My HPLC-DAD method works great with the methylcobalamin (Vitamin B12) standard but the concentration of methylcobalamin in the sample I'm trying to analyze (a thick, sticky semi-solid gel containing 0.0008% methylcobalamin W/W) is below the limit of detection. The sample also contains vitamins B3 and B6 at concentrations 120-150 fold higher than the methylcobalamin. My method has no issues detecting and quantifying the B3 and B6 in the sample with accuracy and precision.
The sample is soluble in water up to 0.1 g/mL but poorly soluble in organic solvents such as methanol and acetonitrile. In attempt to concentrate the methylcobalamin, I've tried increasing the total concentration of the sample solution, increasing the injection volume, and solid phase extraction. Samples are prepped in the dark with foil on the containers to minimize photodegradation and injected immediately after they are prepared. There are vitamin B12 immunoaffinity columns (IAC) designed specifically for problematic matrices like the one I'm dealing with, but all the columns I've found so far only work for the synthetic form of B12, cyanocobalamin and I need a method that works for methylcobalamin. Apparently methyocobalamin isn't stable enough for IAC. Is there a sample prep technique or any other tricks I can try on the HPLC to improve detection of methylcobalamin in these tricky samples?
Hi Monique Speirs what wavelength are you using in analysis? I belive you should use analysis in visible region for improve your signal, and avoid intereference for another vitamins. Did you test your recovery in the methodology? In attachment has a methodology for analysis methylcobalamin with spectrophotometer in 353 nm.
Article A simple spectrophotometric method for the estimation of Mec...
I haven't extracted/purified samples of B12 myself, but I have seen many articles before using SPE (solid phase extraction). I run a lot of SPE of viscous solutions as purification and/or concentration and one line of SPE columns that works with viscous samples is Strata from Phenomenex.
Check this link and fill the required info and it will recommend you conditions and type of SPE column for your analyte: https://www.phenomenex.com/Tools/SPEMethodDevelopment
pKa values and LogP are available on different technical sites such as PubMed, DrugBank, etc. I think for methylcobalamin these are pKa 1.82 (acidic), pKa -0.44 (basic) and LogP = -14. The sample volume you enter doesn't matter. You can request free samples directly from their site. They send you between 3 and 5 cartridges for free to try, depending on the size of the cartridge. Note however that often they recommend LC-MS methods and the final diluent will have formic acid, so make sure that is compatible with your working wavelength or just adjust your final pH using another acid.
If your sample is very thick or has other particulates, you can centrifuge first or filter through 1-5 um filter and then run it through centrifugal units at 13 g before applying onto the SPE catrdige. https://www.sigmaaldrich.com/catalog/product/mm/ufc5003?lang=en®ion=US&cm_sp=Insite-_-caContent_prodMerch_gruCrossEntropy-_-prodMerch10-4). It may require some further dilution in water or buffer to be able to run free through the column. In such case, collect your sample at the end evaporate the diluent and dissolve in 0.5-1.0 mL diluent (if you had started with 10 mL sample volume and ended up with 1.0 mL, you would have concentrated it 10 times).
Waters, Supelco, Agilent and others also have their brands of SPE columns that can do this. It's likely a regular C18 SPE will work depending on how much other crap you have in your sample.
Mrs/Miss Speirs,
You do not need any sample-pretreatment steps. There is needed only to use mass spectrometry for quantification. Please, consider this work:
1. Steroids, 164 (2020) 108750
Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II, Bojidarka Ivanova, Michael Spiteller
I agree that MS is favorable for your analysis.
If the MS analysis is not applicable at your organization, you can try to use advanced analytical sample prep techniques as an alternative in the case of typical LLE SPE evaporation, etc techniques do not work.
Try to synthesis the MIP sorbent for methylcobalamin. Use this compound as a dummy template and compose a bulk polymer for selective enrichment.
Lyophilization may also work,
derivatization of the compound and converting it to a more responsive state and shifting its polarity to more lipophilic can also improve chromatographic behavior and sample enrichment efficacy of the molecule.
Please look at the following below links which may help you in your analysis:
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/328848/B12_in_foods.pdf
https://hal.archives-ouvertes.fr/hal-00895730/document
Thanks
Mr. Eigailani,
What a Government ''says'' (''orders'') does not matter, because of they are in power today; tomorrow - they are not.
What matters is what the Law in front of, ''says'' through Its Directives - or what is being discussed publicly. Government regulations are not public very frequently, while the Parliamentary debates are public. In the conditions of Democracy and transparency, only the public debates are those that are authoritative for the respective Judicial Institutions.
The Judicial Institutions are those judging in Europe, not the Governments. These Institutions are divided and operate completely independently from each other. The public Laws are obligatory to all citizens without exception. In addition to, the Public Laws are completely different from the religious laws/rules. The latter ones are not mandatory to the citizens.
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