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A method for the simultaneous determination of aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), taurine (Tau) and gamma-aminobutyric acid (GABA) in animal blood and brain by two-dimensional liquid chromatography (2D-LC) combined with ultraviolet detection was established for the first time. First, the amino acid neurotransmitters (AANTs) were labeled on the corresponding fluorescent derivatives with 4-fluoro-7-nitrobenzofurazan (NBD-F), enriched on the extraction column and automatically transferred to the analytical column to achieve on-line extraction and complete separation of the target components. This method exhibited good selectivity, and the correlation coefficients for the analyte calibration curves of were > 0.99. The intra- and inter-day precisions were ≤ 16.03, and the accuracies were in the range of 70.59-116.20%. The system realizes the rapid detection and stability quantification of the five AANTs, which proves that the alternative dilution method is feasible. The results show that the system has high loading capacity, excellent resolution, and good peak shape and is not affected by other endogenous substances. Moreover, the developed method has been successfully applied to the analysis of biological samples in the blood and whole brain of rats and pigs. The content of AANTs in the hippocampus and cortex of rats was higher than that in those of pigs. This method is expected to provide applicability for the determination of AANTs in pharmacological, pharmaceutical and clinical research in nervous science., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anabolic androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GC-MS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL -1 in 20 μL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse., Competing Interests: Declaration of Competing Interest The authors declare that N. Wojek is present employment by the UK Anti-Doping, the funder of this study. The funder does not have a role in the data collection, analysis, decision to publish, or preparation of the manuscript. The authors take complete responsibility for the integrity of the data and the accuracy of the data analysis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Tacrolimus whole-blood concentrations imperfectly reflect concentrations at the effect site. Tacrolimus concentrations in the transplanted organ could be more relevant to predict rejection events. Because liver biopsy cannot be repeatedly performed after liver transplantation, we suggested measuring tacrolimus in the bile to have a cost-effective and clinically implementable surrogate marker of intra-hepatic tacrolimus concentration. We developed and fully validated a liquid chromatography-tandem mass spectrometry method for the determination of tacrolimus in human bile. Sample purification was achieved using protein precipitation and liquid-liquid extraction with ethyl-acetate. Gradient elution was performed using a C18 analytical column with a 5min run-time. The method was linear from 0.5ng/mL to 20ng/mL. In this concentration range, within-day and between-day precisions as well as overall bias were within ±15%. Matrix effect was fully corrected by the internal standard (ascomycin). The assay was optimized to achieve good selectivity in this complex biological matrix. Tacrolimus was found to be stable in bile stored 6 months at -80°C, after 3 freeze and thaw cycles, 20h at room temperature and 24h in extracts kept at 15°C in the auto-sampler. The method was applied to quantify tacrolimus in bile from liver transplant recipients. It allowed getting preliminary data about tacrolimus excretion profile in bile and showed the lack of correlation between tacrolimus whole blood concentration and tacrolimus liver exposition. This alternative and innovative analytical approach of tacrolimus bio-analysis appears suitable for further studies evaluating relevance of biliary tacrolimus concentration as a new pharmacological marker of immunosuppressive activity., (Copyright © 2016 Elsevier B.V. All rights reserved.)