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

Pressurized capillary electrochromatography is a variant of capillary elect rochromatography (CEC) in which the driving force is both electroosmotic an d hydraulic. The inlet of the CEC capillary is pressurized using an HPLC pu mp, 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 deri vative. The reaction mixture was injected onto a capillary packed in-house with 3 mu m C-ls Luna particles and separation was carried out at 25 kV usi ng 70% acetonitrile/30% phosphate (10 mM, pH = 4.8) as the mobile phase. Th e derivatization reaction was optimized by the investigation of parameters such as reaction time, temperature and concentration of label in order to i ncrease the yield of the derivative. The optimal conditions were determined to be 30 min, 80 degrees C and 50 nmol/mL, respectively. Doxorubicin was e xtracted from plasma using solid-phase extraction under alkaline conditions , derivatized and injected onto the CEC-VDLIF system. The selectivity of th e assay was demonstrated by a lack of interfering peaks due to plasma const ituents across the elution window of the derivative peak in blank plasma ex tracts (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 th e assay determined at a concentration of 167.7 ng/mL (n = 5) was found to b e within 7.04 %RSD. Copyright (C) 2000 John Wiley & Sons, Ltd.