2.4.1 High Performance Liquid Chromatography (HPLC)
The quantification of serum fructose levels was determined by High Performance Liquid Chromatography (HPLC) (Rahman et al., 2008) and was carried out in serum samples from pre-pregnancy (week 8) and pregnant mothers (week 12) and their offspring (males and females) at 7 and 60 days of life. Serum samples were obtained by centrifugation. The chromatographic system was a Shimadzu Prominence HPLC system, equipped with an LC-20AT pump coupled to a SIL-20AHT autosampler, a CTO-20A thermostat and a SPD-20A UV-Vis detector, in addition to the LC Solution software (Shimadzu, Kyoto, Japan). Chromatographic separations were performed on an Ascentis® C18 chromatographic column, reference code 581305 (250 mm × 2.1 mm, 5 μm; Supelco®, Bellefonte, PA, USA) and the column temperature was 30°C. The injection volume of the samples was 20 μL. The mobile phase was eluted in isocratic profile and consisted of HPLC grade acetonitrile and ultra-pure water (Milli-Q) (55:45, v/v). The mobile phase flow rate was 0.4 mL/min and detection was performed at a wavelength of 195 nm. The identification of fructose in the samples was by comparing the retention time in the standard solution. The results were quantified by means of a calibration curve (r² ≥0.99) prepared with 1.25, 2.5, 5, 10 and 20 mg/mL of fructose standards and the respective peak areas.