Journal of Chromatography B, 854 (2007) 211–218 Simultaneous assay of metformin and glibenclamide in human plasma based on extraction-less sample preparation procedure and LC/(APCI)MS Cristina Georgita a , Florin Albu a , Victor David b , Andrei Medvedovici a,b,∗ a S.C. Labormed Pharma S.A., Splaiul Independentei No. 319E, Bucharest 060044, Romania b University of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, Sos. Panduri, No. 90, Bucharest 050663, Romania Received 26 February 2007; accepted 20 April 2007 Available online 1 May 2007 Abstract Separation of metformin and glibenclamide was achieved within a single chromatographic run on a Zorbax CN column, under isocratic conditions, using acetonitrile and aqueous component (0.01 moles/L ammonium acetate adjusted at pH 3.5 with acetic acid) in volumetric ratio 1/1. Plasma sample preparation is based on protein precipitation by means of organic solvent addition. 1,3,5-Triazine-2,4,6-triamine (IS1) was used as internal standard for metformin, while gliquidone (IS2) played the same role for glibenclamide. Detection was performed with an ion trap mass analyzer, using atmospheric pressure chemical ionization (APCI). A single MS stage was used for detection of metformin and IS1, by extracting ion chromatograms corresponding to molecular ions. MS/MS detection in the SRM mode was used for glibenclamide (m/z transition from 494 to 369 Da) and IS2 (m/z transition from 528 to 403 Da). The method produces linear responses up to 2000 ng/mL for metformin and 400 ng/mL for glibenclamide, respectively. Low limits of quantification were found in the 40 ng/mL range for metformin and at the 4 ng/mL level for glibenclamide. Precision was characterized by relative standard deviations (RSD%) below 9%. The analytical method was successfully applied to a single dose, open-label, randomized, two-period, two-sequence, crossover bioequivalence study of two commercially available anti-diabetic combinations containing 400 mg metformin and 2.5 mg of glibenclamide per coated tablet. © 2007 Elsevier B.V. All rights reserved. Keywords: Metformin; Glibenclamide; Anti-diabetic combination; Simultaneous assay; Human plasma; Extraction-less sample preparation; APCI; MS detection; Method validation; Bioequivalence; Pharmacokinetic parameters 1. Introduction Metformin (1-(diaminomethylidene)-3,3-dimethyl-guani- dine) is an anti-diabetic drug from the biguanide class [1]. Glibenclamide (5-chloro-N-[2-[4-(cyclohexylcarbamoyl-sulfa- moyl) phenyl]ethyl]-2-methoxy-benzamide), also known as glyburide, is an anti-diabetic drug belonging to the sulfonylurea class, used in the treatment of type 2 diabetes [2]. Metformin combined with glibenclamide is a second-line drug designed for type 2 diabetes mellitus treatment when either drug alone does not improve glycaemic control [3]. ∗ Corresponding author at: University of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, Sos. Panduri, No. 90, Bucharest 050663, Romania. Tel.: +40 723 330 423; fax: +40 21 221 36 92. E-mail addresses: avmedved@yahoo.com, qc@labormedpharma.ro (A. Medvedovici). Literature data report several individual methods for the determination of metformin and glibenclamide in biological samples. HPLC separations with UV or fluorescence detection are usually not suitable for the assay of glibenclamide in human plasma after administration of its therapeutic doses since these methods do not have enough sensitivity and/or specificity. There- fore, LC/MS methods using either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) modes are mainly used to assay glibenclamide in plasma samples [4–8]. For the assay of metformin in human plasma, different types of HPLC-UV methods (based on ion-exchange, ion-pair, or reversed-phase separation mechanisms) are used with different types of complex, time-consuming sample preparation proce- dures, such as chemical derivatisation, liquid–liquid extraction or solid phase extraction [9–11]. The analytical challenge is due to the high polarity of metformin which makes it difficult to extract from biological fluids and causes its rapid elution by 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.04.032