Objective: To develop a novel and efficient assay for the determination of free formaldehyde content in vaccines, and to compare the results with the method of the Chinese Pharmacopoeia(ChP) 2020 edition. Methods: Vaccine samples were added directly into headspace vials without any pretreatment and tested after mixed with a new derivatization reagent, 1% p-toluenesulfonic acid-ethanol solution (1% TsOH-EtOH solution). The concentration of the reagent, the dosage and the optimal derivatization conditions (headspace incubation temperature and time) were tested and confirmed. A DB-624 gas capillary column was used with temperature programming. The detector was a hydrogen-ion flame detector (FID) and the sample was injected in headspace. Results: The linear range was 0.25-100 μg·mL-1(r=0.999 5), the detection limit and the quantification limit were 0.10 μg·mL-1 and 0.25 μg·mL-1, respectively. The recovery rates of several kinds of vaccines were all over 94%, and the RSDs were all less than 6%, which were in line with the requirements of specificity, precision and durability. There were no significant differences between the results of multiple batches and those determined by ChP 2020 methods. Conclusion: In this paper, a novel derivatization reagent (1% TsOH-EtOH solution) was developed and validated for the determination of free formaldehyde content in vaccines, which is simple, efficient and can achieve high-throughput detection. The method can just solve the problems in other current detection methods. The method of derivatization headspace gas chromatography for the determination of free formaldehyde content is applicable to vaccines and other biologics, and provide a reference for chemicals, traditional Chinese medicine and excipients.
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