Why would our Standard Produce 2 resolved peaks

[jjp2985]

Hello all,
We are obtaining some weird LC/MS/MS peaks. We are using a Shimadzu-8050 LC/MS/MS system and are running 0.1% Formic in MeOH and 0.1% Formic in Water for our mobile phase. We are starting gradient at 40%B and then will raise to 80%B. If you need more information on our set-up let me know.
We are analyzing Vitamin D or are starting to try to at least. We ran into a problem when we inject even a standard into our LC and obtain double peaks. The Peaks as you can see from the attached picture are 2 very good shaped peaks with a little time between. The MS profile for both peaks gives the correct information for the standard. We just did a Q3 scan and injected 1 standard at a time into the system. What could be causing this issue?

For the standards we are injecting we are doing no processing of the standard besides the following:
1. Take an aliquot from the standard solution the vendor gave us into an LC vial
2. Dry down the standard solution in the LC vial since it came in ethanol
3. Re-constitute in 40% MeOH:Water solution
4. Inject on the LC

If you would like more information on the system or our process let me know.

Thanks for any help

Image of the 2 resolved peaks for 1 standard (25-Hydroxyvitamin-D3-d3)

MS of a Q3 Scan of First Peak

MS of Q3 Scan of Second Peak

[James_Ball]

https://www.phenomenex.com/ViewDocument ... y+lc_ms_ms

It could be Vitamin D3 and Pre-Vitamin D3 as seen in this paper.

I seem to remember the same thing when I was working on Vitamin D about ten years ago. Your standard may contain both isomers of the target analyte.