摘要
【目的】建立超高效液相色谱快速、同时测定柑橘中主要酚酸和类黄酮组成及含量的方法,为柑橘中酚类物质的开发提供技术支撑。【方法】首先对上机条件优化,检测波长选择基于全扫描(190—400 nm),选择所有物质都有最大吸收光谱的波长;柱温、流动相类型和流速优化参考相关文献,为了能使基线分离,采用梯度洗脱方式;再从提取剂类型、提取次数和时间进行单因素比较试验,对柑橘样品处理方法进行优化;柑橘样品经乙酸乙酯振荡提取30 min,果皮和果肉分别提取4次和3次,蒸发浓缩,甲醇定容上机进行测定。【结果】以ACQUITY UPLC BEH C18液相色谱柱(2.1 mm×100 mm,1.7μm)为分离柱,柱温为35℃,进样量为3.0μL,流速为0.3 m L·min-1;梯度洗脱,以0.3%乙酸水溶液(A)/甲醇(B)为流动相:95%—80%(0—3 min)A,80%—80%(3—8 min)A,80%—70%(8—12 min)A,70%—20%(12—17 min)A,20%—95%(17—20 min)A;定量波长为283 nm。19种物质在18 min内基线分离,线性范围为0.01—500 mg·L-1,线性相关系数均大于0.999,精密度、重复性和稳定性良好(相对标准偏差小于5%)。两个水平下加标,果皮回收率为85.8%—109.4%(相对标准偏差为0.86%—6.06%),果肉回收率为88.4%—112.7%(相对标准偏差为1.05%—5.23%),方法检出限为0.001—0.09 mg·kg-1(S/N=3)。采用此方法对实际样品进行检测,样品包括5大类柑橘:宽皮柑橘类(沙糖橘和椪柑)、甜橙类(纽荷尔脐橙)、柚类葡萄柚类(鸡尾葡萄柚)、枸橼柠檬类(柠檬)和金柑类。不同品种类黄酮和酚酸物质含量和种类差异较大,就总酚类物质而言,椪柑和鸡尾葡萄柚果皮中最高,椪柑是金柑的5倍,是其他品种的1—1.5倍,果皮为果肉的3—5倍;就类黄酮物质而言,鸡尾葡萄柚果皮中总含量最高(1 813.22 mg·kg-1),其次是椪柑、纽荷尔脐橙、沙糖橘、柠檬、金柑,果肉总含量变化趋势与果皮一致,果皮明显高于果肉;黄烷酮(圣枸橼苷、柚皮苷、橙皮苷、新橙皮甙、香蜂草甙、柚皮芸香甙、柚皮素和橙皮素)是柑橘中主要的类黄酮,鸡尾葡萄柚果皮中最多(1 491.8mg·kg-1),其次是柠檬和纽荷尔脐橙,最少的是金柑,而果肉含量明显低于果皮;就酚酸而言,沙糖橘果皮中最多(515.21 mg·kg-1),其次是椪柑、柠檬、鸡尾葡萄柚和纽荷尔脐橙,金柑最少,果肉中变化趋势与果皮一致,果皮含量为果肉的3—5倍;绿原酸,阿魏酸是主要的酚酸。【结论】利用超高效液相色谱仪,建立了同时快速检测柑橘中主要酚酸和类黄酮物质的方法。该方法高效、精确、低耗、环保,并经实际样品(5大类柑橘)验证,表明该方法可作为柑橘中酚酸和类黄酮同时、快速检测的常规分析方法。
[Objective] The fast method based on ultra performance liquid chromatograph （UPLC） for the analysis of main phenolic acids and flavonoids in citrus was developed in order to provide technological supports for exploitation of phenolic substance of citrus. [Method] First, the instrument condition optimized the detection wavelength selection based on full-scan （190-400 nm）, all material have maximum absorption spectrum at around 283 nm. The optimization of column temperature, mobile phase and flow rate refer to the relevant literature. In order to make the baseline separation using a gradient elution; to optimize the citrus sample processing method, the extracting agent type, extraction times and time comparing by single-factor comparison test. Then citrus samples were extracted with ethyl acetate for 30 min, evaporated and concentrated. The residue was redissolved in methanol for UPLC analysis, peel samples were extracted for 3 times and pulp for 4 times. [Result] The chromatographic separation was carried on a ACQUITY UPLC BEH C 18 （2.1 mm×100 mm, 1.7μm） in gradient elution with a mobile phase of acetic acid aqueous solution （0.3%, w/w） （A） and methanol （B） as follows： 95%-80% （0-3 min）B, 80%-80% （3-8 min）B, 80%-70% （8-12 min）B, 70%-20% （12-17 min）B and 20%-95% （17-20 min）B. The column temperature was 35~C, the injection volume was 3 μL and the flow rate was 0.3 mL·min^-1. DAD spectra was recorded and 283 nm was the detection wavelength of phenolic compounds. A total of 19 analytes were separated to the baseline within 18 min with the correlation coefficients higher than 0.999 and linear range from 0.01 to 500 mg·L^-1. The precision, reproducibility and stability were satisfactory with the relative standard deviations （RSDs） less than 5%. Peel samples were extracted for 3 times and pulp for 4 times. Recoveries achieved from the spiked peel and pulp at two concentrations ranged from 85.8% to 109.4% （RSDs, 0.86%-6.06%） and 88.4% to 112.7% （RSDs, 1.05%-5.23%）, respectively. The limits of detection （LODs, S/N=3） ranged from 0.001 to 0.09 mg·kg^-1. The proposed method was applied to the determination of phenolic compounds in citrus samples including five categories, which were mandarin orange （sugar orange and ponkan）, sweet orange （Newhallnavel orange）, pomelo grapefruit （cocktail）, citron lemon （lemon） and kumquat. There were different flavonoids and phenolic acids and their different contents in various types of citrus. The total contents of the phenolic compounds in ponkan and cocktail grapefruit peels were the highest, ponkan was about 1.5 times of other species, 5 times of kumquat and the peel was about 3-5 times of the pulp. The content of ,he flavonoids in cocktail peel with 1 813.22 mg.kg1 was the highest, followed by ponkan, Newhallnavel orange, sugar orange, lemon and kumquat. The variation trend of the flavonoid content in the pulp was similar to the peel with the peel significantly higher than the pulp. Flavanones including eriocitrin, naringin, hesperidin, neohesperidin, didymin, narirutin, naringenin and hesperetin are the main flavonoids in citrus. The content of the flavanones in cocktail peel with 1 491.8 mg＇kg1 was the highest, followed by lemon and Newhallnavel orange and kumquat was the least with the pulp significantly lower than the peel. The total content of the phenolic acids in sugar orange peel with 515.21 mg·kg^-1 was the highest, followed by ponkan, lemon, cocktail and Newhall navel orange and kumquat was the least. The phenolic acid contents of the pulp and peel had a consistent trend and the content of peel was about 3-5 times of pulp. Chlorogenic and ferulic acids were the main acid in citrus.[ Conclusion ] A rapid method for the simultaneous detection of the major phenolic and flavonoid substances by UPLC was established. This method was efficient, accurate, low-loss, environment friendly and authenticated by the actual citrus samples including five categories. The flavonoids and phenolic acids were detected in different varieties of citrus and their contents and types. This method can be used for the conventional analysis of the fast and simultaneous detection of flavonoids and phenolic acids in citrus.
出处
《中国农业科学》
CAS
CSCD
北大核心
2014年第23期4706-4717,共12页
Scientia Agricultura Sinica
基金
"十一五"国家科技支撑计划(2007BAD47B07)
农业部现代农业(柑桔)产业技术体系建设专项(CARS-27)
重庆市自然科学基金重点项目(cstc2013jj B80009)
重庆市自然科学基金(一般)项目(cstc2013jcyj A80004)
关键词
超高效液相色谱
柑橘
同时测定
酚酸
类黄酮
ultra performance liquid chromatography
citrus
simultaneous determination
phenolic acids
flavonoids
