Development and validation of a high-performance liquid chromatography method for determining metformin hydrochloride in dried blood spots
Abstract
Objective: This study aimed to develop an analytical method for determining metformin concentrations in human dried blood spots (DBS) usingoptimal chromatographic conditions and DBS preparation methods and validate the analytical methods.Methods: Analysis metformin HCl performed using C-18 columns (250 mm × 4.6 mm) at a column temperature of 40°C with a mobile phase acetonitrilephosphate buffer (40:60% v/v) at pH 7.0 using a flow rate of 0.8 mL/min, a photodiode array detector at a wavelength of 234 nm, and atorvastatincalcium as an internal standard. Samples were prepared by protein precipitation with 60% methanol and then drying using nitrogen gas at 60°C for15 min before reconstituting in 200 μL of the mobile phase.Results: Invalidation procedures, the present methods met the acceptance criteria stated in the EMEA Bioanalytical Guideline 2011. The method wasalso linear at the concentration range of 25.0–5000.0 ng/mL (r=0.9997).Conclusion: The developed method for determination of metformin HCl in DBS sample was valid with a concentration range of 25.0–5000.0 ng/mLand can be applied to in vivo studies.
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