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浙江大学学报(农业与生命科学版)  2019, Vol. 45 Issue (1): 47-53    DOI: 10.3785/j.issn.1008-9209.2017.12.192
食品科学     
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
王丽丽(),杨军国,林清霞,项丽慧,宋振硕,张应根,陈林()
1. 福建省农业科学院茶叶研究所,福建 福安 355015
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
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摘要:

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    
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 words: tea    organic acid    high performance liquid chromatography    diode array detector
收稿日期: 2017-12-19 出版日期: 2019-03-28
CLC:  TS 272  
基金资助: 福建省属公益类科研院所专项(2015R1012-3);福建省自然科学基金(2016J01121,2017J01042)
通讯作者: 陈林     E-mail: 875720551@qq.com;82785676@qq.com
作者简介: 王丽丽https://orcid.org/0000-0002-7433-1844)|陈林(https://orcid.org/0000-0002-4208-421X),Tel: +86-593-2210997
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引用本文:

王丽丽,杨军国,林清霞,项丽慧,宋振硕,张应根,陈林. 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[J]. 浙江大学学报(农业与生命科学版), 2019, 45(1): 47-53.

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.

链接本文:

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

图1  10种有机酸的紫外光谱扫描图
图2  流动A相的不同pH值对10种有机酸保留时间的影响
图3  序列进样下茶样中10种有机酸的HPLC图
图4  不同柱温对10种有机酸保留时间的影响
图5  不同流速对10种有机酸保留时间的影响
图6  在适宜色谱条件下10种有机酸的HPLC图

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
表1  标准品的回归方程

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
表2  精密度、重复性、稳定性与回收率试验
图7  绿茶样中10种有机酸的HPLC图

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
表3  茶样中10种有机酸质量分数
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