Application of dried spot cards as a rapid sample treatment method for determining hydroxytyrosol metabolites in human urine samples. Comparison with microelution solid-phase extraction

Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (mu SPE), 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 mu L of preconcentrated urine volume and 100 mu L 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 mu SPE 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 mu M for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 mu M and 2.5-50 mu M respectively. With regards to mu SPE, the linearity range was 0.5-5 mu M for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 mu M. The quantification limits (LOQs) were 0.3-2.5 mu M and 0.08-0.5 mu M in dried spot cards and in mu SPE, 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.