Amino Acid Separation (L-Cysteine)


I'm currently do the development of the method for L-Cysteine separation by HPLC on tablet product. My reference method is based on the Agilent Method which is the Amino Acid Analysis Using Zorbax Eclipse-AAA Columns and the Agilent 1100 HPLC by John W. Henderson, Robert D. Ricker, Brian A. Bidlingmeyer, and Cliff Woodward. Before this, we already developed the method for L-Lysine based on this reference method also. 

I'm facing the problem on the derivatization of the L-Cysteine with the OPA as well as the FMOC. My concern now is either the derivatization was not complete or the reaction was not happened since there were no peak being eluted out when i tried with the API (L-Cysteine HCL Anhydrous). FYI, trials had been done using exactly the reference method i mentioned earlier. 

Next, may i know more details on DTDPA Reagent for analysis of cysteine. (Since it was mentioned on the ordering information but there was not mentioned in the procedure of the method)

Hoping someone can enlighten me why this problem happened or is there any precautions steps i need to take after this. 

Here i put my details for further discussions,

Muhammad Amran

Contact details:

  • Hi Muhammad,

    derivatizing amino acids can be tricky business. We just did it in a food application and you can find it in this newly published article:

    Our methods for derivatization with OPA, FMOC and DTDPA  are in the files section (or just follow the links).

    There is also a Chinese Patent describing the processes, CN101893611B.

    Good pH control is crucial, and so is fresh reagent.

    Hope this helps.



  • Thank you so much Dr No for sharing the official guide for the derivatization with DTDPA that meant for Agilent system. 

    I have been searching for the right solvent since 3 years ago and I can't wait to try out the described buffer A for my application. 

    I previously tried reconstitute my rotavap dried digested samples in either 0.1 M HCl or the  borate buffer sold by Agilent, both have solubility issues that later resulted in crystallization of the samples. 

    Just a few questions. So according to the guide, I will just need to digest my food samples in 6M HCl (with phenol) with added DTDPA, then dried the solvent with rotary evaporator, finally reconstitute the samples in buffer A, and ready to be analyzed as per the autosampler OPA protocol. Is that correct? 

    If I'm using a 6 ml of 6M HCl for my food samples, does that mean I just need to scale-up the volume of DTDPA solution accordingly? 

    Thank you and looking forward to your respond. 

  • You are absolutely right. You might have far more agent in there, but you never know the amount of amino acids in the sample, so that's the way to go.

  • Thanks! 

    I also want to ask what is your approach to making your own Cys-X (DTDP) standard for calibration? I do have pure L-cysteine in the lab. And how long can you keep the standard in freezer for different analysis session? 

    Do you mix your Cys-X standard with the ready-made mix standard or you inject it separately? I wish to know how you do the maths here, using just the right amount. Cause in the original paper by Tuan, moles ratio between Cys and DTDP is an important factor, else there maybe residual peak of Cystine in case of incomplete conversion to Cys-X. 

  • To be safe we keep derivatized AA standards at -20 °C for no longer than a week, but are running a stability test at different temperatures just now. We inject the derivatized standard prior to an analysis. And you always need more reagent than the expected 1:1 amount.

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