Oguz CAKIR, Enes ARICA, Mustafa Abdullah YILMAZ, Abdulselam ERTAS, İsmail YENER, Birol OTLUDİL, Mehmet Hakki ALMA

An LC-MS/MS Method Validation for the Phytochemical Quantification of Four Edible Plants

Bitkilerde 8 fitokimyasal bileşiğin (sinarin, kafeik asit, sirinjik asit, p-kumarik asit, o-kumarik asit, vanilik asit, ferulik asit, klorojenik asit) miktarsal tayini için kapsamlı bir LC-MS/MS metodu geliştirildi ve validasyon çalışmaları yapıldı. Geliştirilen analitik metot lineerite, gerçeklik (geri kazanım), gün içi ve günlerarası kesinlik (tekrarlanabilirlik ve tekrar üretilebilirlik), tespit ve tayin limitleri (LOD/LOQ) ve bağıl standart belirsizlik (% 95 güven aralığında (k=2)) gibi parametreleri içerek şekilde tam validasyon çalışmaları yapıldı. Kromatografik ayrım ters faz UHPLC sistemi ile yapıldı. Kütle dedeksiyonu ise her analit için negative veya pozitif iyonlaşma modları optimize edilerek üçlü kuadrupol kütle spektrometresi ile gerçekleştirildi. Analitlerin miktarsal tayini için çoklu reaksiyon görüntüleme (MRM) kullanıldı, moleküler iyonlar ve ilgili geçiş iyonları ise optimize edildi. Metot validasyonu sonrası, enginar (Cynara scolymus L.), brokoli (Brassica Oleracea var. Italica), karnabahar (Brassica Oleracea var. Botrytis) ve kenger gibi (Gundelia Tournefortii) bazı yenilebilir bitkilerin methanol ekstrelerinin fitokimyasal içerikleri geliştirilen ve valide edilen LC-MS/MS metodu ile tespit edildi. Analiz edilen bitkiler arasında enginar, açık ara farkla fenolik yönünden en zengini olarak belirlendi. Ek olarak, analiz edilen tüm bitkilerde klorojenik asit en bol bulunan bileşen olarak görüldü. Çalışılan örnekler analiz için seçilmiş bitkiler olsa da, geliştirilen LC-MS/MS metodu bitki dünyasındaki pekçok bitkiye uygulanabilirdir

Yenilebilir Dört Bitki Türünün Fitokimyasal İçeriğinin Miktarsal Tayini için LC-MS/MS Metot Validasyonu

A comprehensive LC-MS/MS method was developed and validated for the quantification of eight plant phytochemicals (cynarin, caffeic acid, syringic acid, p-coumaric acid, o-coumaric acid, vanillic acid, ferulic acid, chlorogenic acid) in plants. The developed analytical method was fully validated in terms of linearity, accuracy (recovery), inter and intra-day precision (repeatability), limits of detection and quantification (LOD/LOQ) and relative standard uncertainty (U% at 95% confidence level (k=2)). Chromatographic separation was performed on a reverse phased UHPLC. MS detection was performed using a triple quadrupole mass spectrometer and negative or positive ionization modes were optimized for each analyte. Multiple reaction monitoring (MRM) was used to quantify the analytes, related molecular ions and transition ions were optimized. After method validation, the phytochemical composition of methanolic extracts of some edible plants including artichoke (Cynara scolymus L.), broccoli (Brassica Oleracea var. Italica), cauliflower (Brassica Oleracea var. Botrytis) and tumble thistle (Gundelia Tournefortii) were investigated by the developed and validated LC-MS/MS method. Among the analysed plants, artichoke was by far the richest one in terms of phenolics. Additionally, chlorogenic acid was the most abundant phenolic compound in all plants. Although the studied edible plants were chosen as real samples, the developed LC-MS/MS method is applicable to a wide range of species in plant kingdom

PDF

___

Ares AM, Nozal MJ, Bernal J, 2013. Extraction: chemical characterization and biological activity determination of broccoli health promoting compounds. Journal of Chromatography A, 1313: 78–95.
Bahorun T, Luximon-Ramma A, Crozier A and Aruoma, OI, 2004. Total phenol, flavonoid, proanthocyanidin and vitamin C levels and antioxidant activities of Mauritan vegetables. Journal of the Science of Food and Agriculture, 84: 1553–1561.
Balasundram N, Sundram K, Samman S, 2006. Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chemistry, 99: 191-203.
Beckman C H, 2000. Phenolic-storing celss: keys to programmed cell death and periderm formation in wilt disease resistance and in general defence responses in plants? Physiological Molecular Plant Pathol 57: 101-110.
Bianco VV, 2005. Present sıtuatıon and future potentıal of artıchoke ın the medıterranean basın. Acta Hortic. 681: 39-58.
Brat P, Georgé S, Bellamy A, Chaffaut LD, Scalbert A, Mennen L, Arnault N, Amiot MJ, 2006. Daily polyphenol intake in france from fruit and vegetables. The Journal of Nutrition, 136: 2368–2373.
Coruh N, Sagdicoglu Celep AG, Ozgokce F, Iscan M, 2007. Antioxidant capacities of Gundelia tournefortii L. extracts and inhibition on glutathione-Stransferase activity. Food Chemistry, 100(3): 1249-1253.
Del Rio D, Rodriguez-Mateos A, Spencer JP, Tognolini M, Borges M, Crozier A, 2013. Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxidants Redox Signaling 18: 1818–1892.
EURACHEM CITAC Guide CG4. 2004. Quantifiying Uncertainty in Analytical Measurement, 3rd ed.; Ellison, S. L. R., Williams, A.,, Eds.; available from www.eurachem.org.
Feliciano RP, Pritzel S, Heiss C, Rodriguez-Mateos A, 2015. Flavonoid intake and cardiovascular disease risk. Current Opinion Food Science 2: 92–99.
Ferreres F, Valentão P, Llorach R, Pinheiro C, Cardoso L, Pereira JA, Seabra RM, Andrade PB, 2005. Phenolic compounds in external leaves of tronchuda cabbage (Brassica oleracea L. Var. costata DC). Journal of Agricultural and Food Chemistry, 53: 2901–2907.
Haghi G, Hatami A, Arshi R, 2011. Distribution of caffeic acid derivatives in Gundelia tournefortii L. Food Chemistry, 124: 1029-1035.
Harbaum B, Hubbermann EM, Wolff C, Herges R, Zhu Z and Schwarz K, 2007. Identification of flavonoids and hydroxycinnamic acids in Pak Choi varieties (Brassica campestris L. spp. Chinensis var. communis) by HPLC–ESI-MSn and NMR and their quantification by HPLC-DAD. Journal of Agricultural and Food Chemistry, 55: 8251–8260.
Häusler M, Ganzera M, Abel G, Popp M and Stuppner H, 2002. Determination of caffeoylquinic acids and flavonoids in Cynara scolymus L. by high performance liquid chromatography. Chromatographia, 56: 407–411.
Jun NJ, Jang KC, Kim SC, Moon DY, Seong KC, Kang KH, Tandang L, Kim PH, Cho SK, Park KH, 2007. Radical scavenging activity and content of cynarin (1,3-dicaffeoylquinic acid) in Artichoke (Cynara scolymus L.). Journal of Applied Biological Chemistry, 50: 244–248.
Kumar S, Andy A, 2012. Health promoting bioactive phytochemicals from Brassica. International Food research Journal, 19: 141–152.
Lattanzio V, Kroon PA, Linsalata V and Cardinali A, 2009. Globe artichoke: A functional food and source of nutraceutical ingredients. Journal of Functional Foods, 1: 131–144.
Latte KP, Appel K, Lampen A, 2011. Health benefits and possible risks of broccoli—an overview. Food and Chemical Toxicology. 49: 3287–3309.
Llorach R, Gil-Izquierdo A, Ferreres F and Tomás-Barberán FA, 2003. HPLCDAD-MS/MS ESI characterization of unusual highly glycosylated acylated flavonoids from cauliflower (Brassica oleracea L. var. botrytis) agroindustrial byproducts. Journal of Agricultural and Food Chemistry, 51: 3895–3899.
Lombardo S, Pandino G, Mauromicale G, Knödler M, Carle R and Schieber A, 2010. Influence of genotype, harvest time and plant part on polyphenolic composition of globe artichoke [Cynara cardunculus L. var. scolymus (L.) Fiori]. Food Chemistry, 119: 1175–1181.
Mulinacci N, Prucher D, Peruzzi M, Romani A, Pinelli P, Giaccherini C and Vincieri F F. 2004. Commercial and laboratory extracts from artichoke leaves: Estimation of caffeoyl esters and flavonoidic compounds content. Journal of Pharmaceutical and Biomedical Analysis, 34: 349–357.
Negro D, Montesano V, Grieco S, Crupi P, Sarli G, De Lisi A, Sonnante G, 2012. Polyphenol compounds in artichoke plant tissues and varieties. Journal of Food Science, 77: 244-252.
Rodriguez-Mateos A, Vauzour D, Krueger CG, Shanmuganayagam D, Reed J, Calani L, Mena P, Del Rio D, Crozier A, 2014. Bioavailability, bioactivity and impact on health of dietary flavonoids and related compounds: an update. Archives of Toxicology, 88: 1803–1853.
Sánchez-Rabaneda F, Jáuregui O, Lamuela-Raventós RM, Bastida J, Viladomat F and Codina C, 2003. Identification of phenolic compounds in artichoke waste by high-performance liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 1008: 57–72.
Schütz K, Muks E, Carle R and Schieber A, 2006. Quantitative determination of phenolic compounds in artichoke-based dietary supplements and pharmaceuticals by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry, 54: 8812–8817.
Shahidi F, 1997. Natural Antioxidants: An Overview, in ‘Natural Antioxidants: Chemistry, Health Effects and Applications’, IL, AOCS Press, Champaign, 1-7.
Sikora E, Bodziarczyk I, 2012. Composition and antioxidant activity of kale (Brassica oleracea l: Var. acephala) raw and cooked. Technologia Alimentaria, 11: 239–248.
Tiveron AP, Melo PS, Bergamaschi KB, Vieira TM, Regitano-d’Arce MA, Alencar SM, 2012. Antioxidant activity of Brazilian vegetables and its relation with phenolic composition. International Journal of Molecular Sciences, 13: 8943-8957.
Turati F, Rossi M, Pelucchi C, Levi F, La Vecchia C, 2015. Fruit and vegetables and cancer risk: a review of southern European studies. The British of nutrition, 2: 102–110.
Vallejo F, Tomás-Barberán FA and Ferreres F, 2004. Characterisation of flavonols in broccoli (Brassica oleracea L. var. italica) by liquid chromatography-UV diodearray detection-electrospray ionization mass spectrometry. Journal of Chromatography A, 1054: 181–193.
Wang M, Simon JE, Aviles IF, He K, Zheng QY and Tadmor Y, 2003. Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.). Journal of Agricultural and Food Chemistry, 51: 601–608.