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 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.
Serum Institute of India Pvt. Ltd, Hadapsar, Pune, Maharashtra 411028, India. Electronic address: sunil.gairola@seruminstitute.com.
Sunil
S
Gairola
Serum Institute of India Pvt. Ltd, Hadapsar, Pune, Maharashtra 411028, India.
Manish
M
Gautam
Serum Institute of India Pvt. Ltd, Hadapsar, Pune, Maharashtra 411028, India.
Dada
D
Patil
Serum Institute of India Pvt. Ltd, Hadapsar, Pune, Maharashtra 411028, India.
Krishna
K
Manoj Kumar
Doctors' Analytical Laboratories Pvt Ltd - TTC Rabale MIDC, Mumbai 400 701, India.
Pravin
P
Shinde
Serum Institute of India Pvt. Ltd, Hadapsar, Pune, Maharashtra 411028, India.
S K
SK
Jana
Serum Institute of India Pvt. Ltd, Hadapsar, Pune, Maharashtra 411028, India.
Rajeev
R
Dhere
Serum Institute of India Pvt. Ltd, Hadapsar, Pune, Maharashtra 411028, India.
Suresh
S
Jadhav
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003840:Deoxycholic Acid / Q000032:analysis*; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D013296:Streptococcus pneumoniae / Q000737:chemistry*
D011135:Polysaccharides, Bacterial; D003840:Deoxycholic Acid
HPLC-MS/MS; HPLC-RI; Pneumococcal polysaccharides; Process-related residuals; Sodium deoxycholate (DOC); Vaccines