Chromatography Forum

Hydrolysis of polysorbate gives oleic acid, witch can be determined by HPLC. What hydrolysis should be performed and what HPLC conditions required? Thank you!

we are using this for polysorbate 20:


Quantitation of polysorbate 20 in protein solutions using mixed-mode chromatography and evaporative light scattering detection
Journal of Chromatography A
Volume 1215, Issues 1-2, 26 December 2008, Pages 156-160

You can test Polysorbates with ELSD. However, for degradation products, perhaps MS or NMR will serve you better than UV/PDA/ELSD.
For non-derivatived sample, what you see in UV/PDA may be other species trapped within the micelles of Polysorbates. UV cannot 'see' the Peroxides.

Please check out these papers:

1/ Structure Elucidation, Synthesis, and Contact Allergenic Activity of a Major Hydroperoxide Formed at Autoxidation of the Ethoxylated Surfactant C12E5
Anna Bodin et al. Chem. Res. Toxicol., 2003, 16 (5), pp 575–582

Short extract: Ethoxylated alcohols, widely used as surfactants, are known to be susceptible to oxidation when exposed to air. At autoxidation, a complex mixture is formed, in which alkyl poly(ethylene glycol) aldehydes, alkyl poly(ethylene glycol) formates, hydroxyaldehydes, and formaldehyde have previously been identified. These compounds are all secondary oxidation products, some of which have been shown to be skin sensitizers and irritants. The primary oxidation products from ethoxylated alcohols are described in the literature as peroxides and hydroperoxides, but their structures have not been elucidated more closel
(Source: http://pubs.acs.org/doi/abs/10.1021/tx025609n)

2/ Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: Structure and degradation pathways.
Bruce A. Kerwin. Journal of Pharmaceutical Sciences. Volume 97 Issue 8, Pages 2924 - 2935. Published Online: 31 Oct 2007

Short Extract: The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate.
(Source: http://www3.interscience.wiley.com/jour ... 1&SRETRY=0)

What industry you are in?
Alfred

Try this:
900 uL sample+100 uL 1M KOH
incubate 70C 6 hrs then dilute w/ an equal amount of methanol (needed for OA solubility).
C18 is too retentive for OA, try C4 w/ water/acetonitrile/formic acid
we use MS for detection

Good luck

Hi vicking, to quantify polysorbates in my protein formulations I used the method of KOH hydrolyisis at 70 C for 6 hrs, but my major concern here is interference of protein. The protein precipitation step was followed with hydrolysis, but I can not make 100% precipitaion or I assumen that the mechanism of precipitation is holding some amount tween into it and my results are underestimating the amounts of tween.

Can you suggest any other alternative for separating out the protein from tween molecule in biotherapeutics

Sunil,

You won't have any protein left after KOH hydrolysis. I recommend you skip the precipitation step. Also, use glass. We saw significant interference when the hydrolysis was performed in polypropylene.

I suggest taking a look at the CAD detector.

http://www.corona-ultra.com/Application ... Levels.pdf

Thanks Vicking, I follow your suggestion of skipping precipitation step and try the hydrolysis for protein samples.

Are you sure that the detection by PAD will not show any protein interference

Vicking one Quick question to you, do you have any idea on hydrolysis of polysorbate 80 at different temperatures, I have data at 40 C and at 70 C both are giving different profiles.

Do I need to optimize the temperature for hydrolysis conditions, I have one paper suggesting 40 C and you suggested at 70 C, after seeing these two results, now I feel we should go for some optimization in regard to this, what is your opinion?

Hi,
I would like to analyze polysorbate 20 at a concentration of 0.05 mg/ml, or slightly lower, in a solution that contains protein, 10 mg/ml. If possible, I would like to use a simple method, including hydrolysis of PS20 by KOH followed by RPC analysis of the released lauric acid and quantification by using UV absorbance at 195-200 nm.
Is this possible?
Have you seen published methods that describes this? References?

I suppose that alternatives are including fluorescence detection after labelling of the released fatty acid? Do you have references to useful methods?

I am grateful for your input.

Best regards,

Goran Karlsson, PhD
Xbrane Biopharma
Sweden