Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography–mass spectrometry with electrospray ionization

We have developed a liquid chromatographic–mass spectrometric method for the simultaneous determination of nitroglycerin (NTG) and its active metabolites, glyceryl 1,2-dinitrate (1,2-GDN) and glyceryl 1,3-dinitrate (1,3-GDN), for metabolism studies in cell cultures. 1,2,4-Butanetriol-1,4-dinitrate was chosen as an internal standard. Using a linear gradient of water/methanol containing 0.025 mM NH4Cl, the compounds were eluted within 12.5 min on an Allure Aqueous C18 column (100 mm × 2.1 mm). Detection and quantification was achieved with multiple reaction monitoring in the negative ion mode. Intra- and inter-day variabilities for simultaneous determination of the three nitrates were below 10 and 18%, respectively, over a range of NTG and GDN concentrations of 0.5–15 ng/ml. The lower limit of quantification was found to be about 0.01 ng on column. Application of this method was illustrated through in vitro metabolism studies of NTG in culture media bathing LLC-PK1 cells and human vascular smooth muscle cells (HA-VSMC) at 37 °C. The degradation half-life of NTG was found to be 4.5 ± 0.4 h and 39.2 ± 0.02 h, respectively, for LLC-PK1 cells versus HA-VSMC. At 5 h, the 1,2-GDN versus 1,3-GDN metabolite distribution ratio in the bathing medium was found to be 1.5 ± 0.1 and 0.2 ± 0.02 for LLC-PK1 and HA-VSMC cells, respectively. With this method, the degradation half-life of NTG in rat plasma at 37 °C was shown to be 26.8 ± 1.8 min, consistent with previous values obtained using gas chromatography.