Chromatography Forum

I’m having a problem of separating one impurity and an excipient peak. I would appreciate any comments or suggestions

HPLC conditions:
Mobile Phase A: For every 990 mL of USP water, add 9.35g of octanesulfonic acid, sodium salt, monohydrate, 10 mL of perchloric acid 69-72%, and 1 mL of phosphoric acid, 85%.

Mobile Phase B: Methanol.
Column Temperature: 30oC
Injection Volume: 10 μL
Detector: 217 nm
Gradient Conditions:
Time (min) %A %B Flow Rate (mL/min)
0.0 98 2 0.75
4.0 98 2 1.00
10.0 40 60 1.00
12.0 40 60 1.00
13.0 98 2 1.00
20.00 98 2 1.00
Column: Ace 5 C18, 4.6 X 150 mm

Retention time of Zoledronic acid = 2.2 min
Retention time of excipient (sodium citrate) = 2.8min
Retention time of impurity = 2.7 min

It looks like you aren't getting enough retention. Perhaps an "AQ" type column used with 100% aqueous mobile phase would improve the retention. Or you could try a HILIC method.

I'm curious about your flow rate being reduced there at the beginning. I've not seen this done before.

I would suggest trying a ROA organic acid column. Although they are a polymer based column, with a little tuning you can often get great separations of organic acids.

These are very very polar compounds. I don't think you have a chance with straight RP retention. I am also not sure if ion-paring will work, but you definitely have to do it without the gradient.

So the simplest thing to do at this time is to get rid of the gradient, do a lengthy equilibration with the ion-pair reagent while injecting the analyte to see if retention increases. If it does, then you can work out an isocratic procedure. If it doesn't I would go to HILIC.

LTNguy3,

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Regards,

Vlad

Thank you for all your advices.
The purpose of reducing the flow rate at the beginning is to get better resolution between the zoledronic acid peak and the sodium citrate peak. I did try to cut octanesulfonic salt in half with this gradient run. The resolution between those two peaks was a lot better but the sodium citrate peak always co-eluted with an impurity. I think the gradient run with ion-pair reagent is a bad choice. Please let me know if you have any more suggestions.
SIELC_Tech, I want to try the other guys’ suggestions first. If they do not work, I will give you a call.
Thanks again for your helps.

Hmmm, all commercial talks,

The method u r using should be with Luna Phenyl-hexyl column and not the C18 . try using that and u can get RT of zolendronic acid at 5 min.

or
Try Pamidronic Disodium method official in BP but it uses RI detector with anion-exchange column.

Good luck!!

My reason for suggesting to delete the gradient is that it disturbs the equilibrium with the ion-pair reagent. Equilibration of a column with an ion-pair reagent also takes a long time, often several hundred milliliters of mobile phase. This is why I suggested to continue the injection of your analyte during isocratic column equilibration. I also would do all this in the presence of 2% methanol to prevent a dewetting of the stationary phase. If indeed you still do not yet have retention after this, then switch to 100% aqueous, all other concentrations remaining the same. If it still does not work after this, HILIC on a silica column would be my next choice.

PS: I disagree that at this moment you need to go to another column. I do not see why a Phenylhexyl would work better for this application than a C18.

The mobile phase used for zolendronic acid is from a published application which is isocratic method with out any gradient.

Uwe, you are right that using gradient is not useful for this application. whether you agree or not but fact remains that this molecule( more of a inorganic part) does not retain on C18 whatever we do. The only column, one can get retention is Phenyl-hexyl or enion-exchange column.

JM

I’ve just analyzed the zoledronic acid and placebo samples again with isocratic elution method.
MP: 0.025M KH2PO4 + 0.02M 1-Octanesufonic cid sodium salt monohydrate. Adjusted pH = 2 with perchloric acid.
Flow rate: 0.75ml/min

The zoledronic acid, impurities and sodium citrate peaks separate from one another . The problem is the mannitol peak co-elutes with the zoledronic acid peak . I also tried 95% buffer and 5% Methanol but it did not work.
Retention time of zoledronic acid peak is 2.2 min

ltnguy3,

At 0.75 ml/min 4.6x150 mm column has a void of approx. 2 min so you are barely off the void of the column. That is why mannitol co-elutes with zoledronic acid - they both are not retatined. I found in the lab ethidronic acid and we will try to see if we can retain it. From my experience with such compounds you can retain it on Primesep columns with no problem, sometimes strongly acidic compounds (phosphoric diacids) is hard to elute.
I will update ou on ethidronic acid in a few days.

Thanks SIELC_Tech,

hi ltnguy3,

Any improvement in the hplc analisys? I'm gonna start to analize zoledronic acid in a while and I'd like to know if you managed to develope the method.

thanx.

Zompa,
I’m still working on it. The problem is excipients (sodium citrate, mannitol) and zoledronic acid in dosage formulations elute right next to each other.

hi ltnguy3,

Which method are you working on at the moment? cuz I've just started to try to develope the method..
My method will be used to analize the api. I won't have excipients to resolve from the main peak.
Any suggestion would be appreciated.
Thanx a lot.