Chromatography Forum

Dear All.,

I tried the Fat Soluble Vitamins by ESI LCMSMS. But one Literature given Precusor ions for Vitamin A 269.20. Acutally Vitamin A Acetate Molecular weight 328.0 (Retinol Acetate). After Saponification, Molecular Weight of Vitamin A 286.45 (Retinol). After Saponification, Vitamin A acetate (Retinol Acetate) conver to Vitamin A (Retinol). No Response for the 269.20, 328.0 & 286.45 precursor ions in Q1 with tuning (ESI LC MSMS) (+ and - mode).

Please reply or any other reference for Fat Soluble Vitamins by ESI LCMSMS for me

Have you tried using APCI mode?

See page 22, 23

http://books.google.ca/books?id=rJAWmOW ... &q&f=false

Sorry for the necropost but this seemed relevant to bump up for my question.

I see Agilent published a method for vitamins D2, D3 by LC-ESI-MS but most fat-soluble vitamins by LC-MS are in APCI mode. This is all well and good but our Agilent 6460 only has an ESI source installed. It is compatible with adding an APCI module but that costs money that my boss probably won't be spending anytime soon.

If we want to see the full gamut of fat-soluble vitamins A, D, E and K, is there any chance we can get them all working on ESI. I figure it might be a stretch for the esters like A-palmitate or E-acetate... but smaller esters do ionize just fine on ESI even without other polar groups. Faint hope?

Kanagavel,

See

Marwah, A., Marwah, P. and Lardy, H. J. Chromatog. (2002) A 964: 137-151

for ESI LC/MS using VERY low (50-200 ppb) levels of formic or TFA for these vitamins.

Kanagavel,

See

Marwah, A., Marwah, P. and Lardy, H. J. Chromatog. (2002) A 964: 137-151

for ESI LC/MS using VERY low (50-200 ppb) levels of formic or TFA for these vitamins.

TFA can be a pain with LCMS, I usually use Formic. It has been a few years but I did some work using Formic with Vitamins, both water and fat soluble, up to 0.1% but not sure if that included the VitAs, it did work well for the VitDs though.

OK I found that paper on ergosteroids, but I don't see any of the structures exactly matching the vitamins in question, unless I'm missing something obvious here?

Dear All.,

I tried the Fat Soluble Vitamins by ESI LCMSMS. But one Literature given Precusor ions for Vitamin A 269.20. Acutally Vitamin A Acetate Molecular weight 328.0 (Retinol Acetate). After Saponification, Molecular Weight of Vitamin A 286.45 (Retinol). After Saponification, Vitamin A acetate (Retinol Acetate) conver to Vitamin A (Retinol). No Response for the 269.20, 328.0 & 286.45 precursor ions in Q1 with tuning (ESI LC MSMS) (+ and - mode).

Please reply or any other reference for Fat Soluble Vitamins by ESI LCMSMS for me

Journal of Chromatography A, Volume 964, Issues 1–2, 26 July 2002, Pages 137–151
Analysis of ergosteroids: VIII: Enhancement of signal response of neutral steroidal compounds in liquid chromatographic–electrospray ionization mass spectrometric analysis by mobile phase additives
Ashok Marwah, Padma Marwah, Henry Lardy

This reference answers the OP exactly; if vit. D is not a neutral, steroidal, fat-soluble vitamin I don't know what is.

OK I found that paper on ergosteroids, but I don't see any of the structures exactly matching the vitamins in question, unless I'm missing something obvious here?

You specifically mention vit. D--don't you think that structure of Vit. D is quite similar to those in the paper ?? In addition, vits. A, E and K all have the typically water-insoluble, hydrocarbon structure that makes them prime candidates for this methodology. In very general terms it is the structural type, not the specific structure, that will determine the applicability of anygiven ionization method.

Also, your post suggests that you have not actually tried the methodology (ESI with v. low formic acid) for any of your analytes. Has this methodology for vits. A, E and K already been tried and dismissed in the literature ?

Kanagavel,

You need first to establish ESI conditions (conc. of formic acid 50-200 ppm in MeCN) and analyte at ~0.01-0.1 mg/mL, that will give a strong [M+H]+ signal at m/z 329 (NOT 328) for the retinol acetate or [M+H]+ signal at m/z 287 (NOT 286) for the retinol when sample is introduced by direct infusion at low flow rate (~5-10 uL/min) (no HPLC).{ low flow rate to give yourself plenty of tuning time}.

This gives you ESI-MS through Q1.

Then set-up ESI-MS through Q3 the same way ( no collision gas).

Then go back to ESI-MS through Q1, pressurize Q2 with collision gas, set collision energy to get about 80-90% fragmentation of [M+H]; tune to optimize and record spectrum with Q3 full-scan.

Now (if all this works) you are ready for HPLC-ESI-MS/MS.

Note that kanagavel's original post was more than two years ago. Hence why I added, "sorry for the necropost".

You specifically mention vit. D--don't you think that structure of Vit. D is quite similar to those in the paper ?? In addition, vits. A, E and K all have the typically water-insoluble, hydrocarbon structure that makes them prime candidates for this methodology. In very general terms it is the structural type, not the specific structure, that will determine the applicability of anygiven ionization method.

Also, your post suggests that you have not actually tried the methodology (ESI with v. low formic acid) for any of your analytes. Has this methodology for vits. A, E and K already been tried and dismissed in the literature ?

Similar is one thing. But with LC-MS/MS it's nice to have an exact match in the literature in order to have a starting point to speed up method development. ESI with low levels of volatile modifier is already what we do here.

I spent a day or two looking at some stock standards on ESI but I have not returned to it as our lab has other priorities for now. Will come back to it in due course. I think my aqueous phase was 5 mM ammonium acetate with 0.01% formic acid, and organic was MeOH.

Going back to my original question - is there literature available where ESI-MS is successful on the vitamin esters such as A-palmitate and E-acetate? I'm asking this because we are looking into doing a method where we would not be doing any saponification. All the papers I've found that did LC-MS on the vitamin esters used APCI instead.

See

Journal of Pharmaceutical and Biomedical Analysis, John M. Ballard, Limin Zhu, Eric D. Nelson, , Randal A. Seburg
Volume 43, Issue 1, 4 January 2007, Pages 142–150
Degradation of vitamin D3 in a stressed formulation: The identification of esters of vitamin D3 formed by a transesterification with triglycerides.

for use of Ag+ cationization LC-ESI/MS. This involves post-column addition of a silver salt, and was applied for ID purposes, not quantitation.

This will also certainly work with esters of vit. A (a polyene) and may also work with the vit. K esters.

See

Journal of Pharmaceutical and Biomedical Analysis, John M. Ballard, Limin Zhu, Eric D. Nelson, , Randal A. Seburg
Volume 43, Issue 1, 4 January 2007, Pages 142–150
Degradation of vitamin D3 in a stressed formulation: The identification of esters of vitamin D3 formed by a transesterification with triglycerides.

for use of Ag+ cationization LC-ESI/MS. This involves post-column addition of a silver salt, and was applied for ID purposes, not quantitation.

This will also certainly work with esters of vit. A (a polyene) and may also work with the vit. K esters.

Good to know and thanks. We do not have access to a syringe pump for post-column addition at the moment. I will keep it in mind. Silver seems like something you don't want to be pumping into an ion source though anyway...