SIMULTANEOUS DETERMINATION OF DOXORUBICIN AND IRINOTECAN IN CONJUNCTION WITH THEIR MAJOR METABOLITES BY ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

An accurate UPLC method was developed and validated for the simultaneous determination of irinotecan and doxorubicin extracted from murine plasma, along with their major metabolites SN-38 and doxorubicinol, respectively. The parent drugs and metabolites were extracted from plasma by protein precipitation method. Camptothecin was used as the internal standard. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (150 × 2.1 mm, particle size of 1.7 µm) at 40°C by means of gradient elution. The mobile phases for gradient elution were consisted of 0.1 % v/v formic acid in either acetonitrile or water. The flow rate was 0.5 mL/min, and the total run time was 7 min. The linear quantitation ranges for parent drugs (irinotecan and doxorubicin) and for metabolites (doxorubicinol and SN-38) were 250–10000 ng/mL and 125–5000 ng/mL, respectively (r² > 0.99). The lower limit of quantification (LLOQ) for irinotecan and doxorubicin was 250 ng/mL, and the LLOQ for SN-38 and doxorubicinol was 125 ng/mL. The intra- and inter-day relative standard deviation was <10 %, and accuracy was >90%.

Keywords:

• doxorubicin
• doxorubicinol
• irinotecan
• simultaneous
• SN-38
• UPLC

ACKNOWLEDGMENT

This work was supported by Singapore's Ministry of Education through the National University of Singapore (NUS) Academic Research Fund FRC-Tier 1 grant (R-148-000-098-112). IM Shaikh and KB Tan are supported by the NUS graduate research scholarship.

Notes

^a Values are mean ± S.D.

^b r is the correlation coefficient obtained from the seven-point standard curve. The concentrations across the range were evenly distributed.

^a Concentrations shown represent the low, medium and high QC samples. Intra-day and inter-day accuracy and precision were determined with replicates (n = 5) for each concentration. C.V. (coefficient of variation) = (S.D./mean) × 100%.

^a Concentrations represent the low, medium and high QC samples. Analytical recovery was determined with triplicates (n = 3) for each concentration. C.V. (coefficient of variation) = (S.D./mean) × 100%.

^a Concentrations represent the low, medium and high QC samples. The stability of samples was determined with triplicates (n = 3) for each concentration. C.V. (coefficient of variation) = (S.D./mean) × 100%.

^a Concentrations represent the low, medium and high QC samples. The stability of samples was determined with triplicates (n = 3) for each concentration. C.V. (coefficient of variation) = (S.D./mean) × 100%.

^a Concentrations represent the low, medium and high QC samples. The stability of samples was determined with triplicates (n = 3) for each concentration. C.V. (coefficient of variation) = (S.D./mean) × 100%.