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

Abstract

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.

Keywords

• Edible plants LC-MS/MS,method validation,phytochemicals

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

Abstract

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.

Keywords

• Fitokimyasal,LC-MS/MS,metot validasyonu,yenilebilir bitkiler

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