Internal standard

[aspirinha]

Hello Chromatographers,

I am confronted with a method that uses Doxylaminesuccinate as internal Standard on a CN column and eluent pH 3,5 for the quantification of Dextrometorphanhydrobromide and Chlorpheniraminemaleate. The Doxs. peak has poor shape and the areas tend to fluctuate. Now, the whole thing is running on a robot and the internal standard is dosed gravimetrically. In theory the observed behaviour should not appear, should it?
I suspect that the compound is not a suitable internal standard.
I am thankful for hints and tips- and would be delighted if anyone could name a more suitable compound.

aspirinha

[tom jupille]

Something is wrong with the method. Insufficient buffer, perhaps? A poor columns with lots of active sites? A poor choice of column (all three of those compounds have been run on C8 or C18 columns with good peak shape)?

[mattmullaney]

I concur with Tom...can you provide a bit more information concerning your column (type of silica, endcapped or not), eluent composition (any modifiers, ion-pairing agents, pH, organic/buffer ratio or gradient profile) and preparation of the samples--a bit more detail about how the robot adds the IS to the samples and standards?

Are the IS peaks poor in shape in both standards and samples as well?

My thanks. With more info, it may be easier to try to figure things out.

Otherwise, here is a link to a recent paper that may be worth a look--even if you may not choose the method described therein, there are quite a number of other references to look at.

http://www.sciencedirect.com/science/ar ... 6410000204

Actually, there seem to be quite a number of articles on ScienceDirect for at least two of these analytes...you may find that an IS may not even be required...please feel free to look and let me know what you think.

Matt

[aspirinha]

Hello Tom and Matt,

thank you very much for your replies.
I cannot understand why we are using an internal standard anyway- for historical reasons, I suppose.
Here are the method informations:
The eluent is 500mg Potassiumdihydrogenphosphate 1 L Water and 1875 ml Acetonitrile (adjusted to pH 3,5 using phosphoric acid).
The column is a Waters Spherisorb CN-RP 150x4,6; 5µ; flow is 1,2 ml/min, column temperature 40°C; 10 µl injection volume and detection is done at 215 nm.
The internal standard is prepared in water and phosphoric acid.
The sample (which consists mostly of sugar) is dissolved in 75 ml water and 25 ml internal standard is added to the aqueous sample solution. Peak shape is awful in samples and calibration standards and the later a peak elutes the worse the shape. The Doxs. being the last and ugliest (tailing 1,8). Will upload a chromatogram for you to shake your heads in disbelief tomorrow.
Thank you for helping already,

A

[mattmullaney]

Hi again,

All analytes are bases, not terribly strong ones. Spherisorb is an older, Type A silica that is non-endcapped...if you're stuck with the older-type silica and cannot switch to a newer Type B silica with better coverage, or something like Waters Atlantis T3 or Agilent Bonus-RP (and for these probably C8 would be fine, but you may not be able to switch), why not further acidify the eluent, changing from buffer to acidic conditions, more like pH 2 or so, and add TEA to the eluent as well...for the older Type A kind of silica...that is, if regs permit you to try things like this...you shouldn't need any TEA with the Atlantis or the Bonus-RP, I would guess naively.

Doxylamine is a pretty nice mimic for Chlorpheniramine...interesting that Dox is "Stickier than Chlor, in the separation you describe.

See what you think, and in any case, best luck!

Matt

[aspirinha]

Hello again,

as I promised :

Makes me cringe everytime I see it.
thank you for you input so far, now I have a starting point to work from.

A

[mattmullaney]

My Goodness...agreed, peak shape isn't terribly good at all. Wasn't thinking clearly enough before. If the column choice isn't negotiable, TEA will likely repair the peak shapes...Spherisorb is just an older-type phase with lots of free silanols. With CN, you rightly won't want to make the eluent much more acidic than it already is. As Tom Jupille rightly suggests, you could probably also up the buffer concentration...you're running on the warm side, which will help keep the buffer in solution, and nearly 50:50 water-to-ACN, too.

Best Luck and Wishes!

[tom jupille]

Check the reproducibility of the peak areas. It it's good enough, get rid of the internal standard. I suspect it's adding rather than cancelling error.

You will have to revalidate the method in any case if you change the internal standard.

[mattmullaney]

Tom's correct...if there's not an easy fix for the Dox IS peak shape...you'll save time, remove a source of uncertainty, one less thing to add to the mobile phase...all of these are pluses. There are other possibilities for additives...best avoided if you can do it.

I'm just too cowardly to say it...

What are the area/height precisions on Dextromethorphan HBr and Chlorpheniramine Maleate by themselves...also, if height precision is better, you can go with that instead of area...

Matt