Authors:D. Antic, S. Filipic, B. Ivkovic, K. Nikolic, and D. Agbaba
A simple and rapid TLC method using β-cyclodextrin as a chiral mobile phase additive (CMPA) was developed for direct separation of S-clopidogrel and its impurity R-clopidogrel. The influence of different factors (stationary phases, organic modifiers, chiral selectors and their concentrations in the mobile phase, and optimal saturation time of the chamber) on enantioseparation was studied. The best resolution of clopidogrel enantiomers was achieved on Polygram®cel 300 Ac-10% plates using isopropanol-0.5 mM β-cyclodextrin (6:4, υ/υ) as mobile phase in TLC chamber previously equilibrated with the mobile phase for 20 min. The spots were detected under UV light and using iodine vapours. The method enables rapid separation of clopidogrel enantiomers and can be successfully used in control of stereoselective synthesis of clopidogrel and in control of its purity. Finally, the molecular modelling of the inclusion complexes between the analytes and α-, β-, and γ-cyclodextrin was performed to investigate the mechanism of the enantiorecognition.
Authors:B. Ivković, J. Brborić, V. Dobričić, and O. Čudina
A simple and convenient reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous separation, identification, and determination of sodium metabisulfite and sodium benzoate in pharmaceutical formulation has been developed and validated. Chromatographic separation was achieved on RP column Zorbax Extend C-18 (150 × 4.6 mm i.d., 3.5 μm particles), and mixture of 0.1% phosphoric acid and acetonitrile in the ratio 62:38 (v/v) was used as a mobile phase. The flow rate was set at 1.0 mL/min with detection wavelength of 275 nm. The method was successfully validated according to International Conference on Harmonization (ICH) guidelines acceptance criteria. The method is selective, as no interferences were observed at retention times corresponding to these analytes. Results of regression analyses (r) and statistical insignificance of calibration curve intercepts (p) proved linearity of the method in defined concentration ranges for sodium metabisulfite and sodium benzoate (0.05–0.15 mg/mL). Relative standard deviations calculated for both analytes in precision testing were below the limits defined for active pharmaceutical ingredients (analysis repeatability: <2%; intermediate precision: <3%). Recovery values were between 98.16% and 101.94%. According to results of robustness testing, chromatographic parameters are not significantly influenced by small variation of acetonitrile content in mobile phase, column temperature, and flow rate. Finally, the method was applied for quantitative determination of investigated preservatives in real sample analysis.
Authors:Biljana K. Tubić, Bojan D. Marković, Sandra S. Vladimirov, Slavica M. Ristić, Branka M. Ivković, Miroslav M. Savić, Jelena M. Poljarević, and Tibor J. Sabo
A series of new (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate esters has shown cytotoxic activity towards human leukemic cell lines. The aim of this study was to develop and validate a bioanalytical method for quantification of (S,S)-O,O-diethyl-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate dihydrochlorides (DE-EDCP) and its metabolite, substituted propanoic acid (EDCP), in mouse serum by ultra high-performance liquid chromatography—tandem mass spectrometry (UHPLC—MS/MS). Structural analog, derivative of 1,3-propanediamine, was used as an internal standard (IS). Sample preparation employed protein precipitation by acetonitrile and subsequent centrifugation. Optimal UHPLC separation conditions were set to achieve simultaneous determination of both compounds in a short run time of 6 min. Additionally, the selected reaction monitoring (SRM) mode developed in this method allowed a highly sensitive, accurate, and precise identification of compounds of interest. The lower limit of quantitation (LOQ) was 1.3 ng mL−1 for DE-EDCP and 0.3 μg mL−1 for EDCP. The calibration curves were linear over the concentration range of 1.3–26.7 ng mL−1 and 0.3–6.7 μg mL−1 for DE-EDCP and EDCP, respectively. Precision (%CV) and accuracy (%RE) for DE-EDCP and EDCP ranged from 3.5% to 16.0% and from 1.8% to 14.4%, respectively.
The validation process was performed in accordance with the regulatory guidance/guideline, and all of the obtained results met the established acceptance criteria. The newly developed and validated UHPLC—MS/MS method is rapid, sensitive, and selective, and it can be successfully applied to drug monitoring in nonclinical studies.