A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Chemicals
2.3. Solutions
2.4. Sample Preparation
2.5. Optimisation of the Extraction Solution
2.6. Optimisation of Saponification Temperature and Reaction Time
2.7. Optimisation of Carotenoid Recovery After Saponification
2.8. Method Validation
2.8.1. Principles of Analytical Calibration and Carotenoid Recovery
2.8.2. Limit of Detection (LOD) and Quantification (LOQ)
2.8.3. Matrix Effect
2.8.4. Precision and Accuracy
2.9. Carotenoid Analysis
2.10. Carotenoid Identification and Quantification
2.10.1. Carotenoid Identification
2.10.2. Carotenoid Quantification
2.11. Statistical Analysis
3. Results
3.1. Optimisation of the Extraction Solution and pH Levels for the Recovery of Carotenoids During the Saponification Procedure
3.2. Optimised Saponification Temperature and Saponification Duration
3.3. Method Validation
3.3.1. Recovery of Carotenoids During Saponification
3.3.2. LOD, LOQ, and Matrix Effect
3.3.3. Precision and Accuracy
3.4. Identification of Carotenoids in Chili/Capsicum Fruit
3.5. Quantification of Carotenoids in Pigmented Chili/Capsicums
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
UV/VIS | ultraviolet–visible spectroscopy |
KOH | potassium hydroxide |
HPLC | High-performance liquid chromatography |
QLD | Queensland |
BHT | butylated hydroxytoluene |
MTBE | methyl tert-butyl ether |
DCM | dichloromethane |
NATA | National Association of Testing Authorities, Australia |
LOD | limit of detection |
LOQ | limit of quantification |
ME | matrix effect |
UHPLC | Ultra high performance liquid chromatography |
DAD | Diode array detector |
APCI | Atmospheric pressure chemical ionization |
MS | Mass spectrometer |
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Peak No | Carotenoids | rt (min) | λ (nm) | m/z (−) | Fragment Ion (m/z) (−) | m/z (+) | Fragment Ion (m/z) (+) | ||
---|---|---|---|---|---|---|---|---|---|
1 | Unknown | 6.27 | 379 | 398 | 420 | 618.4 | 582.3; 277.2; 219.1 | 585.4 | 391.3; 270.3; 256.3 |
2 | Violaxanthin | 6.72 | 399 | 439 | 469 | 600.4 | 582.3; 448.9; 407.7 | 601.3 | 583.4; 565.5; 509.3 |
3 | Violaxanthin isomer | 6.91 | 399 | 439 | 469 | 600.4 | 582.3; 448.9; 407.7 | 601.3 | 583.4; 565.5; 509.3 |
4 | (9-Z)-Luteoxanthin | 7.41 | 399 | 421 | 448 | 600.4 | 448.8; 407.4 | 601.3 | 554.5; 536.4 |
5 | (all-E)-Violaxanthin | 7.81 | 420 | 441 | 468 | 600.4 | 582.3; 448.9; 407.7 | 601.3 | 583.4; 565.5; 509.3 |
6 | Unknown | 8.09 | 400 | 420 | 440 | 600.4 | 575.3; 407.9; 575.3 | 601.3 | 575.4; 397.3 |
7 | Unknown | 8.8 | 400 | 422 | 448 | 600.4 | 491.3; 448.9 | 601.3 | 573.4; 335.3 |
8 | Unknown | 9.7 | 380 | 401 | 426 | 600.4 | 582.4; 465.2; 448.4 | 601.3 | 583.4 |
9 | (all-E)-Antheraxanthin | 10 | 401 | 426 | 471 | 584.3 | 566.3; 281.3 | 585.4 | 577.4, 339.3, 313.3 |
10 | (9-Z)-Antheraxanthin | 10.6 | 401 | 426 | 471 | 584.3 | 566.3; 281.3 | 585.4 | 577.4, 339.3, 313.3 |
11 | Unknown | 10.9 | 382 | 401 | 426 | 600.4 | 255.2 | 601.3 | 583.4; 551.4; 509.4 |
12 | (all-E)-Lutein | 11.7 | 422 | 444 | 472 | 568.4 | 455.4; 450.0 | 569.4 | 551.4; 459.3; 335.3 |
13 | (all-E)-Mutatoxanthin | 12.2 | 400 | 427 | 452 | 584.4 | 568.4; 464.5; 449.7 | 585.4 | 567.4; 409.4; 575.5 |
14 | (all-E)-Zeaxanthin | 13.6 | 425 | 450 | 475 | 568.4 | 465.5; 449.6; 430.1 | 569.4 | 551.4; 177.6 |
15 | (all-E)-Cryptoxanthin * | 18.5 | 423 | 445 | 472 | 552.4 | 784.6; 654.6; 466.6 | 553.4 | 767.5; 533.2, 453 |
16 | Luteoxanthin laurate * | 19.2 | 400 | 427 | 448 | 782.5 | 465.9; 277.2 | 783.6 | 765.5; 597.4; 335 |
17 | Luteoxanthin myristate * | 19.6 | 399 | 427 | 448 | 810.5 | 782.5 | 811.5 | 811.5 |
18 | β-Cryptoxanthin | 22.6 | 422 | 446 | 470 | 552.4 | 536.4; 464.9 | 553.4 | 595.4 |
19 | Luteoxanthin myristate * | 23.8 | 400 | 422 | 448 | 810.5 | 684.8; 464.9 | 811.6 | 793.6; 599.5 |
20 | Luteoxanthin myristate isomer * | 24.1 | 400 | 422 | 448 | 810.5 | 536.4 | 811.6 | 537.3; 793.6 |
21 | (all-E)-carotene * | 27.1 | 379 | 399 | 423 | 536.4 | 794.5; | 537.4 | 851.6; 449.6, 383.3 |
22 | Antheraxanthin myristate * | 27.5 | 425 | 443 | 471 | 794.6 | 810.5; 552.4 | 795.5 | 851.6; 553.4 |
23 | β-Carotene * | 29 | 423 | 446 | 473 | 536.4 | 810.6; 766.5; 838.5 | 537.4 | 853.6; 839.6; 811.6 |
24 | Violaxanthin butyrate-laurate * | 30 | 400 | 441 | 468 | 853.7 | 750.5; 766.5; 810.5 | 855.6 | 733.5; 767.5; 811.6 |
25 | Antheraxanthin myristate * | 31.1 | 425 | 444 | 470 | 794.5 | 932.6 | 795 | 933.6 |
26 | Violaxanthin caprate-myristate * | 31.4 | 425 | 448 | 475 | 964.6 | 838.6; 794.6; 750.5 | 965.6 | 839.6; 795.6; 751.5 |
27 | β-Carotene isomer * | 31.7 | 425 | 451 | 476 | 536.4 | 750.5; 964.7; 855.6 | 537.4 | 965.7; 857.7; 795.6; 751.5 |
28 | Antheraxanthin myristate * | 32.1 | 425 | 446 | 470 | 794.6 | 838.5 | 795.6 | 839.6; 601.5; 533.4 |
29 | Lutein laurate-myristate * | 32.6 | 415 | 444 | 470 | 959.7 | 992.6; 750.5; 734.5 | 961.8 | 993.7; 751.5; 735.6 |
30 | Unknown * | 32.9 | 422 | 444 | 470 | 881.7 | 992.7; 959.7 | 883.7 | 993.7; 961.8 |
31 | Zeaxanthin myristate * | 33.1 | 422 | 448 | 475 | 778.5 | 536.4 | 779.6 | 577.5; 551.4; 537.4 |
32 | Lutein laurate-laurate * | 34.2 | 422 | 444 | 471 | 992.6 | 1020.7; 883.6; 734.5 | 993.6 | 1021.8; 885.7; 735.6 |
33 | Lutein myristate-myristate * | 34.5 | 400 | 423 | 448 | 987.7 | 948.6; 992.6 | 989.8 | 993.7; 965.6; 949.6 |
34 | Antheraxanthin laurate-laurate * | 34.7 | 423 | 448 | 472 | 948.7 | 1048.7; 1021.7, 992.7 | 949.7 | 1049.7, 1021.7, 993.7 |
35 | Unknown * | 35.0 | 404 | 426 | 446 | 1020.7 | 992.6 | 1021.7 | 965.7; 909.7 |
36 | Cryptoxanthin laurate * | 35.4 | 425 | 449 | 475 | 734.5 | 1048.7; 1021.7 | 735.5 | 1049.7, 1021.7 |
37 | Violaxanthin myristate-palmitate * | 35.5 | 426 | 446 | 472 | 1048.7 | 1020.7; 762.5; 734.5 | 1049.8 | 1021.8; 735.6; 765.5 |
38 | Cryptoxanthin myristate * | 35.6 | 425 | 447 | 473 | 762.6 | 1020.7; 734.5 | 763.6 | 1021.8; 735.5 |
39 | Violaxanthin myristate-myristate * | 35.8 | 427 | 444 | 469 | 1020.7 | 762.6; 734.5 | 1021.8 | 763.6; 735.7 |
40 | Antheraxanthin laurate-myristate * | 36.1 | 425 | 446 | 472 | 1004.6 | 948.7; 734.5 | 1005.6 | 949.8; 735.6 |
41 | Violaxanthin palmitate-palmitate * | 36.5 | 425 | 448 | 475 | 1076.7 | 1004.7; 1048.7 | 1077.6 | 1049.8; 965.7; 1005.8; 763.6 |
42 | Violaxanthin myristate-myristate * | 36.9 | 425 | 446 | 472 | 1020.6 | 1004.7; 976.7 | 1021.7 | 1005.8, 977.7; 733.5 |
43 | Zeaxanthin laurate-laurate * | 37.1 | 425 | 450 | 477 | 932.6 | 1076.8, 761.5 | 933.7 | 1076.8; 762.5 |
44 | Lutein myristate-palmitate * | 37.7 | 425 | 448 | 472 | 1048.7 | 1032.7; 1004.7; 986.7 | 1049.8 | 1033.8; 1005.8; 987.7 |
45 | Zeaxanthin laurate- myristate * | 37.9 | 425 | 450 | 477 | 960.7 | 1004.7; 760.6 | 961.7 | 1005.8, 761.6 |
46 | Zeaxanthin myristate-myristate * | 38.5 | 425 | 450 | 476 | 988.7 | 1032.7; 1016.8 | 989.8 | 1033.8; 1016.8 |
47 | Zeaxanthin myristate-palmitate * | 39.3 | 425 | 451 | 477 | 1016.7 | 1060.6; 449.7 | 1017.8 | 1061.9; 789.6 |
48 | Zeaxanthin palmitate-palmitate * | 39.9 | 425 | 451 | 478 | 1044.8 | 1016.7; 963.5 | 1045.9 | 1017.8; 789.6; 761.6; 533.4 |
Carotenoids | RT (min) | λmax (nm) | m/z | MS/MS |
---|---|---|---|---|
Luteoxanthin | 7.4 | 400, 424, 450 | 601.5 | 583.4; 554.5; 536.4 |
Violaxanthin | 6.72; 6.91; 7.8 | 420, 439, 468 | 601.5 | 583.4; 565.5; 509.3 |
Capsorubin | 9.2 | 480 | 601.5 | 583.5; 430.4; 176.9 |
Antheraxanthin | 9.8 | 420, 444, 471 | 585.5 | 561.5; 545.5; 401.3 |
Cis-capsanthin | 10.6 | 468 | 585.5 | 567.4 |
(all-E)-Lutein | 11.5 | 422, 444, 472 | 569.4 | 552.5; 431.2; 176.8 |
(all-E)-Mutatoxanthin | 12.0 | 400, 427, 452 | 585.5 | 567.4; 409.4; 575.5 |
(all-E)-Capsanthin | 12.7 | 473 | 585.5 | 567.5; 493.4; 479.4 |
(all-E)-Zeaxanthin | 13.3 | 425, 450, 478 | 568.4 | 552.4; 431.2; 176.8 |
Cis-Capsanthin | 14.8 | 468 | 585.5 | 567.4 |
α-Cryptoxanthin | 18.1 | 425, 450, 484 | 533.4 | 177.8 |
β-Cryptoxanthin | 22.2 | 425, 451, 484 | 533.4 | 178.0; 120.7 |
α-Carotene | 28.1 | 420, 447, 474 | 537.5 | 177.6 |
Cis-α-Carotene | 29.7 | 420, 447, 474 | 537.5 | 177.6 |
β-Carotene | 31.4 | 425, 451, 478 | 537.5 | 177.6 |
Cis-β-Carotene | 32.7 | 423, 447, 472 | 537.5 | 178.3 |
Samples | RSC | RC | OSC | OC | GC | YSC | BgC |
---|---|---|---|---|---|---|---|
mg/100 g FW | |||||||
Violaxanthin | 1.79 ± 0.11 | 1.89 ± 0.08 | 0.12 ± 0.01 | 0.05 ± 0.00 | 0.29 ± 0.02 | 0.21 ± 0.01 | 2.53 ± 0.41 |
Luteoxanthin | 1.07 ± 0.06 | 0.97 ± 0.02 | 0.71 ± 0.06 | nd | nd | 0.22 ± 0.02 | 1.37 ± 0.33 |
Antheraxanthin | 1.82 ± 0.14 | 1.53 ± 0.05 | 0.04 ± 0.01 | 0.26 ± 0.03 | 0.04 ± 0.00 | 0.02 ± 0.00 | 1.51 ± 0.24 |
Capsorubin | 1.68 ± 0.08 | 1.88 ± 0.02 | nd | nd | nd | nd | nd |
Cis-Capsanthin | 1.31 ± 0.07 | 1.63 ± 0.02 | 0.03 ± 0.00 | nd | nd | 0.01 ± 0.00 | nd |
Lutein | 0.43 ± 0.07 | 0.39 ± 0.03 | 3.20 ± 0.20 | 2.76 ± 0.17 | 0.50 ± 0.02 | 1.78 ± 0.10 | 15.55 ± 0.48 |
Capsanthin | 17.93 ± 0.88 | 15.92 ± 0.2 | nd | nd | 0.33 ± 0.01 | nd | nd |
Mutatoxanthin | 0.61 ± 0.05 | 0.51 ± 0.04 | 3.85 ± 0.18 | 0.69 ± 0.11 | 0.11 ± 0.01 | 0.36 ± 0.10 | 2.75 ± 0.05 |
Zeaxanthin | 1.97 ± 0.24 | 0.54 ± 0.02 | 13.15 ± 1.01 | 11.51 ± 1.09 | 0.21 ± 0.03 | 0.47 ± 0.01 | 3.99 ± 0.18 |
α-Cryptoxanthin | 0.07 ± 0.00 | 0.06 ± 0.00 | 0.24 ± 0.02 | 0.79 ± 0.11 | nd | 0.14 ± 0.01 | 0.66 ± 0.19 |
β-Cryptoxanthin | 0.78 ± 0.09 | 0.17 ± 0.02 | 1.01 ± 0.09 | 0.76 ± 0.09 | nd | 0.05 ± 0.01 | 0.56 ± 0.06 |
α-Carotene | 0.71 ± 0.15 | 0.46 ± 0.06 | 1.31 ± 0.21 | 0.53 ± 0.08 | 0.03 ± 0.00 | 0.41 ± 0.05 | 0.24 ± 0.12 |
β-Carotene | 1.91 ± 0.39 | 1.15 ± 0.22 | 1.61 ± 0.14 | 0.76 ± 0.08 | 0.10 ± 0.02 | 0.25 ± 0.03 | 1.01 ± 0.05 |
Total carotenoid concentration | 32.08 ± 2.25 | 27.10 ± 0.42 | 25.29 ± 1.50 | 18.09 ± 1.76 | 1.63 ± 0.11 | 3.92 ± 0.32 | 30.17 ± 2.11 |
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Hong, H.T.; Agarwal, R.; Takagi, T.; Netzel, M.E.; Harper, S.M.; O’Hare, T.J. A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum. Agriculture 2025, 15, 646. https://doi.org/10.3390/agriculture15060646
Hong HT, Agarwal R, Takagi T, Netzel ME, Harper SM, O’Hare TJ. A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum. Agriculture. 2025; 15(6):646. https://doi.org/10.3390/agriculture15060646
Chicago/Turabian StyleHong, Hung Trieu, Rimjhim Agarwal, Tatsuyoshi Takagi, Michael E. Netzel, Stephen M. Harper, and Tim J. O’Hare. 2025. "A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum" Agriculture 15, no. 6: 646. https://doi.org/10.3390/agriculture15060646
APA StyleHong, H. T., Agarwal, R., Takagi, T., Netzel, M. E., Harper, S. M., & O’Hare, T. J. (2025). A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum. Agriculture, 15(6), 646. https://doi.org/10.3390/agriculture15060646