Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia.

Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-CreERT2/loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.

Metabolism of the tryptamine-derived new psychoactive substances 5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT and their detectability in urine studied by GC-MS, LC-MSn , and LC-HR-MS/MS.

Many N,N-dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5-methoxy-2-methyl-N,N-diallyltryptamine (5-MeO-2-Me-DALT), 5-methoxy-2-methyl-N-allyl-N-cyclohexyltryptamine (5-MeO-2-Me-ALCHT), and 5-methoxy-2-methyl-N,N-diisopropyltryptamine (5-MeO-2-Me-DIPT) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC-MSn ), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC-HR-MS/MS. 5-MeO-2-Me-DALT (24 phase I and 12 phase II metabolites), 5-MeO-2-Me-ALCHT (24 phase I and 14 phase II metabolites), and 5-MeO-2-Me-DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O-demethylation, hydroxylation, N-dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O-demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N-dealkylation. For SUSAs, GC-MS, LC-MSn , and LC-HR-MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC-MS SUSA, both LC-MS SUSAs were able to detect an intake of 5-MeO-2-Me-ALCHT and 5-MeO-2-Me-DIPT via their metabolites following 1 mg/kg BW administrations and 5-MeO-2-Me-DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.

Power of Orbitrap-based LC-high resolution-MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on-spot cleavage in comparison to established LC-MSn or GC-MS procedures.

Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC-HR-MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on-spot cleavage in comparison to established LC-MSn or GC-MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on-spot cleavage followed by liquid extraction (DUSglucE), and one worked-up by acid hydrolysis, liquid-liquid extraction, and acetylation for GC-MS analysis. The 3 series of LC-HR-MS/MS results were compared among themselves, to corresponding published LC-MSn data, and to screening results obtained by conventional GC-MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC-HR-MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one-third more hits in comparison to the corresponding published LC-MSn results with ≤49% detected hits. The GC-MS approach identified 56% of all detected hits. In conclusion, LC-HR-MS/MS provided the best screening results after conjugate cleavage and precipitation.

A multi-analyte approach to help in assessing the severity of acute poisonings - Development and validation of a fast LC-MS/MS quantification approach for 45 drugs and their relevant metabolites with one-point calibration.

Diagnosis, monitoring of the efficiency of detoxification, and estimating the prognosis of acute poisonings are important tasks in emergency toxicology. Comprehensive screening and quantification of relevant substances by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) help in assessing the severity of most acute poisonings. Turnaround time for such analyses must be short enough to impact on clinical decisions. Therefore, a multi-analyte LC-MS/MS approach with a 5-minute gradient was developed and validated for 45 drugs and their active metabolites as a complement to an existing GC-MS approach using the same liquid-liquid extraction. The determination ranges were defined by quality control samples of low and high, representing concentrations from low therapeutic to highly toxic levels. To shorten the turnaround time, one-point calibration was used. Validation showed low matrix effects and ionization effects of co-eluting analytes thanks to APCI source as well as sufficient recoveries, precisions, and selectivities. For accuracy, 32 of the 45 compounds fulfilled the criteria for quantification in lower therapeutic and 41 in overdosed and toxic concentrations, considering limits of ±30% deviation. The reuse of the processed calibrator for a period of 30 days was possible for 32 compounds, showing sufficient stability at 8°C. In addition, analysis of authentic blood samples showed the applicability and yielded drug levels, which were comparable to those determined by fully validated therapeutic drug monitoring methods. In conclusion, the present approach in combination with the GC-MS approach should provide sufficient support for clinical assessment of the severity of poisonings with 68 compounds in an acceptable turnaround time.

Paper Spray Ionization Coupled to High Resolution Tandem Mass Spectrometry for Comprehensive Urine Drug Testing in Comparison to Liquid Chromatography-Coupled Techniques after Urine Precipitation or Dried Urine Spot Workup.

Screening procedures using high resolution (HR)-mass spectrometry (MS) are getting more and more important, e.g., for drug testing or adherence monitoring. Approaches usually include time-consuming sample preparation and compound separation by liquid chromatography (LC). The paper spray ionization (PSI) technique coupled to MS might overcome these steps by direct analysis of complex mixtures without extraction and separation. In recent years, this technology proved its potential for quantification and/or qualitative screening in biofluids. However, so far, PSI-MS was only applied to procedures covering a limited number of targets. Therefore, a PSI-HR-MS/MS approach was developed and successfully validated for comprehensive urine screening. The procedure showed high matrix effects for most drugs but still acceptable limits of identification. Applicability was tested by analyses of three proficiency tests for systematic toxicological analysis and of 103 authentic human urine samples. Its screening power was compared to that of published LC-HR-MS/MS procedures after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup by conjugate cleavage and liquid extraction (DUSglucE). In the authentic samples, 73% of all 777 drug intakes were detectable with the new approach. The LC-HR-MS/MS screening approaches detected more drugs, particularly in samples with low analyte concentrations, with values of 88% after UglucP or 76% after DUSglucE. In conclusion, the new PSI-HR-MS/MS screening approach was suitable for comprehensive urine screening of different drug classes and might be a promising alternative to conventional procedures. However, limitations should be considered such as detection of drugs in low concentrations and risk of false positive or negative results caused by mixed spectra.

Dried urine spots - A novel sampling technique for comprehensive LC-MSn drug screening.

Dried matrix spot (DMS) technique as alternative sampling strategy, especially dried urine spots (DUS), might be an alternative for drug screening. So far only particular drugs or drug classes were covered in DMS screenings. Therefore, workup of DUS for a broad comprehensive library-based LC-MSn screening was developed. It consisted of enzymatic on-spot deconjugation followed by liquid extraction and LC-MSn analysis. This workup was compared to established urine precipitation (UP) and validated according to international guidelines for qualitative approaches, using exemplary compounds of several drug classes (antidepressants, benzodiazepines, cardiovascular drugs, neuroleptics, opioids, stimulants, etc.) with a broad range of (physico-)chemical properties and chromatographic behaviors. On-spot conjugate cleavage and liquid extraction was sufficient for most compounds and the validation results comparable to those obtained after simple UP. For demonstrating the applicability, 103 authentic urine samples, six rat urine samples after low dose substance administrations, and two proficiency tests for systematic toxicological urinalysis were worked up with the new DUS approach or by UP without or with conjugate cleavage. In the authentic urine samples, 112 different drugs out of 43 categories plus metabolites were identified via the used LC-MSn library. With the new DUS approach, 5% less positive hits could be found compared to the UP approach and 15% less than the latter after conjugate cleavage. The differences should be caused mainly by smaller urine volumes used for DUS. In the two proficiency tests, all 15 drugs could be detected. Unfortunately, all three approaches were not able to detect very low-dosed substances in rat urine samples. However, they could be detected using a more sensitive LC-high resolution-MS/MS approach showing that the DUS workup was also suitable for those. In conclusion, DUS might be an alternative sampling technique for comprehensive drug testing or adherence monitoring.

Liquid chromatography-high resolution-tandem mass spectrometry using Orbitrap technology for comprehensive screening to detect drugs and their metabolites in blood plasma.

Orbitrap technology was successfully applied for broad metabolite-based LC-high resolution (HR)-MS screening of drugs in clinical and forensic toxicology. This paper aims to elucidate whether this technology can also be used for a corresponding blood plasma screening after simple precipitation without or with consecutive on-line extraction using turbulent flow chromatography (TurboFlow). The analytes were separated within 10 min and detected by a Q-Exactive mass spectrometer in full scan mode after positive/negative switching. In one single run, a target screening for about 700 relevant compounds was developed in parallel with data-dependent acquisition for unknowns. A compound was positively identified when the corresponding accurate mass precursor ion and the five most intense fragment ions were detected and the MS/MS spectrum fits well with the corresponding full HR-MS/MS reference library currently containing over 2000 parent drugs and 2500 metabolites. All over all run times were 17 min for precipitation and 21 min for TurboFlow after precipitation. Method validation was successfully performed for representative drugs and metabolites concerning recovery, matrix effect, process efficiency, and limits of detection and identification. Process efficiency data ranged for most analytes from 3 to 138% with coefficients of variation (CV) ≤ 20% for precipitation and from 1 to 156% with CV ≤ 20% for TurboFlow. Applicability studies showed that the developed method provided fast, simple, and robust screening and identification of a broad range of drugs within therapeutic ranges.

LC-HR-MS/MS standard urine screening approach: Pros and cons of automated on-line extraction by turbulent flow chromatography versus dilute-and-shoot and comparison with established urine precipitation.

Comprehensive urine screening for drugs and metabolites by LC-HR-MS/MS using Orbitrap technology has been described with precipitation as simple workup. In order to fasten, automate, and/or simplify the workup, on-line extraction by turbulent flow chromatography and a dilute-and-shoot approach were developed and compared. After chromatographic separation within 10min, the Q-Exactive mass spectrometer was run in full scan mode with positive/negative switching and subsequent data dependent acquisition mode. The workup approaches were validated concerning selectivity, recovery, matrix effects, process efficiency, and limits of identification and detection for typical drug representatives and metabolites. The total workup time for on-line extraction was 6min, for the dilution approach 3min. For comparison, the established urine precipitation and evaporation lasted 10min. The validation results were acceptable. The limits for on-line extraction were comparable with those described for precipitation, but lower than for dilution. Thanks to the high sensitivity of the LC-HR-MS/MS system, all three workup approaches were sufficient for comprehensive urine screening and allowed fast, reliable, and reproducible detection of cardiovascular drugs, drugs of abuse, and other CNS acting drugs after common doses.

Biotransformation and detectability of the new psychoactive substances N,N-diallyltryptamine (DALT) derivatives 5-fluoro-DALT, 7-methyl-DALT, and 5,6-methylenedioxy-DALT in urine using GC-MS, LC-MSn, and LC-HR-MS/MS.

Derivatives of N,N-diallyltryptamine (DALT) can be classified as new psychoactive substances. Biotransformation and detectability of 5-fluoro-DALT (5-F-DALT), 7-methyl-DALT (7-Me-DALT), and 5,6-methylenedioxy-DALT (5,6-MD-DALT) are described here. Their metabolites detected in rat urine and pooled human liver microsomes were identified by liquid chromatography (LC)-high resolution (HR)-tandem mass spectrometry (MS/MS). In addition, the human cytochrome-P450 (CYP) isoenzymes involved in the main metabolic steps were identified and detectability tested in urine by the authors' urine screening approaches using GC-MS, LC-MSn, or LC-HR-MS/MS. Aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations could be proposed for all compounds as main pathways. Carboxylation after initial hydroxylation of the methyl group could also be detected for 7-Me-DALT and O-demethylenation was observed for 5,6-MD-DALT. All phase I metabolites were extensively glucuronidated or sulfated. Initial phase I reactions were catalyzed by CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5. Rat urine samples were analyzed following two different low-dose administrations. GC-MS was not able to monitor consumption reliably, but all three compounds are predicted to be detectable in cases of overdose. The LC-MSn and LC-HR-MS/MS approaches were suitable for detecting an intake of all three compounds mainly via their metabolites. However, after the lowest dose, a reliable monitoring could only be achieved for 5-F-DALT via LC-MSn and LC-HR-MS/MS and for 7-Me-DALT via LC-HR-MS/MS. The most abundant targets in both LC-MS screenings were one of two hydroxy-aryl metabolites and both corresponding glucuronides for 5-F-DALT, one N-deallyl hydroxy-aryl, the carboxy, and one dihydroxy-aryl metabolite for 7-Me-DALT, and the demethylenyl metabolite, its oxo metabolite, and glucuronide for 5,6-MD-DALT.

New Psychoactive Substances 3-Methoxyphencyclidine (3-MeO-PCP) and 3-Methoxyrolicyclidine (3-MeO-PCPy): Metabolic Fate Elucidated with Rat Urine and Human Liver Preparations and their Detectability in Urine by GC-MS, "LC-(High Resolution)-MSn" and "LC-(High Resolution)-MS/MS".

BACKGROUND: 3-Methoxyphencyclidine (3-MeO-PCP) and 3-methoxyrolicyclidine (3-MeOPCPy) are two new psychoactive substances (NPS). The aims of the present study were the elucidation of their metabolic fate in rat and pooled human liver microsomes (pHLM), the identification of the cytochrome P450 (CYP) isoenzymes involved, and the detectability using standard urine screening approaches (SUSA) after intake of common users' doses using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-multi-stage mass spectrometry (LC-MSn), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). METHODS: For metabolism studies, rat urine samples were treated by solid phase extraction or simple precipitation with or without previous enzymatic conjugate cleavage. After analyses via LC-HR-MSn, the phase I and II metabolites were identified. RESULTS: Both drugs showed multiple aliphatic hydroxylations at the cyclohexyl ring and the heterocyclic ring, single aromatic hydroxylation, carboxylation after ring opening, O-demethylation, and glucuronidation. The transferability from rat to human was investigated by pHLM incubations, where Odemethylation and hydroxylation were observed. The involvement of the individual CYP enzymes in the initial metabolic steps was investigated after single CYP incubations. For 3-MeO-PCP, CYP 2B6 was responsible for aliphatic hydroxylations and CYP 2C19 and CYP 2D6 for O-demethylation. For 3-MeO-PCPy, aliphatic hydroxylation was again catalyzed by CYP 2B6 and O-demethylation by CYP 2C9 and CYP 2D6 Conclusions: As only polymorphically expressed enzymes were involved, pharmacogenomic variations might occur, but clinical data are needed to confirm the relevance. The detectability studies showed that the authors' SUSAs were suitable for monitoring the intake of both drugs using the identified metabolites.

Diphenidine, a new psychoactive substance: metabolic fate elucidated with rat urine and human liver preparations and detectability in urine using GC-MS, LC-MSn , and LC-HR-MSn.

Diphenidine is a new psychoactive substance (NPS) sold as a 'legal high' since 2013. Case reports from Sweden and Japan demonstrate its current use and the necessity of applying analytical procedures in clinical and forensic toxicology. Therefore, the phase I and II metabolites of diphenidine should be identified and based on these results, the detectability using standard urine screening approaches (SUSAs) be elucidated. Urine samples were collected after administration of diphenidine to rats and analyzed using different sample workup procedures with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-(high resolution)-mass spectrometry (LC-(HR)-MS). With the same approaches incubates of diphenidine with pooled human liver microsomes (pHLM) and cytosol (pHLC) were analyzed. According to the identified metabolites, the following biotransformation steps were proposed in rats: mono- and bis-hydroxylation at different positions, partly followed by dehydrogenation, N,N-bis-dealkylation, and combinations of them followed by glucuronidation and/or methylation of one of the bis-hydroxy-aryl groups. Mono- and bis-hydroxylation followed by dehydrogenation could also be detected in pHLM or pHLC. Cytochrome-P450 (CYP) isozymes CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were all capable of forming the three initial metabolites, namely hydroxy-aryl, hydroxy-piperidine, and bis-hydroxy-piperidine. In incubations with CYP2D6 hydroxy-aryl and hydroxy-piperidine metabolites were detected. After application of a common users' dose, diphenidine metabolites could be detected in rat urine by the authors' GC-MS as well as LC-MSn SUSA. Copyright © 2016 John Wiley & Sons, Ltd.

Orbitrap technology for comprehensive metabolite-based liquid chromatographic-high resolution-tandem mass spectrometric urine drug screening - exemplified for cardiovascular drugs.

LC-high resolution (HR)-MS well established in proteomics has become more and more important in bioanalysis of small molecules over the last few years. Its high selectivity and specificity provide best prerequisites for its use in broad screening approaches. Therefore, Orbitrap technology was tested for developing a general metabolite-based LC-HR-MS/MS screening approach for urinalysis of drugs necessary in clinical and forensic toxicology. After simple urine precipitation, the drugs and their metabolites were separated within 10 min and detected by a Q-Exactive mass spectrometer in full scan with positive/negative switching, and subsequent data dependent acquisition (DDA) mode. Identification criteria were the presence of accurate precursor ions, isotopic patterns, five most intense fragment ions, and comparison with full HR-MS/MS library spectra. The current library contains over 1900 parent drugs and 1200 metabolites. The method was validated for typical drug representatives and metabolites concerning recovery, matrix effects, process efficiency, and limits showed acceptable results. The applicability was tested first for cardiovascular drugs, which should be screened for in poisoning cases and for medication adherence of hypertension patients. The novel LC-HR-MS/MS method allowed fast, simple, and robust urine screening, particularly for cardiovascular drugs showing the usefulness of Orbitrap technology for drug testing.

Metabolism of the new psychoactive substances N,N-diallyltryptamine (DALT) and 5-methoxy-DALT and their detectability in urine by GC-MS, LC-MSn, and LC-HR-MS-MS.

N,N-Diallyltryptamine (DALT) and 5-methoxy-DALT (5-MeO-DALT) are synthetic tryptamine derivatives commonly referred to as so-called new psychoactive substances (NPS). They have psychoactive effects that may be similar to those of other tryptamine derivatives. The objectives of this work were to study the metabolic fate and detectability, in urine, of DALT and 5-MeO-DALT. For metabolism studies, rat urine obtained after high-dose administration was prepared by precipitation and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HR-MS-MS). On the basis of the metabolites identified, several aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations thereof are proposed as the main metabolic pathways for both compounds. O-Demethylation of 5-MeO-DALT was also observed, in addition to extensive glucuronidation or sulfation of both compounds after phase I transformation. The cytochrome P450 (CYP) isoenzymes predominantly involved in DALT metabolism were CYP2C19, CYP2D6, and CYP3A4; those mainly involved in 5-MeO-DALT metabolism were CYP1A2, CYP2C19, CYP2D6, and CYP3A4. For detectability studies, rat urine was screened by GC-MS, LC-MS(n), and LC-HR-MS-MS after administration of low doses. LC-MS(n) and LC-HR-MS-MS were deemed suitable for monitoring consumption of both compounds. The most abundant targets were a ring hydroxy metabolite of DALT, the N,O-bis-dealkyl metabolite of 5-MeO-DALT, and their glucuronides. GC-MS enabled screening of DALT by use of its main metabolites only.

Studies on the metabolism and toxicological detection of the new psychoactive designer drug 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) in human and rat urine using GC-MS, LC-MS(n), and LC-HR-MS/MS.

25I-NBOMe, a new psychoactive substance, is a potent 5-HT2A receptor agonist with strong hallucinogenic potential. Recently, it was involved in several fatal and non-fatal intoxication cases. The aim of the present work was to study its phase I and II metabolism and its detectability in urine screening approaches. After application of 25I-NBOMe to male Wistar rats, urine was collected over 24 h. The phase I and II metabolites were identified by LC-HR-MS/MS in urine after suitable workup. For the detectability studies, standard urine screening approaches (SUSA) by GC-MS, LC-MS(n), and LC-HR-MS/MS were applied to rat and also to authentic human urine samples submitted for toxicological analysis. Finally, an initial CYP activity screening was performed to identify CYP isoenzymes involved in the major metabolic steps. 25I-NBOMe was mainly metabolized by O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations of these reactions as well as by glucuronidation and sulfation of the main phase I metabolites. All in all, 68 metabolites could be identified. Intake of 25I-NBOMe was detectable mainly via its metabolites by both LC-MS approaches, but not by the GC-MS SUSA. Initial CYP activity screening revealed the involvement of CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The presented study demonstrated that 25I-NBOMe was extensively metabolized and could be detected only by the LC-MS screening approaches. Since CYP2C9 and CYP3A4 are involved in initial metabolic steps, drug-drug interactions might occur in certain constellations.

Direct analysis of the mushroom poisons α- and ß-amanitin in human urine using a novel on-line turbulent flow chromatography mode coupled to liquid chromatography-high resolution-mass spectrometry/mass spectrometry.

Poisonings with Amanita phalloides toxins require fast diagnosis in order to avoid expensive and unnecessary therapies. Initial clinical assessment in combination with urinary amanitin analysis is necessary for a definite diagnosis. Therefore, a simple, fast, and robust method was developed for reliable detection of α- and ß-amanitin as well as for fully validated quantification of α-amanitin in human urine. After simple dilution and centrifugation of the urine sample, a fast on-line extraction using a Transcend TLX-II system based on turbulent flow chromatography (TurboFlow) was established. A new TurboFlow mode was introduced, the pseudo quick elute mode (PQEM), which had more options for method optimization than the generic quick elute mode (QEM). It allowed running several modes in one valve arrangement. The PQEM showed better practicability in routine and emergency analysis than the previously used methods. After extraction, the fast 15min LC-high resolution (HR)-MS/MS analysis allowed reliable identification of α- and ß-amanitin based on fragments identified using so-called HR pseudo MS(3) experiments. According to international recommendations, the requirements for full validation including the parameters selectivity, calibration, accuracy, precision, recovery, matrix effects, and stability were fulfilled for α-amanitin. The method was successfully applied to the analysis of authentic urine samples containing amatoxins. In conclusion, this method allowed the determination of amatoxins using the novel PQEM in a faster, robust, and more reliable way than existing methods, making it suitable for daily routine and especially emergency toxicological analysis.