Validated RP-LC Methods for Investigation the Degradation Behavior of Acefylline: Application for Analysis in Two Binary Mixtures

Background: Acefylline (Acef) is a derivative of theophylline that has bronchodilator effects. Two binary mixtures were marketed for Acef: Acefylline piperazine/ Phenobarbitone (Acef-P/Phen) and Acefylline heptaminol/ Cinnarizine (Acef-H/ Cinn). To our knowledge none of the reported methods had the capacity to determine Acef in its binary mixture in presence of its degradation products and potential impurity theophylline (Theo). Methods: Two validated RP-LC methods were established for the determination of Acef-P/Phen and Acef-H/ Cinn in presence of Acef degradation products and its potential impurity Theo. A complete study of the forced acidic, alkaline and oxidative degradation of Acef was presented. The methods were based on LC separation on RP C18 columns using isocratic and gradient elution for Acef-P /Phen and Acef-H /Cinn mixtures, respectively. Different chromatographic conditions were examined and optimized. Results: Linear responses were attained over concentration ranges of 75-500/15-1000 μg/mL and 100-1000 /50- 500 μg/mL with mean percentage recoveries of (100.72±1.23)%/ (99.29±1.12)% and (100.44±1.27)%/ (99.01±0.97)% for Acef-P/Phen and Acef-H /Cinn, respectively. ICH guidelines were used for methods validation and all parameters were found to be acceptable. Conclusion: The methods showed to be accurate, precise and specific for the analysis of Acef-P/Phen and AcefH /Cinn in drug substance, drug product and in laboratory prepared mixtures in presence of Theo and up to 50% of degradation products. The structures of the main degradation products and the expected degradation pathway were suggested using the MS data.

Chromatography of Hedysarum extracts on modified Sepharose CL sorbents

Aim. To study the chromatographic efficiency of modified Sepharose CL-4B sorbents for separation, purification, and preparation of major components from Hedysarum theinum Krasnob. and Hedysarum neglectum Ledeb. root extracts.Materials and Methods. Sorption capacity of the native and modified sepharose was evaluated in both isocratic and gradient elution modes. 4-pyridinecarboxylic acid derivative [(4-hydroxy-3-methoxyphenyl)methylene]hydrazide (ftivazide) was used as a sorbent modifier ligand. We studied chromatographic properties of Sepharose CL-4B, Sepharose CL-6B, and azoepoxyadsorbent Sepharose CL-4B-DEP-p-nitrobenzohydrazide-ftivazide, a chemically modified analogue of Sepharose CL-4B.Results. Sepharose CL-4B and Sepharose CL-4B-DEP-p-nitrobenzohydrazide-ftivazide were found efficient carriers for liquid column chromatography of water-ethanol Hedysarum extracts. Complete elution of bioactive substances has been achieved exclusively by using sodium tetraborate as an eluent. We were able to identify flavonoids and tannins of different condensation in chromatographic fractions.Conclusion. Comparative chromatography of bioactive substances from Hedysarum root extracts is more efficient if employing Sepharose CL-4B instead of Sepharose CL-6B as a sorbent. Sepharose CL-4B-DEP-p-nitrobenzohydrazide-ftivazide demonstrates excellent separation capacity for tannins and flavonoids.

Strategic Combination of Isocratic and Gradient Elution for Simultaneous Separation of Polar Compounds in Traditional Chinese Medicines by HPLC

A simple high-performance liquid chromatography (HPLC) method for the simultaneous separation of the highly polar and weakly polar components of traditional Chinese medicines was developed via a strategic combination of isocratic and gradient elution methods. Liu-Shen-Wan and Liu-Wei-Di-Huang-Wan were used as representative examples of traditional Chinese medicines. This is the first time that 6 components of varying degrees of polarity in Liu-Shen-Wan had been successfully resolved in a single chromatographic run using an ultraviolet-visible detector with a fixed wavelength of 296 nm. In contrast to conventional gradient separation methods, this novel method offered a viable route for separation of the highly and weakly polar fractions simultaneously, thus greatly reducing the time and cost of analysis. This method therefore provides a more efficient way to determine the polar components present in traditional Chinese medicines. It would find potential application in drug screening, drug authentication, and product quality control.