I need help verifying an analyser's work

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

11 posts Page 1 of 1
Greetings,
Cerebrotendinous xanthomatosis is a genetic disease that requires permanent use of medication chenodeoxycholic acid to avoid critical effects such as mental retardation, cataracts, mobility and others. My wife unfortunately suffers from this disease. Unfortunately it is prohibitively expensive (>$20k/month) so in order for her not to suffer and die I must purchase raw API, encapsulate and give to her. Although I try my best to verify that the suppliers I purchase from are trustworthy I still cannot trust them, so I need to test the API.

There's one guy that provides this service, but unfortunately in the past he has been caught lying to customers (saying he tested something he actually didn't) and his trust is shattered.

He sent me chromatogram of chenodeoxycholic acid standard he said purchased from sigma and the sample I sent him. I found it odd because the baseline isn't straight, there were multiple peaks eluted, some even very close to the retention time of the drug. Still he said it's 98%+ pure. When I asked him about it he said "chenodeoxycholic acid has extremely weak signal of 5" which I found very odd because there are several CDCA chromatograms on the internet with normal characteristics (straight baseline, only one peak except for impurities). To make it worst, I don't believe this is a 98%+ pure sample (perhaps 95%+) because I didn't store it 100% properly and it's months old already.

I need help verifying if he actually did the work and if he did it was a competent one for this matter is important.
His test on standard from sigma: https://www.docdroid.net/9suhIXC/chenod ... 19-002.pdf
His test on my sample: https://www.docdroid.net/QMqdUB1/394-39 ... 19-002.pdf

Thank you very much!
The HPLC trace you have might not represent the true content of API as some of the impurities might have much higher UV activity at 210 nm than your API. So unless he is done quantitation I would not rely on the numbers. You might need a different than UV detection technique. You also want to verify your source of API, sometimes alternative route API have a different impurity profile and you need to make sure that these different impurities are not harmful.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
Thank you very much for your interest in helping. Every input is much appreciated.

I know little to nothing regarding HPLC, but my main concern is that I have never seen a pure substance with multiple peaks. Is this possible? I'm starting to wonder if his column is contamined thus these results for both standard from sample from sigma aldrich (min. 97% pure) and my sample. He sent me pictures of my sample and purchased sample from sigma aldrich (in a table, not being tested).

Both substances were raw pharmaceutical powder with no other compound, filler or anything. It was just raw API. Not encapsulated yet. My supplier has claimed it to be minimum 98% pure and sigma aldrich hardly would send him anything less than 97% pure. Is there an explanation for it not to be a normal single peak like other chromatograms I'm used to? Instead of these multiple peaks, some even very close to the claimed drug peak. The tester has said my sample is 98%+ pure.

I need to know whether this tester has actually tested or if he's done something wrong, and I have no idea where to look or ask. This subject has taken my entire week due to its severe importance and I deeply appreciate any help at all.
From the index of the chromatograms it seems that a wide screening gradient from 5-95% organic has been used, involving TFA as modifier. It is not unusual to see gradient ghost peaks in such gradients, i.e. peaks originating from trace impurities in the eluents. These peaks will be visible in any chromatogram, no matter if it's a sample, a standard, a solvent or a blank injection. So yes, it is possible with gradients to see a bunch of peaks even for a pure substance.
From the chromatograms of sample and standard alone it's impossible to judge if the extra peaks are gradient ghost peaks, though. You'd need a blank run or solvent injection to compare.
I would not trust the analysis, especially not based on the presented chromatograms.

UV is the wrong detector to analyze bile acids - the guy is right in this respect, they give a very weak signal. Especially to analyze impurities of the API, you need mass spectroscopy (HPLC-MS/MS).

It is questionable to rely on only two chromatograms (standard and test sample) for the analysis, especially if they look like the ones you posted.

Ask your guy, if he can show you a method validation according to ICH guidelines. Before giving out results, he has to show, that his method is selective, accurate, precise and robust.

Since the health of your wife depends on this substance, I would either look for an accredited lab to do the analysis, or for a supplier that can provide a Certificate of Analysis for his product.
Dear Vlad, HPLCaddict and Thomas,
thank you very much for your opinions.

Still appreciate very much any input on the subject from anyone.
Apples, I have a friend with access to MS detection, they charge about $70 per sample, so you might need to be ready to spend $300-500 to test your samples. Alternatively you can find a better standard and try to redo quantitation.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
Vlad Orlovsky wrote:
Apples, I have a friend with access to MS detection, they charge about $70 per sample, so you might need to be ready to spend $300-500 to test your samples. Alternatively you can find a better standard and try to redo quantitation.

Thank you very much, Vlad. I'll contact you as soon as I have funds available.
You wrote he charges $70 for testing but told me to be ready to spend $300-500. Is that to purchase the standard? Because the standard from Sigma is really cheap for this substance.

Your friend can do identity and purity test for API?

Thank you very much once again!
You will need more than one injection. Once you are ready I will provide you with a contact information. It is a university lab in Arizona with LC/MS capabilities.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
Dear Apples,

why don't you just buy your bulk API from Sigma, instead of just the standard? It comes with a CoA and costs about 500$/25g, which should be good for one month of medication.
HPLCaddict wrote:
From the index of the chromatograms it seems that a wide screening gradient from 5-95% organic has been used, involving TFA as modifier. It is not unusual to see gradient ghost peaks in such gradients, i.e. peaks originating from trace impurities in the eluents. These peaks will be visible in any chromatogram, no matter if it's a sample, a standard, a solvent or a blank injection. So yes, it is possible with gradients to see a bunch of peaks even for a pure substance.
From the chromatograms of sample and standard alone it's impossible to judge if the extra peaks are gradient ghost peaks, though. You'd need a blank run or solvent injection to compare.

Well, you are exactly right. Those are in the blank as well.

Source: I ran the analysis.
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