Simple and fast UPLC-MS method for quantifying the anti-inflammatory candidate 5′-methoxynobiletin in rat plasma: Validation and application in a preliminary pharmacokinetic study

Abstract

This study presents the development and validation of a fast and simple bioanalytical ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method intended for quantifying the anti-inflammatory candidate 5′-methoxynobiletin (5′-MeONB) in rat plasma. Standard of 5′-MeONB was purified from A. conyzoides extract by using preparative HPLC. After a pretreatment of plasma samples with acetonitrile, chromatographic separations were efficiently achieved with a C18 column using a 9 min gradient system of 0.1% aqueous formic acid and acetonitrile as eluent. Drug candidate 5′-MeONB and chrysin (internal standard, IS) detection were carried out using ESI+ through the extracted ion chromatograms approach, monitored at m/z 433.1494 (for 5′-MeONB, tR:1.78 min) and m/z 255.0657 (for IS, tR:1.57 min). Method was validated according to US FDA guidelines, presenting linearity (R2 > 0.999) over concentration range of 30–750 ng/mL. Relative standard deviation (RSD) of repeatability and intermediary precision respectively ranged between 1.93–3.65% and 2.16–7.54%, considering lower limit of quantitation (30 ng/mL) and quality control (90, 360 and 600 ng/mL) samples, while accuracy was between 82.51 and 109.44%. Moreover, no interference from plasma endogenous substances, no carryover effect, and no influence of extraction method even in hemolyzed blood samples were observed. Sample stability in auto-sampler and long-term −80 °C storage, as well as matrix effect were within acceptable limits. For the first time, using the validated UPLC-MS bioanalytical method, the plasma pharmacokinetics of 5′-MeONB following 2 mg/kg intravenous bolus dosing to Wistar rats was characterized allowing the determination of the parameters describing drug distribution and elimination.