School of Food Science and Nutrition, University of Leeds, UK.
J Pharm Biomed Anal. 2011 Jul 15;55(5):1245-54. doi: 10.1016/j.jpba.2011.03.023. Epub 2011 Mar 21.
Hydroxycinnamic acids (HCAs) are emerging naturally occurring anti-inflammatory bioactive compounds. Determination of HCA metabolism has been restricted by the lack of authentic standards for the in vivo metabolites, and so the bioavailability of metabolites is often estimated post-hydrolysis. Recently a set of HCA conjugates were chemically synthesized allowing their detection in biological fluids in a very limited number of studies. However, authentic standards are not widely available and for many investigators accurate quantification of HCA conjugates remains a major analytical challenge. Consequently, we have characterized novel physicochemical properties of 14 authentic standards of HCA conjugates; the resulting data will permit for the first time the accurate quantification of HCA conjugates relative to the parent aglycone without the need for standards. MS operating conditions were optimized to achieve excellent sensitivity, and limits of detection by MS ranged from 3 to 15nM for 12 out of 14 conjugates and 30 to 50nM for the remaining. Intra-day and inter-day precision and accuracy was calculated at <±6% and <±10% respectively. For the first time response factors were determined by triple-quadrupole MS and spectroscopic detection methods, providing essential correction factors. Moreover, we present original analysis of UV-absorbance spectral shifts for HCA conjugates with regio-isomerization, which is advantageous for their differentiation. We demonstrate the usefulness of this method to assess the fate of hydroxycinnamic acids in the Caco-2 cell intestinal model and the impact of metabolism on HCA physicochemistry. For the first time, four HCA conjugates have been unequivocally identified as novel Caco-2 monoculture intestinal metabolites: ferulic acid-4-O-glucuronide, dihydroferulic acid-4-O-sulfate, caffeic acid-4-O-sulfate, and caffeic acid-3-O-sulfate. The characterization data presented here will significantly improve quantification and understanding of the bioavailability in vivo.
羟基肉桂酸 (HCAs) 是一类新兴的天然抗炎生物活性化合物。由于缺乏体内代谢物的真实标准，因此对 HCA 代谢物的测定受到限制，代谢物的生物利用度通常在水解后进行估计。最近，一组 HCA 缀合物被化学合成，允许在非常有限的研究中在生物流体中检测到它们。然而，真实的标准并不广泛可用，对于许多研究人员来说，准确量化 HCA 缀合物仍然是一个主要的分析挑战。因此，我们对 14 种 HCA 缀合物的真实标准品进行了新的物理化学性质的表征；由此产生的数据将首次允许在不需要标准品的情况下，相对于母体糖苷准确地量化 HCA 缀合物。优化了 MS 操作条件以实现优异的灵敏度，对于 14 种缀合物中的 12 种，通过 MS 检测到的检测限范围为 3 至 15 nM，对于其余的检测限范围为 30 至 50 nM。日内和日间精密度和准确度分别计算为<±6%和<±10%。首次通过三重四极杆 MS 和光谱检测方法确定了响应因子，提供了必要的校正因子。此外，我们提出了对具有区域异构化的 HCA 缀合物的 UV 吸收光谱位移的原始分析，这有利于它们的区分。我们证明了该方法在评估 Caco-2 细胞肠模型中羟基肉桂酸命运以及代谢对 HCA 物理化学性质的影响方面的有用性。首次明确鉴定出四种 HCA 缀合物为新型 Caco-2 单核培养肠代谢物：阿魏酸-4-O-葡糖苷、二氢阿魏酸-4-O-硫酸盐、咖啡酸-4-O-硫酸盐和咖啡酸-3-O-硫酸盐。本文介绍的表征数据将显著提高体内生物利用度的定量和理解。
