Comparative study of pre⁃column derivatization liquid chromatography and post-column derivatization liquid chromatography for the determination of free formaldehyde residues in vaccines

Abstract

Objective: To establish a pre⁃column derivatization liquid chromatography method and a post⁃column derivatization liquid chromatography method for the determination of residual amount of free formaldehyde in vaccines. Consistency of the results of two methods was investigated. Methods: The pre⁃column derivatization liq⁃ uid chromatography was performed on a Shimadzu LC⁃20AT liquid chromatograph (SPD⁃20A UV detector). Sepa⁃ ration was accomplished on a Kromasil 100⁃5⁃C₁₈(250mm×4.6mm) column with a mobile phase of 60% acetoni⁃ trile solution at a flow rate of 0. 8 mL·min^-1 at 40℃ and the detection wavelength was 360 nm. The post⁃column derivatization liquid chromatography was performed on a Shimadzu LC⁃20AT liquid chromatograph (SPD⁃M20A diode array detector and vector derivative device). Separation was accomplished on a Chrom Core AQ⁃C₁₈ (250mm ×4.6mm) column with a mobile phase of 0.2% (V/V) phosphoric acid solution at a flow rate of 1.0 mL·min^-1 at 25℃ and the detection wavelength was 412 nm. The derivatization solution was acetate buffer, the flow rate was 0. 5 mL·min^-1, and the temperature was 100℃. The precision, repeatability and sample recovery of the two methods were investigated, and the experiment results were tested for significance by F⁃test and t⁃test. Results: The precolumn derivatization liquid chromatography had good linearity in the range of 0. 025 -100μg· mL^-1(R =0. 999 9, n =12). RSD values of precision and repeatability were 0. 06 % and 0.3%-1.4%, respectively. The average recoveries were 97. 3%-104. 8 % with RSD of 0. 7%-2. 9 %. The limit of quantitation was 0. 02μg·mL^-1, and the limit of detection was 0. 01μg·mL^-1. The post⁃column derivatization liquid chromatog⁃ raphy had good linearity in the range of 0.025-100μg·mL^-1 (R =0. 9999, n=12). RSD values of precision and repeatability were 0. 02% and 0. 0.7%-3.5 %, respectively. The average recoveries were 105. 6%-114. 6% with RSD of 0.3% - 1.9%. The limit of quantitation was 0. 02μg·mL^-1, and the limit of detection was 0. 006μg·mL^-1. The F⁃test and the t⁃test results showed there was no significant difference between two methods. Conclusion: Two methods are simple and accurate with high sensitivity and good specificity, which can be applicable to the determination of free formaldehyde residues in vaccines.