Impurity testing problems

[Charles A. Burger]

System Method

HPLC - Shimadzu 10A class vp series

Column - Merck Lichrospher RP-18, 5 µm, 250 mm x 4.0 mm

Mobile phase - 80% Methanol / 20% K phosphate buffer

Flow - 1ml/min @ 40°C

Wave. - 229nm (UV Cell path legth is 10mm) @ 1 AU/V

Inj. vol - 10 µl



I am qualifing a method for Mfg specific Butaconazole impurites. The method uses a 1 µg/ml conc. for the Standard while the sample checks in at 1000 µg/ml. One of the suitability requirements calls for a ratio of response factors to be between 0.9 and 1.1. I have had no luck in achieving this part of the suitability. My ratio is 1.17. The final sample calculation is Area %

I suspect that I am running into linearity problems considering the response factor ratios I have acheived with lesser concentrations. So far I have tried 100 µg/ml and 10 µg/ml, both have been been very close to 1.

I have asked the mfg to let me know what system they have run in case they have something vastly different.

Any thing I am missing?

[tom jupille]

Not that I can see. 3 orders of magnitude linear range is asking a lot.

How big (max absorbance) is your main peak in the sample?

[Charles A. Burger]

It's right at 600 mV.

[Myriam K]

You are right to ask about instrument differences. Differences between instruments may mean detector overload in one instrument and not in another. I have seen differences even when the cell path length and injection volume are supposed to be the same. Your instrument could be designed and/or installed “too wellâ€

[Mark Tracy]

Also check your data collection rate and rise-time settings on your detector.

[Charles A. Burger]

You could try different brands of methanol, buffer components, and mobile phase filters to see if you can make a mobile phase that absorbs less at 229nm.

Thank you for the response, the background absorbance did enter my mind but I sort of ignored it because the peak was only going to 600 mAU. Would using LC-MS grade materials be a better choice or could this be more related to which manufacturer has better solvents and reagents.

[Myriam K]

Would using LC-MS grade materials be a better choice or could this be more related to which manufacturer has better solvents and reagents.

LC-MS is a different requirement. Interferences/responses are detection technique specific. A lot of contaminants give MS signals but do not absorb at 229 nm. Conversely, often the UV-detectable peak I'm after gives me little or no MS signal.
A manufacturer may have several grades of a solvent. I would look at the methanol grades in terms of UV absorbance. Some grades will have lists of UV absorbance values. The old Burdick and Jackson methanol listed 0.020 at 250 nm, 0.160 at 225 nm, and so on. I do not think you can do much better than that. Pure methanol has some end absorbance in the low UV.
If practical, I would try fresh bottles of each reagent, just in case there's contamination in the ones you are using. I do not know about possible impurities in water or buffer. Do you use bottled HPLC water or have a water purification system in your lab? Do you have alternate sources of water? Do you make your own buffers? If practical, I would try fresh bottles of each reagent, just in case there's contamination in the ones you are using. I would just measure absorbance of the mobile phase. I have seen filters leach UV-absorbing contaminants, but I would not expect a high absorbance from that. Theoretically, there should not be anything to cause the trouble you are seeing. The only times I recall noticing high mobile phase absorbances were cases where mistakes were made in the preparation of the mobile phase (e.g. acetone for acetonitrile, acetic acid for methanesulfonic acid).

[DR]

600mV isn't excessive, but that doesn't mean that you're still in the linear range either (I've seen the same thing with acetaminophen).

Do a more detailed linearity check (several different concentrations from ~100-1100) and see how high you can go before the r² falls off. If you must use 1000 for sample, could you make smaller injections? (If you have a well qualified injector, you should be able to).

You could also be suffering from a lack of robustness with respect to integration technique or you could be overloading the column at higher concentrations (which could mess up your integration).

[Charles A. Burger]

This question may be hopelessly general but are there solvents and reagents out there that a 2/3 majority would classify as superior when it comes to HPLC operation and are they centered around 1 manufacturer. I ask this because trying to locate quality solvents for gradient HPLC has been a trying experience to say the least.

I thought about posting a question as to who makes the best solvents for gradient work, but I fear that 20 people would respond with 30 different answers. I saw that you mentioned Burdick and Jackson almost in a past tense, do they not make quality Methanol anymore?

The old Burdick and Jackson methanol listed 0.020 at 250 nm, 0.160 at 225 nm, and so on.

[bkirschil]

Perhaps decreasing your sample injection volume would help - if your main analyte peak is saturating the photomultiplier. It can work the other way if your injecting too little. What is your calculated S/N ratio? You do need to simulate the 0.1% condition with sample concentrations and determine response factor ratios.

Was the development of this method performed on the same chromatographic system?

[Charles A. Burger]

I initially tried going from 10 µl to 5 µl. My ratio went from 1.17 to 1.11 but, my RSD approached 4.5% which "per the method" is NMT 3.0%.

As far as your last question, I wouldn't be able to tell you because I don't know, it's a Manufacturer supplied method for the impurities that I have to qualify on my system.

Thanks

Chuck

[KC]

I have evaluated a couple of different solvents and found Burdick and Jackson to be superior. The HPLC grade of each works well for just about all applications. We have nanopure system for water but I don't use it I use B&J bottled water for all mobile phases. The one solvent we purchase as ultra-pure ir their GC/MS grade hexane.

[KC]

Just finished a ****conazole impurity study where I was looking at 4 impurities ranging in conc from 0.5 ug/mL to 12.0 ug/mL in solutions of the conazole at a conc of 1200 ug/mL. In order to quantify all impurities I prepared combined standards of the 4 impurities from 0.5 to 20 ug/mL and analyzed all solutions disregarding the huge conazole peak (way off scale). To quantify the conazole I did 25X dilutions on all solutions and analyzed the samples again with conazole standards ranging from 10.0 to 100 ug/mL. Worked like a champ, r2 for each of the 5 components was 1.000 and never had a problem with overlaoding of the detector.

[tom jupille]

KC, as far as I'm concerned, that's the right way to do it. While it's tempting to try to use straight area % for impurities, as I said in an earlier post, three orders of magnitude linear range is asking a lot.

[DR]

re: solvents, I rarely find fault w/ B&J or EM Sci. but your UV spec can be quite helpful in this regard. Just do a scan on an aliquot of MeOH from every lot you can find around the lab. Even though they'll all pass the cutoff spec, once in a while, you will find one that's a lot lower than the others...

Save that one for your extra low wavelength work and NEVER do any development work with it, unless you want to be the only one who can run your method.