Vitamin D3 on UPLC/PDA/QDA

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

6 posts Page 1 of 1
Hello Friends,
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!
I use exactly the same analytical column for these vitamins with an MS/MS detector. I know QDa will have a lower sensitivity, but the minimal on-column amount you determined is 0.1µg = 100ng? That equals to a 10µL injection of 10mg/L, which sounds like a lot and this shouldn't be a problem for even for PDA. Talking about PDA, D2 and D3 will be hard to baseline separate on this column, but you can get pretty close. I don't know about the gradient, but 0.7 mL/min is a high flow for this kind of column. D2 and D3 will not start eluting until around 60-70% organic, you can cut on anything below that if time is an issue (start the method at around 50% organic instead of 10% and decrease the steepness of the gradient slope).

About sensitivity in QDa. You might want to use additives in the aqueous mobile phase (try a formate or acetate buffer), but I'm not 100% sure it will be better. It's definitely worth a try. I've tested several variations for the organic mobile phase (different solvent combinations and additives) and in the end plain MeOH gave me the best sensitivity for D2/D3. I didn't do the same tests for the aqueous mobile phase.

Saponification works for vitamin D2 and D3. You can find plenty of protocols online. After extracting with hexane you do a solvent switch by drying it down, in this way you can concentrate as much as you want. I'm not saying you will stay clear of interferences and matrix effects, however.
Are you doing scan or SIR mode on QDa?
SIR can be much more sensitiv than scan mode.
maybe use advanced mode in instrument method.
Hello Rndirk/Hollow,
Thank you for your replies. Do you get a good separation of D3 and signal on the PDA? Do you perform the saponification method for D3 in oil? I think I am more focused on getting good resolution on the PDA now since the signal on the MS channels (385.30 and 397.28 m/z) is not that great, even when in SIR mode.
kristind858 wrote:
Hello Rndirk/Hollow,
Thank you for your replies. Do you get a good separation of D3 and signal on the PDA? Do you perform the saponification method for D3 in oil? I think I am more focused on getting good resolution on the PDA now since the signal on the MS channels (385.30 and 397.28 m/z) is not that great, even when in SIR mode.


About separation: see my post above. Saponification: yes, and it works in terms of recovery. I can't predict what will happen if you use higher concentration factors.
IIRC, the QDa uses an ESI source. Vitamin D has some pretty poor ionization efficiency. Depending on what else might be in your matrix, I'd recommend sample clean up with a C18 SPE using 40% MeOH to load and 60% MeOH to elute. After that, I would recommend PTAD derivation or the like to increase source ionization. PTAD does not tolerate protons (eg water, acids, alcohols), so unfortunately an LLE and/or evaporation step is needed. Another trick you can do is to add methylamine to the MP. It forms a complex with Vitamin D that is somewhat more ionizable. Keep in mind that both PTAD and methylamine will affect your base peak m/z. Between those two changes, you can probably expect to see at least an order of magnitude improvement in sensitivity, and possibly quite a bit more than that.

0.7mL a minute is really really high for a 1.7um column, and pretty high for an ESI source. You'll get much better response from the ESI source at lower flow rates. I'd say drop it to 0.2mL/min and control the gradients to keep run times low.
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