Controlled microwave derivatization reaction for reproducible trace analysis of budesonide in human plasma

Abstract

Microwave reactors with on-line controlled reaction conditions have been recently innovated to improve reaction kinetics and reproducibility. Herein, a modern microwave reactor was dedicated to develop a new, sensitive and reproducible approach for trace analysis of budesonide (BUD) in human plasma. The method was based on fast microwave reaction of BUD with dansyl hydrazine (DNS-HZ) reagent under controlled conditions coupled with high-performance liquid chromatography (HPLC)-fluorescence detection. The microwave irradiation and dansylation conditions were optimized for the best sensitivity and selectivity. The controlled microwave derivatization reaction (CMDR) decreased the reaction time, amplified the reaction yield and enhanced product purities by reducing the unwanted side reactions. The chromatographic separation was attained by isocratic elution on reversed phase column via a mobile phase consisted of methanol and phosphate buffer (10 mM, pH 7.0) at ratio 80:20 (v/v). The fluorescence detector was set at 500 nm after excitation at 330 nm. Betamethasone dipropionate (BDP) was used as an internal standard. CMDR-HPLC method validation was performed in agreement with bioanalytical method validation guidelines by the US food and drug administration (US-FDA). The obtained linearity range was 0.2–100 ng mL−1 with correlation coefficient 0.9991 and the lower limit of quantitation (LLOQ) in human plasma was 0.21 ng mL−1. The developed CMDR -HPLC method was applied successfully for assessment of plasma levels of BUD in allergic rhinitis patients after intranasal administration of the micronized BUD.