(M1230-08-52) Quality Assessment of Polysorbates 80 and 20 Pharmaceutical Raw Materials by Measuring Fatty Acids Composition using HPLC with Mass Detection

Purpose: Polysorbates are non-ionic surfactants widely used as excipients or inactive ingredients in many pharmaceutical products. To assure safety of the finished drug products, the quality and purity of excipients must be assessed using suitable and reliable test methods. A gas chromatography (GC) with flame ionization detector (FID) procedures for the polysorbate 80 and 20 based on the fatty acids composition are recommended^1,2. These procedures require hydrolysis and derivatization of the polysorbates to free fatty acids. In this work, simple and fast HPLC-mass spectrometry (MS) methods were developed for the determination of fatty acids composition in the polysorbates 80 and 20 by direct analysis of the hydrolyzed samples.

Methods: Two separate standard mixtures containing free fatty acids specified by the USP in polysorbates 80 and 80 were prepared in water/ethanol diluent (50:50, v/v). Polysorbates 80 and 20 test samples were hydrolyzed with 1 M potassium hydroxide solution by incubation at 6 hours at 40°C, neutralized with equal volume of 1 M formic acid, and diluted with water/ethanol (50:50, v/v) to 0.1 mg/mL. Chromatographic separation was performed using XBridge™ BEH™ C18 (4.6 x 100 mm, 3.5 µm) column, operated at 60ºC, run on Arc™ HPLC System. The mobile phase consisting of 10 mM ammonium acetate in water and acetonitrile solvent was delivered under gradient elution with a flow rate of 2 mL/min. For MS detection with an ACQUITY™ QDa™ Detector, a single ion recording (SIR) acquisition in negative electrospray ionization (ESI) mode was used for quantitative analysis of free fatty acids.

Results: The developed HPLC-MS method successfully separated all the USP-specified fatty acids in polysorbates 80 and in polysorbate 20 (Figure 1). Different reaction media were investigated during the study to ensure complete extraction of all fatty acids from the test samples. Hydrolysis with base released most free fatty acids and was used to prepare all the test samples. The identity of the unknown peaks found in the polysorbate 80 samples was verified by accurate mass determination using a Xevo™ G2-XS QTof Mass Spectrometer. The composition of the free fatty acids was determined by calculating percent (%) of each fatty and met the USP criteria.

Conclusion: The developed HPLC-MS method for the analysis of free fatty acids enabled fast quality assessment of the polysorbates 80 and 20 pharmaceutical raw materials by direct injection of the hydrolyzed samples.

References: 1. USP Monograph for Polysorbate 80, United States Pharmacopoeia, USP–NF 2021 Issue 1. The United States Pharmacopoeia Convention, Official 01-May-2020.
2. USP Monograph for Polysorbate 20, United States Pharmacopoeia, USP–NF 2021 Issue 1. The United States Pharmacopoeia Convention, Official 01-May-2020

Figure 1.