Chromatography Forum

Hi - would anyone happen to know of a method to directly test for the purity of Arginine. The arginine is a starting material for a GMP product and the method will need to be validated at some point. I have come across several C18 HPLC methods using various MP additives and have had little success with these. I have also tried HILIC with little success so far. SCX by HPLC maybe? But is this good enough for GMP validation? Am I overlooking something easier possibly? Thank you kindly for you response.

Paul K

Are you trying to analyze it in it's underivatized form? If yes what is your detector?

Below is LC-ELSD method for basic amino acids (including arginine)
using Unison UK-Amino:
http://www.imtakt.com/TecInfo/TI329E.pdf

Here are few methods for direct analysis of arginine with reverse phase mixed mode Primesep columns. You can use low UV or ELSD. I know at least three big pharma companies validating and using Primsep 100 for quantification of amino acids using this column (proline, arginine and lysine):
http://www.sielc.com/compound_006.html

With current situation with ACN (price and availability) you might want to forget about HILIC and even switch to a 2 or 3 mm column (ID). With mixed-mode approach you can use almost no ACN to analyze amino acids as they are retained by combnination of weak reverse phase and strong (in case of arginine wwhich has two basic groups) cation-exchange.

If you send me your email I will provide you with a report on quantitation of amino acid in pharmaceutical formulation (including linearity, LOD and LOQ).

Regards

I'm quite surprised with your results using HILIC.
Arginine has a logP value of -4.2, indicating it to be a very ideal analyte for HILIC. We have done applications with arginine, and seen little problems to my knowledge.

The HILIC Retention Prediction tool, show a k'= 3.3 for arginine
http://www.sequant.com/prediction

using a ZIC®-HILIC column (100x4.6 mm, 5 µm, 200 Å) with an
eluent containing 70% acetonitrile and 30% 100mM ammonium acetete buffer, pH 6.7.

To me, this indicates that you may use methanol or any other organic HILIC solvent and still achieve sufficient retention. Perhaps you can elaborate on your findings.

You can use the same conditions as in the paper:

Petritis et al. Validation of an Ion-Interaction Chromatography Analysis of Underivatized Amino Acids in Commercial Preparation Using Evaporative Light Scattering Detection, Chromatographia, 2004, 60, 293–298.

Arginine does not make part of the mixture but is eluted a little bit after Lys at around 11 min (if I remember correctly, but I can look it up).

It would be interesting to see conditions using methanol in HILIC on amino acids. Even ethanol on ZIC as well as Atlantis Silica were a relatively restrictive affair (water content was very touchy . . . ) in the separation of some prolines.