An overview of the currently available international EQA schemes for antibiotics and a literature overview of available RMPs are given. EQAs including gentamicin and vancomycin have been offered by German providers for more than 25 years. The period 2000-2020 was selected for a detailed analysis. The experiences with a new EQA including linezolid, meropenem, and piperacillin are described. RESULTS: EQAs for gentamicin and vancomycin are provided in many countries. Those for linezolid, meropenem, and piperacillin do not seem to be very common. Most of the antibiotics monitored for decades are measured by commercially available assays. EQAs for linezolid, meropenem, and piperacillin introduced in 2018 were rapidly accepted in Germany. Methods reported in this study were HPLC based either with UV or mass spectrometric detection. The number of participants succeeding was comparable between UV and mass spectrometry. Candidate RMPs for gentamicin, vancomycin, and linezolid based on isotope dilution mass spectrometry were published. CONCLUSIONS: EQAs are offered regularly for many antibiotics worldwide. The results of EQAs in Germany generally compare well, but there is potential for improvement. Both immunoassays and HPLC-based methods work properly in EQAs evaluated in Germany. From a quality control perspective, fast and inexpensive methods may be selected without endangering the patient's health based on clinical needs.

Chromatographic separation of R-(-)/S-(+)-enantiomers of amphetamine and methamphetamine: differentiation between single methamphetamine consumption and co-consumption with amphetamine using enantioselective quantitative LC-MS/MS analysis.

The differentiation between single methamphetamine consumption and co-consumption with amphetamine is difficult, however possible by enantioselective analysis due to different preferred synthesis pathways of both substances. We quantified (R)-(-) and (S)-(+)-enantiomers of methamphetamine and amphetamine by a fast liquid chromatographic tandem-mass spectrometric method using a Lux® 3-µm AMP 150 × 3.0 mm analytical column after simple protein precipitation with methanol. Method validation for quantitative detection showed limits of quantification < 5 ng/mL, linearity in a range between 5 and 300 ng/mL and bias and imprecision data < 15%. Overall, 134 plasma samples of police cases from the German regions of Franconia and Northrhine-Westphalia were analyzed for the enantiomers of methamphetamine and amphetamine. In 28 cases, the intake of racemic illicit amphetamine could be demonstrated; (R)-(-) / (S)-(+)-amphetamine concentration ratios in these cases were between 1.38 and 4.50 with most of the ratios being < 2.0. These ratios were compared to a subgroup of 25 consumers with a co-consumption of (S)-(+)-methamphetamine and racemic amphetamine detected by the qualitative proof of (R)-(-)-amphetamine but also by (R)-(-) / (S)-(+)-amphetamine concentration ratios (< 1 in 11 of 25 cases). Within our collective of 106 plasma samples after methamphetamine use, 25 samples showed co-consumption with amphetamine which shows that co-consumption of both stimulants is not a rare scenario. Furthermore, we could show that if non-stereoselective methods are used and the concentration ratio of total methamphetamine/total amphetamine is determined, a reliable estimation of co-consumption is not possible.

Activity-Based Detection of Cannabinoids in Serum and Plasma Samples.

BACKGROUND: Synthetic cannabinoids are the largest group of new psychoactive substances monitored by the European Monitoring Centre of Drugs and Drug Addiction. The rapid proliferation of novel analogs makes the detection of these new derivatives challenging and has initiated considerable interest in the development of so-called "untargeted" screening strategies to detect these compounds. METHODS: We developed new, stable bioassays in which cannabinoid receptor activation by cannabinoids led to recruitment of truncated ß-arrestin 2 (ßarr2) to the cannabinoid receptors, resulting in functional complementation of a split luciferase, allowing readout via bioluminescence. Aliquots (500 µL) of authentic serum (n = 45) and plasma (n = 73) samples were used for simple liquid-liquid extraction with hexane:ethyl acetate (99:1 v/v). Following evaporation and reconstitution in 100 µL of Opti-MEM® I/methanol (50/50 v/v), 10 µL of these extracts was analyzed in the bioassays. RESULTS: Truncation of ßarr2 significantly (for both cannabinoid receptors; P = 0.0034 and 0.0427) improved the analytical sensitivity over the previously published bioassays applied on urine samples. The new bioassays detected cannabinoid receptor activation by authentic serum or plasma extracts, in which synthetic cannabinoids were present at low- or sub-nanogram per milliliter concentration or in which Δ9-tetrahydrocannabinol was present at concentrations >12 ng/mL. For synthetic cannabinoid detection, analytical sensitivity was 82%, with an analytical specificity of 100%. CONCLUSIONS: The bioassays have the potential to serve as a first-line screening tool for (synthetic) cannabinoid activity in serum or plasma and may complement conventional analytical assays and/or precede analytical (mass spectrometry based) confirmation.

Alterations in gene expression after gamma-hydroxybutyric acid intake-A pilot study.

Gamma-hydroxybutyric acid (GHB) acts as an agonist of the GABAB receptor, where GHB induces a depressant effect in the central nervous system. Besides its therapeutic application, GHB is also used as a date rape drug. However, the detection of GHB ingestion proves to be difficult due to its narrow detection window. The aim of this pilot study was to assess differential gene expressions after GHB intake to identify potential biomarkers for the detection of GHB intake. To this aim, alteration in gene expression of ALDH5A1, AKR7A2, EREG, and PEA15 was investigated via quantitative PCR (qPCR). Data normalization was based on a previously established and empirically derived normalization strategy. Blood samples of patients (n = 3) therapeutically taking sodium oxybate solution (GHB) and of donors without GHB intake (n = 49) were analyzed and compared. All qPCR procedures and results are reported according to the MIQE guidelines. Investigation of suitable reference genes using established algorithms suggested PPIB and FPGS as best-suited normalizers. Alterations in gene expression relating to GHB intake could not be confirmed to a forensically sufficient degree. However, significant differences in expression of EREG in the control group were observed, when time-point of sample collection was considered, indicating circadian rhythm. The study's main limitation is the small number of study subjects. Herein, we are first to present an empirically derived strategy for a robust normalization of qPCR data from the analysis of GHB-induced gene expression in human blood. We present results of the analysis of differential expression of ALDH5A1, AKR7A2, EREG, and PEA15 in the GHB-negative population. Finally, we report our findings on the effect of GHB intake on the expression of these genes and their presumable potential as GHB biomarkers.

1,2-Dimethylimidazole-4-sulfonyl chloride (DMISC), a novel derivatization strategy for the analysis of propofol by LC-ESI-MS/MS.

Analysis of the anesthetic agent propofol in biological samples by LC-MS/MS is a great challenge due to weak fragmentation and poor ionization efficacy of propofol resulting in weak signal intensities. Improvements of the ionization and fragmentation efficacy can be achieved by conversion of propofol to its dimethylimidazolesulfonyl (DMIS) derivative by a derivatization reaction using 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC). This DMIS derivative produced intense [M + H]+ ions in positive-ion LC-ESI-MS/MS with the dimethylimidazole moieties representing the most abundant product ions. Derivatization of serum samples is achieved by direct conversion of the acetonitrile supernatant of a protein precipitation with DMISC followed by a double liquid-liquid extraction using n-hexane. Reliability of the method was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, and processed sample stability. Linearity was demonstrated over the whole calibration range from 5 to 1000 ng/ml with the use of a 1/x 2 weighting. Stability of the processed samples was verified for a time period of up to 25 h. Due to its high sensitivity, appropriate quantification and detection limits (LLoQ = 5 ng/ml, LoD = 0.95 ng/ml) for toxicological propofol analyses could be achieved. Applicability of the method to biological samples could be verified by analysis of a human serum sample collected after propofol-induced sedation. Graphical abstract A novel derivatization strategy using 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) was developed to improve the ionization and fragmentation efficacy of propofol for LC-ESI-MS/MS analysis.

Subnuclear lipid-containing vacuolization in cases of ketoacidosis - correlation of morphological findings and ketone body concentrations.

In a study on alcoholics, diabetics, cases of hypothermia, combinations of alcoholism, diabetes and hypothermia as well as 55 controls, ketone body measurements were performed in femoral vein blood, heart blood, vitreous humor, cerebrospinal fluid and urine. Histological investigations were carried out on the kidneys of the deceased. In addition to HE-staining, the cuts were stained with Sudan and PAS to allow differentiation between lipids and glycogens. The degree of stainability in the Sudan stains was correlated with the ketone body concentrations measured. In those cases in which elevated ketone body concentrations were measured, marked fat deposits in the renal tubular epithelial cells could be demonstrated with the Sudan staining method. The higher the stainability the higher the ketone body concentrations. The ketone body concentrations measured in the various body fluids correlated with the intensity of fat stainability.

[An unusual bodily injury]. / Eine ungewöhnliche Körperverletzung.

In most cases, bodily harm results from the use of sharp objects or blunt force. This paper deals with a 42-year-old pharmacist who was known to the police and the courts because of several previous convictions for bodily injury. The man had visited a pub just before it closed and was therefore not served any drinks. He got angry about this and returned to his pharmacy (within walking distance) to fetch three disposable syringes which he filled with phosphoric acid (85%). Through the open pub window, he splattered the acid from the syringes on two guests and the host, who were hit on the upper part of their bodies and the arms. All the victims developed dermal alterations such as focal erythema and small blisters (pemphigus-like efflorescences, as already described by Weyrich). At first, the pharmacist denied the use of phosphoric acid and claimed to have used a mixture of urine and water. Examinations of spots on the still unwashed clothes revealed very low pH-values (ca. 2.0; pH-Indicator-Stripes, Merck; Medi-Test, Machery & Nagel). Tests for substances typical of urine produced completely negative results. However, very high phosphate concentrations were found on the spots in question. Thus, the probability that the pharmacist had used phosphoric acid to commit the offence was very high. The pharmacist was sentenced to one year and two months' imprisonment for dangerous bodily harm according to Section 224 German Criminal Code. In accordance with the law, phosphoric acid was classified as "poison", for which application on the skin is sufficient.

Interrupted (self -)medication with pancuronium(bromide) and fatal outcome.

Pancuronium(bromide) is used because of its relaxing effect on striated muscles and usually requires artificial respiration. A 52-year-old woman suffered from long-standing "generalized dystonia", which had become resistant to conventional therapy. Therefore, an anesthetist established a permanent medication scheme with pancuronium using a PCA pump. This pump had been controlled by the patient herself ensuring an acceptable quality of life with broad personal autonomy. Finally, the woman was found dead in her flat by a member of a home nursing service. The infusion hose showed a fixed knot and further blocking by a clamp. The autopsy findings were non-specific, except for the presence of opioid tablets in the colon. Toxicological analyses showed 72ng/ml pancuronium and 21 ng/ml oxycodone (therapeutic) in the femoral venous blood. The range of published pancuronium levels varies from approx. 80 to 2,000 ng/ml. Thus it had to be assumed that the pancuronium level was too low (72 ng/ml) so that symptoms of dystonia recurred. Based on extensive literature research, the described case can be qualified as unique. The therapy concept had been innovative, sufficient and effective for more than 10 years. It allowed the patient to enjoy a maximum of autonomy. Ultimately, death was due to the blocked pancuronium infusion. The relatively low pancuronium level had provoked the dystonia to return with generalized spasms also involving the respiratory muscles resulting in respiratory arrest. During the police investigations, two previous suicide attempts came to light.

[Sepsis due to fusobacteria in a young adult]. / Fusobakterien-Sepsis bei einem jungen Erwachsenen.

Fusobacteria belong to the normal population of the pharyngeal mucosa as well as the mucosa of the upper airways and the gastrointestinal tract. Infections are comparatively rare. The most common causative organism is Fusobacterium necrophorum. A well-known infection caused by this germ is Lemierre's syndrome. In the presented case, a 19-year-old man (123 kg body weight, 186 cm body length) was found dead in his bed in the morning after having complained of muscular fatigue and vomiting the previous day. Autopsy was carried out only two days after death. At that time, the body showed marked putrefaction with partial greenish discoloration and marbling of the skin although it had been stored in a refrigerator at +2 degrees C in the meantime. While the autopsy itself revealed no cause of death, microbiological examination of a smear from the left lower pulmonary lobe demonstrated Staphylococcus aureus and Fusobacterium necrophorum. Toxicological investigations produced negative results throughout. The cause of death was defined as sepsis caused by Fusobacterium necrophorum.

Driving under the influence of synthetic phenethylamines: a case series.

New psychoactive drugs, so-called legal highs, have gained more and more popularity during the last years. One of the most important groups of these legal high substances are the synthetic phenethylamines that share a common phenethylamine moiety. Based on certain structural characteristics, these synthetic phenethylamines can be divided into further subclasses, among which the synthetic cathinones ('bath salts') are particularly noteworthy. Synthetic cathinones are characterized by an additional carbonyl group attached at the beta position on the amino alkyl chain. Consumption of synthetic phenethylamines can lead to impairments similar to those observed after the use of, for instance, amphetamine or 3,4-methylenedioxy-N-methylamphetamine (MDMA, 'ecstasy'). These impairments include diverse neurological and psychological symptoms which can affect a safe driving behaviour. Although several reports on clinical symptoms and poisonings due to these substances have been published, most of these publications do not contain any analytical data. Additionally, there is still a lack of information concerning pharmacological and toxicological effects of these rather new psychoactive substances. In particular, the knowledge of the impact on the ability to drive following consumption of synthetic phenethylamines is relevant for the police as well as for forensic toxicologists. In this publication, several cases of individuals driving under the influence (DUI) of synthetic phenethylamines (4-fluoroamphetamine, mephedrone (4-methylmethcathinone, 4-MMC), 2-DPMP (desoxypipradol), methylenedioxypyrovalerone (MDPV), benzedrone, N-ethylamphetamine (etilamfetamine), 3-methylmethcathinone (3-MMC)) are presented, focusing on analytical results and signs of impairment.

[Death by alcoholic ketoacidosis--analytical procedure and case report]. / Tod infolge alkoholischer Ketoazidose--analytisches Vorgehen und Fallbeispiel.

Alcoholic ketoacidosis is a medical emergency, which is characterized at first by an elevated level of ketone bodies. The production of these ketone bodies is accompanied by an equimolar quantity of hydrogen ions and thus causes acidosis, which cannot be detected post-mortem due to anaerobic glycolysis, whereas the three ketone bodies beta-hydroxybutyrate, acetoacetate and acetone can be easily identified. However, the limits of ketone body concentrations mentioned in the literature for diagnosing ketoacidosis vary significantly, partly due to inhomogeneous study groups. A 44-year-old woman was found dead with numerous haematomas and partial mummification. Some days before, she had reported her partner to the police for rape. Consequently investigations for homicide were initiated. The autopsy itself did not reveal any morphologically identifiable cause of death. The ketone body concentrations in three matrices (vitreous humour, cerebrospinal fluid, cardiac blood) left no doubt that death was due to alcoholic ketoacidosis.

Clinical and forensic examinations of glycaemic marker methylglyoxal by means of high performance liquid chromatography-tandem mass spectrometry.

The postmortem determination of hyperglycaemic coma is quite difficult because of the lack of morphological findings and the difficult interpretation of biochemical parameters. Methylglyoxal (MG) is a reactive oxoaldehyde, which is mainly derived from glycolysis. An electrospray ionisation liquid chromatography-tandem mass spectrometric procedure for the determination of methylglyoxal in human serum and postmortem blood was developed. It involves protein precipitation with perchloric acid and a derivatisation step with 2,3-diaminonaphthalene. The assay was validated according to international guidelines. Serum samples from diabetics obtained at a diabetes clinic and from non-diabetics were used to assess data about reference concentrations in human serum. The assay showed linearity within the physiological concentrations in serum (5-500 ng/ml). Intraday imprecision at three concentrations was 10.3, 9.2 and 8.3 %, and interday imprecision was 15.3, 14.2 and 9.4 %; the limit of detection was 1.3 ng/ml, and limit of quantification, 3.2 ng/ml. One hundred and eighteen clinical (100 diabetics, 18 non-diabetics) and 98 forensic samples (84 non-diabetics, 14 in a status of hyperglycaemic coma) were measured. During life, diabetics showed significantly (p < 0.001) higher serum concentrations of MG than non-diabetics. After death, concentrations of MG increased significantly (p < 0.001). However, there was no correlation between the sum formula of Traub in vitreous humour and MG femoral blood concentrations (R = 0.237). This indicates that MG concentrations in the deceased cannot distinguish deaths due to a hyperglycaemic coma from other causes of death.

Metabolism of levamisole and kinetics of levamisole and aminorex in urine by means of LC-QTOF-HRMS and LC-QqQ-MS.

The antihelminthic drug Levamisole can enhance cocaine effects by conversion into the amphetamine-like drug aminorex. We describe an LC-MS method for the determination of levamisole and its metabolite aminorex in human urine. Selectivity is given, calibration curves were linear within the calibration range 2.5-250 ng/mL; limits of the method were LoD 0.51 ng/mL, LoQ 1.02 ng/mL for levamisole and LoD 0.65 ng/mL, LoQ 0.76 ng/mL for aminorex. Precision data was in accordance with the guidelines (intraday precision for aminorex ranged between 5.75 and 11.0 % for levamisole between 8.36 and 10.9 %; interday precision for levamisole 10.9-16.9 % and for aminorex 7.64-12.7 %; accuracy data for levamisole -1.96 to -14.3 % and for aminorex-11.9 to-18.5 %). The validated method was successfully applied to study the urinary excretion of levamisole after the administration of 100 mg of levamisole orally. Levamisole and aminorex could be detected in post-administration urine samples. Levamisole could be detected up to 39 h after ingestion, while aminorex was detectable up to 54 h. Maximum aminorex concentrations were 45 ng/mL urine. Further metabolites of levamisole after oral ingestion by means of liquid chromatography hybrid quadrupole time-of-flight high-resolution mass spectrometry (LC-QTOF-HRMS) were identified. Only 0.5 % of the ingested drug was quantified as unchanged levamisole in urine. Besides aminorex, five isomers of aminorex and 4 hydroxy-metabolites of aminorex or its isomers were found. Furthermore, levamisole is also hydroxylated and eliminated free or conjugated with sulfate or glucuronide into urine.

["Legal highs" from the German internet--"bath salt drugs" on the rise]. / "Legal Highs" aus dem deutschen Internet--der Vormarsch von "Badesalz-Drogen".

The appearance of dangerous and insufficiently studied designer drugs has increased substantially within the last few years. Mixtures containing centrally active compounds are often declared as "bath salt", "incense", "plant food", "bong cleaners" and are marketed in head shops and on the Internet. As the majority of the ingredients of such products are not subject to regulations of the German Narcotics Law (Betäubungsmittelgesetz, BtMG), the vendors and consumers mistake the sale of such products for legal. An alternative possibility to prosecute the distribution of so-called "legal highs" arises from the regulations of the German Medicinal Products Act (Arzneimittelgesetz, AMG). Indicating a private address, several products were purchased via the Internet. The products were analyzed by gas chromatography- mass spectrometry using computer-assisted database search and potential hits were checked for plausibility. The analysis of 100 samples revealed centrally acting compounds (including caffeine) in 98 % (75 % of all samples positive for caffeine). In 16 % of the samples, drugs subject to the BtMG at the time of purchase (end of 2011) were found including 2,5-dimethoxy-4-methylamphetamine, amphetamine, etilamphetamine, N-benzylpiperazine, mephedrone, methcathinone, and phenobarbital. In 55 % of the samples, drugs subject to the current BtMG were found (after its amendment on 20 July 2012). In 37 % of the samples, substances subject to the AMG were found (e.g. ephedrine). In 35 % of the samples, drugs with a potential psychotropic effect were found. In 57.3 % of the positive samples, more than one active ingredient was determined and in some cases up to five active components were found. Other interesting pharmacologically active ingredients found were 4-methylcathinone (n=13), flephedrone (n=8), trifluoromethylphenyl-piperazine (n=7), methylone (n=5), butylone (n=2), hordenine (n=2), and harmane (n=2). Most of the substances not covered by the BtMG can be classified as "unsafe" drugs. The distribution of unsafe drugs is illegal in Germany. However, the easy availability of real or potential drugs has to be seen critically. Little is known about the toxicological and pharmacological effects of those single substances, let alone of interactions in mixtures of such substances. In chat rooms, users advertise such drugs and blaze abroad their own experiences.

Analysis of tetramisole metabolites - Is "Aminorex" found in forensic samples of cocaine users actually 4-phenyl-2-imidazolidinone?

Phenyltetrahydroimidazothiazole (PTHIT, tetramisole) is a common adulterant in cocaine samples. Little is known about its human metabolism. p-hydroxy-PTHIT has long been the only proven phase-I-metabolite. Another putative metabolite is the stimulant aminorex. However, data on its analytical proof is rare and contradictory. Even less known is its constitutional isomer 4-phenyl-2-imidazolidinone which has only been proven in animal samples so far. The aim of the study was to get insight into the metabolism of PTHIT after controlled nasal uptake of PTHIT and in real forensic cocaine/benzoylecgonine-positive samples. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated for quantification of 4-phenyl-2-imidazolidinone and p-hydroxy-PTHIT (LOQ 0.05 ng/ml each). Selectivity was ensured for 4-phenyl-2-imidazolidinone and aminorex (LOD 0.05 ng/ml). After controlled nasal uptake of tetramisole (10 mg, n = 3) a shorter half-life for p-hydroxy-PTHIT (3.4-5.8 h) was determined than for 4-phenyl-2-imidazolidinone (14.0-15.9 h). p-hydroxy-PTHIT (33%) and 4-phenyl-2-imidazolidinone (51%) were also detected in serum samples from cocaine users tested previously positive for PTHIT (n = 73). Aminorex was never detected. The potential of misinterpreting 4-phenyl-2-imidazolidinone as aminorex was tested using a gas chromatography-mass spectrometry (GC-MS) method used in the literature and an in-house liquid chromatography-time-of-flight mass spectrometry (LC-QTOF) screening-method. Using GC-MS the analysed bis-trimethylsilyl-derivatives cannot be differentiated due to co-elution. Both substances were chromatographically separated using the LC-QTOF method, but library comparison workflows misinterpreted 4-phenyl-2-imidazolidinone as aminorex. It seems likely that aminorex, which was allegedly identified as a metabolite of PTHIT in samples of cocaine users in previous studies, is in fact 4-phenyl-2-imidazolidinone.

Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry.

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8 µU/ml. Recoveries ranged between 33.2 % and 51.7 %. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301 µU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog®, Novolog®, Apidra®, Lantus®, and Levemir® in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.

Comparative Study: Postmortem Long-Term Stability of Endogenous GHB in Cardiac Blood, Femoral Blood, Vitreous Humor, Cerebrospinal Fluid and Urine with and without Sodium Fluoride Stabilization.

The interpretation of postmortem γ-hydroxybutyric acid (GHB) concentrations is challenging due to endogenous existence and postmortem GHB production in body tissues and fluids. As an additional complication, formation of GHB was also described in stored postmortem samples. We examined cardiac blood, femoral blood, vitreous humor, cerebrospinal fluid and urine of eight different corpses (male/female 5/3, aged 33-92 years, postmortem interval 1-6 days) where no intake of GHB or one of its precursors was assumed. All samples were collected during autopsy and divided into two aliquots. To one of the aliquots, sodium fluoride (NaF, 1% w/v) was added. Both aliquots were vortexed, further divided into seven aliquots and stored at -20°C. GHB concentrations were measured immediately and subsequently 1 day, 7 days, 2 weeks, 4 weeks, 3 months and 6 months, after sample collection using trimethylsilyl derivatization and gas chromatography, coupled to single quadrupole mass spectrometry. Similar progression curves of GHB concentrations were obtained for the different matrices in the individual corpses. Femoral and cardiac blood GHB concentrations were always found to be higher than in vitreous humor, cerebrospinal fluid, and urine irrespective of stabilization and storage time. None of the obtained GHB concentrations exceed the cutoff values for postmortem matrices commonly used for the identification of an exogenous GHB intake (urine, venous blood and cerebrospinal fluid: 30 mg/L, cardiac blood and vitreous humor 50 mg/L). No significant differences were found for the GHB concentrations measured immediately and 6 months after autopsy. However, we found a significant increase for the GHB concentrations 4 weeks as well as 3 months after sample collection, which was followed by a decrease nearly to initial values. There were no significant differences between samples with and without NaF addition. The data presented are useful for the interpretation of GHB concentrations in upcoming death cases, with special attention to storage conditions and different postmortem matrices.

Sensitive Screening of New Psychoactive Substances in Serum Using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

Analysis of new psychoactive substances (NPS) still poses a challenge for many institutions due to the number of available substances and the constantly changing drug market. Both new and well-known substances keep appearing and disappearing on the market, making it hard to adapt analytical methods in a timely manner. In this study we developed a qualitative screening approach for serum samples by means of liquid chromatography--quadrupole time-of-flight mass spectrometry. Samples were measured in data-dependent auto tandem mass spectrometry mode and identified by fragment spectra comparison, retention time and accurate mass. Approximately 500 NPS, including 195 synthetic cannabinoids, 180 stimulants, 86 hallucinogens, 26 benzodiazepines and 7 others were investigated. Serum samples were fortified to 1 ng/mL and 10 ng/mL concentrations to estimate approximate limits of identification (LOIs). Samples were extracted using solid-phase extraction with non-endcapped C18 material and elution in two consecutive steps. Benzodiazepines were eluted in the first step, while substances of other NPS subclasses were distributed among both extracts. To determine LOIs, both extracts were combined. Ninety-six percent (470/492) of investigated NPS were detected in 10 ng/mL samples and 88% (432/492) were detected in 1 ng/mL samples. Stimulants stood out with higher LOIs, possibly due to instability of certain methcathinone derivatives. However, considering relevant blood concentrations, the method provided sufficient sensitivity for stimulants as well as other NPS subclasses. Data-dependent acquisition was proven to provide high sensitivity and reliability when combined with an information-dependent preferred list, without losing its untargeted operation principle. Summarizing, the developed method fulfilled its purpose as a sensitive untargeted screening for serum samples and allows uncomplicated expansion of the spectral library to include thousands of targets.

Chiral pharmacokinetics of tetramisole stereoisomers-Enantioselective quantification of levamisole and dexamisole in serum samples from users of adulterated cocaine.

Phenyltetrahydroimidazothiazole (PTHIT, tetramisole) is the most frequently used adulterant of cocaine and exists in the two enantiomeric forms levamsiole (S) and dexamisole (R). Existing studies show diverse fractions of samples containing enantiopure levamsiole, levamisole-enriched mixtures, and racemic tetramisole as adulterant. However, blood samples have never been enantioselectively tested for PTHIT. Because enantiomers are usually metabolized stereoselectively, chiral analysis of blood samples can help estimate the time of drug use, provided that a racemic substance is ingested. Therefore, an enantioselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed using a chiral column. Validation of the method was carried out for methanolic substance samples as well as serum samples and showed satisfactory selectivity, sensitivity, linearity (0.05-100 ng/mL), precision, and accuracy; 151 cocaine samples seized in Germany between 2018 and 2021 were analyzed. Most (94%, n = 48) of the 51 PTHIT-positive samples contained racemic tetramsiole, whereas there were two samples containing levamisole-enriched mixtures and one sample containing nearly enantiopure levamisole. Furthermore, 157 cocaine and/or benzoylecgonine-positive forensic serum samples were tested with cocaine-positive samples showing the highest frequency of PTHIT detection (43%). All positive samples contained either dexamisole alone or (R)/(S)-concentration ratios >1 (1.05-70.6). Finally, a self-administration study was conducted with three subjects taking 10 mg of racemic tetramisole each. Although peak concentrations and corresponding times did not differ significantly between the enantiomers, dexamisole showed significantly longer apparent elimination half-lives (7.02-10.0 h) than levamisole (2.87-4.77 h). The resulting steadily increasing (R)/(S)-ratios can therefore be helpful in estimating the time of cocaine consumption.

Determination of the enantiomeric composition of amphetamine, methamphetamine and 3,4-methylendioxy-N-methylamphetamine (MDMA) in seized street drug samples from southern Germany.

Amphetamine (speed), methamphetamine (crystal meth), and 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) represent the most frequently abused amphetamine-type stimulants (ATS). Differences in pharmacological potency and metabolism have been shown for the enantiomers of all three stimulants. Legal consequences in cases of drug possession may also differ according to the German law depending on the enantiomeric composition of the seized drug. Therefore, enantioselective monitoring of seized specimens is crucial for legal and forensic casework. Various kinds of samples of amphetamine (n = 143), MDMA (n = 94), and methamphetamine (n = 528) that were seized in southern Germany in 2019 and 2020 were analyzed for their chiral composition using different chromatographic methods. Whereas all samples of amphetamine and MDMA were racemic mixtures, the chiral composition of the methamphetamine specimens was diverse. Although the vast majority (n = 502) was present as (S)-methamphetamine, also specimens containing pure (R)-methamphetamine (n = 7) were found. Furthermore, few samples (n = 8) were of racemic nature or contained non-racemic mixtures of both enantiomers (n = 10). Because methamphetamine appears in varying enantiomeric compositions, any seizure should be analyzed using an enantioselective method. Amphetamine and MDMA, on the other hand, currently appear to be synthesized exclusively via racemic pathways and are not chirally purified. Nevertheless, regular monitoring of the chiral composition should be ensured.