Purpose: 5-amino-1-methyl quinolinium is a potent Nicotinamide N-methyl transferase (NNMT) inhibitor. To facilitate the characterization of the compound, the objective of this study is therefore to develop and validate a simple, rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of 5-amino-1-methyl quinolinium in solution and rat plasma.
Methods: An LC system equipped with an ACE® Excel™ C18 column (2µm, 50×2.1 mm) was used for chromatographic separation with a binary solvent system comprising of water (A) and acetonitrile (B) containing 0.1% formic acid as mobile phase. Sample were analyzed using gradient elution: initial 20% B, 98% B from 1.5−2 minutes, and 20% B from 2.1 – 2.7 minutes at a total flow rate of 0.4 ml/min. Chromatographic analysis was performed using an API 4000 QTRAP hybrid triple quadruple linear ion trap mass spectrometer equipped with a Turbo V™ ion source. The internal standard used in this study is deuterated 5-amino-1-methyl quinolinium. Mass detection was with multiple reaction monitoring at positive ionization mode with m/z transitions of 160.008 → 90.000 (M+H)+ for the compound and 163.042 → 90.100 for the internal standard. The assay was validated according to the FDA’s Guidance for Bioanalytical Method Validation.
Results: The retention time for 5-amino-1-methyl quinolinium and internal standard is 0.36 minutes and the standard curve in solution and plasma is linear in concentration range of 10–2500 ng/mL. The intra- and inter-day accuracy (relative error) ranged from 0.22–1.77% and 0.63 – 4.18%, respectively. The intra- and inter-day precision (coefficient of variation) ranged from 4.19 – 12.4% and 4.49-8.69%, respectively. The mean extraction recovery rates were 108.23%, 110.59% and 99.47% for the low, medium and high QC concentrations in rat plasma, respectively. No measurable matrix effect interfered with the quantification and identification of 5-amino-1-methyl quinolinium.
The short term (6hrs) stability for the compound was 105.69% and 102.04% at the low and high QC concentrations; long term stability (15 days) at -80⁰C was found to be 95.67% and 105.27% at the low and high QC concentrations.
Conclusion: A rapid, simple and sensitive LC-MS/MS method was developed and validated to quantify 5-amino-1-methyl quinolinium in solution and rat plasma. This method was successfully applied to pharmacokinetic studies in rats.
Harshini Neelakantan– University of Texas Medical Branch
Stanley Watowich– University of Texas Medical Branch
Jing Ma– Texas Southern University
Dong Liang– PROFESSOR AND CHAIR, Texas Southern University, Houston, Texas