Chromatography Forum

I’m involved since ten years in analysis of eye drops formulations. Benzalkonium cloride is used as preservative in almost any formulation and I acquired experience in its identification and quantitation in the finished product by HPLC and phase transfer titration. Among other minor analytical problems related to this component, that is a variable mixture of homologues with poor response to UV detection and a low level of concentration in the formulation, I noticed a strange behaviour in HPLC that wasn’t described in literature. Mixing BAC solution with that of many common pharmaceuticals used in eye drops that didn’t show any chromatographic interference with the peak of BAC, causes a strong percentual loss of peak area of BAC.
I collected many data using standard solutions of BAC and pilocarpine hydrocloride at different levels of concentration. The results clearly show a kind of interaction that seems indipendent from pilocarpine concentraton and results in a loss of about 17 – 18 % of the area of BAC at the conc level of eye drops. In the industrial preparations this effect is normally compensated by overdosage. I suppose, but I’m not sure, that there is a partial addition between the two molecules that cannot be eluted. Someone can give me some explanation, thank you.

Hi

You might get better help from an organic chemist but as your question triggered my curiousity I will give it a go

1. BAC strucurallt seems open for reactions in fact you perform a base extraction of the quartinary salt at room temperature when you do the assay of BAC (at least for the API).

2. Is there for instance a pH difference in the pure BAC std solution compared to BAC/pilocarpine hydrocloride mixture and/or other components in the mixture?

3. As you seem to get a quite stable repeateble loss of 17-18% of the BAC in the mixture independant of the concentration, it would appear that an equlibrum between BAC and "degraded" BAC is quite quickly established. Had it been an irreverisble "degradation/side reaction" it should have been concentration depended and the loss should have varied more.

Cheers

If the loss is independent of the pilocarpine concentration, then I would conclude that it has nothing to do with pilocarpine. How about adsorption of the surfactant to the wall of the vial, or the septa?

I agree with Uwe, I don't think that a reaction is happening. For one thing, the loss is independent of pilocarpine conc. Also, I don't think that quaternary amines undergo addition reactions the way that other amines do. They do however sorb very strongly to negatively charged site on soil, glassware, etc. Try using silanized glassware or plastic and see if it makes a difference. It would also be interesting to see if acid washing your glassware has any affect.

If you're using Nylon filters for BAK or BZK assay: don't.

Benzalkonium quats can be lost by absorption during sample preparation (e.g. filter materials) and autosampler vials by hydrophobic interaction (plastic vial) and ion-exchange (glass vial). I prefer to use glass vials and pre-treat the sample vials with a more hydrophobic quat that is not the analyte of interest to mask the silanol groups. Also inject such masking solution to the LC system to mask adsorption sites in the flow path before analysis. As a result, the recovery can be improved.

For analysis of benzalkonium salts, UV detection should provide quite good detection limit (low ppms) although sometimes ELSD is sufficient for higher concentration samples (>10 ppm). However, very often the analysis can be interfered by other components present in the sample. Therefore, to achieve better sensitive (ppb) and selective detection, suppressed conductivity detection is the solution in which case all neutrals and anionics are transparent.

Dionex is offering a specialty column, Acclaim Surfactant, designed for analysis of cationic, anionic, amphoteric and nonionic surfactant analysis. The following link contains some information on cationic surfactants analysis including benzalkonium salts. Please pay attention to Figures 1, 2, and 6-9.
http://www.dionex.com/en-us/webdocs/685 ... 01-ACS.pdf

Please contact me if you need further details.

Hi, thanks for your replies. I try to explain my considerations with order and more details:
1) I don’t have any specific problem with HPLC method for BAC assay. We use an ICH validated HPLC-RP method with UV detection that is perfectly suitable for most of ours eye drops formulations. We work in very standardized conditions and have collected raw data for many years from standards and samples of different matrix. (I know today are available colums and/or detection systems that can give more resolution, sensitivity and shape peaks but my priority is to have a well known and standardized method).
2) When, many years ago, we noticed a loss of recovery of BAC in a little group of our eye drops formulations, the first idea was about some adsorption on the surface of containers materials (glass vials and plastic bottles) and we realized some tests to quantify it. Really this effects exists but, at least in our case, it’s too low to justify a so great loss in recovery and don’t explain the fact that it’s showed only in presence of few API (most formulations gives recoveries from 98 to 100%). Also, if there was any adsorption on the container surface, it would be about the same in standard and samples.
3) pH of mobile phase is about the same as that of the samples (4,5 – 5,5)
4) To exclude the possibility of adsorption in filters, we don’t use any filtration of the sample (eye drops is a quite clean matrix).
5) There is a great difference between concentration levels of BAC and Pilocarpine HCl in eye drops formulation, BAC = 0,1 mg/ml API = 40 mg/ml, and maybe this can explain the low correlation with recovery of BAC. I tested standard solutions with 10, 20, 40 and 60 mg/ml of API and the mean loss in recovery of BAC was –13.0%, -17.5%, -17.6% and -20.8%.
6) About the same loss is verified also in phase transfer titrations.
5) I don’t suppose a degradation of BAC, only a partial bond with API that subtract it to the analysis. Do you think it’s chemically correct? Thank you.

5) There is a great difference between concentration levels of BAC and Pilocarpine HCl in eye drops formulation, BAC = 0,1 mg/ml API = 40 mg/ml, and maybe this can explain the low correlation with recovery of BAC. I tested standard solutions with 10, 20, 40 and 60 mg/ml of API and the mean loss in recovery of BAC was –13.0%, -17.5%, -17.6% and -20.8%.
6) About the same loss is verified also in phase transfer titrations.
5) I don’t suppose a degradation of BAC, only a partial bond with API that subtract it to the analysis. Do you think it’s chemically correct? Thank you.

Hi

The problem I have with a partial bond between BAC and the API is that you actually confirmed the loss of recovery with the phase transfer titration. If performed like in the European pharmacopiea a partial bond (ie equilibrum between free and bonded BAC) the repeated liquid/liquid extraction procedure would interrupt the equilibrum and manage to extract most of the BAC. Unless of course if the "bonded" material is transfered to the phase that is discarded.

If you like you could rule out any degradation with the releated substance methods in the european pharmacopiea for BAC and BAC solutions. I think the test solution is like 50mg/ml there also version 6.4 of the european pharmacopiea just arrived at our office, think there are a few changes to the BACs monographs.

The problem I have with a partial bond between BAC and the API is that you actually confirmed the loss of recovery with the phase transfer titration. If performed like in the European pharmacopiea a partial bond (ie equilibrum between free and bonded BAC) the repeated liquid/liquid extraction procedure would interrupt the equilibrum and manage to extract most of the BAC. Unless of course if the "bonded" material is transfered to the phase that is discarded.

If you like you could rule out any degradation with the releated substance methods in the european pharmacopiea for BAC and BAC solutions. I think the test solution is like 50mg/ml there also version 6.4 of the european pharmacopiea just arrived at our office, think there are a few changes to the BACs monographs.

The phase transfer method we use isn’t that for the assay of BAC in EP monography, it’s an older internal method that was developed and used in our lab before the introduction of HPLC, only for the assay of BAC in eye drops. It is a more direct and quick one, because it doesn’t plan any extraction step. I never applied the EP method on eye drops and you are right about the possibility that repeated liquid/liquid extractions would interrupt the equilibrum and manage to extract most of the BAC. I’ll try it to confirm the existence of such equilibrium.
I received yesterday the 6.4 EP addendum and I’m evaluating the changes in BAC monography, but I don’t think that the HPLC method for determination of the ratio of alkyl components is suitable to elute a potential addition product between BAC and API.
Thank you for your useful suggestion.

Dear all:
For Benzalkonium ions (BAK, or BAC), UV detector is adequate (low UV region). Treating BAK samples with UV light will degrade the signals (from our experiment). So, BAK is UV-sensitive.

When we test aqueous eye drops by HPLC methods, the recovery is very high (~100%), but when we test a polymer-based matrix (e.g. containing Polyvinyl Alcohol), the recovery is lower (~94%). However, the % error never exceeded 10%.
In our tests, we expect to see 3 peaks of 3 homologs: C-12, C-14, and C-16, and we prepare standards by mixing 3 components (purchased from Sigma).
Also, CPG is correct that Nylon syringe filter will absorb BAK. We wrote into our SOPs to use only PTFE filters for BAK. We have banished the "Phase transfer" or "Chloroform extraction" method, to move toward "Green Chemistry." (I wish that we have a substitute for MeCN!).

Could you share info on how you make the in-house BAK standard(s)?

Alfred.

Dear Alfred88, what you write it’s very interesting, really BAK is UV-sensitive? Can we have more details about your experiments?
I verified a lowering of BAK HPLC recovery in formulations with Polyvinyl Alcohol and with jelling agents in the same percentual degree that you found, but I never deepened in the causes because I thought it was in some way connected to the increment of density; lowering of BAK recovery with pilocarpine and other pharmaceuticals is much greater and cannot be so easily explained.
I’m also moving toward a “green chemistryâ€