3.3.1.1. Buffer Preparation:
0.1N HCl was prepared by diluting 8.5 ml of hydrochloric acid with water to 1000 ml. But, pH 3 was prepared by dissolving 6.8 g of potassium dihydrogen phosphate in 900 ml water for HPLC then it was adjusted by diluted o-phosphoric acid and water was added to a final volume of 1000 ml. The same procedure was done for the preparation of buffers pH 6.8 and 7.4, however, the required pH was adjusted using 1 N NaOH.
Intra-conversion of 500 ppm gabapentin ProD 1, ProD 2 and ProD 3 solutions, in 0.1N HCl, buffer pH 3, buffer pH 6.8 and buffer pH 7.4, to its parent drug, gabapentin, was followed by HPLC at a wavelength of 210 nm. Conversion reactions were run mostly at 37˚C.
3.3.1.2. Calibration curves
3.3.1.2.1. Calibration…show more content… concentration of the pharmaceutical was then plotted, and R2 of the plot was recorded.
3.3.1.3. Preparation of gabapentin standard and sample solution: A 500 ppm of gabapentin standard was prepared by dissolving 50 mg of gabapentin in 100 ml of 0.1N HCl, buffer pH 3, buffer pH 6.8 and buffer pH 7.4, then each sample was injected into HPLC to detect the retention time of gabapentin.
A 500 ppm of the linkers standard (Hexahydro-4-methylphthalic anhydride, 1,2-cyclohexanedicarboxylic anhydride, and camphoric acid anhydride) was prepared by dissolving 50 mg of each linker in 100 ml of 0.1N HCl, buffer pH 3, buffer pH 6.8 and buffer pH 7.4, then each sample was injected into HPLC to detect the retention time of linkers.
A 500 ppm of each gabapentin ProD 1-3 was prepared by dissolving 50 mg of the gabapentin ProD 1-3 in 100 ml of 0.1N HCl, buffer pH 3, buffer pH 6.8 or buffer pH 7.4 then each sample was injected into HPLC to detect the retention time.
The progression of the reactions was followed by monitoring the disappearance of the prodrugs and appearance of gabapentin and the linkers attached vs. time.
Gabapentin ProD 1-3 were incubated at 37˚C to be monitored on the HPLC for several days to detect the intraconversion of the three prodrugs to their corresponding parental drugs, to calculate the t1/2 of each