A highly sensitive LC-MS/MS method for the simultaneous quantification of Nitroglycerin and its two major metabolites in human plasma

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Nitroglycerin 1,2 Dintroglycerin 1,3 Dintroglycerin


Introduction: Nitroglycerin is a colorless, oily, explosive liquid produced by nitrating glycerol. Nitroglycerin forms free radical nitric oxide, which activates guanylate cyclase resulting in increased guanosine 3’5’monophosphate in smooth muscles and other tissues. These events lead to dephosphorylation of myosin light chain, which regulate the contractile state in smooth muscles and causes vasodilatation. Nitroglycerin is used to prevent chest pain (angina) in people with coronary artery disease.


Selective and highly sensitive methods at a lower limit of quantification (LLOQ) of about 20 pg/mL for nitroglycerin, 40 pg/mL for both active metabolites i.e 1, 2 dinitroglycerin and 1, 3 dinitroglycerin are required for their estimations in human plasma. This was accomplished with the new method developed at our end.


Method: Briefly, the method for the quantification of nitroglycerin, 1, 2 dinitroglycerin and 1, 3 dinitroglycerin involves solid phase extraction using nitroglycerin-d5, 1, 2 dinitroglycerin-d5 and 1, 3 dinitroglycerin-d5 as internal standards (ISTD).  Chromatographic separation of the drug and both metabolites was achieved on C8 column with a mobile phase consisting of ammonium chloride in methanol and ammonium chloride in water, with gradient time program, and a total run-time of 11 minutes and adequate separation between the drug and metabolites.


This new method was validated in terms of linearity, stabilities, carry-over effect, reinjection reproducibility, precision, accuracy and selectivity. The linearity ranged from 20 to 4000 pg/mL for nitroglycerin and 40 to 8000 pg/mL for both metabolites. As nitroglycerin and its two metabolites are unstable in blood, buffered blood and plasma was used for establishing stability and was found to be stable in plasma under the assay condition. Moreover, the method exhibited good precision and accuracy with precision even at the lower level of quantification. Also, there was no matrix effect observed in normal human lots and no inter-conversion issues.


This validated method is suitable for the analysis of samples from a single oral dose of 0.6 mg Nitroglycerin sublingual tablets in humans under fasting conditions, and will be applied to the analysis of study samples.


Conclusion: A selective, precise, accurate and highly sensitive LC-MS/MS method is now available for the simultaneous quantification of nitroglycerin, 1, 2 dinitroglycerin and 1, 3 dinitroglycerin in human plasma.

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