Comparative study on the determination of free formaldehyde residue in vaccines by pre-column derivative liquid chromatography and post-column derivatization liquid chromatography

Abstract

Objective: To develop and compare methods for quantifying residual free formaldehyde in vaccines using pre-column and post-column derivatization liquid chromatography, and to assess the consistency between these methods. Methods: For pre-column derivatization, we used a Shimadzu LC-20AT liquid chromatograph equipped with an SPD-20A UV detector. Separation was achieved with a mobile phase consisting of 60% acetonitrile at a flow rate of 0. 8 mL·min⁻¹ and a column temperature of 40 ℃. Detection was performed at 360 nm using a Kromasil 100-5-C₁₈ (250 mm × 4.6 mm) column. For post-column derivatization, we employed a Shimadzu LC-20AT liquid chromatograph with an SPD-M20A diode array detector and a vector derivative device. Separation was conducted with a mobile phase of 0.2% (V/V) phosphoric acid at a flow rate of 1.0 mL·min⁻¹ and a column temperature of 25 ℃. Detection was carried out at 412 nm using a Chrom Core AQ-C₁₈ (250 mm × 4.6 mm) column. The derivatization solution was acetate buffer, flowing at 0.5 mL·min−1 and maintained at 100 ℃. Both methods were evaluated for precision, repeatability, and sample recovery, with statistical significance assessed using F-test and t-test. Results: The pre-column derivatization method exhibited linearity over the range of 0.025–100 μg·mL⁻¹ (R = 0.9999, n = 12). Precision showed an RSD value of 0.06%, and repeatability ranged from 0.3% to 1.4%. Average recoveries were between 97.3% and 104.8%, with RSD values from 0.7% to 2.9%. The limit of quantitation (LOQ) was 0.02 μg·mL−1, and the limit of detection (LOD) was 0.01 μg·mL⁻¹. The post-column derivatization method demonstrated linearity in the same range (0.025–100 μg·mL⁻¹, R = 0.9999, n = 12). Precision showed an RSD value of 0.02%, and repeatability ranged from 0.7% to 3.5%. Average recoveries were between 105.6% and 114.6%, with RSD values from 0.3% to 1.9%. The LOQ was 0.02 μg·mL⁻¹, and the LOD was 0.006 μg·mL⁻¹. Statistical analysis indicated that both methods produced comparable results. Conclusion: Both pre-column and post-column derivatization liquid chromatography methods are simple, accurate, and highly sensitive, making them suitable for determining residual free formaldehyde in vaccines.