Reversed–Phase Liquid Chromatographic Determination of Zaleplon in Human Plasma and its Pharmacokinetic Application

Abstract

A simple, rapid, specific, and reliable high performance liquid chromatographic assay of zaleplon in human plasma has been developed. Reversed‐phase chromatography was conducted using a mobile phase of methanol∶ammonium acetate buffer (50∶50) v/v, pH 3.2 adjusted with orthophosphoric acid, UV detection at 232 nm. After extraction from plasma by precipitation the drug was chromatographed using a C₁₈ reversed‐phase analytical column. The average recoveries of zaleplon from spiked plasma in the concentration range from 0.005–0.2 µg/ml were 93.29%, and their respective CV% was 2.557%. Regression analysis for the calibration plot for plasma standards obtained on three different days for the drug concentrations between 0.005–0.2 µg/ml indicated excellent linearity (r>0.999) and the coefficient of variation of the slopes of the three lines was less than 2%. The limit of detection was 5 ng/ml. Analysis of variance of the data showed no detectable difference in the slopes of the three standard plots (F=3.1, P>0.01). The high correlation coefficients and the similarities in the slopes are good indications of the excellent reproducibility and linearity of the proposed method. The proposed method was applied to study the bioequivalence of a commercial product of zaleplon, using as reference standard the innovator drug product. The study was conducted by using one capsule (1×10 mg) of each of the commercial product and the reference standard in a two‐way open randomized crossover design involving 24 volunteers. The criteria used to assess bioequivalence of the products were AUC (0−∞), C_max, t_max, t_1/2, and K. The obtained values for these parameters were 0.246±0.03 µg h/ml, 0.150±0.013 µg/ml, 1 h, 1.26±0.36 h, and 0.5928±0.1732 h⁻¹ for product A whereas, for product B they were 0.256±0.044 µgh/ml, 0.142±0.014 µg/ml, 1 h, 1.18±0.33 h, and 0.63±0.1747 h⁻¹, respectively.

Keywords:

• Zaleplon
• HPLC determination
• human plasma
• bioequivalence
• pharmacokinetics
• bioavailability