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.