Plasma indomethacin assay using high-performance liquid chromatography- electrospray-tandem mass spectrometry: Application to therapeutic drug monitoring and pharmacokinetic studies

The authors report the use of high-performance liquid chromatography- electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) for the quantification of indomethacin (IND) in plasma with microscale sample preparation. Plasma samples (100 μL) and mefanamic acid (internal standard [IS]), buffered to pH 3.5, were prepared using solid phase extraction and chromatographed using a C8 column. The mobile phase composition was 80% methanol to 20% ammonium acetate buffer (40 raM, pH 5.1). A flow rate of 300 μL per minute was used with a 1-to-12 postcolumn split into the mass spectrometer. Selected reaction monitoring with mass transitions m/z 357.9 → 139.0 and m/z 242 → 209.0 were used for IND and IS, respectively. The chromatographic analysis time was 4 minutes. The assay was linear from 5 μg/L to 2000 μg/L with interday imprecision (n = 5) over the analytic range (5%). At four concentrations (10 μg/L, 25 μg/L, 250 μg/L, 1500 μg/L), assay imprecision was 9% (total coefficient of variation [CV]) and accuracy ranged between 96.5% and 102.8% (n = 16). The absolute recovery of IND and IS was 74% (n = 8) and 95% (n = 24), respectively. This method was developed and validated in less than 10 working days, had a lower limit of quantification than reported HPLC- ultraviolet (UV) methods, and uses small sample volumes. These factors illustrate the power of HPLC-ESI-MS/MS for drug analysis. Furthermore, the ability of this method to measure IND over a wide concentration range makes it suitable for therapeutic drug monitoring and pharmacokinetic studies.