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Objective <p>To develop and validate a simple, rapid, high-performance liquid chromatography (HPLC) method for the quantification of caffeic acid using a systematic Quality by Design (QbD).</p> Methods <p>The chromatographic separation was achieved on an Intersil ODS column (250&#xa0;mm × 4.6&#xa0;mm, 5&#xa0;μm particle size) with an isocratic mobile phase consisting of methanol and water. UV detection was performed at 325&#xa0;nm. A three-factor, three-level Box-Behnken Design was used to optimize methanol concentration, flow rate, and injection volume based on their effects on theoretical plate number, tailing factor, and retention time. The optimized method was validated for linearity, accuracy, precision, specificity, limit of detection (LOD), and limit of quantitation (LOQ).</p> Results <p>The QbD approach identified the optimal conditions as 40% (v/v) methanol in water, a 1.0 mL/min flow rate, and a 20 µL injection volume, yielding a theoretical plate number of 5900, a tailing factor of 0.61, and a retention time of 6.3&#xa0;min. The method demonstrated linearity over a concentration range of 20–120 ppm, with a coefficient of determination (R²) of 0.999. The calculated LOD and LOQ were 2.71 ppm and 9.02 ppm, respectively. The method was also found to be specific, with no interference from the blank.</p> Conclusion <p>A robust, accurate, and precise RP-HPLC method for the quantification of caffeic acid was successfully developed and validated using a QbD framework. The method is simple, reliable, and fulfills all ICH requirements, making it highly suitable for routine quality control applications in the pharmaceutical and natural product industries.</p>

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A Validated, High-Performance Liquid Chromatography Method for the Analysis of Caffeic Acid: A Systematic Approach

  • 期刊论文

  • 发表日期：

    2026年5月4日

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  • Amol Patil,
  • Durgacharan Bhagwat

摘要

Objective

To develop and validate a simple, rapid, high-performance liquid chromatography (HPLC) method for the quantification of caffeic acid using a systematic Quality by Design (QbD).

Methods

The chromatographic separation was achieved on an Intersil ODS column (250 mm × 4.6 mm, 5 μm particle size) with an isocratic mobile phase consisting of methanol and water. UV detection was performed at 325 nm. A three-factor, three-level Box-Behnken Design was used to optimize methanol concentration, flow rate, and injection volume based on their effects on theoretical plate number, tailing factor, and retention time. The optimized method was validated for linearity, accuracy, precision, specificity, limit of detection (LOD), and limit of quantitation (LOQ).

Results

The QbD approach identified the optimal conditions as 40% (v/v) methanol in water, a 1.0 mL/min flow rate, and a 20 µL injection volume, yielding a theoretical plate number of 5900, a tailing factor of 0.61, and a retention time of 6.3 min. The method demonstrated linearity over a concentration range of 20–120 ppm, with a coefficient of determination (R²) of 0.999. The calculated LOD and LOQ were 2.71 ppm and 9.02 ppm, respectively. The method was also found to be specific, with no interference from the blank.

Conclusion

A robust, accurate, and precise RP-HPLC method for the quantification of caffeic acid was successfully developed and validated using a QbD framework. The method is simple, reliable, and fulfills all ICH requirements, making it highly suitable for routine quality control applications in the pharmaceutical and natural product industries.