LC-ESI-MS-MS Determination of Rat Plasma Protein Binding of Major Flavonoids of Flos Lonicerae Japonicae by Centrifugal Ultrafiltration

A simple, precise, accurate, selective, and sensitive reversed-phase LC–UV method has been developed for simultaneous analysis of diltiazem and non-steroidal anti-inflammatory drugs (NSAIDs) in the bulk drug, tablet dosage forms, and human serum. Chromatographic separation of the drugs was performed at ambient temperature on a C₁₈ stationary phase with 80:20 (v/v) methanol–water, pH 3.1 ± 0.02, as isocratic mobile phase. The mobile phase flow rate was initially 0.5 mL min^?1 then increased to 1 mL min^?1. All the NSAIDs were well separated from each other and from diltiazem. Total run time was 10 min. The assay was successfully applied to pharmaceutical formulations and serum and there was no chromatographic interference from tablet excipients. The method was linear in the range 1.25–50 μg mL^?1 both for diltiazem and the NSAIDs. The suitability of this HPLC method for quantitative analysis of the drugs was proved by validation in accordance with International Conference on Harmonization (ICH) guidelines. The validation results, and results from statistical analysis of the data, demonstrated the method was reliable.