Acute death associated with Citrobacter freundii infection in an African elephant (Loxodonta africana).

A 21-year-old male African elephant (Loxodonta africana) died suddenly with no previous medical history. Grossly, there were severe multifocal epicardial and endocardial hemorrhages of the atria and ventricles, hydropericardium, multifocal pleural hemorrhages, and severe pulmonary congestion and edema. Histologically, there was fibrinoid vasculitis and thrombosis in the heart and lung and myocardial necrosis. Citrobacter freundii was isolated in abundance in pure culture from liver and heart samples. Low levels of multiples types of elephant endotheliotropic herpesvirus (EEHV-6, EEHV-2B, and EEHV-3A) were detected in spleen samples, but not in heart samples. The levels of EEHV DNA found were much lower than those usually associated with acute EEHV hemorrhagic disease, and many other genomic loci that would normally be found in such cases were evidently below the level of detection. Therefore, these findings are unlikely to indicate lethal EEHV disease. Polymerase chain reaction for encephalomyocarditis virus (EMCV) and toxicology for oleander (Nerium oleander) were negative. Stress, resulting from recent transport, and antimicrobial therapy may have contributed to the death of this animal.

Liquid chromatography, in combination with a quadrupole time-of-flight instrument, with sequential window acquisition of all theoretical fragment-ion spectra acquisition: validated quantification of 39 antidepressants in whole blood as part of a simultaneous screening and quantification procedure.

Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is a data independent acquisition (DIA) method for very fast scanning quadrupole time-of-flight (QTOF) instruments. SWATH repeatedly cycles through 28 consecutive 20 Da precursor isolation windows detecting all precursor ions and fragments MS ALL like and yet fast enough to generate more than 10 data points over the chromatographic peak. It was already shown in previous publications that SWATH, despite its wide Q1 windows, allows the identification of different substances and that SWATH has a higher identification rate than data dependent acquisition approaches. The aim of this study was a proof of concept study whether these same data sets can also enable validated quantification according to international guidelines, exemplified for 39 antidepressants. The validation included recovery, matrix effects, process efficiency, ion suppression, and enhancement of coeluting ions, selectivity, accuracy, precision, and stability. The method using SWATH acquisition proved to be selective, sensitive, accurate, and precise enough for 33 out of the 39 antidepressants. The applicability of SWATH for screening and validated quantification in the same run was successfully tested with authentic whole blood samples containing different antidepressants and other drugs thus proving the QUAL/QUAN abilities of SWATH. In an additional systematic investigation, it could be shown that calibration curves injected a few days after or before the actual sample can be used for quantification with acceptable accuracy.

Comparison of conventional liquid chromatography-tandem mass spectrometry versus microflow liquid chromatography-tandem mass spectrometry within the framework of full method validation for simultaneous quantification of 40 antidepressants and neuroleptics in whole blood.

Microflow liquid chromatography (MFLC) coupled to mass spectrometry (MS) is claimed to improve analysis throughput, reduce matrix effects and lower mobile phase consumption. This statement was checked within the framework of method validation of a multi-analyte procedure in clinical and forensic toxicology employing MFLC-MS/MS and conventional LC-MS/MS. 200 µL whole blood were spiked with 50 µL internal standard mixture and extracted by protein precipitation. The concentrated extract was separated into two vials. One was analyzed using a Thermo Fisher Ultimate liquid chromatography system coupled to an ABSciex 5500 QTrap mass spectrometer (LC-MS/MS) and one by an ABSciex Eksigent Microflow LC system coupled to an ABSciex 4500 linear ion trap quadrupole MS (MFLC-MS/MS). Both methods were fully validated and compared in terms of selectivity, stability, limits, calibration model, recovery (RE), matrix effects (ME), bias, imprecision and beta tolerance interval for 40 antidepressants and neuroleptics including 9 metabolites. Both methods had comparable LODs, LOQs and calibration models with some exceptions. The MFLC system showed slightly higher coefficients of variation (CVs) in the RE experiments. ME were reproducible in both systems but with lower CVs in the conventional LC system. Acceptance criteria for imprecision and bias were fulfilled for 32 analytes on the LC and for 28 analytes on the MFLC system. Beta tolerance intervals indicated better reproducibility in terms of narrower intervals for the conventional LC system. The advantages of the MFLC system were low mobile phase consumption, short run time, and better peak separation. The systems were comparable in terms of peak interference, LOD, ME, bias and imprecision. The advantages of the conventional LC system were more data points per peak, linear calibration models, stable retention times and better beta tolerance intervals. Due to higher robustness, the conventional LC system was finally chosen for routine application in forensic toxicology.

Liquid chromatography, in combination with a quadrupole time-of-flight instrument (LC QTOF), with sequential window acquisition of all theoretical fragment-ion spectra (SWATH) acquisition: systematic studies on its use for screenings in clinical and forensic toxicology and comparison with information-dependent acquisition (IDA).

Forensic and clinical toxicological screening procedures are employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques with information-dependent acquisition (IDA) approaches more and more often. It is known that the complexity of a sample and the IDA settings might prevent important compounds from being triggered. Therefore, data-independent acquisition (DIA) methods should be more suitable for systematic toxicological analysis (STA). The DIA method sequential window acquisition of all theoretical fragment-ion spectra (SWATH), which uses Q1 windows of 20-35 Da for data-independent fragmentation, was systematically investigated for its suitability for STA. Quality of SWATH-generated mass spectra were evaluated with regard to mass error, relative abundance of the fragments, and library hits. With the Q1 window set to 20-25 Da, several precursors pass Q1 at the same time and are fragmented, thus impairing the library search algorithms to a different extent: forward fit was less affected than reverse fit and purity fit. Mass error was not affected. The relative abundance of the fragments was concentration dependent for some analytes and was influenced by cofragmentation, especially of deuterated analogues. Also, the detection rate of IDA compared to SWATH was investigated in a forced coelution experiment (up to 20 analytes coeluting). Even using several different IDA settings, it was observed that IDA failed to trigger relevant compounds. Screening results of 382 authentic forensic cases revealed that SWATH's detection rate was superior to IDA, which failed to trigger ∼10% of the analytes.

Single hair analysis of small molecules using MALDI-triple quadrupole MS imaging and LC-MS/MS: investigations on opportunities and pitfalls.

Single hair analysis normally requires extensive sample preparation microscale protocols including time-consuming steps like segmentation and extraction. Matrix assisted laser desorption and ionization mass spectrometric imaging (MALDI-MSI) was shown to be an alternative tool in single hair analysis, but still, questions remain. Therefore, an investigation of MALDI-MSI in single hair analysis concerning the extraction process, usage of internal standard (IS), and influences on the ionization processes were systematically investigated to enable the reliable application to hair analysis. Furthermore, single dose detection, quantitative correlation to a single hair, and hair strand LC-MS/MS results were performed, and the performance was compared to LC-MS/MS single hair monitoring. The MALDI process was shown to be independent from natural hair color and not influenced by the presence of melanin. Ionization was shown to be reproducible along and in between different hair samples. MALDI image intensities in single hair and hair snippets showed good semiquantitative correlation to zolpidem hair concentrations obtained from validated routine LC-MS/MS methods. MALDI-MSI is superior to LC-MS/MS analysis when a fast, easy, and cheap sample preparation is necessary, whereas LC-MS/MS showed higher sensitivity with the ability of single dose detection for zolpidem. MALDI-MSI and LC-MS/MS segmental single hair analysis showed good correlation, and both are suitable for consumption monitoring of drugs of abuse with a high time resolution.

Purple discoloration of the colon found during autopsy: Identification of betanin, its aglycone and metabolites by liquid chromatography-high-resolution mass spectrometry.

During autopsy of a 38-year-old man the forensic pathologist noted an atypical purple discoloration of the colon membrane. Hypothesis was that the discoloration could have been caused by ingestion of red beetroot. In order to exclude other toxicological causes for this finding and to analytically verify this hypothesis, colon membrane, blood and urine were screened not only for the typical forensically relevant substances but also for the main chromophoric beetroot compounds employing liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Betanin (m/z 551.1495) and its aglycone betanidin (m/z 389.0973) were found in the extracts of colon membrane and urine. Betanin was detected in whole blood, and urinary analysis additionally revealed two metabolites: betanidin glucuronide (m/z 565.1294) and betanidin sulfate (m/z 469.0541) - showing the same fragmentation pattern as betanidin after the characteristic neutral loss of m/z 176.0315 and m/z 79.9554 for glucuronic acid and sulfate, respectively. This is the first time that betacyanins could be analytically confirmed as cause for a purple discoloration of the colon. Urine analysis further revealed that besides betanin itself betanidin phase II metabolites could be detected in human urine.