How to identified the purity of standard substance?

[wxmwqr]

Hi:
Recently I submitted a papper about the analysis of active compounds in medicinal herb by HPLC method.
Because the compouds could not be purchased, so we prepared them by ourselives. All these compounds were identified by direct comparison of their spectral data (UV, IR, NMR and MS) with those reported in the literature. The purity of the standards was checked by HPLC/DAD at three different wavelengths (210, 252, 300 nm), and the results suggested their purity to be above 98%.
But the reviewer gave us some advices as below:
"it indicated the purity of four standards (prepared by authors) was checked by HPLC/DAD and got a result of >98% purity. To the best of our known, similarity test of spectra at various places in a peak can be employed to calculate the peak purity, but it is imprecise, and there are many other methods of purity test on HPLC/DAD reported. So, the author should give some explanation on the calculation method of peak purity test, and some literatures involved expected to be cited."

Could you give some literatures or some good method for how to identified the purity of srandard substances

[james little]

could do Carbon/hydrogen/nitrogen analysis (elemental).

Could do internal standard NMR where you add a known amount of very pure component and use its molar response to calculate the purity of unknown. Might have to do determine your precision and accuracy with some known material if reviewer is pickey..

Should make sure material is dry to minimize error in proton NMR analysis.

Example found at

http://www.ncbi.nlm.nih.gov/entrez/quer ... t=Abstract

[wxmwqr]

Thanks for you reply!
But the reviewer asked me to use the HPLC method to test the purity of reference standards and to cite the literature. So I need the literature urgently.

[james little]

with hplc, could do area percent, but not too good since don't know molar extinction coefficient of impurities and major component.

Also, everything might not absorb at wavelengths monitored.

Furthermore, everything might not elute from column.

I doubt if they would mind using another technique since most important point is proving the purity of your prepared standards.

[JZeszotarski]

I'm not familiar with any literature on peak purity analysis by HPLC/DAD (try a lit search maybe to see if anything is out there). In my experience, each HPLC and/or software vendor has their own specific implementation of peak purity calculations. For example, Waters uses a peak purity angle and Agilent uses a numerical rating.

I think the important point is to detail the exact method used (i.e. exactly how the purity is calculated) and possibly reference the software version used.

Just my 2 cents...

[Peter Apps]

The referee might be refering to the "Peak Purity" procedures that are available in some HPLC software packages, but these do not provide a measure of the purity of the substance that was injected, only the proportion of the peak area that is made up by the major component, which is still not purity in the chemical sense.

How did you calculate 98 % purity from absorption at three wavelengths ?

Peter

[ta068]

My suggestion from peak purity part in a HPLC software (Agilent).
%peak purity from software depend on concentration, if concentration is high peak purity from software calculation is high and if concentration is low the peak purity is low, althrough it is reference standard.
But I'm not much sure, but you can try this.

[JM]

The purity of standard and Peak purity are two different things. Peak purity by DAD gives you assurance that no other impurity peak is co-eluting in your method.

The purity of reference standard is calculated by deducting following things from the chromatographic purity of standard ( method should be capable of showing all impurities present in the standard ),

1. Moisture
2. solvent present
3. Inorganic impurities ( residue on ignition value)

hope it is clear.

JM

[HW Mueller]

We have been through this several times. There is no unequivocal way to tell purity with UV alone if you don´t know the relevant parameters, and even then it might be impossible.
For instance, JM, what if different impurities have the same UV spec? How will a DAD distinguish them?

[JZeszotarski]

This subject has gone around and around discussing two topics as if they were one and the same...

There are two issues here:

peak purity - the spectral analysis of a chromatographic peak to determine if it is truly representative of one and only one compound (or more rigorously, that the peak consists of only 1 specific spectrum)

standard purity - the assay of how pure the compound studied truly is

In my estimation, wxmwqr reported the standard purity in his paper, but the reviewer seems concerned with peak purity. They are really two separate issues. Wxmwqr may well have 100% peak purity and 98% standard purity, as I am assuming the >98% purity is obtained as a mean area % for a chromatographic run analyzed at the three different wavelengths.

I would suggest wording to clarify the issue as an average HPLC-UV purity from three different wavelengths as this purity could just as easily been calculated using three separate runs on a LC-UV system as opposed to a DAD system.

Just my thoughts, perhaps someone else has a better idea.

[Bruce Hamilton]

I was assuming the reviewer was concerned about the suggestion that the peak purity at the specific wavelengths from the same analytical run was sufficient to validly assign a purity to the standard.

The reviewer appears to want evidence that such a use of peak purity can be related to the standard purity, and is supported by references to others who have calculated purity the same way. I assume they want to know that the use of the specific peak purity measurement, the selected wavelengths, and chromatographic conditions were conventional and best practice.

I'm not sure that I'd accept such a determination without further confirmation of the standard or technique.

If all else fails, copy this thread to the journal, and ask for a clarification .

Bruce Hamilton

[wxmwqr]

The purity of standard and Peak purity are two different things. Peak purity by DAD gives you assurance that no other impurity peak is co-eluting in your method.

The purity of reference standard is calculated by deducting following things from the chromatographic purity of standard ( method should be capable of showing all impurities present in the standard ),

1. Moisture
2. solvent present
3. Inorganic impurities ( residue on ignition value)

hope it is clear.

JM

Thanks alot to everyone

I think I am agree with JM's opioion.

I think this problem over and over. And I think the reviewer want to me to explain how did I get the purity of the standards. And the reviewer think that the method (DAD/HPLC) is not accuracy.

I think the official method has strong persuasion. So I browsed the web of FDA and ICH, but I failed to find official document about this problem.
If you have the document, please sent it to me, I would to be very much obliged.
My E-mail: WXMWQR@56.com.

[Uwe Neue]

Another interpretation: the reviewer wants you to find publications published by him and cite them. Look in the journal for which you have submitted this publication for references on the subject.

[JM]

HW,
DAD gives you "Assurance" about peak purity and is acceptable in regulated environment. for that matter NO single technique can give you absolute proof of peak purity. LC-MS does not distinguish between isomers, LC-NMR does not distinguish between optical isomers and so on.

There is no end to produce confirmatory data but we should restrict to what is acceptable to regulatory bodies. It is even acceptable to have non-specific method provided that we are controlling that perticular impurity by some other method ( a impurity of 0.2 % does not affect the assay even if it is coeluting as RSD of 2 % is allowed for assay ).

Just for lighter side of it, 80 % of chemistry was developed without DAD/LC-MS

JM

JM

[HW Mueller]

My concern is that there are a lot of younger people that don´t know the difference, anymore, between what is pure according to regulation and what is pure according to the best humans can muster at the present. In other words, regulation seems to have the tendency to corrupt.