Luna 5u nh2 100a column

Lactose concentrations were determined using liquid chromatography‐mass spectrometry (LCMS). Breast milk samples were diluted in the mobile phase (80% Acetonitrile and 20% H₂O), then filtered using a syringe filter (0.45 μm, 40 hydrophilic nylon syringe filter, Millipore Corporation) and transferred into sealed glass vials. The standard lactose analysis reported previously (Liu et al., 2019) was used. The LC equipment (1100 Series, Agilent) consisted of a degasser (G1322A, Agilent), a pump (G1312A, Agilent) and an auto‐sampler (G1313A, Agilent). The system was interfaced with a Quattro Ultima mass spectrometer (Micromass, UK Ltd.) fitted with an electrospray ion source. The Luna 5u NH2 100A column (250 × 3.20 mm, 5 μm, Phenomenex) was used to separate lactose at room temperature. Chromatographic separation was conducted using a mobile phase of 80% acetonitrile. The flow rate was set at 0.7 ml/min, and the volume injected was 5 μl. Peaks were determined by comparing retention times to the standard solution made from the lactose monohydrate standard. A calibration curve using a range of six standard solutions (10–150 mg/L of lactose in 80% acetonitrile) was created for quantification of lactose level in the breast milk samples. Samples and standards were run in duplicate or triplicate in a randomised order.

The composition of the test meals was analysed as follows. Fructose, glucose and sucrose were analysed using a high-performance liquid chromatography method with evaporative light scattering detection (HPLC-ELSD) [18 (link)]. Total acid contents were estimated by potentiometric titration [19 ]. Pectin were hydrolysed using the method of Ahmed and Labavitch [20 (link)]. The uronic acid contents were determined using a colorimetric method according to Filisetti-Cozzi and Carpita [21 (link)]. Total dietary fibre contents were examined gravimetrically according to AOAC 985.28 [22 (link)]. Total phenolic compound contents were determined with Folin–Ciocalteu reagents according to the method of Singleton et al. [23 ], and gallic acid was used as a standard phenolic compound. The analysis was carried out on a Phenomenex Luna 5u NH2 100 A column with isocratic elution of acetonitrile: water (82.5:17.5, v/v). The oxygen radical absorbing capacity (ORAC) were determined according to Ou et al. [24 (link)] using Trolox as a standard compound.