HPLC data acquisition and processing this website was performed by Shimadzu LC Solutions Ver 1.23 SP 1 software. PZA belongs to the basic class of drugs due to its amide functional group. Therefore adjusting the pH of mobile phase to the acidic side ionizes the PZA present in plasma thereby leading to poor recovery. In order to extract the un-ionized form of the drug, it is imperative to adjust the pH to the alkaline side, however, alkaline mobile phase characteristics causes deterioration of the bonded phase in the column due to alkaline hydrolysis of end-capped silica. Compared to acid catalyzed hydrolysis, the hydrolysis of end-capped
silica in alkaline conditions is usually very rapid. Therefore experiments were performed using potassium dihydrogen phosphate in a limited range of pH 7.0–8.0. The response was checked at the detector using a connector (without the column). A pH value of 7.4 ± 0.1 gave maximum VE822 response for the analyte at 268 nm. The run time of analysis was higher when a longer reverse phase column (250 × 4.6 mm i.d,) was used. The resolution
between the peaks was decreased and peaks were not of acceptable peak shape when the experiment was performed using a shorter column (50 × 4.6 mm i.d,). However better resolution, less tailing and high theoretical plates were obtained with Phenomenex column C18 150 × 4.6 cm 5 μm column. The mobile consists of 15:85 v/v methanol and 10 mM potassium dihydrogen phosphate (pH 7.4). The flow rate of the method was 1.0 ml/min. The column temperature was maintained at 25 °C. At the reported flow rate peak shape was excellent,
however, increasing or decreasing the from flow rate increased the tailing factor and resulted in poor peak shape and in decreased resolution between the drug and internal standard. There was no interference in the drug and the internal standard, from the extracted blank. The peak shape and symmetry were found to be good when the mobile phase composition of 15:85 v/v was used with better resolution of the drug and internal standard. Increasing the organic portion of the mobile phase caused PZA to elute with high tailing and also merging of the peaks for PZA and MTZ. A mobile phase containing aqueous portion greater than 85% led to very late elution and very poor peak shape for MTZ. The peaks were also broad and had unacceptable asymmetry factor. Extraction methods were initially attempted using protein precipitation technique. Organic solvents such as acetonitrile and/or methanol were used as reagents for protein precipitation.13 Initial experiments of protein precipitation were done using 1:3 ratio of plasma:organic solvents. The recovery of the PZA was poor while that of the internal standard was relatively unchanged as compared with liquid–liquid extraction. Since the noise effects in solid phase extraction (SPE) method are similar to that of liquid–liquid extraction, the final analysis was carried out using liquid–liquid extraction (LLE).