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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