Dear All,
I would like to assay L-Citrulline and L-Arginine, however, I am not sure if I should use RP-HPLC or Enzymatic Assays. How many samples can I get through in a day with these techniques ? Are there better ways to do this instead of RP-HPLC and Enzymatic Assays ? How sensitive and specific are these techniques ? What is the nature of the analyte(s) ?
Regards,
Nina Smith.
The fastest you can measure a sample by RP-HPLC is probably about 2-3 minutes if you have an autosampler and use an isocratic solvent system. If you use a gradient, it is probably about 5 minutes because of the time it takes to re-equilibrate to the starting conditions. Samples must be measured sequentially.
With a microplate-based enzyme assay, you can measure many samples simultaneously. With a 96 or 384-well plate and suitable multichannel dispensing equipment (manual 8- or 12-channel pipettors at a minimum), you can measure 96 or 384 samples at once, respectively. Many plates of samples can be done in a day, even without automation.
The advantage of the HPLC method is that includes separation, which can alleviate the problem of interference in the assay readout by substances in impure samples.
You also have to consider any differences in sensitivity of product detection by various assay methods. Fluorescence methods are much more sensitive than absorbance methods. Fluorescence plate readers are pretty common, but not all HPLCs are equipped with them.
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285646/
https://www.jst.go.jp/erato/asano/activity/AAM_Kameya_2014_1.pdf
https://www.fda.gov/media/109619/download
Thanks
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