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Fatty acid composition analysis in polysorbate 80 with high performance liquid chromatography coupled to charged aerosol detection.

European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V (2014-12-03)
David Ilko, Alexandra Braun, Oliver Germershaus, Lorenz Meinel, Ulrike Holzgrabe
ABSTRACT

The fatty acid (FA) composition of polysorbate 80 (PS80), a sorbitan oleic acid ester copolymerized with about 20mole of ethylene oxide, is typically characterized by gas chromatography. Here, an alternative method was developed. After saponification with potassium hydroxide the FA fraction was collected with liquid-liquid extraction using methyl-tert-butyl ether. HPLC in combination with a Corona® charged aerosol detector (CAD) was applied for the separation and detection. The method was fully validated in terms of specificity, repeatability, limits of quantification, linearity, range, accuracy and robustness. The characterization of 16 different PS80 batches demonstrated variability regarding their FA composition, with e.g. the amount of oleic acid ranging from 67.8±0.7% to 96.6±1.4%. Furthermore, we identified petroselinic acid, a double-bond positional isomer to oleic acid in all batches, an FA not known to pharmacopoeias at present. In addition, 11-hydroxy-9-octadecenoic acid, an oxidation product of oleic acid was identified. Structure elucidation was performed by means of HPLC-MS/MS. In addition, the method was expanded to the evaluation of the free FAs. Having determined the entire FA composition, the acid value according to EP and USP can be calculated.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methanol, anhydrous, 99.8%
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Acetonitrile, anhydrous, 99.8%
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Oleic acid, ≥99% (GC)
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tert-Butyl methyl ether, anhydrous, 99.8%
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Palmitic acid, ≥99%
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Oleic acid, technical grade, 90%
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Linoleic acid, ≥99%
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Stearic acid, Grade I, ≥98.5% (capillary GC)
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Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
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Stearic acid, reagent grade, 95%
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Oleic acid, suitable for cell culture, BioReagent
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Octanoic acid, ≥99%
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Myristic acid, Sigma Grade, ≥99%
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Decanoic acid, ≥98.0%
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Lauric acid, ≥98%, FCC, FG
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Stearic acid, ≥95%, FCC, FG
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Palmitoleic acid, ≥98.5% (GC), liquid
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Palmitic acid, BioXtra, ≥99%
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Oleic acid, natural, FCC
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Lauric acid, natural, ≥98%, FCC, FG
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Heptadecanoic acid, ≥98%
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Dodecanoic acid, 98%
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Octanoic acid, ≥98%
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Potassium hydroxide, anhydrous, ≥99.95% trace metals basis
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Decanoic acid, ≥99.5%, FCC, FG
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Palmitic acid, ≥95%, FCC, FG
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Formic acid, ≥95%, FCC, FG