Dried blood spot as biosampling method offers a less invasive and easier procedure. This study aimed to develop the validated analytical method of doxorubicin hydrochloride and doxorubicinol simultaneously in dried blood spot with hexamethylphosphoramide as the internal standard. A total of 30 μL blood was spotted on DBS paper and dried for 3 hours before it was extracted by protein precipitation method using water and methanol. The separation was performed on column Acquity UHPLC BEH C-18 (2.1 × 100 mm; 1.7 μm), with 0.15 mL/min flow rate and using 0.1% acetic acid and acetonitrile as mobile phase in gradient elution for 7 min. Quantification analysis was performed by a triple quadrupole mass spectrometry with electrospray ionization (ESI) in positive ion mode. The multiple reaction monitoring (MRM) was set at m/z 544.22 > 397.06 for doxorubicin hydrochloride; m/z 546.22 > 361.05 for doxorubicinol; and m/z 180.03 > 135.16 for hexamethylphosphoramide. The lower limit of quantitation was 10 ng/mL for doxorubicin and 4 ng/mL for doxorubicinol. Concentration range acquired was 10-200 ng/mL for doxorubicin and 4-100 ng/mL for doxorubicinol. The precision and accuracy were within acceptable criteria of <15%. Dried blood spot samples acquired was stable for at least 30 days before analysis. This method fulfilled the validation requirement refers to Bioanalytical Method Validation Guideline of European Medicines Agency 2011 and US Food and Drug Administration 2018.
Keywords: Bioanalytical method validation; doxorubicin; doxorubicinol; dried blood spot; hexamethylphosphoramide; ultra-high-performance liquid chromatography–tandem mass spectrometry.
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