Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escuela Técnica Superior de Ingeniería Agraria, University of Lleida, Av/Alcalde Rovira Roure 191, 25198, Lleida, Spain.
Anal Bioanal Chem. 2013 Nov;405(28):9179-92. doi: 10.1007/s00216-013-7322-2. Epub 2013 Oct 6.
Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (μSPE), with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) have been developed and validated for the determination of hydroxytyrosol and its metabolites in spiked human urine samples. Hydroxytyrosol, hydroxytyrosol-3'-O-glucuronide, hydroxytyrosol-4'-O-glucuronide, hydroxytyrosol-3-O-sulphate, and homovanillic alcohol-4'-O-glucuronide were used as the target compounds. Using the FTA DMPK-A dried urine spot card under optimum conditions, with 5 μL of preconcentrated urine volume and 100 μL of methanol/water (50/50, v/v) as the elution solvent, the extraction recovery (%R) of the compounds studied was higher than 80%, and the matrix effect (%ME) was less than 8%. The stability of these cards and punching at the centre or side of the card were also studied, obtaining an excellent stability after 7 days of storage and complete homogeneity across the surface of the dried drop. The different μSPE parameters that affect the efficiency were also studied, and under optimum conditions, the %R and the %ME were higher than 70% and lower than 17%, respectively. The linearity range in dried urine spot cards was 2.5-20 μM for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 μM and 2.5-50 μM respectively. With regards to μSPE, the linearity range was 0.5-5 μM for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 μM. The quantification limits (LOQs) were 0.3-2.5 μM and 0.08-0.5 μM in dried spot cards and in μSPE, respectively. The two developed methods were then applied and compared for determining hydroxytyrosol and its metabolites in human 24 h-urine samples after a sustained consumption (21 days) of a phenol-enriched virgin olive oil. The metabolites identified were hydroxytyrosol in its glucuronide and sulphate forms, homovanillic alcohol in its glucuronide and sulphate forms, homovanillic acid sulphate and hydroxytyrosol acetate sulphate.
两种不同的快速样品预处理策略，即干斑卡片和微洗脱固相萃取板（μSPE），与超高效液相色谱串联质谱（UPLC-MS/MS）相结合，已被开发和验证用于测定人尿液中羟基酪醇及其代谢物。羟基酪醇、羟基酪醇-3'-O-葡糖苷酸、羟基酪醇-4'-O-葡糖苷酸、羟基酪醇-3-O-硫酸盐和香草酸-4'-O-葡糖苷酸被用作目标化合物。在最佳条件下，使用 FTA DMPK-A 干尿斑卡，浓缩尿液体积为 5 μL，洗脱溶剂为甲醇/水（50/50，v/v），研究化合物的提取回收率（%R）高于 80%，基质效应（%ME）低于 8%。还研究了这些卡片的稳定性和在卡片中心或侧面打孔，结果表明储存 7 天后稳定性极好，且干滴表面完全均匀。还研究了影响效率的不同 μSPE 参数，在最佳条件下，%R 和 %ME 分别高于 70%和低于 17%。在干尿斑卡中，所有代谢物的线性范围为 2.5-20 μM，除羟基酪醇-3-O-硫酸盐和羟基酪醇外，其线性范围分别为 0.3-70 μM 和 2.5-50 μM。对于 μSPE，所有研究化合物的线性范围为 0.5-5 μM，除羟基酪醇-3-O-硫酸盐外，其线性范围为 0.08-5 μM。在干斑卡和 μSPE 中，定量限（LOQ）分别为 0.3-2.5 μM 和 0.08-0.5 μM。然后应用和比较了这两种开发的方法，用于测定持续摄入（21 天）富含苯酚的特级初榨橄榄油后人体 24 小时尿液中羟基酪醇及其代谢物。鉴定的代谢物为羟基酪醇的葡糖苷酸和硫酸盐形式、香草酸的葡糖苷酸和硫酸盐形式、香草酸硫酸盐和羟基酪醇乙酸硫酸盐。
