Cystine assay development difficulty

[MestizoJoe]

Hello,

I've been working on developing an assay for Cystine. We have a previous method which worked but the column has been unreliable. That method was:

Mixed mode column (strong cation and anion on C18)
gradient:
30%ACN|70%h2o ----------> 30%ACN|70%[0.5%H2SO4 in h2o]

I have tried ion pair in acidic mobile phase with C18 and had no luck. I also tried mixed mode with the following:
30%ACN|70%[0.05%H2SO4 in h2o] ----------> 30%ACN|70%[0.05%H2SO4 & 100mM Na2SO4 in h2o]

Any idea why a sodium gradient and ion pair didn't work?
Any suggestion for assay method?

We have been having a lot of difficulty with this method.

[HPLC chemist]

You didn't indicate what type of detector you are using or whether the Cysteine is derivatized. However, I note that you are using a strong but variable pH gradient (Sulfuric Acid) in your separation which negates any ion pairing you are using.

I would search the internet for methods and use more stable pH buffers
(2 or 3>7).

[MestizoJoe]

The detector is UV at 210 nm. Cystine (not cysteine) is not derivatized in this case.

The method we want to replace uses the acid gradient. There is no acid in the gradient initially. I thought the cystine may elute because as acid increases the negative functional group gets neutralized and kicks off the cystine. That would explain why my sodium gradient didn't work. I don't know.

Cystine is similar to EDTA. I am looking at methods for EDTA assay to try.

[HPLC chemist]

Use the same methodology as you are currently using but use more stable pH buffers. 0.10 M (100 mM) pH 3 buffer is Potassium Dihydrogen Phosphate with the counterion being Phosphoric Acid. 0.10 M (100 mM) buffer pH 7 is Disodium Hydrogen Phosphate with the counterion being Phosphoric Acid. Phosphate buffers are invisible in the UV.