Chromatography Forum

In what circumstances HPLC assay is more than 100%

Dear Friend,

If you are getting values close to 100%, such as 101%, then it could be that just some experimental errors are being accumulated and the values turn out higher than expected. Most assay techniques by HPLC have standard deviations close to 1%.

If your values are much higher than 100%, such as 110%, then the reason very likely is that your standard and your samples are not the same. In other words the purity of your standard is less than 100%, or lower than stated in the certificate of analysis.

A common situation that causes high assay values is when the standard has a higher water content than the samples. Obviously the water in those cases could be just accumulated moisture or crystallization water.

Good luck,

josebenjamin

The possibilities here are numerous. You should really specify the type of assay and the calibration method involved. I used to work for a bulk pharmaceutical company that just used the area percent for a rough measure of purity. That method at least had the advantage of never yielding a purity above 100%.

One significant possibility that comes to mind is an unresolved impurity.

ravenwork

josebenjamin wrote:
Most assay techniques by HPLC have standard deviations close to 1%.

Not in bio-analysis So, kumaresan, as ravenwork asked, please specify.

Regards Bert

If you're weighing standard by difference, and spill a bit during the transfer,

If you qualify a very good in-house standard against a marginal, old USP lot (100%, by definition),

If your standard has shed some water since it was last tested,

you introduce a high bias.

If you have a batch of product that was slightly overcharged with X, you have a high result.

(I had to say something different - I thought I was channeling Jeff Foxworthy for a minute there).

thanks benjamin, actully i got assay around 106%. I confirmed the moisture also

I know a story of a substance which could contain up to 10% impurities (Dequalinium chloride). But a high purity standard is available. When you have bad separation (co-elution) it might be possible that the sum of the areas (analyte plus impurities) is higher than the area of the standard.

Florian

Dear Kumaresan,

I am glad my comments were of help. The situation you found is very commmon. Over the years I found it at least 10 times.

Once you know the water contents of samples and standrds, then you can correct your calculations as required.

Good luck,

josebenjamin