Metformin d6 hydrochloride

For PK analysis, serial blood samples were collected at 0, 0.5, 1, 2, 4, and 24 hours after the administration of metformin on day -7 or day -14 and day 1. Metformin plasma levels were quantitated using a liquid chromatography–mass spectrometry method. The Thermo Fisher Scientific LTQ FT Ultra mass spectrometer, a hybrid mass spectrometer consisting of a linear ion trap and a Fourier-transform ion cyclotron resonance mass spectrometer, was used for the detection of metformin. Metformin was separated from other analytes by reverse phase chromatography using a Waters XBridge Amide column (2.1 mm × 100 mm, 3.5 μm). The composition of the mobile phase was 25:75 (95:5 water:acetonitrile 0.1% HCOOH; and 95:5 acetonitrile:water 0.1% HCOOH). Metformin and the deuterated internal standard metformin-d6 hydrochloride (Toronto Research Chemicals) were detected by multiple reaction monitoring of the 130.11 m/z → 60 m/z and 136.15 m/z → 60 m/z quantifying transitions for metformin and metformin-d6 hydrochloride, respectively. PK analysis was performed using noncompartmental analysis (Phoenix 64 WinNonlin; Certara USA Inc).

Metformin (MET), glyburide (GLB), and glipizide were purchased from Sigma (Poole, England). Glyburide-d₁₁ and metformin-d₆ hydrochloride were purchased from Toronto Research Chemicals Inc. (Toronto, Canada). The metabolites of glyburide: 4-trans-hydroxycyclohexyl glyburide (M1) (purity ~ 99.9%), 4-cis-hydroxycyclohexyl glyburide (M2a) (purity ~ 96.0 %), 3-cis-hydroxycyclohexyl glyburide (M2b) (purity ~ 98.9%), 3-trans-hydroxycyclohexyl glyburide (M3) (purity ~ 97.8%), and 2-trans-hydroxycyclohexyl glyburide (M4) (purity ~ 96.0 %) were synthesized by Dr. Hill and made available to our laboratory (Hill et al., 2003 (link)). Acetonitrile, methanol, ammonium acetate, ethyl acetate and acetic acid were of HPLC grade and purchased from Fisher Scientific (Pittsburgh, USA). Blank human plasma was purchased from Innovative Research (Peary Court, USA). Blank human urine was collected from 6 healthy volunteers.