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.

Spectrophotometric Determination of Anthracycline Anticancer Agents with Aluminum(III) and Chromazurol S in a Nonionic Surfactant Micellar Medium

“…HPLC with fluorescence, [7][8][9] laser-induced fluorometry, 10 electrochemical, 11 and MS, 12 detections are used nowadays. However, there is little analysis reported that used visible spectrophotometry.…”

Spectrophotometric Determination of Anthracycline Anticancer Agents with Aluminum(III) and Chromazurol S in a Nonionic Surfactant Micellar Medium

“…HPLC with fluorescence, [7][8][9] laser-induced fluorometry, 10 electrochemical, 11 and MS, 12 detections are used nowadays. However, there is little analysis reported that used visible spectrophotometry.…”

Microfluidic immunoaffinity separations for bioanalysis

“…The analysis of biological matrices by visible diode LIF is advantageous because of the minimal biological sample background exhibited in the far-red (>620 nm) spectrum [80]. Since most sample antigens do not fluoresce in the far-red region of the spectrum, derivatization of functional groups with fluorochrome labels is often required [81]. Aminereactive dyes excitable by lasers may be used to readily label protein antigens, antibodies, or peptides for immunoaffinity separations.…”

Determination of anthracycline antibiotics doxorubicin and daunorubicin by capillary electrophoresis with UV absorption detection

“…Several classical methods have been used for quantitation, including measurement of total fluorescence after the liquid extraction [2,3], thin-layer chromatography with fluorescence detection [3,4], radioimmunoassay ( 3 H) [2,5], spectrophotometry [6], and voltammetry [7]. HPLC with fluorescence [8][9][10], laser-induced fluorescence [11], electrochemical [12] and MS [13] detection are exclusively used nowadays. Capillary electrophoresis (CE) appears to be an attractive alternative method.…”

Spectrophotometric Determination of Anthracycline Anticancer Agents with Aluminum(III) and Chromazurol S in a Nonionic Surfactant Micellar Medium

“…HPLC with fluorescence, [7][8][9] laser-induced fluorometry, 10 electrochemical, 11 and MS, 12 detections are used nowadays. However, there is little analysis reported that used visible spectrophotometry.…”

Microfluidic immunoaffinity separations for bioanalysis

“…The analysis of biological matrices by visible diode LIF is advantageous because of the minimal biological sample background exhibited in the far-red (>620 nm) spectrum [80]. Since most sample antigens do not fluoresce in the far-red region of the spectrum, derivatization of functional groups with fluorochrome labels is often required [81]. Aminereactive dyes excitable by lasers may be used to readily label protein antigens, antibodies, or peptides for immunoaffinity separations.…”

Determination of anthracycline antibiotics doxorubicin and daunorubicin by capillary electrophoresis with UV absorption detection

“…Several classical methods have been used for quantitation, including measurement of total fluorescence after the liquid extraction [2,3], thin-layer chromatography with fluorescence detection [3,4], radioimmunoassay ( 3 H) [2,5], spectrophotometry [6], and voltammetry [7]. HPLC with fluorescence [8][9][10], laser-induced fluorescence [11], electrochemical [12] and MS [13] detection are exclusively used nowadays. Capillary electrophoresis (CE) appears to be an attractive alternative method.…”

Spectrophotometric Determination of Anthracycline Anticancer Agents with Aluminum(III) and Chromazurol S in a Nonionic Surfactant Micellar Medium

“…HPLC with fluorescence, [7][8][9] laser-induced fluorometry, 10 electrochemical, 11 and MS, 12 detections are used nowadays. However, there is little analysis reported that used visible spectrophotometry.…”

Microfluidic immunoaffinity separations for bioanalysis

“…The analysis of biological matrices by visible diode LIF is advantageous because of the minimal biological sample background exhibited in the far-red (>620 nm) spectrum [80]. Since most sample antigens do not fluoresce in the far-red region of the spectrum, derivatization of functional groups with fluorochrome labels is often required [81]. Aminereactive dyes excitable by lasers may be used to readily label protein antigens, antibodies, or peptides for immunoaffinity separations.…”

Determination of anthracycline antibiotics doxorubicin and daunorubicin by capillary electrophoresis with UV absorption detection

“…Several classical methods have been used for quantitation, including measurement of total fluorescence after the liquid extraction [2,3], thin-layer chromatography with fluorescence detection [3,4], radioimmunoassay ( 3 H) [2,5], spectrophotometry [6], and voltammetry [7]. HPLC with fluorescence [8][9][10], laser-induced fluorescence [11], electrochemical [12] and MS [13] detection are exclusively used nowadays. Capillary electrophoresis (CE) appears to be an attractive alternative method.…”