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. [ABSTRACT FROM AUTHOR]