Validation of chromatographic method (area % result)

Hi all!

Could you please give me some suggestions on how (if possible) to validate an HPLC method that gives results in Area %?
I was asked to execute a validation of this method. There is no reference std whatsoever of the impurity I have to detect and quantitate in area %.
Nowadays, this method is used. The impurity quantity is given in area %.
I think that one can’t execute any linearity test or accuracy if doesn’t have a reference standard available.

Thanks a lot for your kind help.

Could you please let me know about the structure of the protocole? I also need to know how to validate an HPLC analytical method, only the lnearity suppose that I have the same concentration of the sample and the standard because I have to give back my homework as soon as possible,

Thanks in advance

I think if there is no Reference std then it is impossible to perform method validation. Because the validation is a process of confiming the capability of the method to detect and quantify of the substance. If reference std is unavailable, how to prove the capability of the method?

Then, what you can do is try to determine what is potential impurity and try to get the reference std so as to check the practical impurities identification and quantitation. Since the impurities are usually related substances, they can be obtained by synthesis or isolation.
Any comments will be appreciated!

I agree with crystalover point of view, without refernce standard it is useless to perform method validatio or in my view it is impossible to do validation!

Any comments will be appreciated!

If you erforming test for related substance or impurity, validation can be carry out only for parameters of specificity and LOD/LOQ.

for unknown impurity, specificity (by degradation study, to ascertain that no peak is interfering each other) + LOD

for known impurity, specificy (by comparing the retention time) + LOQ

Dear all :slight_smile:
I have a few questions and a big hope that some of you could help me. I am working in the lab and now I have got the task to do the validation for the fatty acid in the fish oil sample using GC chromatography. I do not know with what I should start.
I have run the std containing most of the fatty acid that I am interested in. So the identification of the peaks and correct ret. time of the fatty acids I know. But what to do after this? What should I consider? How to find the impurities, how to find the different matrices.

Thanks in advance.


how many compound you have?

  1. specificity
    in pharma, we will force-degrade (acid + base hydrolysis, oxidation etc)
    , each compound and analyse. if you are having multiple compound, then it will be a huge task to you.

  2. linearity
    can be done by prepare fatty acids and (fatty acids + matrices) at your working range (for axample, 100% +/- 50%)

  3. accuracy & recovery
    can be done with (fatty acids + matrices). Use % active detected to check your result

  4. intermediate precision
    perform two set of experiment, comprises differents (day, analyst, column, reagents, instrument)

  5. robustness
    you only play with temperature, i think so.

the basic should be somehow like this. However, it can still be improve from here.

Thanks for the replay,

I work with the fish oil sample which contains more than 50 different fatty acids. I am very interested in omega 3 fatty acid. Some of then I have identified using standards. Now the aim is to validate the method I am using to analyze the fatty acid in the sample.
So I think I have to start step by step. I am looking for an good advices and some examples since I have never done it before. So if You please can explain me step by step what to do I would be appreciate. How to check that no peak is interfering each other, that the tops I am seeing is always due to one components, how to check different matrix and what it should be. How to know that the method is accurate, What is the quantitative limit? What impurities can be present in a sample, how to analyze them? With that kind of questions I am struggling.


50 compounds is a lot… I had done max 3 compounds only.

My suggestion is, you may try do the specificity by comparing the retention time for each of the compound, meaning inject one by one of your fatty acid standard solution (primary is expensive, so you can use working standards). Here, you will generate abt 50 chromatograms.

Since you compound is a lot, then you may not needto do spike sample. Just use your fish oil as your sample, inject. Check the retention time.

Linearity: perform different dilution from a concentrated sample solution (stock), then dilute accordingly to get ex: 50%, 75%, 100, 125, 150% to see if you can get a linear curve out of them. If possible, do this together with your standard solution.

Once you quantitate the sample against the std solution, you may get accuracy, and thus the recovery.

you need to take a simpler approach, because your compound is too many.

Dear All
What is the difference between validation of compendial and non compendial method. Which points involved in both.