Visible diode laser induced fluorescence detection of doxorubicin in plasma using pressurized capillary electrochromatography

Abstract

Pressurized capillary electrochromatography is a variant of capillary electrochromatography (CEC) in which the driving force is both electroosmotic and hydraulic. The inlet of the CEC capillary is pressurized using an HPLC pump, and an electric field is simultaneously applied. This work describes a method for the analysis of doxorubicin. Doxorubicin was reacted with Cy5.29.OSu in acetonitrile. The derivative was confirmed by RP-TLC. A CEC system equipped with a VDLIF detector was constructed and used to analyze the derivative. The reaction mixture was injected onto a capillary packed in-house with 3 microm C-18 Luna particles and separation was carried out at 25 kV using 70% acetonitrile/ 30% phosphate (10 mM, pH = 4.8) as the mobile phase. The derivatization reaction was optimized by the investigation of parameters such as reaction time, temperature and concentration of label in order to increase the yield of the derivative. The optimal conditions were determined to be 30 min, 80 degrees C and 50 nmol/mL, respectively. Doxorubicin was extracted from plasma using solid-phase extraction under alkaline conditions, derivatized and injected onto the CEC-VDLIF system. The selectivity of the assay was demonstrated by a lack of interfering peaks due to plasma constituents across the elution window of the derivative peak in blank plasma extracts (n = 6 sources). The limit of detection (LOD) of the assay in plasma calculated as 3 s(b)/m was determined to be 1.7 ng/mL. The precision of the assay determined at a concentration of 167.7 ng/mL (n = 5) was found to be within 7.04 %RSD.