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, t_R:1.78 min) and m/z 255.0657 (for IS, t_R: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 82.51–109.44%. 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.

这项研究提出了一种快速，简单的生物分析超高性能液相色谱-串联质谱（UPLC-MS）方法的开发和验证，该方法旨在定量大鼠血浆中的抗炎候选物5'-甲氧基诺比林汀（5'-MeONB）。通过使用制备型HPLC从A. conyzoides提取物中纯化5'-MeONB的标准品。用乙腈对血浆样品进行预处理后，使用C ₁₈色谱柱，使用0.1％甲酸水溶液和乙腈的9分钟梯度系统作为洗脱液，可以有效地实现色谱分离。使用ESI +通过提取的离子色谱图方法对候选药物5'-MeONB和chrysin（内标，IS）进行检测，以m / z 433.1494监测（对于5'-MeONB，t_R：1.78min）和m / z 255.0657（对于IS，t _R：1.57min ）。方法已根据美国FDA指南进行了验证，呈线性关系（R ²> 0.999）的浓度范围为30-750 ng / mL。考虑到定量下限（30 ng / mL）和质量控制（90、360和600 ng / mL）样品，重复性和中间精密度的相对标准偏差（RSD）分别在1.93-3.65％和2.16-7.54％之间，而准确性在82.51–109.44％之间。此外，即使在溶血的血液样品中也没有观察到来自血浆内源性物质的干扰，没有残留效应，也没有观察到提取方法的影响。自动进样器和-80°C的长期存储中的样品稳定性以及基质效应均在可接受的范围内。首次使用经过验证的UPLC-MS生物分析方法，静脉推注2 mg / kg后5'-MeONB的血浆药代动力学 对Wistar大鼠给药的特征在于可以确定描述药物分布和消除的参数。