Purity problem with HPLC analysis ?

[Narendra]

Dear Friends

I have atypical problem. I am determining related substances by HPLC using Area normalisation method and concentration of sample is 1mg/ml injection volume 10microlitre, Purity is 98.30% and other is impurities
(i.e 1.7%). I am doing Assay also using same chromatogrphic condition, with same concentration of sample 1mg/ml. only change is using External standard method and assay value I am getting 96.00% on anhydous basis?

My problem is why I am not getting 98.30% assay value with external standard method whereas method and chromatographic condition is same?

Where I am going wrong or what could be the reason for such results ?
From Narendra - India - Mumbai

[Mark]

Narendra,

One good possibility is that an area percent measurement is LC with a UV detector is very wavelength dependant. To get the "correct" purity from an area percent measurement all of the impurities must have the same extinction coefficient (response) as the API. In my humble experience this is rarely the case, there is always some slight mismatch where one impurity has 1/2 the response of the API and appears to be about 1/2 as much as there really is there. The way around this is to inject each impurity and determine its relative response and then include these in the calculations.

Regards,
Mark

[msrinureddi]

Dear Narendra,

Which chromatography software you are using. Probably you can also try with the Max plot where all the impurities will give the peak response at their lambda max. This option - macro is available with Agilent Chemstation and Dionex - Chromelion softwares.

best of luck

Srinivas Reddy M

[GT]

Dear Narendra,
one possibility is that you have in your compound about 2% of unknown impurities that you can't see by HPLC purity. The mass balance between purity and assay it's not so easy.
bye

[Narendra]

Dear Mark Thanks for reply.

As you said I agree to it but I don't have impurities. I used standard having similar impurity profile with my sample and it also has same impurity percentage and retention time as of sample. Even though I am getting assay value 96.0%. In assay weight of standard and sample almost same but area of standard and sample not similar (water content in standard and sample is same). Can you suggest in this case anything or shall I fails the batch?

Dear Reddy Thanks for reply

I am using Winchrom software. I don't think my software contains such facility or I don't known.

Dear GT Thanks for reply

My Standard and sample have same impurity profile or I would say it is same. In such case standard and sample area should be the same (weight taken for standard and sample almost same.).

From - Narendra - India - Mumbai