Analysis of Nitroglycerin Metabolites in Blood Plasma Using NCI-GC/M

Significance of the Topic

Nitroglycerin serves dual roles as a pharmaceutical vasodilator and explosive. Accurate quantification of its metabolites in blood is essential for optimizing therapeutic regimens while minimizing hypotensive side effects. Additionally, monitoring downstream nitric oxide metabolites (nitrite and nitrate) provides insight into drug bioactivation and physiological responses.

Study Objectives and Overview

This work aims to establish a unified NCI-GC/MS method to quantify nitroglycerin and its primary metabolites (1,2-glyceryl dinitrate, 1,3-glyceryl dinitrate, and glyceryl mononitrate) alongside nitric acid metabolites (nitrite and nitrate) in blood plasma. The goal is to achieve low-nanomolar to micromolar sensitivity and assess the potential of metabolite profiles for personalized dosing guidance.

Methodology

Sample preparation involves liquid–liquid extraction from plasma using toluene and incorporation of an internal standard (o-iodobenzyl alcohol for nitroglycerin series; 15N-labeled nitrite/nitrate for nitric acid derivatives). Nitroglycerin metabolites are extracted and treated with triethylamine to prevent analyte adsorption, then concentrated and transferred to GC vials. Nitrite/nitrate are derivatized with pentafluorobenzyl bromide in acetone at 50 °C, dried, and extracted into toluene.

Instrumentation Used

• GC-MS system: Shimadzu GCMS-QP2020 NX
• Auto-injector: AOC-30i/20s
• Column: InertCap 17MS (30 m × 0.25 mm × 0.25 µm)
• Ionization: Negative chemical ionization (NCI) with methane reagent gas
• Injection mode: Splitless (150 °C for nitroglycerin; 250 °C for nitrate/nitrite)
• Carrier gas: Helium at linear velocity ~40 cm/s
• Data acquisition: Scan and SIM modes monitoring key m/z values (e.g., 62, 46, 91, 127)

Main Results and Discussion

Nitroglycerin and its glycerol nitrates were detectable at 5 nM in water standards, with calibration curves linear (R2 > 0.997) up to low micromolar levels. Omission of triethylamine led to complete analyte loss due to adsorption. Time-course analysis of plasma samples post-dosing revealed clear trends in GDN and GMN concentrations.

For nitric acid metabolites, NCI sensitivity exceeded EI by over two orders of magnitude. Derivatized nitrite and nitrate achieved limits of detection at 0.75 µM and 7.5 µM respectively, with linear calibration (R2 > 0.997). Plasma measurements showed nitrite ~47 µM and nitrate ~2.5 µM after nitroglycerin administration, demonstrating feasibility for in vivo monitoring.

Benefits and Practical Applications

• High sensitivity quantification of nitroglycerin metabolites at nanomolar levels
• Single GC-MS configuration covers both organic nitrates and nitric acid derivatives
• Potential to tailor nitroglycerin dosing and reduce adverse effects via metabolite profiling
• Robust sample workup minimizes analyte loss and simplifies clinical implementation

Future Trends and Possibilities

The integrated approach may expand to real-time pharmacokinetic studies and personalized medicine. Further enhancements could include automated PTV inlets for thermally labile analytes and application to other nitrate drugs or biomarkers of nitric oxide pathways. Combining this method with high-throughput sample handling may support large clinical trials and therapeutic monitoring.

Conclusion

An NCI-GC/MS workflow was developed for simultaneous measurement of nitroglycerin, its liver-formed metabolites, and nitric acid products in plasma. By leveraging derivatization and masking strategies, the method achieves nanomolar to micromolar sensitivity, paving the way for metabolite-guided dosing of nitroglycerin in clinical settings.

Reference

• Dimitrios Tsikas, Analytical Chemistry, 2000, 72, 4064–4072
• Peter Akrill, John Cocker, Journal of Chromatography B, 778 (2002) 193–198
• Martin Jorgensen, Michael P. Andersen, Journal of Chromatography, 517 (1992) 167–170