2.4. Triacylglycerol composition analysis
Extracted human milk fat was dissolved in n -hexane to obtain a concentration of 0.3 mg/mL. The composition of TAGs in human milk fat was determined using UPLC-Q-TOF-MS following our previous studies (Zhang et al., 2021). Column was BEHC18 (2.1 mm × 50 mm, 1.9 μm, Waters, USA). The UPLC condition was set as: column temperature is 45 ℃, injection temperature of 20℃ and injection volume of 1 μL. Mobile phase A was acetonitrile / isopropanol (1:9, v /v ), and mobile phase B was acetonitrile/water (4:6, v /v ). Both mobile phases contain ammonium acetate (10 mmol/L). Mobile phase of 70% A was used as the starting gradient for 1 min; then increase to 87% A at 30 min and hold for 1 min; then return to initial 70% A at 32 min and maintain for 4 min. The flow rate was 300 μL/min. After each sample, the column was washed with 5% mobile phase A for 5 min before starting detection for the next sample.
Q-TOF-MS condition was set as 10 mmol/L of ammonium formate in methanol as inclusion with a shunt ratio of 1:3 and a flow rate of 0.2 mL/min; electrospray ionization source (ESI), positive ion mode; capillary voltage of 3.5 kV, cone hole voltage of 30 V; ion source temperature 100 ℃, desolvent temperature 400℃; collision gas is argon, flow rate of 50 L/h; dessolvated gas is nitrogen, flow rate of 700 L/h. The molecular weight scans ranged from 200 to 1500 m/z with a scan time of 0.2 s and a time interval of 0.02 s. TAGs were qualitative according to mass spectrometry information and quantified by area normalization. Relative concentrations were calculated by dividing the peak area of a single TAG by the sum of all peak areas within the sample.