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

doi:10.1016/j.jchromb.2014.05.048

Journal of Chromatography B

Volume 963, 15 July 2014, Pages 83-89

https://doi.org/10.1016/j.jchromb.2014.05.048 Get rights and content

Highlights

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

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 × 2.1 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.

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[5.4.0.0^2,6.0^3,10.0^5,9]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[5.4.0.0^2,6.0^3,10.0^5,9]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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View full text

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

Highlights

Abstract

Introduction

Section snippets

Chemicals and materials

Mass spectra and liquid chromatography

Conclusion

Acknowledgements

Pharmacol. Res. Commun.

Bioorg. Med. Chem.

Brain Res.

Eur. J. Pharmacol.

Bioorg. Med. Chem.

Brain Res.

Neurosci. Lett.

Neurochem. Res.

Stroke

Physiol. Rev.