Materials and Reagents

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.

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)

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

-80 °C freezer
Analytical balance (Mettler Toledo, model: AB54-S )
Centrifuge (Beckman Coulter, model: Allegra X-15R )
Centrifuge (Thermo Scientific, model: SL 16 )
GC/FID (Shimadzu, model: GC 2010 plus ) with RTx 5DA column (0.25 mm × 0.25 mm × 30 m) (Restek; catalog number: 10523 )
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 )
Knife grinder (Retsch, model: GM200 )
Lyophilizer (Christ, model: Epsilon 1-4 LSC )
Orbital shaker (IKA, model: KS260 )
Vacuum evaporator (Labconco, model: Centrivap )
Vacuum filtration (Do-Chrom, model: FB01 )

Software

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

Procedure

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.

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.

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:

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
Add 0.05% trimethylamine to acetonitrile. Filter methanol and acetonitrile through membrane filter using vacuum filtration.
Use methanol:acetonitrile at 90:10 (v:v) ratio as the mobile phase for isocratic elution.
Use a RP column and set the column temperature at 30 °C. Set the flow rate as 1 ml/min.
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.
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

Prepare standard solutions of vitamin C in ultrapure water at 10, 50, 100, 150, 200 and 250 ppm concentrations.
Filter methanol and phosphate buffer through membrane filter using vacuum filtration.
Use methanol: KH₂PO₄ 10:90 (v:v) ratio as the mobile phase for isocratic elution.
Use RP column and set the column temperature at 40 °C. Set the flow rate as 1 ml/min.
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.
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

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.
Filter methanol and acetonitrile through membrane filter using vacuum filtration.
Use methanol:acetonitrile 25:75 (v:v) ratio as the mobile phase for isocratic elution.
Use RP column and set the column temperature at 40 °C. Set the flow rate as 1.5 ml/min.
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.
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

Prepare standard solutions of phenolic acids in methanol at 1, 5, 10, 20, 30, 40, and 50 ppm concentrations.
Filter NH₄H₂PO₄, acetonitrile and H₃PO₄through membrane filter using vacuum filtration.
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
Use RP column and set the column temperature at 35 °C. Set the flow rate as 1 ml/min.
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.
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

Prepare standard solutions of phenolic acids in methanol at 10, 50, 100, 150, 200, 250 ppm concentrations.
Filter trifluoroacetic acid and methanol through membrane filter using vacuum filtration.
Use trifluoroacetic acid:methanol (97:3, v:v) as the mobile phase for isocratic elution.
Use RP column and set the column temperature at 35 °C. Set the flow rate as 0.2 ml/min.
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.
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.

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

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.
Filter trifluoroacetic acid and methanol through membrane filter using vacuum filtration.
Use acetonitrile: water (90:10, v:v) as the mobile phase for isocratic elution.
Use RP column and set the column temperature at 40 °C. Set the flow rate as 1 ml/min.
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.
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.

GC Analysis of Metabolites
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

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.
It is better to centrifuge the sample (25,830 × g, 1-2 min) once more before syringe filtration to extend column life.
A RP C30 column can provide better resolution but is more expensive than a RP C18 column.
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).
Helium can be used instead of nitrogen as the carrier gas.
Freshly prepare methoxamine hydrochloride in pyridine.
Use only ultrapure water for extraction and analysis.

Recipes

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
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
0.1 M KH₂PO₄, pH = 7.0
1. Weigh 13.609 g KH₂PO₄in a beaker
2. Add 800 ml of water and mix until KH₂PO₄dissolves
3. Bring the pH to 7.0
4. Make up volume to 1,000 ml with water
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

Alenazi, M. M., Shafiq, M., Alsadon, A. A., Alhelal, I. M., Alhamdan, A. M., Solieman, T. H. I., Ibrahim, A. A., Shady, M. R. and Al-Selwey, W. A. (2020). Improved functional and nutritional properties of tomato fruit during cold storage. Saudi J Biol Sci 27(6): 1467-1474.
Bakre, S. M., Gadmale, D. K., Toche, R. B. and Gaikwad, V. B. (2015). Rapid determination of α- tocopherol in olive oil adulterated with sunflower oil by reversed phase high-performance liquid chromatography. J Food Sci Technol 52(5): 3093-3098.
Capanoglu, E., Beekwilder, J., Boyacioglu, D., Hall, R. and de Vos, R. (2008). Changes in antioxidant and metabolite profiles during production of tomato paste. J Agric Food Chem 56(3): 964-973.
Celik, I., Gurbuz, N., Uncu, A. T., Frary, A. and Doganlar, S. (2017). 942 3" target="_blank">Genome-wide SNP discovery and QTL mapping for fruit quality traits in inbred backcross lines(IBLs) of Solanum pimpinellifolium using genotyping by sequencing. BMC Genomics 18(1): 1. doi 10.1186/s12864-016-3406-7.
Dumont, D., Danielato, G., Chastellier, A., L., Hibrand Saint Oyant, Fanciullino, A. L. and Lugan, R. (2020). Multi-targeted metabolic profiling of carotenoids, phenolic compounds and primary metabolites in goji(Lycium spp.) berry and tomato(Solanum lycopersicum) reveals inter and intra genus biomarkers. Metabolites 10(10). doi:10.3390/metabo10100422.
Figueira, J. A., Pereira, J. A. M. and Camara, J. S. (2017). Quantification of δ-, γ- and α-tocopherol in tomatoes using an improved liquid-dispersive solid-phase extraction combined with ultrahigh pressure liquid chromatography. Food Anal Methods 10(7): 2507-2517.
Gómez-Alonso, S., García-Romero, E. and Hermosỉn-Gutiẻrrez, I. (2007). HPLC analysis of diverse grape and wine phenolics using direct injection and multidetection by DAD and fluorescence. J Food Compos Anal 20(7): 618-626.
Gürbüz Çolak, N., Eken, N. T., Ulger, M., Frary, A. and Doganlar, S. (2020). Mapping of quantitative trait loci for antioxidant molecules in tomato fruit: Carotenoids, vitamins C and E, glutathione and phenolic acids. Plant Sci 292: 110393.
Çolak, Gürbüz, N., Tek Eken, Ülger, N., Frary, M. and Doğanlar, A. (2020). Exploring wild alleles from Solanum pimpinellifolium with the potential to improve tomato flavor compounds. Plant Sci 298: 110567.
Ingallina, C., Sobolev, A. P., Circi, S., Spano, M., Giusti, A. M. and Mannina, L. (2020). New hybrid tomato cultivars: An NMR-based chemical characterization. Appl Sci 10: 1887. doi:10.3390/app10051887.
Ishida, B. K., Ma, J. and Chan, B. (2001). A simple, rapid method for HPLC analysis of lycopene isomers. Phytochem Anal 12(3): 194-198.
Kader, A. A., (2008). Flavor quality of fruits and vegetables. J Sci Food Agric 88(11): 1863-1868.
Khan, A., Khan, M. I., Iqbal, Z., Shah, Y., Ahmad, L., Nazir, S., Watson, D. G., Khan, J. A., Nasir, F. and Khan, A. (2011). A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection. Talanta 84(3): 789-801.
Li, H. B. and Chen, F. (2001). Simultaneous determination of twelve water- and fat-soluble vitamins by high-performance liquid chromatography with diode array detection. Chromatographia 54: 270-273.
Li, H. B. and Chen, F. (2001). Simultaneous determination of nine water-soluble vitamins in pharmaceutical preparations by high- performance liquid chromatography with diode array detection. J Sep Sci 24(4): 271-274.
Lobo, V., Patil, A., Phatak, A. and Chandra, N. (2010). Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev 4(8): 118-126.
Martínez, J. P., Fuentes, R., Farías, K., Lİzana, C., Alfaro, J. F., Fuentes, L., Calabrese, N., Bigot, S., Quinet, M. and Lutts, S. (2020). Effects of salt stress on fruit antioxidant capacity of wild(Solanum chilense) and domesticated(Solanum lycopersicum var. cerasiforme) tomatoes. Agronomy 10: 1481. doi:10.3390/agronomy10101481.
Masetti, O., Nisini, L., Ciampa, A. and Dell’Abate, M. T. (2020). ¹H NMR spectroscopy coupled with multivariate analysis was applied to investigate Italian cherry tomatoes metabolic profile. J Chemom 3191.
Migalatev, O., (2017). Optimisation of operating parameters for supercritical carbon dioxide extraction of lycopene from industrial tomato waste. Ukr Food J 6(4): 698-716.
Nagarajan, J., Kay, H. P., Krishnamurthy, N. P., Ramakrishnan, N. R., Aldawoud, T. M. S., Galanakis, C. M. and Wei, O. C. (2020). Extraction of carotenoids from tomato pomace via water-induced hydrocolloidal complexation. Biomolecules 10(7): 1019.
Namgung, H. J., Park, H. J., Cho, I. H., Choi, H. K., Kwon, D. Y., Shim, S. M. and Kim, Y. S. (2010). Metabolite profiling of doenjang, fermented soybean paste, during fermentation. J Sci Food Agric 90(11): 1926-1935.
Pellicanò, T. M., Sicari, V., Loizzo, M. R., Leporini, M., Falco, T. and Poiana, M. (2019). Optimizing the supercritical fluid extraction process of bioactive compounds from processed tomato skin by-products. Food Sci Technol 40(3): 692-697.
Petkova, T. N., Pascal, B.A., Annick, M. and Pantele, D. P. (2013). HPLC analysis of mono- and disaccharides in food products. Presentation at the meeting of the Scientific Works Volume LX, Food Science, Engineering and Technology, Plovdiv, Bulgaria, 761-765.
Roessner, U., Wagner, C., Kopka, J., Trethewey, R. N. and Willmitzer, L. (2000). Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. Plant J 23(1): 131-142.
Serino, S., Gomez, L., Costagliola, G. and Gautier, H. (2009). HPLC assay of tomato carotenoids: validation of a rapid microextraction technique. J Agric Food Chem 57(19): 8753-8760.
Tan, S., Ke, Z., Chai, D., Miao, Y., Luo, K. and Li, W. (2021). Lycopene, polyphenols and antioxidant activities of three characteristic tomato cultivars subjected to two drying methods. Food Chem 338: 128062.
Tohge, T., Scossa, F., Wendenburg, R., Frasse, P., Balbo, I., Watanabe, M., Alseekh, S., Jadhav, S. S., Delfin, J. C., Lohse, M., Giavalisco, P., Usadel, B., Zhang, Y., Luo, J., Bouzayen, M. and Fernie, A. R. (2020). Exploiting natural variation in tomato to define pathway structure and metabolic regulation of fruit polyphenolics in the lycopersicum complex. Mol Plant 13(7): 1027-1046.
Treutler, H., Tsugawa, H., Porzel, A., Gorzolka, K., Tissier, A., Neumann, S. and Balcke, G. U. (2016). Discovering regulated metabolite families in untargeted metabolomics studies. Anal Chem 88(16): 8082-8090.
Turner, T. and Burri, B. J. (2012). Rapid isocratic HPLC method and sample extraction procedures for measuring carotenoid, retinoid, and tocopherol concentrations in human blood and breast milk for intervention studies. Chromatographia 75: 241-252.