2015 Seminar, Institute of Food Science & Biotechnology, NCHU
Title:
Rapid method for the determination of 14 isoflavones in food using UHPLC coupled to photo
diode array detection
Authors:
You-Shin Shim , Won-Jin Yoon , Jin-Bong Hwang , Hyun-Jin Park , Dongwon Seo , Jaeho Ha
Source: Food Chemistry 187 (2015) 391-397
Speaker:白佳哲
Moderator:蔡維倫
Date: 2015/12/20
Advisor: Prof. Pei-Fen Liu
Abstract
A rapid method for the determination of 14 types of isoflavones in food using ultra-high
performance liquid chromatography (UHPLC) was validated in terms of precision, accuracy,
sensitivity and linearity. The UHPLC separation was performed on a reverse-phase C18 column
(particle size 2 lm, i.d. 2 mm, length 100 mm) using a photo diode array detector that was fixed to
260 nm. The limits of detection and quantification of the UHPLC analyses ranged from 0.03 to
0.33 mg/ kg. The intra-day and interday precision of the individual isoflavones were less than
11.77% and calibration curves exhibited good linearity (r2 = 0.999) within the tested ranges. These
results suggest that the rapid method used in this study could be available to determine 14 types of
isoflavones in a variety of food such as soy bean, black bean, red bean and soybean paste.
1. Introduction
Isoflavones are regarded as biologically important compounds as they are precursors of
phytoalexins, which play an important role in plant disease resistance and antifungal activity.
Much work has focused on the assembly and data analysis of 12 isoflavone isomers, three
aglycones (daidzein, genistein, and glycitein) and nine glucosides (daidzin, genistin, glycitin,
acetyl-daidzin, acetylgenistin, acetylglycitin, malonyldaidzin, malonylgenistin, and
malonylglycitin) found in soyfood. In the pharmacokinetic studies, studies,
daidzein-7-O-glucuronide and genistein-7-O-glucuronide have been shown to be important
compounds. The glucuronides of genistein and daidzein are the major circulating metabolites in
human subjects; therefore, they merit the most attention in terms of their biological activities.
Therefore, our study was focused on the evaluation of a rapid analytical method for the
determination of 14 types of isoflavones in food using UHPLC coupled to photo diode array
detection.
2. Materials and methods
3. Results and discussion
3.1. Optimization of UHPLC detection:
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2015 Seminar, Institute of Food Science & Biotechnology, NCHU
In this study, we used C18 as the separation column, which exhibited satisfactory separation
efficiency for 14 types of isoflavones. The optimized run time was 25 min and the consumption of
mobile phase was only 7.5 mL, which is very fast compared with conventional HPLC detection
(Shen et al., 2012). The runtime of conventional HPLC/UVD detection was 40 min and consumes
40 mL of organic solvents as the mobile phase.
3.2. Optimization of extraction condition:
To evaluate each sample pretreatment method, we considered using DMSO as a pre-extraction
solvent. A 3.6 mL of DMSO were added to 1 g of homogenized sample and vortex mixed for 5
min followed by the addition of 8.4 mL of 70% methanol to act as the main extraction solvent in
the vial. The extracts were filtered through a 0.2-lm syringe filter after vortex mixing for 5 min
again. Using DMSO allowed for the omission of the sonication without the loss of target
compounds and reduced the pretreatment time threefold compared with the conventional method.
3.3. Linearity, limit of detection and limit of quantification:
To determine the linearity, seven different concentrations (0.109, 0.219, 0.438, 0.875, 1.750,
3.500, and 7.000 mg/ kg) of standards were used for each of isoflavones. Individual solutions were
injected 10 times and the averages of the results are presented in Table 1. All calibration curves
showed good linearity (r2 = 0.999) within the tested ranges. The limits of detection and
quantification of the UHPLC analyses ranged from 0.03 to 0.33 mg/ kg.
3.4. Precision and accuracy:
To determine the precision and accuracy of this method, intraday (n = 4) and inter-day (n = 3)
tests were performed on the spiked red bean and soybean paste. The RSD values for the intra-day
and inter-day repeatability were below 11.77% for red bean and were below 11.76% for soybean
paste. The recovery values were ranged from 85.6% to 113.5% for red bean and 87.8% to 114.6%
for soybean paste.
3.5. Application tests:
The application tests of the UHPLC detection coupled with rapid pretreatment method presented
in this study were examined with various samples such as soy bean, black bean, red bean, and
soybean pastes. In the results, we could not observed DAI-Gc and GEN-Gc in tested samples but
we expect that this rapid method using UHPLC could be used for analysis of food as well as
related pharmacokinetic studies.
4. Conclusions
A precise and rapid extraction method using DMSO was developed for the reliable, simple, and
rapid analysis of 14 types of isoflavones in food using UHPLC. Benefits of the rapid method
coupled with UHPLC detection are much faster analysis (approximately 2.6 times) and lower
consumption of organic solvents (approximately 3.3 times) than the conventional method coupled
with HPLC. The rapid method coupled with UHPLC detection developed in this study could be
applicable to the various food, including soybean, red bean, black bean and soybean paste.
5. References
1.Shen, D., Wu, Q., Sciarappa, W. J., & Simon, J. E. (2012). Chromatographic fingerprints and
quantitative analysis of isoflavones in Tofu-type soybeans. Food Chemistry, 130, 1003–1009.
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