611 / 670
μ
g/mL, 4 IU/mL), and 20 mL of 50% KOH solution. The flask was flushed with nitrogen for 1
min to remove the air in the headspace, and the saponification reaction was then conducted by
incubating the reaction solution at room temperature overnight with shaking at the speed of 120
rpm. The solution was then transferred into a 250 mL separation funnel. The saponification flask
was rinsed 2 times with water (25 mL per time), and the rinsed solution was also transferred into
the separation funnel. The BHT hexane solution (30 mL, 12.5 mg/mL) was added into the
separation funnel followed by shaking vigorously for 1 min. After the layer separation, the lower
aqueous phase was discarded, and the upper hexane phase was then washed with lab water for 4
times (20 mL per time). The hexane phase was then cooled under - 20
o
C for 60 min. Aliquot (6
mL) of the hexane extraction solution was accurately transferred into a small vial, and the solvent
was removed by nitrogen flushing. PTAD acetonitrile solution (0.6 mL, 1.0 mg/mL) was then
added, followed with vortexing (5 s) and sonication (5 min). The reaction solution was incubated
at room temperature for 30 min. After the derivatization reaction, 0.4 mL of lab water was added
and mixed well. The solution was filtered with 0.22
μm P
VDF membranes to obtain derivatized
sample solution for the UPLC-MS-MS analysis.
2.3 UPLC parameters
Instrument: 1290 Infinity UPLC; Column: Waters BEH C18
᧤
50 mm
2.1 mm, 1.7
μ
m
᧥
;
Oven temperature
᧶
30
o
C
᧷
mobile phase A: 0.1% formic acid aqueous solution
᧨
mobile phase B:
acetonitrile
᧨
the gradient program is shown in Table 2; injection volume
᧶
10 μL
.
Table 2. The gradient program of the mobile phase
Time (min)
A%
B%
Flow rate (mL/min)
0.0
40
60
0.3
1.0
40
60
0.3
1.5
5
95
0.3
5.0
5
95
0.3
5.1
0
100
0.6
8.0
0
100
0.6
8.5
40
60
0.3
10.0
40
60
0.3
2.4 MS parameters
Instrument
᧶
6490 Triple Quad MS-MS
᧷
ionization mode
᧶
ESI+ (Electrospray, positive ion)
᧷
drying gas temperature: 180
ć
᧷
drying gas flow
᧶
11 L/min; cone voltage
᧶
500 V
᧷
nebulizer
pressure
᧶
50 psi
᧷
capillary voltage
᧶
4000 V
᧷
desolvation gas temperature
᧶
400
ć
᧷
desolvation
VitD-17 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE