Establishment of Analytical Methods Validation for Thimerosal and Phenol Content in Preservatives for Animal Vaccines
Hsun-Lung Chan
Abstract
In order to prevent microbial contamination during the manufacturing process and multi-dose injection, it is permitted to add appropriate amounts of preservatives to vaccines for animal use. Since the preservatives are toxic, the pharmacopoeia and regulations of each country specify the minimum effective safety concentration. According to the test standards established in Chapter 3 of the Test Standards for Veterinary Drugs, the preservative content limits for thimerosal and phenol in different types of animal vaccines are 0.02-0.01 % or less and 0.5-0.1% or less, respectively. In the current pharmacopoeia and official compendium, the methods for testing the preservatives thimerosal and phenol mainly include titration and UV/VIS spectrophotometer. However, these methods lack specificity for complex matrix vaccines. By using high performance liquid chromatography (HPLC) to effectively separate the matrix from the analyte, the accuracy of the test can be improved. The analytical methods developed and established in this study were validated in accordance with the GL02 Analytical Validation Guideline published by the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH). For the liquid extraction method of animal vaccine pretreatment solution, Tetrahydrofuran was used as the extraction solution for phenol extraction, while Dichloromethane was used for thimerosal extraction and deionized water was used for extraction of fat-soluble substances. For phenol analysis, Acetonitrile-deionized water (55:45, v/v) was used as the mobile phase and a Waters Symmetry® C18 (4.6 × 250 mm, 5 μm) column with a detection wavelength of 217 nm was used for analysis. Thimerosal analysis was performed using Methanol-0.02 M ammonium acetate (35:65, v/v) as mobile phase and HypersilTM GOLD (4.6 × 250 mm, 5 μm) column with a detection wavelength of 215 nm. The items required for validation of analytical methods include accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ), linearity, and range. The results are in accordance with the standard requirements of the guideline, and this testing technique can be applied to the national validation of animal vaccines to ensure the quality of animal vaccines.
Differential diagnosis of lumpy skin disease from other bovine diseases
Yen-Wen Chen
Abstract
Lumpy skin disease (LSD) is a poxviral disease which can cause significant morbidity but usually results in low mortality rates among infected cattle. The economic losses resulting from bovine LSD outbreaks stem primarily from losses in condition, reductions in milk yield, as well as increases in abortions, cases of infertility, and damaged hides. An outbreak of LSD in Taiwan was first reported in Kinmen County on July 10 2020 and 38 cattle farms were confirmed to have experienced LSD outbreaks by the end of August, 2020, at which time all cattle had been vaccinated with LSDV vaccines in Kinmen County. From August to September 2020, three LSD-suspected cases in cattle from Taiwan and the Penghu Islands were also investigated. These cattle displayed irregularly sized papules, nodules, pustules or plaques that were ubiquitously distributed on the cutis. Outside of the skin lesions, no other significant clinical signs were noted, and no mortality occurred. By using skin impression smears, bacterial culturing, histopathological examinations, electron microscopy, and PCR, LSD was ruled out and the three cases were subsequently diagnosed as bovine demodicosis, dermatophilosis, and pseudo-lumpy skin disease, respectively. The immediate and correct differential diagnosis of LSD plays an important role within the cattle industry of LSD-affected countries, which can avoid culling and other economic losses caused by misdiagnosis.