Simultaneous determination of nicotinic acid and its four metabolites in rat plasma using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS).

A sensitive and specific liquid chromatography electrospray ionization–tandem mass spectrometry method for the simultaneous quantitation of nicotinic acid (NicA) and its metabolites nicotinamide (NA), 1-methylnicotinamide (MNA), 1-methyl-2-pyridone-5-carboxamide (M2PY) and 1-methyl-4-pyridone-5-carboxamide (M4PY) in rat plasma has been developed and validated. As an internal standard, 6-chloronicotinamide was used. The samples (100μL) were subjected to deproteinization with acetonitrile (200μL) and then, after centrifugation, 150μL of the supernatant was transferred into conical vial and evaporated. Dry residue was reconstituted in 100μL of the ACN/water (10:90, v/v) mixture. Chromatography was performed on a Waters Spherisorb® 5μm CNRP 4.6×150mm analytical column with gradient elution using a mobile phase containing acetonitrile and water with 0.1% of formic acid. The full separation of all compounds was achieved within 15min of analysis. Detection was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer set at unit resolution. The mass spectrometer was operated in the selected reactions monitoring mode (SRM), monitoring the transition of the protonated molecular ions m/z 153–110 for M2PY, 153–136 for M4PY, 124–80 for NicA, 123–80 for NA and 137–94 for MNA. The mass spectrometric conditions were optimized for each compound by continuously infusing the standard solution at the rate of 5μL/min using a Harvard infusion pump. Electrospray ionization (ESI) was used for ion production. The instrument was coupled to an Agilent 1100 LC system. The precision and accuracy for both intra- and inter-day determination of all analytes ranged from 1.3% to 13.3% and from 94.43% to 110.88%. No significant matrix effect (ME) was observed. Stability of compounds was established in a battery of stability studies, i.e. bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for various applications. In particular using this method we detected increased concentration of MNA and its metabolites in rat plasma after treatment with exogenous MNA (100mg/kg), as well as increased concentration of endogenous NA and MNA in rat plasma in the early phase of hypertriglyceridemia development in rats fed high-fructose diet.