Advances in testing for sample manipulation in clinical and forensic toxicology - Part A: urine samples.

In many countries, adherence testing is used to monitor consumption behavior or to prove abstinence. Urine and hair are most commonly used, although other biological fluids are available. Positive test results are usually associated with serious legal or economic consequences. Therefore, various sample manipulation and adulteration strategies are used to circumvent such a positive result. In these critical review articles on sample adulteration of urine (part A) and hair samples (part B) in the context of clinical and forensic toxicology, recent trends and strategies to improve sample adulteration and manipulation testing published in the past 10 years are described and discussed. Typical manipulation and adulteration strategies include undercutting the limits of detection/cut-off by dilution, substitution, and adulteration. New or alternative strategies for detecting sample manipulation attempts can be generally divided into improved detection of established urine validity markers and direct and indirect techniques or approaches to screening for new adulteration markers. In this part A of the review article, we focused on urine samples, where the focus in recent years has been on new (in)direct substitution markers, particularly for synthetic (fake) urine. Despite various and promising advances in detecting manipulation, it remains a challenge in clinical and forensic toxicology, and simple, reliable, specific, and objective markers/techniques are still lacking, for example, for synthetic urine.

Advances in testing for sample manipulation in clinical and forensic toxicology-part B: hair samples.

As a continuation of part A, focusing on advances in testing for sample manipulation of urine samples in clinical and forensic toxicology, part B of the review article relates to hair, another commonly used matrix for abstinence control testing. Similar to urine manipulation, relevant strategies to manipulate a hair test are lowering drug concentrations in hair to undercut the limits of detection/cut-offs, for instance, by forced washout effects or adulteration. However, distinguishing between usual, common cosmetic hair treatment and deliberate manipulation to circumvent a positive drug test is often impossible. Nevertheless, the identification of cosmetic hair treatment is very relevant in the context of hair testing and interpretation of hair analysis results. Newly evaluated techniques or elucidation of specific biomarkers to unravel adulteration or cosmetic treatment often focused on specific structures of the hair matrix with promising strategies recently proposed for daily routine work. Identification of other approaches, e.g., forced hair-washing procedures, still remains a challenge in clinical and forensic toxicology.

Comparative study on the metabolism of the ergot alkaloids ergocristine, ergocryptine, ergotamine, and ergovaline in equine and human S9 fractions and equine liver preparations.

1. Ergopeptine alkaloids like ergovaline and ergotamine are suspected to be associated with fescue toxicosis and ergotism in horses. Information on the metabolism of ergot alkaloids is scarce, especially in horses, but needed for toxicological analysis of these drugs in urine/feces of affected horses. The aim of this study was to investigate the metabolism of ergovaline, ergotamine, ergocristine, and ergocryptine in horses and comparison to humans. 2. Supernatants of alkaloid incubations with equine and human liver S9 fractions were analyzed by reversed-phase liquid-chromatography coupled to high-resolution tandem mass spectrometry with full scan and MS2 acquisition. Metabolite structures were postulated based on their MS2 spectra in comparison to those of the parent alkaloids. All compounds were extensively metabolized yielding nor-, N-oxide, hydroxy and dihydro-diole metabolites with largely overlapping patterns in equine and human liver S9 fractions. However, some metabolic steps e.g. the formation of 8'-hydroxy metabolites were unique for human metabolism, while formation of the 13/14-hydroxy and 13,14-dihydro-diol metabolites were unique for equine metabolism. Incubations with equine whole liver preparations yielded less metabolites than the S9 fractions. 3. The acquired data can be used to develop metabolite-based screenings for these alkaloids, which will likely extend their detection windows in urine/feces from affected horses.

Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system.

Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.

Optimization of metabolomics of defined in vitro gut microbial ecosystems.

The metabolic functionality of a microbial community is a key to the understanding of its inherent ecological processes and the interaction with the host. However, the study of the human gut microbiota is hindered by the complexity of this ecosystem. One way to resolve this issue is to derive defined communities that may be cultured ex vivo in bioreactor systems and used to approximate the native ecosystem. Doing so has the advantage of experimental reproducibility and ease of sampling, and furthermore, in-depth analysis of metabolic processes becomes highly accessible. Here, we review the use of bioreactor systems for ex vivo modelling of the human gut microbiota with respect to analysis of the metabolic output of the microbial ecosystem, and discuss the possibility of mechanistic insights using these combined techniques. We summarize the different platforms currently used for metabolomics and suitable for analysis of gut microbiota samples from a bioreactor system. With the help of representative datasets obtained from a series of bioreactor runs, we compare the outputs of both NMR and mass spectrometry based approaches in terms of their coverage, sensitivity and quantification. We also discuss the use of untargeted and targeted analyses in mass spectroscopy and how these techniques can be combined for optimal biological interpretation. Potential solutions for linking metabolomic and phylogenetic datasets with regards to active, key species within the ecosystem will be presented.

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.

Assessment of maternal drug intake by urinary bio monitoring during pregnancy and postpartally until the third perinatal year.

PURPOSE: Although sales of prescribed and over-the-counter (OTC) medication are rising, little is known about individual drug intake. This study was aimed to obtain complementary information about drug intake. METHOD: Information on drug utilization was obtained in a female cohort for five different time points (TP): 36th week of pregnancy (n = 622), 7th perinatal week (n = 533), 3rd perinatal month (n = 340), and 1st perinatal (n = 534) and 3rd perinatal year (n = 324) by a validated urine screening method. RESULTS: Drugs were detected 807 times among all analyzed samples (n = 2353) with less drug intake for early TP compared with later TP (~24.4%, n = 152; ~33.8%, n = 180; ~23.2%, n = 79; ~42.5%, n = 227; and ~52.2%, n = 169). The diversity of drugs increased from 25 up to 40 different drugs for the investigated period. OTC drugs were detected most frequently reflected by the top three drugs: acetaminophen (~37%, n = 292), ibuprofen (~23%, n = 183), and xylometazoline (~12%, n = 98). Mainly guideline-orientated drug therapy was observed. However, contraindicated ibuprofen intake during third trimester urine samples (n = 26) and a repeated usage of acetaminophen and/or ibuprofen (n = 9), as well as xylometazoline (n = 7), reveal missing information about drug safety. CONCLUSION: Bio monitoring was applied for detection of drug intake revealing a lack of information about OTC products and their health risks. Hence, information about health risks for certain drugs and patient groups must be improved for and by pharmacists, to avoid (i) usage of contraindicated drugs and (ii) abuse of OTC drugs.

False-positive MDA findings in HRMS-based screening of putrefied postmortem blood samples-Identification of the interference as N-acetyltyramine.

An unidentified compound in putrefied postmortem blood samples showed identical accurate mass and chromatographic behavior as 3,4-methylenedioxyamphetamine (MDA) and led to false-positive preliminary screening results. The aim of the study was to identify this unknown interference. Postmortem blood samples were analyzed after protein precipitation on a QExactive Focus high-resolution mass spectrometer (Thermo Fisher, Germany) coupled to a RP C18 column (Macherey-Nagel, Germany). Based on the analysis of mass spectrometry (MS) adducts and isotope ratios using fullscan (m/z 134-330) information, the empiric formula of the protonated molecule [M + H]+ of the unknown compound was found to be C10H14O2N (+ 0.6 ppm). Product ion spectra recorded using normalized collision energy 22% showed a base peak of C8H9O1 (+ 1.5 ppm) and a low-abundant water loss to C7H9 (+ 1.9 ppm), neutral losses of C2H2O and NH3 were found. Based on fullscan and MS-MS information and under consideration of the observed order of neutral losses, the compound was presumptively identified as N-acetyltyramine. This assumption was supported by SIRIUS software showing a SIRIUS score of 99.43% for N-acetyltyramine. Finally, the putative structure annotation was confirmed by a reference compound. The described false-positive MDA findings could be attributed to the presence of N-acetyltyramine in putrefied blood samples. Being an isomer of MDA, N-acetyltyramine could not be distinguished by high-resolution data of the protonated molecules. The presented results once again highlight that false-positive findings may occur even in hyphenated high-resolution mass spectrometry (HRMS) when using full-scan information only.

Evaluation of biochemical assays and optimization of LC-MS-MS analysis for the detection of synthetic urine.

Ensuring specimen validity is an essential aspect of toxicological laboratories. In recent years, substituting authentic urine specimens for synthetic urine (SU) has become increasingly popular. Such SU products consist of components expected in normal urine and show physiological values for specific gravity and pH. Thus, standard specimen validity testing may fail in revealing adulteration by SU. The present study investigated three methods to distinguish authentic and SU specimens: enzymatic detection of uric acid, the commercially available Axiom Test True SU and liquid chromatography coupled with (tandem) mass spectrometry (LC-MS-MS) analysis of 10 endogenous biomolecules. Additionally, novel direct markers of SU were investigated. Two specimen sets were analyzed by each method. Specimen set A consisted of eight SU products purchased from the Austrian/German market and 43 urine specimens from volunteers of known authenticity, which underwent double-blind analysis. Specimen set B consisted of 137 real urine specimens submitted for drug testing, which were selected due to initial suspicious test results in adulteration testing and reanalyzed by all three methods. Uric acid and LC-MS-MS-based endogenous biomolecule testing showed 100% sensitivity and specificity for set A. The commercial test had 87.5% sensitivity and 97.7% specificity for set A. For set B, uric acid and LC-MS-MS analysis showed almost similar results, even if uric acid was missing one presumptive authentic urine specimen according to LC-MS-MS findings. Nearly half of the SU assignments for the commercial test were presumptive false positives. New SU markers were observed for SU products from the Austrian/German market. One specimen in set B had both an endogenous biomolecule pattern and SU markers suggesting urine dilution with SU. In conclusion, several analytes or methods should be used rather than one, and the most reliable results are achieved if both indirect and direct markers of urine substitution are analyzed.

A liquid chromatographic-mass spectrometric procedure for analysis of pentaerythrityl tetranitrate metabolites - Development, validation and application to ovine serum and human plasma samples.

Pentaerythrityl tetranitrate (PETN) is an established drug in the treatment of coronary heart disease and heart failure. It is assumed, that the vasodilative and vasoprotective effects of PETN also have a positive impact on pregnant patients with impaired placental perfusion and studies evaluating the effect of PETN in risk pregnancies have been carried out. In the context of these clinical trials, measuring of serum levels of PETN and its metabolites pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDN), pentaerythrityl mononitrate (PEMN) and pentaerythritol (PE) were required. To evaluate the transfer of PETN and its metabolites (PEXN) from the mother to the fetus using samples from a human clinical trial and animal study, the present work aimed to develop a rapid and simple method to simultaneously analyze PEXN in human and ovine samples. A method employing a rapid and simple liquid-liquid extraction followed by reversed-phase (C18) liquid chromatography coupled to high-resolution mass spectrometry with negative electrospray ionization was developed and validated for the detection of PETN and PEXN in human and ovine samples. PE could only be qualitatively detected at higher concenrations. Method validation requirements, including accuracy, repeatability and intermediate precision were fulfilled in ovine and human samples for all other PEXN with exception PETriN in human samples. The recovery (RE) in ovine samples was 76.7 % ± 12 % for PEMN, 98 % ± 23 % for PEDN, 94 % ± 22 % for PETriN, in human samples RE was 59 % ± 16 % for PEMN, 67 % ± 19 % for PEDN, 71 % ± 17 %. The matrix effects (ME) in ovine samples were 90 % ± 11 % for PEMN, 70 % ± 30 % for PEDN, 107 % ± 17 % for PETriN, in human samples the ME were 93 % ± 13 % for PEMN, 84 % ± 17 % for PEDN, 98 % ± 16 % for PETriN. The limits of quantification (LOQ) in ovine samples were 1.0 ng/mL for PETriN and 0.1 ng/mL for PEMN and PEDN. The LOQs in human samples were 5.0 ng/mL for PETriN and 0.3 ng/mL for PEMN und PEDN. The newly developed method was used to analyze 184 ovine serum samples and 18 human plasma samples. In ovine maternal samples, the highest observed PEDN concentration was 3.5 ng/mL and the highest PEMN concentration was 10 ng/mL, the respective concentrations in fetal serum samples were 4.9 ng/mL for PEDN and 5.4 ng/mL for PEMN. PETriN was only detected in traces in maternal and fetal samples, whereas PETN could not be detected at all. In human maternal samples, the highest concentration for PEDN was 27 ng/mL and for PEMN 150 ng/mL. In umbilical cord plasma, concentrations of 2.3 ng/mL for PEDN and 73 ng/mL for PEMN were detected. Although the PEMN and PEDN concentrations in the human samples were several times higher than in ovine samples, neither PETN nor PETriN signals could be detected. These results demonstrated that the metabolites were transferred from mother to fetus with a slight time delay.

Subtoxic and toxic concentrations of benzene and toluene induce Nrf2-mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549.

Since people in industrialized countries spend most of their time indoors, the effects of indoor contaminants such as volatile organic compounds become more and more relevant. Benzene and toluene are among the most abundant compounds in the highly heterogeneous group of indoor volatile organic compounds. In order to understand their effects on lung epithelial cells (A549) representing lung's first line of defense, we chose a global proteome and a targeted metabolome approach in order to detect adverse outcome pathways caused by exposure to benzene and toluene. Using a DIGE approach, 93 of 469 detected protein spots were found to be differentially expressed after exposure to benzene, and 79 of these spots were identified by MS. Pathway analysis revealed an enrichment of proteins involved in Nrf2-mediated and oxidative stress response glycolysis/gluconeogenesis. The occurrence of oxidative stress at nonacute toxic concentrations of benzene and toluene was confirmed by the upregulation of the stress related proteins NQO1 and SOD1. The changes in metabolism were validated by ion chromatography MS/MS analysis revealing significant changes of glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate, and NADPH. The molecular alterations identified as a result of benzene and toluene exposure demonstrate the detrimental effect of nonacute toxic concentrations on lung epithelial cells. The data provided here will allow for a targeted validation in in vivo models.

Qualitative studies on the metabolism and the toxicological detection of the fentanyl-derived designer drugs 3-methylfentanyl and isofentanyl in rats using liquid chromatography-linear ion trap-mass spectrometry (LC-MS(n)).

The opioid 3-methylfentanyl, a designer drug of the fentanyl type, was scheduled by the Controlled Substance Act due to its high potency and abuse potential. To overcome this regulation, isofentanyl, another designer fentanyl, was synthesized in a clandestine laboratory and seized by the German police. The aims of the presented study were to identify the phase I and phase II metabolites of 3-methylfentanyl and isofentanyl in rat urine, to identify the cytochrome P450 (CYP) isoenzymes involved in their initial metabolic steps, and, finally, to test their detectability in urine. Using liquid chromatography (LC)-linear ion trap-mass spectrometry (MS(n)), nine phase I and five phase II metabolites of 3-methylfentanyl and 11 phase I and four phase II metabolites of isofentanyl could be identified. The following metabolic steps could be postulated for both drugs: N-dealkylation followed by hydroxylation of the alkyl and aryl moiety, hydroxylation of the propanamide side chain followed by oxidation to the corresponding carboxylic acid, and, finally, hydroxylation of the benzyl moiety followed by methylation. In addition, N-oxidation of isofentanyl could also be observed. All hydroxy metabolites were partly excreted as glucuronides. Using recombinant human isoenzymes, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were found to be involved in the initial metabolic steps. Our LC-MS(n) screening approach allowed the detection of 0.01 mg/L of 3-methylfentanyl and isofentanyl in spiked urine. However, in urine of rats after the administration of suspected recreational doses, the parent drugs could not be detected, but their common nor metabolite, which should therefore be the target for urine screening.

Long time stability of 35 small endogenous biomolecules in dried urine spotted on various surfaces and environmental conditions.

Analysis of endogenous biomolecules is an important aspect of many forensic investigations especially with focus on DNA analysis for perpetrator/victim identification and protein analysis for body fluid identification. Recently, small endogenous biomolecules have been used for differentiation of synthetic "fake" urine from authentic urine and might be also useful for biofluid identification. Therefore, the aim of this study was to adapt and optimize a method for analysis of small EBs and to investigate long time stability of 35 small endogenous biomolecules (including acylcarnitines with their isomers and metabolites as well as amino acids with their metabolites) in spotted urine samples. Urine samples were spotted on seven different surfaces (Whatman 903 Protein Saver Cards, cotton swabs, cotton glove, denim, underwear, and smooth and rough flagstone) and stored under six environmental conditions (reference condition, sunlight, LED light, 4 °C, 37 °C, humidity of 95%). At certain time points (d0, d7, d28 and d56) samples were analyzed in triplicates by an optimized extraction and LC-HRMS approach. In addition, the urine marker Tamm-Horsfall-Protein was determined on cotton swabs at the same time points using a commercial lateral flow test. Twenty-one of 35 small endogenous biomolecules were stable on most materials/surfaces and under most storage conditions. Significant lower endogenous biomolecule peak areas were found for rough flagstone and underwear as well as for high humidity storage. Kynurenic acid proved to be photo labile. While high long time stabilities were found for 19 of 28 acylcarnitines, nine acylcarnitines showed aberrant stability patterns without evident structural reason. For Tamm-Horsfall-Protein degradation within 28 days was observed even under reference conditions. The presented study demonstrated the value of sensitive LC-HRMS analysis for small endogenous biomolecules / pattern. However, further studies will be indispensable for unambiguous body fluid identification by small endogenous biomolecules.

Liquid Chromatography High-Resolution Mass Spectrometry in Forensic Toxicology: What are the Specifics of Method Development, Validation and Quality Assurance for Comprehensive Screening Approaches?

The use of high-resolution mass spectrometry (HRMS) has increased over the past decade in clinical and forensic toxicology, especially for comprehensive screening approaches. Despite this, few guidelines in this field have specifically addressed HRMS issues concerning compound identification, validation, measurement uncertainty and quality assurance. To fully implement this technique, certainly in an era in which the quality demands for laboratories are ever-increasing due to various norms (e.g. the International Organization for Standardization's ISO 17025), these specific issues need to be addressed. This manuscript reviews 26 HRMSbased methods for qualitative systematic toxicological analysis (STA) published between 2011 and 2021. Key analytical data such as samples matrices, analytical platforms, numbers of analytes and employed mass spectral reference databases/libraries as well as the studied validation parameters are summarized and discussed. The article further includes a critical review of targeted and untargeted data acquisition approaches, available HRMS reference databases and libraries as well as current guidelines for HRMS data interpretation with a particular focus on identification criteria. Moreover, it provides an overview on current recommendations for the validation and determination of measurement uncertainty of qualitative methods. Finally, the article aims to put forward suggestions for method development, compound identification, validation experiments to be performed, and adequate determination of measurement uncertainty for this type of wide-range qualitative HRMSbased methods.

Ultra high performance liquid chromatographic-tandem mass spectrometric multi-analyte procedure for target screening and quantification in human blood plasma: validation and application for 31 neuroleptics, 28 benzodiazepines, and Z-drugs.

For fast and reliable screening, identification, and quantification of as many analytes as possible, multi-analyte approaches are very useful in clinical and forensic toxicology. Using ultra high performance liquid chromatography-tandem mass spectrometry, such an approach has been developed for blood plasma analysis after simple liquid-liquid extraction. In the present paper, validation and application is described for 31 neuroleptics, 28 benzodiazepines, and Z-drugs (zaleplone, zolpidem, and zopiclone). The validation parameters included recovery, matrix effects, process efficiency, ion suppression/enhancement of co-eluting analytes, selectivity, crosstalk, accuracy and precision, stabilities, and limits of quantification and detection. The results showed that the approach was selective, sensitive, accurate, and precise for 24 neuroleptics and 21 benzodiazepines and Z-drugs. The remaining analytes were unstable and/or too low dosed. Cost- and time-saving one-point calibration was applicable only for half of the analytes. The applicability was successfully shown for most of the drugs by analyzing authentic plasma samples and external quality control samples.

Full validation and application of an ultra high performance liquid chromatographic-tandem mass spectrometric procedure for target screening and quantification of 34 antidepressants in human blood plasma as part of a comprehensive multi-analyte approach.

Multi-analyte procedures are of great interest in clinical and forensic toxicology making the analytical process much simpler, faster, and cheaper and allow monitoring of analytes of different drug classes in one single body sample. The aim of the present study was to validate an ultra high performance liquid chromatographic-tandem mass spectrometric approach for fast target screening and quantification of 34 antidepressants in plasma after simple liquid-liquid extraction as part of a multi-analyte procedure for over 130 drugs. The validation process including recovery, matrix effects, process efficiency, ion suppression/enhancement of co-eluting analytes (already published), selectivity, cross talk, accuracy and precision, stabilities, and limits of quantification and detection showed that the approach was selective, sensitive, accurate, and precise for 28 of the 34 tested drugs. The applicability was successfully tested by analyzing authentic plasma samples and external quality control samples. Furthermore, it could be shown that time- and cost-saving one-point calibration was applicable for 21 drugs for daily routine and especially in emergency cases.

Development of the first metabolite-based LC-MS(n) urine drug screening procedure-exemplified for antidepressants.

In contrast to GC-MS libraries, currently available LC-MS libraries for toxicological detection contain besides parent drugs only some main metabolites limiting their applicability for urine screening. Therefore, a metabolite-based LC-MS(n) screening procedure was developed and exemplified for antidepressants. The library was built up with MS(2) and MS(3) wideband spectra using an LXQ linear ion trap with electrospray ionization in the positive mode and full-scan information-dependent acquisition. Pure substance spectra were recorded in methanolic solution and metabolite spectra in urine from rats after administration of the corresponding drugs. After identification, the metabolite spectra were added to the library. Various drugs and metabolites could be sufficiently separated. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs were determined using protein precipitation. Automatic data evaluation was performed using ToxID and SmileMS software. The library consists of over 700 parent compounds including 45 antidepressants, over 1,600 metabolites, and artifacts. Protein precipitation led to sufficient results for sample preparation. ToxID and SmileMS were both suitable for target screening with some pros and cons. In our study, only SmileMS was suitable for untargeted screening being not limited to precursor selection. The LC-MS(n) method was suitable for urine screening as exemplified for antidepressants. It also allowed detecting unknown compounds based on known fragment structures. As ion suppression can never be excluded, it is advantageous to have several targets per drug. Furthermore, the detection of metabolites confirms the body passage. The presented LC-MS(n) method complements established GC-MS or LC-MS procedures in the authors' lab.

Metabolism studies of the Kratom alkaloid speciociliatine, a diastereomer of the main alkaloid mitragynine, in rat and human urine using liquid chromatography-linear ion trap mass spectrometry.

Mitragyna speciosa (Kratom) is currently used as a drug of abuse. When monitoring its abuse in urine, several alkaloids and their metabolites must be considered. In former studies, mitragynine (MG), its diastereomer speciogynine (SG), and paynantheine and their metabolites could be identified in rat and human urine using LC-MS(n). In Kratom users' urines, besides MG and SG, further isomeric compounds were detected. To elucidate whether the MG and SG diastereomer speciociliatine (SC) and its metabolites represent further compounds, the phase I and II metabolites of SC were identified first in rat urine after the administration of the pure alkaloid. Then, the identified rat metabolites were screened for in the urine of Kratom users using the above-mentioned LC-MS(n) procedure. Considering the mass spectra and retention times, it could be confirmed that SC and its metabolites are so far the unidentified isomers in human urine. In conclusion, SC and its metabolites can be used as further markers for Kratom use, especially by consumption of raw material or products that contain a high amount of fruits of the Malaysian plant M. speciosa.

Monitoring of kratom or Krypton intake in urine using GC-MS in clinical and forensic toxicology.

The Thai medicinal plant Mitragyna speciosa (kratom) is misused as a herbal drug. Besides this, a new herbal blend has appeared on the drugs of abuse market, named Krypton, a mixture of O-demethyltramadol (ODT) and kratom. Therefore, urine drug screenings should include ODT and focus on the metabolites of the kratom alkaloids mitragynine (MG), paynantheine (PAY), speciogynine (SG), and speciociliatine (SC). The aim of this study was to develop a full-scan gas chromatography-mass spectrometry procedure for monitoring kratom or Krypton intake in urine after enzymatic cleavage of conjugates, solid-phase extraction, and trimethylsilylation. With use of reconstructed mass chromatography with the ions m/z 271, 286, 329, 344, 470, 526, 528, and 586, the presence of MG, 16-carboxy-MG, 9-O-demethyl-MG, and/or 9-O-demethyl-16-carboxy-MG could be indicated, and in case of Krypton, with m/z 58, 84, 116, 142, 303, 361, 393, and 451, the additional presence of ODT and its nor metabolite could be indicated. Compounds were identified by comparison with their respective reference spectra. Depending on the plant type, dose, administration route, and/or sampling time, further metabolites of MG, PAY, SG, and SC could be detected. The limits of detection (signal-to-noise ratio of 3) were 100 ng/ml for the parent alkaloids and 50 ng/ml for ODT. As mainly metabolites of the kratom alkaloids were detected in urine, the detectability of kratom was tested successfully using rat urine after administration of a common user's dose of MG. As the metabolism in humans was similar, this procedure should be suitable to prove an intake of kratom or Krypton.