Increasing response

Chromatography Forum: GC Archives: Increasing response
Top of pagePrevious messageNext messageBottom of pageLink to this message  By Ravi on Wednesday, June 12, 2002 - 07:06 am:

Hello all,
Recently, I developed a HPLC stability-indicating method for a tricyclic anti-depressant drug product and I am in the process of validating it. The method uses 25% (10 mM) Na2HPO4 (pH=7.7):45% CH3CN:30% CH3OH mobile phase, Luna C-8 column, 254 nm UV detection, and 1.0 mL/min flow. The working concentration is 500 g/ml for the active, (std., and sample) 0.5 g/mL for the six impurities. I understand that 500 g/mL is a high concentration, but my peak shape is good for the active ingredient and I am not overloading the column. My suitability parameters are well within the specs. After validation, this method will serve for both drug substance and drug product for assay and purity, hence the high concentration. However, I observe an increasing trend in the standard response for all the components (including the impurities) in six successive injections. The rare standard (3) also has this trend. If I am overloading the column, I should only see increasing trend for the active ingredient, but not for the impurities. Any thoughts?
Thank you.


Top of pagePrevious messageNext messageBottom of pageLink to this message  By A.Nonymous on Wednesday, June 12, 2002 - 11:20 am:

Maybe sample carryover, when you inject a blank after your standards, you may see some peak. If this is so, you have change your needle wash solvent to something stronger. Another possible reason is that when you have injected your componend, the seal from your vial is perforated, so your sample is evaporating. You can avoid this when you use silicon seals and a sample temperature of 10-15 C ( be sure that your component doesn't precipitate).

I hope this will help you, lot of success.


Top of pagePrevious messageNext messageBottom of pageLink to this message  By Ravi on Wednesday, June 12, 2002 - 12:01 pm:

Thank you for the response. The solvent is pretty clean after the rear std. injections. Therefore, it could be from the evaporation. Given that the mobile phase is 75% organic, this could be the culprit. I can check this very quickly by recapping the std vial with new caps after every injection. Definitely, this should not be a practice in the final method:-), but at least will answer the question.


Top of pagePrevious messageNext messageBottom of pageLink to this message  By Anonymous on Wednesday, June 12, 2002 - 01:57 pm:

If it is evaporation just make each injection from a new vial.


Top of pagePrevious messageNext messageBottom of pageLink to this message  By Anonymous on Wednesday, June 19, 2002 - 01:44 am:

What about further dissolution on autosampler? We had some standards dissolved in methanol, and there was clear increasing response throughout, even each standard was injected from a new vial. Solutions showed no visible undissolved material but we got away with carefull sonication before analysis.


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