EverY substance doesn’t have UV absorbing

property or UV active so that u have if standard than u can spike it in your sample by this u can verify or identify the peak retention time and response or else u can perform the UV scan to find out the wavelength of substance

finally u can perform your analysis in photo diod array (PDA) detector so that if it elute at same retention time you can find out your required substance

EverY substance doesn’t have UV absorbing

property or UV active so that u have if standard than u can spike it in your sample by this u can verify or identify the peak retention time and response or else u can perform the UV scan to find out the wavelength of substance

finally u can perform your analysis in photo diod array (PDA) detector so that if it elute at same retention time you can find out your required substance

I think you should give more information about the kind of metabolites (SCFA short chain fatty acids) and indeed the detection sytem and operational system used in your HPLC. The detection system may be inappropiate as dr Sutatiya pointed out.

Besides, are you sure that the samples with the missing peaks come from very similar experiments as the other ones. And the sample preservation from the very beginning was also equal?

Thank you Vishnu and Jos for your answers. I'l specify more about the conditions of the experiment:

All the samples tested were from 2 bacteria (a Bacteroides and a Clostridium) where the Bacteroides is known for producing the metabolites I analyzed. All samples were treated and preserved together, so it shouldn't be a difference given by sample treatment inequality. From 24 samples (12 in biological replicate), only 5 samples had this problem (1 was a negative control and 4 were from the Bacteroides). Three of these samples had a biological replicate that did give a result, and two were from the same condition so I don't have information about that one.

About the metabolites measured, they are acetic acid, lactic acid, butyrate, succinate, and propionate. We included serial dilutions of standards from 30 g/L to 0.155 g/L and all of them gave a beautiful calibration curve, so it was possible to detect the metabolites used in the study (and 18 samples had peaks for almost every metabolite). The metabolites used for the standards were of high purity and for analytical purposes and the peaks given for them match with the data that was previously registered for these metabolites (from literature and from experiments done in the past). This should discard the methodology or the column used as the origin of the problem.

Then, the reason why only 5 random samples didn't give any peaks is still a mystery for me. As I need those results, I don't know what to change in order to obtain peaks with these 5 samples. Any advice will be very useful and thank you for taking the time in helping me.

Belén wrote; "This should discard the methodology or the column used as the origin of the problem." -

Please do not generalize and assume. What you do not yet know is interfering with your explanation. That the methodology or column used do not relate to the problem are not a logical conclusions to come to (we do not know if this is true). You have not provided the actual analysis method used (we do not even know what type of detection is used) so there is no way for those viewing your post to verify that the method you used was selective for the compounds under analysis or met good chromatography fundamentals at all. We review many methods where calibration curves are drawn, but the results obtained are invalid because the method is shown to NOT be selective for the sample(s) analyzed. Please do not assume. Many literature methods are flawed (a common problem that I see come across my desk every week as a reviewer) so can not be relied upon without additional review and information. Sample prep, instrument set-up and operation are all complex tasks (variables). The "instrument" is just a tool. The system will eagerly provide data (or no data, which is still data) that is inaccurate unless operated and setup by someone with the training to do so (something that takes many years to learn, at just a basic level).

Jos Wielders's concerns are well founded and we would need more detailed information about your method to suggest specific reasons for your observations. We can only help if you help us to understand what you have actually done.

You're right, I'm sorry. I'm new to this topic, but I'll give all the details I know.

In my laboratory, we usually send the samples to analyze, and an expert does the measurement and delivers the results. The details of the methodology used are detailed in the work of Mendoza et al. (2017):

"(...) the content from each flask was centrifuged, the supernatant was collected and an aliquot was injected in a Lachrom L-700 HPLC system (Hitachi, Japan) equipped with a Diode Array and a Refractive Index detectors (Merck Hitachi, Japan). Organic acids, alcohols, and sugars were separated using an Aminex HPX-87H ion-exchange carbohydrate-organic acid column (Bio-Rad, USA) and quantified, as described previously (Varela et al., 2003). "

( Article Genome-Scale Reconstruction of the Metabolic Network in Oeno...

This procedure usually gives results that make biological sense, and the people of my laboratory have never had this problem before.

The preparation of the samples was just as mentioned above, but with a storage period at -80°C before its analysis.

Please tell me if there is an important detail missing.