[논문]HPLC Method for Simultaneous Quantitative Detection of Quercetin and Curcuminoids in Traditional Chinese Medicines

Ang, Lee Fung; Yam, Mun Fei; Fung, Yvonne Tan Tze; Kiang, Peh Kok; Darwin, Yusrida

doi:10.3831/kpi.2014.17.035

HPLC Method for Simultaneous Quantitative Detection of Quercetin and Curcuminoids in Traditional Chinese Medicines 원문보기

Journal of pharmacopuncture, v.17 no.4, 2014년, pp.36 - 49

Ang, Lee Fung (School of Pharmaceutical Sciences, Universiti Sains Malaysia) , Yam, Mun Fei (School of Pharmaceutical Sciences, Universiti Sains Malaysia) , Fung, Yvonne Tan Tze (School of Pharmaceutical Sciences, Universiti Sains Malaysia) , Kiang, Peh Kok (School of Pharmaceutical Sciences, Universiti Sains Malaysia) , Darwin, Yusrida (School of Pharmaceutical Sciences, Universiti Sains Malaysia)

Abstract ▼ AI-Helper

Objectives: Quercetin and curcuminoids are important bioactive compounds found in many herbs. Previously reported high performance liquid chromatography ultraviolet (HPLC-UV) methods for the detection of quercetin and curcuminoids have several disadvantages, including unsatisfactory separation times and lack of validation according the standard guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Methods: A rapid, specific, reversed phase, HPLC-UV method with an isocratic elution of acetonitrile and 2% v/v acetic acid (40% : 60% v/v) (pH 2.6) at a flow rate of 1.3 mL/minutes, a column temperature of $35^{\circ}C$, and ultraviolet (UV) detection at 370 nm was developed. The method was validated and applied to the quantification of different types of market available Chinese medicine extracts, pills and tablets. Results: The method allowed simultaneous determination of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin in the concentration ranges of $0.00488-200{\mu}g/mL$, $0.625-320{\mu}g/mL$, $0.07813-320{\mu}g/mL$ and $0.03906-320{\mu}g/mL$, respectively. The limits of detection and quantification, respectively, were 0.00488 and $0.03906{\mu}g/mL$ for quercetin, 0.62500 and $2.50000{\mu}g/mL$ for bisdemethoxycurcumin, 0.07813 and $0.31250{\mu}g/mL$ for demethoxycurcumin, and 0.03906 and $0.07813{\mu}g/mL$ for curcumin. The percent relative intra day standard deviation (% RSD) values were $0.432-0.806{\mu}g/mL$, $0.576-0.723{\mu}g/mL$, $0.635-0.752{\mu}g/mL$ and $0.655-0.732{\mu}g/mL$ for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and those for intra day precision were $0.323-0.968{\mu}g/mL$, $0.805-0.854{\mu}g/mL$, $0.078-0.844{\mu}g/mL$ and $0.275-0.829{\mu}g/mL$, respectively. The intra day accuracies were 99.589%-100.821%, 98.588%-101.084%, 9.289%-100.88%, and 98.292%-101.022% for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and the inter day accuracy were 99.665%-103.06%, 97.669%-103.513%, 99.569%-103.617%, and 97.929%-103.606%, respectively. Conclusion: The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of commercial Chinese medicine products containing the flour flavonoids as their principle components in the extracts.

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제안 방법

5. The mean values of the six replicate injections of 160 µg/mL QC standards were used to evaluate the retention time, the peak area, the resolutions for the analyte peaks, the tailing factor, the number of theoretical plates and the capacity factor.
HPLC analysis was performed using a Shimadzu-LC system (Shimadzu, Japan) equipped with an CBM-20A controller, LC-20AT pump, DGU-20A5 prominence degasser, SIL-20A auto sampler, SPD-20AV detector and CTO10ASvp column oven.
In addition, each parameter showed good repeatability of the retention time and response. In conclusion, the proposed method is simple, easy and cost effective, no specific solvent is involved and it utilizes common HPLC instruments with UV detectors. Hence, this UV-HPLC method is suitable for routine analysis of quercetin and curcuminoid formulations or products.
Intra day precisions and accuracies were determined by using a replicate analysis (n = 6) of the QC samples on the same day under the same analytical conditions. Inter day precisions and accuracies were tested by using a replicate analysis (n = 3) of the same QC samples on six consecutive days. The precision is calculated from the mean of the accuracy and the relative standard deviation (RSD).
830 for quercetin-bisdemethoxycurcumin, bisdemethoxycurcumin-demethoxycurcumin and demethoxycurcumin-curcumin, respectively. No interferences or excipient peaks co eluted with the analytes were observed, indicating the method is selective and specific in relation to the medium and excipients used in this study (Fig. 2, Table 2).
25, 5, 20, 40, 80, 140 and 200 µg/mL. Separate calibration curves were constructed for quercetin, bisdemethoxycurcumin demethoxycurcumin and curcumin by plotting the peak areas against the concentrations, and the methods were evaluated by determining the coefficient of determination (R²). Unknown assay samples were quantified by referencing them to these calibration curves.
The method developed in this study was used to quantitatively determination the quercetin and the curcuminoid contents of extracts, pills and tablets made from Chinese medicinal plants.
The proposed method was applied to quantitatively detect the quercetin and curcuminoids in Chinese medicines such as plant granule extracts, tablets and pills. The results of 19 samples are summarized in Table 7.
The robustness parameters tested were the mobile phase’s composition, the concentration of acetic acid (pH effect), the flow rate and the column temperature.
As defined in the United States Pharmacopeia/National Formulary (USP/NF) [54] system suitability parameters were established as a direct result of the ruggedness and the robustness of the experiments. The system suitability testing proved that the proposed method will allow the separation of all four analytes and will produce satisfactory peak shapes.

데이터처리

The differences were analyzed by using SPSS version 20, and a one way analysis of variance (ANOVA), followed by Tukey’s test.

성능/효과

A regression analysis of the data showed a linear relationship for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, with excellent R2 values of 0.99993, 0.99984, 0.99985 and 0.99993 μg/mL, respectively.
The RSD values for the intraday and the inter-day precisions were < 1%. For the accuracy test, the intraday and the inter day accuracies ranges from 98.292% to 103.617%, confirming the accuracy of the method.
This method was validated for specificity, linearity, precision, accuracy and robustness as per ICH guidelines. The data showed good selectivity and sensitivity, a wide linear range, precision and accuracy. The method was sensitive to HPLC conditions; that is, changes in the mobile phase’s composition, the pH, the column temperature and the flow rate affected the retention time and response, but did not affected the separation of the compounds.
The method was sensitive to HPLC conditions; that is, changes in the mobile phase’s composition, the pH, the column temperature and the flow rate affected the retention time and response, but did not affected the separation of the compounds.

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