Chromatography Forum

Having reproducibility issues w/ ELSD. Using conditions from Alltech website achieved separation of various phospholipids, however std peak areas decreased over course of analysis(14hrs) from 5000 units to 3000 units.
Found nebulizer blocked and significant deposits at base of drift tube. Cleaned all but was a short-lived solution.

Appears that, based on ELSD conditions in conjunction w/ large, waxy compounds, this problem is inevitable. Need method for weekly release of API.

Also tried literature conditions of ACN, MeOH, H3PO4(85%) (90,30,10 v/v/v), various silica columns w/ detection at 203nm. We can not reproduce the literature results - essentially no peaks detected.

Comments/suggestions

Thanks,
Robotjock

You're going to hate me for this, but what about going back to RI?

About 6ish years ago i tried VERY hard to get a normal phase ELSD method for a phospholipid to work but could not get past the reproducibility issues. I tried all sorts of mobile phase additives to try and keep the nebulizer clear... but nothing helped

The matrix i was using meant I had to use normal phase and a gradient so RI was out of the question and i could not use UV because the mobile phase was opaque at the wavelength I needed.

Eventually I went to a normal phase Sep-Pak for initial cleanup then quantitated by RP HPLC using UV (concentration was high enough)


- Karen

Do you have a caustic mobile phase?

Virtual Colleagues

The mobile phase is non-caustic consisting of Hexane:IPA:H20, but we do need to run a gradient. This eliminates/reduces success w/ RI.

The ACN,MeOH, H3PO4 MP is used in isocratic mode so may be do by RI. However, it bothers me that these conditions (w/ 203 nm detection)were "successful" in three literature articles but I can't reproduce them.



Kind regards,
Robotjock

Does anybody have a reference to any reverse phase method for phospholipids with LC/MS detection-I just want to compare our method with whatever exists. I did a quick Google search and did not find anything, although there are a bunch of normal phase methods.

Regards,

Vlad

Reverse phase HPLC of lipids:

LC-MS:
Analytical Chemistry, 76, 1935-1941 (2004)

LC-ELSD:
Journal of Pharmaceutical and Biomedical Analysis, 30, 391-403 (2002)

MG,

I'll request the ELSD article. Review of abstract indicates phospholipid derivative w/ UV detection. Does article actually address separation/detection of phosphatidylcholine/ethanolamine using ELSD with long term precision, accuracy data?

Thanks,
Robotjock

Robotjock,

The article although mentions ELSD it does not do to much with it, other than mentioning that the method developed with UV can work with ELSD and/or MS. The long term precision/accuracy data are for UV detection only (at 220 nm). Their S/N with UV detection were not great though...

If your question is more general about the possibility of validating methods with ELSD you may look at:

Petritis et al. Chromatographia, 2004, 60, 293-298

and hot just out of the press:

Rodriguez et al. American Laboratory, October 2005, 9-14

Both articles of course provide precision/accuracy data...

I checked the AC method and it is gradient reverse phase into normal phase. Traces look good in terms of peaks shape and resolution but mobile phase is rather complicated.
I would like to compare apples and apples-our reverse phase method with ACN-water-Ammonium acetate with the corresponding RP application.

Kostas,

Can you send me reprints from your publication on phospholipids? Thank you in advance. My email is vlad.orlovsky@sielc.com



Regards,



Vlad

I checked the AC method and it is gradient reverse phase into normal phase.

It looks to me like they were doing reverse phase the entire time, and are using 10% hexane in MeOH as their strong solvent on the C18. They were getting separation by chain length, and it is my understanding that normal phase separates by head-group and not by chain length. I agree, the step-gradient is a little weird.

Vlad,

The publication I was referring is not on phospholipids, as I said I refer to it as a reference on validation by using ELSD (there are not a lot of validated methods which use ELSD). Mine is on amino acids, the American Laboratory one is on "an Excipient in a sSelf-Emulsifying drug delivery system formulation".

I will send it to you by e-mail anyway.

Kostas

Kostas,

Thank you for the article. I am looking of a simple RP method for lipids without ACN-water going into MeOH hexane? Something like ACN-water-ammonium formate or acetate (isocratic or gradient of ACN or buffer gradient or combination of two). We developed a method/ column for analysis of phospholipids in RP conditions and I would like to compare it to something similar in terms of retention control, peak shape and efficiency.

regards,

Vlad

Re: the original posting about phospholipids and ELSD, has anyone tried the ESA Corona charged aerosol detector? I saw it last year at the EAS, and they claimed to have improved reproducibility and precision for phospholipids, though it still uses the same nebulizer and drift tube.

Thanks for all the comments. Unfortunately, I'm no closer to the needed separation than when I started. But I have learned some things... which is good.

Robotjock