Development and application of UHPLC–MS/MS method for the determination of phenolic compounds in Chamomile flowers and Chamomile tea extracts
Introduction
Chamomile (Matricaria recutita L.) is a medicinal plant often used for its analgesic, anti-allergic, anti-spasmodic, antibacterial, anti-inflammatory and sedative properties. Its essential oil containing volatile compounds including terpenoids such as azulene, chamazulene and α-bisabolol is the most commonly used [1]. Recently, the attention has been paid also to non-volatile fraction of the extract, especially phenolic compounds for their spasmolytic and antiphogistic activity [2] and for antioxidant activity. Antioxidant activity of Chamomile was found to be 0.42 of quercetin equivalent or 1.30 of trolox equivalent [3].
As described in literature, flavonoid glycosides represent the major fraction of water-soluble components in Chamomile. Apart from the glycosides, flavonoid aglycones were found in great variety among lipophilic constituents [2]. Apigenin and later apigenin-7-glucoside were the first flavonoid compounds isolated from Chamomile [4], [5]. Phenolic fraction of Chamomile might further contain phenolic acids: chlorogenic acid, caffeic acid, vanillic acid, syringic acid and anisic acid, coumarins: umbelliferone and herniarin and flavonoids including aglycones and/or glycosides from: isorhamnetin, luteolin, quercetin, apigenin, patuletin and some others [2], [3]. There are differences among individual Chamomile plant types and similarly, the process of drying and tea product preparation might influence the content of phenolic compounds.
Only few analytical methods for qualitative and quantitative evaluation of the Chamomile extracts have been published so far. Phenolic fraction of Chamomile was previously analyzed by means of reversed phase chromatography with UV detection [6], [7], [8], [9], [10] or MS detection [6], [7], [11], [12] or by capillary electrochromatography (CEC), capillary zone electrophoresis (CZE) and μ-HPLC, which were compared by Fonseca et al. [13]. CEC was found to be a powerful tool enabling high efficiency and resolution however on the other hand an impractical approach which requires very long column conditioning and use of fragile, often self-made capillary columns, that makes CEC time-consuming and non-robust method [13]. HPLC methods on the other hand were typically very time-consuming, which means about 50 min for one analytical run [6], [7], [10], [11] and they were not validated for quantitative purposes, which might be caused also by quite old origin of the methods [6], [7], [10], [11]. A combination of two different methods was often used for the quantitation of various groups of phenolics such as phenolic acids and coumarins [7] or phenolic acids and flavonoids [14] or solely phenolic acids were determined in Chamomile samples [15].
Only identification and description of phenolic profile of the Chamomile tea was performed by means of HPLC–ESI-MS [3]. Some of developed methods were focused on the determination of one of Chamomile's the most abundant compounds – apigenin and its derivatives using CZE [16], HPLC [17] or HPLC–MS [18].
Sensitive, efficient and selective methods for the quantitative evaluation of phenolic fraction of extracts of Chamomile (M. recutita) are still missing in scientific literature as it is demonstrated by low number of given references and also by their old origin. The development of such method might be a difficult task due to variability of phenolic compounds present in phenolic fraction and due to great differences in their concentration and polarity. Moreover, individual Chamomile varieties may significantly differ in quantitative phenolic profile as well. High selectivity of detection, high separation efficiency and wide linear range of the determination are therefore necessary. In this paper, an original method using coupling of high separation efficiency of UHPLC with tandem mass spectrometry using triple quadrupole analyzer for its wide linearity range was developed. ESI in both positive and negative ion mode was compared in analysis of 12 phenolic compounds (Fig. 1). Finally, the method was applied for the comparison of phenolic content of Chamomile flowers and Chamomile tea extracts.
Section snippets
Chemicals and reagents
Working standards of chlorogenic acid, caffeic acid, umbelliferone, rutin, quercetin-3-glucoside, apigenin-7-glucoside, quercitrin, quercetin, luteolin, kaempherol, apigenin and isorhamnetin were used for the purpose of this study. All compounds were obtained from Sigma–Aldrich (Prague, Czech Republic). Formic acid LC–MS grade was purchased by Sigma–Aldrich as was LC–MS grade methanol. Ultra-pure water was obtained with a Milli-Q reverse osmosis Millipore (Bedford, MA, USA) and met the
Development of UHPLC–MS/MS method
Acquity BEH C18 analytical column was chosen for the separation of 12 selected phenolic compounds based on our previous experience [22], [23]. Development of separation method addressed two important issues – the separation of luteolin and kaempherol, as the molecular weight 286 is the same for both structures and secondly, the separation of caffeic and chlorogenic acid, as in fact, chlorogenic acid contains caffeic acid in its structure and this ester bond might easily be fragmented. Efficient
Conclusion
A novel UHPLC–MS/MS method for the quantitation of 12 phenolic compounds in methanolic extracts of Chamomile flowers and Chamomile tea was developed. The method combined high separation efficiency of UHPLC together with wide linear range of triple quadrupole mass analyzer therefore it possessed high sensitivity and selectivity. One of the key issues of the method development was the right choice of dissolution media for standard mixtures and dilution of Chamomile samples. 60% methanol in water
Acknowledgements
The authors gratefully acknowledge research projects MSM0021620822, MSM6046070901 and technical support of Waters, Czech Republic.
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