Development and validation of an LC–MS/MS method for determination of the L-type voltage-gated calcium channel/NMDA receptor antagonist NGP1-01 in mouse serum

• First reported LC–MS/MS technique for the quantification of NGP1-01.
• Method was validated for determination of NGP1-01 in mouse serum according to FDA bio analytical method guidelines.
• Ammonium acetate in the mobile phase enhanced the signal and improved the peak shapes of analyte and internal standard.

NGP1-01 (8-benzylamino-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane) is a heterocyclic cage compound with multifunctional calcium channel blocking activity that has been demonstrated to be neuroprotective in several neurodegenerative models. A sensitive internal standard LC–MS/MS method was developed and validated to quantify NGP1-01 in mouse serum. The internal standard (IS) was 8-(2-phenylethylamino)-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane. Sample preparation involved a protein precipitation procedure by addition of acetonitrile. Chromatographic separation was carried out on a Phenomenex Kinetex phenyl-hexyl column (100 mm × 2.1 mm, 2.6 μm) employing a gradient (45% isocratic 3 min, 45–95% linear gradient 6 min, 95% isocratic 3 min) of an elution mobile phase of 5 mM ammonium acetate in 100% acetonitrile mixing with an application mobile phase of 5 mM ammonium acetate in 2% acetonitrile. Detection was achieved by a QTrap 5500 mass spectrometer (AB Sciex) employing electrospray ionization in the positive mode with multiple-reaction-monitoring (MRM) for NGP1-01 (m/z 266→91) and IS (m/z 280→105). The method validation was carried out in accordance with Food and Drug Administration (FDA) guidelines. The method had a linear range of at least 0.5–50 ng/mL with a correlation coefficient 0.999. The intra-assay and inter-assay precisions (%CV) were equal to or within the range of 1.0–4.3% and the accuracies (% relative error) equal to or within −2.5% to 3.4%. The analyte was stable for at least 2 months at −20 °C, for at least 8 h at room temperature and for at least three freeze–thaw cycles. The extraction recovery was 94.9 to 105.0%, with a %CV ≤ 9.5%. The technique was found to be free of any matrix effects as determined by experiments involving five different lots of mouse serum. Cross-talk interferences were not present. Two different gradient slope chromatography runs were done on dosed mouse serum samples to assess a possible positive error in peak area determination from in-source fragmentation of metabolites generating the same MRM transitions as the parent drug or IS. No such interference was found in the NGP1-01 peak, while a minor interference was identified in the IS peak. The optimized method was applied to the measurement of NGP1-01 in serum of dosed mice.