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Purpose <p>Chlorogenic acid (CA), a naturally occurring immunomodulatory compound, has shown considerable promise in cancer treatment. Our group has developed an oral self-microemulsion formulation of CA. The stability studies have revealed that prolonged storage at room temperature and under accelerated conditions leads to the accumulation of impurities. Therefore, it is critical to establish an effective method for impurity separation and identify the structures of the principal impurities to ensure the quality and safety of the formulation.</p> Methods <p>By examining the liquid phase conditions and method validation, this study establishes a novel high-performance liquid chromatography method using a gradient method, designed to separate significant impurities generated from both CA active pharmaceutical ingredient and the chlorogenic acid oral self-microemulsion formulation under conditions such as acidic, alkaline, oxidative, thermal, photolytic, and stability testing. In addition, high performance liquid chromatography-mass spectrometry and nuclear magnetic resonance were employed to identify and characterize the major impurities formed during the degradation of CA formulations under extreme conditions or prolonged storage.</p> Results <p>Method validation verified satisfactory performance across all critical parameters. Excellent linearity was achieved over the concentration range of 1.0–1500.0&#xa0;µg/mL (R<sup>2</sup> = 1.000), with the limit of detection and limit of quantification measured at 0.07&#xa0;µg/mL and 0.2&#xa0;µg/mL, respectively. Accuracy was achieved with recovery rates of 101.96%–102.58%. The accelerated test of CA-SME at 6-month yielded 18 impurity peaks, nine of which contained more than 0.1% of impurities. By contrast, the 24-month long-term test produced nine impurity peaks, among which only Impurity I and Impurity II surpassed the 0.1% threshold, and their chemical structures were successfully characterized.</p> Conclusion <p>The establishment of this method is not only expected to contribute to the quality control of CA-SME products, but also provide a valuable reference for the development and quality control of natural product preparations.</p> Graphical Abstract <p></p>

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Development and Validation of An HPLC Method for Isolation and Structural Elucidation of Impurities in Chlorogenic Acid Self-Microemulsion Formulations

  • 期刊论文

  • 发表日期：

    2026年2月28日

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  • Yue Gao,
  • Yanan Wang,
  • Jing Xu,
  • Ming Ji,
  • Dongyang Zhao,
  • Yanqi Han,
  • Longhui Hao,
  • Yi Zhang,
  • Ying Sun,
  • Zhenshuai Qin,
  • Jun Ye,
  • Hongliang Wang,
  • Yuling Liu,
  • Yanfang Yang

摘要

Purpose

Chlorogenic acid (CA), a naturally occurring immunomodulatory compound, has shown considerable promise in cancer treatment. Our group has developed an oral self-microemulsion formulation of CA. The stability studies have revealed that prolonged storage at room temperature and under accelerated conditions leads to the accumulation of impurities. Therefore, it is critical to establish an effective method for impurity separation and identify the structures of the principal impurities to ensure the quality and safety of the formulation.

Methods

By examining the liquid phase conditions and method validation, this study establishes a novel high-performance liquid chromatography method using a gradient method, designed to separate significant impurities generated from both CA active pharmaceutical ingredient and the chlorogenic acid oral self-microemulsion formulation under conditions such as acidic, alkaline, oxidative, thermal, photolytic, and stability testing. In addition, high performance liquid chromatography-mass spectrometry and nuclear magnetic resonance were employed to identify and characterize the major impurities formed during the degradation of CA formulations under extreme conditions or prolonged storage.

Results

Method validation verified satisfactory performance across all critical parameters. Excellent linearity was achieved over the concentration range of 1.0–1500.0 µg/mL (R2 = 1.000), with the limit of detection and limit of quantification measured at 0.07 µg/mL and 0.2 µg/mL, respectively. Accuracy was achieved with recovery rates of 101.96%–102.58%. The accelerated test of CA-SME at 6-month yielded 18 impurity peaks, nine of which contained more than 0.1% of impurities. By contrast, the 24-month long-term test produced nine impurity peaks, among which only Impurity I and Impurity II surpassed the 0.1% threshold, and their chemical structures were successfully characterized.

Conclusion

The establishment of this method is not only expected to contribute to the quality control of CA-SME products, but also provide a valuable reference for the development and quality control of natural product preparations.

Graphical Abstract