, Volume 81, Issue 4, pp 669–675 | Cite as

Rapid Analysis of Fatty Acid Composition in Polysorbate 80 by Gas Chromatography with On-line Pyrolytic Methylation Technique

  • Zhongping Huang
  • Ruofeng Qiu
  • Yilei Huang
  • Huijun Liu
  • Zaifa Pan
  • Lili Wang


A novel method of on-line pyrolytic methylation–gas chromatography was developed for the rapid analysis of fatty acid composition in Polysorbate 80 without any tedious pre-treatment steps. Fatty acids in Polysorbate 80 were converted into their corresponding fatty acid methyl esters in the presence of trimethylsulfonium hydroxide with a vertical microfurnace pyrolyzer at 300 °C. The premixing procedure of sample and organic alkali reagent was necessary before the on-line pyrolytic methylation to improve the repeatability. The relative standard deviations for peak areas of fatty acids in Polysorbate 80 were over the range of 0.3–9.1% (n = 5). Six Polysorbate 80 samples, consisting of three samples of pharmaceutical grade and three samples of non-pharmaceutical grade, were analyzed to evaluate the feasibility of the proposed method. The relative percentages (%) of fatty acids for samples of pharmaceutical grade meet the Chinese Pharmacopoeia requirements with the amount of oleic acid varying from 78.4 to 89.3%. On the other hand, the relative percentages (%) for palmitic acid and stearic acid in samples of non-pharmaceutical grade were out of the specification limits, with the amount of oleic acid varying from 62.0 to 63.5%. The quantitative results determined by on-line pyrolytic methylation were in agreement with those obtained by off-line methylation. The result proved that gas chromatography with on-line pyrolytic methylation technique is of great value for rapid screening analysis of Polysorbate 80 samples in bulks.


Fatty acids Polysorbate 80 Pyrolytic methylation Gas chromatography 



This research was financially supported by National Natural Science Foundation of China (No. 51,503,182), Analysis and Measurement Foundation of Zhejiang Province (No. 2017C37064), Key Laboratory Item of Furniture Inspection Technology of Zhejiang Province (No. 2016J01) and Zhejiang University of Technology Natural Science Foundation (No. 2014XY002).

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflicts of interest in relation to this work.


  1. 1.
    Thakur RK, Villette C, Aubry JM, Delaplace G (2007) Spectrophotometric method associated with formulation scans for application of hydrophilic–lipophilic deviation concept in food emulsions. Coll Surf A 301:469–474CrossRefGoogle Scholar
  2. 2.
    Dimitrijevic D, Shaw AJ, Florence AT (2000) Effects of some non-ionic surfactants on transepithelial permeability in caco-2 cells. J Pharm Pharmacol 52:157–162CrossRefGoogle Scholar
  3. 3.
    Nair LM, Stephens NV, Vincent S, Raghavan N, Sand PJ (2003) Determination of Polysorbate 80 in parenteral formulations by high-performance liquid chromatography and evaporative light scattering detection. J Chromatogr A 1012:81–86CrossRefGoogle Scholar
  4. 4.
    Pan JH, Ji Y, Du ZX, Zhang JW (2016) Rapid characterization of commercial Polysorbate 80 by ultra-high performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry. J Chromatogr A 1465:190–196CrossRefGoogle Scholar
  5. 5.
    Coors EA, Seybold H, Merk HF, Mahler V (2005) Polysorbate 80 in medical products and nonimmunologic anaphylactoid reactions. Ann Allergy Asthma Immunol 95:593–599CrossRefGoogle Scholar
  6. 6.
    Oszi Z, Petho G (1998) Quantitative determination of Polysorbate 20 in nasal pharmaceutical preparations by high-performance liquid chromatography. J Pharm Biomed 18:715–720CrossRefGoogle Scholar
  7. 7.
    Hu M, Niculescu M, Zhang XM, Hui A (2003) High-performance liquid chromatographic determination of Polysorbate 80 in pharmaceutical suspensions. J Chromatogr A 984:233–236CrossRefGoogle Scholar
  8. 8.
    Fekete S, Ganzler K, Fekete J (2010) Fast and sensitive determination of Polysorbate 80 in solutions containing proteins. J Pharm Biomed 52:672–679CrossRefGoogle Scholar
  9. 9.
    Nayak VS, Tan Z, Ihnat PM, Russell RJ, Grace MJ (2012) Evaporative light scattering detection based HPLC method for the determination of Polysorbate 80 in therapeutic protein formulations. J Chromatogr Sci 50:21–25CrossRefGoogle Scholar
  10. 10.
    Ayorinde FO, Gelain SV, Johnson JH, Wan LW (2000) Analysis of some commercial Polysorbate formulations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 14:2116–2124CrossRefGoogle Scholar
  11. 11.
    Abrar S, Trathnigg B (2011) Separation of Polysorbates by liquid chromatography on a HILIC column and identification of peaks by MALDI-TOF MS. Anal Bioanal Chem 400:2119–2130CrossRefGoogle Scholar
  12. 12.
    Hewitt D, Alvarez M, Robinson K, Ji J, Wang Y, Kao YH, Zhang T (2011) Mixed-mode and reversed-phase liquid chromatography–tandem mass spectrometry methodologies to study composition and base hydrolysis of Polysorbate 20 and 80. J Chromatogr A 1218:2138–2145CrossRefGoogle Scholar
  13. 13.
    Zhang R, Wang Y, Tan L, Zhang HY, Yang M (2012) Analysis of Polysorbate 80 and its related compounds by RP-HPLC with ELSD and MS detection. J Chromatogr Sci 50:598–607CrossRefGoogle Scholar
  14. 14.
    Zhang R, Wang Y, Ji Y, Shi B, Zhang Z, Zhang H, Yang M, Wang Y (2013) Quantitative analysis of oleic acid and three types of polyethers according to the number of hydroxy end groups in Polysorbate 80 by hydrophilic interaction chromatography at critical conditions. J Chromatogr A 1272:73–80CrossRefGoogle Scholar
  15. 15.
    Christiansen A, Backensfeld T, Kühn S, Weitschies W (2011) Stability of the non-ionic surfactant Polysorbate 80 investigated by HPLC-MS and charged aerosol detector. Pharmazie 66:666–671Google Scholar
  16. 16.
    Ilko D, Braun A, Germershaus O, Meinel L, Holzgrabe U (2015) Fatty acid composition analysis in Polysorbate 80 with high performance liquid chromatography coupled to charged aerosol detection. Eur J Pharm Biopharm 94:569–574CrossRefGoogle Scholar
  17. 17.
    Hvattum E, Yip WL, Grace D, Dyrstad K (2012) Characterization of Polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: specific determination of oxidation products of thermally oxidized Polysorbate 80. J Pharm Biomed 62:7–16CrossRefGoogle Scholar
  18. 18.
    Zhang Q, Wang A, Meng Y, Ning T, Yang H, Ding L, Xiao X, Li X (2015) NMR method for accurate quantification of Polysorbate 80 copolymer composition. Anal Chem 87:9810–9816CrossRefGoogle Scholar
  19. 19.
    European Pharmacopoeia (2014) European directorate for the quality of medicines and healthcare (EDQM), eighth edn. Strasbourg, FranceGoogle Scholar
  20. 20.
    Chinese Pharmacopoeia (2015) China pharmacopoeia committee, tenth edn. Beijing, ChinaGoogle Scholar
  21. 21.
    Robb EW, Westbrook JJ (1963) Preparation of methyl esters for gas liquid chromatography of acids by pyrolysis of tetramethylammonium salts. Anal Chem 35:1644–1647CrossRefGoogle Scholar
  22. 22.
    Kossa WC, MacGee J, Ramachandran JS, Webber AJ (1979) Pyrolytic methylation/gas chromatography: a short review. J Chromatogr Sci 17:177–187CrossRefGoogle Scholar
  23. 23.
    Huang Z, Zhang P, Sun Y, Huang Y, Pan Z, Wang L (2015) Determination of glyceride and free fatty acid residuals in biodiesel by thin layer chromatography combined with on-line pyrolytic methylation gas chromatography. J Anal Appl Pyrolysis 113:288–295CrossRefGoogle Scholar
  24. 24.
    Matter L, Schenker D, Husmann H, Schomburg G (1989) Characterization of animal fats via the GC pattern of fame mixtures obtained by transesterification of the triglycerides. Chromatographia 27:31–36CrossRefGoogle Scholar
  25. 25.
    Dron J, Linke R, Rosenberg E, Schreiner M (2004) Trimethylsulfonium hydroxide as derivatization reagent for the chemical investigation of drying oils in works of art by gas chromatography. J Chromatogr A 1047:111–116CrossRefGoogle Scholar
  26. 26.
    Firi N, Kienberger H, Hauser T, Rychlik M (2013) Determination of the fatty acid profile of neutral lipids, free fatty acids and phospholipids in human plasma. Clin Chem Lab Med 51:799–810Google Scholar
  27. 27.
    Shadkamia F, Helleur R (2010) Recent applications in analytical thermochemolysis. J Anal Appl Pyrolysis 89:2–16CrossRefGoogle Scholar
  28. 28.
    Ishida Y, Wakamatsu S, Yokoi H, Ohtani H, Tsuge S (1999) Compositional analysis of polyunsaturated fatty acid oil by one-step thermally assisted hydrolysis and methylation in the presence of trimethylsulfonium hydroxide. J Anal Appl Pyrolysis 49:267–276CrossRefGoogle Scholar
  29. 29.
    Ishida Y, Katagiri M, Ohtani H (2009) Reaction efficiency of organic alkalis with various classes of lipids during thermally assisted hydrolysis and methylation. J Chromatogr A 1216:3296–3299CrossRefGoogle Scholar

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© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Chemical EngineeringZhejiang University of TechnologyHangzhouChina

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