Method for the determination of polysorbate 80

1. A method for the determination of polysorbate in a protein-containing sample, comprising the steps:

(a) subjecting the sample to alkaline hydrolysis;

(b) neutralizing the sample after alkaline hydrolysis;

(c) optionally removing denatured protein precipitate from the neutralized sample by filtration or centrifation to form a filtered sample;

(d) adding an aqueous mixture of a thiocyanatometal complex to the optionally filtered sample to form a sorbitan polyoxyethylenethiocyanatometal complex;

(e) extracting said sorbitan polyoxyethylenethiocyanatometal complex formed in step (d) into a non-water miscible organic solvent;

(f) measuring the absorbance of the extract obtained in step (e) to quantify the amount of said sorbitan polyoxyethylenethiocyanatometal complex formed in stop (d); and

(g) calculating the amount of the polysorbate contained in the sample from the amount of said sorbitan polyoxyethylenethiocyanatometal complex determined in step (f).

2. method according to claim 1 , wherein the polysorbate is polyoxyethylene sorbitan monooleate (polysorbate 80).

3. The method according to claim 2 , wherein the polysorbate 80 originates from animals.

4. The method according to claim 2 , wherein the polysorbate 80 originates from plants.

5. The method according to claim 1 , wherein the thiocyanatometal complex added in step (d) is an aqueous mixture of Co(NO 3 ) 2 . 6 H 2 O and NH 4 SCN and the sorbitan polyoxyethylenethiocyanatometal complex formed is a sorbitan polyoxyethylenethiocyanatocobaltate (II )complex.

6. The method according to claim 5 , wherein said aqueous mixture contains about 3% (w/v) Co(NO 3 ) 2 . 6 H 2 O and about 20% (w/v)NH 4 SCN.

7. The method according to claim 1 , wherein the sample further contains isooctylnhenol polyoxyethylene ether.

8. The method according to claim 1 , wherein the sample further contains tri-n-butyl phosphate (TNBP).

9. The method according to claim 1 , wherein alkaline hydrolysis in step (a) comprises hydrolysis with an alkaline agent, selected from the group consisting of NaOH, KOH, LiOH, Ba (OH) 2 , Sr(OH) 2 , Ca(OH) 2 , tetrabuthylammonium hydroxide, tetracthylammonimn hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, and homologous or heterologous substituted quaternary alkyl- and cycloalkylammonium hydroxides.

10. The method according to claim 1 , wherein alkaline hydrolysis in step (a) is carried out at a temperature in the range of about 80° C. to about 100° C. for at least 15 minutes.

11. The method according to claim 10 , further comprising, after step (a) and before step (b) the step:

(a2) cooling the sample after alkaline hydrolysis.

12. The method according to claim 11 , further comprising after step (b) and before step (c) the step:

(b2) keeping the sample at room temperature for at least 30 minutes to allow the formation denatured protein precipitate.

13. The method according to claim 1 , wherein alkaline hydrolysis in step (a) comprises hydrolysis with at least 3 N NaOH at a temperature of about 95° C. to about 100° C. for at least 45 minutes.

14. The method according to claim 1 , wherein the filter used in step (c) does not bind polyoxyethylene sorbitan.

15. The method according to claim 1 , wherein the non-water miscible organic solvent used in step (e) is selected from the group consisting of methylene chloride, chloroform, o-dichloroberizene, bromoform, and trichloroethylene.

16. The method according to claim 13 , wherein the non-water miscible organic solvent is methylene chloride.

17. The method according to claim 1 , wherein the absorbance of the extract obtained in step (e) is measured in step (f) at about 324 nm.