title: Simultaneous determination of glipizide and rosiglitazone unbound drug concentrations in plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry.
abstract: Glipizide and rosiglitazone are widely used to treat Type 2 diabetes. In order to investigate drug-drug protein binding interaction between glipizide and rosiglitazone, a method was developed and validated for simultaneously determining the free (unbound) fraction of glipizide and rosiglitazone in plasma employing equilibrium dialysis for the separation of free drug and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitation. Post-dialysis human plasma or buffer samples of 0.2 ml were extracted using a liquid-liquid extraction procedure and analyzed by a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Zorbax SB-Phenyl column, ionized using an atmospheric pressure electrospray ionization source and analyzed in positive ion mode with multiple reaction monitoring. The ion transitions monitored were m/z 446-->321 for glipizide, m/z 358-->135 for rosiglitazone, and m/z 271-->155 for tolbutamide (internal standard, IS). The chromatographic run time was 5 min per injection, with retention times of 2.3, 3.4 and 2.3 min for glipizide, rosiglitazone and IS, respectively. The calibration curves of glipizide and rosiglitazone were over the range of 1-2000 ng/ml (r(2)>0.9969) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v). The inter-assay precision and accuracy of the quality control samples were <10.9% of coefficient of variability and >93.5% and 94.5% of nominal concentration for glipizide and rosiglitazone, respectively. The lower limit of quantitation of both glipizide and rosiglitazone was 1.0 ng/ml. Both glipizide and rosiglitazone bound to plasma protein extensively (>99% bound). Glipizide and rosiglitazone free fraction averaged 0.678+/-0.071 and 0.389+/-0.061%, respectively, at plasma concentration of 1000 ng/ml. This developed method proves reproducible and sensitive and its application to clinical samples is also reported.
authors.affiliation: Avantix Laboratories, Inc., 57 Read's Way, New Castle, DE 19720, USA. linj@avantixlabs.com
authors.forename: Zhongping John
authors.initials: ZJ
authors.lastname: Lin
authors.forename: Daksha
authors.initials: D
authors.lastname: Desai-Krieger
authors.forename: Linyee
authors.initials: L
authors.lastname: Shum
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003956:Dialysis; D004355:Drug Stability; D005913:Glipizide / Q000097:blood*; D006801:Humans; D007004:Hypoglycemic Agents / Q000097:blood*; D011786:Quality Control; D015203:Reproducibility of Results; D000077154:Rosiglitazone; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D045162:Thiazolidinediones / Q000097:blood*
chemical_list: D007004:Hypoglycemic Agents; D045162:Thiazolidinediones; D000077154:Rosiglitazone; D005913:Glipizide