Development and verification of RP-HPLC for determination of preservative content in multi-dose bivalent HPV vaccine
Objective To develop a reverse phase-high-performance liquid chromatography(RP-HPLC)method for the determination of three candidate preservatives(methyl paraben,benzyl alcohol and m-cresol)in bivalent human papilloma-virus(HPV)vaccine,and to verify and preliminarily apply the method.Methods Waters Acquity Peptide BEH C18 300 Å(150 mm × 2.1 mm,1.7 μm)chromatographic column was used;mobile phase A(0.1％trifluoroacetic acid aqueous solution)and mobile phase B(0.1％trifluoroacetic acid acetonitrile solution)were used for gradient elution;the flow rate was 0.2 mL/min,column temperature was 40 ℃,and the detection wavelength was 254 nm.The specificity,linearity,accuracy,precision and stability of the developed method were verified,and the concentration of each preservative in bivalent HPV vaccine containing three preservatives was detected using this method.Results Under the selected chroma-tographic conditions,the peak times of methyl paraben,benzyl alcohol and m-cresol were different(14.6 min,7.2 min and 15.3 min respectively),which was not affected by other components in the sample and detection reagents.The linearity of methyl paraben was good in the range of 0.005％-0.020％(R2=0.997 3),the average recovery was 99.60％,and the RSD was 2.31％(n=5);the linear relationship of benzyl alcohol was good in the range of 0.1％-0.5％(R2=0.999 9),the average recovery was 100.46％,and the RSD was 2.70％(n=5);the linear relationship of m-cresol was good in the range of 0.4％-2.0％(R2=0.999 4),the average recovery was 100.12％,and the RSD was 0.74％(n=5).When methyl paraben and m-cresol were placed at 2-8 ℃ for 3 d and benzyl alcohol for 2 d,the RSDs of the detection results were all less than 5％,and the stability was good.The developed method was used to detect the concentration of three preservatives in bivalent HPV vaccine samples,all the results of which met the error range of±20％.Conclusion The developed RP-HPLC method is simple for operation with good specificity,accuracy,precision and stability,which can effectively detect and control the quality of three preservatives,methyl paraben,benzyl alcohol and m-cresol,in bivalent HPV vaccines.
Bivalent human papillomavirus(HPV)vaccinePreservativeMethyl parabenBenzyl alcoholM-cresolReverse phase-high-performance liquid chromatography(RP-HPLC)
NETL
NSTL
万方数据
