Chromatography Forum

Greetings,

I have been tasked with troubleshooting a USP HPLC method for our lab. It is the assay and limit of other polyols analysis according to the USP monograph for Xylitol. Previously, the USP method for Xylitol and other polyols involved derivatization and analysis by GC. They have changed over to a HPLC-UV method monitoring at 192 nm. Currently we are struggling with baseline issues. Does anyone have any suggestions for running this USP method successfully? We have tried running on multiple instruments and cleaning the instrument...but the baseline is still problematic.

Thanks in advance for any ideas!

192 nm no wonder you're having baseline issues.

Does your assay meet any other criteria of this method?

It may be that a crappy baseline is par for the course.

Since its a USP monograph, I don't have any any ability to change the wavelength I am monitoring at. According to USP, this method works, but I am having a hard time getting it to.

The devil is in the details:
- degassing (see, for example: http://www.shimadzu.com/an/hplc/support ... 5/042.html)
- solvent purity
- flow consistency
- detector operation (N2 purge of the monochromator optics)
- data system settings (e.g., time constant).

What were they thinking!

That assay should have been developed using RI....

- Karen

What were they thinking!

USP. That explains a lot. Most likely that's the set-up that the contributor had, and what worked for him. Welcome to the flat-earth world of USP.

A perusal of the extant literature disclosed that 100% of the "rest of the world" uses Refractive Index as the Detector, and USP water only as the eluant and sample prep medium. *Even the manufacturer of specialty type sugar alcohol LC columns always use RI detection with 100% USP water as the m.p..
Not sure why monitoring of a UV signal @ 192 nm is cited as the means of conducting the HPLC run. The multiple OH groups of the alditols could present small pi-pi interactions--maybe that is where the UV signal is sourced. However, there are no chromophores or double bonded activity to create a strong enough signal.
So, whomever sponsored the UV method for the USP evidently has an elaborate LC setup that cannot be reproduced by anyone else in the world since eryone else has reported extreme problems trying to perform it.
Also, with regard to quantitation, GC method involving acetic anhydride and pyridine as derivitizing agents is frequently used.

My take:
1. The USP and FDA "thinks" they understand HPLC

2. The USP "thinks" that more labs would have UV detectors than RI detectors.

What were they thinking!

USP. That explains a lot. Most likely that's the set-up that the contributor had, and what worked for him. Welcome to the flat-earth world of USP.

Look at some of the other USP chapters. Like Volumetric Solutions, for sodium hydroxide: it states "standardize frequently", so just what does that mean? And standardize in singleton, duplicate, triplicate, whatever? How about some real guidance? Like can certified titrant solutions from real companies like Baker or Aldrich be used without standardizing? I'm not so sure...

Where is the chromophore? An aldehyde tautomer?