Determination of flavonoids and their metabolites by chromatographic techniques

Accepted Manuscript Determination of flavonoids and their metabolites by chromatographic techni‐ ques Małgorzata Szultka, Katarzyna Papaj, Aleksandra Rusin, Wiesław Szeja, Bogusław Buszewski PII: DOI: Reference:

S0165-9936(13)00075-7 http://dx.doi.org/10.1016/j.trac.2013.02.008 TRAC 14056

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Trends in Analytical Chemistry

Please cite this article as: M. Szultka, K. Papaj, A. Rusin, W. Szeja, B. Buszewski, Determination of flavonoids and their metabolites by chromatographic techniques, Trends in Analytical Chemistry (2013), doi: http://dx.doi.org/ 10.1016/j.trac.2013.02.008

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Determination of flavonoids and their metabolites by chromatographic techniques Małgorzata Szultka, Katarzyna Papaj, Aleksandra Rusin, Wiesław Szeja, Bogusław Buszewski This article presents an overview of biological properties and biotransformation of isoflavones in vivo and describes methods of their identification, with special attention given to investigation by liquid chromatography. We discuss the advantages and the disadvantages of different methods of sample preparation, and the choice of suitable columns, solvents and detectors used for identifying isoflavones in different samples. Keywords: Chromatography; Chromatographic column; Detector; Determination; Isoflavone; Liquid chromatography (LC); Metabolite; Sample preparation; Solvent

Flavonoid;

*

Małgorzata Szultka, Bogusław Buszewski Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87 100 Torun, Poland Katarzyna Papaj, Wiesław Szeja Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, Gliwice, Poland Aleksandra Rusin Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch

∗

Corresponding author. Tel.: +48 566114308; Fax: +48 566114837; E-mail: [email protected]

1.

Introduction

Flavonoids occur as secondary metabolites in plants, in which they play important function as antioxidants, fungicides, insecticides, dyes and UV protectants [1]. These compounds are classified as benzo-γ-pyrone derivatives (ring A+C), due to the presence of a heterocyclic compound containing oxygen (γ-pyrone, ring C) between the rings A and B. The phenyl substituent (ring B) is attached to the ring C at the position C-2 or C-3 [2,3]. Due to differences in the structure of the ring C (presence of a double bond between atoms at position C-2 and C-3, the number and the position of hydroxyl groups and the presence of a carbonyl group at C-4), these compounds are divided into 1

several classes: flavones, isoflavones, flavonols, flavanonols, flavanones, flavanols and anthocyanidins [2]. Flavonoids differ from each other by type and place of attachment of different substituents {e.g., methoxyl groups (-OCH3), hydroxyl groups (-OH) [2,3], acyl groups (-C (= O) R) [4] and saccharides [2]}. The chemical structures of flavonoids belonging to different classes, with their dietary sources, are presented in the Table 1. One of the best known isoflavones is genistein, which is present in high concentrations in plants of the legume family. The content of genistein in soy food is in the range 0.2–1 mg/g [6]. Epidemiological data indicating positive correlation between consumption of genistein-rich soy products and reduced mortality from certain cancers in Asian countries initiated intensive research on the biological activity of genistein [6,9]. Many cellular processes regulated by genistein, relevant to its anti-metastatic, pro-apoptotic and antiproliferative activity have been described [10]. Genistein reduces the proliferation of cancer cells by inhibiting the activity of tyrosine kinases and topoisomerase II. Moreover, it significantly influences the expression of genes regulating cell proliferation [6,10]. Some of genistein derivatives also show anti-proliferative activity, and they can inhibit cell cycle. Genistein analogues that contained a sugar substituent at C-7 position showed the ability to inhibit the cell cycle in the G2 and M phases [11,12]. Compounds in which the sugar was attached at the C-4' position inhibited the cell cycle in G1 phase [Rusin, unpublished] Genistein is also considered to be used as a supplementary drug in treatment of osteoporosis and alleviating symptoms of the menopause [6]. These potential applications of genistein result from its structural similarity to 17-β-estradiol – the natural ligand of the ER receptor [6,13] and binding to estrogen receptors (ER) [6,13–17]. Genistein can bind to both sub-types of this receptor, ER-α and ER-β [13], but with stronger affinity to ER-β [6]. Genistein induces agonistic or antagonistic effects, depending on its concentration, the target tissue and the hormonal status of the organism [18].

2.

Transformation of flavonoids in living organisms

2.1. Absorption and distribution Absorption of isoflavonoids (including genistein) in the gastrointestinal tract depends on their chemical structure [19]. Aglycones (flavonoids not substituted with saccharides, or sulfuric or glucuronic acid) are absorbed differently from their hydrophilic carbohydrate derivatives. Aglycones have lower molecular weight and more hydrophobic character, so they are transported fast. They show the ability to interact with lipids of enterocyte membranes and pass through the small intestine mucosa as a result of a passive diffusion [9,20]. Glycosides of flavonoids administered by the oral route are absorbed in the small intestine mostly after their prior hydrolysis to aglycones [5,19]. Flavonoids connected with saccharides can be absorbed only to a small extent in an unchanged form via active transport using sodium-dependent glucose transporter-1 SGLT1 [19] and Breast Cancer Resistance Protein [21]. 2.2. Metabolism and elimination Biotransformation of flavonoids in the human body comprises two stages: • the first involves disconnection of functional groups from isoflavone molecules, and, next, the released isoflavone is O-methylated or conjugated to glucuronic or sulfuric acid; this stage occurs in the small intestine, kidneys and liver; and, 2

• the second, leading to total degradation of isoflavonoids, occurs in the large intestine (Fig. 1). Glycosides of flavonoids ingested orally are exposed in the digestive tract to the enzymes that remove the saccharide from the molecule and release the aglycone. The enzymes primarily involved in this process are the bacterial β-glucosidases and, to a lesser extent, intestinal β-glucosidases. The β-glucosidases, present in the epithelium of the small intestine, which are able to break down the β-glycosidic bond, include phlorizin hydrolase, cytosolic β-glycosidase C and glucocerebrosidase [19]. Next, polar groups of flavonoids are conjugated with glucuronic acid (as a result of glucuronosyl-transferase activity) or sulfuric acid (due to the activity of sulfo-transferase). This type of conjugation may occur in the wall of the small intestine during the transport of these compounds, and in the liver [9,19]. The metabolism of isoflavonoids in the liver is catalyzed by enzymes responsible for the hydroxylation and demethylation (enzymes of phase I) and enzymes leading to O-methylation or to conjugation with glucuronic acid or sulfuric acid (enzymes of phase II) [9,19,20]. Flavonoids may also undergo hydroxylation in microsomes of hepatocytes. This reaction is a result of the activity of cytochrome P-450 [20,22]. Genistein is converted by P450 into two dihydroxylated and hydroxylated bioactive derivatives, which, due to their toxicity, are neutralized by conjugation with sulfate or glucuronic acid [23]. The liver may also carry out methylation of isoflavonoids with catechol-O-methyltransferase [19], but the methylated derivatives are produced only in trace amounts [20,23]. Hydrophilic compounds formed in the liver are next excreted with urine [9]. The part of isoflavonoids that has not been modified in the liver is excreted with bile to the intestine. After the excretion to the intestine, flavonoids may be re-absorbed (the process being called enterohepatic circulation) [9,23]. This route is interrupted when the substance loses its lipophilic nature as a result of biotransformation in the liver, and the products are excreted with the urine. This circulation extends the half-time of isoflavonoid elimination and prolongs the duration of their activity [9,19]. The final stage of biotransformation of isoflavonoids takes place in the large intestine. Microorganisms present in this part of the alimentary canal split the nucleus of a flavonoid molecule [9,19]. Genistein is converted to dihydrogenistein. Then, the ring C is decomposed, resulting in formation of 6'-hydroxy-O-desmethylangolensin (6'-OH-ODMA). 6'-OH-ODMA is converted by colonic bacteria to 4-hydoxyphenyl-2-propionic acid and trihydroxybenzene. Decarboxylation of the propionic acid derivative leads to 4-ethylphenol [9,20]. Some metabolites may be re-absorbed again, but absorption in the colon is less efficient than in the small intestine. Unabsorbed metabolites are excreted in the feces [9,19].

3.

Sample preparation techniques for flavonoids analysis

Over the years, many sample pre-treatment methods have been developed to determine flavonoids in different matrices. For the purpose of isolating analytes, solvent extraction, which may be followed by solid-phase extraction (SPE), is still the most widely used technique, mainly because of its ease of use and wide-ranging applicability. Soxhlet extraction is used less frequently to isolate flavonoids from solid samples. From liquid samples, analytes are isolated by liquid-liquid extraction (LLE) or SPE. In case of LLE, extraction is usually directed at the isolation of aglycones, while the other methods can have isolation of both aglycones and conjugates as their goal. 3

Intensive research on the medicinal properties of flavonoids and their derivatives contributed to the progress of analytical methods, allowing fast, reliable detection of these substances in various samples [24,25]. The technique used most often for the analysis of different flavonoids, including genistein, is high-performance liquid chromatography (HPLC) or ultra-HPLC (UHPLC) in combination with various detection methods, described in detail below [26,27]. For both quantitative and qualitative analyses, a very important step is proper preparation of the sample, which differs depending on: (1) the physical state of the sample (fluid or solid) [26,28]; (2) the concentration of the compound in the sample (flavonoids in biological samples are usually at a fairly low level, so it may be necessary to enrich the sample by extraction) [26,28]; (3) the type of the matrix containing flavonoids (particular components of a matrix may interfere with the analyte, making the analysis difficult) [26,28]; and, (4) the chemical nature of the analyzed compound [26,28] (flavonoids in physiological human material and in plant samples are most frequently in the form of conjugates: in food samples, they mainly occur as glycosides, whereas in the material isolated from animals and humans, the most common forms are glucuronic or sulfuric derivatives [24,27]. The samples analyzed include plant material, food and drinks, biological fluids or feces, so the methods of sample preparation depend primarily on the physical state of the sample [26,28]. For determination of flavonoid content in solid samples, the tested material is first homogenized [29], lyophilized [30] or frozen in liquid nitrogen [31]. Then, isolation of the analyte is carried out by LLE, using diethyl ether, ethyl, acetate or methylene chloride with a small amount of acid [8,30]. SPE is used equally often in the samplepreparation stage [24]. Depending on the sample adsorption to the surface of the sorbent, different types of SPE packing and elution solvents are used. For purification of flavonoids by SPE, silica gel modified with an octadecyl group (C18) is suitable as a packing material and aqueous solutions of methanol, ethanol or acetonitrile are used as extractants [24,28]. The efficiency of isolating flavonoids from a biological matrix can be improved through the use of supercritical-fluid extraction (SFE), which is combined with liquid-solid extraction in the final stage of analyte isolation [25]. For the analysis of flavonoids in the liquid matrix, the sample is prepared by filtration [26] or centrifugation [8]. In some cases, this type of sample preparation is sufficient for the analysis, although some samples may require additional SPE or LLE [27]. The choice of the method of sample preparation also depends on the chemical nature of isoflavonoids (aglycones or conjugates) to be detected [8]. Whereas aglycones are easily detected in the samples using direct analysis, the detection of flavonoid conjugates is more complicated due to the presence of substituents [32]. 3.1. Preparation of sample including flavonoid conjugates In the literature concerning analysis of conjugates of isoflavonoids present in biological samples, two different approaches are described. The first method requires the use of a standard (flavonoid connected with an appropriate acid or saccharide) and a suitable detector [15]. Identification of the metabolite is performed by comparing the standard’s retention time (RT) with the RT of compounds present in a sample [33]. Moreover, the molecular mass of sample components is determined using a mass spectrometer [33,34]. 4

However, another method, based on the quantitative determination of aglycones released after hydrolysis of flavonoid conjugates, is used more frequently. The first step is determination of the amount of free aglycones present in the sample, and the next is identification of the amount of aglycones in the hydrolyzed sample. The amount of flavonoid conjugates is calculated by subtracting the amount of free aglycones from the amount of aglycones present in the hydrolyzed sample. Usually in this method, the enzymatic hydrolysis is used, enabling the selective hydrolysis of analyzed conjugates (e.g., to analyze flavonoids conjugated with sulfuric acid, samples are treated with sulfatases) [35]. The alternative to enzymatic hydrolysis of flavonoid conjugates is acidic processing, also leading to release of the aglycone. Acid hydrolysis is usually carried out under reflux, using hydrochloric acid [36] or formic acid in the presence of ethanol [25]. Because in most cases hydrolysis in an acidic environment leads to changes of the structure of flavonoid molecules, enzymatic hydrolysis is used instead [24]. The most frequently used enzymes (glucuronidase and arylsulfatase) are acquired from the intestines of mollusks. β-glycosidase is obtained from almonds, cellulase from Aspergillus niger and recombinant β-glucuronidase from Escherichia coli [24]. The cheapest source of sulfatase and β-glucuronidase is Helix pomatia gut [37]. Samples are hydrolyzed by a mixture of enzymes dissolved in sodium-acetate buffer (0.14 M at pH = 5.0) [58] during incubation at 37° C for 2–24h [24]. For verification of enzyme activity, phenolophthalein glucuronide and 4-methylumbelliferone glucuronide or sulfate are used [24,28]. After the enzymatic hydrolysis, samples are further processed by LLE [38], using diethyl ether [39] or ethyl acetate [35], or SPE [40]. In order to determine the content of particular forms of flavonoids in the tested sample, it may be divided into several aliquots, each treated with an enzyme to release the aglycone from a specific type of conjugate (Fig. 2). As far as the analysis of untouched glycoside conjugates of flavonoids is concerned, mild extraction conditions, including low temperature and inhibition of endogenous enzymes digesting the glycosidic bond, are essential. In the first stage of material preparation, samples are lyophilized and then extracted at room temperature or 4°C. The choice of proper solvent used for extraction is another factor crucial in analysis of glycosides of flavonoids and aglycones. Glycosides are extracted with a mixture of methanol (ethanol) and water (80:20, v/v) or with a mixture of acetonitrile and hydrochloric acid (0.1 M) or water [24], whereas, for extraction of aglycones, ethyl acetate is commonly used [8]. In order to perform quantitative analysis, it is extremely important to determine the amount of analyte that is degraded during sample preparation, so the sample is enriched with a defined amount of the compound, called the internal standard (IS). Next, the final concentration of the IS in samples is determined [8,25]. The compound that serves as the IS should have a structure and properties similar to the analyte. Moreover, it should give the analytical signal within the range of wavelengths used for detecting the compounds tested [24]. For the analysis of flavonoids by HPLC combined with ultraviolet detection (HPLCUV), fluorescein or flavone is used as the IS [24]. For HPLC-MS, the IS can be deuterated genistein, daidzein [35], biochanin A [39] or formononetin [33]. Due to the 13 2 C or H isotopes, other chemical limited availability of isoflavonoids labeled with compounds of structure similar to the analyte are quite often applied as the IS [24,28].

4.

Chromatographic investigations of flavonoids and their metabolites

Several separation techniques [e.g., gas chromatography (GC), capillary electrophoresis (CE) and LC] have been applied in analysis of flavonoids, their derivatives and metabolites, yet 5

HPLC is used most commonly. HPLC separates components of a mixture on the basis of their differential distribution between the mobile and the stationary phase. For detection of genistein, adsorption chromatography is most commonly used. For improving the selectivity of separation of flavonoids, packings containing polar groups incorporated into hydrophilic chain (e.g., acrylamide, cholesterol or phospholipid phase) are used. Using the stationary phases described above, separation is carried out by reversedphase LC (RP-HPLC or RP-UHPLC) [8,55]. Separation can be also achieved using hydrophilic interactive liquid chromatography (HILIC) with properly selected ion stationary phases and excess of acetonitrile as a component of the mobile phase [41]. Another important factor is the selection of an appropriate mobile phase. For separation of isoflavonoids by RP-HPLC, the universal mobile phases used are mixtures of methanol or acetonitrile with water [8,25]. Because isoflavones are weak acids, in order to increase retention rates and improve the shape of the peaks, the mobile phase is acidified with 0.1–1% formic acid, acetic acid [25], phosphoric (V) acid [25], trifluoroacetic acid [25], 10-mM ammonium acetate or formate [25]. At present, phosphoric acid is rarely used because of its negative influence on the ion source in the mass spectrometer [25]. For RP-HPLC analysis, the selection of an appropriate column temperature and injection volume is another factor influencing detection. For genistein, analysis is carried out at room temperature [25], and the injection volume is from 5–100 µL [26]. The next element of the detection system that requires detailed description is the selection of a suitable detector. The detectors used most often are: o UV absorbance [55]; o UV-diode array (UV-DAD) [54]; o electrochemical (ED) [56]; o fluorescence (FLD) [42]; or, o MS [25]. The detectors used most often are based on UV absorbance or, less frequently, visible light absorbance. In analysis of flavonoids, UV detectors can be used, due to the presence of aromatic rings in the molecule. These rings absorb light in the wavelength range 230–280 nm [8,25]. Flavonoids have two characteristic ranges of absorption: o range II with absorption maximum at λ = 240–285nm associated with the ring A; and, o range I, with the maximum occurring at λ = 300–550 nm, associated with the ring B. For genistein and other isoflavones, range II is usually more intensive than range I [25,26]. The use of a UV detector that performs the analysis only at one wavelength is limited to studies on mixtures of flavonoids, because the absorption maxima of different classes of these compounds may differ [26]. The solution to this problem is the use of a UV-DAD, allowing for continuous adjustment of the wavelength across the UV and visible parts of the spectrum. Slightly higher sensitivity, compared to the UV or the UV-DAD detector, is exhibited by an electrochemical detector (ED) [8]. Flavonoids and their metabolites can be determined by an ED because of the presence of electroactive phenolic groups in the molecule. Phytoestrogens are oxidized, and their oxidation potential is in the ranges 0–+1000 mV. The maximum potential is usually close to +700 mV [8]. Moreover, the presence of different substituents in analogous structures influences their voltamperometric properties. This makes the use of an ED suitable for detection of derivatives and metabolites of flavonoids [32]. In flavonoid analysis, the FLD is used only occasionally, because the number of flavonoids that exhibit native fluorescence is limited. For these compounds, the limits of 6

detection (LODs) in LC and CE are typically about an order of magnitude lower than in UV detection. Moreover, their fluorescence facilitates selective detection in complex mixtures [42]. Classes of flavonoids that show native fluorescence include isoflavones, flavonoids with an –OH group in the 3-position. Since most flavonoids are electroactive due to the presence of phenolic groups, ED can also be used [25]. Compared to the above detectors used in flavonoids analysis, HPLC-MS offers higher sensitivity, selectivity and versatility. This type of detection is especially useful for reliable identification of flavonoids in complex matrices. The use of HPLC-MS obtains simultaneously information regarding not only the type, the amount and the RT of the particular compounds, but also their molecular weight and fragmentation pathways [28]. The analysis with MS detector may be performed with different types of ionization in the ion source. In case of flavonoids, electrospray ionization (ESI) and chemical ionization under pressure, called atmospheric pressure chemical ionization (APCI), are most commonly used. Although flavonoids may be determined in both positive [M +H]+ and negative [M-H]ion modes, the negative ionization mode is used in most cases [28,34]. An important element of the MS detector is the analyzer. In the analysis of flavonoids, the most commonly used analyzers are quadrupole and triple quadrupole [25,28,29]. Due to the low resolving power of a single device, tandem mass spectrometers are much more frequently used. Such systems are not only more selective and sensitive, but also allow for quantitative analysis, provide information about the structure of the compound and characterize the fragmentation pathways of a single compound present in a complex matrix. Currently, in the analysis of flavonoids, a combination of several detectors, mainly UV-MS, is most commonly used [25,33]. The application of matrix-assisted laser desorption/ionization (MALDI) and time-offlight (TOF) in flavonoid MS has also been reported [43]. MALDI-TOF-MS is a simple, fast technique for the analysis of large biomolecules, but is not suitable for detection of low molecular weight molecules and compounds (e.g., flavonoids), mainly due to the lack of an appropriate matrix. However, Liu et al. [44] developed a quick, simple LDI-TOF-MS method for the detection of flavonoids. Analytes were spotted onto a matrix of graphene-based nanoparticles and then analyzed in the negative ion mode. Ptekovic et al. [45] applied flavonoids (apigenin, kaempferol and luteolin) as matrices for MALDI-TOF-MS analysis of a transition-metal complex. Such application of flavonoids as matrices is recommended for monitoring of synthesis processes because the acquired spectra are simple, and they have low background signal. Moreover, the flavonoid matrices appeared to have a higher tolerance for MALDI-TOF-MS analysis of samples with higher amounts of inorganic salts. This approach has great potential for reliable analysis of new metallo-drugs. 4.1. Gas chromatography Flavonoids became a subject of interest in GC, thanks to their low volatility, nonflammability, good solvent properties and high viscosity. Also, GC-based methods provide high resolution and low LODs. However, they possess some disadvantages, because they are labor intensive and time consuming. Moreover, a derivatization step is required to increase the volatility of the flavonoids and to improve their thermal stability. Magiera et al. [46] reported separation and detection of dihydrobiochanin, dihydrodadzein, dihydrogenistein, chrysin, pelargonidin, biochanin, 2’-hydrobiochanin, (±)catechin, daidzein, (-)-epicatechin, genistein, hesperetin, glycitein, 8-hydroxydaidzein, apigenin, desmethylglycitein, quercetin and myricetin by GC-MS in human urine. Also, this work was devoted to separation of different flavonoids ingested in combination with βblockers, which have a narrow therapeutic range. The results obtained showed that GC-MS 7

could be used effectively for the analysis of flavonoids and their metabolites in real urine samples. The method proposed provides a convenient index of metabolism of compounds in urine and could be used to explore the effect of dietary polyphenols on pathways involved in drug metabolism. Finally, this new approach may be extended to determine the pharmacokinetics of drugs and flavonoids and to examine drug-flavonoid interactions in combination therapy. Gao et al. [47] presented for the first time the applicability of comprehensive twodimensional GC (GC×GC) coupled to both flame ionization (GC×GC-FID) and TOF-MS detectors (GC×GC-TOF-MS) for 34 flavonoids in natural samples with complex matrices. The results obtained demonstrated the potential of the analytical procedure described with full spectral information for rapid, sensitive structure elucidation and identification of previously unidentified compounds in a sample. 4.2. Electromigration techniques Chemical properties of flavonoids often depend more on the anionic part of the molecule, which makes them good candidates for electroosmotic flow (EOF) modifiers in organic solvents. Although capillary electrophoresis (CE) allows higher separation efficiency than LC, it has found very limited application in flavonoid analysis. The CE modes primarily used are capillary-zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Besides GC and LC, CE also appears to be a powerful tool in the analysis of flavonoids. The main advantages over HPLC are that run times are relatively short and only minimal reagents, mostly of a non-toxic nature, are consumed during analysis, resulting in a more cost effective and environment-friendly technique that both rivals and complements HPLC. Lee et al. [48] developed a rapid CE method, involving large-volume-sample stacking (LVSS), suitable for sensitive detection and quantitation of selected flavonoids in food samples. This procedure allows for extractions of flavonoids at low concentration levels without the need for expensive detectors. In addition, short analysis time along with greatly reduced solvent consumption associated with this method made it a viable alternative to traditional HPLC. CE based on the principles of frontal analysis (FA) was found to be a good method for the study of the interaction of flavonoids [flavones in the basic form (e.g., monoglycoside quercitrin and diglycoside rutin)] with human serum albumin (HSA). Binding constants and thermodynamic parameters were characterized and the specific binding site for flavonoids to HSA was identified. Finally, it was concluded that the presence of sugar moieties, and the number of saccharides, had a marked effect on the binding properties of flavonoids in the case of HSA. Moreover, glycosylation of flavonoids decreased their binding capacity to HSA [49]. Chi et al. [50] reported the simultaneous determination of kaempferol, apigenin, rutin, ferulic acid, quercetin and luteolin in herbal samples by CZE, because these compounds possess similar structure and chemical characteristics. β-cyclodextrin in the running buffer was chosen for the separation. 4.3. High-performance liquid chromatography The technique most widely used in flavonoid analysis is based on RP-HPLC coupled to photodiode-array (PDA) detection and/or MSA or tandem MS with atmospheric pressure ionization techniques (i.e. ESI and APCI). PDA is an indispensable tool for provisional identification of the main phenolic structures present in plant samples, since they show characteristic UV-Vis spectra. LC-MS has, however, become the best option for their identification and structural characterization, because, for the investigation of structure8

activity relationships and food-quality control, it is important to have access to rapid, reliable methods for the analysis and identification of phenolic compounds in all their numerous forms. Since flavonoids exist in complex natural matrices, these analytical methods should also be selective and sensitive. HPLC analysis of flavonoids is usually carried out in the RP mode employing octyl C8-bonded or octadecyl C18-bonded silica columns. RP columns are used because these compounds are weak acids that can be separated as neutral, relatively hydrophobic compounds in a weak acid matrix. RP C18 phases with the following conventional dimensions are almost exclusively employed: column length, 100–250 mm; internal diameter, 3.9–4.6 mm; particle diameter, 3–10 µm. With regards to the selection of a suitable RP C18 column, a well end-capped column is preferred, as it has been demonstrated that residual silanol groups impair the separation of flavonoid glycosides. The choice of column also depends on the sample-preparation technique because fairly crude plant extracts could damage the column. The main column is therefore usually protected by adding a small in-line guard column containing the same stationary phase. Gradient elution is generally performed with a binary solvent system comprising an organic modifier and a slightly acidified aqueous phase. Instead of linear elution gradients, complicated gradient profiles, comprising several steps and applying various slopes, are often used. In the HPLC analysis of flavonoids, separations are obtained by acidifying the mobile phase. A weakly acidic mobile phase suppresses ionization, thereby increasing retention and decreasing the peak broadening that is caused by formation of the deprotonated form, especially of phenolic acids. Most of them have pKa values of ~4, while the phenolic groups of flavonoids have pKa values >9. The recommended pH range for the HPLC assay of phenolic compounds is therefore 2–4 [25]. LC-MS represents a powerful tool for the analysis of natural products, since MS ensures high sensitivity. Furthermore, it provides information on the molecular weight and structural features of the sample analytes. With regard to structural characterization of flavonoids, information can be obtained on: o the aglycone moiety; o the types of carbohydrates or other types of substituent present; o the stereochemical assignment of terminal monosaccharide units; o the sequence of the glycan part; o the interglycosidic linkages; and, o the linkage point of the substituent to the aglycone. Chen et al. [51] developed a method for simultaneous separation and identification of flavonoids from lotus leaves by HPLC-MS. Optimization of flavonoid extraction from biological matrix was studied via an orthogonal experimental design by considering extraction conditions, including solvent, solvent-to-sample ratio, extraction time and extraction temperature. The proposed method has potential to aid the quality control of this important medicinal plant, and could also be useful in the development of high-flavonoid content leaves for use as a foodstuff or medicine. The recoveries obtained in this study were in the range 85.12–97.63%. Mikulic-Petkovsek et al. [52] described HPLC-MSn identification and quantification of flavonol glycosides in 28 different berry species with quercetin, myricetin, kaempferol, isorhamnetin, syringetin and laricitrin aglycones. Knowledge of specific flavonols in berry species can provide accurate quantification of the dietary intake of phenolic compounds in various clinical studies and, with that, better estimation of the potential health benefits of berries. Stanoeva et al. [53] studied the analytical capabilities of an ion trap as a mass analyzer and aimed to estimate its potential and limits for quantitative analysis. For that purpose, 9

HPLC-ESI/MSn using three different MS acquisition methods was performed for quantification of the flavonoids hypolaetin, 4’-O-methylhypolaetin, isoscutellarein and 4’-Omethylisoscutellarein, apigenin and luteolin in the forms of various glycosides present in the extracts of mountain tea. The results of this thorough study provided evaluation of the capability of the ion trap to detect, to identify and to quantify various flavonoid glycosides without preliminary information concerning their nature and presence in the sample. A comparison of various methods of flavonoid determination in physiological samples, including sample preparation and conditions of chromatographic analyses is presented in Table 2.

5.

Conclusions

Flavonoids attract the attention of physicians and nutritionists and aware consumers due to their beneficial influence on human health. Functional food containing flavonoids is regarded as an important factor that helps prevent or cure cardiovascular diseases, diabetes and cancer, as well as relieve post-menopausal syndrome. From a medical point of view, precise analysis of flavonoids in food and in body fluids, and metabolites of these compounds is very important for understanding their activity and safety in use. All the methods proposed for the determination of selected flavonoids and their metabolites appear to be the latest trends in bioanalysis, aiming at selecting a method capable of extracting and analyzing the highest possible number of samples and different flavonoids at one time, with additional purpose of shortening sample preparation and analysis time. The application of micro-scale and nanoscale extraction and separation techniques is the most likely future development, resulting in quick, sensitive analytical methods for sample preparation and analysis of flavonoids and their metabolites. Miniaturization, high-throughput systems utilizing new sorbents and automation of chromatographic system are of great interest in clinical, pharmaceutical, environmental and food fields. Our increasing analytical capabilities due to implementation of appropriate analytical techniques (e.g., HPLC in combination with UV and MS) enable us to determine the metabolic pathways of these compounds in human organism at the cellular level. Acknowledgements This work was supported by the European Union Structural Funds in Poland (UDAPOKL.04.01.01-00-114/09-01), National Science Center (Cracow, Poland) (No. 2011/01/N/ST4/03178) and Kujawsko-Pomorskie Voivodship Budget Krok w przyszlosc 2011/2012. References [1] D. Treutter, Environ. Chem. Lett. 4 (2006) 147. [2] K.E. Heim, A.R. Tagliaferro, D.J. Bobilya, J. Nutr. Biochem. 13 (2002) 572. [3] L.H. Yao, Y.M. Jiang, J. Shi, T. Tomás-Barberán, N. Datta, R. Singanusong, S. Chen, Plant Foods Hum. Nutr. 59 (2004) 113. [4] Q. Wu, M. Wang, J.E. Simon, J. Chromatogr., B 812 (2004) 325. [5] I. Erlund, Nutr. Res. 24 (2004) 851. [6] K. Polkowski, A.P. Mazurek, Acta Pol. Pharm. 57 (2000) 135. [7] L.H. Yao, Y.M. Jiang, J. Shi, R.A. Tomás-Barberán, N. Datta, R. Singanusong, S. Chen, Plant Food Hum. Nutr. 59 (2004) 113. [8] A. Saleem, H. Kivelä, K. Pihlaja, Z. Naturforsch. 58c (2003) 351. 10

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12

Figure 1 FLAVONOIDS

SMALL INTESTINE

COLON

LIVER

FECES

OTHER TISSUES

KIDNEY

URINE

13

Figure 2

Sample

Determination of aglycones

Determination of aglycones and aglycones conjugated with sulphuric acid

Extraction

HPLC analysis

Determination of aglycones and aglycones conjugated with glucuronic acid

Determination of aglycones and aglycones conjugated with sulphuric acid and aglycones connected glucuronic acid

Incubation with sulfatase in buffer (37°C, 2–18 h)

Incubation with β-glucuronidase in buffer (37°C, 2–18 h)

Incubation with sulfatase/ β-glucuronidase in buffer (37°C, 2–18 h)

Extraction

Extraction

Extraction

HPLC analysis

HPLC analysis

HPLC analysis

14

Table 1. Main classes of flavonoids and their dietary sources

Main classes of flavonoids FLAVONE

Dietary occurrence

Ref.

Celery

[3]

Red pepper

[5]

Parsley

[3]

Lemon

[3]

Soya bean

[6]

O

O

ISOFLAVONE O

Legumes

[7]

Kudzu root

[4]

Red clover

[4,6]

Onion

[3]

Apple

[3]

Tea

[5]

O

FLAVONOL

O

Red wine

[2,5]

OH O

FLAVANONOL

O

Pine, larch

[8]

Orange grapefruit,

[3,7]

Apple

[3,5]

Pear

[5]

Tea

[2]

Red wine

[5]

Cherry

[3,7]

Strawberry

[3,5]

OH O

FLAVANONE

O

O FLAVANOL

O

OH

ANTHOCYANIDIN O+

15 OH

Blueberry

16

[5,7]

Table 2

Table 2. Analytical methods for the determination of selected flavonoids Analytes

Methods of sample preparation

Chromatographic analysis Rat urine  Column chromatography  Mobile phase

 daidzein

Flow rate Injection volume

 dihydrodaidzein  genistein



  

 dihydrogenistein  glycitein

SPE: Supelco LC-18 cartridge (2g):



conditioning: MeOH:H2O (1:1,v/v), × 2 application of sample: 5 ml of urine elution: 20 ml of MeOH

Gradient elution

evaporate, dissolve in 250 µl of 75% MeOH (50µl/mL urine)

column: Eclipse XDB-C8 dimensions of column: 150×4.6 mm A: 0.1% HCOOH in H2O B: 25% ACN in MeOH 1.0 ml/min 5µl time [min] A% 0 92 40 75 70 45 75 0 80 92 UV-DAD:

LOQ or LOD

B% 8 25 55 100 8

Ref.

No data

[54]

No data

[55]

No data

[56]

λ=265nm, λ=315nm, MSn:

 ODMA  biochanin A

      

Detection

and their metabolites

capillary voltage: 3200V nebulizing pressure: 33.4 psi dry gas flow: 10 ml/min dry gas temperature: 340°C [M+H]+, [M-H]ionization: electrospray (ESI) full scan

Rat plasma

 apigenin

  

0.5ml of plasma + 0.8 ml of MeOH vortex (60s), centrifuge (2200 × g, 4°C, 15min) evaporate under a stream of nitro gen (N2) dissolve in 250µl MeOH Determination of aglycones:

 quercetin



 isorhamnetin and their metabolites



50µl of plasma + 50µl of 0.2 M CH3COONa buffer (pH 5.0) + 900µl mixture of MeOH:CH3COOH (100:5,v/v), vortex (30s), sonicate (30s), vortex (30s), centrifuge (5000×g, 4°C, 10 min) supernatant + H2O (1:1,v/v) Determination of free aglycones and aglycones connected with Glu and Su acid



50µl of plasma + 50µl of sulfatase H-5 in 0.2 M CH3COOH

Column chromatography

  

Mobile phase (v/v/v) Flow rate Detection Column chromatography Mobile phase (v/v/v) Flow rate Injection volume Detection

column: Separon SGX C18 dimensions of column: 150×3mm size of particle: 7µm 2% HCOOH:ACN:MeOH (40:35:25)

  

1.0 ml/min UV-Vis: λ=349nm column: TSKgel ODS-80TS dimensions of column: 150×4.6 mm size of particle: 5µm H2O:MeOH:CH3COOH (53:45:2,v/v/v)+50 mM CH3COOLi 0.9 ml/min 20µl ED: amperometry, +800mV



buffer (pH 5.0; 500U of β-glucuronidase + 25U of sulfatase), incubate (37°C, 50 min) add 900µl of mixture MeOH:CH3COOH (100:5,v/v), another operation as describe above Determination of free aglycones and aglycones connected with Glu acid

 

50µl of plasma + 50µl of β-glucuronidase type VII-A in 0.2 M CH3COONa buffer (pH 5.0; 25U of β-glucuronidase), incubate (37°C, 2h) add 900µl of mixture MeOH:CH3COOH (100:5,v/v), another operation as describe above Rat blood Column chromatography Mobile phase

   daidzein  genistein



and their metabolites   

blood (4°C), centrifuge (3000×g, 25 min) 75µl of serum + ACN (1:1,v/v),vortex, sonicate (10min.), centrifuge (15000rpm, 5 min) 100µl of supernatant + 1.0ml of sodium citrate buffer (25mM, pH 5.0) + sulfatase/ glucuronidase from Helix pomatia (460 U of glucuronidase, 23U of sulfatase) + 0.84U of sulfatase from Aerobacter aerogenes + 3.24U of recombinant glucuronidase (G2035), incubate (37°C, 30min) add I.S. (5-150 pmol deuterated genistein and deuterated daidzenin), extract with 3×1.0 ml EtOAc, evaporate (N2) dissolve in 25-150 µl MeOH

Isocratic elution

Gradient elution

Flow rate

  

column: Luna C18 dimensions of column: 150×2 mm size of particle: 3µm A: 0.1% HCOOH B: ACN A:B 65%:35%

aglycones

Coniugates with glucuronic acid

time[m in] 0 10 15 20

A%

B%

80 50 50 80

20 50 50 20

No data

[35]

No data

[57]

0.2 ml/min MSn:

 ion source temperature: 150°C  [M+H]+  ionization: ESI  sampling cone-skimmer potential: 30V mode: SIM Human plasma  column: TSK-gel ODS-80TS QA Column  dimensions of column: 150×4.6 mm chromatography  size of particle: 5µm Temperature 40°C analysis H2O : MeOH : CH3COOH (57:41:2) +50mmol Mobile phase CH3COOLi (v:v:v) 0.9 ml/min Flow rate ED: +950mV Detection Detection

 daidzein



 genistein  aglycones and conjugates with glucose



50µl of plasma + 50µl of acetate buffer (0.2 mol/L, pH 5.0) + 500U of β-glucuronidase + 40U of sulfatase, incubate (37°C , 1 h) add 0.3ml MeOH:CH3COOH (100:5,v/v), mix, sonicate, centrifuged (4000×γ, 4°C, 5 min) supernatant +150 mmol of CH3COOLi (1:0.5;v/v)

 genistin  genistein  daidzin  daidzein

 50µl of plasma + 50µL of acetate buffer (0.2mol/L; pH 5.0) + 500U of H-5 sulfatase, incubate (37°C, 1 h)  extract with 0.9ml of mixture MeOH:CH3COOH (100:5,v/v), sonicate, centrifuged (5000×g, 4°C, 5 min)  supernatant + 2 volume of 100mmol/L CH3COOLi in H2O

Column chromatography

  genistein

 

0.5 ml of plasma + 1000U of β-glucuronidase or 100U of sulfatase or 1000U/100U of β-glucuronidase/ sulfatase, incubate (37°C, 3h) extract with 2×5ml (C2H5)2O, evaporate (N2, 55°C) add 0.5ml of mobile phase A+ I.S. : biochanin A

column: TSK-gel ODS-80TS QA dimensions of column: 150×4.6 mm size of particle: 5µm H2O:MeOH:CH3COOH (58:40:2) + 50 mmol/L CH3COOLi

Mobile phase

No data

[38]

ED: amperometric, +950 mV

Detection

Column chromatography:

 daidzein

  

  

column: Supelco Discovery RPamide-C16 dimensions of column: 250×4.6 mm size of particle: 5µm No data

A: 25% MeOH + 10mM CH3COONH4 + 71nM (C2H5)3N (pH 4.5) B: 95% MeOH + 10mM CH3COONH4 + 71nM (C2H5)3N (pH 4.5)

Mobile phase:

[58] Flow rate: time[min] 0 10 11 12

and their metabolites Gradient elution

Detection

1.0 ml/min A% 35 5 5 35 MSn:

B% 65 95 95 65

 [M-H] Atmospheric pressure-chemical ionization: (APCI) \

 quercetin (Q) 

kaempferol (KA) from Gingko biloba extract tablets

 daidzein (DA)  genistein (GE)

   



4.0ml of urine + 1ml of 25% HCl, incubate (30min, 80°C) extract with 5.0 ml ether (5min), centrifuge (10min) evaporate under nitrogen stream (N2) dissolve in 100µl mobile phase

filtrate the urine (0.45µm) add 0.15M of acetate buffer (1:1,

Human urine  column: Platinum EPS C18 Column  dimensions of column: 250×4.6 mm chromatography:  size of particle: 5µm  pre-column C18 Pre-column  dimensions of column: 10 × 4.6 mm  size of particle: 5µm phosphate buffer (pH 2.0):THF:MeOH:isopropanol Mobile phase: (70:15:10:20) (v/v/v/v) 0.7 ml/min Flow rate: 20 µl Injection volume UV: Detection λ=380nm  column: Atlantis dC18 Column  dimensions of column: 150×2.1 mm chromatography:  size of particle: 3.5µm

LOD

Ng/mL

Q

1,0

KA

1.1

LOQ Q

Ng/mL 1.61

KA

1.85

LOD

pg/mL

[36]

[59]

1:10, 1:100; v/v)

Analysis temperature:

25°C

Mobile phase:

A: ACN B: 0.15 M acetate buffer (pH 5.5) time [min] 0 2 22 27 31 47

Gradient elution



0.5ml of urine + 100 ng I.S.: [ 13C] daidzein, [13C] genistein, 7,4’-dihydroxyflavone + 10 vol. of 0.5mol triethylamine sulfate (pH 5.0); heated 64°C



SPE: C18 cartridge

Flow rate: Injection volume Detection Column chromatography: Mobile phase

 washing: 10ml H2O  elution: 5 ml MeOH 

 daidzein  genistein



evaporate under nitrogen (N2) + 0.5mol acetate buffer (pH 4.5) + 10.000U of β-glucuronidase/ sulfatase from Helix pomatia, incubate (1518h, 37°C) SPE:





SPE: C18 cartridge  washing: 10ml H2O  elution: 5 ml MeOH



 daidzein  genistein

GE

390

Gradient elution

A% 100 100 50 50 100

B% 0 0 50 50 0 No data

[40]

No data

[60]

10µl MSn:

Detection

      

ionization: electrospray (ESI) desolvation temperature 300°C Source temperature 100°C Voltage of sampling cone 50V Voltage of extractor 2V [M+H]+ mode: SIM

evaporate under nitro gen (N2), dissolve in mobile phase urine



Time [min] 0 2 24 29 >29

Injection volume

evaporate under nitrogen (N2), dissolve in 100 µl of mobile phase 0.5ml of urine+β-glucuronidase/ sulfatase from Helix pomatia, incubate (15-18h 37°C) + I.S.: 500ng dihydroflavone, incubate (15-18h, 37°C)

B% 87 87 60 50 45 87

6.4

A: 10mmol CH3COONH4 in H2 O (0.1% CF3COOH) B: ACN

C18 cartridge

 washing: 10ml H2O  elution: 5 ml MeOH 

 

A% 13 13 40 50 55 13 0.4ml/min 5µl ED: coulometric 450mV column: ODS (C18) dimensions of column: 250×4.6 mm

DA

2 ml of urine + 0.5ml of 0.5M buffer CH3COONH(C2H5)3 (pH 7.0) + I.S.: 20µl of flavone (120 ppm in 96% EtOH), mix

Human urine and plasma  column: NovaPak C18 Column  dimensions of column: 150×3.9 mm chromatography:  size of particle: 4µm  pre-column: Adsorbosphere C18 Pre-column:  dimensions of column: 10×4.6 mm

  



 

add 10 µl of β-glucuronidase (200 U/mL) + 10 µl of arylsulfatase (5U/mL), incubate (1h, 37°C) extract with 3×2ml (C2H5)2O, evaporate under nitrogen (N2), dissolve in 140µl of MeOH + 60µl of 0.2M CH3COONa buffer (pH 4.0) Plasma 1ml of plasma + 0.25ml of 0.5 M triethylamine acetate buffer(pH 7.0) + 80µl of β-glucuronidase ( 200 U/mL) + 80 µl of arylsulfatase (5U/mL) + I.S.: 20µl of flavon (120 ppm in 96% EtOH) incubate (17h, 37°C) add 0.25 ml of 10% CCl3COOH in H2O, extract with 3×2ml EtOAc evaporate under a stream of nitrogen (N2), dissolve in 100µl of MeOH + 100µl of 0.2M acetate buffer (pH 4.0), sonicate (30s)

 Mobile phase: (v/v/v) Flow rate: Injection volume

Gradient elution

Detection

Column chromatography: 

1 ml of plasma or 5 ml of urine + β-glucuronidase/ sulfatase + I.S.: 50µl THB incubate (20h, 37°C)



SPE: Extrelut QE cartridge

 daidzein  genistein  glicitein

 

  

Mobile phase: Flow rate:

extraction : EtOAc,

evaporate under a stream of nitrogen, dissolve 2ml 80% MeOH in H2O

Gradient elution Injection volume Detection 

Determination of aglycones: 

 daidzein

  

 genistein  glicytein

200 µl I.S.: 4-HBPH ( 10µg/mL sol. in MTBE) + 1ml of plasma or urine add 6ml of MTBE, mix (30min.), centrifuge (2000×g, 10 min) evaporate under a stream of nitrogen (N2, 45-50°C) dissolve in 250 - 4000µl mixture of MeOH:0.05M HCOONH4 (20:80,v/v, pH 4.0), Determination free aglycones and aglycones conjugate with Glu and Su acids

   

250µl of plasma or urine + 0.5 of β-glucuronidase/sulfatase from Helix pomatia incubate (15-18h, 37°C) + I.S.4-HBPH add 6ml of MTBE, mix (30min.), centrifuge (2000×g, 10 min) evaporate under a stream of nitrogen (N2, 45-50°C) dissolve in 250- 4000µl of mixture MeOH:0.05M HCOONH4 (20:80,v/v, pH 4.0),

Column chromatography:

  

Pre-column: Analysis temperature

Mobile phase:

Flow rate: Injection volume

 

size of particle: 5µm A: CH3COOH:H2O (10:90,v/v) B: MeOH:ACN:CH2Cl2 (10:5:1,v/v/v) 0.8 ml/min 20µl of urine 100 µl of plasma time [min] A% B% 0 95 5 5 95 5 25 55 45 31 30 70 34 95 5 UV-DAD: λ=260nm, λ=280nm λ=190-400 nm column: YMC-Pack ODS-AM C18 dimensions of column: 250×4.6 mm size of particle: 5µm A: 0.1g/L of CH3COOH (glacial) in H2O B: MeOH 1.0 ml/min time [min] A% B% 0 70 30 45 50 50 50 50 50 20µl UV-DAD: λ=200-350 nm column: Luna Phenyl-Hexyl/Zorbac Eclipse XDB-phenyl (plasma/urine) dimensions of column: 150×4.6 mm/ 75×4.6 mm (plasma/urine) size of particle: 5µm/3.5µm (plasma/ urine) pre-column:: Zorbax Eclipse XDB-phenyl guard column dimensions of column: 12.5×4.6 mm size of particle: 5µm 40°C plasma: MeOH : 0.05M HCOONH4 in H2O (pH 4.0) urine: MeOH/ACN (1:1 v/v): 0.05M HCOONH4 in H2O (pH 4.0) 2.0 ml/min 100µl

No data

[61]

No data

[61]

Detection Determination of aglycones: 

3ml of urine or plasma +1.5ml of 1.5 mol CH3COONa buffer (pH 5.5) + I.S.: 50µl THB



SPE: Sep-Pak C18 cartridge

  

Mobile phase:

 washing: 2ml of 0.15mol/L CH3COONa buffer (pH 3.0)  elution: 4 ml of MeOH (plasma)or 2ml mixture of MeOH in H2O (urine) 

Column chromatography:

evaporate under a stream of nitrogen (N2), dissolve in 0.25 ml mixture of 80% MeOH in H2O

Flow rate: Gradient elution Injection volume

UV: λ=260nm column: YMC-Pack ODS-AM C18 dimensions of column: 250×4.6 mm size of particle: 5µm A: 0.1g/L of CH3COOH (glacial) in H2O B: MeOH 1.0 ml/min time [min] A% B% 0 70 30 45 50 50 50 50 50 20µl

Determination of free aglycones and aglycones conjugated with Glu and Su acid  daidzein



1 ml of plasma or 5 ml of urine + β-glucuronidase/sulfatase H-2 from Helix pomatia (5000U/2.5ml urine 2000U/1.0ml plasma) + I.S.: 50µl of THB, incubate (20h, 37°C)



SPE: Extrelut QE cartridge

 genistein and their metabolites

 



extraction : EtOAc,

SPE: Extrelut QE cartridge extraction : EtOAc,

evaporate, dissolve, in 2ml of 80% mixture MeOH in H2O

Determination of aglycones conjugated with  daidzein



 genistein and their metabolites

 

UV-DAD: λ=200-350 nm

Detection

1 ml of plasma or 5 ml of urine + β-glucuronidase B-3 (5000U/ 2.5ml urine, 2000U/ 1.0ml of plasma) + I.S.: 50µl of THB, incubate (20h, 37°C) 



[37]

Evaporate, dissolve in 2ml of mixture 80% MeOH in H2O Determination of free aglycones and aglycones conjugates with Glu acid



No data

Glu acid

0.5 ml of plasma or 1 ml of urine + 1000 U of βglucuronidase, incubate (37°C, 3h) with or without 100mmol/L D-saccharic,1,4-lactone ( inhibitor of enzyme ), spit into 2 equal portions: I portion: extract with 2×5ml (C2H5)2O, evaporate under a stream of nitro gen (N2, 55°C) dissolve in 0.5-1ml of mobile phase A, +I.S.: biochanin A, analyze

Column chromatography:

Mobile phase: (v/v)

Flow rate: Gradient elution

  

column: Supelco Discovery RPamide-C16 dimensions of column: 250×4.6 mm size of particle: 5µm A: 25% MeOH + 10mM CH3COONH4 + 71mM (C2H5)3N (pH 4.5) B: 95% MeOH + 10mM CH3COONH4 + 71mM (C2H5)3N (pH 4.5)

time [min]

1.0 ml/min A%

B%

No data

[39]

0 10 11 12

Determination of aglycones conjugates with Su acid 

II portion: + 100 U sulfatase, incubate (37°C, 3h) with or without 100mmol/L D-saccharic,1,4-lactone ( inhibitor of enzyme ) extract as describe above

Determination of free aglycones and aglycones conjugated with Glu and Su acid 

 daidzein  genistein

50 µl of plasma + H2O (1:2 v/v) or 100µl urine + βglucuronidase/ sulfatase from Helix pomatia (15h, 37°C) + 1ml of H2O



SPE: Baker cartridge

 glicytein and their metabolites and glycosides

   

Column chromatography: Column temperature Mobile phase: (v/v) Flow rate:

Gradient elution:

conditioning: MeOH:H2O elution: MeOH:H2O (8:2,v/v/)

evaporate under stream of nitrogen (N2, 60°C) dissolve in 200µl of H2O

65 95 95 65

MSn: Detection

Plasma or urine + β-glucuronidase/sulfatase from Helix pomatia (100/1000U), incubate (37°C, 3h), extract as describe above



35 5 5 35

Injection volume Detection

 [M-H]  Atmospheric pressure-chemical ionization: (APCI) -

 

column Nucleosil: C18 dimensions of column: 120 × 3 mm 30°C A: 999.5g H2O+ 2g H3PO4 B: ACN 0.8 ml/min time [min] A% B% 0 90 10 20 90 10 25 72 28 35 50 50 45 90 40 100µl UV: λ= 257 daidzein, glycitein, genistein λ= 290 dihydrogenistein λ= 276nm dihydrodaidzein Fluorimetric detector:

No data

[63]

- wavelength of light excitation : 280nm - wavelength of light emission: 310nm

 daidzein  genistein

Sample preparation: 

 ODMA  equol (EQ)  another



2

1 ml of serum or 2 ml of urine+I.S.: ([ H6]-enterolactone, [2H6]-enterodiol, [2H6]-matairesinol, [2H4]-equol, [2H5]-ODMA, [2H4]-genistein, [2H4]-daidzein dissolve in DMSO and 96% EtOH, 4-methylumbelliferone glucuronide and 4-methylumbelliferone sulfate and 4-MU glucoronide in 96% EtOH) serum+1ml of CH3COONH4 (250mM, pH 5,0)

Urine and serum  column: Prism RP Column  dimensions of column: 50 × 3.0 mm chromatography:  size of particle: 5µm CH3COONH4:ACN:MeOH Mobile phase Time [min] CH3COONH4 ACN 0 6.5 mM 18% Gradient elution 7 0.5mM 47.5% 9.5 6.5 mM 18% 0.8 ml/min Flow rate:

serum

LOD MeOH 18% 47.5% 18%

ng/mL 0.5 0.6 0.3 2.4

DA GE ODMA EQ urine

[64]

phytoestrogens



to serum and urine add β-glucuronidase/ sulfatase from Helix pomatia (1mg/mL in 500µl 1M CH3COONH4¸ pH 5,0), incubate (15-18h, 37°C)

25µl

Injection volume

extraction of phytoestrogens from urine 

add 300 µl of MeOH to preparation samples



SPE: C18 cartridge  washing: 3ml of 0,1% CH3COOH in H2O:MeOH (9:1, v/v)  elution: 2ml MeOH  dry (50°C), dissolve in 100µl mixture (70% 10mM CH3COONH4, 15% ACN, 15% MeOH) + 200ng 4nitrophenol

MSn:  Detection

extraction of phytoestrogens from serum 

add 300 µl of MeOH to preparation samples



SPE: Oasis HLB (60mg) cartridge

   

ng/mL

DA

9.3

GE

4.2

ODMA

0.3

EQ

1.1

atmospheric-pressure chemical ionization: (APCI) collision gas (Ar) 375 molecules/cm2 temperature of the nebulizer probe: 500°C [M-H]mode: MRM

 washing: 0,1% NaOH in 10% MeOH elution: 2ml MeOH  elution: 2ml MeOH 

LOD

dry (55°C), dissolve in 100µl mixture (70% 10mM CH3COONH4, 15% ACN, 15% MeOH) + 200ng 4nitrphenol

 After culture medium (Caco-2 cell line)

 daidzein

Column chromatography:

 genistein

Mobile phase: (v/v)

 biochanin A  prunetin

 

growth medium (HBSS buffer + isoflavones ) + I.S.: 100µl 100µM testosterone in mixture MeOH:ACN centrifuge (16000×g, 8 min)

 glycitein  formononetin and their metabolites

Gradient elution

 column: Aqua  dimensions of column: 150×4.5 mm  size of particle: 5µm A: 0.04% (v/v) H3PO4+ 0.06 % N(CH2CH3)3 (pH 2.8) B: ACN genistein, biochanin A, prunetin, formononetin Time [min] 0 80 20 3 80 20 22 51 49 24 51 49 daidzein, glycitein Time[min] 0 90 10 3 90 10 19 66 34 28 48 52

No data

[65]

Injection volume Detection

200µl UV-DAD: λ=254nm

List of abbreviations: Aglycone: flavonoid not connected with sugar or acid moiety; DMSO: Dimethyl sulfoxide; MRM: Multiple reaction monitoring; MTBE: Methyl tert-butyl-ether; ODMA: Odesmethylangolensin; SIM:. Selected ion monitoring; THB: 2,4,4'-trihydroxydeoxybenzoin; THF: Tetrahydrofuran; 4-HBPH: 4-hydroxybenzophenone; MSn: Tandem mass spectrometry; 4-MU: 4methylumbelliferone; UV-Vis: Ultraviolet–visible detector; CH3OH: Methanol (MeOH); C2H5OH: Ethanol (EtOH); CH3COOH: Acetic acid; CH3COOCH2CH3: Ethyl acetate (EtOAc); (C2H5)2O: Diethyl ether; CH3COONa: Sodium acetate; CH3COONH(C2H5)3: Triethylamine acetate buffer; CH3CN: Acetonitrile (ACN); CH3COOLi: Lithium acetate; CCl3COOH: Trichloroacetic acid; CF3COOH: Trifluoroacetic acid; CH2Cl2: Dichloromethane; HCOOH: Formic acid; HCOONH4: Ammonium formate; CH3COONH4: Ammonium acetate; H3PO4:Pphosphoric acid

Biological properties and biotransformation of flavonoids and their metabolites Different methods for sample preparation of flavonoids and their metabolites Analytical methods for identification of flavonoids and their metabolites

17