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Simultaneous analysis of Gemcitabine and Irinotecan hydrochloride drugs.HPLC-DAD validated method for pharmaceutical applications and biomedical analysis.Validated method in rat plasma.Pharmacokinetic of Gemcitabine and Irinotecan hydrochloride in rat plasma.In this manuscript we aimed at the simultaneous separation and quantification of Gemcitabine and Irinotecan hydrochloride (injected both as single components and in combination) from Sprague Dawley rat plasma by using a validated method obtained through the use of a High Performance Liquid Chromatography (HPLC)-diode array detector (DAD).Gemcitabine and Irinotecan hydrochloride were detected and quantified using a Zorbax Extend C-18 column (250 mm × 4.6 mm; 5 μm particle size) in gradient elution mode. The chromatographic analyses were carried out in 15 min. The analytical mode was calibrated and validated in the concentration range from 0.1 to 18 μg/mL both for Gemcitabine and Irinotecan hydrochloride. Sprague Dawley rat plasma was used to perform the analysis. 3-methylxanthine was the internal standard. The weighted-matrix matched standard curves of Gemcitabine and Irinotecan hydrochloride showed a good linearity up to 18 μg/mL. Parallelism tests were also performed to evaluate whether the over-range samples could be analyzed after dilution without affecting the analytical performance. The intra- and inter-day precision (RSD%) values of Gemcitabine and Irinotecan hydrochloride were ≤7.14% and ≤11.5%, respectively. The intra- and inter-day trueness (Bias%) values were in the range from −11.5% to 1.70% for both drugs.The analytical mode performance was further tested after collecting Sprague Dawley rat plasma following a single-dose administration of chemotherapeutics or their association. The validated HPLC-DAD method allowed the simultaneous quantification of Gemcitabine and Irinotecan hydrochloride in the rat plasma, besides the evaluation of the pharmacokinetic parameters and drug delivery.