Analytical and Bioanalytical Chemistry

, Volume 407, Issue 21, pp 6369–6379 | Cite as

Improvement of chlorophyll identification in foodstuffs by MALDI ToF/ToF mass spectrometry using 1,5-diaminonaphthalene electron transfer secondary reaction matrix

  • Cosima Damiana Calvano
  • Giovanni Ventura
  • Tommaso R. I. CataldiEmail author
  • Francesco Palmisano
Research Paper
Part of the following topical collections:
  1. High-Resolution Mass Spectrometry in Food and Environmental Analysis


Chlorophylls (Chls) are important pigments responsible for the characteristic green color of chloroplasts in algae and plants. In this study, 1,5-diaminonaphthalene (DAN) was introduced as an electron transfer secondary reaction matrix for the identification of intact chlorophylls and their derivatives, by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). DAN was proved to drastically outperform conventional matrices such as α-cyano-4-hydroxycinnnamic acid, dithranol, antracene, and even terthiophene, since loss of the metal ion and fragmentation of the phytol–ester linkage are negligible. Absence of significant fragmentation of radical cations of Chls a and b at m/z 892.529 and 906.513, respectively, makes MALDI MS capable of following natural degradation of intact porphyrin-based pigments whose initial steps are just represented by demetalation and dephytylation. Chl by-products, such as pyropheophytins, have been identified in dried tea leaves showing the potential of MALDI MS to follow chlorophyll biotransformation occurring in processed foodstuffs. Finally, preliminary results show the potential of MALDI MS to detect illegal vegetable oil re-greening practices.


Matrices Chlorophylls High-resolution MS Foodstuffs Electron transfer matrices Tandem MALDI-ToF MS 



This work was supported by the project PONa3_00395/1 “BIOSCIENZE & SALUTE (B&H)” of Italian Ministero per l'Istruzione, l'Università e la Ricerca (MIUR). Simoncarlo Giacummo is acknowledged for his technical support.

Supplementary material

216_2015_8728_MOESM1_ESM.pdf (186 kb)
ESM 1 (PDF 185 kb)


  1. 1.
    Wrolstad RE, Acree TE, Decker EA, Penner MH, Reid DS, Schwartz SJ, Shoemaker CF, Smith D, Sporns P (2005) Chlorophylls. Handbook of food analytical chemistry. Wiley, New Jersey, pp 153–199Google Scholar
  2. 2.
    Willstätter R, Asahina Y (1910) Untersuchungen über Chlorophyll. Oxydation der Chlorophyllderivate. Justus Liebig’s Ann Chem 373:227–238. doi: 10.1002/jlac.19103730205 CrossRefGoogle Scholar
  3. 3.
    Woodward RB, Ayer WA, Beaton JM, Bickelhaupt F, Bonnett R, Buchschacher P, Closs GL, Dutler H, Hannah J, Hauck FP, Itô S, Langemann A, Le Goff E, Leimgruber W, Lwowski W, Sauer J, Valenta Z, Volz H (1960) The total synthesis of chlorophyll. J Am Chem Soc 82:3800–3802. doi: 10.1021/ja01499a093 CrossRefGoogle Scholar
  4. 4.
    Ballschmiter K, Katz JJ (1968) Long wavelength forms of chlorophyll. Nature 220:1231–1233. doi: 10.1038/2201231a0 CrossRefGoogle Scholar
  5. 5.
    Mangos TJ, Berger RG (1997) Determination of major chlorophyll degradation products. Z Lebensm Forsch A 204:345–350. doi: 10.1007/s002170050088 CrossRefGoogle Scholar
  6. 6.
    Humphrey AM (1980) Chlorophyll. Food Chem 5:57–67. doi: 10.1016/0308-8146(80)90064-3 CrossRefGoogle Scholar
  7. 7.
    Von Elbe JH, Huang AS, Attoe EL, Nank WK (1986) Pigment composition and color of conventional and Veri-Green canned beans. J Agric Food Chem 34:52–54. doi: 10.1021/jf00067a014 CrossRefGoogle Scholar
  8. 8.
    Jones ID, White RC, Gibbs E, Butler LS, Nelson LA (1977) Experimental formation of zinc and copper complexes of chlorophyll derivatives in vegetable tissue by thermal processing. J Agric Food Chem 25:149–153. doi: 10.1021/jf60209a030 CrossRefGoogle Scholar
  9. 9.
    Segner WP, Ragusa TJ, Nank WK HW (1984) Process for the preservation of green color in canned vegetables. U.S. Patent 4,473,591Google Scholar
  10. 10.
    LaBorde LF, von Elbe JH (1996) Treatment with zinc or copper ions. U.S. Patent 5482727 A 08,279Google Scholar
  11. 11.
    Lauro MF (1934) Olive oil adulteration and the analyst. Oil Soap 11:253–254. doi: 10.1007/BF02640936 CrossRefGoogle Scholar
  12. 12.
    Roca M, Gallardo-Guerrero L, Mínguez-Mosquera MI, Gandul Rojas B (2010) Control of olive oil adulteration with copper-chlorophyll derivatives. J Agric Food Chem 58:51–56. doi: 10.1021/jf902084d CrossRefGoogle Scholar
  13. 13.
    Edelenbos M, Christensen LP, Grevsen K (2001) HPLC determination of chlorophyll and carotenoid pigments in processed green pea cultivars (Pisum sativum L.). J Agric Food Chem 49:4768–4774CrossRefGoogle Scholar
  14. 14.
    Eijckelhoff C, Dekker JP (1997) A routine method to determine the chlorophyll a, pheophytin a and β-carotene contents of isolated Photosystem II reaction center complexes. Photosynth Res 52:69–73. doi: 10.1023/A:1005834006985 CrossRefGoogle Scholar
  15. 15.
    Grimm B, Porra RJ, Rüdiger W, Scheer H (2006) Chlorophylls and bacteriochlorophylls. doi:  10.1007/1-4020-4516-6
  16. 16.
    Grese RP, Cerny RL, Gross ML, Senge M (1990) Determination of structure and properties of modified chlorophylls by using fast atom bombardment combined with tandem mass spectrometry. J Am Soc Mass Spectrom 1:72–84. doi: 10.1016/1044-0305(90)80008-B CrossRefGoogle Scholar
  17. 17.
    Huang SC, Hung CF, Wu WB, Chen BH (2008) Determination of chlorophylls and their derivatives in Gynostemma pentaphyllum Makino by liquid chromatography-mass spectrometry. J Pharm Biomed Anal 48:105–112. doi: 10.1016/j.jpba.2008.05.009 CrossRefGoogle Scholar
  18. 18.
    Egner PA, Stansbury KH, Snyder EP, Rogers ME, Hintz PA, Kensler TW (2000) Identification and characterization of chlorin e(4) ethyl ester in sera of individuals participating in the chlorophyllin chemoprevention trial. Chem Res Toxicol 13:900–906CrossRefGoogle Scholar
  19. 19.
    Mendes-Pinto MM, Silva Ferreira AC, Caris-Veyrat C, Guedes de Pinho P (2005) Carotenoid, chlorophyll, and chlorophyll-derived compounds in grapes and port wines. J Agric Food Chem 53:10034–10041. doi: 10.1021/jf0503513 CrossRefGoogle Scholar
  20. 20.
    Aparicio-Ruiz R, Riedl KM, Schwartz SJ (2011) Identification and quantification of metallo-chlorophyll complexes in bright green table olives by high-performance liquid chromatrography-mass spectrometry quadrupole/time-of-flight. J Agric Food Chem 59:11100–11108. doi: 10.1021/jf201643s CrossRefGoogle Scholar
  21. 21.
    Srinivasan N, Haney C, Lindsey J, Zhang W, Chait B (1999) Investigation of MALDI-TOF mass spectrometry of diverse synthetic metalloporphyrins, phthalocyanines and multiporphyrin arrays. J Porphyrins Phthalocyanines 03:283–291. doi: 10.1002/(SICI)1099-1409(199904)3:4<283::AID-JPP132>3.0.CO;2-F CrossRefGoogle Scholar
  22. 22.
    Vieler A, Wilhelm C, Goss R, Süss R, Schiller J (2007) The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC. Chem Phys Lipids 150:143–155. doi: 10.1016/j.chemphyslip.2007.06.224 CrossRefGoogle Scholar
  23. 23.
    McCarley TD, McCarley RL, Limbach PA (1998) Electron-transfer ionization in matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 70:4376–4379. doi: 10.1021/ac980527i CrossRefGoogle Scholar
  24. 24.
    Macha SF, McCarley TD, Limbach PA (1999) Influence of ionization energy on charge-transfer ionization in matrix-assisted laser desorption/ionization mass spectrometry. Anal Chim Acta 397:235–245. doi: 10.1016/S0003-2670(99)00408-0 CrossRefGoogle Scholar
  25. 25.
    Wyatt MF, Stein BK, Brenton G (2006) Characterization of various analytes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile matrix. Anal Chem 78:199–206. doi: 10.1021/ac050732f CrossRefGoogle Scholar
  26. 26.
    De Winter J, Deshayes G, Boon F, Coulembier O, Dubois P, Gerbaux P (2011) MALDI-ToF analysis of polythiophene: use oftrans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile—DCTB—as matrix. J Mass Spectrom 46:237–246. doi: 10.1002/jms.1886 CrossRefGoogle Scholar
  27. 27.
    Suzuki T, Midonoya H, Shioi Y (2009) Analysis of chlorophylls and their derivatives by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Anal Biochem 390:57–62. doi: 10.1016/j.ab.2009.04.005 CrossRefGoogle Scholar
  28. 28.
    Boutaghou MN, Cole RB (2012) 9,10-Diphenylanthracene as a matrix for MALDI-MS electron transfer secondary reactions. J Mass Spectrom 47:995–1003. doi: 10.1002/jms.3027 CrossRefGoogle Scholar
  29. 29.
    Calvano CD, Monopoli A, Ditaranto N, Palmisano F (2013) 1,8-bis(dimethylamino)naphthalene/9-aminoacridine: a new binary matrix for lipid fingerprinting of intact bacteria by matrix assisted laser desorption ionization mass spectrometry. Anal Chim Acta 798:56–63. doi: 10.1016/j.aca.2013.08.050 CrossRefGoogle Scholar
  30. 30.
    Shroff R, Rulísek L, Doubsky J, Svatos A (2009) Acid–base-driven matrix-assisted mass spectrometry for targeted metabolomics. Proc Natl Acad Sci U S A 106:10092–10096. doi: 10.1073/pnas.0900914106 CrossRefGoogle Scholar
  31. 31.
    Calvano CD, Zambonin CG, Palmisano F (2011) Lipid fingerprinting of gram-positive lactobacilli by intact—matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge based matrix. Rapid Commun Mass Spectrom 25:1757–1764. doi: 10.1002/rcm.5035 CrossRefGoogle Scholar
  32. 32.
    Demeure K, Quinton L, Gabelica V, De Pauw E (2007) Rational selection of the optimum MALDI matrix for top-down proteomics by in-source decay. Anal Chem 79:8678–8685. doi: 10.1021/ac070849z CrossRefGoogle Scholar
  33. 33.
    Molin L, Seraglia R, Dani FR, Moneti G, Traldi P (2011) The double nature of 1,5-diaminonaphthalene as matrix-assisted laser desorption/ionization matrix: some experimental evidence of the protonation and reduction mechanisms. Rapid Commun Mass Spectrom 25:3091–3096. doi: 10.1002/rcm.5201 CrossRefGoogle Scholar
  34. 34.
    Thomas A, Charbonneau JL, Fournaise E, Chaurand P (2012) Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition. Anal Chem 84:2048–2054. doi: 10.1021/ac2033547 CrossRefGoogle Scholar
  35. 35.
    Willows RD, Lake V, Roberts TH, Beale SI (2003) Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus. J Bacteriol 185:3249–3258CrossRefGoogle Scholar
  36. 36.
    Sievers G, Hynninen PH (1977) Thin-layer chromatography of chlorophylls and their derivatives on cellulose layers. J Chromatogr 134:359–364CrossRefGoogle Scholar
  37. 37.
    Mínguez-Mosquera MI, Gallardo-Guerrero L, Hornero-Méndez D, Garrido-Fernández J (1995) Involvement of copper and zinc ions in green staining of table olives of the variety Gordal. J Food Protect 5:473–578Google Scholar
  38. 38.
    Mirza SP, Raju NP, Vairamani M (2004) Estimation of the proton affinity values of fifteen matrix-assisted laser desorption/ionization matrices under electrospray ionization conditions using the kinetic method. J Am Soc Mass Spectrom 15:431–435. doi: 10.1016/j.jasms.2003.12.001 CrossRefGoogle Scholar
  39. 39.
    Burton RD, Watson CH, Eyler JR, Lang GL, Powell DH, Avery MY (1997) Proton affinities of eight matrices used for matrix-assisted laser desorption/ionization. Rapid Commun Mass Spectrom 11:443–446. doi: 10.1002/(SICI)1097-0231(199703)11:5<443::AID-RCM897>3.0.CO;2-3 CrossRefGoogle Scholar
  40. 40.
    Maier JP (1974) Photoelectron spectroscopy of peri-amino naphthalenes. Helv Chim Acta 57:994–1003. doi: 10.1002/hlca.19740570405 CrossRefGoogle Scholar
  41. 41.
    Nakato Y, Chiyoda T, Tsubomura H (1974) Experimental determination of ionization potentials of organic amines, b-carotene and chlorophyll a. Bull Chem Soc Jpn 47:3001CrossRefGoogle Scholar
  42. 42.
    Demeure K, Gabelica V, De Pauw EA (2010) New advances in the understanding of the in-source decay fragmentation of peptides in MALDI-TOF-MS. J Am Soc Mass Spectrom 21:1906–1917. doi: 10.1016/j.jasms.2010.07.009 Google Scholar
  43. 43.
    Glasovac ZV, Margetić D (2009) Proton affinities of didehydroporphyrin and subporphyrin in ground and excited states obtained by quantum chemical calculations. Croat Chem Acta 82:63–70Google Scholar
  44. 44.
    Wei J, Li H, Barrow MP, O’Connor PB (2013) Structural characterization of chlorophyll-a by high resolution tandem mass spectrometry. J Am Soc Mass Spectrom 24:753–760. doi: 10.1007/s13361-013-0577-1 CrossRefGoogle Scholar
  45. 45.
    Roberts EAH (1958) The chemistry of tea manufacture. J Sci Food Agric 9:381–390Google Scholar
  46. 46.
    Schwarts SJ, Eelbe JH (1983) Kinetics of chlorophyll degradation to pyropheophytin in vegetables. J Food Sci 48:1303–1306. doi: 10.1111/j.1365-2621.1983.tb09216.x CrossRefGoogle Scholar
  47. 47.
    Jerz G, Arrey TN, Wray V, Du Q, Winterhalter P (2007) Structural characterization of 132-hydroxy-(132-S)-phaeophytin-a from leaves and stems of Amaranthus tricolor isolated by high-speed countercurrent chromatography. Innov Food Sci Emerg Technol 8:413–418. doi: 10.1016/j.ifset.2007.03.024 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Cosima Damiana Calvano
    • 1
    • 2
  • Giovanni Ventura
    • 1
  • Tommaso R. I. Cataldi
    • 1
    • 2
    Email author
  • Francesco Palmisano
    • 1
    • 2
  1. 1.Dipartimento di ChimicaUniversità degli Studi di Bari “Aldo Moro”BariItaly
  2. 2.Centro di Ricerca Interdipartimentale S.M.A.R.T.Università degli Studi di Bari “Aldo Moro”BariItaly

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