Please wait a minute...
Journal of Zhejiang University (Agriculture and Life Sciences)  2019, Vol. 45 Issue (1): 47-53    DOI: 10.3785/j.issn.1008-9209.2017.12.192
Food sciences     
Determination of 10 organic acid contents in tea using high performance liquid chromatography-diode array detector
Lili WANG(),Junguo YANG,Qingxia LIN,Lihui XIANG,Zhenshuo SONG,Yinggen ZHANG,Lin CHEN()
1. Tea Research Institute, Fujian Academy of Agricultural Sciences, Fu’an 355015, Fujian, China
Download: HTML   HTML (   PDF(928KB)
Export: BibTeX | EndNote (RIS)      

Abstract  

A high performance liquid chromatography (HPLC) was developed to simultaneously determine the content of 10 organic acids (oxalic acid, D/L-tartaric acid, formic acid, L-malic acid, ascorbic acid, lactic acid, acetic acid, citric acid, succinic acid and fumaric acid) in tea. The chromatographic conditions were optimized in the aspects of mobile phase composition, column temperature, flow rate and detection wavelength. The established HPLC method was methodologically assessed and applied to teas, which included maintaining TSKgel ODS-100V column (4.6 mm×250 mm, 5 μm) at 40 ℃, using 0.10% phosphoric acid-water solution (mobile phase A) and 50% acetonitrile-water solution (mobile phase B) with the gradient elution mode of 100% A (0 min)→100% A (12 min)→0% A (17 min)→0% A (25 min)→100% A (30 min)→100% A (33 min) with 2 min post-run at the flow rate of 1.0 mL/min, and detecting at the wavelengths of 210 and 245 nm for ascorbic acid, and 210 nm for other organic acids by diode array detector (DAD). The correlation coefficients of regression equations for all of the components were greater than 0.999 5, and the relative standard deviations (RSDs) of accuracy and repeatability were lower than 1%, and their RSDs of stability were lower than 4% (except ascorbic acid), as well as the recovery rate with the method of 97%-116%. It is appeared that the newly developed method can yield fine linear relationship, desirable precision, repeatability and separability for determinations of 10 organic acids in tea.



Key wordstea      organic acid      high performance liquid chromatography      diode array detector     
Received: 19 December 2017      Published: 28 March 2019
CLC:  TS 272  
Corresponding Authors: Lin CHEN     E-mail: 875720551@qq.com;82785676@qq.com
Cite this article:

Lili WANG,Junguo YANG,Qingxia LIN,Lihui XIANG,Zhenshuo SONG,Yinggen ZHANG,Lin CHEN. Determination of 10 organic acid contents in tea using high performance liquid chromatography-diode array detector. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(1): 47-53.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2017.12.192     OR     http://www.zjujournals.com/agr/Y2019/V45/I1/47


高效液相色谱-二极管阵列检测器法测定茶叶中10种有机酸含量

从流动相配比、柱温、流速、检测波长等方面优化色谱条件,并进行方法学考察及验证性试验,从而建立了一种同时分离和定量测定茶叶中10种有机酸(草酸、D/L-酒石酸、甲酸、L-苹果酸、抗坏血酸、乳酸、乙酸、柠檬酸、琥珀酸和富马酸)的高效液相色谱法。优化后的色谱条件为:采用TSKgel ODS-100V(4.6 mm×250 mm,5 μm)色谱柱,流动相为0.10%磷酸水溶液(A相)和50%乙腈水溶液(B相),洗脱方式为100% A(0 min)→100% A(12 min)→0% A(17 min)→0% A(25 min)→100% A(30 min)→100% A(33 min),后运行2 min,柱温为40 ℃,流速为1.0 mL/min,二极管阵列检测器同时在210和245 nm波长下检测抗坏血酸,在210 nm波长下检测其他9种有机酸。结果显示,各有机酸组分回归方程的相关系数均在0.999 5以上,其精密度和重复性的相对标准偏差(relative standard deviation, RSD)均小于1%,稳定性的RSD小于4%(除抗坏血酸外),回收率为97%~116%。说明该方法的线性关系、精密度和重复性良好,对组分分离效果较好,可用于茶叶中10种有机酸含量的测定。


关键词: 茶叶,  有机酸,  高效液相色谱法,  二极管阵列检测器 
Fig. 1 Ultraviolet spectra of 10 organic acids
Fig. 2 Effect of mobile phase A with different pH values on the retention time of 10 organic acids
Fig. 3 HPLC chromatograms of 10 organic acids in tea during sequence sampling
Fig. 4 Effect of different column temperatures on the retention time of 10 organic acids
Fig. 5 Effect of different flow rates on the retention time of 10 organic acids
Fig. 6 HPLC chromatogram of 10 organic acids under the suitable chromatographic condition

组分

Component

回归方程

Regression equation

相关系数

Correlation coefficient

线性范围

Linear range/(μg/mL)

检出限

LOD/(μg/mL)

定量限

LOQ/(μg/mL)

草酸 Oxalic acidY=7.675 3X+1.054 31.000 015.0~215.01.3332.665
D/L-酒石酸 D/L-tartaric acidY=0.927 9X+0.108 61.000 06.0~105.02.0315.080
甲酸 Formic acidY=0.507 4X-0.548 20.999 57.5~125.03.0507.625
L-苹果酸 L-malic acidY=0.468 2X-1.414 61.000 025.0~500.05.2288.714
抗坏血酸 Ascorbic acidY=58.138 1X-7.345 71.000 02.5~40.00.5150.644
乳酸 Lactic acidY=0.262 5X+0.232 01.000 060.0~2 000.012.09024.180
乙酸 Acetic acidY=0.326 2X+0.180 01.000 015.0~210.06.97910.469
柠檬酸 Citric acidY=0.638 8X-0.018 10.999 95.0~80.02.0554.110
琥珀酸 Succinic acidY=0.333 8X-0.010 20.999 95.0~205.06.38125.522
富马酸 Fumaric acidY=74.201 6X-0.228 60.999 90.05~0.10.0420.104
Table 1 Regression equation of standards

组分

Component

相对标准偏差Relative standard deviation (RSD)

回收率

Recovery (n=3)

精密度Accuracy重复性Repeatability稳定性Stability
草酸 Oxalic acid0.4210.3611.52598.289
D/L-酒石酸 D/L-tartaric acid0.0360.1430.673104.277
甲酸 Formic acid0.0090.6570.077106.688
L-苹果酸 L-malic acid0.1070.2403.73897.802
抗坏血酸 Ascorbic acid0.4480.24473.84398.677
乳酸 Lactic acid0.2340.3221.00698.081
乙酸 Acetic acid0.3570.2220.591115.904
柠檬酸 Citric acid0.1590.1991.07099.202
琥珀酸 Succinic acid0.0820.0810.164108.900
富马酸 Fumaric acid0.6510.1722.447100.237
Table 2 
Fig. 7 HPLC chromatogram of 10 organic acids in green tea samples

组分

Component

绿茶

Green tea

白茶

White tea

乌龙茶

Oolong tea

红茶

Black tea

普洱茶

Pu-erh tea

草酸 Oxalic acid6.60±0.478.01±0.586.15±0.487.55±0.2212.89±0.73
D/L-酒石酸 D/L-tartaric acid2.61±0.652.43±0.330.99±0.0373.89±0.0450.57±0.044
甲酸 Formic acid0.028±0.002 10.10±0.0130.14±0.0280.095±0.001 20.045±0.001 3
L-苹果酸 L-malic acid1.10±0.101.42±0.313.40±0.231.98±0.101.13±0.22
抗坏血酸 Ascorbic acid0.45±0.110.12±0.0120.10±0.003 20.095±0.005 10.072±0.006 6
乳酸 Lactic acid13.33±0.7616.52±1.476.18±0.3812.18±1.221.44±0.20
乙酸 Acetic acid5.44±0.4213.90±0.724.63±0.506.00±0.01215.84±0.33
柠檬酸 Citric acid2.10±0.232.55±0.471.66±0.0424.01±0.421.50±0.098
琥珀酸 Succinic acid6.10±0.82NDND15.22±1.705.01±0.025
富马酸 Fumaric acid0.042±0.001 10.054±0.001 20.051±0.002 10.021±0.001 50.032±0.011
总量 Total content37.80±3.5645.10±3.9123.30±1.7051.04±3.7238.53±1.67
Table 3 Content of 10 organic acids in teas
[1]   刘盼盼,钟小玉,许勇泉,等.茶叶中有机酸及其浸出特性研究.茶叶科学,2013,33(5):405-410.
LIUP P, ZHONGX Y, XUY Q, et al. Study on organic acids contents in tea leaves and its extracting characteristics. Journal of Tea Science, 2013,33(5):405-410.(in Chinese with English abstract)
[2]   谭和平,叶善蓉,陈丽,等.茶叶中有机酸的测试方法概述.中国测试技术,2008,34(6):77-80.
TANH P, YES R, CHENL, et al. Determination overview of organic acids in tea. China Measurement & Testing Technology, 2008,34(6):77-80. (in Chinese with English abstract)
[3]   何水平,郭春芳,孙云.茶叶有机酸的研究进展.亚热带农业研究,2015,11(1):63-67.
HES P, GUOC F, SUNY. Research progress of organic acids in tea. Subtropical Agriculture Research, 2015,11(1):63-67. (in Chinese with English abstract)
[4]   张光军,许建文,丁明玉.离子交换色谱法同时分析绿茶中有机酸和无机阴离子的含量.新乡医学院学报,2002,19(1):36-38.
ZHANGG J, XUJ W, DINGM Y. Simultaneous analysis with ion-exchange chromatography for the contents of organic acids and inorganic anions in green tea. Journal of Xinxiang Medical College, 2002,19(1):36-38. (in Chinese with English abstract)
[5]   何水平,郭春芳,孙云,等.白茶中的有机酸高效液相色谱分析方法的建立.中国茶叶加工,2015(3):37-42.
HES P, GUOC F, SUNY, et al. Determination of organic acids in white tea by HPLC method. China Tea Processing, 2015(3):37-42. (in Chinese with English abstract)
[6]   张静,蒋华军,刘仲华,等.亲水性C18硅胶反相色谱柱同时分离测定红茶中的有机酸.食品安全质量检测学报,2014,5(8):2476-2481.
ZHANGJ, JIANGH J, LIUZ H, et al. Simultaneous separation and determination of organic acids in black tea by high performance liquid chromatography using polar-enhanced C18 column. Journal of Food Safety and Quality, 2014,5(8):2476-2481. (in Chinese with English abstract)
[7]   袁玲,黄建安,龚志华,等.茯砖茶有机酸反相高效液相色谱分析方法建立及其应用.中国农学通报,2011,27(30):246-252.
YUANL, HUANGJ A, GONGZ H, et al. Study on development and application technology of analysis method for organic acid in Fu-brick tea by RP-HPLC. Chinese Agricultural Science Bulletin, 2011,27(30):246-252. (in Chinese with English abstract)
[8]   丁玲,屠幼英,陈晓敏.高效液相色谱测定紧压茶中有机酸条件研究.茶叶,2005,31(4):224-227.
DINGL, TUY Y, CHENX M. Simultaneous determination of various organic acids in compressed tea by high performance liquid chromatography. Journal of Tea, 2005,31(4):224-227. (in Chinese with English abstract)
[9]   乔方,黄略略,方长发,等.不同产区的妃子笑及怀枝荔枝的甜酸滋味物质比较及电子舌分析.食品与生物技术学报,2012,31(9):984-990.
QIAOF, HUANGL L, FANGC F, et al. Comparison of taste-related compounds and analysis using electronic tongue of Feizixiao and Huaizhi lychee fruits from different planting area. Journal of Food Science and Biotechnology, 2012,31(9):984-990. (in Chinese with English abstract)
[10]   刘小莉,周剑忠,董明盛,等.高效液相色谱-二极管阵列检测器法测定水蜜桃中水溶性有机酸和维生素的含量.南京农业大学学报,2009,32(1):151-154.
LIUX L, ZHOUJ Z, DONGM S, et al. Determination of water-soluble organic acids and vitamins in juicy peach using HPLC-DAD. Journal of Nanjing Agricultural University, 2009,32(1):151-154. (in Chinese with English abstract)
[11]   MAGDALENAJ S, AGNIESZKAZ G. Analysis of antioxidant activity, chlorogenic acid, and rutin content of Camellia sinensis infusions using response surface methodology optimization. Food Analytical Methods, 2014,7(10):2033-2041.
[12]   KELEBEKH, SELLIS, CANBASA, et al. HPLC determination of organic acids, sugars, phenolic compositions and antioxidant capacity of orange juice and orange wine made from a Turkish cv. Kozan. Microchemical Journal, 2009,91(2):187-192.
[13]   杨焱,谷镇,刘艳芳,等.反相高效液相色谱法测定食用菌中7种有机酸的研究.菌物学报,2013,32(6):1064-1070.
YANGY, GUZ, LIUY F, et al. Determination of seven organic acids in edible fungi by reversed-phase high performance liquid chromatography. Mycosystema, 2013,32(6):1064-1070. (in Chinese with English abstract)
[14]   刘晨明,曹宏斌,曹俊雅,等.梯度洗脱高效液相色谱法快速检测厌氧菌代谢物中的有机酸.分析化学研究报告,2006,34(9):1231-1234.
LIUC M, CAOH B, CAOJ Y, et al. Fast detection of organic acid in anaerobe medium with gradient elution-high performance liquid chromatography. Chinese Journal of Analytical Chemistry, 2006,34(9):1231-1234. (in Chinese with English abstract)
[1] Fumei XIN, Yuting WANG, Shengmao LI, Danzengluobu, Pubuciren. Effects of different temperatures on photosynthesis and rooting of Cupressus gigantea seedlings.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(1): 102-108.
[2] QI Dandan, CHEN Wei, MIAO Aiqing, WANG Wenwen, PANG Shi, MA Chengying. Effects of rapid ageing technology on the aroma quality of white tea using gas chromatographymass spectrometry/mass spectrometry combined with chemometrics[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(6): 704-710.
[3] KONG Dedong, ZHAO Yueling, WANG Yuefei, XU Ping. Review on inhibition mechanism of tea polyphenols against tumor immune escape[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(5): 539-548.
[4] NI Jiadan, PAN Shizhe, DAN Fan, JIANG Yihong. Effects of tea polyphenols-chitosan complex solution on changes of quality indicators under storage in Miichthys miiuy[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(5): 594-600.
[5] LI Qingqing, LU Shanshan, ZHANG Hong, YANG Yan, XIAO Jiaxin. Physiological response to different soil pH values between Vaccinium bracteatum and Vaccinium ashei[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(4): 469-475.
[6] XU Pengcheng, FAN Fangyuan, LU Debiao, JIN jing, GONG Shuying1. Infusion color and taste characteristic of Dafo Longjing tea processed from different cultivars[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(3): 317-328.
[7] FAN Fangyuan, CHEN Ping, LUO Wenwen, XU Wenwu, GU Zhaoqi, MAO Zufa, GONG Shuying.  Preliminary study on processing technology of white tea“Chunyu 2”from Zhejiang Province.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(2): 229-238.
[8] LU Peng, WANG Xiaochang, WANG Yingqi . Overview of tea constituents against type 2 diabetes[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(3): 358-367.
[9] ZHANG Dan, LU Ying, LI Bo, YU Huajun, TU Youying. Development and study on properties of tea flower soap[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(3): 333-339.
[10] KONG Zhangliang. Carbon sequestration potential of tea garden soil in northwest Zhejiang and its accumulation characteristic on different fractions of organic carbon.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(2): 209-219.
[11] TAN Liangliang, SHEN Lirong. Determination of the metabolites, eicosanoids derived from polyunsaturated fatty acids in Drosophila tissue with high performance liquid chromatography tandem mass spectrometry[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(1): 8-16.
[12] HOU Ling, SHEN Xian, CHEN Lin, KIM Eunhye, WU Yuanyuan, TU Youying. Optimization on alkaline and enzymatic extraction of tea flower protein and its functional properties[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(04): 442-450.
[13] Zhang Jun, Gong Shuying, Tang Desong, Zhang Yingbin, Chen Meili. Quality evaluation and price discrimination of jasmine-scented tea[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(5): 577-585.
[14] Xu Yuanjun, He Jing, Jia Lingyan, Wu Yuanyuan, Tu Youying. Differentiation of aroma compositions in different regions and special varieties of black tea[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(03): 323-330.
[15] Chen Meili1,2, Tang Desong1*, Gong Shuying1*, Yang Jie1, Zhang Yingbin3. Quantitative analysis and correlation evaluation on taste quality of green tea.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2014, 40(6): 670-678.