Chromatography Forum

Dear all,

I am looking for a method to determine both viamin B1 (thiamin pyrophosphate) and B6 (pyridoxal-5-phosphate) in whole blood by LC-MS/MS.
I tried already different separation methods:

1. reversed phase (good peak shape for B6 but much tailing for B1)
2. HILIC (response of both compounds decrease dramatically by using high percentage of organic modifier)
3. Ion pairing (lot of ion suppression)

I found only a LC-MS/MS method for vit B6 in literature where they using reversed phase

Thank you

Bas

I developed a method for several water soluble vitamins by LCMSMS once and based most of it off of this paper:

Pei Chen, Wayne R. Wolfe LC/UV/MS-MRM for Simultaneous Determination of Water Soluble Vitamins in Multi-Vitamin Dietary Supplements.

Anal Bioanal Chem (2007) 387:2441-2448 DOI 10.1007/s00216-006-0615-y

I was able to use that as a starting point and tweak out a method using reverse phase to run most of the B vitamins all together. It worked for extracts of vitamin capsules, cereal, and gummy vitamins. (gummy vitamins are a pain to extract, but found the secret is to place them in a small beaker of DI Water and hit them for a few seconds in a microwave, dissolves them really well)

Regarding tailing for B1, what were your mobile phase additives? Did you try a variety of options?

Thank you for answering my question. The vitamins from the article are the non-phophorylated ones. The phosphate group makes them much more polar. I am using meoh/water + 0.1% formic acid as mobile phase. I think the tailing from vitamin B1 is due to metal complexation of the diphosphate group with Fe in the hplc system, but I am not sure.

Thank you for answering my question. The vitamins from the article are the non-phophorylated ones. The phosphate group makes them much more polar. I am using meoh/water + 0.1% formic acid as mobile phase. I think the tailing from vitamin B1 is due to metal complexation of the diphosphate group with Fe in the hplc system, but I am not sure.

Perhaps then it would help to have some cations in the system? E.g. 5 mM ammonium formate + 0.05% formic acid.

B1 has little retention on a non-polar reverse phase column, try polar-embedded one