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Mar 2020

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Chromatographic Analysis for Targeted Metabolomics of Antioxidant and Flavor-Related Metabolites in Tomato
番茄抗氧化剂和风味相关代谢产物靶向代谢组学的色谱分析   

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Abstract

Targeted metabolomics is a useful approach to evaluate crop breeding studies. Antioxidant and flavor-related traits are of increasing interest and are considered quality traits in tomato breeding. The present study presents chromatographic methods to study antioxidants (carotenoids, vitamin C, vitamin E, phenolic compounds, and glutathione) and flavor-related characters (sugars and organic acids) in tomato. Two different extraction methods (for polar and apolar entities) were applied to isolate the targeted compounds. The extraction methods developed in this work were time and cost-effective since no further purification was needed. Carotenoids, vitamin C, glutathione, and phenolic acids were analyzed by HPLC-PDA using a RP C18 column at an appropriate wavelength for each compound. Vitamin E and sugars were analyzed by HPLC with RP C18 and NH2 columns and detected by FLD and RI detectors, respectively. In addition, organic acids were analyzed with GC-FID using a Rtx 5DA column after derivatization with MSTFA. As a result, sensitive analytical methods to quantify important plant metabolites were developed and are described herein. These methods are not only applicable in tomato but are also useful to characterize other species for flavor-related and antioxidant compounds. Thus, these protocols can be used to guide selection in crop breeding.

Keywords: HPLC (高效液相色谱法), GC (气相色谱), Carotenoids (类胡萝卜素), Vitamins (维生素), Phenolic compounds (酚类化合物), Glutathione (谷胱甘肽), Sugars (糖类), Organic acids (有机酸)

Background

Metabolomics is an applied biochemical approach which has gained attention for its potential to aid crop breeding studies. In tomato breeding, both antioxidant and flavor-related metabolites are of increasing interest because of consumer preferences for improved crop quality. Humans cannot synthesize antioxidant molecules themselves; therefore, these molecules must be provided by the external daily diet (Lobo et al., 2010). Flavor is a complex trait that also affects the consumer marketability of tomato (Kader, 2008). Thus, the quantification of these traits is important. Because the metabolome is complex and consists of a wide variety of compounds including lipid-soluble metabolites, aqueous polar metabolites, stable and unstable metabolites, as well as acidic and basic metabolites, many methods for both extraction and detection of plant metabolites are available. These methods encompass a range of different chromatographic techniques with different extraction methods depending on the type of metabolite. In the literature, different techniques including ultrasonication (Tan et al., 2021), supercritical CO2 extraction (Pellicanò et al., 2020), water-induced hydrocolloidal complexation (Nagarajan et al., 2020), and solid phase extraction (Figueira et al., 2017) have been used to extract antioxidant molecules or flavor-related metabolites. Most of these extraction methods include many steps, sometimes require special chemicals and equipment, or entail pre-purification procedures. As a result, many of these methods are expensive and time consuming. Similarly, different chromatographic techniques have been used to quantify metabolites. The most commonly used method for both targeted (Dumont et al., 2020, Tohge et al., 2020) and untargeted metabolic profiling (Capanoglu et al., 2008, Treutler et al., 2016) is mass spectrophotometry coupled with either liquid or gas chromatography. The nuclear magnetic resonance technique (NMR) can also be used for metabolomics (Ingallina et al., 2020, Masetti et al., 2020). These methods not only require very expensive equipment but also require expertise. An alternative is the use of spectrophotometric methods which can be applied to evaluate lycopene content (Migalatev, 2017), total antioxidant capacity (Martínez et al., 2020), total phenolic acids and flavonoid content (Alenazi et al., 2020). Although spectrophotometric methods are easy and cheap, they are not as sensitive as chromatographic methods. Moreover, quantification of individual molecules such as specific phenolic acids or sugars is not feasible.

Given the limitations of the available extraction and analysis protocols, the current work was designed to establish rapid, easy and relatively cheap extraction methods for two groups of compounds, polar and apolar metabolites. In addition, cheap, easy and sensitive chromatographic methods (HPLC and GC) were developed to detect and quantify the different types of antioxidant and flavor-related metabolites. Apolar extracts were used for analysis of carotenoids with a HPLC method modified from those described by Ishida et al. (2001) and Serino et al. (2009). Polar extracts were used for the analysis of vitamin C, vitamin E, phenolic acids, reduced and oxidized glutathione, and sugars using HPLC. The HPLC methods for these compounds were modified from those described in previous studies for vitamin C (Li and Chen, 2001a and 2001b), vitamin E (Turner and Burri, 2012; Bakre et al., 2015), phenolic acids (Gómez-Alonso et al., 2007), glutathione (Khan et al., 2011), and sugars (Petkova et al., 2013). Organic acids were also detected from polar extracts using a GC method modified from those described in previous studies (Roessner et al., 2001; Namgung et al., 2010). All protocols were developed using fully ripe fruits (at market stage) from a tomato inbred backcross line population as samples. This interspecific population was derived from S. lycopersicum by S. pimpinellifolium cross (Celik et al., 2017) and provided sufficient variation for the metabolites of interest to test the new protocols.

¥¥¥¥¥¥¥¥¥¥ Materials and Reagents

  1. Plant Material

    An interspecific IBL (inbred backcross line) population (BC2F6) derived from the cross S. lycopersicum cv. Tueza (recurrent parent) × S. pimpinellifolium (LA1589) (donor parent) was used as plant material. This population was developed in previous studies and is described more fully in the literature (Celik et al., 2017; Gürbüz Çolak et al., 2020a and 2020b). Tueza is a cultivated fresh market tomato line with large (150-160 g), red, slightly flattened round fruits. LA1589 is a wild tomato accession with small (0.8-1 g), red, round fruits. The IBL population and parents were grown in the greenhouse in Antalya, Turkey. Ten plants per genotype were grown in double rows with 140 and 30 cm between wide and narrow rows, respectively. Plants were spaced at 40 cm intervals within rows. For basal fertilization, 500 kg 15:15:15 (N:P:K) fertilizer and 50 t of composted manure were applied per ha. Drip irrigation was used with fertigation (1.4 dS m-1 EC value) at each irrigation using 1-2-1 fertilizer until first fruit set, 2-1-1 fertilizer until first fruit ripening and 1-1-2 fertilizer after first fruit ripening.


  2. Major Ingredients

    1. C18 column (GL Sciences, catalog number: 5020-03946 )

    2. NH2 column (GL Sciences, catalog number: 5020-05546 )

    3. RTx 5DA column (Restek, catalog number: 10523 )

    4. Syringe (BD, Emerald, catalog number: 307742 )

    5. Syringe filter (Millipore, catalog number: Z227412 )

    6. Polyamide membrane filter (45 µm, 47 mm) (Sartorius, catalog number: 25006-47-N )

    7. Acetonitrile (VWR, catalog number: 97065 )

    8. Ammonium dihydrogen phosphate (Merck, catalog number: 1.01126.0500 )

    9. Butylated hydroxytoluene (BHT) (Sigma-Aldrich, catalog number: W218405 )

    10. Chloroform (VWR, catalog number: JT9175 )

    11. Dichloromethane (VWR, catalog number: BDH23373 )

    12. Ethyl acetate (VWR, catalog number: JT9282 )

    13. Hexane (VWR, catalog number: BDH24575 )

    14. Methanol (VWR, catalog number: BDH20864 )

    15. Methoxamine hydrochloride (Sigma-Aldrich, catalog number: M6524 )

    16. N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) (Sigma-Aldrich, catalog number: 69479 )

    17. Ortho-phosphoric acid (Merck, catalog number: 100573 )

    18. Potassium dihydrogen phosphate KH2PO4 (Riedel-de Haën, catalog number: 04243)

    19. Pyridine (Sigma-Aldrich, catalog number: 270970 )

    20. Triethylamine (Merck, catalog number: 8083520500 )

    21. Trifluoroacetic acid (Sigma-Aldrich, catalog number: T6508 )

    22. 0.1 M KH2PO4, pH = 7.0 (see Recipes)

    23. 0.05% Trifluoroacetic acid(aq) (see Recipes)


  3. Chemicals used as control molecules

    1. β-carotene (Carl Roth, catalog number: 5669.1 )

    2. Apigenin (Applichem, catalog number: A3641 )

    3. Caffeic acid (Sigma-Aldrich, catalog number: C0625 )

    4. Catechin (Applichem, catalog number: A4325 )

    5. Chlorogenic acid (Sigma-Aldrich, catalog number: 00500590)

    6. Chrysin (Fluka, catalog number: 9582 )

    7. Cinnamic acid (Sigma-Aldrich, catalog number: W228826 )

    8. Citric acid (Carl Roth, catalog number: X863.1 )

    9. Coumaric acid (Carl Roth, catalog number: 9906.1 )

    10. Cyanidine (Carl Roth, catalog number: 4545.1 )

    11. Epicatechin (Applichem, catalog number: A3424 )

    12. Epigallocatechin (Applichem, catalog number: A2010 )

    13. Ferulic acid (Sigma-Aldrich, catalog number: 128708 )

    14. Fructose (Sigma-Aldrich, catalog number: F0127 )

    15. Fumaric acid (Sigma-Aldrich, catalog number: 47910 )

    16. Gallic acid (Sigma-Aldrich, catalog number: 91215 )

    17. Glucose (Sigma-Aldrich, catalog number: G8270 )

    18. Hydroxybenzoic acid (Sigma-Aldrich, catalog number: W398608 )

    19. Lactic acid (Sigma-Aldrich, catalog number: L1750 )

    20. Lutein (Applichem, catalog number: A1283 )

    21. Luteolin (Applichem, catalog number: A3424 )

    22. Lycopene (Carl Roth, catalog number: 5670.1 )

    23. Malic acid (Carl Roth, catalog number: 8684.1 )

    24. Malvidin (Applichem, catalog number: A8720 )

    25. Myricetin (Carl Roth, catalog number: 6461.1 )

    26. Oxidized glutathione (Sigma-Aldrich, catalog number: G4376 )

    27. Pelargonidin (Carl Roth, catalog number: 4540.1 )

    28. Peonidin (R&D, catalog number: 0 942 )

    29. Pterostilben (Sigma-Aldrich, catalog number: P1499 )

    30. Quercetin (Applichem, catalog number: A3415 )

    31. Reduced glutathione (Sigma-Aldrich, catalog number: G4251 )

    32. Resveratrol (Sigma-Aldrich, catalog number: R5010 )

    33. Salicyclic acid (Sigma-Aldrich, catalog number: W398500 )

    34. Shikimic acid (Carl Roth, catalog number: 7305.2 )

    35. Sinapic acid (Carl Roth, catalog number: 5317.1 )

    36. Sucrose (Sigma-Aldrich, catalog number: S5016 )

    37. Syringic acid (Carl Roth, catalog number: 5361.1 )

    38. Tartaric acid (Carl Roth, catalog number: K302.1 )

    39. Vanillic acid (Carl Roth, catalog number: 3685.1 )

    40. Vitamin C (Sigma-Aldrich, catalog number: A5960 )

    41. Vitamin E (Sigma-Aldrich, catalog number: T3251 )

    42. Zeaxanthin (Carl Roth, catalog number: 5672.2 )

    43. Extraction solvent 1 (see Recipes)

    44. Extraction solvent 2: Chloroform: methanol: water (1:3:1, v:v:v) (see Recipes)

¥¥¥¥¥¥¥¥¥¥ Equipment

  1. -80 °C freezer

  2. Analytical balance (Mettler Toledo, model: AB54-S )

  3. Centrifuge (Beckman Coulter, model: Allegra X-15R )

  4. Centrifuge (Thermo Scientific, model: SL 16 )

  5. GC/FID (Shimadzu, model: GC 2010 plus ) with RTx 5DA column (0.25 mm × 0.25 mm × 30 m) (Restek; catalog number: 10523 )

  6. HPLC (Shimadzu, model: LC20-AT ; refractive index (RI) detector model: RID 10A; photodiode array (PDA) detector model: SPD M20A; fluorescence detector model: RF 20A) with C18 column (5 µm-25 × 4.6 mm) (GL Sciences, catalog number: 5020-03946 ) and NH2 column (5 µm-25 × 4.6 mm) GL Sciences; catalog number: 5020-05546 )

  7. Knife grinder (Retsch, model: GM200 )

  8. Lyophilizer (Christ, model: Epsilon 1-4 LSC )

  9. Orbital shaker (IKA, model: KS260 )

  10. Vacuum evaporator (Labconco, model: Centrivap )

  11. Vacuum filtration (Do-Chrom, model: FB01 )

¥¥¥¥¥¥¥¥¥¥ Software

  1. LC Solution (Shimadzu, https://www.shimadzu.com/an/lcms/opensolution/opensol4.html)

  2. GC Solution (Shimadzu, https://www.shimadzu.com/an/gc/advflowtech/sw-dl.html)

¥¥¥¥¥¥¥¥¥¥ Procedure

  1. Extraction of Metabolites

    1. Collect 10 ripe tomato fruits (average fresh weight was 65 g), bulk and dice. Take 100 g sample and lyophilize for two days (make sure they are completely dry). Grind the dried samples with a knife grinder and obtain a fine powder.

    2. Weigh 1 g of dried sample and add 5 ml of extraction solvent 1 (see Recipes) to extract the apolar metabolites. Extract the metabolites by shaking on an orbital shaker at 400 rpm at 18 °C overnight.

    3. Centrifuge the samples at 3,724 × g at 4 °C for 20 min. Save the supernatant and do a second overnight extraction with the pellet using the same solvent.

    4. Centrifuge the samples again and combine the extracts. Aliquot the extracts (1 ml) and store at -80 °C until the analysis.

    5. To extract the polar metabolites, use the pellet obtained in Step A4 and follow the same steps 1-4 using extraction solvent 2 (see Recipes). Aliquot the extracts (1 ml) and store at -80 °C until the analysis.

    6. See Note 1.


  2. HPLC Analysis of Metabolites

    Carotenoids

    Two different methods were used for the analysis of carotenoids: method 1 for the analysis of lycopene and β-carotene; method 2 for the analysis of lutein and zeaxanthin.


    Method 1:

    1. Prepare standard solutions of lycopene in methanol:acetone (1:1, v:v) at 1, 5, 10, 25, 50 and 100 ppm concentrations, and β-carotene in dichloromethane at 1, 5, 10, 25, 50, 100 ppm concentrations.

    2. Add 0.05% trimethylamine to ethyl acetate and acetonitrile. Filter methanol, ethyl acetate and acetonitrile through membrane filter using vacuum filtration.

    3. Use methanol:ethyl acetate:acetonitrile at 50:40:10 (v:v:v) ratio as the mobile phase for isocratic elution.

    4. Use RP column and set the column temperature at 30 °C. Set the flow rate as 1.5 ml/min.

    5. Filter the sample and the standard solutions through syringe filter. Inject 20 µl of the sample or the standard solution. Detect lycopene and β-carotene at 469 nm.

    6. See Notes 2 and 3.

    A chromatogram for 100 ppm standard molecules (Figure 1), standard curves (Figure 2) and the sample chromatogram for tomato (Figure 3) are shown.



  3. Figure 1. Chromatogram of lycopene and β-carotene standards



    Figure 2. Standard curves of the carotenoids



    Figure 3. Sample chromatogram for tomato sample showing lycopene and β-carotene peaks


    Method 2:

    1. Prepare standard solutions of lutein and zeaxanthin in dichloromethane containing 0.01% BHT at 0.5, 1, 5, 12.5, 50, 100, 200 and 300 ppm concentrations

    2. Add 0.05% trimethylamine to acetonitrile. Filter methanol and acetonitrile through membrane filter using vacuum filtration.

    3. Use methanol:acetonitrile at 90:10 (v:v) ratio as the mobile phase for isocratic elution.

    4. Use a RP column and set the column temperature at 30 °C. Set the flow rate as 1 ml/min.

    5. Filter the sample and the standard solutions through syringe filter. Inject 20 µl of the sample or the standard solution. Detect lutein and zeaxanthin at 469 nm.

    6. See Notes 2 and 3.

    Chromatogram for 50 ppm standard molecules (Figure 4), standard curves (Figure 5) and the sample chromatogram for tomato (Figure 6) are shown.



    Figure 4. Chromatogram of lutein and zeaxanthin standards



    Figure 5. Standard curves of the carotenoids



    Figure 6. Sample chromatogram for tomato with lutein and zeaxanthin peaks


    Vitamin C

    1. Prepare standard solutions of vitamin C in ultrapure water at 10, 50, 100, 150, 200 and 250 ppm concentrations.

    2. Filter methanol and phosphate buffer through membrane filter using vacuum filtration.

    3. Use methanol: KH2PO4 10:90 (v:v) ratio as the mobile phase for isocratic elution.

    4. Use RP column and set the column temperature at 40 °C. Set the flow rate as 1 ml/min.

    5. Filter the sample and the standard solutions through syringe filter. Inject 20 µl of the sample or the standard solution. Detect vitamin C at 265 nm.

    6. See Note 2.


    Chromatogram for 100 ppm standard molecule (Figure 7), standard curve (Figure 8) and the sample chromatogram for tomato (Figure 9) are shown.



    Figure 7. Chromatogram of vitamin C standard



    Figure 8. Standard curve for vitamin C



    Figure 9. Sample chromatogram for tomato with vitamin C peak


    Vitamin E

    1. Prepare standard solutions of vitamin E in acetonitrile:methanol (80:20, v:v) at 10, 50, 100, 150, 200, 250, 300 and 500 ppm concentrations.

    2. Filter methanol and acetonitrile through membrane filter using vacuum filtration.

    3. Use methanol:acetonitrile 25:75 (v:v) ratio as the mobile phase for isocratic elution.

    4. Use RP column and set the column temperature at 40 °C. Set the flow rate as 1.5 ml/min.

    5. Filter the sample and the standard solutions through a syringe filter. Inject 20 µl of the sample or the standard solution. Detect vitamin E with the fluorescence detector at 300 nm excitation and 360 nm emission.

    6. See Note 2.


    Chromatogram for 500 ppm standard molecule (Figure 10), standard curve (Figure 11) and the sample chromatogram for tomato (Figure 12) are shown.



    Figure 10. Chromatogram of vitamin E standard



    Figure 11. Standard curve of vitamin E



    Figure 12. Sample chromatogram for tomato with vitamin E peak


    Phenolic acids

    1. Prepare standard solutions of phenolic acids in methanol at 1, 5, 10, 20, 30, 40, and 50 ppm concentrations.

    2. Filter NH4H2PO4, acetonitrile and H3PO4 through membrane filter using vacuum filtration.

    3. Use NH4H2PO4 (Mobile phase A), acetonitrile (Mobile phase B), and H3PO4 (Mobile phase C) as the mobile phases for gradient elution. See Table 1 for gradient elution parameters.


      Table 1. Gradient elution parameters

      Time (min) Flow rate (ml/dk) Mobile Phase A % Mobile Phase B % Mobile Phase C %
      0 1 100 0 0
      2 1 100 0 0
      5 1 92 8 0
      17 1 0 14 86
      22 1 0 18 82
      29.5 1 0 21 70
      55 1 0 33 67
      70 1 0 50 50
      75 1 0 50 50
      78 1 20 80 0
      81 1 20 80 0
      86 1 100 0 0


    4. Use RP column and set the column temperature at 35 °C. Set the flow rate as 1 ml/min.

    5. Filter the sample and the standard solutions through a syringe filter. Inject 20 µl of the sample or the standard solution. Detect phenolic acids with PDA detector at different wavelengths. Epigallocatechin, epicatechin and chrysin are detected at 204 nm. Hydroxybenzoic acid, vanillic acid, myricetin and quercetin are detected at 254 nm. Gallic acid, catechin, syringic acid and cinnamic acid are detected at 280 nm. Chlorogenic acid, caffeic acid, coumaric acid, ferulic acid, sinapic acid, resveratrol, apigenin and pterostilben are detected at 320 nm. Luteolin is detected at 360 nm, while cyaniding, pelargonidin and peonidin are detected at 520 nm.

    6. See Note 2.


    Chromatogram for 50 ppm standard molecules (Figure 13), standard curves (Figure 14) and the sample chromatogram for tomato (Figure 15) are shown.



    Figure 13. Chromatograms of standard molecules



    Figure 14. Standard curves of phenolic acids



    Figure 15. Sample chromatogram for tomato showing peaks for phenolic acids


    Glutathione

    1. Prepare standard solutions of phenolic acids in methanol at 10, 50, 100, 150, 200, 250 ppm concentrations.

    2. Filter trifluoroacetic acid and methanol through membrane filter using vacuum filtration.

    3. Use trifluoroacetic acid:methanol (97:3, v:v) as the mobile phase for isocratic elution.

    4. Use RP column and set the column temperature at 35 °C. Set the flow rate as 0.2 ml/min.

    5. Filter the sample and the standard solutions through a syringe filter. Inject 20 µl of the sample or the standard solution. Detect oxidized and reduced form of glutathione with PDA detector at 208 nm.

    6. See Note 2.


    Chromatogram for 50 ppm standard molecules (Figure 16), standard curves (Figure 17) and the sample chromatogram for tomato (Figure 18) are given below.


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    Figure 16. Chromatogram of reduced and oxidized glutathione standards



    Figure 17. Standard curves reduced and oxidized glutathione



    Figure 18. Sample chromatogram for tomato with glutathione results


    Sugars

    1. Prepare standard solutions of sugars (glucose, fructose and sucrose) in ultrapure water at 100, 150, 200, 250, 300, 500, 750, 1,000, 1,500, 2,000, 2,500 ppm concentrations.

    2. Filter trifluoroacetic acid and methanol through membrane filter using vacuum filtration.

    3. Use acetonitrile: water (90:10, v:v) as the mobile phase for isocratic elution.

    4. Use RP column and set the column temperature at 40 °C. Set the flow rate as 1 ml/min.

    5. Filter the sample and the standard solutions through a syringe filter. Inject 20 µl of the sample or the standard solution. Detect sugars with RI detector using positive mode.

    6. See Notes 2 and 4.


    Chromatogram for 50 ppm standard molecules (Figure 19), standard curves (Figure 20) and the sample chromatogram for tomato (Figure 21) are given below.



    Figure 19. Chromatogram of glucose, fructose and sucrose standards



    Figure 20. Standard curves of sugars



    Figure 21. Sample chromatogram for tomato showing peaks for sugars. Sucrose was not detected in fully ripe fruit.


  4. GC Analysis of Metabolites

      Organic acids
    1. Weigh standard molecules and dissolve them in methoxamine hydrochloride (40 µl, 20 mg/ml in pyridine) at 0.25, 0.5, 1, 2.5, 5, 7.5, 10 mg/ml concentrations.

    2. Take 100 µl of the sample. Evaporate the organic solvent of the sample using a vacuum evaporator at 30 °C until completely dry.

    3. Dissolve the tomato sample in methoxamine hydrochloride (40 µl, 20 mg/ml in pyridine) in an ultrasonic bath for 5 min.

    4. Derivatize both the samples and standard molecules at 37 °C for 90 min.

    5. Perform the second derivatization with MSTFA. Add 60 µl MSTFA to the samples and the standard molecules, and incubate at 37 °C for 30 min.

    6. Centrifuge the samples at 25,830 × g, 5 min.

    7. Inject the supernatant into GC-FID.

    8. Analyze the organic acids on an Rtx 5DA column with a thermogradient program. Set the column temperature from 100 °C (1 min hold) to 150 °C at a rate of 5 °C min-1, from 150 °C (1 min hold) to 280 °C at a rate of 5 °C min-1, 2 min hold at the final temperature.

    9. Hold the injection port temperature at 250 °C, and detector temperature at 300 °C. Use nitrogen as the carrier gas, and adjust the split ratio 1/25.

    10. See Notes 5 and 6.

    Chromatogram for 1 mg/ml standard molecules (Figure 22), standard curves (Figure 23) and the sample chromatogram for tomato (Figure 24) are given below.



    Figure 22. Chromatogram of organic acid standards



    Figure 23. Standard curves of organic acids



    Figure 24. Sample chromatogram for tomato showing peaks for organic acids

¥¥¥¥¥¥¥¥¥¥ Data analysis

Experiments were repeated and data recorded for three replicates for control molecules. Draw standard curves using LC solution for HPLC data and GC Solution for GC data. These software programs calculate the average value of the triplicate analysis and plot the curves based on the area and concentrations automatically. Also, the software programs calculate metabolite concentrations in the samples automatically using the standard curves. Average metabolite contents of the parental lines and population are shown in Table 2.

Table 2. Mean metabolite content measured in the IBL population and parents

Metabolites S. lycopersicum S. pimpinellifolium Population
Carotenoids
Lycopene 16141.58 26733.95 16919.46
β-carotene 56.62 36.06 45.98
Lutein 3.25 5.06 3.83
Zeaxanthin 3.26 2.78 3.60
Vitamins
Vitamin C 19.11 20.17 17.84
Vitamin E 3.61 20.28 20.40
Phenolic acids
Epigallocatechin 1.92 2.19 2.61
Epicatechin 0.24 5.89 2.45
Chrysin 0.42 2.07 80.12
Hydroxybenzoic acid 6.60 1.37 46.80
Vanillic acid 12.8 2.05 61.12
Myricetin 10.00 40.00 3.06
Quercetin 1.88 1.67 1.025
Gallic acid 31.46 1.06 5.07
Catechin 0.37 0.59 26.53
Syringic acid 3.96 20.40 89.46
Cinnamic acid 0.06 0.11 0.98
Chlorogenic acid 19.20 0.83 0.53
Caffeic acid 9.54 3.32 3.99
Coumaric acid 0.24 2.13 1.87
Ferulic acid 2.08 31.00 3.29
Sinapic acid 1.47 1.29 1.60
Malvidin 0.76 0.75 3.48
Resveratrol - - -
Apigenin 2.18 1.24 13.04
Pterostilbene - - -
Luteolin 0.62 0.20 0.72
Cyanidin - - -
Pelargonidin - - -
Peonidin -
Glutathione
Reduced glutathione 17.75 10.79 52.17
Oxidized glutathione 5.18 0.09 56.14
Sugars
Glucose 8738.04 4153.29 6596.45
Fructose 8401.38 3967.70 5839.52
Sucrose - - -
Organic acids
Salicylic acid 0.01 0.02 0.03
Fumaric acid - - -
Lactic acid 0.31 0.04 0.18
Malic acid 6.19 0.86 3.58
Tartaric acid - - -
Shikimic acid 0.94 0.00 1.17
Citric acid 10.40 8.51 7.59

* Quantities are given as mg 100 g-1 DW.
** Resveratrol, pterostilbene, cyanidin, pelargonidin, peonidin, sucrose, fumaric acid and tartaric acid were not detected in the tomato samples.

¥¥¥¥¥¥¥¥¥¥ Notes

  1. It is better to do extraction in the dark and at low temperatures (+4 °C) since some metabolites are affected by light and high temperatures.

  2. It is better to centrifuge the sample (25,830 × g, 1-2 min) once more before syringe filtration to extend column life.

  3. A RP C30 column can provide better resolution but is more expensive than a RP C18 column.

  4. It is better to wait for conditioning of the RI detector before analysis. Sometimes it is better to leave the detector overnight with mobile phase at low flow rate (e.g., 0.1 ml/min-0.5 ml/min).

  5. Helium can be used instead of nitrogen as the carrier gas.

  6. Freshly prepare methoxamine hydrochloride in pyridine.

  7. Use only ultrapure water for extraction and analysis.

¥¥¥¥¥¥¥¥¥¥ Recipes

  1. Extraction solvent 1

    1. Dichloromethane: hexane (1:1, v:v) containing 0.01% BHT to avoid oxidation reactions while extracting the metabolites

    2. Measure 50 ml of dichloromethane

    3. Add 0.01 g BHT into the dichloromethane and mix until BHT dissolves

    4. Add 50 ml of hexane and shake well

  2. Extraction solvent 2: Chloroform: methanol: water (1:3:1, v:v:v)

    1. Measure 20 ml of chloroform

    2. Add 60 ml of methanol and mix well

    3. Place the solution on a shaker and add water slowly into the solution to avoid phase separation

  3. 0.1 M KH2PO4, pH = 7.0

    1. Weigh 13.609 g KH2PO4 in a beaker

    2. Add 800 ml of water and mix until KH2PO4 dissolves

    3. Bring the pH to 7.0

    4. Make up volume to 1,000 ml with water

  4. 0.05% Trifluoroacetic acid(aq)

    1. Measure 950 ml of water into a volumetric flask

    2. Add 0.5 ml of trifluoroacetic acid and bring the volume to 1,000 ml with water

¥¥¥¥¥¥¥¥¥¥ Acknowledgments

This work was supported by funds from the Scientific and Technological Research Council of Turkey (TÜBİTAK, project number 114Z116) and the Republic of Turkey Ministry of Agriculture and Forestry, General Directorate of Agricultural Research Institute (TAGEM-16/AR-GE/03). This protocol was derived from Gürbüz Çolak et al. (2020a and 2020b) manuscripts.

¥¥¥¥¥¥¥¥¥¥ Competing interests

The authors declare no financial and non-financial competing interests.

¥¥¥¥¥¥¥¥¥¥ Ethics

No human and/or animal subjects were used in these protocols.

¥¥¥¥¥¥¥¥¥¥ References

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简介

[摘要]靶向代谢麦克风是评估作物育种研究的有用方法。抗氧化剂和与风味有关的性状受到越来越多的关注,被认为是番茄育种中的品质性状。本研究提供了色谱方法来研究番茄中的抗氧化剂(类胡萝卜素,维生素C,维生素E,酚类化合物和谷胱甘肽)和与风味相关的特征(糖和有机酸)。应用了两种不同的提取方法(针对极性和非极性实体)来分离目标化合物。由于不需要进一步纯化,因此这项工作中开发的提取方法既省时又经济。使用RP C18色谱柱,通过RP-C18色谱柱在适当波长下对每种化合物进行类胡萝卜素,维生素C,谷胱甘肽和酚酸的分析。用RP C18和NH2色谱柱通过HPLC分析维生素E和糖,并分别通过FLD和RI检测器进行检测。此外,MSTFA衍生化后,使用Rtx 5DA色谱柱使用GC-FID分析有机酸。结果,开发了定量重要植物代谢物的灵敏分析方法,并在本文中进行了描述。这些方法不仅适用于番茄,但都还表征其他物种的香味相关的和抗氧化的化合物。因此,这些协议可用于指导作物育种的选择。

[背景]代谢组学是其受到关注的其潜在的援助作物育种研究施加生化途径。在番茄育种中,由于消费者对提高作物品质的偏好,抗氧化剂和与风味有关的代谢产物都受到了越来越多的关注。人类无法自己合成抗氧化剂分子; 因此,这些分子必须由外部日常饮食提供(Lobo等,2010)。风味是一个复杂的特征,它也影响了番茄的消费者可销售性(Kader,2008)。因此,这些特征的量化很重要。由于代谢组很复杂,并且由多种化合物组成,包括脂溶性代谢物,水性极性代谢物,稳定和不稳定的代谢物以及酸性和碱性代谢物,因此有许多提取和检测植物代谢物的方法都可以使用。这些方法包括一系列不同的色谱技术,取决于代谢物的类型,提取方法也不同。在文献中,不同的技术,包括超声处理(谈等人。,2021),超临界CO 2萃取(Pellican ò等人,2020年),水引起的水解胶体络合(Nagarajan等人,2020),和固相萃取( Figueira et al。,2017)已用于提取抗氧化剂分子或与风味相关的代谢产物。这些提取方法大多数都包含许多步骤,有时需要特殊的化学药品和设备,或者需要进行预纯化程序。结果,这些方法中的许多昂贵且耗时。同样,已使用不同的色谱技术对代谢物进行定量。靶向(Dumont等,2020,Tohge等,2020)和非靶向代谢谱分析(Capanoglu等,2008,Treutler等,2016)的最常用方法是质谱联用液体或光度法气相色谱法。核磁共振技术(NMR)也可用于代谢组学(Ingallina等人,2020年,Masetti等人,2020)。这些方法不仅需要非常昂贵的设备,而且还需要专业知识。一种替代方法是使用分光光度法,该方法可用于评估番茄红素含量(Migalatev ,2017),总抗氧化能力(Martínez等人,2020),总酚酸和类黄酮含量(Alenazi等人,2020)。尽管分光光度法既简便又便宜,但它们不如色谱法灵敏。此外,定量单个分子(例如特定的酚酸或糖)是不可行的。

考虑到可用提取和分析方案的局限性,当前的工作旨在为两种化合物(极性和非极性代谢物)建立快速,简便和相对便宜的提取方法。此外,开发了廉价,简便且灵敏的色谱方法(HPLC和GC)来检测和定量分析不同类型的抗氧化剂和与风味相关的代谢物。使用非极性提取物通过HPLC方法对类胡萝卜素进行分析,该方法是由Ishida等人(2002年)描述的方法改进而来的。(2001)和Serino等。(2009)。极性提取物用于通过HPLC分析维生素C,维生素E,酚酸,还原型和氧化型谷胱甘肽和糖。这些化合物的HPLC方法进行从那些在维生素C的先前研究(Li和陈述改性,2001A和2001 b)中,维生素E(Turner和布里,2012 ; Bakre学习等人,2015),酚酸(Gómez- Alonso等,2007),谷胱甘肽(Khan等,2011)和糖类(Petkova等,2013)。还使用从以前的研究中描述的方法(Roessner等,2001 ;Namgung等,2010)改进的GC方法从极性提取物中检测到有机酸。所有规程均使用来自番茄自交回交系群体的完全成熟的果实(在市场阶段)开发的,作为样品。该种间种群通过S. pimpinellifolium cross(Celik et al。,2017)衍生自lycopersicum (S . lycopersicum)(Celik et al。,2017),并为感兴趣的代谢产物提供了足够的变异来测试新方案。

关键字:高效液相色谱法, 气相色谱, 类胡萝卜素, 维生素, 酚类化合物, 谷胱甘肽, 糖类, 有机酸

材料和试剂
植物材料
 种间的IBL(近交回交系)种群(BC 2 F 6 ),来自杂交S. lycopersicum cv。将Tueza(轮回亲本)× P . pimpinellifolium (LA1589)(供体亲本)用作植物材料。该种群在先前的研究中得到发展,并在文献中有更完整的描述(Celik等,2017;GürbüzÇolak等,2020a和2020b )。Tueza是栽培新鲜市场番茄符合大(150 - 160克),红,稍扁平圆水果。LA1589是具有小野生番茄加入(0.8 - 1克),红色,圆形水果。IBL人口和父母都是在土耳其安塔利亚的温室中种植的。每个基因型十株植物分别在宽行和窄行之间分别长140 cm和30 cm的双行中生长。在行中以40cm的间隔将植物间隔开。对于基础施肥,每公顷施用500 kg 15:15:15(N:P:K)肥料和50 t堆肥。在每次灌溉中使用滴灌施肥(1.4 dS m-1 EC值),每次灌溉使用1-2-1肥料直至结实,2-1-1肥料直到果实成熟,1-1-2肥料后成熟。


主要成分
C18柱(GL Sciences ,目录号:5020-03946)
NH2柱(GL Sciences ,目录号:5020-05546)
RTx 5DA色谱柱(Restek ,目录号:10523)
注射器(BD ,翡翠,目录号:307742)
注射器过滤器(Millipo re,目录号:Z227412)
聚酰胺膜滤器(45 µm,47 mm)(S artorius ,目录号:25006-47-N)
乙腈(VWR ,目录号:97065)
磷酸二氢铵(Merck ,目录号:1.01126.0500)
丁羟甲苯(BHT)(Sigma - Aldrich ,目录号:W218405)
氯仿(VWR ,货号:JT9175)
二氯甲烷(VWR ,目录号:BDH23373)
乙酸乙酯(VWR ,目录号:JT9282)
己烷(VWR ,目录号:BDH24575)
甲醇(VWR ,目录号:BDH20864)
盐酸甲氧明(Sigma - Aldrich ,目录号:M6524)
N-甲基-N-(三甲基甲硅烷基)三氟乙酰胺(MSTFA)(Sigma - Aldrich ,目录号:69479)
正磷酸(Merck ,目录号:100573)
磷酸二氢钾KH 2 PO 4 (Riedel-deHaën ,目录号:04243)
吡啶(Sigma - Aldrich ,目录号:270970)
三乙胺(Merck ,目录号:8083520500)
三氟乙酸(Sigma - Aldrich ,目录号:T6508)
0.1 M KH 2 PO 4 ,pH = 7 .0 (请参阅配方)
0.05%三氟乙酸(水溶液)(请参阅食谱)

用作控制分子的化学品
β-胡萝卜素(Carl Roth ,目录号:5669.1)
芹菜素(Applichem ,目录号:A3641)
咖啡酸(Sigma - Aldrich ,目录号:C0625)
儿茶素(Applichem ,目录号:A4325)
绿原酸(Sigma - Aldrich ,目录号:00500590)
Chry sin(Fluka ,目录号:9582)
肉桂酸(Sigma - Aldrich ,目录号:W228826)
柠檬酸(Carl Roth ,目录号:X863.1)
香豆酸(Carl Roth ,目录号9906.1)
氰胺盐(Carl Roth ,目录号4545.1)
表儿茶素(Applichem ,目录号:A3424)
表没食子儿茶素(Applichem ,目录号:A2010)
阿魏酸(Sigma - Aldrich ,目录号:128708)
果糖(Sigma - Aldrich ,目录号:F0127)
富马酸(Sigma - Aldrich ,目录号:47910)
没食子酸(Sigma - Aldrich ,目录号:91215)
葡萄糖(Sigma - Aldrich ,目录号:G8270)
羟基苯甲酸(Sigma - Aldrich ,目录号:W398608)
乳酸(Sigma - Aldrich ,目录号:L1750)
叶黄素(Applichem ,目录号:A1283)
木犀草素(Applichem ,目录号:A3424)
番茄红素(Carl Roth ,目录号:5670.1)
苹果酸(Carl Roth ,目录号8684.1)
Malvidin(Applichem ,目录号:A8720)
杨梅素(Carl Roth ,目录号:6461.1)
氧化型谷胱甘肽(Sigma - Aldrich ,目录号:G4376)
Pelargonidin(Carl Roth ,目录号:4540.1)
Peonidin(R&D ,目录号:0942)
翼龙(Sigma - Aldrich ,目录号:P1499)
槲皮素(Applichem ,目录号:A3415)
还原型谷胱甘肽(Sigma - Aldrich ,目录号:G4251)
白藜芦醇(Sigma - Aldrich ,目录号:R5010)
水杨酸(Sigma - Aldrich ,目录号:W398500)
ki草酸(Carl Roth ,目录号:7305.2)
芥子酸(Carl Roth ,目录号:5317.1)
蔗糖(Sigma - Aldrich ,目录号:S5016)
丁香酸(Carl Roth ,目录号:5361.1)
酒石酸(Carl Roth ,目录号:K302.1)
香草酸(Carl Roth ,目录号3685.1)
维生素C(Sigma - Aldrich ,目录号:A5960)
维生素E(Sigma - Aldrich ,目录号:T3251)
玉米黄质(Carl Roth ,目录号5672.2)
萃取溶剂1(请参见配方)
萃取溶剂2:氯仿:甲醇:水(1:3:1,v:v:v)(请参见配方)

设备


-80°C冷冻室
分析天平(梅特勒-托利多,型号:AB54-S)
离心机(贝克曼库尔特(Beckman Coulter),型号:Allegra X-15R)
离心机(T hermo Scientific,型号:SL 16)
带有RTx 5DA色谱柱(0.25 mm × 0.25 mm × 30 m)的GC / FID(Shimadzu ,型号:GC 2010 plus )(Restek;目录号:10523)
HPLC(Shimadzu ,型号:LC20-AT;折射率(RI)检测器型号:RID 10A;光电二极管阵列(PDA)检测器型号:SPD M20A;荧光检测器型号:RF 20A)带C18色谱柱(5 µm - 25 × 4.6 mm) )(GL Sciences ,目录号:5020-03946)和NH2色谱柱(5 µm - 25 × 4.6 mm)GL Sciences; 目录号:5020-05546)
磨刀机(Retsch ,型号:GM200)
冻干机(基督,型号:Epsilon 1-4 LSC)
轨道振动筛(IKA ,型号:KS260)
真空蒸发器(L一bconco ,型号:Centrivap)
真空过滤(Do-Chrom ,型号:FB01)

软件


LC解决方案(Shimadzu,https://www.shimadzu.com/an/lcms/opensolution/opensol4.html)
GC解决方案(Shimadzu ,https://www.shimadzu.com/an/gc/advflowtech/sw-dl.html)

程序


代谢物的提取
收集10个成熟的Tom Ato水果(平均新鲜重量为65克),散装并切成小方块。取100克样品,冻干两天(确保它们完全干燥)。用刀磨机研磨干燥的样品,得到细粉。
              称量1克干燥样品,然后加入5毫升萃取溶剂1 (参见R ecipes)以萃取非极性代谢物。通过在18℃下以400 rpm的速度在振荡器上振荡过夜,提取代谢物。 
              离心3样品,724 ×克在4℃下进行20分钟。保存上清液,并使用相同的溶剂对沉淀进行第二次过夜萃取。
再次离心样品,合并提取物。分装提取物(1 ml)并保存在-80°C直至分析。 
              要提取的极性代谢物,使用在得到的颗粒步骤A4和按照相同的步骤1-4使用萃取溶剂2(参见ř ecipes)。分装提取物(1 ml)并保存在-80°C直至分析。
见注1。

代谢物的HPLC分析
类胡萝卜素

两种不同的方法用于分析类胡萝卜素:方法1用于分析番茄红素和β-胡萝卜素;方法2用于分析叶黄素和玉米黄质。


方法1:

制备番茄红素在甲醇:丙酮(1:1,v:v)中浓度分别为1,5,10,25,50和100 ppm的标准溶液,以及β-胡萝卜素在二氯甲烷中的浓度为1,5,10,25,50, 100 ppm浓度。
向乙酸乙酯和乙腈中添加0.05%三甲胺。使用真空过滤,通过膜滤器过滤甲醇,乙酸乙酯和乙腈。
以50:40:10(v:v:v)的比例使用甲醇:乙酸乙酯:乙腈作为流动相进行等度洗脱。
使用RP色谱柱并将色谱柱温度设置为30°C。将流速设置为1.5 ml / min。
通过注射器过滤器过滤样品和标准溶液。进样20 µl样品或标准溶液。在469 nm处检测番茄红素和β-胡萝卜素。
参见注释2和3。
一种用于100ppm的标准molecul色谱ES(图1),标准曲线(图URE 2一和2b )和番茄样品色谱(图3)被示出。



图1.番茄红素和β-胡萝卜素标准品的色谱图



图2.类胡萝卜素的标准曲线。a )番茄红素和b)β-胡萝卜素。



图3.番茄样品的色谱图,显示番茄红素和β-胡萝卜素峰


方法2:

配制叶黄素和玉米黄质在二氯甲烷中的标准溶液,其中含有0.01%BHT,浓度为0.5、1、5、12.5、50、100、200和300 ppm
向乙腈中添加0.05%三甲胺。使用真空过滤器通过膜滤器过滤甲醇和乙腈。
以90:10(v:v)的比例使用甲醇:乙腈作为流动相进行等度洗脱。
使用RP色谱柱,并将色谱柱温度设置为30°C。将流速设置为1 ml / min。
通过注射器过滤器过滤样品和标准溶液。进样20 µl样品或标准溶液。在469 nm处检测叶黄素和玉米黄质。
参见注释2和3。
色谱用于50ppm的标准分子(图4),标准曲线(图5A和5 B)和番茄样品色谱(图6)中示出。

图4.叶黄素和玉米黄质标准品的色谱图



图5.类胡萝卜素的标准曲线。a)叶黄素和b)玉米黄质。



图6.具有叶黄素和玉米黄质峰的番茄样品色谱图


维生素C

在超纯水中以10、50、100、150、200和250 ppm的浓度制备维生素C的标准溶液。
使用真空过滤通过膜滤器过滤甲醇和磷酸盐缓冲液。
使用甲醇:KH 2 PO 4 10:90(v:v)的比例作为等度洗脱的流动相。
使用RP色谱柱并将色谱柱温度设置为40°C。将流速设置为1 ml / min。
通过注射器过滤器过滤样品和标准溶液。进样20 µl样品或标准溶液。在265 nm处检测维生素C。
见注2。
显示了100 ppm标准分子的色谱图(图7),标准曲线(图8)和番茄的样品色谱图(图9)。



图7.维生素C标准品的色谱图



图8.维生素C的标准曲线



图9.带有维生素C峰的番茄的样品色谱图


维生素E

制备浓度为10、50、100、150、200、250、300和500 ppm的维生素E在乙腈:甲醇(80:20,v:v)中的标准溶液。
使用真空过滤器通过膜滤器过滤甲醇和乙腈。
使用25:75(v:v)比例的甲醇:乙腈作为流动相进行等度洗脱。
使用RP色谱柱并将色谱柱温度设置为40°C。将流速设置为1.5 ml / min。
通过注射器过滤器过滤样品和标准溶液。进样20 µl样品或标准溶液。用荧光检测器在300 nm激发和360 nm发射下检测维生素E。
见注2。
显示了500 ppm标准分子的色谱图(图10),标准曲线(图11)和番茄的样品色谱图(图12)。



图10.维生素E标准品的色谱图



图11.维生素E的标准曲线



图12.带有维生素E峰的番茄的样品色谱图


酚酸

制备浓度为1、5、10、20、30、40和50 ppm的酚酸在甲醇中的标准溶液。
使用真空过滤器通过膜滤器过滤NH 4 H 2 PO 4 ,乙腈和H 3 PO 4 。
使用NH 4 H 2 PO 4 (流动相A),乙腈(流动相B)和H 3 PO 4 (流动相C)作为梯度洗脱的流动相。有关梯度洗脱参数,请参见表1。



表1.梯度洗脱参数


使用RP色谱柱并将色谱柱温度设置为35°C。将流速设置为1 ml / min。
通过注射器过滤器过滤样品和标准溶液。进样20 µl样品或标准溶液。使用PDA检测器在不同波长下检测酚酸。在204 nm处检测到表没食子儿茶素,表儿茶素和chrysin。在254 nm处检测到羟基苯甲酸,香草酸,杨梅素和槲皮素。在280 nm处检测到没食子酸,儿茶素,丁香酸和肉桂酸。绿原酸,咖啡酸,香豆酸,阿魏酸,芥子酸,白藜芦醇,芹菜素和pterostilben都在320nm处检测。木犀草素在360 nm处检测到,而氰化物,pelargonidin和peonidin在520 nm处检测到。
见注2。
显示了50 ppm标准分子的色谱图(图13),标准曲线的色谱图(图14a-v)和番茄的样品色谱图(图15)。


图13.标准分子的色谱图

图14.酚酸的标准曲线。a)表没食子儿茶素b)表儿茶素c)胰蛋白酶d)羟基苯甲酸e)香草酸f)杨桃素g)槲皮素h)没食子酸i)儿茶素j)丁香酸k)肉桂酸l)绿原酸m)咖啡酸n)香豆酸o)阿魏酸p)芥子酸r)麦维菌素s)白藜芦醇t)芹菜素u)萜品戊烯v)木犀草素w)花青素x)pelargonidin y)peonidin 。


图15.番茄样品色谱图,显示酚酸峰


谷胱甘肽

准备浓度为10、50、100、150、200、250 ppm的酚酸在甲醇中的标准溶液。
使用真空过滤,通过膜滤器过滤三氟乙酸和甲醇。
使用三氟乙酸:甲醇(97:3,v:v)作为流动相进行等度洗脱。
使用RP色谱柱并将色谱柱温度设置为35°C。将流速设置为0.2 ml / min。
通过注射器过滤器过滤样品和标准溶液。进样20 µl样品或标准溶液。用PDA检测器在208 nm处检测谷胱甘肽的氧化还原形式。
见注2。
下面列出了50 ppm标准分子的色谱图(图16),标准曲线(图17a -b)和番茄的样品色谱图(图18)。



图16.还原和氧化的谷胱甘肽标准品的色谱图



图17. a)还原的和b)氧化的谷胱甘肽的标准曲线



图18.带有谷胱甘肽结果的番茄样品色谱图


糖类

在100、150、200、250、300、500、750、1000、1500、2000、2500 ppm浓度的超纯水中制备糖(葡萄糖,果糖和蔗糖)的标准溶液。
使用真空过滤,通过膜滤器过滤三氟乙酸和甲醇。
使用乙腈:水(90:10,v:v)作为流动相进行等度洗脱。
使用RP色谱柱并将色谱柱温度设置为40°C。将流速设置为1 ml / min。
通过注射器过滤器过滤样品和标准溶液。进样20 µl样品或标准溶液。使用正模式的RI检测器检测糖。
见注释2和4。
色谱用于50ppm的标准分子(图19),标准曲线(FIGUR Ë图20A-C)和番茄样品色谱(图21)在下面给出。



图19.葡萄糖,果糖和蔗糖标准品的色谱图



图20.糖的标准曲线。a)葡萄糖b)果糖c)蔗糖。



图21.番茄的样品色谱图,显示糖的峰。在完全成熟的水果中未检测到蔗糖。


代谢物的GC分析
有机酸

称量标准分子,然后将它们溶解在浓度为0.25、0.5、1、2.5、5、7.5、10 mg / ml的盐酸甲恶胺(40 µl,在吡啶中为20 mg / ml )中。
取100 µl样品。使用真空蒸发器在30°C下蒸发样品的有机溶剂,直到完全干燥。
将番茄样品在超声浴中溶解于盐酸甲恶胺(40 µl,在吡啶中为20 mg / ml)中,溶解5分钟。
将样品和标准分子在37°C衍生90分钟。
使用MSTFA执行第二次衍生化。向样品和标准分子中加入60 µl MSTFA,并在37°C下孵育30分钟。
在离心样品2 5 ,830 ×克,5分钟。
将上清液注入GC-FID。
使用热梯度程序分析Rtx 5DA色谱柱上的有机酸。从100设定的柱温℃(保持1分钟)至150℃以5的速率℃每分钟-1 ,150℃(保持1分钟)至280℃以5℃每分钟的速率-在最终温度下保持1到2分钟。
将进样口温度保持在250°C,将检测器温度保持在300°C。使用氮气作为载气,并调整分流比1/25。
见注释5和6。
色谱对1mg / ml的标准分子(图22),标准曲线(FIGUR Ë 23A-G)和番茄样品色谱(图24)在下面给出。



图22.有机酸标准品的色谱图



图23.有机酸的标准曲线。a)水杨酸b)富马酸c)乳酸d)苹果酸e)酒石酸f)iki草酸g)柠檬酸。



图24.番茄的色谱图,显示有机酸的峰


数据分析


重复实验并记录对照分子三个重复的数据。使用LC溶液(用于HPLC数据)和GC溶液(用于GC数据)绘制标准曲线。这些软件程序会计算三次分析的平均值,并根据面积和浓度自动绘制曲线。同样,软件程序会使用标准曲线自动计算样品中的代谢物浓度。亲本系和群体的平均代谢物含量见表2。




表2.在IBL人群和父母中测得的平均代谢物含量

*数量以mg 100 g -1 DW给出。

**在番茄样品中未检出白藜芦醇,萜烯,花青素,pelargonidin,peonidin,蔗糖,富马酸和酒石酸。


笔记


最好在黑暗和低温(+4°C)下进行提取,因为某些代谢物会受到光照和高温的影响。
这是更好地离心样品(25 ,830 ×克注射器过滤前一次,1-2分钟),以延长柱寿命。
RP C30色谱柱可以提供更好的分离度,但比RP C18色谱柱昂贵。
最好在分析之前等待RI检测器的调节。有时,最好是离开与流动相的检测器过夜低流速(例如,为0.1ml /分钟- 0.5ml /分钟)。
可以使用氦气代替氮气作为载气。
在吡啶中新鲜制备盐酸甲氧明。
仅使用超纯水进行提取和分析。

菜谱


萃取溶剂1
二氯甲烷:含有0.01%BHT的己烷(1:1,v:v)避免在提取代谢物时发生氧化反应
量取50毫升二氯甲烷
向二氯甲烷中加入0.01克BHT,混合直至BHT溶解
加入50毫升己烷并摇匀
萃取溶剂2:氯仿:甲醇:水(1:3:1,v:v:v)
量取20毫升氯仿
加入60毫升甲醇并充分混合
将溶液放在振荡器上,并向溶液中缓慢加入水,以避免相分离
0.1 M KH 2 PO 4 ,pH = 7 .0
在烧杯中称量13.609 g KH 2 PO 4
加800毫升水,搅拌直至KH 2 PO 4溶解
使pH值达到7.0
用水补足至1,000毫升
0.05%三氟乙酸(水溶液)
在容量瓶中计量950毫升水
加入0.5毫升三氟乙酸,用水将体积调至1,000毫升

致谢


这项工作得到了土耳其科学技术研究委员会(TÜBİTAK,项目号114Z116)和土耳其共和国农林部,农业研究所总局(TAGEM-16 / AR-GE / 03)的资助。 。该协议源自GürbüzÇolak等。(2020 a,b )手稿。


利益争夺


作者声明没有任何金融和非金融竞争利益。


伦理


这些规程中未使用任何人类和/或动物受试者。


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引用:Gürbüz Çolak, N., Eken, N. T., Frary, A. and Doganlar, S. (2021). Chromatographic Analysis for Targeted Metabolomics of Antioxidant and Flavor-Related Metabolites in Tomato. Bio-protocol 11(5): e3929. DOI: 10.21769/BioProtoc.3929.
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