Chromatography Forum

We are trying to quantitate the amount of ergocalciferol in energy drinks. The results are not accurate when analysing samples without pretreatment. When comparing results to an accredited laboratory, our results are about 1 ug/100 ml lower (in samples containing approximately 2 ug/100 ml D2).

We do not have access to rotavap and can not perform saponification but the matrix is water based so i guess saponification is not needed.

We have tried extraction with different solvents.
Energy drink:ethyl acetate
Energy drink:heptane heptane:methanol
Energy drink: isooctane Isooctane: methanol
Energy drink: butanol

But it does not seem to work, no peak for D2 is obtained. We use LCMS with APCI and C18 column. MeOH/Water as mobile phase.

Does anyone analyze ergocalciferol in similar matrix without extensive sample preparation? Or any ideas?

If you haven't already done so, you should try to find out whether the loss is during extraction, or due to issues in the actual ionisation/MS. Have you done a spiking experiment? (i.e. take some energy drink, spike half of it with a known amount of D2 in roughly the same ball-park as the expected level, extract it and extract in parallel the unspiked half, and compare the two: do you get the expected difference?). Simultaneously, you should also run a third sample-type: extract of unspiked energy-drink, spiked before running: this will help to determine whether the loss is a problem post-extraction.

Instead of an extraction method, can you not just load the aqueous drink sample onto a RP pre-column/cartridge , wash off the sugars etc with H2O, then elute the analyte onto an analytical RP column with an MeCN gradient??

J Pharm Biomed Anal
. 2007 Jan 4;43(1):142-50. doi: 10.1016/j.jpba.2006.06.036. Epub 2006 Aug 9.
Degradation of vitamin D3 in a stressed formulation: the identification of esters of vitamin D3 formed by a transesterification with triglycerides
John M Ballard 1, Limin Zhu, Eric D Nelson, Randal A Seburg
Affiliations expand
PMID: 16901672 DOI: 10.1016/j.jpba.2006.06.036
Abstract
Four unknown degradants in the LC-UV profile of a stressed experimental tablet formulation that contains vitamin D3 have been identified by a combination of Ag+-cationization electrospray ionization (ESI) LC/MS and atmospheric pressure chemical ionization (APCI) LC/MS/MS.

See the above paper for some work with cholecalciferol, especially for details of LC/MS and LC/MS/MS analysis.