Development of green and high throughput microwell spectrophotometric and spectrofluorimetric assays for quantitation of roscovitine in its bulk form and capsules

Abstract

Roscovitine (RCV) is a drug with potential for the treatment of different types of cancer, neurodegenerative disease, renal dysfunction, viral infections, and chronic inflammatory disorders. This study describes, for the first time, the development and validation of three novel microwell-based assays for quantitation of RCV in its bulk form and capsules. These assays are two spectrophotometric assays and one spectrofluorimetric assay. The first spectrophotometric assay (UV-SPA) is based on measuring the native ultraviolet (UV) radiation absorption of RCV. The second assay (CT-SPA) is based on the formation of colored charge transfer (CT) complex upon the reaction of RCV, as n-electron donors, with 2,5-dichloro-3,6-dihydroxybenzoquinone (CHBQ), as a π-electron acceptor. The spectrofluorimetric assay (NF-SFA) is based on measuring the native fluorescence of RCV. These three assays were carried out in 96-microwell assay plates, and the signals were measured by a microplate reader. The optimum conditions of the three assays were established and their analytical performances were validated according to the guidelines of the International Council for Harmonization (ICH) for validation of analytical procedures. The linear ranges for the three assays were 10–300 µg mL1, 25–800 µg mL1 and 25–500 ng mL1 for UV-SPA, CT-SPA and NF-SFA, respectively. The limits of detection (LOD) of the assays were 4.1 µg mL1, 8.8 µg mL1 and 9.2 ng mL1 for UV-SPA, CT-SPA and NF-SFA, respectively. The accuracy and precision of the three assays were proved as the recovery values were ≥98.8%, and the relative standard deviations (RSD) did not exceed 2.32%. The three assays were successfully applied to the quantitation of RCV in its bulk form and capsules; the mean recovery values were ≥99.5%. The greenness of the assays was evaluated by three different metric tools, and the results proved that the three assays fulfill the requirements of green analytical approaches. In addition, the simultaneous handling of large number of samples with micro-volumes in the proposed assays gave these assays the property of high throughput analysis. In conclusion, the three assays are valuable for the rapid routine application in the pharmaceutical quality control units for the quantitation of RCV.

Keywords

• Roscovitine
• Microwell assays
• Spectrophotometry
• Spectrofluorimetry
• Green analytical approach
• High throughput analysis