Hydrophilic interaction liquid chromatography-tandem mass spectrometric approach for simultaneous determination of safingol and D-erythro-sphinganine in human plasma

Abstract

A simple and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for the simultaneous determination of C18-L-threo-sphinganine (safingol, an anti-neoplastic in phase I trials) and its diastereomer, C18-D-erythro-sphinganine (sphinganine), in human plasma. Sample pretreatment involved a protein precipitation with methanol using 25 μL aliquots of plasma. Chromatographic separation of the diastereomers and C17-D-erythro-sphinganine, an internal standard, was achieved on a Xbridge HILIC (3.5 μm, 100 × 2.1 mm) using isocratic elution with the mobile phase of 2 mM ammonium bicarbonate in water (pH 8.3) and acetonitrile at a flow rate of 0.3 mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were linear over the concentration range of 0.2-100 ng/mL with a lower limit of quantification of 0.2 ng/mL. The relative standard deviation of intra-day and inter-day precision was below 8.27%, and the accuracy ranged from 92.23 to 110.06%. The extraction recoveries were found to be higher than 93.22% and IS-normalized matrix effect was higher than 90.92%. The analytes were stable for the durations of the stability studies. The validated method was successfully applied to the analyses of pharmacokinetic samples from patients treated with safingol and all-trans-N-(4-hydroxyphenyl)retinamide; (fenretinide, 4-HPR) in a current phase I clinical trial (SPOC-2010-002, ClinicalTrials.gov Identifier: NCT01553071).

Keywords: HILIC; LC-MS/MS; Safingol; Sphinganine.

Conflict of interest statement

Conflict of interest

C. P. Reynolds and B.J. Maurer are co-inventors on issued patents for intravenous formulations of fenretinide and safingol with financial interests through institutional intellectual property revenue sharing agreements, and are also consultants to, and own stock in, CerRx, Inc., that licenses this technology. M. H. Kang is a consultant for CerRx, Inc

Copyright © 2019 Elsevier B.V. All rights reserved.

Figures

Fig. 1.

Product ion mass spectra of C18-L-threo-sphinganine (safingol, A), C18-D-etylhro-sphinganine (sphinganine, B) and the internal standard (IS) C17-sphinganine (C).

Fig. 2.

Separation of safingol and sphinganine using different buffers based on ammonium formate at pH 6.4 (A), ammonium acetate at pH 6.9 (B), or ammonium bicarbonate at pH 8.3 (C). HILIC was performed using 90:10 (v/v) acetonitrile:2 mM buffer at 30 °C.

Fig. 3.

Effects of the buffer concentration at 30 °C in acetonitrile: ammonium bicarbonate buffer (90:10, v/v) (A) and the column temperature in acetonitrile:2 mM ammonium bicarbonate buffer (90:10, v/v) (B) on the retention factors. Observed data are indicated by the closed circles for safingol and the closed squares for sphinganine.

Fig. 4.

Extracted ion chromatograms of safingol, sphinganine and C17-sphinganine (IS) in human plasma (top to bottom): (A) blank plasma; (B) plasma spiked with analytes at 0.2 ng/mL (LLOQ) and IS at 10 ng/mL; (C) patient sample after intravenous administration of safingol at 210 mg/m2/day as 48 hr infusion, RT: safingol (10.8), sphinganine (9.2) and IS (9.4).

Fig. 5.

Individual plasma concentration-time profile of safingol and fenretinide following continuous intravenous co-administration. Dose: mg/m2/day.