Bioanalytical Method Development and Validation of Ibuprofen Using RP-HPLC

An accurate, simple, precise and sensitive HPLC method with UV detection was developed and validated to separate and detect ibuprofen in human plasma using Nimesulide as an internal standard. Ibuprofen and Nimesulide were extracted from human plasma using acetonitrile protein precipitation and HPLC analysis was performed using Waters 515 Series pumps combined with a Waters PDA 2998 series photo diode array detector (DAD). The column used was Agilent C18 column (150mm×4.6mm, particle size 5-micron Agilent, USA). Analysis was isocratic at 1.5 ml/min flow rate with ACN: Buffer (0.025M Potassium dihydrogen ortho phosphate) pH 4.5 (55:45, v/v) as mobile phase. The mobile phase was premixed, filtered through a 0.2 µm nylon membrane filter to remove any particulate matter and degassed by sonication before use. The elution was detected at 230 nm. Each solution was injected in triplicate, and the relative standard deviation (R.S.D.) was measured. The retention times of Ibuprofen 2.24 min and for I.S. 1.72 min respectively. The method was validated over the range of 0.5-8.0 μ/ml. The limit of detection was 0.06μg/ml and the limit of quantification was 0.193μg/ml for ibuprofen. Inter-day as well intra-day replicates of Ibuprofen, gave % R.S.D. below 2.07 and 2.001 respectively  The absolute recovery of ibuprofen was greater than 90% were achieved. This method of analysis for Ibuprofen determination using RP-HPLC was applied for determination of Ibuprofen in plasma.