Quantification of Hydroxytyrosol and Metabolites

Hydroxytyrosol and its metabolites
in plasma were determined by liquid–liquid extraction followed
by LC-MS/MS analysis as previously described.^{11 (link)} Briefly, 200 μL of plasma was mixed with 10 μL of freshly
prepared 10% ascorbic acid followed by 10 μL of 0.5% acetic
acid and 10 μL of 2-(3-hydroxyphenyl) ethanol (10 μM,
I.S.). Then, 2 mL of ethyl acetate was added to the samples that were
vigorously mixed in a vortex (5 min), placed into an ultrasonic bath
(10 min), and centrifuged at 1500g at 4 °C (15
min) (Centrifuge Megafuge 1.0R). A second extraction of the pellet
with 2 mL of ethyl acetate was carried out. Subsequently, the two
pooled supernatants were evaporated to dryness, reconstituted with
100 μL of 80% methanol, placed into amber vials, and immediately
analyzed by LC-MS/MS.
An Agilent 1260 liquid chromatograph (Agilent
Technologies, Santa Clara, USA) coupled to a QTRAP 4000 mass spectrometer
(AB Sciex, Toronto, Canada) equipped with a Turbo V electrospray ionization
(ESI) source was used for the determination of hydroxytyrosol and
its metabolites. Analyst software, version 1.6.2. (AB Sciex) operated
the instrument and was employed for data analysis. The equipment was
located at the Scientific and Technological Centre of the Universitat
de Barcelona (CCiTUB).
Injections of 2 μL of each sample
were performed by an automated
autosampler that maintained vials at 10 °C to avoid degradation.
Chromatographic separation of hydroxytyrosol and its metabolites was
performed in a Zorbax Eclipse XDB-C18 reversed-phase column (150
mm × 4.6 mm, 5 μm) protected with a guard cartridge of
the same material (Zorbax Eclipse XDB-C18, 12.5 mm × 4.6 mm,
5 μm) with the temperature set at 30 °C. The mobile phase
consisted of phase A formed by Milli-Q water with 0.025% acetic acid
and phase B containing acetonitrile with 5% acetone delivered at a
flow of 0.8 mL/min. The following gradient elution was used: 0 min,
95% A and 5% B; 1 min, 90% A and 10% B; 10 min, 35% A and 65% B; 10.5
min, 0% A and 100% B. Solvent B was maintained at 100% for 5 min to
prevent carryover prior to returning to initial conditions. A 6 min
delay was programmed before the next injection to ensure the equilibration
of the system. Moreover, the injector needle was washed with 1:1:1
(v/v) 2-propanol, tetrahydrofuran, and Milli-Q water to avoid further
carryover.
The ESI source, operating in negative mode, was set
as follows:
temperature, 600 °C; curtain gas (N₂), 25 arbitrary
units (au); ion source gas 1 (source heating gas, N₂);
50 au; ion source gas 2 (drying gas, N₂); 50 au, and ionization
spray voltage, −3500 V. The MS analysis was performed in multiple
reaction monitoring (MRM) mode and the specific parameters are shown
in Figure 1. Within
each analytical run, a full set of calibration standards was injected
including a reagent blank and blank plasma.
The plasmatic concentrations were calculated by
the interpolation
of the peak area ratio of hydroxytyrosol versus I.S. on a calibration
curve. Calibration standards were constructed with blank plasma obtained
by cardiac puncture from overnight fasted rats that had never received
either table olives or hydroxytyrosol. Then, 190 μL of blank
plasma was spiked with 10 μL of working standards at 0, 200,
500, 1000, 2000, 3000, and 5000 nmol/L to obtain the final concentrations
of 0, 10, 25, 50, 100, 150, and 250 nmol/L. Metabolites were identified
with the m/z indicated in Figure 1 and were assumed
to possess a LC-MS/MS response similar to that of hydroxytyrosol.
Hence, the concentrations of the sulfate and glucuronide derivatives
were quantified using the standard curve of the parent compound. Results
were expressed in nmol per liter of plasma (nmol/L). The method was
validated following the EMA guidelines³³ at six different concentrations ranging from 0 to 250 nmol/L analyzed
on three different days. Validation results indicated that the analytical
method is linear (R² > 0.998), precise
(CV < 15%), with satisfactory recovery (98.4 ± 1.64%), absence
of matrix effect (96.7 ± 2.75%), no carry-over, and adequate
sensibility with a limit of quantification (LOQ) of 0.2 nmol/L.