Validation of a sensitive LC-MS assay for quantification of glyburide and its metabolite 4-transhydroxy glyburide in plasma and urine: an OPRU Network study

Abstract

Glyburide (glibenclamide, INN), a second generation sulfonylurea is widely used in the treatment of gestational diabetes mellitus (GDM). None of the previously reported analytical methods provide adequate sensitivity for the expected sub-nanogram/mL maternal and umbilical cord plasma concentrations of glyburide during pregnancy. We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for simultaneous determination of glyburide (GLY) and its metabolite, 4-transhydroxy glyburide (M1) in human plasma (0.5 mL) or urine (0.1 mL). The limits of quantitation (LOQ) for GLY and M1 in plasma were 0.25 and 0.40 ng/mL, respectively whereas it was 1.06 ng/mL for M1 in urine. As measured by quality control samples, precision (% coefficient of variation) of the assay was <15% whereas the accuracy (% deviation from expected) ranged from -10.1 to 14.3%. We found that the GLY metabolite, M1 is excreted in the urine as the glucuronide-conjugate.

Fig. 1

The chemical structure of GLY, M1 or M2 and the internal standard, GP (bottom).

Fig. 2

Full mass spectrum of GLY in positive ionization mode with fragmentor voltage of 50 V using direct injection of GLY (25 μg/mL) in 1:1 methanol: water Positive ionization mode gave 2 times better sensitivity as compared to negative ionization mode (A). Full mass spectrum of M1 in positive ionization mode with fragmentor voltage of 250 V using direct injection of M1 (10 μg/mL) in 1:1 methanol: water. M2 showed similar mass spectrum as M1. Abundance of sodium adducts (m/z 532.2) for M1 or M2 was 3-fold greater than that of the pseudomolecular ions (m/z 510.2) (B). Full mass spectrum of GP in positive ionization mode with fragmentor voltage of 50 V using direct injection of GP (25 μg/mL) in 1:1 methanol: water (C).

Fig. 3

Selected ion monitoring chromatogram for plasma-calibrator containing 2.5 ng/mL of GLY (m/z, 494.2) (A). Selected ion monitoring chromatogram for blank plasma (m/z, 494.2). The chromatogram shows no interference at the retention time of GLY (B). Selected ion monitoring chromatogram for clinical urine sample treated with 1000 U/mL of β-glucuronidase for 16 hr, showing M1, M2 and M3 (m/z, 532.2) (C). Selected ion monitoring chromatogram for blank urine (m/z, 532.2). The chromatogram shows no interference at the retention time of M1, M2/M3 (D). Selected ion monitoring chromatogram for 50 ng/mL of GP (m/z, 446.2) (E).

Fig. 4

Plasma concentration-time profile of glyburide and its metabolite M1 over a dosing interval in a gestational diabetes subject who received 1.25 mg oral dose of glyburide every 12 hrs. The amount of M1 excreted in the 12 hr-urine was 0.47mg.