Isolation and Identification of Three New Isomer Impurities in Milbemycin Oxime Drug Substance

Chemistry Analytical Chemistry Medicinal Chemistry Pharmacy Medicine
Milbemycin Oxime Isomer Impurities Isolation Identification Drug Analysis HPLC NMR

Isolation and Identification of Three New Isomer Impurities in Milbemycin Oxime Drug Substance

Academic Background

Milbemycins are a class of 16-membered macrolides produced by Streptomyces, exhibiting potent anthelmintic and insecticidal activities with low mammalian toxicity. Milbemycins and their derivatives are widely used in agriculture and veterinary fields to control various insects and parasites. Milbemycin Oxime (MO), a semi-synthetic product prepared by the oxidation and oximation of milbemycins A3 and A4, has been marketed since 1990 primarily for preventing heartworm disease and controlling other endoparasites in dogs.

However, as a semi-synthetic product derived from natural compounds, MO inevitably forms impurities during the synthesis process, including residual starting materials, intermediates, degradation products, and by-products. These impurities may affect the quality and safety of the drug. Although the European Pharmacopoeia and the United States Pharmacopoeia have specified limits for known impurities in MO, some unknown impurities remain unidentified. Therefore, the research team aimed to isolate and identify unknown impurities in MO to further optimize drug quality control standards.

Source of the Paper

This paper was co-authored by Xiaohan Ren, Jianwei Lu, Yefei Wu, Shaoyong Zhang, Huan Qi, Hui Zhang, Jidong Wang, and Linghui Zheng. The authors are affiliated with the College of Life Science, Huzhou University, Zhejiang Qianjiang Biochemical Co., Ltd., Taizhou Vocational College of Science and Technology, and Zhejiang Huida Biotech Co., Ltd. The paper was published online on November 28, 2024, in The Journal of Antibiotics, with the DOI 10.1038/s41429-024-00791-7.

Research Process and Results

1. Detection and Isolation of Impurities

The research team first analyzed laboratory-prepared MO samples and two commercially available MO drugs (Milpro® and Hailechong®) using high-performance liquid chromatography-mass spectrometry (HPLC-MS). They detected two unknown impurity peaks with molecular weights 14 Da higher than MO A4. According to the International Council for Harmonisation (ICH) guidelines, the content of these impurities ranged from 0.10% to 0.15%, exceeding the identification threshold of 0.10%, thus requiring further identification.

To isolate these impurities, the research team developed a two-dimensional (2D) preparation method. First, the Agilent Zorbax-C3 preparative column was used for the first-dimensional separation, employing isocratic elution (water/methanol/acetonitrile = 30/35/35, v/v/v) at a flow rate of 1.5 mL/min. Subsequently, the Sepax Amethyst C18-H preparative column was used for the second-dimensional separation, employing isocratic elution (water/methanol = 15⁄85, v/v) at a flow rate of 1.5 mL/min. Using this method, the research team successfully isolated three impurities, named impurity 1, impurity 2, and impurity 3.

2. Structural Identification of Impurities

To determine the structures of these three impurities, the research team employed high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) techniques. The specific steps were as follows:

  • Impurity 1: Through 1H NMR and 13C NMR analysis, it was found that impurity 1 shared the same scaffold as MO A4 but lacked a methyl group at the C-14 position, which was replaced by an ethyl group. Further 1H-1H COSY and HMBC spectral analyses confirmed this structure. Ultimately, impurity 1 was identified as 14-desmethyl-14-ethyl-MO A4.

  • Impurity 2: Similar NMR analysis revealed that impurity 2 was structurally related to MO A4 but lacked a methyl group at the C-24 position, which was replaced by an ethyl group. Therefore, impurity 2 was identified as 24-desmethyl-24-ethyl-MO A4.

  • Impurity 3: NMR analysis showed that impurity 3 was structurally related to MO A4 but lacked a methyl group at the C-12 position, which was replaced by an ethyl group. Therefore, impurity 3 was identified as 12-desmethyl-12-ethyl-MO A4.

3. Analysis of Impurity Origins

The research team further analyzed the origins of these impurities. By comparing the structures of natural milbemycin homologs, it was found that these impurities might be generated through the oxidation and oximation of natural milbemycin homologs present in the fermentation broth. This discovery provides an important theoretical basis for the quality control of MO drugs.

Research Conclusions and Significance

This study successfully isolated and identified three new isomer impurities in MO drugs: 14-desmethyl-14-ethyl-MO A4, 24-desmethyl-24-ethyl-MO A4, and 12-desmethyl-12-ethyl-MO A4. The identification of these impurities not only fills a gap in the quality control of MO drugs but also provides important references for impurity control during drug production.

Additionally, the two-dimensional preparation method developed by the research team significantly improved the efficiency of impurity separation, offering a new technical approach for the isolation of high-purity impurities from complex samples. This method is not only applicable to the quality control of MO drugs but can also be extended to the separation and identification of impurities in other complex drugs.

Research Highlights

  1. Identification of New Impurities: For the first time, three new isomer impurities in MO drugs were isolated and identified, filling a gap in drug quality control.
  2. Two-Dimensional Preparation Method: An efficient two-dimensional preparation method was developed, significantly improving the separation efficiency of impurities in complex samples.
  3. Analysis of Impurity Origins: By comparing the structures of natural milbemycin homologs, the generation mechanism of these impurities was revealed, providing a theoretical basis for impurity control during drug production.

Other Valuable Information

This study was supported by the Zhejiang Provincial Natural Science Foundation of China (LGN22B020002), the Huzhou Public Welfare Research Project (2021GZ25), and the Taizhou Science and Technology Plan Project (24NYB10). The research team also acknowledges the support from Zhejiang Huida Biotech Co., Ltd. and Zhejiang Qianjiang Biochemical Co., Ltd.

Through this research, the quality control standards for MO drugs have been further improved, providing important support for enhancing drug safety and efficacy. In the future, the research team plans to further optimize the two-dimensional preparation method and apply it to the separation and identification of impurities in more complex drugs.