Hello

I have to analyze lozenges containing Lidocain-HCl and Dequalinium chloride. I developped a suitable method for the part of dissolving the lozenges (I obtain a clear solution) and one for the chromatographic separation (Column: Merck RP-Select B-5 µm-250 mm, Mobile phase: mix 660 mL of a solution of 10 g Na2HPO4 and 2 g Na-Heptansulfonic acid per litre with 340 mL Acetonitrile and adjust the pH to 4 with H3PO4, Detection: UV 215 nm/240 nm, Flow: 1.5 mL/Min.)

On the first instrument, I obtain good reproducibility between the injections, but very bad reproducibility between the samples (RSD: up to 10 % and more). The mean assay is about 100%.
On the second instrument, I obtain a good reproducibility between the injection, but a very bad sample to sample reproducibility and the mean assay is about 80%.

Does anybody see a reason for this behaviour ?

Thank you very much for every answer

Florian

is it random variation or can you see a trend in your results? Random variation would indicate an extraction problem, trend could be a stability issue...

Hi Anon

I checked if there is a correlation between the sample weight and the obtained assay. There's no correlation. I measured the pH value of standard solution and every sample solution: exactly the same for all solutions.

Hi Florian,

You've mentioned that you were getting good reproducibility between the injections and bad reproducibility between the samples.

Have you ever checked the stability of your sample solution? Possibility is there for the degradation of the samples in the mobile phase.

Please check. This may be one of the reasons.

Regards,

Navaneetha Kannan.

Hello Navaneetha

I can't check the stability because I'm not able to determine the assay in these solutions. But I know that for example lidocain injection solutions (sterilized using steam 121°C) are available and these are stable. I don't think that my solutions are unstable in the mobile phase, I know similar HPLC methods for lidocaine and dequalinium chloride. My matrix contains mannit, xylit, Sodium cyclamate, povidone, citric acid, SiO2 and magnesium stearate.

Greetings
Florian

Florian,
Maybe there is some sample viscosity issues here. Samples are maybe a bit too viscous for the autoinjector and even slight variations might cause a larger variation in the actual volume injected and hence the assay? Just a thought.

Regards,
Mark

I bet that the key issue here is the fact that you do not use a buffer to control the pH. Try to adjust the pH to get a real buffer (e.g. around pH 2 for phosphate or substitute phosphate with acetate to get a buffer at pH 4)! If you do that, you should get more reproducible results.

Hi Uwe

Thanks a lot for your hint. I'm going to try an acetate buffer now and hope this could bring better results.

Best regards from Switzerland
Florian

Hello Uwe

I tried to use sodium and ammonium acetate. The result was that I get a big noise when I use the detection wavelength of 215 nm.
Do you (or does anybody) know another established possibility to prepare a buffer in the area of pH 4 considering the fact I have a detection wavelength of 215 nm ?

Thank you very much
Florian

Florian,
215 nm is difficult with all carboxylic acids, and these are the buffers that are good for this pH range. You are - what English speakers call - between a rock and a hard place. Either you live with the reproducibility difficulties, or you can change the wavelength or you redevelop the method at a different pH that is compatible with phosphate.
Maybe your retention won't change if you go to pH 2 to 3?
Uwe

Uwe
Thank you for your short explanation. I think it might be possible to work around a pH of 2 to 3 where I can use KH2PO4 which should be suitable to have a good buffer capacity. In former experiments, the retention on the column was only slightly depending on the pH of the eluent, but I used a wrong buffer substance then (HPO4 salt instead of H2PO4 salt: no real buffer !)

Regards
Florian