Original Papers 559 A First Step in the Quest for the Active Constituents in Filipendula ulmaria (Meadowsweet): Comprehensive Phytochemical Identification by Liquid Chromatography Coupled to Quadrupole-Orbitrap Mass Spectrometry Authors Sebastiaan Bijttebier1, Anastasia Van der Auwera1, Stefan Voorspoels2, Bart Noten2, Nina Hermans1, Luc Pieters1, Sandra Apers1 Affiliations 1 University of Antwerp, Natural Products & Food Research and Analysis (NatuRA), Antwerp, Belgium 2 Flemish Institute for Technological Research (VITO), Business Unit Separation and Conversion Technology (SCT), Mol, Belgium Key words Abstract ray-accurate mass mass spectrometry methods. l" Filipendula ulmaria ! Selective ion fragmentation experiments with a l" Rosaceae Filipendula ulmaria (meadowsweet) is tradition- hybrid quadrupole-orbital trap mass spectrome- l" meadowsweet ally used for the treatment of inflammatory dis- ter significantly contributed to compound identi- l" LC‑PDA‑amMS eases and as a diuretic and antirheumatic. Ex- fication: a total of 119 compounds were tenta- l" flavonoids l" salicylic acid derivatives tracts of Filipendulae herba are on the market in tively identified, 69 new to F. ulmaria. A rich di- l" tannins the European Union as food supplements. Never- versity of phenolic constituents was detected theless, its active constituents remain to be re- and only a few non-phenolic phytochemicals vealed. During this study, the phytochemical were observed. Metabolisation and pharmacolog- composition of Filipendulae Ulmariae Herba was ical studies should be conducted to investigate comprehensively characterised for the first time which of these constituents or metabolites there with two complementary generic ultrahigh-per- of contribute to the activity of F. ulmaria after oral formance liquid chromatography-photodiode ar- intake. Introduction [3–9]. Only a limited number of non-phenolic ! constituents such as phytosterols, carotenoids, Preparations from the herb and/or flowers of Fili- triterpenes, and chlorophyll derivatives have pendula ulmaria (L.) Maxim (Rosaceae) (meadow- been reported [7,10,11]. In view of the phenolic sweet) have been used traditionally since the late nature of the main constituents reported in 16th and 17th century for the treatment of in- meadowsweet, extensive metabolisation after flammatory diseases and as a diuretic and anti- oral intake before absorption can be expected rheumatic. F. ulmaria is administered as herbal [12]. Indeed, natural products are often pro- received August 21, 2015 revised Dec. 7, 2015 tea, as a powdered herbal substance in solid dos- drugs, e.g., glycosides, which must undergo in accepted Dec. 14, 2015 age form for oral use, and as a tincture. In some vivo metabolic conversion (activation). Salicylic countries of the European Union, tinctures or acid, the in vivo metabolite of salicylic alcohol de- Bibliography This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. DOI http://dx.doi.org/ possible tincture-based products containing alco- rivatives present in the plant extract, is responsi- 10.1055/s-0042-101943 holic extracts of Filipendulae Herba are on the ble for part of the pharmacological activity [5,13]. Published online February 4, market as food supplements used for complaints Nevertheless, the chemistry of meadowsweet has 2016 such as rheumatic and arthritic pain [1]. Because not been studied in a comprehensive manner and Planta Med 2016; 82: 559–572 of its anti-inflammatory properties, Harbourne et its active constituents remain to be revealed. © Georg Thieme Verlag KG Stuttgart · New York · al. studied the phenolic content of aqueous F. ul- Phytochemical studies have often dedicated their ISSN 0032‑0943 maria extracts for their incorporation into a bev- efforts towards the identification of a limited erage as a functional ingredient [2]. number of metabolites, which renders the avail- Correspondence Dr. Sebastiaan Bijttebier It has been shown that meadowsweet contains able information fragmented. Usually detectors Natural Products & Food phenolic constituents such as flavonoid aglycons designed for targeted analysis such as photodiode Research and Analysis (NatuRA) (e.g., quercetin, kaempferol), glycosylated flavo- array detectors (PDA) and triple quadrupole mass University of Antwerp – – Universiteitsplein 1 noids (e.g., rutin, hyperoside, quercitrin, avicular- spectrometry detectors are used [2 5, 7 9,14]. 2610 Antwerp in, astragalin), and hydrolysable tannins (tellima- These detectors often do not provide sufficient Belgium grandin I and II, rugosin A, B, D, and E) as well as structural information for compound identifica- Phone: + 3232652733 sebastiaan.bijttebier@ salicylates (salicylic acid, methyl salicylate, sa- tion in complex mixtures without the use of ana- uantwerpen.be licylaldehyde, salicylalcohol and their glycosides) lytical standards [15]. Due to the lack of commer- Bijttebier S et al. A First Step… Planta Med 2016; 82: 559–572 560 Original Papers cially available analytical standards, identification of unknown Although data-dependent fragmentation (ddMS2) provides vast metabolites, therefore, often result in tedious multistep purifica- amounts of structural information, in some cases, the generated tion protocols involving solvent extractions followed by sequen- product ions may not suffice for full characterisation of the sub- tial fractionations with open-bed liquid chromatography (LC) structures (e.g., aglycon moieties of flavonoid glycosides). In and/or (semi) preparative high-performance LC (HPLC) [6,16]. these cases, in-source collision-induced dissociation (CID) frag- To obtain unambiguous structure elucidation and compound mentation was used to generate substructure product ions in a identification, nuclear magnetic resonance (NMR) still remains first step. These in-source product ions were subsequently se- indispensable. NMR is, however, not a very sensitive technique lected with the quadrupole for higher energy collisional dissocia- and requires compound purification before detection to allow tion (HCD) fragmentation to obtain pseudo MS3 spectra for their proper structure elucidation. One of the possible strategies to cir- tentative identification. cumvent this sensitivity issue is the use of a solid-phase extrac- Structures were assigned to unknown peaks only when both the tion (SPE) interface in an LC‐SPE‐NMR configuration [17]. mass/charge (m/z) ratios and molecular formulae of the precur- In the past decade, the usage of analytical instrumentation has sor and product ions were in agreement. PDA spectra and reten- shifted from simple analytical instrumentation, such as HPLC- tion times often provided additional confirmation of the pro- PDA, towards more selective and more complex separation and posed structures. The information obtained by analysis is, how- detection systems, such as ultrahigh-performance liquid chroma- ever, not always sufficient for peak identification at an acceptable tography-photodiode array-accurate mass mass spectrometry confidence level. Additional information for successful dereplica- (UHPLC‑PDA-amMS), to achieve more definite compound identi- tion was often acquired from in-house and commercial com- fication [18]. Accurate mass MS detectors facilitate the tentative pound databases and peer reviewed publications. l" Tables 1 and identification of unknown metabolites without the use of analyt- 2 show the diagnostic amMS and PDA data used for chroma- ical standards. Orbitrap MS detectors can routinely generate tographic peak identification. These tables also specify the litera- mass spectra with a resolving power up to 140 000 at full width ture consulted for confirmation of compound identity. at half maximum (FWHM) and obtain mass accuracies within 1– Application of a generic LC‑PDA-amMS method for the identifica- 2 ppm; this enables the calculation of the most probable molecu- tion of moderately polar phytochemicals enabled the identifica- lar formulae of the generated precursor and product ions [19]. tion of a multitude of phenolic constituents, many of which have This utility combined with the selectivity and sensitivity of cur- never been reported before in F. ulmaria. Compounds identified rent hyphenated UHPLC‑PDA-amMS systems has paved the way for the first time in F. ulmaria are indicated in italics in l" Tables towards generic phytochemical analysis and has led to a very sig- 1 and 2. l" Fig. 1 provides an overview of the retention times and nificant impact of MS-based technologies in the field of meta- m/z values in the heated electrospray ionisation (HESI) negative bolomics [18]. However, a wide diversity of phytochemical struc- mode of the compounds identified with this method. The data tures is produced in nature. Consequently, the complete metabo- labels match the compound numbers in l" Table 1. Predomi- lite profile cannot be extracted with one solvent nor be analysed nantly deprotonated molecules ([M – H]−) were formed in the with one analytical method. Comprehensive phytochemical char- negative ionisation mode. Formic acid adducts ([M – H+FA]−), acterisation should, therefore, be performed with several analyt- deprotonated dimers ([2 M – H]−), doubly deprotonated mole- ical methods to (ideally) cover the whole range of plant metabo- cules ([M – 2H]2−) and product ions caused by in-source frag- lites present in plants. mentation were also observed (l" Table 1). In the positive ionisa- The goal of the current study was to explore the phytochemical tion