HPLC determination of pirenzepine dihydrochloride in rabbit aqueous humor

Pirenzepine was considered as a pharmacologic agent of preventing form-deprivation myopia. To assess the ocular bioavailability of pirenzepine, a HPLC method for determination of pirenzepine in rabbit aqueous humor was developed. An HPLC system was used in the reverse phase mode for the determination of pirenzepine. A Luna RP18 5 μm 4.6 mm × 150 mm column was employed at 35 °C. The mobile phase was methanol/0.02 M KH2PO4/sodium 1-pentanesulfonate (350/650/1, v/v/m, pH was adjusted to 8.0 by dropping 1 M NaOH). The flow rate was 1 ml/min. Pirenzepine was monitored at 280 nm. Sample treatment procedure consists of deproteinisation with methanol. Calibration curves fitted by plotting the peak area versus concentration were linear in the range 20-400 ng/ml. The limit of quantification (LOQ) of present method was 20 ng/ml. Within-day and inter-day coefficient of variation was lower than 10%. Analytical recoveries were determined as 92.4, 95.4 and 101.4% at concentrations of 40, 200 and 400 ng/ml. In conclusion, this HPLC method using a simple sample treatment procedure appears suitable for monitoring ocular concentration of pirenzepine.