Chromatography Forum

Hi

I trying to develop a HPLC method to quantify Benzalkonium Chloride in an opthalmic solution that also contains Polysorbate 80 at 0.2% (w/v). (Benzalkonium chloride concentration is 0.004% (w/v).) I am having trouble with Polysorbate 80 peaks interfering with the Benzalkonium Chloride peaks. I have tried many conditions but there is always with interference with one or two or all of the C12, C14 and C16 homologue peaks. Does anybody have any suggestions?

Thanks in advance.

Maybe an SPE method, I think a size exclusion cardridge can do it, will help. Tween 80 removal is critical. Many many years ago I ended up with a photometric method to do the quantification of Benzalkonium Chloride, and it was also an ophtalmic product. Over the weekend I will check if I can find the old files.

Hi
meanwhile an archive search (top right corner) might be helpful, seems to recall a bunch of Benzalkonium related threads in the past.

Polysorbates are mixtures of polymers and they usually produce very wide peak. We developed method for benzalkonium chloride and Tween 20 for one of our customers. Unfortunately I cannot share details, but it is similar to this approaches:
http://www.sielc.com/Compound-Polysorbate-80.html (second chromatogram)

What you do is you try to group all peaks related to polysorbate in much narrower peak by doing step gradient. You will use buffer concentration to adjust retention time of benzalkonium chloride.

How about getting rid of the polysorbate with ultrafiltration?

While Benzalkonium Chloride, a cationic surfactant, contains C12, C14 and C16 homologues, polysorbate 80 (or Tween 20), a nonionic surfactant, is a complex mixture of many components with various capacity. Thus analysis of one in the presence of the other can be challenging. In addition, obtaining good peak shapes of cationic surfactants on a silica based column can be difficult, especially when a MS-compatible mobile phase is desired.

One strategy is to remove polysorbate first by trapping the cationic surfactant on the cation-exchange SPE material (on-line or off-line). Then elute the cationic surfactant out by pH and/or ionic strength onto an analytical column under suitable conditions.

Another strategy is to use an application-specific column - Dionex Acclaim Surfactant (Thermo Scientific) and surpressed conductivity detection using acetic acid aq. solution and acetonitrile as the mobile phase. There are several benefits. First, conductivity detection selectively "see" charged analytes without any interferences from neutral analytes such as polysorbate 80. Thus, sample preparation requires nothing except simply filter and inject. Second, the Acclaim Surfactant column provides ideal selectivity and good peak shape for cationic surfactants. Third, the mobile phase is MS compatible.

Attached link gives more details of the Acclaim Surfactant column: http://www.dionex.com/en-us/webdocs/258 ... et_V24.pdf. Please pay attention to Figure 9. If you have any questions, feel free to contact me at xiaodong.liu@thermofisher.com.

Good luck.

Hi
The easiest way = > to drive the pH in strong side (<3) in start gradient , the component to benzalkonium will fly a bullet.