2.4. Tartaric Acid Determination

Formic acid (approximately 98%), picric acid (98%, moistened with approximately 33% water), and sodium hydroxide (≥98%) were obtained from Panreac. L-(+)-Tartaric acid and creatinine were purchased from Sigma. The labelled internal standard DL-(±)-tartaric-2,3-d2 acid was obtained from C/D/N Isotopes. Solvents were high-performance liquid chromatography grade, and all other chemicals were analytical reagent grade. Ultrapure water was obtained from a Milli-Q Gradient water purification system (Millipore, Bedford, MA, USA).

Stock solutions of tartaric acid were prepared in water. Working standard solutions that ranged from 0.01 to 5 µg/mL were made by appropriate dilution in 0.5% formic acid in water and then stored in amber glass vials at −20 °C.

Determination of urinary tartaric acid was performed following a previously validated stable-isotope dilution LC-ESI-MS/MS method by our research group [27]. Briefly, urine samples (20 µL) were diluted 1:50 (v:v) with 0.5% formic acid in water, and 10 µL of a deuterated isotope standard solution in water (DL-(±)-tartaric-2,3-d2 acid, 200 µg/mL) were added. The sample dilution was passed through a 0.20 µm filter and analyzed by LC–ESI-MS/MS. Urinary tartaric acid data were corrected by urine creatinine, measured according to the adapted Jaffé alkaline picrate method for thermo microtiter 96-well plates, according to Medina-Remón et al. [28]. Finally, urinary tartaric acid was expressed as µg of tartaric acid per mg of creatinine. According to previous data, the cut-off of 8.84 μg/mg creatinine was used to discriminate daily consumers and non-consumers of wine [21].

After filtration, tartaric acid was analyzed using an Atlantis TE C18, 100 mm × 2.1 mm, 3 µm (Waters, Milford, MA, USA) reversed-phase column coupled for detection to the triple quadrupole mass spectrometer API 3000 (Applied Biosystems, Foster City, CA, USA). The mass spectrometer was operated in negative electrospray ionization mode. The column was maintained at 25 °C throughout the analysis. Mobile phases A and B were 0.5% formic acid in water and 0.5% formic acid in acetonitrile, respectively. The following linear gradient was used: holding at 100%A for 3.5 min, decrease to 10%A over 2 min and holding for 2 min, return to initial conditions for 1.5 min, and re-equilibration for 6 min. The flow rate was set at 350 µL/min and the injection volume was 10 µL. Post-column addition of acetonitrile (250 µL/min) was carried out to improve analyte ionization efficiency. The detection was accomplished in multiple reaction monitoring (MRM) mode, and the following MS/MS transitions were used for quantification and confirmation, respectively: m/z 149/87 and m/z 149/73 for tartaric acid, and m/z 151/88 and m/z 151/74 for the deuterated isotope.