Ammonium acetate linearity problem

[mojo]

We're validating a gradient LC method for related substances determination (quantitation by area %). We have examined linearity across the range 0.05-10.0% five times: different HPLC's, different analysts, etc. Correlation coefficient and residuals are fine each time. However, the intercept is significantly negative. When we further analyse the data by calculating recovery at 0.05% and plotting response factor versus concentration, the results are alarming. Response factor at 0.05% is about 60% of the RF at 0.5% and above. RF increases with concentration. Our sample diluent is pH3.5 ammonium acetate buffer, wavelength 280 nm. Might the buffer inhibit response at low concentrations? Has anyone seen this phenomenon before?

[Kostas Petritis]

No, ammonium acetate will not inhibit the response of your analyte at low concentrations. Did you considered that you might have absorption of your analyte on the walls of your vial or something similar? It will be much more apparent in very diluted samples as the one that you describe.

If you can, try to plot the calibration curve by using higher concentration samples but inject smaller volumes (assuming your autosampler can handle it) or just try different type of vials...

[mohan_2008]

You said it is a gradient method.

Are you mixing online - 100% Aq.Buffer (Amm.acetate) with 100% organic (Methanol/ACN/THF) to attain the desired mobilephase composition level for the analyte elution.

Amm.acetate is a pretty soluble salt. However, what concentration (in mM) are you preparing.

What is your diluent composition.

What is the solubility of the compound in the diluent. Did you check the threshold solubility level?

Need to know these answers prior to guestimating the loss of analyte due to the vials.