Method optimization and validation to assess the in vivo bioavailability and metabolism of raspberry ketone

项目摘要 乌藨子酮[4-（p-hydroxyphenyl）-2-butanone]（RK）是主要的天然 红树莓（Rubus idaeus L.）果实中的一种芳香化合物。目前， RK在美国作为一种受欢迎的膳食补充剂销售， 体重。父母补助金的主要目的之一是审查 RK在小鼠模型中的生物利用度和代谢结局。的目的 补充是优化和充分验证我们的综合分析方法 将液-液酚萃取、微洗脱固相萃取（µSPE）、 脂质去除和样品净化，以及使用UHPLC-QqQ- MS/MS，用于高效可靠地分析血浆、脑、肝脏中的RK及其代谢物 和WAT样本，以支持RK药代动力学和生物利用度研究。为 建议的补充项目，首先，我们将进一步优化开发的方法， 血浆的生物分析并将应用扩展到富含脂质的组织，例如脑、肝 和白色脂肪组织（WAT）（目标1）;然后我们将对 开发的方法包括检测限（LOD）的可靠性特征， 定量限（LOQ）、校准线性、化合物稳定性、准确度， FDA生物分析后的重复性/精密度、回收率和基质效应 行业方法验证指南（目标2）。本次行政会议的成果 补充研究将确保灵敏、准确和可重复的定量 肥胖症患者口服RK后RK和衍生代谢物的分析 预防，并为其他人提供创新和可靠的方法来检查RK， RK结构相关的酚类化合物在RK-丰富的植物制剂在体内。的 拟议的项目也与NIH支持绩效的总体目标相关 和出版物的正式单一实验室验证研究的定量分析 方法.

PROJECT SUMMARY Raspberry ketone [4-(p-hydroxyphenyl)-2-butanone] (RK) is the principal naturally occurring aroma compound from the fruit of red raspberries (Rubus idaeus L.). Currently, RK is marketed and sold in the US as a popular dietary supplement for the management of body weight. One of the primary aims of the parent grant is to examine the bioavailability and metabolic fates of RK in mouse models. The objective of this supplement is to optimize and fully validate our comprehensive analytical methods integrating liquid-liquid phenolic extraction, microelution solid-phase extraction (µSPE), lipid removal and sample clean-up, and targeted metabolomics using UHPLC-QqQ- MS/MS, for efficient and reliable analysis of RK and its metabolites in plasma, brain, liver and WAT specimens to support the RK pharmacokinetic and bioavailability studies. For the proposed supplement project, first we will further optimize the method developed for bioanalysis of plasma and expand the application to the lipid-rich tissues e.g. brain, liver and white adipose tissue (WAT) (Aim 1); we will then conduct full validation for the methods developed including the reliability characteristics of Limit of detection (LOD), Limit of quantification (LOQ), Calibration linearity, Compound stability, Accuracy, Repeatability/Precision, Recovery, and Matrix effects following the FDA's Bioanalytical Method Validation Guidance for Industry (Aim 2). Outcomes from this Administrative Supplement studies will ensure sensitive, accurate and reproducible quantitative analysis of RK and derived metabolites following oral administration of RK in obesity prevention and provide innovative and reliable approaches for others to examine RK and RK structurally-related phenolics in RK-enriched botanical preparations in vivo. The proposed project is also relevant to the overall NIH goal of supporting the performance and publication of formal single-laboratory validation studies of quantitative analytical methods.