Hello, my lab is trying to separate amino acids using an Agilent HPLC with diode array detector. We are able to separate 17 amino acids, and 16 of these are perfectly accurate with the known concentrations of our samples. But Histidine continues to be far lower than the known concentration! This is using calculations from both standard curves and internal standards (Norvaline). We have tried many things but nothing works. Does anyone have any insight for problems relating to histidine?? Details below, let me know if you need more. Thanks very much.
We are acid hydrolysing samples containing 5.0 mg of protein with 1.0 mL of 6 N HCl in vacuum-sealed hydrolysis vials at 110°C for 22 h. The amino acids are determined by reverse phase HPLC after pre-column derivatization of o-phthaldialdehyde (OPA) and fluorenylmethyloxycarbonyl chloride (FMOC). 2.5µL of 0.4M Borate buffer (pH = 10.4), 1.0µL of sample, 0.5µL of OPA reagent [purchased from Agilent], 0.4µL of FMOC reagent (purchased from Agilent) and 32µL of injection diluent were mixed prior to run with 20µL of the final solution injected into the HPLC.
John R Carney thanks for the response. No, the standard mix came out fine. This is with actual samples. Fish feed and blood meal. Thank you.
This might be very well be a matrix effect. Have you performed matrix effect analysis and recovery study? Spike the matrix with the amino acid mix and run them in parallel with aa mix in standard solvent. What you are seeing may be a matrix suppression effect. And if you have that, then you need to clean up the matrix. In case you find this helpful:
https://link.springer.com/article/10.1007%2Fs13361-018-2093-9
John S Wishnok
thank you for the comment. The samples were analysed for amino acids by the Australian Proteome Analysis Facility, so we are confident that the anticipated histidine concentrations are correct.Navid Jubaer thank you for the paper. I had not heard of matrix effects. I see a paragraph in your paper 'A 24h incubation would be sufficient for all amino acids except His. DL-His can be determined by using a very long incubation of > 78 h for formation and quantification of the bis-adduct.'. This is very interesting. Do you think this is relatable to our low detection of histidine? Would increasing the incubation time in 6N HCl at 110 degrees C from 22 hours to >78 work for our method, do you think? If not, how else would you recommend cleaning up the matrix, in the context of our current method and reagents? Thank you so much. :)
Dear Jessica,
I think the best approach would be to take a specific concentration of histidine and do a reaction kinetics to determine the optimum derivatization time with your method. This will tell you how much time it takes for histidine to undergo complete derivatization.
In terms of matirx effect, you should determine if there is any, after you spike the matrix of your interest and comparing it to standard solvent system. This is very important to validate your method. What is your matrix i.e. in what sample type you want to determine the amino acid composition? Example of matrix: cell lysate, plasma, serum, tissue etc.
Navid Jubaer thank you again for responding. I see in your previous post you said to spike the matrix with the amino acid mix and run them in parallel with aa mix in standard solvent. Can you please be more specific? I'm not sure what we would be looking for here.
Can you also be more specific about how we would go about doing reaction kinetics for histidine? Sorry, we are a lipids lab, amino acids are fairly new to us!
The sample matrices we generally look at are fish diets, fish faeces and protein meals such as fish meal, blood meal, vegetable meal etc.
I can't thank you enough for helping out here. :)
Jessica, matrix is the sample environment where you want to detect the presence of the amino acids. For example, in the paper i attached, our matrix was bacterial cell extract. Let me ask you one question: you want to detect these amino acids in which sample? Right now you are developing the method with standard solvent system. But your goal is to detect the presence of these amino acids in some type of sample right? What is that sample?
Navid Jubaer, the type of samples we want to detect amino acids in are those listed above: fish diets, fish faeces and protein meals such as fish meal, blood meal, vegetable meal etc. They are generally freeze-dried and ground. Thank you!
Jessica, so those are your matrix. Take a grounded fish meal, dissolve it in water, make a 10 uM amino acid mix in this grounded fish meal water with firmic acid and 10 uM amini acid mix in water with formic acid, run these two samples side by side. For each amino acid if the difference in signal is bug than that means there is a matrix effect. If the intensity for each amino acids are very similar in between your grounded fish meal and water then there is no matrix effect. Now, as your grounded fish meal will have amino acid in it, you will see an increased signal in it, in order to determine the tru matrix effect, you have to dikute the fish meal solution till you dont get to a point where the amino acid signal is negligible before you can make the 10 u amino acid in it. If you need more info or need to talk to sort it out, please contact me on skype at [email protected]
Jessica, I am sorry for the typo in my last post. Was writing from my cell phone. Let me know if you have any more inquiries.
Did you ever find a solution to this problem? We are seeing something very similar to what you describe. we do classic acid digestion then derivatize with AQC as per aoac methods from 2018 and 2019. your posting is the first I have found of this issue anywhere and hoping you found the source of the problem
thanks,
Ken Riedl, PhD
Kannapolis, North Carolina, USA
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