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.