what is the cause for the diffusing peak

[ym3142]

I mean the peak was going up and going down by diffusing peak, which partially overlap to my methscopolamine peak around 20 min
I have to come to ask for suggestions after I read into many threads about negative peaks but my peak was not just negative.
And this diffusing peak was absent at the C-gram for the blank which was the diluent without API's.
The parameters:
Luna c18, 150X4.6mm, 5u; 30C, 1.5ml/min, isocratic 34% methanol and 66% Buffer(10mM Octanesulfonic acid, 20mM KH2PO4, pH 2.55);
sample 10% to 120% of 4mg Phenylephrine.HCl and 0.25 mg Methscopolamine nitrate in 100 mL 0.2M (yes, 0.2M!) KH2PO4 pH2.8(adjusted by H3PO4);
wavelength 225nm;
injection volume 200ul.
run time 30min.

this diffusing peaks was present at all ten consecutive injections with the concentrations of 10% to 120% but not the blank.

By the way, the purpose for me to inject 200ul was to increase the methscopolamine UV absorption.

Thanks in advance,

[pliva]

it seems that you are not saturating the column well. the case i had with luna for ion paring agent. sometimes one hrs pf saturation is not enough. try to wash the column with methanol and then saturate properly. all the best

[ym3142]

Pliva, '
thank you for your suggestions but I had 13 injection and run time for each was 30min. Should those later injections have been saturated? Or I have to saturate the column without injections? Thanks again,

[tom jupille]

Is the same interfering peak present when you run standards of the APIs only?

[SIELC_Tech]

Excel,

We are doing method development for one of our customers and the mixture (3 compounds) also have methscopolamine (bromide). We are using one of our Primesep columns with UV/ELSD/LC/MS compatible mobile phase(s) without IP reagent. If you would like we can use phenylephrine/methscopolamine mixture...I mean we will look at it anyway. So far the method looks good retention, peak shape, resolution)
I’ll update you next week.

regards,

Vlad

[michaldousa]

Hello,
I would try to change ion-pair reagent (what about pentasulfonic acid) or pH mobile phase. The concentration of ion pair reagent seems to be relatively high... (what about 5 mM ?). I know no more.

[ym3142]

Thanks for everyone for your thinking and advice.

I am sorry I need to clarify one more thing. The diffusing peak was absent with my standard reference and blank.
The difference between the standard reference and the samples was the latter contained some placebo and the former not.

The excipents : methocel, dicalcium phosphate, talc, dye, steric acid, magnisium stearate.

I will inject the plain placebo. Any other suggestions? Thanks again,

[ym3142]

I just tested and the up-and-down peak did show in the suspension of my placebo and diluent.
So why the placebo gave this kind of peak? By the way, my dye is orange.
Thanks

[ym3142]

My story does not end here.
As I said that the diffusing peak was in my placebo when I tested on LC with Double wavelength detector. BUt, after I moved all the sample vials, buffer and column to another LC with PDA the peak dissappeared.

Besides the detector, the other difference was needle wash changed from 20% to 50% methanolic aqueous.

Any suggestions?

Thanks

[tom jupille]

Was the other LC the same model as the first?

[ym3142]

Tom, thank you for asking.
Both are waters 2695.

[ym3142]

I was suspecting maybe the needle wash may be contaminated. I got a fresh bottle of needle wash, injected di-water for ten times and tested my placebo on the third LC(waters 2695 with double wavelength detector). The peak was present.

[tom jupille]

Well, that eliminates detector design differences. When you tried on the third system, did you use the 20% or the 50% MeOH wash solution?

[ym3142]

Tom, thank you. I was using the 20% methonal as the needle wash for the third LC.
But 1) I do not know how the third LC test eliminated the detector design difference since the third had the same detector as the 1st. Both are double wavelength and both gave the diffusing peak while the 2nd did not gave and was PDA.

2) I did try to switch the needle wash from LC 1 to LC 2 after I did not see the diffusing peak for the injections in LC 2. USing the needle wash from LC1 did not give the diffusing peak. Though I just tested for one injection.
I May need to test 10 or 50 injection since the "old" need wash was filling the lines and it took some injection to replace the liquid with the "new" needle wash.
Thanks again,

[ym3142]

so far, I found 1) all my dual wavelength LC gave this peak and none of my PDA(three) did. Maybe coincidently, all my PDA had longer tube from column to the detector than the dual ones;
2) after changed flow or organic: buffer ratio, the peak survived.
I am still seeking suggestions,
Thanks