Method Validation for Determination of Haemophilus influenzae Type B Vaccine’s Antigen Content by High Performance Anion Exchange Chromatography with Pulse Amperometric Detection

Abstract

The objective of this study was to validate the method for determination of Haemophilus influenzae type B vaccine’s antigen content by HPAEC-PAD. The validation steps were comprised of laboratory preparedness, method optimization and method validation. Parameters of method validation included linearity and range, specificity, precision, accuracy, limit of detection and limit of quantitation. The results showed that the ribitol standard curve had linearity where the ranges of concentration were between 0.15 and 10.50 µg/ml. The linear regression equation was Y=2E+06X+5946 and value of coefficience of correlation was 1. The F-test value was more than F-critical value at alpha 0.05. The method had specificity for ribitol which showed peak eluted at retention time of mean±standard deviation (SD) of 16.45±0.30 min and the percentage of relative standard (%RSD) was 1.83. The precision presented a mean±SD of antigen content determined by six tests within the first day and then six single daily tests thereafter were 16.94±0.40 and 16.95±0.79 µg/ml, respectively, while %RSD were 2.38 and 4.66, respectively. Accuracy showed percent recovery of ribitol content which was added to 3 brands of diphtheria, tetanus, whole cell pertussis, Hepatitis B and Hib combined vaccine, they were between 83.56 and 97.14. The limit of ribitol detection was 0.07 µg/ml and limit of ribitol quantitation was 0.22 µg/ml. The result of all parameters met the pre-defined acceptance limit. In conclusion, HPAEC-PAD method is precise, accurate and reliable for determination of Hib’s antigen when combined in diphtheria, tetanus, whole cell pertussis and Hepatitis B vaccines. It can be used as a standard method for national laboratory quality control of Hib vaccine’s potency. Consumers should only get the qualified vaccines which have met both product and international standards.