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
Keywords:
Gemcitabine, dFdc, LC-MSMS, Dried blood spot, TetrahydrouridineAbstract
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.
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