Analysis of Midazolam in Small Volumes of Plasma Using High Performance Liquid Chromatography and UV-Detection Method:Pharmacokinetics of Midazolam in Rats

This research work aims to exploit the high selectivity and sensitivity of UV detector to develop and validate a high performance liquid chromatography (HPLC) method having very small sampling volume, much better mass-sensitive detection limit and lower operating cost for the determination of midazolam, known to have low oral bioavailability, in rat plasma. The chromatographic separation was achieved using a Vydac C₁₈ monomeric (250 × 4. 6 mm inner diameter × 5-μm particle size) column with mobile phase comprising of acetonitrile and potassium dihydrogen phosphate buffer (50:50 v/v), delivered isocratically at a flow rate of 1. 0 ml min^-1. Diazepam was used as an internal standard (I. S.). The chromatographic peak-area ratio, based on UV absorbency at 245 nm, was used for quantitative analysis. The statistical evaluation of the method was examined and the method was found to be precise and accurate with a linearity range of 5–3000 ng ml^-1 (r > 0. 9980). The intra-day and inter-day precision studies showed good reproducibility with coefficients of variation (C. V.) less than 4. 70%. The developed method is simpler and more sensitive than previously reported methods. The analytical sensitivity and accuracy of this assay were adequate for characterization of midazolam in rat plasma and the assay has been applied successfully to the in vivo pharmacokinetic study of midazolam in rats. After midazolam (5 mg kg^-1) was given orally, the maximum concentration (C_max) and the area under curve (AUC) were 120 ± 35. 38 ng ml^-1 and 446. 52 ± 49. 04 ng h ml^-1, respectively. The oral bioavailability, F (%), was approximately 24. 75 ± 2. 72%. The developed method holds upper hand over other methods reported in literature so far in terms of both, a small sample volume (200 μl), short analysis time (12. 5 min). Our laboratory is actually involved in a study to investigate the drug-drug interaction studies using oral midazolam as one of the cytochrome P450 (CYP3A) marker compounds in the rat model. The established method provides a reliable bioanalytical methodology to carry out midazolam pharmacokinetics in rat plasma. The small sample volume required makes it possible to study the full pharmacokinetic profile in individual small animals, like the rat.