Column:

HP-C18, 5 µm, 120 Å,4.6 × 150 mm

Mobile phase:

A. ACN : H₂O (5 mM K₂HPO₄, 3 mM KH₂PO₄, pH7.5) = 125 : 875;  B.  ACN : H₂O : H₃PO₄ = 499: 499: 2 (v/v)

Time (min):

0   13   28   38

B (%):

0    0   100   0

 Flow rate:

1.2  1.2  1.5   1.2    (mL/min)

Temperature:

40 ^oC

Detection:

UV 360 nm

Injection volume:

20 µL

Sample:

Vitamin B12 （维生素B12）