Diurnal variation in biofluid metabolome as observed in a healthy human may alter in perturbed conditions. Biofluids like urine are rich in molecular constituents including metabolites, and infectious disease conditions like tuberculosis (TB) may influence diurnal differences for which limited reports are available in the literature. In this study, we present an optimized gas chromatography coupled to a quadrupole mass spectrometry (GC-MS) method to analyze processed and trimethylsilyl (TMS)

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... ivatized urine metabolites. Urine samples were collected at four time points (0, 6, 12 and 24 hours) of study subjects [n=15; mean age 37 (24-70) in years] including controls [n=7; mean age 29.3 (24-35) years] and culture-confirmed active TB patients [ATB; n=8; mean age 43.7 (25-70) years] receiving treatment in the intensive phase. Global urine metabolite profiling was carried out using the optimized GC-MS method. Higher urine analyte diversity was observed in ATB patients (74) than in controls (36) during the day. Diurnal variations of the parent anti-TB drugs and their breakdown products (pyrazinamide, pyrazinoic acid, 5-hydroxy pyrazinoic acid, isonicotinic acid and alpha amino butyric acid) were observed with maximum abundance at 6 h. Interestingly, urine of ATB subjects at 6 h showed the highest metabolic diversity, whereas it was at 12 h in controls. Many analytes including glycine and alanine amino-acids showed diurnal variation in ATB and controls. These changes could be attributed to the altered host metabolic activities due to disease, treatment-associated decrease in total body bacterial burden and gut microbiota dysbiosis. And the optimized spiked-in internal standard, urine sample volume and GC-MS method could be used for global urine metabolome analysis in healthy and different perturbed conditions.