Developing an analytical method by HPLC for simultaneous quantification of methylene blue and metformin applied to in...

The aim of this study was to develop an HPLC method for simultaneous quantification of metformin (MET) and methylene blue (MB) in in vitro skin permeation/retention studies, in which retention was evaluated in the different layers of the skin [stratum corneum (SC) and the viable epidermis + dermis (VE + D)]. The method was validated considering the following parameters: specificity, linearity, quantitation limit (LOQ), recovery, precision and accuracy. Calibration curves were obtained using the following six matrices: methanol, water, methanolic extracts from the SC and VE + D spiked with the drugs and drugs extracted from the SC and VE + D. The precision, accuracy and LOQ of the method were evaluated in water and in VE + D and SC, applying the drug extraction process. The results show that the method is selective and linear for both drugs. The precision and accuracy values, independent of matrix and drug, were below the limit of 15%. The LOQ of MB was defined as 0.4 μg/ml in the VE + D and SC and 0.8 μg/ml in water. The LOQ of MET was defined as 0.8 μg/ml in the VE + D and SC and 0.4 μg/ml in the water. The recovery of the method was adequate, consistent and reproducible for the concentration range of 0.4-10 μg/ml for MB (73.3-92.1%) and 0.8-10.0 μg/mL for MET (72.4-94.4%). This method has a potential application in the development of formulation for skin delivery of MB and MET.