Validated and optimized RP-HPLC method for the simultaneous quantification of meloxicam and its major metabolites in biological fluids with liquid liquid and solid phase extraction technique

Meloxicam is the most commonly prescribed non-steroidal anti-inflammatory drug. In this study, a simple, rapid and cost-effective method for the analysis of meloxicam and its major metabolites (5-hydroxy meloxicam and 5-carboxy meloxicam) in biological fluids (human plasma, urine and saliva) was developed and validated using RP-HPLC coupled with UV detector. The samples were analyzed by injecting 20 μl into the HPLC system using supelco analytical C18 (150 mm ×4.6 mm, 5 μm) column, protected by a C18 (30 mm×4.6 mm, 10 μm) Perkin Elmer, guard column. The mobile phase methanol: TFA (0.05% aqueous solution) in 60:40%v/v was pumped with a flow rate of 1.3 mL/min at ambient temperature and the eluents were checked at 353nm using Piroxicam as internal standard. Meloxicam and the metabolites were extracted from biological fluids using dichloromethane and the percent recovery for meloxicam, 5-hydroxy meloxicam and 5- carboxy meloxicam were 98.8%, 97.3%, 97% in plasma, 99%, 98.6%, 95.1% in urine and 95.8%, 92.9%, 92.7% in saliva, respectively. The limits of detection of meloxicam, 5-hydroxy meloxicam and 5-carboxy meloxicam were 3 ng, 10 ng and 8 ng, whereas limit of quantification were 9 ng, 30 ng and 25 ng, respectively. The method was linear over the concentration range of 10 - 2000 ng/mL for meloxicam, 30 - 1000 ng/mL, 25 - 1000 ng/mL for 5-hydroxy meloxicam and 5-carboxy meloxicam, respectively. The developed method was validated according to standard guidelines, various experimental parameters and chromatographic conditions such as mobile phase composition, flow rate, linearity, accuracy, precision, sensitivity etc. were optimized and were successfully applied for the pharmacokinetic studies in the plasma samples of the healthy human volunteers.