Development and application of HPLC-RI and HPLC-MS/MS based methods for quantification of residual deoxycholate levels in pneumococcal polysaccharides
The analysis of residual sodium deoxycholate (DOC); a detergent of biological origin used in manufacturing of polysaccharide vaccines is challenging due to complex sample matrices and the lack of suitable methods. Here we report, rapid and sensitive high-performance liquid chromatography-refractive...
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Published in | Biologicals Vol. 44; no. 6; pp. 517 - 525 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Elsevier Ltd
01.11.2016
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Subjects | |
Online Access | Get full text |
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Summary: | The analysis of residual sodium deoxycholate (DOC); a detergent of biological origin used in manufacturing of polysaccharide vaccines is challenging due to complex sample matrices and the lack of suitable methods. Here we report, rapid and sensitive high-performance liquid chromatography-refractive index (HPLC-RI) and tandem mass spectrometry (HPLC-MS/MS) methods for estimation of residual DOC in pneumococcal polysaccharides. For HPLC-RI method, separation was achieved using Luna C18 column and mobile phase compositions of acetonitrile: methanol: 20 mM sodium acetate (60:05:35% v/v). For HPLC-MS/MS method, separation was achieved using a Hypersil BDS C18 column with gradient elution of methanol and water (0.1% formic acid). MS/MS method showed linearity (r2 = 0.997) over the range of 10–320 ng/mL with limits of detection (LOD) and lower limit of quantitation (LOQ) of 3 and 10 ng/mL respectively. Precision (% RSD) and accuracy (% recovery) for both methods were in the range of 0.74–8.29% and 82.33–117.86% respectively. Sample matrices interferences were addressed following novel sample clean-up method based on liquid–liquid extraction. Both methods enabled traceable quantitation of DOC in intermediate and purified pneumococcal polysaccharides of serotypes: 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1045-1056 1095-8320 |
DOI: | 10.1016/j.biologicals.2016.08.004 |