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Development of an Efficient HPLC Fluorescence Detection Method for Brassinolide by Ultrasonic-Assisted Dispersive Liquid–Liquid Microextraction Coupled with Derivatization

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Abstract

A novel hyphenated technique based on ultrasonic-assisted dispersive liquid–liquid microextraction (UA-DLLME) coupled with derivatization has been established for the determination of brassinolide (BL, a representative of brassinosteroids) by HPLC fluorescence detection. 9-Phenanthreneboronic acid is used as labeling reagent of BL. UA-DLLME parameters containing type and volume of extraction and disperser solvent, pH and ultrasonication time are optimized. Derivatization parameters are optimized included amount of 9-phenanthreneboronic acid, volume ratio of pyridine, derivatization time and temperature. Under optimal conditions, quantitative linear range of BL is 50–1,000 ng L−1 and excellent linear response is observed with correlation coefficient of 0.9996. Limit of detection and limit of quantification are calculated as 8.0 and 25.0 ng L−1, respectively. RSDs of retention time and peak area are in the range of 0.68–0.97 % and 4.61–6.54 % for intra-day precision, 1.32–1.94 % and 7.28–9.75 % for inter-day precision, respectively. Accuracy is satisfactory in the range of 82.3–125.1 %. RSDs’ values of repeatability are in the range of 0.82–1.79 and 3.95–8.53 % for retention time and peak area, respectively. Enrichment factor for BL is 189. The results of recovery and matrix effect are in the range of 82.0–108.6 and 90.0–115.3 %, respectively. The proposed method has been applied for the determination of BL in Arabidopsis thaliana, Daucus carota and Brassica campestris L. leaves with much higher sensitivity than many other methods.

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Acknowledgments

The authors acknowledge the financial support from the National Natural Science Foundation of China (81303179, 21275089 and 21475075), and the Foundation of Qufu Normal University (BSQD2012019).

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Authors and Affiliations

  1. Shandong Provincial Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, 273165, Shandong, People’s Republic of China

    Tao Lv, Xian-En Zhao, Shuyun Zhu, Zhongyin Ji, Guang Chen, Zhiwei Sun, Cuihua Song & Jinmao You

  2. Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Qufu Normal University, Qufu, 273165, Shandong, People’s Republic of China

    Tao Lv, Xian-En Zhao, Shuyun Zhu, Guang Chen, Zhiwei Sun, Cuihua Song & Jinmao You

  3. Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Science, Xining, 810001, Qinghai, People’s Republic of China

    Zhongyin Ji, Jinmao You & Yourui Suo

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  1. Tao Lv
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  2. Xian-En Zhao
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Correspondence to Xian-En Zhao or Jinmao You.

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Lv, T., Zhao, XE., Zhu, S. et al. Development of an Efficient HPLC Fluorescence Detection Method for Brassinolide by Ultrasonic-Assisted Dispersive Liquid–Liquid Microextraction Coupled with Derivatization. Chromatographia 77, 1653–1660 (2014). https://doi.org/10.1007/s10337-014-2767-9

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  • DOI: https://doi.org/10.1007/s10337-014-2767-9

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