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.
Papers of special note have been highlighted as: ▪ of interest ▪▪ of considerable interest
Bibliography
- 1 Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. (3rd Ed.). J. Wiley & Sons, Inc., NJ, USA 69–74; 253–303; 403–475 (2010).
- 2 Snyder LR, Kirkland JJ, Glajch JL. Practical HPLC Method Development. Wiley, NY, USA (1988).
- 3 Dolan JW. Broad peaks. LC–GC North Am.22,26–30 (2004).
- 4 Pedersen K, Dolan JW. A picture is worth a thousand words. LC-GC Europe24(2),72–76 (2011).
- 5 Keunchkarian S, Reta M, Romero L et al. Effect of sample solvent on the chromatographic peak shape of analytes eluted under reversed-phase liquid chromatographic conditions. J. Chromatogr. A1119(1–2),20–28 (2006).
- 6 Castells CB, Castells RC. Peak distortion in reversed-phase liquid chromatography as a consequence of viscosity differences between sample solvent and mobile phase. J. Chromatogr. A805(1–2),55–61 (1998).
- 7 Cherrak D, Guernet E, Cardot P et al. Viscous fingering: a systematic study of viscosity effects in methanol–isopropanol systems. Chromatographia46(11–12),647–654 (1997).
- 8 Snyder LR, Dolan JW. High Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model. 188–195; John Wiley & Sons, Inc., NJ, USA (2007).
- 9 Loeser E, Drumm P. Using strong injection solvents with 100% aqueous mobile phase in RPLC. J. Sep. Sci.29(18),2847–2852 (2006).▪ Highlights the possibility of using strong sample diluents with full aqueous mobile phases, without peak broadening and/or distortion.
- 10 Medvedovici A, Vasile D, Victor D et al. Retention phenomena induced by large-volume injection of solvents non-miscible with the mobile phase in reversed-phase liquid chromatography. J. Liq. Chromatogr. Relat. Technol.30(2),199–213 (2007).▪ Presents some of the basic aspects relating with the injection of large volumes from diluents not miscible with the mobile phase.
- 11 Udrescu S, Medvedovici A, David V. Effect of large-volume injection of hydrophobic solvents on the retention of less hydrophobic pharmaceutical solutes in RP-LC. J. Sep. Sci.31,2939–2945 (2008).
- 12 David V, Barcutean C, Georgita C et al. Non-miscible solvent large-volume injection – PLC–DAD method for determination of butylated hydroxyanisole in lovastatin and simvastatin pharmaceutical formulations. Rev. Roum. Chim.5,445–451 (2006).
- 13 Udrescu S, Sora ID, David V et al. Large-volume injection of hexane solutions in RPLC–UV to enhance on sensitivity of the assay of ginkgolic acids in Ginkgo Biloba standardized extracts. J. Liq. Chromatogr. Relat. Technol.33(1),133–149 (2010).
- 14 Loeser E, Babiak S, Drumm P. Water-immiscible solvents as diluents in reversed-phase liquid chromatography, J. Chromatogr. A.1216(15),3409–3412 (2009).▪▪ Describes an application in drug discovery of injection from diluents not miscible with the mobile phase.
- 15 Udrescu S, Sora ID, Albu F et al. Large-volume injection of 1-octanol as sample diluent in reversed-phase liquid chromatography: application in bioanalysis for assaying of indapamide in whole blood. J. Pharm. Biomed. Anal.54(5),1163–1172 (2011).▪▪ Describes the application of high-volume injection from immiscible solvents in bioanalysis considering whole blood samples.
- 16 Montes B, Catalan M, Roces A et al. Single dose pharmacokinetics of fenspiride hydrochloride: phase I clinical trial. Eur. J. Clinical. Pharmacol.45(2),169–172 (1993).
- 17 Janniot JP, Chezaubernard C. Pharmacokinetic properties of fenspiride: an update. Eur. Resp. Rev.1,101–104 (1991).
- 18 Sauveur C, Baune A, Vergnes N et al. Quantitative determination of fenspiride in human plasma and urine by liquid chromatography with electrochemical or ultraviolet detection. J. Pharm. Biomed. Anal.7(12),1799–1804 (1989).
- 19 Dumasia MC, Houghton E, Hyde W et al. Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography–mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose. J. Chromatogr. B767(1),131–144 (2002).
- 20 Guideline on Validation of Bioanalytical Methods. European Medicines Agency EMEA/CPMP/EWP/19221 (2009).
- 21 Lytle FE, Julian RK, Tabert AM. Incurred sample reanalysis: enhancing the Bland–Altman approach with tolerance intervals. Bioanalysis1(4),705–714 (2009).
