Xdb c8

The plant extract was filtered through 0.2 µm PVDF filters (Sterivex, Millipore, Bedford, MA, USA). Determination of phytochemical composition was performed on a UHPLC (model 1260) coupled to a 6530 model Accurate-Mass QTOF LC/MS; Agilent Technologies (Palo Alto, CA, USA) equipped with an ESI interface operating in positive ion mode and an Agilent XDB-C8 2.7 μm 3 × 50 mm, 2.7 column. The mobile phase was 0.2% formic acid in water as eluent A and 0.1% formic acid in acetonitrile as eluent B, with the following set of operation parameters: Capillary voltage, 3500 V; nebulizer pressure, 35 psi; dry gas flow, 8l/min; dry gas temperature, 300 °C ; LC–MS mass spectra were recorded across the range mass 100–1,700 m/z; fragmentor 135 V; column temperature 40 ^oC; solvent gradient conditions: 0 min, 0% B; 5 min, 10% B; 10 min, 80% B; 12 min, 100% B and then 15 min, 0%B. Compound identification was performed through MassHunter Workstation software using libraries G3874-60007 Massahunter METLIN PCDL B.08.00.

EHFAo analysis was performed using an Agilent 1290 (Agilent®) Liquid Chromatograph with DAD detector, coupled to an Agilent G400 Triple Quadrupole Electrospray Mass Spectrometer in positive ionization mode.
Samples containing 5 mg/mL of the extract were prepared with methanol, filtered in microfilters, and then analyzed on a reverse phase column (ZORBAX XDB C8; 2.1 x 50 mm 3.5 micron), eluted with water and (A) 0.1% acetic acid and (B) acetonitrile (40:60) in isocratic mode, with 2 μL of injection volume, flow rate of 0.05 mL/min, and 1,200 bars of pressure limit, in 13 min. of analysis time. The column temperature was kept at 40°C, the thermostat at 20°C and the samples were kept at room temperature. The compounds were detected at 230 nm. Mass spectrometry was performed through electrospray ionization in full scan mode, operating between 50 and 700 m/z, with 50 V of collision energy. Nitrogen gas was used as nebulizer (45 psi), with a flow rate of 5 L/min in positive mode. The mass found was registered in positive ionization mode, and the spectra of the fragments were identified according to the literature.

Plasma TAC, activity of SOD and GPx were measured using commercial kits (Randox Laboratories Ltd., United Kingdom). Vitamin C was quantified applying a colorimetric method ²⁴. Spectrophotometric readings of these antioxidant indicators and TBARS were obtained with a Stat Fax Millenium III.
The normative ranges given by manufacturers in the kit are: 1.30-1.77 mmol/L for TAC, 1102-1601 U/g Hb or 164-240 U/mL for SOD, 27.5-73.6 U/g Hb or 4171-10881 U/L for GPx. TAC, SOD and GPx were considered low when they were below their 10th percentile calculated in the total group, because not available normal values ​​were found in Venezuelan children.
Vitamins A and E were analyzed through high performance liquid chromatography, using the methodology of Bieri et al. ²⁵, with a Hewlett Packard 1050 chromatograph, under the following conditions: reverse phase column of octadecyl silica as the stationary phase (Zorbax Eclipse, XDB-C8 x 150 mm, 5 mm) and a 95:5 methanol:water mixture as mobile phase. The flow rate was maintained at 1.5 mL/min. The vitamin E/TC index was calculated. As antioxidants, vitamin C, A and E values and the vitamin E/TC index were deficient when they were lower than 0.9 mg/dL, 74.4 (g/dL, 1.3 mg/dL and 4.85 umol/mmol ²⁶.