title: 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.
abstract: NGP1-01 (8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]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[5.4.0.0(2,6).0(3,10).0(5,9)]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×2.1mm, 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 5mM ammonium acetate in 100% acetonitrile mixing with an application mobile phase of 5mM 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 8h 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.
authors.affiliation: Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA.
authors.forename: Harini
authors.initials: H
authors.lastname: Jogiraju
authors.affiliation: Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA.
authors.forename: Ashta Lakshmi Prasad
authors.initials: AL
authors.lastname: Gobburi
authors.affiliation: Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA.
authors.forename: Kiran K
authors.initials: KK
authors.lastname: Pedada
authors.affiliation: Department of Pharmaceutical Sciences, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA.
authors.forename: Werner J
authors.initials: WJ
authors.lastname: Geldenhuys
authors.affiliation: Graduate School and Department of Biomedical and Pharmaceutical Sciences, Idaho State University, Pocatello, ID 83209-8075, USA.
authors.forename: Cornelis J
authors.initials: CJ
authors.lastname: Van der Schyf
authors.affiliation: Department of Pharmaceutical Sciences, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA.
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Crish
authors.affiliation: Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA. Electronic address: d.anderson@csuohio.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Anderson
mesh_terms: D000818:Animals; D001952:Bridged-Ring Compounds / Q000097:blood*; D002121:Calcium Channel Blockers / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D057230:Limit of Detection; D051379:Mice; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001952:Bridged-Ring Compounds; D002121:Calcium Channel Blockers; D016194:Receptors, N-Methyl-D-Aspartate; C049649:NGP 1-01
keywords: LC–MS/MS; Mouse serum; Multifunctional drug; NGP1-01; Neuroprotective agent; Pentacycloundecylamine