Simultaneous determination of nicotinamide and N1‐methylnicotinamide in human serum by LC–MS/MS to associate their serum concentrations with obesity

A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of nicotinamide and its metabolite N1 -methylnicotinamide in human serum. Serum samples were prepared by protein precipitation with acetonitrile. The chromatographic separation was performed on a Waters Spherisorb S5 CN microbore column (2.0 × 100 mm, 5 μm) with gradient elution within 7 min. Acetonitrile and 5 mm ammonium formate aqueous solution (containing 0.1% formic acid) were used as mobile phases. Nicotinamide, N1 -methylnicotinamide and N'-methylnicotinamide (internal standard) were detected with a triple-quadrupole tandem mass spectrometer in the positive ion mode. Multiple reaction monitoring was used to monitor precursor to product ion transitions of m/z 123.1 → 80.1 for nicotinamide, m/z 137.1 → 94.1 for N1 -methylnicotinamide and m/z 137.1 → 80.1 for the internal standard. The linear ranges of nicotinamide and N1 -methylnicotinamide were 5.000-160.0 and 2.500-80.00 ng/ml, respectively. The intra- and inter-day precisions (RSD) of both analytes were within 6.90%. The recoveries were >88%. The analytes were proven to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to determine the concentrations of nicotinamide and N1 -methylnicotinamide in human serum to investigate the association between their concentrations and obesity in 1160 Chinese subjects.