High-throughput analysis of anthocyanins in horticultural crops using probe electrospray ionization tandem mass spectrometry (PESI/MS/MS)

Abstract

Plant secondary metabolites exhibit various horticultural traits. Simple and rapid analysis methods for evaluating these metabolites are in demand in breeding and consumer markets dealing with horticultural crops. We applied probe electrospray ionization (PESI) to evaluate secondary metabolite levels in horticultural crops. PESI does not require pre-treatment and separation of samples, which makes it suitable for high-throughput analysis. In this study, we targeted anthocyanins, one of the primary pigments in horticultural crops. Eighty-one anthocyanins were detected in approximately 3 minutes in the selected reaction-monitoring mode. Tandem mass spectrometry (MS/MS) could adequately distinguish between the fragments of anthocyanins and flavonols. Probe sampling, an intuitive method of sticking a probe directly to the sample, could detect anthocyanins qualitatively on a micro-area scale, such as achenes and receptacles in strawberry fruit. Our results suggest that PESI/MS/MS can be a powerful tool to characterize the profile of anthocyanins and compare their content among cultivars.

Acknowledgements

This work was supported by the Japan Society for the Promotion of Science KAKENHI [grant numbers 20J00948, 20K20372, 21K19111, and 21H02184]. We are grateful to Drs. Kanako Sekimoto (Yokohama City University), Hidekazu Saiki, Eishi Imoto (Shimadzu Corporation), Yoshinori Fujimura (Kyushu University), Kazuyoshi Terasaka (Nagoya City University), Hidetoshi Ikegami (Fukuoka Agriculture and Forestry Research Center), and Kenta Tsunekawa (Aichi Agricultural Research Center) for helpful discussion.

Author Contributions

K.S. and M.I. conceived and designed the study; K.Z. organized the system construction for the PESI/MS/MS method; M.I. and I.Y. performed the analysis with the support of K.Z.; M.I., I.Y. and K.S. analyzed data; K.Z. S.O., S.M. and A.O. contributed to data analysis; M.I. and K.S. wrote and revised the manuscript accordingly; All authors have read and approved the manuscript.

Data availability

All data supporting the findings in this study are included in this article and supplementary materials.

Conflict of interest statement

The authors declare that they have no conflict of interest.

References

[1] Yahia EM, Carrillo-López A . Postharvest physiology and biochemistry of fruits and vegetables. Elsevier. 2018; 1: 1-476.

[2] Bueno JM, Sáez-Plaza P, Ramos-Escudero F et al. Analysis and antioxidant capacity of anthocyanin pigments. Part II: chemical structure, color, and intake of anthocyanins. Crit Rev Anal Chem. 2012; 42: 126-51.

[3] Kortbeek RWJ, van der Gragt M, Bleeker PM . Endogenous plant metabolites against insects. Eur J Plant Pathol. 2019; 154: 67-90.

[4] Wu X, Prior RL . Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States: fruits and berries. J Agric Food Chem. 2005; 53: 2589-99.

[5] Kruger MJ, Davies N, Myburgh KH et al. Proanthocyanidins, anthocyanins and cardiovascular diseases. Food Res Int. 2014; 59: 41-52.

[6] de Arruda NE, de Lima CL, da Silva CJ et al. In vitro anticancer properties of anthocyanins: a systematic review. Biochim Biophys Acta - Rev Cancer. 2022; 1877: 188748(15 pages).

[7] Wu X, Prior RL . Identification and characterization of anthocyanins by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry in common foods in the United States: vegetables, nuts, and grains. J Agric Food Chem. 2005; 53: 3101-13.

[8] Mikulic-petkovsek M, Slatnar A, Stampar F et al. HPLC-MS n identification and quantification of flavonol glycosides in 28 wild and cultivated berry species . Food Chem. 2012; 135: 2138-46.

[9] Cooks RG, Ouyang Z, Takats Z et al. Ambient mass spectrometry. Science. 2006; 311: 1566-70.

[10] Guo T, Yong W, Jin Y et al. Applications of DART-MS for food quality and safety assurance in food supply chain. Mass Spectrom Rev. 2017; 36: 161-87.

[11] Mason TJ, Bettenhausen HM, Chaparro JM et al. Evaluation of ambient mass spectrometry tools for assessing inherent postharvest pepper quality. Hortic Res. 2021; 8: 160.

[12] Cabral EC, Mirabelli MF, Perez CJ et al. Blotting assisted by heating and solvent extraction for DESI-MS imaging. J Am Soc Mass Spectrom. 2013; 24: 956-65.

[13] Moreno-Pedraza A, Rosas-Román I, Garcia-Rojas NS et al. Elucidating the distribution of plant metabolites from native tissues with laser desorption low-temperature plasma mass spectrometry imaging. Anal Chem. 2019; 91: 2734-43.

[14] McVey PA, Alexander LE, Fu X et al. Light-dependent changes in the spatial localization of metabolites in solenostemon scutellarioides (coleus henna) visualized by matrix-free atmospheric pressure electrospray laser desorption ionization mass spectrometry imaging . Front Plant Sci. 2018; 9: 1348.

[15] Liu Q, Lan J, Wu R et al. Hybrid ionization source combining nanoelectrospray and dielectric barrier discharge ionization for the simultaneous detection of polar and nonpolar compounds in single cells. Anal Chem. 2022; 94: 2873-81.

[16] Hiraoka K, Nishidate K, Mori K et al. Development of probe electrospray using a solid needle. Rapid Commun Mass Spectrom. 2007; 21: 3139-44.

[17] Hiraoka K, Ariyada O, Usmanov DT et al. Probe electrospray ionization (PESI) and its modified versions: dipping PESI (dPESI), sheath-flow PESI (sfPESI) and adjustable sfPESI (ad-sfPESI). Mass Spectrom (Tokyo). 2020; 9: A0092-21.

[18] Zaitsu K, Hayashi Y, Murata T et al. Intact endogenous metabolite analysis of mice liver by probe electrospray ionization/triple quadrupole tandem mass spectrometry and its preliminary application to in vivo real-time analysis. Anal Chem. 2016; 88: 3556-61.

[19] Mandal MK, Chen LC, Hiraoka K . Sequential and exhaustive ionization of analytes with different surface activity by probe electrospray ionization. J Am Soc Mass Spectrom. 2011; 22: 1493-500.

[20] Hu B, Yao ZP . Electrospray ionization mass spectrometry with wooden tips: a review. Anal Chim Acta. 2022; 1209: 339136 (10 pages).

[21] Freund DM, Martin AC, Cohen JD et al. Direct detection of surface localized specialized metabolites from Glycyrrhiza lepidota (American licorice) by leaf spray mass spectrometry . Planta. 2018; 247: 267-75.

[22] Zaitsu K, Eguchi S, Ohara T et al. PiTMaP: a new analytical platform for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry using an R software-based data pipeline. Anal Chem. 2020; 92: 8514-22.

[23] Hisatsune K, Murata T, Ogata K et al. RECiQ: a rapid and easy method for determining cyanide intoxication by cyanide and 2-aminothiazoline-4-carboxylic acid quantification in the human blood using probe electrospray ionization tandem mass spectrometry. ACS Omega. 2020; 5: 23351-7.

[24] Kawakami D, Tsuchiya M, Murata T et al. Rapid quantification of extracellular neurotransmitters in mouse brain by PESI/MS/MS and longitudinal data analysis using the R and Stan-based Bayesian state-space model. Talanta. 2021; 234: 122620(9 pages).

[25] Thirukumaran M, Singh V, Arao Y et al. Solid-phase microextraction-probe electrospray ionization devices for screening and quantitating drugs of abuse in small amounts of biofluids. Talanta. 2021; 231: 122317(8 pages).

[26] Zhao D, Tao J . Recent advances on the development and regulation of flower color in ornamental plants. Front Plant Sci. 2015; 6: 1-13.

[27] Horai H, Arita M, Kanaya S et al. MassBank: a public repository for sharing mass spectral data for life sciences. J Mass Spectrom. 2010; 45: 703-14.

[28] Arvaniti OS, Samaras Y, Gatidou G et al. Review on fresh and dried figs: chemical analysis and occurrence of phytochemical compounds, antioxidant capacity and health effects. Food Res Int. 2019; 119: 244-67.

[29] Jurikova T, Rop O, Mlcek J et al. Phenolic profile of edible honeysuckle berries (genus Lonicera) and their biological effects . Molecules. 2012; 17: 61-79.

[30] Wang Y, Fong SK, Singh AP et al. Variation of anthocyanins, proanthocyanidins, flavonols, and organic acids in cultivated and wild diploid blueberry species. HortScience. 2019; 54: 576-85.

[31] Oh YS, Lee JH, Yoon SH et al. Characterization and quantification of anthocyanins in grape juices obtained from the grapes cultivated in Korea by HPLC/DAD, HPLC/MS, and HPLC/MS/MS. J Food Sci. 2008; 73: C378-89.

[32] Blackhall ML, Berry R, Davies NW et al. Optimized extraction of anthocyanins from Reid fruits’ Prunus avium ‘Lapins’ cherries . Food Chem. 2018; 256: 280-5.

[33] Lopes da Silva F, Escribano-Bailón MT, Alonso JJP et al. Anthocyanin pigments in strawberry. LWT - Food Sci Technol. 2007; 40: 374-82.

[34] Buendía B, Gil MI, Tudela JA et al. HPLC-MS analysis of proanthocyanidin oligomers and other phenolics in 15 strawberry cultivars. J Agric Food Chem. 2010; 58: 3916-26.

[35] Wiczkowski W, Szawara-Nowak D, Topolska J . Red cabbage anthocyanins: profile, isolation, identification, and antioxidant activity. Food Res Int. 2013; 51: 303-9.

[36] Viacava GE, Roura SI, Berrueta LA et al. Characterization of phenolic compounds in green and red oak-leaf lettuce cultivars by UHPLC-DAD-ESI-QToF/MS using MSE scan mode. J Mass Spectrom. 2017; 52: 873-902.

[37] Pérez-Gregorio RM, García-Falcón MS, Simal-Gándara J et al. Identification and quantification of flavonoids in traditional cultivars of red and white onions at harvest. J Food Compos Anal. 2010; 23: 592-8.

[38] Zielińska D, Turemko M . Electroactive phenolic contributors and antioxidant capacity of flesh and peel of 11 apple cultivars measured by cyclic voltammetry and HPLC-DAD-MS/MS. Antioxidants. 2020; 9: 1-18.

[39] Enomoto H, Sato K, Miyamoto K et al. Distribution analysis of anthocyanins, sugars, and organic acids in strawberry fruits using matrix-assisted laser desorption/ionization-imaging mass spectrometry. J Agric Food Chem. 2018; 66: 4958-65.

[40] Wang J, Yang E, Chaurand P et al. Visualizing the distribution of strawberry plant metabolites at different maturity stages by MALDI-TOF imaging mass spectrometry. Food Chem. 2021; 345: 128838(9 pages).

[41] Fait A, Hanhineva K, Beleggia R et al. Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development. Plant Physiol. 2008; 148: 730-50.

[42] Usmanov DT, Mandal MK, Hiraoka K et al. Dipping probe electrospray ionization/mass spectrometry for direct on-site and low-invasive food analysis. Food Chem. 2018; 260: 53-60.

[43] Hiraoka K, Rankin-Turner S, Ninomiya S et al. Component profiling in agricultural applications using an adjustable acupuncture needle for sheath-flow probe electrospray ionization/mass spectrometry. J Agric Food Chem. 2019; 67: 3275-83.

[44] Ishibashi M, Okochi S, Sone K et al. Seasonal variation of the major allergen Fra a 1 in strawberry fruit. Hortic J. 2019; 88: 354-63.

[45] Matsuo T, Tsugawa H, Miyagawa H et al. Integrated strategy for unknown EI-MS identification using quality control calibration curve, multivariate analysis, EI-MS spectral database, and retention index prediction. Anal Chem. 2017; 89: 6766-73.