DEVELOPMENT AND VALIDATION OF A DRIED BLOOD SPOT LC-MS/MS ASSAY TO QUANTIFY GEMCITABINE IN HUMAN WHOLE BLOOD: A COMPARISION WITH AND WITHOUT CYTIDINE DEAMINASE INHIBITOR
Journal Title: International Journal of Pharmacy and Pharmaceutical Sciences - Year 2015, Vol 7, Issue 8
Abstract
Objective: The purpose of this paper is to develop and validate a LC-MS/MS method for the quantification of gemcitabine in whole human blood using dried blood spots.Methods: Gemcitabine fortified blood samples without tetrahydrouridine were spotted (50 µl) onto the DBS cards and dried for 2h at ambient room temperature. 3 mm punched spots were extracted by acetonitrile: water (90:10v/v) containing carbamazepine as internal standard (IS). Analyte and IS were separated on BDS Hypersil C18,(100 X 4.6 mm, 5 µ) column using a mixture of methanol and 2 mM ammonium acetate buffer (65:35 v/v) at a flow rate of 0.5 mL/min. Detection involved API-4000 LC-MS/MS with electrospray ionization in the positive ion mode.Results: The assay was validated over the concentration range of 5-5000 ng/ml. Intra and inters assay precision values (% CV) were less than 6.0% while the accuracy was within±15%. The mean recovery (%CV) of gemcitabine from DBS was ≥83.5% (≤4.0). Hematocrit values ranging between 0.25 and 0.62 were within acceptable limit with accuracy 93.0-103.1% of nominal values and %CV of ≤6.5 across the LQC and HQC levels. Gemcitabine was stable on DBS cards for atleast 90days at room temperature.Conclusions: A cytidine drug like gemcitabine exhibits ex vivo instability and rapidly converts to inactive metabolite in blood. All current published methods for stabilisation include tetrahydrouridine a cytidine deaminase inhibitor in the sample collection tubes. The proposed DBS method can be used as an alternative assay to conventional plasma analysis without adding enzyme inhibitor.Â
Authors and Affiliations
Sujan Kumar Dp, Palavan C, Seshagiri Rao Jvln
Keywords
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