I wonder if anyone could help.
I have been analysing vitamin B1 in foods using HPLC using the method from Finglas and Faulks (1984). The method uses the derivatization of thiamine (as well as TMP/TDP/TTP) with potassium ferricyanide (0.03M) in alkaline conditions (3.75M NaOH). However, I have found that the product (thiochrome) degrades within 2 hours of the derivatization step - meaning I can only run few samples at a time.
I have tried to stop the reaction by the addition of 5M phosphoric acid, however, this has not increased the shelf life of the thiochrome more than the 2 hours.
Has anyone else found a protocol which is able to stabilise the thiochrome compound for at least 6-8 hours? Or have a protocol that forms a stable compound to analyses?
Many thanks in advance,
Robyn
Hi Robyn,
look here:
Article The HPLC Determination of Total Thiamin (Vitamin B1) in Foods
https://www.glsciences.com/pdf/technicalnote_lc/143.pdf
Article Rapid HPLC Measurement of Thiamine and Its Phosphate Esters ...
http://www.chromatographyonline.com/analysis-vitamin-b1-foods-and-dietary-supplements-hplc-post-column-derivatization-and-fluorescence-d
Article Methods for analysis of thiamin and folic acid by HPLC-DAD i...
Article HPLC determination of riboflavin, niacin, and thiamin in bee...
https://watermark.silverchair.com/42-3-117.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAo4wggKKBgkqhkiG9w0BBwagggJ7MIICdwIBADCCAnAGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMnnbq2IvFWWHpPjYcAgEQgIICQZtpbHu9Eaye4-aKzZrx7KEk-F8rW3XHuNUMiCNv0IQsG3S068k6zF1aH5SO8559d_8qDH4Kp0xPcK_hRYto0sWC1pgZcNk3mBlsETEqOR4R1SvGao4wmZlVq3S2IIiQczRi8wkyz6Av5WqMvEmmu-N4h6tJtZWWfslwnwYD0zcdpAD1UalUXp5eJA2nLzk6w7GQTMPKu6-zs1QYXEcoms_Cpd9WVPIOziE84UU2jUcmgb4rzc1wJqbhcCmBet8hryNyIVRlRMyXNj0OubRufHeyu_7X8cinecujlsW2c7kFs6Yh7ReL7HsepEl1bnoBPkndk-M9agI0gcsYhZ_PjV8ZQAMo8wtxEL8AIeW8cXA4JJY1WiZTCSvFFT2NuI3VndAuuc5UJtZCFACscqY0di51HdrMj7vGuJuA2PDP7qjpw97UmF-KtK6xNRy0dJ9NOLk8qmqQqS2NNYbXyUB6MTY1Fh1bIShAZwEmMXkoI7wewPaVb_DRxgz9TvuAFUfClARc9vCG21QtfhRY4kSU3VnnbOJS6DrwffEQjSsF5hfX6v-Boyx_BVDY0trpZdZ-dEQShquMg4PmuUNViMhRuqkGBbFZAxmDHzTj7W64FoCveG4R--DNH5AvfLwRHpusc5l73ez-_NOQU3da_-xKf5ucISNE6Zg5QAe_L67CVP2kYT3-sxrAHHDYAhyMWP3xAaSy2jqah7b1juxTFp5fQkh58QRMg8FLS86pFyAR3kAf8ZQ-ww-TPgvXCdaerNE_qBY
http://www.piwet.pulawy.pl/jvetres/images/stories/pdf/20083/20083435440.pdf
http://www.orientjchem.org/vol32no2/analysis-of-water-soluble-vitamins-thiamine-nicotinamide-and-pyridoxine-in-fortified-infant-food-products-by-hplc/
Article Assessment of thiamine content in some dairy products and rice milk
Regards
Markus
Hi, most of the methods use either UV or fluorescence detection which requires oxidation of thiamine to thiochrome. I am not using that derivatization procedure. I am simply using HPLC-DAD and getting good signal. You can use the same method for the extraction Vitamin B from your food sample (that you are presently following). Before the enzymatic digestion maintain pH 4.5 with 2.5 M sodium acetate. After incubation centrifuge/filter the sample. Make appropriate dilution and apply to HPLC. As I am working with several B vitamins and analyzing in a single run, I have developed my method in different way. I am able to store my extracted sample up to 24 hours at -20 degree C. After that it started degrading. I am getting good results. Pavan has provided an excellent literature.
Companies like Agilent, Thermo Fisher are come up with different types of kits for different water and fat soluble vitamin analysis. You can look into those products also. Please feel free to contact me. All best, Roy
For details of our laboratory work, you can visit the link: Experiment Findings Basudha Biotechnology Laboratory for Conservation Annual Report
Thank you so much everybody for your input! I will get back in touch when I have any feedback!
Many thanks again!
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