Chromatographic separation of derivatives of 4-alkoxy-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione by TLC and HPLC

Five derivatives of 4-alkoxy-6-methyl-pyrrolo[3,4-c]pyridine-1,3-dione were separated by TLC and HPLC methods using silica gel 60 F-254 plates, analytical columns: LiChrosorb (R) RP-8 (250 x 4 mm, 5 mu m) and LiChrospher (R) 100 RP-18 (250 x 4 mm, 5 mu m), respectively. From the seven remaining mobile phases, three were offered for use with two-dimensional TLC. The values of separation parameter (a), constant of the pair separation (R-F(alpha)), and Delta R-F of two neighbouring spots on each chromatogram were defined. The mobile phase C (butan-1-ol-propan-2-ol-cyclo-hexane-acetic acid (1.05 kg/L); 6:3:2:1, v/v/v/v) was used in the first dimension but mobile phase D (chloroform-propan-2-ol-acetic acid (1.05 kg/L); 40:15:6, v/v/v) and/or E (chloroform-cyclohexane-propan-2-ol-acetic acid (1.05 kg/L); 20:20:15:6, v/v/v/v) were used in the second dimension. The main validation parameters (linearity, limits of quantification (LOQ) and determination (LOD), repeatability of retention times) were evaluated for the analysis of all of the investigated derivatives in their mixture, by using RP-HPLC with UV detection at 254 nm (LiChrospher (R) 100 RP-18 column; acetonitrile-methanol-phosphate buffer, pH 7 (45:15:40), 1.5 mL/min).