Analysis of metformin, sitagliptin and creatinine in human dried blood spots.

For analysis of the anti-diabetic drugs metformin and sitagliptin and the renal clearance marker creatinine in the same human dried blood spot (DBS) extract two liquid chromatography methods employing HPLC/UV and LC-ESI-MS/MS have been developed and validated. An accurate volume of 40μL blood was spotted on a sampling paper which was extracted using 90% acetonitrile with 10% formic acid. The new methods were shown to be selective, accurate and precise. The validated ranges were 0.2-5μg/mL for metformin, 1.5-15μg/mL for creatinine and 3-500ng/mL for sitagliptin. Since drug analysis in DBS determines whole blood concentrations as opposed to the typically used plasma levels the partition ratios between human plasma and blood cells, c(P)/c(BC), were elucidated in vitro to gain insight into the significance of blood cells as compartment of distribution for both compounds. The c(P)/c(BC) was found to be 4.65±0.73 for metformin and 5.58±0.98 for sitagliptin. While an accumulation of metformin in erythrocytes was already known we now present the first evidence that sitagliptin distributes into human blood cells. The analytical methods were also successfully applied to authentic capillary blood samples from two diabetic patients regularly taking a combination of metformin and sitagliptin. Both samples revealed analyte trough concentrations well above the lower limit of quantification of the respective compounds. Therefore, the present study offers a methodological basis for the DBS analysis of metformin and sitagliptin in relation to the patients' creatinine concentration.