10 mg sample was digested with 6 N 5ml HCl. Neutralized with 5m of 6N NaOH.
From this 10 micro liter was taken for derivatization with 20 micro liter derivatization reagent and 70 micro liter buffer. From this 100 micro liter, 2 micro liter was injected in hplc.
For this purpose you need to run your standard sample after proper derivatisations either pre or post column derivatisations. Make sure that the derivatised sample has good absobance at particular wavelength. Usually,OPA and FMOC derivatives are good and recommended .You are also using suitable column such as ODS (Shim pack) for this purpose. Once you have prepared standard run replicates of standard to make sure that you are getting good peak resolution with consistency in retention time. Once you are satisfied with the retention time and peak resolution, you can prepare the sample in same way like standard. Run your sample after derivatisation and work up and look for the peak of interest that matches the retention time of standard. You can confirm the presence of amino acid of your interest by spiking the sample with known con of standard .This way you can quantify recovery and confirm the peak of your interest. Hope this helps you.
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