Can anyone help me with this assay?? I've already tried USP method but with no satisfatory results.
The problem is that I have more than one peak (4) and as I'm trying to assay nasal solutions, the concentration is too small (0,1 mg/mL).

As I don't have the same absorbance for all peaks and the concentration is so small I just detect two of them.

And the worst part... The peaks are horrible with the assymetry of 4.

Well, i tried to join all peaks and have a unique area value. But I made the linearity test and it didn't pass.

My mobile phase is Sodium Acetate pH 5,0 and Acetonitrile (15:85)

Column = Cianno 250 x 4 - Merck

my injection volume is 50 mcL and I think that it's already a high quantity to inject.

And one more problem :o)

I can't achive the proper recovery. What I mean is that when I prepare a sample with NaCl (because I have it in my product) plus a Known concentration of Benzalconeo, when I inject i have 80% of recovery.

What can I do? :o?

Thanks for helping!

Renata Amaral

hello renata
have you ever tried to use a reversed phase system (C8 or C18 column)and an ion pairing reagent (heptane sulfonic acid sodium salt, ...) and a higher injection volume (200 µL) in combination with a preconcentration step (solid phase extraction) ?
Kind regards
Heinz

yes I have..... no success at all.....

the SPE is a great idea but there are some inconvenients:

- i don't have the equipament to do that now, and to order it, it'll demand some months.

- i have never used it before, so maybe i would have some dificulties on dealing with it

- i'm not sure but i think that it's not so cheap and, as we analyse this product all the time, it won't be allowed by the company i work for. At least i would have to create a very good explanation for that....

- so, it would be my last choice...

well, i don't know if i have reached my last choice, but anyway.....


But please, i would like to receive some other help..... and thanks a lot for your attention, Heinz....

Renata

Renata - do you mean "benzalkonium chloride"? My company developed and validated an HPLC procedure for this a few years ago (and this was in a fragranced finished product, a more difficult matrix than yours). However, I'm afraid that my company considers our procedure as our intellectual property, but may be interested in "leasing" that information to you, I'd need to check with management types. I will say that it was one of the most difficult test method developments we have done, but validation was as routine as that gets. Or you may want to look into H. Okumura, “Optimization of Analytical Conditions for Surfactants by High-Performance Liquid Chromatography”, J. SCCJ, Vol. 26, No. 1 (1992), pp. 31-38..

You might want to consider these two methods for quaternary amines with good peak symmetry;
http://allsep.com/makeChr.php?chr=Chr_047
http://allsep.com/makeChr.php?chr=Chr_043

Renata

Please clarify several things!

What is your sample dissolved in? Mobile phase? Water? Something else?

Do I read this correctly: the acetonitrile concentration is 85% and the sodium acetate buffer is 15%?

Are you bound by the USP assay, or are you free to use other methods?

Hi Uwe

i will clarify the things for you :o)

I do not dilute the sample with anything.... as the concentration of the product is small I just filter the product, and inject it....

The product is made of: water, NaCl and Benzalkonium Chloride... only

2) you read it correctly

3) I'm free!!!!


thanks.....


and for the message above, i'll check the links out.... thanks


Renata

Hello Renata,
maybe this article can help you
Title
Analysis of benzalkonium chloride and its homologs: HPLC versus HPCE

Author(s)
Prince, S.J., McLaury, H.-J., Allen, L.V., McLaury, P.,

Journal
Journal of Pharmaceutical and Biomedical Analysis


Vol. 19, No. 6, pages 877-882 (1999)

ISSN
0731-7085

Publisher
Elsevier

Abstract
Benzalkonium chloride (BAK) is a mixture of alkylbenzyldimethylammonium chloride homologs with n-C"1"2H"2"5, n-C"1"4H"2"9, and n-C"1"6H"3"3 comprising a major portion of the alkyl groups present. An analytical method for BAK must differentiate and quantitate the homologs in the BAK mixture. Reversed-phase high performance liquid chromatography (HPLC) separates compounds based on their affinity for a nonpolar column, which is a direct correlation to the compounds* polarity. High performance capillary electrophoresis (HPCE), however, separates compounds in an electric field according to their charge and size. The BAK homologs are suitable for separation by either of these methods because their polarity and sizes differ significantly. The HPLC method employed a mobile phase of 60% acetonitrile and 40% 0.1 M sodium acetate buffer pH 5 pumped at 1.0 ml min^-^1, a 4.6x250 mm cyano column with 5 @mm packing, and UV detection at 254 nm. The HPCE method utilized a run buffer of 30% acetonitrile and 70% 0.05 M sodium phosphate pH 3.06, a 50 @mmx20 cm open silica capillary, 7.5 kV electric field and UV detection at 214 nm. Both HPLC and HPCE demonstrated good linearity in the range of 0.025 to 0.8 mg ml^-^1 with r^2 values of approximately 0.99. The HPLC method produced good separation of the homolog peaks with a total analysis time of 25 min. HPCE run time was less than 5 min and demonstrated good separation of the three homologs. The HPLC method, however, was superior to HPCE in the areas of sensitivity and precision. The HPLC has been extensively used in the routine quantitation and qualitation of benzalkonium chloride concentrations in various products; however, long analysis times make this method inefficient. The HPCE method produced comparable results to the HPLC method but with much shorter analysis times. An HPCE analysis method, as presented here, may prove to be a much more useful and efficient method for the analysis of benzalkonium chloride and its homologs.

Heinz

I would use a silica column with the exact same mobile phase. I don't see a reason why the cyano column is supposed to do something wonderful. I do not expect any hydrophobic interaction in this mobile phase, even with the long chain, but rahter hydrophilic interaction. If this is the case, the silica column will do just fine or even better than the CN column.
If you try it, let me know how it worked.

Hi Uwe,

Are you talking about that HILIC column?? Because my mobile phase is composed of water, and also my product.

thanks for answering back

Renata

Yes Renata, this is how HILIC works: a high concentration of organic solvent, and a low concentration of water. Your HPLC method contains 85% organic and 15% aqueous, just as in HILIC.

Can you give an example of a HILIC column for me to buy?

I just work with Waters and Merck columns but if you know any other one, and the name of the column, i thank you.

Since BZK binds to non-woven material like antiseptic towelettes, I am wondering if the BZK would bind to the filtering material (e.g. cellulose) resulting in lower recovery. This may be worth looking into.