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
Introduction
An increasingly important focus in drug discovery research in recent years is development of multifunctional drugs, agents with more than one therapeutic mechanism. A promising multifunctional agent which has shown neuroprotection in neurodegenerative disease systems is NGP1-01, the pentacycloundecylamine 8-benzylamino-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane. NGP1-01 (Fig. 1A) is a heterocyclic cage compound first characterized by the Van der Schyf group in the mid-1980s [1]. NGP1-01 has been shown to produce neuroprotective effects by inhibiting calcium uptake by acting as an uncompetitive antagonist of both the ligand-operated calcium channel [N-methyl-d-aspartate (NMDA) receptor] and the voltage-gated calcium channel in neuronal cells [2], [3]. This inhibition prevents an increase in intracellular calcium, protecting against the excitotoxicity response that would lead to neuronal cell death by necrotic or apoptotic mechanisms caused by increased intracellular calcium [4], [5], [6]. NGP1-01 and derivatives of NGP1-01 have also been shown to have other functional protective effects pertinent to neurodegenerative diseases [7], [8], [9], [10], [11]. NGP1-01 is thus a promising therapeutic candidate for treatment of neurodegenerative disorders through its multimodal effects.
Development of sensitive analytical methodology for the determination of NGP1-01 in biological samples is warranted. The only reported analytical method for NGP1-01 is a HPLC technique employing UV absorbance detection at 210 nm, which was applied to the determination of the compound in aqueous solutions assessing compound stability [12], [13]. This technique is not applicable to biological samples because of the poor limit of detection and low analytical specificity inherent in absorbance detection. Although a LC–MS technique for NGP1-01 was reported in these references, the technique used was an out-of-date particle beam ionization technology and it was only used for mass spectral identification of the HPLC peak and not for the quantification of the compound.
The present work reports the development and validation of a sensitive and specific LC–MS/MS technique for NGP1-01, applied to the determination of NGP1-01 in mouse serum.
Section snippets
Chemicals and materials
NGP1-01 (Fig. 1A) and the internal standard (IS), 8-(2-phenylethylamino)-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane (Fig. 1B), were synthesized and purified [1], [10]. Results of C, H, N elemental analysis of the purified NGP1-01 and IS were at most ±0.4% from the expected percentages, indicating that the compounds were essentially pure. HPLC grade dimethyl sulfoxide (DMSO) and Optima LC/MS grade acetonitrile (ACN) were from Fisher Scientific (Fair Lawn, NJ, USA). ACS reagent grade
Mass spectra and liquid chromatography
Infusion studies identified protonated parent molecular ions at m/z 266 and 280 for NGP1-01 and IS respectively, which generated predominant daughter ions at m/z 91 and 105 respectively. MRM transitions of m/z 266→91 for NGP1-01 and 280→105 for IS were thus chosen for quantification in this study. Infusion studies also identified minor daughter ions at m/z 248 and 65 for NGP1-01 and 262, 159 and 79 for IS.
Various isocratic chromatographic schemes employing different concentrations of ACN or
Conclusion
A rapid and sensitive internal standard LC–MS/MS method has been developed and validated for the quantitative measurement of NGP1-01 in mouse serum. A simple protein precipitation method was used to prepare samples. The method employed a phenyl-hexyl reversed-phase HPLC column for separation of the analyte and the internal standard and a MRM detection mode for sensitive and selective detection of the compounds. An ACN gradient with addition of ammonium acetate to the mobile phase was found to
Acknowledgements
The authors acknowledge the following sources of support for this work:
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“Bloomberg Foundation, Youngstown, Ohio” supported the synthesis of NGP-01 and the purchase and preparation of the mice.
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“Fight for Sight” supported the synthesis of NGP-01 and the purchase and preparation of the mice.
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“The National Science Foundation Major Research Instrumentation Grant (CHE-0923398)” supported the requisition of the AB Sciex QTrap 5500 mass spectrometer instrument.
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