-
- Posts: 58
- Joined: Tue Oct 09, 2012 12:25 am
I am trying to bring in a method for Vitamin D3 on the UPLC. Here is my instrument method:
Column: Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 um
Mobile phase: 10 % A) Water: ACN (9:1)
90% B) ACN: MeOH (1:1)
Column temp: 40 deg C
Flow rate: 0.7 ml/min
UPLC: Acquity UPLC H-class with PDA and QDA detection.
I am using Vitamin D2 as an internal standard. It coelutes with D3 on PDA 265 nm channel and I am hoping to quantitate the D3 using the MS of D3 and D2. I am getting good signal on my 397.28 m/z channel for D2 but the D3 concentration in my sample is too little to detect. I know the ionization of D3 and D2 is not great to begin with....
I spiked my sample with D3 standard and found that to achieve a decent signal on the 385.3 m/z channel, the minimum injection onto the column of D3 needs to be roughly ~0.1035 mcg. I need to concentrate the D3 in my sample.
The amount of D3 in our oil is 1.5 mcg/ml.
Rather than using 14 serving sizes of my oil (equivalent to the amount of D3 I theoretically need to get a good signal), I was hoping saponification would do the trick and I could extract my D3 out with n-hexane after saponifying.
Does anyone have a suggestion on the best method of concentrating my D3? Thanks in advance for your help!