[논문]엉겅퀴에 함유된 Cirsimaritin 분석을 위한 HPLC 분석법 밸리데이션

이주성; 레오; 조이스; 강기성; 함대현; 심재석; 최유진; 이상현

doi:10.22889/kjp.2020.51.3.217

엉겅퀴에 함유된 Cirsimaritin 분석을 위한 HPLC 분석법 밸리데이션
Validation of an HPLC/UV Analysis Method for Cirsimaritin in Cirsium japonicum var. maackii 원문보기

생약학회지, v.51 no.3, 2020년, pp.217 - 221

이주성 (중앙대학교 식물생명공학과) , 레오 (중앙대학교 식물생명공학과) , 조이스 (산카를로스대학교 생물학과) , 강기성 (가천대학교 한의학과) , 함대현 (경희대학교 의학과) , 심재석 (임실생약 영농조합법인) , 최유진 ((재)임실치즈앤식품연구소) , 이상현 (중앙대학교 식물생명공학과)

Abstract ▼ AI-Helper

Cirsium japonicum var. maackii (CM) has been used to treat certain medical conditions such as hemorrhage, hepatitis, and hypertension. Cirsimaritin was previously found as the major flavonoid in CM and is said to contribute to its pharmacological effects. There are currently no reports detailing the qualitative and quantitative detection of phytochemical indicators in the aerial parts of CM. Therefore, we developed a method to rapidly identify and quantify cirsimaritin in CM using HPLC/UV, and we optimized and validated this analytical method. The results showed good linearity in the concentration range tested (0.25-0.015 mg/mL, r2 ≥ 0.9999), accuracy (93.9-111.3%), and precision (RSD ≤ 0.59%). The developed method can therefore be used for the rapid evaluation of cirsimaritin in CM.

주제어

표/그림 (5)

그림 Fig. 1. Chemical structure of cirsimaritin.
그림 Fig. 2. HPLC chromatograms and specificity of CM (A) and cirsimaritin (B).
표 Table I. Linearity, LOD, and LOQ for cirsimaritin
표 Table II. Accuracy for the determination of cirsimaritin
표 Table III. Intra- and inter-day precision for the determination of cirsimaritin

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문제 정의

¹⁹⁾ However, there are no reports available on validated analytical approaches for evaluating the phytochemical indicators in CM. The aim of this study was to develop and establish an optimized technique using HPLC/UV to detect and quantify the flavones in CM for use in the future development of its raw materials.

제안 방법

The quantification parameters for cirsimaritin were examined using the aforementioned HPLC conditions. A calibration curve for the isolated compound was constructed by mapping the peak areas of the prepared concentrations, and linear regression was used to assess the linearity. For the evaluation of linearity, six solutions were used with a concentration range of 0.
Conversely, the LOQ is the minimum concentration of the analyzed compound that can be quantified with great precision and accuracy using the instrument and the analytical method. The LOD value obtained in this analysis was 0.006 mg/mL, while the LOQ value was 0.018 mg/mL, suggesting that the analytical approach used to measure the three compounds extracted from CM achieved high sensitivity (Table I).
To assess inter-day precision, the % relative standard deviation (% RSD) of the standards was determined on three separate days. The accuracy of the HPLC method was determined in recovery tests in which sample extracts spiked with three different standard mixture concentrations (mg/g) were analyzed and the % recovery was calculated. To validate accuracy and precision, standard mixtures with three concentrations of cirsimaritin were used for intra- and inter-day precision analyses.
The validation techniques employed with the analytical methodology indicated that excellent results were obtained in terms of the accuracy, precision, specificity, and quantifying parameters. The findings of our analysis support the use of our method for the identification and accurate quantification of cirsimaritin from CM in routine analyses and large-scale extraction processes and for content determination. Moreover, our study provided an optimized technique that could be applied to the pharmaceutical industrialization of CM leaf extract.
LOD and LOQ were determined as the lowest concentration producing an appropriate peak shape and experimentally calculated by injecting a series of diluted solutions with known concentrations until the signal-to-noise ratio was 3:1 for LOD and 10:1 for LOQ. To assess inter-day precision, the % relative standard deviation (% RSD) of the standards was determined on three separate days. The accuracy of the HPLC method was determined in recovery tests in which sample extracts spiked with three different standard mixture concentrations (mg/g) were analyzed and the % recovery was calculated.

성능/효과

59%, respectively. The values obtained were lower than 2%, indicating that the corresponding results of the analytical method to quantify cirsimaritin derived from CM showed great reliability.

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