Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites

Abstract

A sensitive, specific capillary gas chromatographic-electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs greater than 76% and for GTN and the GDNs greater than 95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1.3-GDN maximum concentrations (Cmax) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the Cmax ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN Cmax ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.