Large-volume injection of sample diluents not miscible with the mobile phase as an alternative approach in sample preparation for bioanalysis: an application for fenspiride bioequivalence

Author:

Medvedovici Andrei,Udrescu Stefan1,Albu Florin1,Tache Florentin2,David Victor2

Affiliation:

1. Bioanalytical Laboratory, SC Labormed Pharma SA, #44B Th. Pallady Blvd, Bucharest – 032266, Romania

2. University of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, #90 Panduri Av., Bucharest – 050663, Romania

Abstract

Background: Liquid–liquid extraction of target compounds from biological matrices followed by the injection of a large volume from the organic layer into the chromatographic column operated under reversed-phase (RP) conditions would successfully combine the selectivity and the straightforward character of the procedure in order to enhance sensitivity, compared with the usual approach of involving solvent evaporation and residue re-dissolution. Large-volume injection of samples in diluents that are not miscible with the mobile phase was recently introduced in chromatographic practice. The risk of random errors produced during the manipulation of samples is also substantially reduced. Results: A bioanalytical method designed for the bioequivalence of fenspiride containing pharmaceutical formulations was based on a sample preparation procedure involving extraction of the target analyte and the internal standard (trimetazidine) from alkalinized plasma samples in 1-octanol. A volume of 75 µl from the octanol layer was directly injected on a Zorbax SB C18 Rapid Resolution, 50 mm length × 4.6 mm internal diameter × 1.8 µm particle size column, with the RP separation being carried out under gradient elution conditions. Detection was made through positive ESI and MS/MS. Aspects related to method development and validation are discussed. Conclusions: The bioanalytical method was successfully applied to assess bioequivalence of a modified release pharmaceutical formulation containing 80 mg fenspiride hydrochloride during two different studies carried out as single-dose administration under fasting and fed conditions (four arms), and multiple doses administration, respectively. The quality attributes assigned to the bioanalytical method, as resulting from its application to the bioequivalence studies, are highlighted and fully demonstrate that sample preparation based on large-volume injection of immiscible diluents has an increased potential for application in bioanalysis.

Publisher

Future Science Ltd

Subject

Medical Laboratory Technology,Clinical Biochemistry,General Pharmacology, Toxicology and Pharmaceutics,General Medicine,Analytical Chemistry

Reference21 articles.

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2. SnyderLR, Kirkland JJ, Glajch JL.Practical HPLC Method Development. Wiley, NY, USA (1988).

3. Effect of sample solvent on the chromatographic peak shape of analytes eluted under reversed-phase liquid chromatogaphic conditions

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