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Phytochemical analysis of isoflavonoids using liquid chromatography coupled with tandem mass spectrometry

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Phytochemical analysis of isoflavonoids using liquid chromatography coupled with tandem mass spectrometry

Kanumuri Siva Rama Raju • Naveen Kadian • Isha Taneja • M. Wahajuddin

Received: 31 December 2014 / Accepted: 12 March 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Isoflavonoids are the biologically active made it possible to analyse and characterize several secondary metabolites of plants that are being used for constituents and their metabolites in a single run along several health promoting and restoring effects medi- with high selectivity and sensitivity. In this review, we ated through different pathways. Isoflavonoids are have summarised the application of LC–MS/MS for structurally similar to estrogens due to which also the identification and quantification of isoflavonoids known as phytoestrogens and have shown potent reported for various plant extracts and food products estrogenic and anti-estrogenic activity. Association along with their general extraction procedures and with large biological activity lead to the need of rapid, factors affecting extraction providing a view towards sensitive and precise quantitation of different iso- the conditions used for their analysis. The most flavonoids in different plant extracts, food materials suitable and widely acceptable extraction solvent and biological matrices as the biological activities system for the isoflavonoids is methanol or ethanol mainly depends on the quantities and nature of in combination with water ranging from 40 to 60 % isoflavonoids present in them. The characterisation, organic solvent based on the type of tissues and the standardisation and quantification of herbal extracts or isoflavonoids to be extracted by different extraction food products require techniques that are highly techniques. ESI ionization with Q-TOF MS was the selective, sensitive and also provide mass measure- most useful detection system for the characterisation ment precisions and structural information. Liquid and quantification of the diverse isoflavonoids with chromatography with tandem mass spectroscopy has molecular insights.

Keywords LC–MS/MS Isoflavonoids Kanumuri Siva Rama Raju, Naveen Kadian and Isha Taneja Phytoestrogens Extraction Polyphenols have contributed equally to this work.

CDRI Communication No. 8948. Abbreviations APCI Atmospheric pressure chemical K. S. R. Raju N. Kadian I. Taneja M. Wahajuddin Pharmacokinetics and Metabolism Division, CSIR- ionization Central Drug Research Institute, Lucknow, India CHS Chalcone synthase CID Collision induced dissociation & K. S. R. Raju I. Taneja M. Wahajuddin ( ) DAD Diode array detector Academy of Scientific and Innovative Research, New Delhi, India DHB 2,5-Dihydroxybenzoic acid e-mail: [email protected]; [email protected] ECD Electron capture dissociation 123 Phytochem Rev

ESI Electro spray ionization Lissin and Cooke 2000). Along with their estrogen EST Expressed sequence tag receptor modulatory effects, other pharmacological FWHM Full peak width at one-half maximum activities such as antifungal, antibacterial, antioxidant, HPLC High performance liquid anti-inflammatory, anti-diabetic, anticancer, cardio- chromatography vascular diseases, menopause, osteoporosis and anti- HPLC– High performance liquid obesity activity have been reported for various MS chromatography coupled with mass isoflavonoids (Barnes 1998; Hsu et al. 2000; Davis spectrometer et al. 1998; Hillman et al. 2001; Davis et al. 2000; Hsu IFS Isoflavone synthase et al. 1999; Kenny and Prestwood 2000; Anthony ILUAE Ionic liquid based ultrasonic assisted 2000; Hsu et al. 2000; Van der Schouw et al. 2000; extraction Kenny and Prestwood 2000; Rostagno et al. 2002). IT Ion trap Along with the above mentioned pharmacological LC Liquid chromatography activities, various anticancer mechanisms (i.e. asso- MALDI Matrix-assisted laser desorption ciation with lower incidences of hormonally dependent MS Mass spectroscopy cancers) were detected by in vitro experiments, using MS/MS Tandem mass spectroscopy cell cultures, and immune-suppressed mice carrying NMR Nuclear magnetic resonance xenotransplants of human cancer cells (Klejdus et al. spectroscopy 2005a). They are believed to have some beneficial PDA Photodiode array effects in humans upon epidemiological studies indi- PLE Pressurized liquid extraction cating that the Asian people are less prone to incidence Q-TOF Quardrupole time-of flight mass of carcinoma of the breast, prostate, large intestine, and spectrometer cardiovascular diseases compared to western people as rDA retro-Diels–Alder the Asian diets contain higher amounts of the RP-HPLC Reverse-phase liquid chromatography isoflavonoids (Adlercreutz et al. 2000). But there are SFE Supercritical fluid extraction no experimental evidences to prove the same till now. SPE Solid phase extraction Isoflavonoids occur predominantly in their precursor SPME Solid-phase micro extraction forms such as acetate, malonyl and glycoside e.g. SRM Selected reaction monitoring daidzin, the precursor of diadzein (the glycosidic form TLC Thin layer chromatography that contains a carbohydrate moiety) (Sharma and UAE Ultrasound assisted extraction Ramawat 2013). UHPLC Ultra high performance liquid The basic pathways for the synthesis of core chromatography isoflavonoid skeletons have been established both UV Ultraviolet–Visible spectroscopy enzymatically and genetically. Many flavonoids and isoflavonoids are derived from the flavonoid biosyn- thesis pathway using liquiritigenin or naringenin as precursors (Database; Ralston et al. 2005). The other Introduction precursors of flavonoid biosynthesis include shikimic acid, phenylalanine, cinnamic acid, and p-coumaric Isoflavonoids are heterocyclic phenolic secondary acid. Shikimic acid acts as an intermediate in the metabolites present in plants which are biogenetically biosynthesis of aromatic acid (Sharma and Ramawat related to flavonoids but constitute a distinctly separate 2013). The entry point enzymes are the polyketide class containing rearranged C15 skeleton and may be synthase, chalcone synthase (CHS) and isoflavone regarded as derivatives of 3-phenylchroman while synthase (IFS), more correctly termed as 2-hy- flavonoids are 2-phenylchromans (Cheng et al. 1997). droxyisoflavanone synthase, a cytochrome P450 that Most of the isoflavonoids structurally resemble en- catalyzes the aryl migration reaction that converts a dogenous 17b-estradiol and has the ability to modulate 2-phenylchroman to a 3-phenylchroman (Database). the estrogen receptors, showing both estrogenic and The structural diversity of isoflavonoids is derived by anti-estrogenic effects, due to which they are com- substitution of these basic carbon skeletons through monly known as phytoestrogens (Casanova et al. 1999; further hydroxylation, glycosylation, methylation, 123 Phytochem Rev acylation and prenylation as well as, in the case of procedure according to the type of plant tissue from proanthocynadins (also known as condensed tannins) which they are to be sought i.e. heartwood, seeds, and phalobaphenes, by polymerization (Dixon and leaves, rhizomes, roots etc. The need for sample Pasinetti 2010; Dixon and Steele 1999). The enzymes preparation depends strongly on the sample type and that catalyses the substitution reactions are often the analysis techniques to be employed (Bajer et al. encoded by large gene families, which can be recog- 2007). Extraction technique, extraction solvent, pH of nized in expressed sequence tag (EST) and genome the solvent, extraction time, temperature, sample to data sets through family-specific conserved sequence solvent ratio, sample characteristics are the defining motifs (Dixon and Pasinetti 2010). In comparison to factors that affect extraction efficiency, isoflavonoids the flavonoids (2-phenylchromans), which are found profile and recovery (Rostagno et al. 2010). The main widely in higher plants, the isoflavonoids appear in a objective of sample treatment is to eliminate the limited occurrence of taxon, probably owing to the undesired matrix components and the enrichment of limited occurrence of IFS enzyme, being confined samples. The extraction procedures must allow quan- essentially to the subfamily Papilionoideae (Lo- titative recovery of isoflavonoids in their native form, toideae) of the leguminosae (Klejdus et al. 2005a; whilst avoiding any chemical modification or degra- Veitch 2007, 2009). There are however, occasional dation during the processes. In addition to the examples of their occurrence in the subfamily Cae- traditional extraction methods i.e. maceration, perco- salpinioidae, and in other families (Rosaceae, Moraceae, lation, soxhilation which have been used for decades, Amaranthaceae, Podocarpaceae, Chenopodiaceae, Cu- newer techniques like accelerated solvent extraction, pressaceae, Iridaceae, Myristicaceae and Stemonaceae) extraction using ultra sonication, microwave assisted together with some reports of their isolation from extraction (Rostagno et al. 2007), supercritical fluid microbial sources (Dewick 1982; Reynaud et al. 2005). extraction (Cheng et al. 2011; Klejdus et al. 2005a, Structurally, the isoflavonoids may be subdivided 2010; Rostagno et al. 2002), ionic liquid based into several sub-classes according to the degree of ultrasonic assisted extraction (ILUAE), solid phase oxidation in the skeleton, and the complexity of the extraction (SPE) and pressurized liquid extraction skeleton, for instance, formation of further hetero- (PLE) (Bajer et al. 2007; Rostagno et al. 2004) have cyclic rings. Major sub-classes include isoflavones, been increasingly reported for the isolation of isoflavanones, isoflavans, 6a-hydroxypterocarpans, isoflavonoids from plant products. These techniques coumestans, rotenoids, dehydro-rotenoids, isoflav-3- were developed for application in natural product enes, 2-arylbenzofuran, isoflavanquinones, other extraction owing to their advantages with regard to the classes of isoflavonoids are relatively rare. The basic extraction time, reproducibility and better yields due structure of isoflavonoids is illustrated in Fig. 1 to elevated pressure and temperature which lead to (Dewick 1982). Around 840 new isoflavonoids have better penetration of extraction solvent to the matrix been reported over last 15 years indicating the pores. The difficulties associated with the large increasing interest in search for the natural isoflavo- particle size of plant cells and applying traditional or noids (Sharma and Ramawat 2013; Veitch 2013). The SPE methods in continuous mode for large scale aim of this article is to provide an insight into the steps production, has stimulated the usage of ultrasonic involved and the processes used for the extraction, techniques for natural product extraction (Bajer et al. identification and characterization of isoflavonoids 2007; Xu et al. 2010). Ultrasound techniques have from plant sources. shown to aid extraction in various plant materials by significantly reducing extraction time and increasing extraction yields (Rostagno et al. 2003; Xu et al. Extraction of isoflavonoids 2007). Additionally, several studies have been con- ducted to compare the effect of solvents and extraction The analysis of Isoflavonoids in different matrices techniques (Luthria et al. 2007), The use of SPE either requires a prior step to extract and concentrate the alone or in combination with other methods for analytes, sometime including a clean-up procedure to extraction from liquid foods has been reported (Cor- reduce matrix effects. Isoflavonoids have structural radini et al. 2011; Delmonte and Rader 2006; Rostag- diversity, which leads to adaption of extraction no et al. 2005). The evaporation of solvent to 123 Phytochem Rev

Fig. 1 Structures of the subclasses of the Isoflavonoids concentrate the extracts increases detection and quan- ethyl acetate, diethyl ether and dichloromethane for tification levels, which is an attractive approach for the the medium polar compounds and; n-hexane, heptane, analysis of samples with low isoflavonoids content. petroleum ether and chloroform for the non-polar Many methods have been reported such as Tradi- compounds (Luthria et al. 2007). A number of studies tional extraction methods with different solvents on have used successive extraction with up to four of the basis of polarity of the compounds to be isolated. these solvents starting with the least polar and The solvents used by the workers mainly include, progressing to the most polar. This is probably a good water, ethanol and methanol for the polar compounds; method when the number and structures of the 123 Phytochem Rev isoflavonoids present is unknown. Partitioning be- Longer exposure to extraction solvent may affect tween organic and aqueous phase solvents has been isoflavonoids stability. However, if the samples are reported to be a useful means of cleaning up the initial liquid or dissolvable, a very useful alternative to avoid plant extracts (Luthria et al. 2007). In a study tedious extraction procedures is the precipitation of conducted by Luthria et al. (2007) the effect of protein or fats. In a study conducted by Lee and Row extraction solvents and techniques on the assay of (2006) on the extraction of Korean soybean by isoflavones from soybean was investigated and they ultrasonic extraction they found almost negligible concluded that the extraction efficiencies for shaker, effect of frequency on the extraction efficiency while vortex, stirring and soxhlet were between 65 and 70 % the efficiency increased with extraction time (Lee and as compared to PLE. Total isoflavone content extract- Row 2006). Lee et al. conducted the comparison ed by the sonication procedure was 93.3 % as between traditional extraction ultrasonic extraction compared to PLE (Luthria et al. 2007). The classical and PLE on the roots of Radix puerariae for the methods are not recommended as they are time preparation of ethanolic extract and observed higher consuming and requires large amount of extraction yields with ultrasonic techniques. Also, the extraction solvents. In contrast, the modern techniques provide yield became higher as the mean particle size of the advantage of reduction in extraction time and low Radix puerariae particles decreased (Lee and Lin solvent consumption. Modern techniques also have 2007). For the extraction of isoflavonoids from the the ability to provide highly reproducible quantitative species Matricaria recutita, Roasmarinus officinalis, recoveries, without changing the isoflavonoids profile Foeniculum vulgare and Agrimonia eupatoria, Bajer (Rostagno et al. 2007; Vacek et al. 2008). For routine et al. (2007) used supercritical fluid extraction (SFE), analysis of solid samples with an approximate knowl- pressurized fluid extraction (PFE), matrix solid phase edge of concentration and distribution of isoflavo- dispersion, ultrasound assisted extraction (UAE), noids, using modern techniques such as ultrasound, extraction by soxhlet apparatus and SPE. The max- including sequential extractions are time and cost imum yields of different isoflavones varied with the effective. If there is a large variety of samples with technique used. It has been observed that increasing in unknown isoflavonoid concentration and distribution, temperature in UAE lead to increase in the efficiency more efficient techniques such as PLE is preferred of the extraction process due to the increase in the (Rostagno et al. 2004). The amount of sample is an number of cavitation bubbles formed (Bajer et al. important parameter for the extraction to achieve 2007). Newer extraction techniques such as ionic better contact between the sample and the solvent. liquid ultrasonic assisted extraction (ILUAE), SFE Smaller amounts of sample favours the movement of and solid-phase micro extraction (SPME) have also the stirrer inside the tube containing sample, facilitat- found their way into isoflavonoid extraction. They can ing contact with extraction solvent and thereby easily be automated as solvent consumption and increasing extraction efficiency. It has been observed analysis time are reduced resulting in higher through- that the use of pure solvent is not recommended for the put and minimizing the alteration and degradation of isolation of isoflavonoids and the presence of water in sample (Griffith and Collison 2001). Some of the a concentration of 40–60 % have shown to increase extraction method used for the extraction of isoflavo- the recovery rates. A slight increase in temperature noids has been listed in Table 1. Recently, Bustaman- also favours the extraction of isoflavonoids, but it te-Rangel et al. (2014) studied the effect of agitation should be noted that the temperature chosen must not techniques such as vortex agitation, ultrasound probe causing degradation (Rostagno et al. 2009). Optimiza- and stirring agitation, thermostattedtrayshaking tion of extraction temperature is also important, as and the use of an ultrasound bath with temperature high temperature increase the solubility of the com- control on the extraction of Cicer arietinum, Lens pounds, thus improving the extraction efficiency and culinaris and Phaseolus vulgaris and concluded that reducing the extraction time (Rostagno et al. 2010). It thermostatted tray shaking is be the best method for has been observed that some isoflavonoids require the extraction of Cicer arietinum and Phaseolus sequential extraction with same solvent or different vulgaris, whereas ultrasound probe is the best for the solvent for better recovery. The extraction time is also extraction of Lens culinaris (Bustamante-Rangel an important parameter for an efficient extraction. et al. 2014). 123 Phytochem Rev

Table 1 Extraction Procedures for the Isolation of Isoflavonoids Source Tissue Extraction procedure Reference

Cicer arietinum Beans Shaken with acetonitrile:water (70:30 %v/v) followed by Bustamante-Rangel Lens culinaris acetonitrile. Then magnesium sulphate and sodium et al. (2014) chloride were added and shaken. Centrifuge and collect Phaseolus vulgaris supernatant Glycine max Powder Extracted with 80 %methanol, 80 % methanol in 0.1 N Lee et al. (2015) HCl, 80 % acetonitrile, 80 % acetontirile in 0.1 N HCl and shaken for 2 h and centrifuged further to collect supernatant for analysis Radix astragali Powder Extracted by PLE using 70 % ethanol at 100 °C for 15 min; Qiu et al. (2015) static cycle 3; pressure, 1500 psi; and 60 % flush volume Glycyrrhiza uralensis Roots and Rhizomes Extraction was performed twice with ethanol–water (5:5 % Fan et al. (2014) v/v) at 80 °C. The extract was filtered and dissolved in 50 % methanol at -18 °C for analysis which was fractionized into 4 parts with ethyl ether, ethyl acetate, n-butanol, the fractions were dried and analysed Ginkgo biloba Leaf, fruit and stems Extracted by sonication with ethanol for 30 min at room Pandey et al. (2014) temperature using ultrasonic bath Chinese herbal Plant extract of Sample solution was prepared by soaking the powdered Shi et al. (2015) Medicine Radix puerariae, extract in water and boiling for 2 h. The sample solution Coptis chinensis was further mixed well with acetonitrile by vortexing, the and Radix mixed solution was frozen and the acetonitrile layer was glycyrrhizae removed and diluted for analysis praparata Flos lonicerae Herb Ultrasonic extraction for 20 min with methanol Zhou et al. (2015) japonicae Mixture of Cortex Powder Extracted with 75 % methanol Cao et al. (2014) mangnoliae officinalis, Fructus aurantii immaturus and Radix et rhizoma rhei Green Tea, Grape – Dilute and shoot approach was used where samples were Sapozhnikova (2014) Juice and Ground diluted ten times with 0.05 % formic acid in water. 0.2 ml coffee of the diluted samples were filtered Phaseolus vulgaris L. Beans Extracted with 60 ml of 70 % ethanol (pH = 2, adjusted de Lima et al. (2014) with formic acid) for 24 h at ambient temperature. Extract were partitioned with n-hexane, and the aq. phase was evaporated under reduced pressure to dryness. The residue of each extract was resuspended in methanol– water (1:1) and filtered Iris crocea, Iris Rhizomes The powdered materials were extracted with methanol in an Bhat et al. (2014) germanica and Iris ultrasonic bath for 30 min each time spuria Monokin Soyabean Grounded powder Extraction was carried out with 12 ml of six different Lin and Giusti (2005) solvents (83 % acetonitrile, 83 % acidified acetonitrile, 80 % methanol,80 %acidified methanol, 58 % acetonitrile, and 58 %acidified acetonitrile). Found 58 % acetonitrile as the best solvent Soyabean Food samples Extracted with 4 different acidified solvents 53 % Murphy et al. (2002) acetonitrile, 53 % methanol, 53 % ethanol and 53 % acetone. Mixture stirred for 2 h Soyabean Defated soy flakes High pressure hot water process (110 °C and 641psig over Li-Hsun et al. (2004) 2.3 h of extraction)

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Table 1 continued Source Tissue Extraction procedure Reference

Soyabean Soyabean Compared different extraction condition (extraction time, Klejdus et al. (2005a, temperature, pressure, cycles, solvents) and techniques 2005b) (PLE, PLE? sonication, sonication and Soxhlet) with 90 % methanol Radix puerariae Roots Compared the extraction techniques i.e. Traditional Lee and Lin (2007) Method, Pressurized Solvent Extraction and Ultrasonic Extraction using ethanol Pueraria lobata Stem Liquid–Liquid Extraction using n-butanol/water two phase Li-Hsun et al. (2004) system Iris tectorum Dried roots ILUAE method Sun et al. (2011) Red clover Plant Dried leaves SFE with Carbon dioxide as an external fluid Klejdus et al. (2005a) Pueraria lobata Stem UAE Xu et al. (2007) Red clover Plant Dried leaves SPE Klejdus et al. (1999) Soyabean Flour Refluxing 180 ml of 80 % methanol for 4 h at 80 °C Aussenac et al. (1998) Soyabean Soy Protein Ultrasonic extraction using 50 % Ethanol Rostagno et al. (2003) Soyabean Beans Centrifugation with different solvents 66 % acetonitrile, Shihabi et al. (1994) 0.4 % NaCl, and 30 ml/L of Methyl isobutyl xanthine Lupinus albus Roots Ultrasonic extraction Stobiecki et al. (1999) Soyabean Soyabean Extraction from five different techniques (vortexing, Luthria et al. (2007) shaking, stirring, sonication and Soxhlet) using dimethyl sulphoxide:acetonitrile:water (5:58:37 v/v/v) as solvent Soyabean Soyabean Microwave assisted extraction Careri et al. (2007) Glycyrrhiza glabra Roots Root powder was extracted with either 70 % (v/v) aq. Simons et al. (2009) ethanol or Ethyl Acetate. Further extraction was performed by two step sequential extraction in sonication bath Medicago truncatula Leaves Ultrasonication with 80 % methanol Jasinski et al. (2009) Hedysarum Leaves Extracted with water/acetone (3:7) containing ascorbic acid Tibe et al. (2011) coronarium (1 g/L) Lupinus albus Leaves Extracted with 80 % methanol and the extract was defatted Kuhn et al. (2003) with n-hexane Arabidopsis thaliana Leaves Extracted in 10 ml of 80 % methanol and the suspension Kachlicki et al. was placed in an ultrasonic bath for 30 min (2008) Lupinus angustifolius Leaves Extracted in 10 ml of 80 % methanol and the suspension Kachlicki et al. was placed in an ultrasonic bath for 30 min (2008) Pueraua lobata Roots Extracted by sonication for 45 min with 40 ml of 70 % Fang et al. (2006) methanol Radix puerariae Roots Extracted with 50 % ethanol for 4 h at room temperature Nguyen et al. (2009) Glycine max Seedlings Defatted by refluxing with hexane for 4 h. The defatted Simons et al. (2011a) powder was extracted with 50 ml of ethanol by sonication and shaking at 20 °C Medicago truncatula Roots Frozen plant material was homogenized with 80 % Staszkow et al. methanol and the suspension was placed in an ultrasonic (2011) bath for 30 min Glycine max Powder Sample was sonication with 80 % methanol for 30 min. Lojza et al. (2012) The supernatant was extracted twice with 10 ml and 5 ml of 80 % Methanol twice

123 Phytochem Rev

Table 1 continued Source Tissue Extraction procedure Reference

Lupinus albus et al. Seeds Plant Material was homogenized in 80 % methanol and the Kachlicki et al. Lupinus angustifolius suspension was ultrasonicated for 30 min (2005) Lupinus luteus Glycyrrhiza glabra Dried roots Ultrasonicate with 30 ml of 70 % methanol solution for Li et al. (2011) Glycyrrhiza inflata 30 min in dark Glycyrrhiza pallidiflora Spatholobus Stems Extracted with different techniques including Simultaneous Cheng et al. (2011) suberectus Ultrasonic/microwave assisted extraction, Ultrasonic assisted extraction, Microwave assisted extraction Soxhlet extraction and heat reflux extraction Glycine max Beans Defatted by hexane by sonication at 30 °C and extracted Simons et al. (2011b) with ethanol by a two-step sequential extraction, each for 30 min in a sonication bath at 30 °C Dalbergia odorifera Heartwood Extracted with 60 % methanol aqueous solution by Liu et al. (2005) ultrasonication for 1 h Astragalus Roots Extracted with 80 % ethanol by maceration at room Lin et al. (2000a) mongholicus temperature, the concentrate was diluted with water and the solution was successively extracted with hexane, chloroform, ethyl acetate and n-butanol Radix astragali Plant Extracted by refluxing with ethanol for 3 times (2 h each). Tang et al. (2010) After filtration and concentration, the dried extract was re-dissolved in water and separated by eluting with water and 30 % ethanol and then washing with 70 % ethanol Lupin albus Root Extracted with 80 % aqueous acetone by ultrasonication for Stobiecki et al. (1999) 30 min. The extract was filtered and the residue is washed with acetone. The filtrates were combined and concentrated which are further processed by solid phase extraction to obtain isoflavonoids Lens culinaris Bean Extracted with methanol and 0.1 N HCl (5:1 %v/v) by Konar et al. (2012) Phaseolus vulgaris Bean sonication for 2 h at room temperature Phaseolus vulgaris Bean Cicer arientinum Chickpea Trifolium pratense Leaves Extracted by ultrasonication with methanol–water (9:1) at (de Rijke et al. 2001) room temperature and 350 mM Tris buffer Trifolium repense Leaf, stem, root and For qualitative studies—extracted with 80 % methanol by Wu et al. (2003) Trifolium hybridum flower sonication for 1 h at room temperature Trifolium campestre For quantitative studies—extracted with ethanol, water and HCl. the mixture was refluxed for 2 h Radix pueraria Root Extracted with 70 % ethanol for more than 24 h at room Zhang et al. (2005) temperature for 3 times Radix pueraria Root Extracted with 70 % methanol by sonication for 45 min Fang et al. (2006) Pueraria lotaba Root Trifolium pratense Flowers and leaves Extracted with methanol/water (9:1) using sonication for Lin et al. (2000b) 60 min at room temperature

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Table 1 continued Source Tissue Extraction procedure Reference

Trifolium pratense Aerial parts Extracted with 50 % ethanol in a high-intensity ultrasound Hanganu et al. (2010) Cytisus nigrican probe system Trifolium repens Medicago lupulina Genistella sagittalis Melilotus officinalis Belamcanda chinesis Roots Extracted twice with ethanol at room temperature for 1 h Kang et al. (2008)

LC–MS/MS analysis of isoflavonoids specific transition pair will only be detected in the MS (Lojza et al. 2012; Rostagno et al. 2009). Rapid identification and characterization of unknown For the efficient separation of the required iso- compounds present in natural extracts can be achieved flavonoids from complex mixtures different separation by various analytical techniques. Different analytical techniques have been employed. Presently reverse techniques such as TLC, UV, HPLC, capillary elec- phase-HPLC (RP-HPLC) is widely used for the trophoresis have been employed for analysis of analysis of most of the herbal extracts. Normal phase isoflavonoids over the past decades (Drasˇar and HPLC is also used for the analysis of polar Moravcova 2004; Liu et al. 2008; Zhou et al. 2009). isoflavonoids whose retention in reverse phase is low LC methods with UV detection have questionable (Barnes et al. 1994, 1998, 1999, 2006, 2002; Coward specificity for distinguishing compounds of very et al. 1993; Eldridge 1982; Franke et al. 1995; similar structures as they often exhibit fairly similar Fukutake et al. 1996; Griffith and Collison 2001; UV absorption characteristics (Bilia et al. 2001; Cao Liggins et al. 1998; Wang and Murphy 1994). The et al. 2008; Dachtler et al. 2001; Krause and Galensa longer the alkyl chain attached to the adsorbent, the 1991; Pietta et al. 1991; Qi et al. 2006; Rehwald et al. higher will be the retention of the components. From 1994; Tian et al. 2006; Uchiyama et al. 2008; Wu and last decade, the use of fast HPLC with less than 2 lm Thompson 2006; Zhou et al. 2008, 2009). Also these particle size has increased due to greater speed of methods are time consuming and labour-intensive. analysis without compromising the sensitivity and The structural characterisation of the newer isoflavo- resolution of the chromatography. In a study Church- noids that are present in the plant extracts require more well et al. (2005) compared the differences in HPLC– amounts of the pure compounds for NMR, mass MS performance by conducting a parallel UHPLC– spectrometry and crystallography. To alleviate this MS analysis for several methods previously optimized drawback, hyphenated techniques such as combining for HPLC-based separation and quantification of the separation power of HPLC with structural infor- multiple analytes with maximum throughput. The mation that can be provided by NMR and mass results showed that UHPLC produced significant spectrometry (MS) are commonly used for the phyto- improvements in method sensitivity, peek shape and chemical analysis of the plant extracts (Maul et al. resolution (Churchwell et al. 2005).Coupling of HPLC 2008). These techniques will increase the speed and with mass spectrophotometer has proved to be selectivity of analysis (Barnes et al. 2006; Vukics and extremely powerful tools in natural product analysis Guttman 2010). The use of HPLC with conventional and characterization as they permit the fast screening detection requires a typical purification step (e.g. SPE) of crude natural product extracts or fraction for of crude extracts and/or careful optimization of HPLC detailed information, using a minimum amount of separation to avoid co-elution of target analytes with sample. matrix co-extracts. Some degree of simplification of Various reviews have been published showing the sample preparation procedure is enabled by employ- advantages and application of mass spectroscopy for ing MS detection as the peak corresponding to a natural product quantitation and characterization from

123 Phytochem Rev different crude extracts and biological matrices. Along arrangement i.e. analyzer-modified-analyzer corre- with the identification and characterization of iso- sponds to tandem in space, where ions are treated in flavonoids, mass spectrophotometer enables the iden- different regions of space (de Hoffmann 1996; tification and quantitation of their metabolites in Pinheiro and Justino 2010). plasma, urine and faeces, helping in the pharmacoki- m/z modification can be achieved by various netic profiling of the constituents. For ionization of techniques, but the most common is Collision Induced molecules, different techniques such as fast-atom (or Activated) Dissociation (CID or CAD), where bombardment (FAB), liquid secondary ion mass precursor ions undergo collisional activation with spectrometry (LSIMS), electrospray ionization (ESI), neutral atoms or molecules (such as inert gases) in the atmospheric pressure chemical ionization (APCI) and gas phase. CID is an example of a post-source matrix-assisted laser desorption/ionization (MALDI) fragmentation, in which energy is added to the already are available. The ionized ions from the different vibrationally excited ions. An alternative to CID is sources are analysed by mass analysers which are ion ECD (Electron Capture Dissociation), in which mul- type, quadrupole type, time of flight analysers (TOF), tiply charged positive ions are submitted to a beam of Fourier transform ion cyclotron resonance (FT-ICR) low energy electrons, producing radical cations. In and FT orbitrap. The nebulizer systems ESI and APCI opposition to post-source fragmentation, in in-source are most commonly used. Of these ESI is the most fragmentation, ions already possess sufficient internal widely used, while APCI is preferred for some special energy to fragment spontaneously within the mass cases where ESI is not efficient enough for the spectrometer. Although usually this is an undesired ionisation. ESI method of ionization is sufficiently effect, because it leads to lower abundance of precur- ‘‘soft’’ that even intact proteins can be transferred sor ions, it may in some cases become useful (Abranko´ successfully as multiple-charged ions into the gas et al. 2011; Pinheiro and Justino 2010). Four general phase (Barnes et al. 1998). Barnes et al. (1998) for the types of tandem MS scans are possible, and all may first time described the application of HPLC–MS with generate valuable information. A Product Ion Scan heated nebulizer-atmospheric pressure chemical ion- analyses all the fragment ions resulting from a single ization and ion-spray interfaces to analysis which selected precursor ion (these are usually called MS2 showed that the isoflavone composition could be spectra). Conversely, a Precursor Ion Scan will changed by variation in temperature (Barnes et al. identify all the precursors of a selected product ion; 1994). ESI–MS is well suited to investigations of a Neutral Loss Scan is performed from a selected metabolites of isoflavonoids in physiological fluids. neutral fragment and will identify the fragmentations Sulfate esters and glucuronides, which are thermally leading to the loss of that neutral fragment; these two labile in the APCI interface, form molecular ions in techniques cannot be performed in time-based analy- ESI. A particular advantage of the ESI interface is that sers (de Hoffmann 1996; Pinheiro and Justino 2010). it is much sensitive. Both positive mode and negative More selective than these three techniques, Selected mode of ionisation can be used based on the nature of Reaction Monitoring (SRM) will analyse if a specific the isoflavonoid to be analyzed. Tandem mass spec- product ion comes from the fragmentation of a specific troscopy, abbreviated as MS/MS or MSn for nth order precursor ion. fragmentation, involves minimum two stages of mass In the MS/MS analysis we will obtain the informa- analysis, in conjugation to a fragmentation process, tion about quasi molecular ion [M?H]? at first. Then either dissociation of a reaction, which causes a the fragmentation pattern of the quasi molecular ion change in the m/z ratio on an ion. Commonly a mass can be studied by retro-Diels–Alder (rDA) reaction. analyser is used for the isolation of a precursor ion, Maul et al. (2008) explained the possible fragmenta- which is then further fragmented to yield product ions tion patterns for the isoflavonoids for structural that will be detected in the second mass analysis. This information predictions (Maul et al. 2008). In case of process can be expanded further to successive modifi- isoflavones, firstly the ring C is usually cleaved from cation and detection giving rise to MSn. Only selected the structure forming a protonated diene fragment ions which are detected in one analyzer, move further from ring A (ESI?) and neutral alkyne from ring B to second analyzer, so MS3 is usually considered to be (Fig. 2). Only diene will be detected in positive mode the highest order achieved in the MSn analysis. This giving very week signal. The substitution on the basic 123 Phytochem Rev

Fig. 2 a Fragmentation of isoflavanones in ESI-(?) MS/MS fragmentation formononetin. The parenthesis indicates the % leading to two different cleavages of the C-ring by rDA relative intensity of each fragment possible. [Adopted with fragmentation and non-rDA fragmentation. b rDA fragmentation permission from Maul et al. Anal Bioanal Chem (2008)] (Maul of the isoflavone, daidzein in ESI-(?)MS/MS. c rDA et al. 2008)

rings A and B can be easily studied by the changes in additional product ion of 1amu less can be expected the m/z values. In case of the isoflavonones, the because of the differences in the protonation sites. In fragmentation is similar with slight modification that case of formononetin the fragments of 137 and 136 are the ring B forms alkenes (Fig. 2). In case of methoxy- formed from ring A and 133 from ring B. Besides the lated isoflavones such as formononetin and Biochanin rDA reaction cleavage of the ring C there is also the A the same rDA fragmentation takes place but the possibility of the non-rDA fragmentation by cleavage

123 Phytochem Rev

123 Phytochem Rev b - Fig. 3 Fragmentation Scheme for (M-H) ion (m/z = 268) fragments were neutral losses of CO, CO2,C3O2 and [Adopted with permission from March et al. Int J Mass C H O (Kang et al. 2007). Even though fragments Spectrom (2004)] (March et al. 2004) 2 2 resulting from rDA reaction were found in much lower abundance, they were in particular useful for structural of the C2–C3 bond for the formation of hydroxy elucidation as they allowed not only the determination benzyl cation fragment from ring B as illustrated in of -OH groups on different rings, but also to identify Fig. 2. March et al. (2004) studied the fragmentation the position of glucosidic bonds using fragments with pattern of the isoflavone glucosides by characterising intact glycoside bonds. Recently Zhang et al. (2014) the fragmentation of the genistein-7-O-b-D-glucoside. explored the fragmentation behavior of isoflavones The product ion spectra shows the neutral loss of using electrospray ionization-ion trap-time of flight glycan moiety from [M-H]- and the fragmentation mass spectrometry (ESI-IT-TOF-MSn). They found pattern of remaining moiety is depicted in Fig. 3. They that the isoflavone glycoside bond was easily broken. proposed a seven-membered ring C structure inter- The fragmentation occurred mostly on the C ring, and mediate, which successfully explained the mechanism the fragment ions of A (1, 3?) produced by the rDA of eliminating CO2 at ring C on an isoflavones cracking will predict the hydroxylation replacement glycoside [M-H]- radical anion (March et al. on A ring or B ring (Zhang et al. 2014). The most 2004). The was idea found to be useful and adapted commonly observed fragments for some common for the isoflavones [M-H]- ion, proposing a similar isoflavonoids are listed in Table 2. seven-member ring C structure with a cycloacetone. Isoflavonoid derivatives such as pterocarpans This intermediate structure was thought to be formed show much more complex fragmentation pathways; as part of the process of ring C loss of CO2 or MS studies of different deuterated peterocarpan sometimes CO or CHO–. The individual fragmenta- derivatives, as well as of pterocarpan glycosides, tion pattern depends on several factors and might points out that these are dominated by various and therefore differ among different instruments. In case successive ring openings and/or contractions (Zhang of glucosides, the loss of the malonyl, acetyl and et al. 2007). A rare 2/4 rDA fragmentation, like that glucosyl groups are common fragmentation reactions of isoflavan-derived flavonoids, has also been that are very valuable for the structural elucidation of observed for pterocarpans in the negative mode isoflavonoids conjugates (Wu et al. 2003). Using (Simons et al. 2011a). multistage fragmentation experiments (MSn), Kang In a study conducted by Kuhn et al. (2003) the et al. (2007) investigated the fragmentation mechan- fragmentation behaviour of seven pairs of isomeric ism in negative ion mode and the predominant flavone/isoflavone aglycones (solely hydroxylated

Table 2 MS/MS fragments Compound Tandem MS fragment ion of some common isoflavonoids Daidzin 415 [M-H]-; 253 [M-H-Glc]-3 Malonyl daidzin 457 [M-COOH]-; 253 [M-H-MalGlc]-3 Glycitin 445 [M-H]-; 283 [M-H-Glc]-3 Malonyl glycitein 487 [M-COOH]-; 283 [M-H-MalGlc]-3 Genistin 431 [M-H]-; 269 [M-H]- Malonyl genistin 473 [M-COOH]-; 269 [M-H-MalGlc]-3 Acetyl daidzin 457 [M-H]-; 253 [M-H-AcGlc]-3 Acetyl glycitin 487 [M-H]-; 283 [M-H-AcGlc]-3 Daidzein 133 (0,3 A-); 117 (1,3B-); 197; 181b Glycitein 163 (0,3 A-); 117 (1,3B-)3 Acetyl genistin 473 [M-H]-; 269 [M-H-AcGlc]-3 Genistein 133 (0,3 A-); 107 (0,4A-); 163 (0,4B-); 197b Biochanin A 177 (0,4 B-); 213 (1,2A-); 254 (1,3A-); 252; 195b Formononetin 107 (0,4 A-); 177 (0,4B-); 229 (1,2A-); 270 (1,3A-); 268; 211b

123 Phytochem Rev and/or methoxylated) was studied using ion trap mass qualitative analysis of 26 phenolic compounds in plant spectroscopy with atmospheric pressure ionisation material, including 15 isoflavonoids (daidzein, genis- (API, both electrospray and APCI) in the positive and tein, isoformononetin, formononetin, prunetin, negative ion modes. It was observed that neutral loss biochanin A, daidzin, genistin, ononin, sissotrin), 2 of 56 amu was a common feature of all isoflavones in prenylated Isoflavonoids(osajin, pomiferin), 5 fla- ESI(?) and was identified as double loss of CO by vones (apigenin, kaempferol, quercetin, quercetin-3- accurate mass tandem mass spectroscopic measure- glucoside, rutin), 4 flavanones (hesperetin, naringenin, ment using quardrupole time-of-flight (Q-TOF) in- naringenin-7-glucoside, naringin), a coumestan strument (Kuhn et al. 2003). (coumestrol) and a coumarin (scopoletin). The appli- Some of the various LC–MS/MS determination of cation of this method was also demonstrated by the isoflavonoids in the plant extracts were summarised analysis of mung bean (Vigna radiata) sprout extract, along with their extraction methods in Table 3. in which 14 of the 26 analytes were detected and Simons et al. (2011a) identified isoflavonoids in quantified (Prokudina et al. 2012). Rhizopus spp. elicited soya bean seedlings by using Klejdus et al. (2010) developed a hyphenated RP-UPHLC associated with ESI–MS and successfully ultrasound assisted super critical fluid extraction identified 13 isoflavones (Daidzin, Genistin, Glycitin, method for the extraction and determination of Malonyl-glycitin, Malonyl-daidzin, Acetyl-daidzin, isoflavones in sea and freshwater algae and cyanobac- 20-Hydroxydaidzein, Malonylgenistin, Acetylgenistin, teria. Different extraction conditions were tried and Glycitein, Daidzein, 20-Hydroxygenistein and Genis- ultrasound assisted super critical fluid extraction was tein) and 5-pterocarpans (Glycinol, Glyceollidin I/II, found to give better recovery. Eight isoflavone com- Glyceollin III, Glyceollin II and Glyceollin I) in a pounds were found for the first time in seven real single run (Simons et al. 2011a). Lin et al. (2000a, b) samples of sea algae and in three control samples of identified eight isoflavonoids calycosin-7-O-b-D-glu- freshwater algae and cyanobacteria (Klejdus et al. coside, calycosin-7-O-b-D-glucoside-600-O-malonate, 2010). ononin, (6aR,11aR)-3-hydroxy-9,10-dimethoxyptero- LC–MS methods utilising use of time-of-flight carpan-3-O-b-D-glucoside, calycosin, (3R)-7,20-dihy- (TOF) detection system have been used for the analysis droxy-30,40-dimethoxyisoflavan-7-O-b-D-glucoside, of isoflavonoids as TOF analyzer is well suited to formononetin-7-O-b-D-glucoside-60-O-malonate and perform structure elucidation or confirmation, espe- formononetin. Also the existence of (6aR,11aR)-3- cially for non-target compounds, due to its unique hydroxy-9,10-dimethoxypterocarpan, (3R)-7,20-dihy- features (vide infra). TOF instruments are capable of droxy-30,40-dimethoxy isoflavan, astrapterocarpan achieving 5,000–10,000 resolving power expressed in glucoside-60-O-malonate and astraisoflavanglucoside- terms of full peak width at one-half maximum 60-O-malonate was detected using LC–ESI–MS. (FWHM), which provides better confirmatory ability Wu et al.(2004) in a study characterised Edamame than quadrupole, triple quadrupole or ion trap (IT) mass and Tofu soybeans using LC–UV–ESI–MS/MS and analyzer (Zhou et al. 2009). Wang and Sporns (2000) identified a total of 16 isoflavones, including three were the first to demonstrate the application Matrix- aglycones, three glycosides, two glycoside acetates assisted laser desorption/ionization time-of-flight mass and eight glycoside malonates by employing positive spectrometry (MALDI–TOF–MS) in the identification ESI–MS/MS for collecting molecular mass informa- of isoflavones in soy samples. 2,5-dihydroxybenzoic tion (Wu et al. 2004). Wu et al. (2003) in another study acid (DHB) was used as the matrix for isoflavones. detected 31 isoflavones in Trifolium pratense L. and Fragmentation occurred only through loss of glycosidic related species using HPLC–UV–ESI–MS. The residues (Wang and Sporns 2000). separated isoflavones including aglycones, glycosides Addition of MS/MS with liquid chromatographic and glycoside malonates, were individually analyzed has added much accuracy and precision towards the and identified by their molecular ions and character- analytical method. Tandem mass experiments can be istic fragment ion peaks using LC–MS under MS and performed in most instruments except for those MS/MS mode, in comparison with the standard equipped with a single quadrupole mass analyzer. isoflavones (Wu et al. 2003). Prokudina et al. (2012) Most commonly, triple quadrupole mass analyzers are used UPLC–ESI–MS/MS for the quantitative and used that allow the fragmentation of selected ions by 123 htce Rev Phytochem Table 3 Liquid chromatography with Tandem Mass Spectroscopy method for the determination and quantification of Isoflavonoids in different plant extracts and food materials Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Glycine max Daidzin, Glycitin, Genistin, LC–ESI– C18 column (150 9 2.1 mm; TOF–MS An effective analytical method Lee et al. Malonyl daidzin, Malonyl MS 5 lm) for the simultaneous (2015) glycitin, Acetyl daidzin, Acetyl Mobile Phase—0.05 % formic characterization and glycitin, Malonyl genistin, acid and Methanol quantification of isoflavones Daidzein, Glycitein, Acetyl with exceptionally short time, genistin and Genistein high selectivity and high linearity was developed for estimation of Isoflavonoids from Soyabean products

Radix astragali Calycosin, Calycosin-7-O-b-D- LC–ESI– C18 column (50 9 2.1 mm; Qtrap MS A simple, rapid and sensitive Qiu et al. glucoside, Formononetin and MS/MS 3.5 lm) method has been developed for (2015) Formononetin-7-O-glycoside Mobile Phase—0.1 % formic the simultaneous estimation of acid and acetonitrile seven bioactive components in containing 0.1 % formic acid Radix astragali

Glycyrrhiza uralensis Liquiritin, Ononin, LC-DAD- C18 Column (250 9 4.6 mm; PDA and MS Identified 13 compounds in Fan et al. Glycycoumarin ESI–MS/ 5 lm) which 10 exhibited free radical (2014) Isoglycyrol, Liquiritin apioside MS Mobile Phase—acetonitrile and scavenging activity and 2.0 % acetic acid Isoliquiritin apioside

Chinese Herbal Puerarin, Daidzin and Daidzein UPLC– C18 column (100 9 2.1 mm; Qtrap MS A separation method for 12 Shi et al. Medicine ESI–MS/ 5 lm) components within 10 min (2015) MS Mobile Phase—ammonium providing the quality control of formate buffer and Gegen-Qinlian decoction and acetonitrile related chinese medicines

Cicer arietinum Daidzin, Glycitin, Genistin, LC–MS– C18 column (50 9 4.6 mm; Q-TOF Comparison of five different Bustamante- Lens culinaris Daidzein, Glycitein, Genistein, MS 1.8 lm) agitation techniques for Rangel Formononetin and Biochanin- extraction was compared and et al. Phaseolus vulgaris Mobile Phase—acetonitrile and A 0.01 % aq. formic acid evaluated using Quick Easy (2014) Cheap Effective Rugged Safe (QuEChERS) approach

Mixture of Cortex 5,7-Dihydroxylcoumarin, UHPLC– C18 column (50 9 2.1 mm; LTQ Orbitrap Identified various constituents Cao et al. mangnoliae officinalis, Auraptene and Marmin MS–MS 1.9 lm) MS including 3 coumarin from (2014) Fructus aurantii acetonid Mobile Phase—methanol and Chinese traditional medicine immaturus and Radix 0.1 % formic acid (For 123 et rhizoma rhei Positive mode) Methanol and Water (For Negative mode) 123 Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Grape Juice, Green Tea Diadzin, Genistin, Daidzein, LC–ESI– C8 Column (100 9 4.6 mm; MS Analyzed isoflavonoids and Sapozhnikova and Ground Coffee Genistien, Glycitein, Biochanin MS 3 lm) with C8 column flavonoids in grape juice, green (2014) A, Formononetin and (4 9 3 mm) tea and ground coffee Coumestrol Mobile Phase—0.2 mM ammonium formate buffer at pH 4.7 and methanol

Phaseolus vulgaris L. Daidzein and Genistein LC–ESI– C18 Column (150 9 2 mm; Q-TOF MS Identified isoflavonoids in de Lima et al. QTOF– 5 lm) Phaseolus vulgaris which is a (2014) MS Mobile Phase— common diet in many countries methanol/formic acid, (95:5v/v) and 5 % formic acid

Ginkgo biloba Genistein UHPLC– C18 Column (50 9 21 mm; API 4000 A Validated method for Pandey et al. ESI– 1.77 lm) QTrap determination of flavonoids in (2014) MS–MS Mobile Phase—0.1 % formic leaf, stem and fruit extracts of acid and 0.1 % formic acid Ginkgo biloba in acetonitrile

Flos lonicerae J. Genistein and Genistin UPLC– C18 Column (100 9 2.1 mm; Qtrap MS Developed a method for Zhou et al. ESI–MS/ 1.8 lm) determination of different (2015) MS Mobile Phase—acetonitrile/ constituent in a run time of methanol (4:1 % v/v)— 8 min 0.4 % formic acid

Rhizomes of Iris Apocynin, Picied, Tectoridin, UHPLC– C18 Column (250 9 4.5 mm; PDA and MS About 27 constituent including Bhat et al. crocea, Iris germanica Tectorigenin-40-glucoside, DAD– 5 lm) isoflavones, isoflavone (2014) and Iris spuria Iridin, Tectorigenin, Irigenin, ESI–MS/ Mobile Phase—acetonitrile glycosides, xathones, flavones, 0 0 5,2 ,3 -Trihydroxy-7- MS and 0.05 % acetic acid rotenoids, iridalglycosides, methoxyflavanone, alkylated benzoquinones and Irisflorentin, 5,7-Dihydroxy- stilbenes, were identified based 0 6,2 -dimethoxyisoflavone and on retention time (tR), UV and Crocetenone MS spectra compared with those of authentic compounds and literature data htce Rev Phytochem htce Rev Phytochem Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Sprout Extract of Mung Daidzein, Genistein, UPLC– C8 column (150 9 2.1 mm; Qtrap MS 26 phenolic compounds in plant Prokudina bean (Vigna radiata) Isoformononetin, ESI–MS/ 1.7 lm) material, including 15 et al. (2012) Formononetin, Prunetin, MS Mobile Phase—Methanol and isoflavonoids, 5 flavones, 4 Biochanin A, Daidzin, 10 mM aqueous formic flavanones, a coumestan and a Genistin, Ononin, Sissotrin, acid coumarine were analyzed Osajin, Pomiferin and together in a very shorter run Coumestrol time 0 0 Dried Roots of 5,6,7,3 -tetrahydroxy-4 -methoxy LC–NMR– C18 column (150 9 4.6 mm; NMR and Ion LC-NMR and LC–MS were used Kang et al. Belamcanda chinensis isoflavone, Tectorigenin, MS 5 lm) trap MS together for the identification (2008) Iristectorigenin A, Irigenin and Mobile Phase—30 % and quantification of Irisflorentine acetonitrile and 70 % isoflavonoids Water

Kudzu Root Daidzein, Daidzin, Genistein, LC–APCI– C18 column (250 9 4.6 mm; DAD and The isoflavones were identified Zhang et al. Genistin, Formononetin and MS 5 lm) APCI–MS based on their UV spectra, (2005) Puerarin Mobile Phase—0.1 % acetic mass spectra of protonated and acid (v/v) in acetonitrile deprotonated molecules, and and 0.1 % acetic acid MS–MS data Freeze dried food Biochanin A, Daidzein, LC–ESI– Diphenyl column Qtrap MS Describes a generic method for Zhang et al. Formononetin, Genistein, MS/MS (150 9 2 mm; 3 lm) the analysis of dietary (2007) Glycitein and Coumestrol Mobile Phase—40 % phytoestrogens, using ammonium acetate in automated solid-phase methanol and methanol extraction and liquid chromatography-tandem mass spectrometry

Leaves and Flowers of Daidzin, Glycetin, Calycosin LC–ESI– C18 column (150 9 2.1 mm; PDA and MS Identified around 46 compounds Lin et al. Trifolium prantense 7-O-b-D-glucoside, Genistin, MS 5 lm) with sentry guard from the methanolic extract of (2000b) Ononin, Glycetein, Sissotrin, column (5 lm, red clover including 20 Daidzein, Calycosin, Genistein, 3.9 9 20 mm) flavonoid glycoside malonates. Formononetin, Prunetin and Mobile Phase—0.25 % acetic It concludes that the flowers Biochanin A acid and acetonitrile contained flavones as the major containing 0.25 % acetic flavonoids, whereas the leaves acid had isoflavones as the major flavonoids

35 Dietary supplements Genistein, Daidzein, Glycitein, LC–ESI– C18 column (150 9 2.1 mm, MS The method described here is the Clarke et al. containing Genistein, Coumestrol, MS/MS 5 lm) first generic LC/MS method for (2008) 123 Phytoestrogen in Formononetin, Biochanin A, Mobile Phase—acetonitrile the determination of Canada and Italy Daidzin, Genstin and Puerarin and water phytoestrogens in commonly used food supplements 123 Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Leaves of Trifolium Daidzin, Daidzein, Genistin, LC–APCI– C18 column (250 9 4.6 mm; UV and Ion Isoflavones, their glucoside and de Rijke et al. prantense Genistein, Ononin, MS 5 lm) Trap MS their glucoside malonates were (2001) Formononetin, Sissotrin and Mobile Phase—methanol- determined in red clover using Biochanin A 10 mM ammonium formate RHPLC coupled with APCI– buffer, pH 4.0 MS

Lentis (Red, Green and Biocanin A, Daidzein, LC–ESI– C18 column (50 9 2.1 mm; MS Determined the Isoflavone Konar et al. Yellow), Chickpea, Formononetin, Genistein, MS/MS 3 lm) content in legumes which are (2012) Haricot beans and Dry Glycitein, Sissotrin, Daidzin, Mobile Phase—0.01 % formic commonly consumed in Red Kidney beans Ononin, Genistin and Glycitin acid and 5 mM ammonium Western diets and are not good formate and 5 mM alternative to soy and soy ammonium formate products as source of isoflavones

Flowering of Trifolium Daidzin, Glycitin, Genistin, LC–ESI–MS C18 column (150 9 3.2 mm; PDA and MS A total of 31 isoflavones were Wu et al. repense, Trifolium Daidzein-Glucosyl-Malonyl, 5 lm) detected in red clover and (2003) hybridum, Trifolium Calycosin-Glucosyl-Malonyl, Mobile Phase—0.1 % formic several isoflavones were campestre Pratensein-Glucosyl-Malonyl, acid and 0.1 % formic acid identified for the first time in the Pesudobaptigenin-Glucosyl, in acetonitrile related species Ononin, Pratensein-Glucosyl- Malonyl, Daidzein, Glycitein, Irilone-Glucosyl, Calycosin, Pesudobaptigenin-Glucosyl- Malonyl, Formononetin- Glucosyl-Malonyl, Biochanin— A-glucosyl, Prunetin-Glucosyl, Genistein, Irilone-Glucosyl- Malonyl, Biochanin-A- Glucosyl-Malonyl, Prunetin- Glucosyl-Malonyl, Pseudobaptigenin, Biochanin- A-Glucosyl-Malonyl, Formononetin, Irilone, Prunetin and Biochanin A

Seeds of Lupinus albus Genistein-7-O-glucoside, LC–ESI– C18 column (200 9 200 mm) UV and MS Demonstrated the applicability of Stobiecki 0 Genistein, 2 -hydroxygenistein, MS/MS Mobile Phase— LC–ESI–MS for the et al. (1999) Wighteone, Lupalbigenin, 20- simultaneous analysis of water:acetonitrile:acetic Rev Phytochem 0 hydroxyly palbigenin, 2 - acid, (79.9:20:0.1) and secondary metabolites hydroxygenistein-7-O- water:acetonitrile:acetic 00 glucoside-6 -malonate and acid, (20:79.9:0.1) Genistein-7-O-diglucoside htce Rev Phytochem Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Grains of Chickpea Daidzein, Glycitein and Genistein LC–ESI–MS C18 column Q-TOF MS Evaluated the presence of Aguiar (cicer arientinum), Mobile Phase—Methanol and isoflavones and their conjugated et al. Pigeonpeas (Cajanus Water forms in grains and leaves of (2007) cajan), Snapbean several leguminous plant (Phaseolus vulgaris), utilized largely in Brazilian Favabean (Vicia faba), cuisine Gaint Peruvian lima (Phaseolus limensis), English peas (Pisum sativum), Soyabean (Glycine max) Sea Algae Samples Daidzin, Genistin, Ononin, LC–MS/MS CN column (100 9 2.1 mm; PDA and MS New Hyphenated technique for Klejdus (Sargassum muticum, Daidzein, Sissotrin, Genistein, 3.5 lm) the extraction and determination et al. Sargassum Vulgare, Formononetin and Biochanin A Mobile Phase—0.2 % acetic of isoflavones in sea and (2010) Hypnea spinella, acid and acetonitrile freshwater algae and Porphyra sp, Undaria cyanobacteria was developed pinnatifida, Chondrus and fast chromatography crispus, Halopytis analysis was performed using incurvus) RHPLC–MS

Dried roots of Calycosin-7-O-b-glucoside, LC–ESI–MS/ C18 column (50 9 2.0 mm; MS Determined 3 major isoflavonoids Kim et al. Astragalus Formononetin, Astragaloside I MS 3 lm) and a precolumn filter and 2 main saponins using LC– (2007) membranaceous and Astragaloside IV (0.5 lm) ESI–MS/MS by isocratic Mobile Phase—acetonitrile elution and water (40:60) 00 Dried Roots of Pueraria Puerarin, Daidzin-6 -O- LC–ESI–MS– C18 column (250 9 4.6 mm; PDA and Ion Major components from the Fang et al. lobata acetylester, Genistin-600-O- MS 5 lm) Trap Mass Methanolic extract from P. (2006) malonylester, Biochanin A-7-O- Mobile Phase—0.5 % acetic Spectrometer lobata callus cultures and major 00 glucoside-6 -O-malonylester acid and acetonitrile isoflavonoids components of P. and Daidzein lobata cell suspension were identified using LC–MS/MS Soybean Genistien, Genistin, Daidzin, UHPLC–ESI– Hypersil Gold Analytical Orbitrap MS Provided method for the Lojza et al. (Glycine max) Acetyl-daidzin, Malonyl- Orbitrap MS Column (100 9 2.1 mm; quantitative analysis of (2012) daidzin, Acetyl-Genistin, 1.9 lm) Isoflavones isolated from Malonyl-genistin, Diadzein, Mobile Phase—0.1 % acetic soybean using direct analysis in Glycitein, Glycitin, Acetyl- acid and 0.1 % acetic acid in real time ion source coupled glycitin and Malonyl-glycitin methanol with high resolution orbitrap 123 mass spectrometer 123 Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Seeds of Daidzein, Genistein, Glycitein,Daidzin, LC–ESI–MS Prodigy column UV and Ion The report provides a reliable Wu et al. Edamame and Glycitin, Genistin, Daidzin-Malonate, (150 9 3.2 mm; 5 lm) Trap MS analytical technique for (2004) Tofu Soyabean Glycitin-Malonate, Daidzin-Acetate, Mobile Phase—0.1 % formic screening of authenticated fresh Genistin-Acetate and Genistin- acid and 0.1 % formic acid immature Edamame soybeans Malonate in acetonitrile and Tofu soybeans 0 Fresh Leaves of 2 -Hydroxygenistein, Genistein, LC–ESI–MS C18 column (125 9 2 mm; PDA and Provides the fragmentation Kuhn et al. Lupinus albus Kaempferol, Isorhamnetin, 20- 3 lm) Q-TOF MS behaviour of seven pairs of (2003) 0 Hydroxygenistein, 3 -O- Mobile Phase—acetonitrile isomeric flavone/isoflavones 0 Methylorobol, 7,3 -Di-O- and water aglycones methylorobol, Luteone, Wighteone, Daidzein, Genistein and Glycitein Seeds of Lupinus 20-hydroxygenistein-40,7-O- LC–ESI–MS/ RP-18 column (250 9 2 mm) Ion Trap MS Analyzed numerous flavonoid and Kachlicki albus, Lupinus diglucoside, Genistein 8-C-7-O- MS Mobile Phase—acetonitrile/ isoflavonoid glycosides with et al. (2005) 0 angustifolia, diglucoside,Genistein4 ,7-O- water/acetic acid (95:4.5:0.5 different degree of 0 0 Lupinus luteus diglucoside,2 -hydroxygenistein 4 ,7- v/v/v) and water/ glycosylation and various O-diglucoside malonate I, Genistein acetonitrile/acetic acid pattern and location of 0 8-C-7-O-diglucoside malonate,-2 - (95:4.5:0.5 v/v/v) glycosidic moieties hydroxygenistein 8-C-glucoside, Genistein 7-O-glucosylglucoside,20- hydroxy genistein 40,7-O-diglucoside maloylated I, 20-hydroxygenistein 7-O-glucoside, Genistein40.7-O- diglucoside malonate II, Genistein 8-C-glucoside, 20-hydorxygenistein 8-C-glucoside malonate, Genistein 6-C-glucoside, Genistein 7-O- glucosylglucoside malonate, Genistein 8-C-glucoside malonate I, 20-hydroxygenistein 7-O-glucoside malonate I, Genistein 7-O-glucoside, 20-hydroxygenistein 7-O-glucoside malonate II, Genistein 8-C-glucoside malonate II, Genistein 40-O- glucoside, Genistein 7-O-glucoside malonate I and Genistein 7-O- glucoside malonate II Rev Phytochem htce Rev Phytochem Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Tea and Shiitake Rotenone LC–ESI–MS/ C18 column (100 9 2.1 mm; MS LC–MS/MS method for the Xu et al. mushroom MS 3.5 lm) determination of rotenone in (2010) Mobile Phase—water with food stuff with the LOQ within the range of 0.001 to 0.1 % formic acid (w/v) and -1 acetonitrile. 0.005 mg kg in variety of food matrices

Roots of Calycosin-7-O-b-D-glucoside, LC–ESI–MS C18 column (150 9 2.1 mm; PDA and MS First to report the use of LC–ESI– Lin et al. Astragalus Calycosin-7-O-b-D-glucoside-600-O- 5 lm) with a sentry guard MS for the identification of (2000a) mongholicus malonate, Ononin, Calycosin (3R)- column C18 (20 9 3.9 mm; flavonoids of A. mongholicus and Astagalus 7,20-dihydroxy-30,40- 5 lm) and A. membranaceus membranaceus dimethoxyisoflavan-7-O-b-D- Mobile Phase—0.25 % acetic glucoside and Formononetin-7-O-b- acid and acetonitrile D-glucoside-6-O-malonate, containing 0.25 % acetic formononetin acid 0 0 Heartwood of 3 -hydroxydaidzein, Koparin,2 ,7- LC–ESI–MS C18 column (250 9 4.6 mm; PDA and Ion Provides a method for the Liu et al. Dalbergia dihydroxy-40,50-dimethoxyisoflavone, 5 lm) with a C18 guard trap MS simultaneous identification of (2005) odorifera 20-hydroxy formononetin, column (20 9 4 mm; 5 lm) major bioactive flavonoids Formononetin, Prunetin, Violanone, Mobile Phase—acetonitrile present in D. odorifera 0 Vestitone, 3 -O-methylviolanone, and 0.3 % acetic acid Sativanone, (3R)-40-methoxy-20,3,7- trihydroyisoflavonone and Medicarpin

Rhizopus Daidzin, Glycitin, Glycinol, Genistin, UPLC–ESI– C18 column (150 9 2.1 mm; PDA and The screening for Isoflavonoids Simons et al. microsporus Malonyl-daidzin, malony-genistin, MS 1.7 lm) Linear Ion was performed using LC–MS. It (2011b) elicted Glycine Biochanin A, Glycitein, Glyeofuran, Mobile Phase—0.1 % acetic Trap MS was estimated that 40 % of total 0 max seedlings Daidzein, 2 -OH-genistein, Genistein, acid and acetonitrile with isoflavonoids were prenylated Glyceollidin I/II, Glyceollin III, 0.1 % acetic acid pterocarpans Glyceollin II, Glyceollin I, Aprenyl- 20-OH-daidzein, Bprenyl-glycitein, Glyceollin V/VI, Aprenyl-daidzein, Aprenyl-20-OH-genistein, Bprenyl- daidzein, Glyceollin IV, Aprenyl- genistein andBprenyl-genistein 123 123 Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Rhizopus Daidzin, Glycitin, Glycinol, Genistin, UPLC–ESI– C18 column (150 9 2.1 mm; PDA and Identified the effect of Rhizopus Simons et al. microsporus Malonyl-daidzin, malony-genistin, MS 1.7 lm) Linear Ion microsporus induction on (2011b) elicted Glycine Biochanin A, Glycitein, Glyeofuran, Mobile Phase—0.1 % acetic Trap MS Glycine max and concluded that 0 max seedlings Daidzein, 2 -OH-genistein, Genistein, acid and acetonitrile with there was increase in the Glyceollidin I/II, Glyceollin III, 0.1 % acetic acid isoflavonoid content Glyceollin II, Glyceollin I, Aprenyl- accompanied by a gradual 20-OH-daidzein, Bprenyl-glycitein, increase in agonistic activity Glyceollin V/VI, Aprenyl-daidzein, towards estrogen receptors. The Aprenyl-20-OH-genistein, Bprenyl- Identification of 27 daidzein, Glyceollin IV, Aprenyl- Isoflavonoids was conducted genistein and Bprenyl-genistein using LC–MS 0 Roots of Formononetin, Glabridin, 3 -Hydroxy- LC–ESI–MS C18 column (2.1 9 5 mm; PDA and Provided a UHPLC–ESI–MS Simons et al. Glycyrrhiza 40-O-methylglabridin, 40-O- 1.7 lm) Linear Ion method for the screening of (2009) glabra methylglabridin, Hispaglabridin A, Mobile Phase—0.1 % acetic Trap MS prenylated flavonoids in multi- Hispaglabridin B, Glabrone and acid and acetonitrile with component plant extracts Glabrene 0.1 % acetic acid

Dried Roots and Licoisoflavone A, Licoisoflavone B, HPLC–ESI– C18 column (50 9 4.6 mm; PDA and TOF Describes the LC–MS/MS (Li et al. Rhizomes of G. Glyurallin B, Angustone B, MS/MS 1.8 lm) MS method for the determination of 2011) inflata, kanazonol X and Kanazonol H Flavonoids and Isoflavonoids Glycyrrhiza from Glycyrrhiza Species glabra and Glycyrrhiza pallidiflora

Leaves of Formononetin-7-glucoside-6- LC–APCI– C18 column (150 9 4.6 mm; Linear Ion Described the detailed chemical Tibe et al. Hedysarum omalonate MS 1.5 lm) Trap MS composition of phenols and (2011) coronarium Mobile Phase—0.1 % formic Condensed Tannins in Sulla acid and acetonitrile with leaves 0.1 % formic acid htce Rev Phytochem htce Rev Phytochem Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

0 Seeds of Lupinus Genistein 4 ,7-di-O-glucoside, LC–ESI–MS/ C18 column (100 9 2.1 mm; TOF MS Developed LC–MS/MS method Muth et al. angustifolius Genistein 40,7-di-O-glucoside MS 1.8 lm) capable for resolution of (2008) 0 malonate I, Genistein 4 ,7-di-O- Mobile Phase—water/ Isomeric malonate glucoside malonate II, Genistein 8-C- acetonitrile/formic acid glycoconjugates of Flavonoids 0 glucoside, Genistein 4 ,7-di-O- (95:4.5:0.5 %) and and recognition of structural 0 glucoside malonate III, 2 - acetonitrile/water/formic difference Hydroxygenistein 7-O-glucoside, acid (95:4.5:0.5 %) Genistein 40,7-di-O-glucoside dimalonate I, Genistein 7-O- glucoside, Genistein 40,7-di-O- glucoside dimalonate II, Genistein 40,7-di-O-glucoside dimalonate III, Genistein 8-C-glucoside malonate, Genistein 7-O-xylosylglucoside, 20- Hydroxygenistein 7-O-glucoside malonate, Genistein 7-O-glucoside malonate I, Genistein 7-O- xylosylglucoside malonate and Genistein 7-O-glucoside malonate II

P. Medicanginis Formononetin 7-O-glucoside, HPLC–ESI– C18 column (250 9 2 mm) PDA and TOF Investigated the effect of P. Jasinski et al. infected seeds Formononetin 7-O-glucoside MS/MS Mobile Phase—0.5 % formic MS medicaginis on the phenolic (2009) of M.truncatula malonate and Medicarpin acid) and acetonitrile with profile of Medicago trunculata 0.5 % formic acid and analyzed the phenolic constitutes via LC–MS/MS

Seedlings of Daidzein and its glycoconjugates, HPLC–ESI– C18 column (100 9 2.1 mm; PDA and TOF Analysis conducted provided an Staszkow Medicago Genistein and its glycoconjugates, MS 1.8 lm) MS overview of flavonoids and their et al. (2011) truncatula Biochanin A and it glycoconjugates; conjugates in different plant Formononetin and its material representing the model glycoconjugates; Irisolidone and its legume, M. truncatula glycoconjugates and Medicarpine and its glycoconjugates

Dried stems of Ononin, Daidzein, Calycosin, HPLC–ESI– C18 column (250 9 4.6 mm; PDA and TOF A potential combined method Cheng et al. S.suberectus Genistein, Formononetin, MS 5 lm) MS using ultrasonic/microwave (2011) Afrormosin, Prunetin and assisted extraction and a Biochanin A diagnostic ion filtering strategy with LC-Q-TOF–MS is

123 proposed for the rapid characterization of flavonoids 123 Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Roots of Neopuerarin A and Neopuerarin B LC–ESI–MS C18 column (250 9 4.6 mm) NMR with Ion Identified two new isoflavonoids Zhang et al. Pueraria lobata Mobile Phase—0.1 % formic Trap MS and characterized them with (2010) acid and methanol LC–MS and NMR

Roots of Radix Glucosyl-a-1,6-puerarin LC–ESI–MS/ C18 column (150 9 3.9 mm) UV and TOF Identified a novel compound in Nguyen et al. puerariae MS Mobile Phase—0.1 % formic MS the roots and determined the (2009) acid and acetonitrile structure using LC–MS and NMR data

Roots of Aureol, Sissotrin, LC–ESI–MS C18 column (250 9 4.6 mm) PDA with Ion Studies the fragmentation Yang et al. Hedysarum Demethylhedysarimpterocarpene A, Mobile Phase—acetonitrile Trap MS behaviour and identified the (2007) mulijugum Hedysarimcoumestan E, and 0.2 % acetic acid constituents in Methanolic Hedysarimcoumestan B, 1,3- extracts and identified 29 Dihydroxy-9-methoxypterocarpene, compounds Hedysarimpterocarpene A, Hedysarimcoumestan F, 7-Hydroxyhedysarimpterocarpene B, Hedysarimcoumestan D, Hedysarimcoumestan G, 1,7- Dihydroxy-3,9-dimethoxy-10-(3- methylbut-2-enyl)-pterocarpene, Hedysarimpterocarpene B, Kanzonol K, 1,7-Dihydroxy-3,9-dimethoxy-8- (3-methylbut-2-enyl)pterocarpene, 2-(2,6-Dihydroxy-4-methoxyphenyl)- 3-formyl-4-hydroxy-5/6-methoxy-6/ 5-(3-methyl-but-2-enyl)benzofuran, Hedysarimcoumestan H, Gancaonin M, Gancaonin A, Methylhedysarimcoumestan H and 5,7-Dihydroxy-40-methoxy-6,8- dipenylisoflavone htce Rev Phytochem htce Rev Phytochem Table 3 continued Sample Isoflavonoids analyzed Analytical Instrument condition Detector Remarks References technique condition

Roots and Cell Genistein b-D-di-glucoside, Genistein LC–ESI–MS C18 column (250 9 4.6 mm) PDA and TOF Simultaneously identified and Farag et al. Culture of M. b-D-di-glucoside-malonate, Daidzin, Mobile Phase—0.1 % acetic MS quantified 35 polyphenols (2007) truncatula Daidzin malonates, Biochanin A-b-D- acid and acetonitrile di-glucoside, Genistin, Biochanin A b-D-diglucoside-malonate, Genistein 7-O-b-D-glucoside-60’-O-malonate, 20-Hydroxyformononetin b-Dglucoside, Ononin, 20- Hydroxyformononetin b-Dglucoside- Malonates, Formononetin 7-O-b-D- glucoside-60’-O-malonate, Medicarpin 3-O-b-D-glucoside, Biochanin A 7-O-b-D-glucoside, Medicarpin 3-O-b-D-glucoside- Malonates, Biochanin A 7-O-b-D- glucoside-60’-O-malonate, Formononetin and Medicarpin

Seeds of OH-Acetylpuerarin, 2-OH-Puerarin, HPLC–ESI– C8 Column (250 9 4.6 mm) PDA and MS A total of 48 metabolites were Prasain et al. Pueraria lobata OH-Puerarin, Malonylpuerarin, MS/MS with RP8 guard column detected in extracts of root (2007) Acetylpuerarin, Acetyldiadzin, (15 9 3.2 mm) culture by LC–MS/MS Malonyldaidzin, Daidzin, 2-OH- Mobile Phase—10 % aq. daidzin, Daidzein-2-O-hexoside, OH- acetonitrile containing acetyldiadzein, Genistin, OH- 0.1 % trifluoroacectic acid Genistin, Genistein and Biochanin A and 90 % aq. acetonitrile containing 0.1 % trifluoroacectic acid 0 0 Roots of Sophora Kushenol O,3 ,7-Dihydroxy-4 - LC–ESI–MS/ C18 column (200 9 4.6 mm; PDA and Ion Identified 24 Flavonoids from Zhang et al. flavescens methoxy isoflavones, Formononetin, MS 5.0 lm) Trap MS Sophora flavescens based on (2007) Sophoraisoflavanone A, Trifolirhizin, Mobile Phase—acetonitrile UV and MS Spectra 00 Trifolirhizin6 -malonate, Maachiain and 0.1 % acetic acid and Kushecarpins A 123 Phytochem Rev

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