cholecalciferol-D3

[balzachugo]

Hi,

I have derivatized vitamin D3 (cholecalciferol = MW 384) with PTAD (MW 154) for a UPLC-MS/MS analysis. I expect to have a parent ion of 558 and a transition to 298 as many papers report it. however I haven't seen any paper for D3 not 25(OH)D3 please.

for a 1 ng/ mL solution I get a fantastic transition however very unusual 374 > 254 which was not expected!!!!
Has anybody done this before??

Many thanks

[JMB]

Dear balzachugo,

You will have a long wait to see the 558----> 298 transition, since the PTAD adduct is C27H44O + C8H5N3O2 = C35H49N3O2, MW 559.4 Da (monoisotopic). The [M+H]+ is therefore at m/z 560.4 and this should be your parent ion.

I used this reaction for vitamin D3 about 8-10 yrs ago, but cannot remember the MS/MS fragmentation. If you separate the reaction mixture by UPLC you should see two isomeric adducts, roughly 95:5, I believe.

Good Luck.

[Alp]

Hi, I did a google search and came across this link on PTAD and Vit. D3.

From your reply, I see you have done this sort of thing 8 to 10 years ago.
What sort of instrument were you using?

I am trying to see the analytes (cholecalciferol and ptad-deriv) on a Sciex API-III triple quad.

SOMETIMES I see Vitamin D3 (m/z 385.3), sometimes I do not when scanning 1ug/mL solutions (75/25 meoh/10mM AmAc, infused, ionspray/electrospray).

After taking steps to derivatize (as described in various sources, Ptad in ACN, PTAD in Acetone, ...) I have yet to see any peak at m/z 560.

Do you think the API-III is capable of this analysis?
Has anyone looked at this type of derivatization using an API-III??

Alp

[JMB]

I used a Finnigan LCQ system; as I recall from publications that I no longer have, the PTAD derivatization goes very readily at ambient temp.

As stated, there are two isomeric products formed in the ratio of ~ 95:5.

VD3 is difficult to detect by ESI*; it is more readily detected by APCI-MS, BUT loses H2O easily to give m/z 367 as the base peak, unless the source/probe temps. are reduced significantly from normal operating condns.

See, J.M. Ballard et al, Identification of Degradates of Vit. D3, J.Pharm. & Biomed. Anal. 43 (2007) 142-150.

This has literature refs to,

1) derivatization with PTAD and subsequent MS/MS analysis
2) *use of VERY low formic acid concns. to detect steroids (which VD3 is) by ESI.

JMB

[Alp]

Thanks for the reply JMB.

The two products make sense, I was wondering why all the lit I found only mentioned one PTAD derivative of Vit D3. I was also aware of the loss of water, your reply describes the possibilities rather well.

If the reaction goes as readily as described, then there may be issues seeing it with an API-III. From the structure (all those nitrogens) I would think that it would give a strong M+H peak with ESI, but I am not seeing it. Perhaps it is breaking down in the source, maybe the optics of the API-III are too harsh. I should look for logical PTAD-VitD3 fragments in MS mode.

As for the formic acid.... I did notice with my early experiements that 75/25 MeOH/0.1% formic acid gave a stronger vitamin D peak than MeOH/10mM AmAc. I went with the AmAc because keeping the Vit D3 infusion solution in formic acid seemed to give smaller peaks over time, I thought it might be degrading. Would you remember any issues with the stability of the derivative in acidic solution (like the mobile phase above)?

I will check to see if I have or can get that article you mentioned. Thanks again.

Alp

[Devaiah]

Hi
I have derivatized Vitamin D2 (caliciferol- molecular weight 396.6) with PTAD (MW 154) for direct injection ESI/MS. I expect to have have parent ion of 572 and a transition to 298 as per literature. I am getting a 551 > 301

Please let me know how should I address the issue.

Thanks

[JMB]

Your [M+H] at m/z 551 means that MW 550 has Even number of N atoms. Suspect that the PTAD adduct has undergone a ring-opening or other rxn to lose one N.
Suggest that you run the PTAD rxn with vit D3 (if cheaper than vit D2) until you can make the correct adduct with no decomposition.
Regards,
JMB