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Citrus are among the most relevant sources of vitamin C (ascorbic acid + dehydroascorbic acid). Recent studies have revealed that it increases in the peel as fruit ripens and remains constant or even decreases in the pulp tissue. Moreover, important differences on ascorbic acid content exist among citrus varieties in both tissues. Here we describe a simple method for vitamin C analysis/quantification in the peel and pulp tissues of citrus fruit.

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Citrus Fruit Ascorbic Acid Extraction and Quantification by HPLC
柑橘类水果中的抗坏血酸提取及高效液相色谱法测定

植物科学 > 植物生物化学 > 其它化合物
作者: Enriqueta Alós
Enriqueta AlósAffiliation: Postharvest Laboratory, Food Science Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain
Bio-protocol author page: a2042
Joanna Lado
Joanna LadoAffiliation: Postharvest Laboratory, Food Science Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain
Bio-protocol author page: a2043
María Jesús Rodrigo
María Jesús RodrigoAffiliation: Postharvest Laboratory, Food Science Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain
Bio-protocol author page: a2044
 and Lorenzo Zacarías
Lorenzo ZacaríasAffiliation: Postharvest Laboratory, Food Science Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain
For correspondence: lzacarias@iata.csic.es
Bio-protocol author page: a2045
Vol 5, Iss 5, 3/5/2015, 4121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1416

[Abstract] Citrus are among the most relevant sources of vitamin C (ascorbic acid + dehydroascorbic acid). Recent studies have revealed that it increases in the peel as fruit ripens and remains constant or even decreases in the pulp tissue. Moreover, important differences on ascorbic acid content exist among citrus varieties in both tissues. Here we describe a simple method for vitamin C analysis/quantification in the peel and pulp tissues of citrus fruit.
Keywords: Citrus(柑橘), Ascorbic acid(抗坏血酸), Vitamin C(维生素C), HPLC(高效液相色谱法), Dehydroascorbic acid(脱氢抗坏血酸)

[Abstract]

Materials and Reagents

  1. Citrus fruit tissue (ground frozen flavedo or pulp tissue)
  2. Metaphosphoric acid (MPA) (65%) (Sigma-Adrich, catalog number: 79615 )
  3. Orthophosphoric acid (Sigma-Adrich, catalog number: W290017 )
  4. Tris base (Sigma-Adrich, catalog number: T1503 )
  5. Methanol HPLC-grade (Sigma-Adrich, catalog number: 34860 )
  6. Ascorbic acid (Sigma-Adrich, catalog number: A902902 )
  7. C18 Sep Pak (Waters, catalog number: WAT091139 )
  8. DTT (Sigma-Aldrich, catalog number: D0632 )
  9. Sterile Mili-Q water
  10. 0.1% MPA (see Recipes)
  11. 2% MPA (see Recipes)
  12. 8.5% orthophosphoric acid (see Recipes)
  13. 400 mM Tris base (see Recipes)
  14. 200 mM DTT in 400 mM Tris base (see Recipes)
  15. Methanol:Milli-Q water (15:85, v/v) (see Recipes)
  16. Ascorbic acid stock solution I for standard curve (see Recipes)

Equipment

  1. Disposable 15 ml plastic tubes
  2. 0.45 μm nylon filter (25 mm diameter) (Análisis Vínicos, catalog number: E0036 )
  3. Refrigerated centrifuge (15 ml tubes)
  4. Polytron (Kinematica AG, model: PT-1035 GT; http://www.kinematica.ch/en.html)
  5. HPLC system with a photodiode array detector (PDA, Dionex)
  6. Ultrabase C18 column (100 x 4.6 mm, 2.5 μm)

Software

  1. Chromeleon version 6.80 (Dionex)

Procedure

  1. Ascorbic acid extraction
    1. Resuspend frozen ground citrus flavedo or pulp tissue (0.5 g) in 4 ml 0.1% MPA in a 15 ml disposable plastic tube and keep it on ice. The frozen tissue was previously ground using mortar and pestle chilled with liquid nitrogen.
    2. Homogenize the tissue for 1 min using a Polytron homogenizer at maximum speed (30,000 rpm).
    3. Centrifuge the homogenate for 10 min at 4,000 x g at 4 °C.
    4. Collect the supernatant and keep it in a fresh 15 ml disposable plastic tube on ice.
    5. Activate the Sep Pak C18 cartridges using 4 ml methanol and subsequently wash them with 4 ml Milli-Q water and 4 ml 2% MPA. Discard the solution from the washing steps.
    6. For the ascorbic acid determination in flavedo take 1 ml of supernatant and mix it with 3 ml 0.1% MPA (1:4 dilution). For ascorbic acid determination in pulp do not dilute it, just continue with the protocol using the whole supernatant.
    7. Filter the solutions obtained in step A4 (pulp) or A6 (flavedo) through a previously activated C18 Sep Pak cartridge using a syringe. Collect the filtrate in a fresh 15 ml disposable plastic tube.
    8. Wash the C18 Sep Pak cartridge with 4 ml 2% MPA. Collect the eluate together with the solution from step A7.
    9. Filter the solution from step A8 through a 0.45 μm nylon filter using a syringe. This step is added to make sure the solution is clean of any particles that could damage the HPLC column.
    10. Wash the filter with 4 ml 2% MPA and collect it together with the filtrate solution from step A9.
    11. Measure the final volume and take it into account together with the initial dilution (in the case of flavedo samples) for the calculations of ascorbic acid concentration.
    12. Prepare an amber HPLC vial with 1 ml of the final extract (from step A10) for the ascorbic acid measurement.

  2. Dehydroascorbic acid extraction
    Dehydroascorbic acid is converted to ascorbic acid by the addition of DTT, then the amount of total ascorbic acid (dehydroascorbic acid + ascorbic acid) is measured and the dehydroascorbic acid concentration results from the substraction of ascorbic acid to total ascorbic acid (see step D3).
    1. Transfer a 200 μl aliquot of the final extract (step A10) to an amber HPLC vial.
    2. Incubate it for 15 min at room temperature with 100 μl of 200 mM DTT in 400 mM Tris base (pH 8.5).
    3. Stop the reaction by acidification with 100 μl of 8.5 % orthophosphoric acid.

  3. Ascorbic acid and dehydroascorbic acid HPLC measurements
    1. To quantify the ascorbic acid concentration inject 10 μl of the solution obtained from step A10 in a HPLC equipped with a photodiode array detector and an Ultrabase C18 column (100 x 4.6 mm, 2.5 μm). Set column temperature at 35 °C. Use an isocratic mobile phase of methanol: Milli-Q water (pH 2.5) (15:85, v/v, pH adjusted with MPA) at 0.2 ml/min flow.
    2. Quantify the area under the peak that displays a maximum at 248 nm (ascorbic acid absorbs between 210 and 300 nm with a maximum at 248 nm). This area is applied to the formula described in D2 to calculate the ascorbic acid concentration of the sample. Measure the spectrum between 200 and 450 nm with a PDA detector to confirm the quantification of pure ascorbic acid.
    3. To obtain the dehydroascorbic acid concentration inject 10 μl of the solution obtained from step B3 using the same equipment and conditions described in step C1.
    4. Quantify the area under the peak that displays a maximum at 248 nm. Apply this area to the formula described in D3 to calculate the dehydroascorbic acid concentration of the sample. Measure the spectrum between 200 and 450 nm with a PDA detector to confirm the quantification of pure ascorbic acid.

  4. Ascorbic acid and dehydroascorbic acid quantification
    1. Prepare a calibration curve using a series of ascorbic acid dilutions as described in Table 1 and measure the peak area following the same procedure as described in section C.
    2. Adjust the linear function of ascorbic acid concentration vs. peak area and use the slope to calculate the concentration of the unknown samples as described in the formula:

    3. Dehydorascorbic acid concentration:

      The x2 is included in the formula because it is the dilution factor due to the addition of 200 μls of extra volume in steps B2-3.

Recipes

  1. 0.1% MPA
    1.5 g of MPA 65%
    900 ml of Milli-Q water and after completely dissolved complete to 1,000 ml with Milli-Q water
    Solution can be stored at room temperature indefinitely
  2. 2% MPA
    31 g of MPA 65%
    900 ml of Milli-Q water and after completely dissolved complete to 1,000 ml with Milli-Q water
    Solution can be stored at room temperature indefinitely
  3. 8.5% orthophosphoric acid
    10 ml of 85% orthophosphoric acid
    90 ml of Milli-Q water and store at room temperature indefinitely
  4. 400 mM Tris base (pH 9.0)
    Dissolve 12.1 g of Tris base in 200 ml of Milli-Q water
    Add Milli-Q water to a final volume of 250 ml, stored at 4 °C for three months
  5. 200 mM DTT in 400 mM Tris base (pH 8.5)
    Dissolve 0.031 g of DTT in 1 ml of 400 mM Tris base solution and vortex
    Do not store, this solution must be prepared fresh daily
  6. Methanol:Milli-Q water (pH 2.5) (15:85, v/v)
    Mix 850 ml of methanol HPLC grade with 150 ml of Milli-Q water at pH 2.5 (pH is adjusted with 2% MPA)
  7. Ascorbic acid stock solution I for standard curve (100 mg/ml in 2% MPA)

    Table 1. Calibration curve preparation
    Ascorbic acid solution
    Final concentration
    Composition
    Stock solution I
    100 mg/ml
    100 mg ascorbic acid + 1 ml 2% MPA
    Stock solution II
    1 mg/ml
    990 μl MPA 2% + 10 μl stock solution I
    A
    100 μg/ml
    900 μl MPA 2% + 100 μl stock solution II
    B
    50 μg/ml
    500 μl MPA 2% + 500 μl solution A
    C
    25 μg/ml
    500 μl MPA 2% + 500 μl solution B
    D
    12.5 μg/ml
    500 μl MPA 2% + 500 μl solution C
    E
    6.25 μg/ml
    500 μl MPA 2% + 500 μl solution D
    F
    3.125 μg/ml
    500 μl MPA 2% + 500 μl solution E
    G
    1.5625 μg/ml
    500 μl MPA 2% + 500 μl solution F
    H
    0 μg/ml
    500 μl MPA 2%

Acknowledgments

This work was supported by research grants FP7-PEOPLE-2011-CIG-2011-303652 (Marie Curie Actions, European Union), Proyectos de I + D para Grupos de Investigación Emergentes GV/2012/036 (Generalitat Valenciana, Spain). Enriqueta Alós was recipient of a JAE-Doc (CSIC) post-doctoral contract which is co-funded by Fondo Social Europeo de Desarrollo Regional (FEDER). This protocol is based on the methodology used in the manuscript Alós et at. (2014).

References

  1. Alós, E., Rodrigo, M. J. and Zacarías, L. (2014). Differential transcriptional regulation of L-ascorbic acid content in peel and pulp of citrus fruits during development and maturation. Planta 239(5): 1113-1128.

材料和试剂

  1. 柑橘类水果组织(地面冷冻的黄油或纸浆组织)
  2. 偏磷酸(MPA)(65%)(Sigma-Adrich,目录号:79615)
  3. 正磷酸(Sigma-Adrich,目录号:W290017)
  4. Tris碱(Sigma-Adrich,目录号:T1503)
  5. 甲醇HPLC级(Sigma-Adrich,目录号:34860)
  6. 抗坏血酸(Sigma-Adrich,目录号:A902902)
  7. C18 Sep Pak(Waters,目录号:WAT091139)
  8. DTT(Sigma-Aldrich,目录号:D0632)
  9. 无菌Mili-Q水
  10. 0.1%MPA(参见配方)
  11. 2%MPA(参见配方)
  12. 8.5%正磷酸(见配方)
  13. 400 mM Tris碱(见配方)
  14. 200mM DTT在400mM Tris碱(见Recipes)中
  15. 甲醇:Milli-Q水(15:85,v/v)(参见配方)
  16. 标准曲线的抗坏血酸储备溶液I(参见配方)

设备

  1. 一次性使用15 ml塑料管
  2. 0.45μm尼龙过滤器(25mm直径)(AnálisisVínicos,目录号:E0036)
  3. 冷冻离心机(15 ml管)
  4. Polytron(Kinematica AG,型号:PT-1035 GT; http://www.kinematica.ch/en.html
  5. 具有光电二极管阵列检测器(PDA,Dionex)的HPLC系统
  6. Ultrabase C 18柱(100×4.6mm,2.5μm)

软件

  1. Chromeleon版本6.80(Dionex)

程序

  1. 抗坏血酸提取
    1. 重悬在4毫升的冷冻的地面柑橘flavedo或纸浆组织(0.5克) 0.1%MPA在15ml一次性塑料管中并保持在冰上。 的 冷冻的组织先前用研钵和杵捣碎 液氮。
    2. 使用Polytron匀浆器以最大速度(30,000rpm)将组织匀浆1分钟
    3. 在4℃下以4,000×g离心匀浆10分钟
    4. 收集上清液,并保存在新鲜的15毫升一次性塑料管在冰上
    5. 使用4 ml甲醇激活Sep Pak C18柱 随后用4ml Milli-Q水和4ml 2%MPA洗涤它们。 丢弃 从洗涤步骤的解决方案
    6. 对于抗坏血酸 在flavedo中的测定取1ml上清液并将其与3ml混合 0.1%MPA(1:4稀释)。 对于抗坏血酸在纸浆中的测定没有 稀释,只要继续使用全部上清液的协议
    7. 过滤在步骤A4(纸浆)或A6(淡黄色)中获得的溶液, 通过使用注射器预先活化的C18 Sep Pak柱。 将滤液收集在新鲜的15ml一次性塑料管中
    8. 用4 ml 2%MPA洗涤C18 Sep Pak柱。 将洗脱液与来自步骤A7的溶液一起收集
    9. 通过0.45μm尼龙过滤器过滤步骤A8的溶液 使用注射器。 添加此步骤以确保解决方案是干净的 的任何可能损坏HPLC色谱柱的颗粒
    10. 用4ml 2%MPA洗涤过滤器,并与步骤A9的滤液一起收集
    11. 测量最终体积,并将其与   初始稀释(在flavedo样品的情况下)用于计算 的抗坏血酸浓度
    12. 用1ml最终提取物(来自步骤A10)制备琥珀色HPLC小瓶用于抗坏血酸测量。

  2. 脱氢抗坏血酸提取
    通过加入DTT将脱氢抗坏血酸转化成抗坏血酸,然后测量总抗坏血酸(脱氢抗坏血酸+抗坏血酸)的量,并且从抗坏血酸减去总抗坏血酸得到脱氢抗坏血酸浓度(参见步骤D3) 。
    1. 将200μl等分试样的最终提取物(步骤A10)转移到琥珀色HPLC小瓶中
    2. 在室温下用100μl200mM DTT在400mM Tris碱(pH8.5)中孵育15分钟。
    3. 通过用100μl的8.5%正磷酸酸化停止反应。

  3. 抗坏血酸和去氢抗坏血酸HPLC测量
    1. 为了量化抗坏血酸浓度,注入10μl的溶液   在装备有光电二极管阵列的HPLC中从步骤A10获得 检测器和Ultrabase C 18柱(100×4.6mm,2.5μm)。 设置列 温度在35℃。 使用甲醇的等度流动相:Milli-Q   水(pH 2.5)(15:85,v/v,用MPA调节的pH),流速为0.2ml/min。
    2. 量化在248 nm处显示最大值的峰下面积 (抗坏血酸吸收在210和300nm之间,最大在248nm)。   该区域应用于在D2中描述的公式以计算 抗坏血酸浓度。 测量之间的光谱 200和450 nm,用PDA检测器确认纯的定量   抗坏血酸
    3. 以获得脱氢抗坏血酸浓度 使用其注射10μl从步骤B3获得的溶液 设备和条件在步骤C1中描述
    4. 量化区域 在248nm处显示最大值的峰下。 应用此区域到   公式描述在D3中以计算脱氢抗坏血酸 样品的浓度。 测量200和450 nm之间的光谱   用PDA检测器确认纯抗坏血酸的定量 酸。

  4. 抗坏血酸和脱氢抗坏血酸定量
    1. 使用一系列抗坏血酸稀释液制备校准曲线   并测量跟在表1中的峰面积 程序,如第C节所述。
    2. 调整线性函数 的抗坏血酸浓度对峰面积,并使用斜率 计算未知样品的浓度 公式:

    3. 脱氢抗坏血酸浓度:

      x2是 包括在公式中,因为它是由于稀释因子 在步骤B2-3中添加200μl的额外体积。

食谱

  1. 0.1%MPA
    1.5g MPA 65%
    900毫升Milli-Q水,并在用Milli-Q水完全溶解后达到1000毫升 溶液可以无限期储存在室温下。
  2. 2%MPA
    31克MPA 65%
    900毫升Milli-Q水,并在用Milli-Q水完全溶解后达到1000毫升 溶液可以无限期储存在室温下。
  3. 8.5%正磷酸 10ml 85%的正磷酸 90毫升Milli-Q水,并无限期储存在室温下
  4. 400mM Tris碱(pH9.0) 将12.1g Tris碱溶解在200ml Milli-Q水中
    加入Milli-Q水至最终体积为250ml,在4℃保存三个月。
  5. 200mM DTT的400mM Tris碱(pH8.5)中 将0.031g DTT溶解在1ml 400mM Tris碱溶液中并涡旋
    不要储存,此溶液必须每天新鲜准备
  6. 甲醇:Milli-Q水(pH 2.5)(15:85,v/v)
    将850ml甲醇HPLC级与150ml pH2.5的Milli-Q水混合(用2%MPA调节pH)
  7. 标准曲线的抗坏血酸储备溶液I(100mg/ml,在2%MPA中)
    表1.校准曲线准备
    抗坏血酸溶液
    最终浓度
    组合
    库存解决方案
    100 mg/ml
    100mg抗坏血酸+ 1ml 2%MPA
    库存解决方案II
    1 mg/ml
    990μlMPA 2%+10μl储备溶液I
    A
    100μg/ml
    900μlMPA 2%+ 100μl储备液II
    B
    50μg/ml
    500μlMPA 2%+500μl溶液A
    C
    25μg/ml
    500μlMPA 2%+500μl溶液B
    D
    12.5μg/ml
    500μlMPA 2%+500μl溶液C
    E
    6.25μg/ml
    500μlMPA 2%+500μl溶液D
    F
    3.125μg/ml
    500μlMPA 2%+500μl溶液E
    G
    1.5625μg/ml
    500μlMPA 2%+500μl溶液F
    H
    0μg/ml
    500μlMPA 2%

致谢

这项工作得到研究补助FP7-PEOPLE-2011-CIG-2011-303652(Marie Curie Actions,European Union),Proyectos de I + D para Grupos deInvestigaciónEmergentes GV/2012/036(Generalitat Valenciana,Spain)的支持。 EnriquetaAlós获得由Fondo Social Europeo de Desarrollo Regional(FEDER)共同资助的JAE-Doc(CSIC)博士后合同。 这个协议是基于手稿Alós等人在(2014)上使用的方法。

参考文献

  1. Alós,E.,Rodrigo,M. J.和Zacarías,L.(2014年)。 柑橘类水果皮和果肉中L-抗坏血酸含量的差异转录调控 在发育和成熟期间。 239(5):1113-1128。
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How to cite this protocol: Alós, E., Lado, J., Rodrigo, M. J. and Zacarías, L. (2015). Citrus Fruit Ascorbic Acid Extraction and Quantification by HPLC . Bio-protocol 5(5): e1416. DOI: 10.21769/BioProtoc.1416; Full Text



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