Two L-menthol peaks with RP C8 150 mm x 4,6 mm

[Nahru]

Greetings,

First time posting here, though I've been browsing the forum from time to time.

I may have a problem. I'm trying to assay L-menthol, using an RP C8 column. The problem is that I have been consistently obtaining 2 peaks. I've ran blanks (both just mobile phase and placebo sample), and they never show those 2 peaks, only the standards and samples which contain L-menthol.

Using UV detection at 197 nm and isocratic elution with a mixture of acetonitrile:water. The peaks are well separated, one at ~ 9,1 min and the other at ~ 12,5 min. The baseline is consistently relatively flat. Both peaks are linear (from 30 - 150 ug/mL), as well as their sum. Recovery tests, using the sum of areas for those 2 peaks, show R% to be around 100 %, when adding known volumes of standard solution to blank samples, and then assaying them (R% is from 97,5 % to 102,5 %).

I've been using the sum of areas of those 2 peaks to assess the content of L-menthol in a sample, and what I get is consistently correct content of L-menthol (with regards to theoretical content).

Could it be that L-menthol changes its configuration during preparation? The solvent for standard and sample is the mobile phase. Perhaps one of the peaks is an impurity (standard is of 99,56 % purity, determined via GC)? Perhaps one of the peaks is a degradation product of L-menthol?

Thanks for all the input in advance.

[mattmullaney]

Hi Nahru,

I don't know whether or not there is a degradation product in the standard you're using, my thought is that it may be another enantiomer of (-)-menthol [maybe (+)-menthol?] that is present in the standard material you're using...or possibly as the standard is separated within the column multiple conformers of (-)-menthol are formed, as you suggest. This kind of thing often happens with Schiff bases--which are not anything like menthol--but maybe the chair form one would expect (-)-menthol to adopt has a companion?

My vote is for the presence of (+)-menthol in the standard and sample...however unlikely that may be.

Please, see what others have to say as well and thank you!

[Nahru]

Hi Nahru,

I don't know whether or not there is a degradation product in the standard you're using, my thought is that it may be another enantiomer of (-)-menthol [maybe (+)-menthol?] that is present in the standard material you're using...or possibly as the standard is separated within the column multiple conformers of (-)-menthol are formed, as you suggest. This kind of thing often happens with Schiff bases--which are not anything like menthol--but maybe the chair form one would expect (-)-menthol to adopt has a companion?

My vote is for the presence of (+)-menthol in the standard and sample...however unlikely that may be.

Please, see what others have to say as well and thank you!

No, thank you for the effort.

The issue may be that I've been using a working standard, which normally is used by my Company's manufacturing sector. I have ordered a true analytical standard, only L-menthol, after which I should know more. To my knowledge, it *should* only be (1R,2S,5R)-5-methyl-2-propan-2-ylcyclohexan-1-ol [or (-)-menthol], as that is what the certificate from the supplier states, but I shall double check again.

I am also considering the presence of (-)-menthol and (+)-menthol in the standard ("standard"), that is - basically a racemate. The peaks really are well separated, (almost) no interference, both are linear, and recovery tests show what they should (on average +/- 1 % of variation)...

Will check more later, when I get the standard (-)-menthol in a few days time.

[mattmullaney]

Hi Again, Nahru,

Understood, and Best Wishes! It'll be interesting to see how things turn out.

[Nahru]

Just wanted to finish this. Obviously, only one peak can really be menthol (D and/or L), because I'm using non-chiral column, and I haven't used derivatization to make them diastereomers. I don't know how this didn't occur earlier to me... Anyway, it turns out it's most likely some sort of impurity, my guess being related substances formed during the production of L-menthol.

[mattmullaney]

Hi Nahru,

Understood, and thank you. As you said, the first standard material you used (if the analytical standard containing 100% (-)-menthol hasn't arrived) was a working standard typically used by the manufacturers...makes some sense that if that source is man-made there could be process impurities within it.

Menthol is interesting...the naturally-occurring stuff is 100% (-) if I believe the Internet. Weird that the man-made stuff isn't necessarily enantiomerically-pure unless a lot of work is done upon the prepared menthol!

My thanks for the discussion, sir, and again, Best Wishes.