interference from placebo

[kavita]

hello
i am facing a problem in method for estimation of benzalkonium chloride by HPLC in one of eye drops contianing polycarbophil and poloxamer , the method is not specific , placebo is showing a interference of approx 60% , i have tried UV , titrimetry methods also but same problem of interference is there , please advise what to do in order to remove this interference from placebo in HPLC method, the eye drop is extremly viscous.

thanks

[Consumer Products Guy]

the method is not specific

Therein lies your problem. You need to provide more details, and I'd be surprised if something isn't already available in literature. Are you using straight RP, or ion-pairing? I'm assuming that you're using UV detector for this. If your sample is viscous try diluting with water or methanol.

[mohan_2008]

Hi Kavitha,

I work with a suspensions on a daily basis - although not eye formulations.

The formulation excipients sometimes show up as valid peaks at the peak of interest.

Give me info regarding:

1. HPLC conditions.
2. Diluent used & Mobile phase used.
3. Working concentration levels.
4. Sample prep & Standard prep
5. Solubility of formulation excipients in water (or vehicle) and the compound in the formulation.

I need as much info as possible. Then I can give you a good method that can solve the problem.

[kavita]

thanks sir ,
here are the chromatographic conditions :
mp: buffer pH5.0 (OPA+ TEA):ACN (40:60)
column: C18(250*4.6)5µ
diluent : ACN: water (2:1)
conc. std & sample : 30ppm (claim is 0.003%)
sample preparation:sample heated on heating mantle till residue, cooled then added diluent , mixed , centrifuged , injected without filteration.
sample is not soluble in water , preservative ,poloxamer and polycarbophil are having similar solublilty ( i think here is the problem) profile in all solvents.

i have also checked the sobility of sample as well as individual excipients also its same ( all are showing similar profile) , sample is very viscous (approx. 2000 cps)

hope you can guide me

regards

[praveenpaliwal]

Can u tell me what is placabo?

regards

praveen

[mohan_2008]

Hi Kavitha,

You have an extensive preparation for your sample. Doesn't it dissolve in the Diluent.

Can you tell me why you adapt this cumbersome procedure instead of just dissolving the sample in the straight diluent.

If your placebo is made in the same way - Maybe it was contaminated with your sample during this prep cycle.

Can you please give me a clear picture of the sample solubility and that of the individual excipients. For eg: if they are not soluble in water then whatelse did you try.

And what exactly is the problem. Is it a recovery issue or you are seeing a significant interference at the peak of interest which might be due to an excipient.

[mohan_2008]

Also,

Is your sample a complex matrix that you need to concentrate by ashing it. I will be concerned if the sample is thermally labile.

What is the whole point of this complex sample extraction. Can you tell me the reason.

[kavita]

hello sir ,
the basic reason behind that tedious method of sample preparation is that the sample is very viscous , white , translucent and the claim of the component of intrest is very low so the solubility of sample is a big concern, so i tried this method after trying to filter it from many filters (glass filter chocked after 1 sample and rest of the filters didn't work as bkc gets absorbed and at this dilution it is impossible to filter) the sample is also not clear after dilution, so i tried to concentrate the sample because after heating the sample becomes a bit clear on dilution, regarding the thermal stability of sample it is thermally stable at 80-100°c ( i have checked it ), one thing more exactly similar peaks due to excipients are coming at the retention time of the component ( in every method) both the poloxamer and polycarbophill are showing same pattern as that of bkc in every method . thats why i am concerned as these are having same solubility also so i am not able to separate bkc from these two excipients ( as recently i have injected individual excipients in the same chromatography ) so please suggest me any method to separate out bkc from these two.
regards

[mohan_2008]

Thanks for the info, Kavitha.

Basically, you are concentrating the sample out of the sample matrix so that you can get most of the sample into the diluent and minimize volatile excipients.

However, the problem is: Both Polycarbophil & Polaxamer are still being extracted into the diluent because they are equally soluble in all diluents tested for the sample.

And these excipients are showing up at the peak of interest in the placebo.

Let us try this instead: (we will avoid the heating procedure and adapt a direct method)

1. Assuming that your workable or hplc injectable concentration is 10 ug/ml or 10 ppm.

2. Use a "To contain pipet" (TC pipet is different from TD) aliquot 1 ml of the sample (whole sample solution containing the excipients) into a 100 ml class A volumetric flask. I assume you know how to use the "TC" pipet. Add the diluent, make up to volume and mix well. Now, you essentially have a 10 ug/ml nominal concentration sample solution.
If aliquoting using a "1mL TC pipet" is difficult since your sample is highly viscous, use a larger 4mL or 5 mL TC pipet and prepare accordingly (say 5 mL in 500 mL).

3. Now you a 10 ppm solution of the sample (including all potential excipients) in the diluent and all being solubilized (Make sure they are all soluble or adjust dilution scheme accordingly).

4. Using a 0.45 uM filter, filter the diluent into the HPLC injection vial and shoot at your HPLC conditions.

5. This might or might not help, and still you see the excipients at the peak of interest.

Now let us modify the HPLC conditions (hopefully you are not following a validated method, if you are - please let me know):

1. Increase %B or ACN concentration plus or minus 5% in your mobile phase and see if this moves the excipient peaks away from the BKC.
If not try, plus or minus 10%.

2. If step 1. doesn't work - use either a Cyano CN column or Phenyl column and perform the separation.

Let me know the results.

[HW Mueller]

I wondered about the water "insolubility" of the chloride, so I checked google, here is the first entree (I didn´t look any further):

http://en.wikipedia.org/wiki/Benzalkonium_chloride

my advice to kavita: Inform yourself of the real properties of the involved substances, check whether someone has handled them in a chromatography context and then start again. (I´ll be darned if the polymers are not easily separable from the benzalkonium chloride via ultrafiltration, SPE, or even SEC).

[mohan_2008]

I agree with Mueller.

The sample seems to be soluble in water, although the dissolution process might be slower.

Maybe a little bit of sonication might help.

[Uwe Neue]

My suggestion is to run a gradient from about 40% MeCN to about 75% MeCN. Often polymers (or oligomers) behave differently than a standard molecule and you could get reasonably sharp peak for your polymers that show up in a different part of your chromatogram than the benzalkonium stuff.