Simultaneous Determination of the Novel Antithrombotic Agent, Acetylsalicylic Acid Maltol Ester (Aspalatone) and its Metabolites in Rat Plasma and Urine by HPLC

Abstract

A rapid and sensitive reversed‐phase high performance liquid chromatography (HPLC) method was developed for the determination of the novel antithrombotic agent acetylsalicylic acid maltol ester (aspalatone, CAS: 147249‐33‐0) and its four metabolites, salicylic acid maltol ester (SME), salicylic acid (SA), salicyluric acid (SUA), and gentisic acid (GA), in rat plasma and urine. After a treatment of plasma or urine sample by liquid‐liquid extraction, the compounds were analyzed on an HPLC system with ultraviolet detection at 229 nm for aspalatone and SME, and at 313 nm for SA, SUA, and GA. HPLC analysis was carried out using reversed‐phase isocratic elution with a C18 column (4.6 mm×250 mm, 5 µm), a mobile phase of a mixture of butanol, acetic acid, doubly deionized water, and sodium sulfate (2∶5∶83∶10, v/v/v/w %) at a flow rate of 1.0 mL/min. The chromatograms showed good resolution and sensitivity and no interference of plasma and urine. The calibration curves for all substances in both plasma and urine samples were linear over the concentration range of 0.05–200 µg/mL for both plasma and urine. The intra‐ and inter‐day assay accuracies of this method were within 100±11% of nominal values and the precision did not exceed 13% of relative standard deviation. The lower limits of quantitation were 50 ng/mL for aspalatone and its metabolites in plasma and urine, which were sensitive enough for pharmacokinetic studies.