HPLC method for determination of cysteine and cystine

We are looking for a method to determine the % cystine in manufactured samples of cysteine.  HPLC would be fine or GC/MS.

  • Based on my reading, Agilent recommends the uses of either Cysteic Acid or DTDPA. 

    Cysteic Acid

    • The AOAC has description for this method. It generally involved leaving samples overnight in a solution of performic acid to allow cysteine and cystine to be converted to cysteic acid before drying of solvent and follow up of digestion with 6M HCl. Analysis of cysteic acid on a HPLC required cysteic acid standards which is commercially available. 
    • There is an application note from Agilent talking about this. You can search for it. 

     

    Cys-DTDPA

    • There is a journal described this in details. See https://pubs.acs.org/doi/abs/10.1021/jf970098%2B 
    • This step involved.preparation of DTDPA solution in NaOH, and adding of the solution into samples mixed with 6M HCl before digestion. You should expect the Cysteine+Cystine peak (which both converted to Cys-DTDPA) to have different retention time than the cystine standard you used. 
    • As far as I'm concerned, Cys-DTDPA standard is not available commercially. But one can easily make their own in the lab with pure Cysteine chemical following the same digestion protocol as described in the journal above. A centrifugal evaporator may come in handy in this situation to allow higher molarity used of DTDPA, in my opinion. 
    • Agilent recommends the uses of borate buffer for your sample with DTDPA to prevent solubility issues. You can buy the buffer directly from Agilent or make your own if you know the recipe. 
    • I have obtained good linearity (r2=0.9999) on a 3 point calibration curves of 125 pmol, 500 pmol and 1 nmol on a DAD. Feel free to experiment or modify it according to your system detector sensitivity and analysis goal. 

     

    Personally, I would be more than happy to see Agilent to have details protocol described for the uses of DTDPA. I have been having some solubility issues in the lab even with the substitution of 0.1M HCl with 0.4N, pH 10.2 Borate buffer. 

     

    That's all I know and I can share. All opinion are my own and do not represent the views or endorsement from Agilent. 

    Best of luck. 

  • If you have found answer to your inquiry elsewhere, I hope you can share it here too. I could used some advise for the analysis of cysteine too. Thank you. 

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