Exactly your sample being a secondary metabolite might have been degraded and converted into other metabolic products. Prepare fresh samples and compare again.

Regards

If the physical and chemical properties of your target compounds have already been known, it is helpful to keep it in mind. In your problem, there would be many reasons.

1.) if the compound is susceptible to auto-oxidation, it is good to keep it in inert atmosphere, e.i. bubbling with N2 or Ar gas followed by keeping the solution at least 4oC without exposure to light.

2.) If your analytical coulmn is still in good condition, analysis with fresh-prepared sample under the same analytic condition can be confirmed.

3.) If new peaks were found in your last analysis, your target compound could be degraded or converted into other products. If so, comparing mass spectra of the new compounds with that of original one you previously found is also helpful.

.....

I agree with the collegues, should you check your solution (sample) and also the cromatographic conditions

I agree with the previous answers, but I think you should also check:

- the extraction procedure;

- the status of your HPLC equipment, including the lamp(s).

After a few months could be a long time for secondary metabolites to remain undegraded. If it's the same sample from few months back.. I would try extracting again.

and one more thing if the sample was stored in -80 C or not.. cause the may help in not letting it degrade or if it was stored properly then sometimes number of freeze thaw may cause degradation.

Firstly, I would try extracting again before coming to any conclusion.

Hope this helps.

I agree with several answers given by many colleagues. I would like to add the following- some may be already addressed. (1) Make sure HPLC conditions used are same. (2) If you know the compound, try injecting the standard. If you did not get peak of the standard, then check your instrument. (3) Probably, your target compound is unstable under conditions you stored. Degradation of the compound may result in no peak.

Marimuthu,

There are some great answers to this question already, and I won't reiterate their answers. I believe the one answer which may be missing is this:

Are you confident in your original analysis? In other words, did you use a brand new column when you saw the peak the first time around? OR, did you use a previously used column... one that had been used before for another analysis?

Years ago I was troubleshooting with a client for months... trying to figure out what was wrong with this chromatograms. Only after investigatig everything under the sun, did we realize that he had developed the method on a column that had been used for a previous project. This ruined all of his results moving forward, and he had to redevelop the entire method from scratch... because the column had contaminants which had affected the chromatography. Of course, this was just when his company needed him to run samples the most.

Paul

First of all you must check your HPLC system (column, Detector etc.) by injecting the standard sample. If it shows the peak properly, then the problem is in your sample.

I agre with the previous answers. You may extract again the sample, keep a portion of the extract under the same conditions in which you kept you this time and analyze it week by week to follow the possible degradation process. A part of the extract should be store at -80°C and analyzed week by week to compare

Agree with previous answers. Best to eliminate the possibility of instrument or column problem by running a standard if available and fresh buffer to make sure instrument is working properly. If standard does not show ( it may be sticking on the column consider cleaning column with appropriate solvents for that column). Trouble shooting your column to see if a similar compound elutes using recommended fresh buffer for that compound, then retry standard if peak shows then try sample. If no peak for sample, see recommendation above for sample prep and storage and sample degradation analysis.

S we used a previously used column intially but later changed to a new column......also the standards show peak

Also agree with all previous answer. I may add a few additional suggestions in order to prevent autodegradation caused by oxydation or photoreaction. Use amber flasks or vials. Avoid unnecessary exposure to environmental lights. Fill headspace of flasks and vial with inert gas. Expecting that your analytical tools are operational, previously cited actions would only increase the shelf life of your extract solution content, however the ultimate solution would presumably be to re-perform an fresh extraction if you are not limited in sample resources.

I suspect the same as other coleagues of degradation of a component. This would be more probable than malfunctioning of HPLC system.

Please check the instrument with standard sample. If everything is fine, then the problem is in the sample and the degradation of sample is the main cause of losing peak in the test sample.

I agree with previous answers,

I also suggest to ALWAYS run some QCs and BLANKS with each run so they will show if there is any instrument problems...

There are 2 possibilities: 1. that those metabolites were slowly catabolised 2. There might have been some sort of other complex formation and there was a shift in RT. Confirm your finding with LC MS with TOF or better still MALDI TOF MS analysis.

Sometimes there is a shift of the retention time due to different room temperatures (summer-winter). Check out with standard substance.

La sustancia se descompuso, mas si estaba en solución

Peak was not disappeared its degraded have you injected freshly prepared extract to conform its so called disappearance. Make a SOP (standard operating procedure) of all working if you don't have SOP then follow any other's SOP to avoid such type of problems.

There are many reasons:

1-the compond itself may be decayed.9check the shelf life of the standard)

2- id you use UV-Vis detector, may be the lamp exhausted.(check the lamp)