A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte <20% for the lowest limit of quantification and <15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1–12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeat analysis during a run consisting of 18 plates. The % CV (coefficient of variation) values obtained for each analyte were <15%. Additional biological testing applied the assay to samples collected from healthy control participants and two groups diagnosed with inflammatory bowel disease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylic acid (5-ASA) and one group untreated), with results showing significant differences in the concentrations of picolinic acid, kynurenine, and xanthurenic acid. The short analysis time and 96-well plate format of the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MS metabolomic analysis in large-scale clinical and epidemiological population studies.

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo.

Capillary scale (100 mm × 150 μm id) UPLC/MS/MS, performed using reversed-phase gradient chromatography on sub 2 μm particles, has been successfully employed for the characterization of the metabolites of the drug tienilic acid (TA) excreted via the urine following oral administration to the rat. The capillary LC system provided a significant increase (range ca. 11-33-fold) in sensitivity compared with a conventional 150 mm × 2.1 mm id UPLC system. An investigation of the effect of the injection volume and sample mass loading on the capillary column on the results obtained for both endogenous metabolites and TA was performed. This demonstrated that the injection of up to 2 μL of rat urine onto the system was permitted whilst still providing excellent chromatographic results and robustness. Qualitative analysis of the urine revealed the presence of TA itself and a total of 15 metabolites of the drug, including those resulting from biotransformations such as hydroxylation or conjugation. The capillary chromatography system was shown to be robust, and capable of providing comprehensive drug metabolite profiles from small format urine samples such as those obtained from preclinical studies in rodents.

A U(H)PLC–MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.