Basic questions from a clueless guy

[mark02]

Hi everybody!

Can some of you experts please answer a few basic questions for me please? I bought a certain medication in powder form from somebody who supplies pharmacies. But now I’m afraid to use it because I’m concerned that the seller sent me something else. That’s why I want to have the powder tested in order to confirm that I received the right medication. I also want to test it for purity. The medication is supposed to be very pure, but I’m concerned that the seller mixed some kind of filler into the medication in order to make bigger profit.

I first wanted to have the powder tested as an unknown substance, but that turned out to be too expensive. It costs around $350. But then I contacted another laboratory where they said they can test the powder with Gas Chromatography-Mass Spectrometry or Liquid Chromatography-Mass Spectrometry which cost only $60 and $50. However, I would first have to tell them what is my medication supposed to be so they can calculate the molecular weight and decide whether to test the powder with GC-MS or LC-MS analysis. But the problem is that if I tell them what is my medication supposed to be, then they might not even test it and just tell me what they think I want to hear. Yes I know I’m paranoid, but you have to understand that my life runs according to Murphy’s Law. Even if there is the tinniest possibility that I’ll get screwed, then I’ll get screwed for sure. LOL!

But maybe I found a way to solve the problem. Can you answer these questions below for me please?:

-What do GC-MS and LC-MS machines actually measure? Do they measure molecular weight of different molecules present in the sample? Do machines show you specific numbers?

-If I understand correctly, GC-MS is used for analyzing low molecular weight molecules, and LC-MS is used for analyzing high molecular weight molecules. So what is the exact range for each analysis? Does this depend on the specific machine that is used for the analysis?

-If I tell the laboratory that the molecular weight of the substance which I want to have tested is between 800 and 1300, will they be able to decide whether to test it with GC-MS or LC-MS?

-And the last question is about purity. Let’s say you mix 20 grams of substance A (in powder form) with 10g of substance B (also in powder form). And let’s say substance A can be analyzed with GC-MS, and substance B can be analyzed with LC-MS. And now let’s say you analyze the mixture with GC-MS. Will the analysis show the molecular weight of the substance A and that the substance A represents only 66.6% of the mixture? I basically want to know whether GC-MS and LC-MS can also show you the percentage of each substance in the sample.

Your help is greatly appreciated.

Mark

PS. If you’re aware of any even cheaper GC-MS and LC-MS laboratory then please let me know. In the future I’ll need to test more medications.

[tom jupille]

-What do GC-MS and LC-MS machines actually measure? Do they measure molecular weight of different molecules present in the sample? Do machines show you specific numbers?

Both of those are "tandem" techniques. GC = gas chromatography; LC = liquid chromatography; MS = mass spectrometry.

To grossly oversimplify:
Chromatography (either GC or LC) separates the compounds in a sample from one another (you inject the sample and the various compounds come out sequentially). Mass spectrometry ionizes compounds and lets you determine molecular weight and may give you a "fingerprint" of each compound.

For qualitative analysis (what compounds are there) the combination of how long it takes to get through the chromatography with the MS pattern provides identification. For quantitative analysis (how much of each compound is there), you have to determine the "response factor" of each compound by running standards of that compound at known concentrations.

-If I understand correctly, GC-MS is used for analyzing low molecular weight molecules, and LC-MS is used for analyzing high molecular weight molecules. So what is the exact range for each analysis? Does this depend on the specific machine that is used for the analysis?

The actual criterion is volatility: in order to run GC, the compound has to be vaporized. In general, small molecules are easier to vaporize than are large molecules, but there is a lot of overlap.

-If I tell the laboratory that the molecular weight of the substance which I want to have tested is between 800 and 1300, will they be able to decide whether to test it with GC-MS or LC-MS?

Not for sure but LC-MS would probably be the choice in that range.

-And the last question is about purity. Let’s say you mix 20 grams of substance A (in powder form) with 10g of substance B (also in powder form). And let’s say substance A can be analyzed with GC-MS, and substance B can be analyzed with LC-MS. And now let’s say you analyze the mixture with GC-MS. Will the analysis show the molecular weight of the substance A and that the substance A represents only 66.6% of the mixture? I basically want to know whether GC-MS and LC-MS can also show you the percentage of each substance in the sample.

In order to quantitate, you need to know the response factor for each compound, which you get by running standards of that compound at known concentrations. That's why they want to know what they're looking for ahead of time.

[mark02]

Thank you very much Tom for your reply.

In order to quantitate, you need to know the response factor for each compound, which you get by running standards of that compound at known concentrations. That's why they want to know what they're looking for ahead of time.

I tried to educate myself on the response factor on Wikipedia but I’m not sure whether I understood anything (I’m in Europe and English is not my first language). If I understand correctly “running standards” would require performing the analysis several times. But I assume that would make it also several times more expensive for me. Am I right?

Would it make any difference if the medication was 99% pure? Would it be still necessary to run standards in order to determine purity?

Do you know any amateur, maybe even on this forum, who does GC-MS and/or LC-MS analysis cheaply? Could you do it Tom?

Thank you again for your help.

Mark

[tom jupille]

An LC-MS or GC-MS system costs anywhere from $100k up, requires specialized skills to set up and operate, and uses expensive reagents. I really doubt that there are any "amateurs" doing this. As far as the cost is concerned, $60/sample is about what a reputable lab would charge if you are committing to run 1,000 samples -- or if they are already set up to do the analysis and could slip your sample into the work flow.

The catch to all of this is that chromatography and mass spectrometry are not "absolute" techniques that spit out the quantity of a compound. They are "comparative" techniques that generate a response which must be compared the response of a known amount of the compound.

There is a tutorial on "getting started in HPLC" hidden on our web site. The section on quantitation that explains how it works is here: http://www.lcresources.com/resources/getstart/4e01.htm

[mark02]

An LC-MS or GC-MS system costs anywhere from $100k up, requires specialized skills to set up and operate, and uses expensive reagents. I really doubt that there are any "amateurs" doing this.

Whaaaaaaat?! I had no idea this machine was so expensive. I read on some forum a post from a person who said that he bought a used Gas Chromatograph for $1600. But obviously he made a mistake, or I didn’t read right. I got a feeling these kinds of systems are something chemistry students keep at home for their own personal use. LOL!

The catch to all of this is that chromatography and mass spectrometry are not "absolute" techniques that spit out the quantity of a compound. They are "comparative" techniques that generate a response which must be compared the response of a known amount of the compound.

There is a tutorial on "getting started in HPLC" hidden on our web site. The section on quantitation that explains how it works is here: http://www.lcresources.com/resources/getstart/4e01.htm

Thank you very much for this link. I’m gonna look it up and try to educate myself. I can see things are more complicated than I imagined.

Thank you very much Tom for your help. I really appreciate it.

Mark

[skunked_once]

I read on some forum a post from a person who said that he bought a used Gas Chromatograph for $1600.

What you read could certainly be true. There are two parts to a GC-MS or HPLC-MS system. The GC or HPLC instruments can be purchased used for less than $10,000. It is the MS part of the instrument that is expensive since it involves strong vacuum components, complex electronics, and computer software. Please continue learning and asking questions. We are here to help.

[mark02]

What you read could certainly be true. There are two parts to a GC-MS or HPLC-MS system. The GC or HPLC instruments can be purchased used for less than $10,000. It is the MS part of the instrument that is expensive since it involves strong vacuum components, complex electronics, and computer software.

So I didn’t read wrong. But I wrongly assumed that GC-MS system would be just a bit more expensive than GC system.

Please continue learning and asking questions. We are here to help.

Okay, thank you. Although I don’t want to ask too many stupid questions and waste your time. LOL

[mark02]

There is a tutorial on "getting started in HPLC" hidden on our web site. The section on quantitation that explains how it works is here: http://www.lcresources.com/resources/getstart/4e01.htm

I think I understand a few things now. The lab would first have to create 3 samples of the medication at different concentrations and analyze them. This way they could establish a calibration plot. And then they could analyze the unknown sample and use the calibration plot to calculate the concentration of the medication in the sample.

But what I don’t understand is how they would even be able to create samples of the medication in 3 known concentrations. I assume they would need to get the medication in perfectly pure form or in a measured form (like tablets or capsules) and create the 3 samples from that. I imagine you have to know exactly how much medication you put into the 3 samples in order for the calibration plot to be useful. But it’s okay, it doesn’t matter. The important thing is that I understand how the process is done, how much work it takes. Now I know why it costs so much more to determine quantity.


I just got an idea how to solve my problem. Could you answer 2 more questions for me please? These are the last ones, I promise. After this I won’t bother you again.

As I said, the medication which I want to have tested is supposed to be 99% pure. So let’s say I have the medication tested with both analysis, LC-MS and GC-MS. If the medication really is 99% pure, would the results show only one peak? Or do you think those 1% of impurities would create an additional peak or peaks? Because if the results showed only one peak, then this would be a confirmation that the medication really is 99% pure.

Thank you very much!

Mark

[tom jupille]

f the medication really is 99% pure, would the results show only one peak? Or do you think those 1% of impurities would create an additional peak or peaks?

Actually, it could be a lot less than that if it were cut with something that didn't show up in the analysis (salt, just for an example).

The thing about chromatography (somewhat less true about the coupled techniques) is that it provides "negative" qualitative information: if you see two peaks, you know you have at least two compounds, but if you see only one peak, you can never be 100% certain that there are not two compounds coming out together (or that some compounds in your sample never come out at all).

[HPLCaddict]

Sorry, but I just have to cut in here.

What the hell is this stuff you want to have analyzed???? If it is a "serious" medication you really need, get it from a reliable source e.g. some sort of serious pharmaceutical company. They are heavily regulated and controlled by authorities on a regular basis. Certainly, you cannot be 100% sure the stuff they're selling is absolutely correct, but at least 99,9% sure. Don't play with your health!!

If this is about vitamin pills or anything to increase the size of certain body parts , well, you must judge for yourself...

[mark02]

Actually, it could be a lot less than that if it were cut with something that didn't show up in the analysis (salt, just for an example).

The thing about chromatography (somewhat less true about the coupled techniques) is that it provides "negative" qualitative information: if you see two peaks, you know you have at least two compounds, but if you see only one peak, you can never be 100% certain that there are not two compounds coming out together (or that some compounds in your sample never come out at all).

Okay, I get it now. This was a very good explanation. Chromatography can give you a proof whether certain substances ARE present in the sample, but it cannot give you a 100% proof there are no other substances present in the sample as well.

The important thing I learned is that I will have to pay more for a quantitative test if I want to be sure how pure the medication is. There’s no other way. It doesn’t matter that the medication is supposed to be 99% pure.

Thank you very much Tom for taking time to help me. I really do appreciate it.

Mark

[mark02]

Sorry, but I just have to cut in here.

What the hell is this stuff you want to have analyzed???? If it is a "serious" medication you really need, get it from a reliable source e.g. some sort of serious pharmaceutical company. They are heavily regulated and controlled by authorities on a regular basis. Certainly, you cannot be 100% sure the stuff they're selling is absolutely correct, but at least 99,9% sure. Don't play with your health!!

If this is about vitamin pills or anything to increase the size of certain body parts , well, you must judge for yourself...

No, this medication doesn’t increase size of certain body part. LOL This medication is called nystatin, and I use it for treating chronic intestinal candida infection, because my immune system doesn’t work properly. I used to get it from my doctor and it was covered by insurance, but now manufacturers in my country no longer make it. It was replaced by newer medications, which give me side effects. So I have to buy it from abroad. If I’d buy it from internet pharmacies I’d pay more than $7.000 for 1kg. But since I bought it directly from a manufacturer in China it cost me only $380/1kg.

Mark

[Pepter]

Sorry, but I just have to cut in here.

What the hell is this stuff you want to have analyzed???? If it is a "serious" medication you really need, get it from a reliable source e.g. some sort of serious pharmaceutical company. They are heavily regulated and controlled by authorities on a regular basis. Certainly, you cannot be 100% sure the stuff they're selling is absolutely correct, but at least 99,9% sure. Don't play with your health!!

If this is about vitamin pills or anything to increase the size of certain body parts , well, you must judge for yourself...

No, this medication doesn’t increase size of certain body part. LOL This medication is called nystatin, and I use it for treating chronic intestinal candida infection, because my immune system doesn’t work properly. I used to get it from my doctor and it was covered by insurance, but now manufacturers in my country no longer make it. It was replaced by newer medications, which give me side effects. So I have to buy it from abroad. If I’d buy it from internet pharmacies I’d pay more than $7.000 for 1kg. But since I bought it directly from a manufacturer in China it cost me only $380/1kg.

Mark

First i would start with compare some of this 7k$ medication with nystatin from China producers.

[MaryCarson]

This paper describes an analysis that would tell you what percent of your powder actually is nystatin:

J AOAC Int. 2001 Jul-Aug;84(4):1050-5.
Liquid chromatographic determination of nystatin in pharmaceutical preparations.
Wilson P, Stewart A, Flournoy V, Zito SW, Vancura A.
Source

US Food and Drug Administration, NRL, Jamaica, NY 11433, USA.
Abstract

A rapid, reversed-phase liquid chromatographic method was developed for the assay of nystatin in the bulk drug and a variety of dosage forms. Analysis was performed on a Symmetry C18 reversed-phase column using a mobile phase of methanol-water-dimethylformamide (DMF; 55 + 30 + 15, v/v/v), with detection by UV at 305 nm. Quantitation is based on the sum of the peak areas of the 2 major isomers of nystatin. The linearity of the assay was determined for a concentration range of 0.05 to 0.2 mg/mL (correlation coefficient > 0.999). Accuracies and precision showed good reproducibility.

The instrument used runs about $30-60K, but is a fairly common piece of analytical equipment. However, this analysis won't tell you if there is bacterial or other types of trace toxic contamination (think current outbreak of fungal meningitis in the U.S.), or if the crystalline form is correct to allow the drug to dissolve in your stomach and be effective, or any of a number of other tests usually run to assure a product is ready for the market. These are really good reasons to buy your drugs from a reputable and regulated source.

[paulw]

These are really good reasons to buy your drugs from a reputable and regulated source.

I can't echo this enough. From what you have said it seems as if the Chinese supplier is selling to you the raw material as though you are a manufacturer, not an individual. Every manufacturer is expected to do the tests that Mary has listed and insure that the material is of good quality. In addition, if you are getting raw material grade, you would then have to compound a mixture to replicate the capsules/tablets you previously received. You would have to make sure you had adequate mixing, that the mixture you made gave you the same efficacy when taken, that you were handling the materials in a sterile manner, including storage and whatever vessels you used to compound.