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Carvalho, D.; Pinho, C.; Oliveira, R.; Moreira, F.; Oliveira, A.I. Chromatographic Methods for Quercetin Quantification from Natural Sources. Encyclopedia. Available online: https://encyclopedia.pub/entry/52328 (accessed on 19 July 2025).
Carvalho D, Pinho C, Oliveira R, Moreira F, Oliveira AI. Chromatographic Methods for Quercetin Quantification from Natural Sources. Encyclopedia. Available at: https://encyclopedia.pub/entry/52328. Accessed July 19, 2025.
Carvalho, Daniel, Cláudia Pinho, Rita Oliveira, Fernando Moreira, Ana Isabel Oliveira. "Chromatographic Methods for Quercetin Quantification from Natural Sources" Encyclopedia, https://encyclopedia.pub/entry/52328 (accessed July 19, 2025).
Carvalho, D., Pinho, C., Oliveira, R., Moreira, F., & Oliveira, A.I. (2023, December 04). Chromatographic Methods for Quercetin Quantification from Natural Sources. In Encyclopedia. https://encyclopedia.pub/entry/52328
Carvalho, Daniel, et al. "Chromatographic Methods for Quercetin Quantification from Natural Sources." Encyclopedia. Web. 04 December, 2023.
Chromatographic Methods for Quercetin Quantification from Natural Sources
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This entry is adapted from the peer-reviewed paper 10.3390/molecules28237714

Quercetin (QUE) is the most widely used flavonoid for therapeutic purposes. To improve the available knowledge about the properties of some natural products, determining the amount of QUE is crucial. Accordingly, the development, optimization and validation of analytical methods capable of featuring the amount of QUE in natural products is not only usefull, but necessary.

analytical methods chromatography flavonoids quercetin

1. Introduction

Flavonoids are antioxidant compounds commonly found in vegetal origin products that show multiple potentialities in human health, substantiated by their antiallergic, antiviral, anticancer, anti-inflammatory, and cardiovascular documented activities [1][2]. This class of secondary metabolites belongs to a group named phenolic compounds, where lignans, tannins, phenolic acids, and stilbenes are also included but where flavonoids remain the major compounds, being widely spread throughout the plant kingdom and found in several fruits and vegetables [3][4][5][6][7][8]. Flavonoids are structurally composed of two benzene (A and B) rings linked to a heterocyclic (C) ring (two aromatic and one oxygenated ring) with a 15-carbon structure (C6-C3-C6). Flavonoids are often found in nature as aglycones but also conjugated with organic acids or sugars [3][5][6][7][8][9][10].
According to their chemical structure, more specifically with the degree of hydroxylation of the central ring, flavonoids can be divided into different subclasses (flavonols, isoflavones, flavones, anthocyanins, flavanones, and flavan-3-ols) [3][5][7][8][9][11].
QUE is a flavonoid with multiple potentialities in human health. It is the most widely used flavonoid in the treatment of various diseases, which may be related to its properties, such as antioxidant, anti-inflammatory, and prevention of cardiovascular and neurodegenerative diseases. This flavonol is present in multiple vegetal sources, namely plants, fruits, and vegetables (e.g., onions, apples, tea, brassicas, grapes, nuts, Hypericum perforatum, Ginkgo biloba, Sambucus canadensis, and Aesculus hippocastanum) [1][2][12][13][14].
Chromatography is an analytical technique that has been one of the most widely used techniques for analyzing compounds or mixtures of compounds, such as products of plant origin, to identify their chemical composition and determine the number of compounds present in each sample [15][16][17][18].
Determining the amount of QUE present in natural sources is an important step towards improving knowledge about the properties related to plant sources, as well as, in the case of therapy, being able to determine the amount of compound to be administered and quantify the amount of QUE present in new formulations.

2. Chromatographic Methods Developed for the Quantification of Quercetin Extracted from Natural Sources

2.1. Quercetin Sources

Table 1 summarizes the publications regarding the analyte, plant source and sample, sample preparation and extraction procedure, and the amount of QUE in real samples.
Table 1. Summary of studies published between 2018 and 2022 describing chromatographic methods for the quantification of quercetin in plant sources regarding sample information, sample preparation, and amount of quercetin in real samples.
Reference Analyte Sample Source Sample Preparation and
Extraction Procedures		 Amount of Quercetin in Real Samples (µg/g)
Du et al. [19] Chlorogenic acid; Cryptochlorogenic acid;
Neochlorogenic acid; Isochlorogenic acid A; Isochlorogenic acid B; Isochlorogenic acid C; Caffeic acid; Hyperin; Isoquercitrin; Quercetin; Campherol; p-coumaric acid; Isorhamnetin;
Rutin; Astragalin; Apigenin;		 Cuscuta chinensis Lam. Undisclosed Pulverization;
Ultra-sonication assisted extraction;
Filtration (0.22 µm);		 0.0735 ± 0.0788
Rajauria [20] Phloroglucinol; Gallic acid;
Cyanidin 3-glucoside; Chlorogenic acid; Rutin; Quercetin;		 Himanthaliaelongata Seaweed Grinding;
Percolation;
Solid-phase extraction;
Filtration (0.22 µm);		 4.2 ± 0.15
Yang et al. [21] Alpinetin; Apigenin-7-O-β-D-glucopyranoside; Quercetin-3-O-β-D-glucopyranoside;
Scutellarein; Apigenin; Wogonoside; Quercetin; Amentoflavone; Wogonin; Chrysin; Luteolin; Rutin; Naringenin; Baicalein; Baicalin;		 Scutellaria barbata
D. Don
and
Hedyotis
diffusa (Willd.) Roxb.		 Dry Grass (Plants) Reflux extraction (twice);
Lyophilization;
Solvent resuspension;
Liquid–liquid extraction;
Filtration (0.22 µm);		 0.02199 ± 0.000618
Zhou et al. [22] Myricetin-3-O-β-D-galactoside;
Myricetin-3-O-glucoside;
Quercetin3-O-β-D-galactoside;
Quercetin-3-O-β-D-glucoside;
Quercetin-3-O-(2″-O-galloyl-β-d-galactoside); Quercetin-3-O(2″-O-galloyl-β-d-glucoside); Kaempferol-3-O-β-D-galactoside;
Kaempferol-3-O-β-D-glucoside;
Kaempferol-3-O(2″-O-galloyl-β-D-galactoside); Kaempferol-3-O-(2″-O-galloyl-β-D-glucoside); Quercetin; Kaempferol;		 Diospyros khaki Leaves (Plant) Grinding;
Reflux extraction (twice);
Defat procedure (twice);
Liquid–liquid extraction (twice);
Gel Column
Chromatography;		 12,700 ± 8000
Srivastava et al. [23] Acteoside; Isoacteoside; Durantoside-I;
Quercetin;
Methylapigenin-7-O-D-glucopyranuronate;		 Duranta erecta L. Undisclosed Pulverization;
Ultra-sonication assisted extraction;
Filtration (0.22 µm);		 2010
Pu et al. [24] Hydroxysafflor yellow A; Safflomin C;
Anhydrosafflor yellow B; Kaempferol; Kaempferol-3-O-glucoside;
Kaempferol-3-O-rutinoside;
Kaempferol-3-O-β-sophoroside;
6-hydroxykaempferol;
6-hydroxykaempferol-3-O-β-D-glucoside;
6-hydroxykaempferol-3,6-di-O-β-D-glucoside; 6-hydroxykaempferol-3,6,7-tri-O-β-D-glucoside; Quercetin; Rutin; Luteoloside; Apigenin;
Quercetin-3-O-β-D-glucoside;		 Carthamus
tinctorius L.		 Undisclosed Pulverization;
Ultra-sonication assisted extraction;
Filtration (0.22 µm);		 65 ± 75
Huang et al. [25] Chlorogenic acid; Rutin; Isoquercetrin;
Nictoflorin; Astragalin; Quercetin;		 Sambucus
formosana		 Stems, leaves, and roots (Plant) Pulverization;
Percolation;
Liquid–liquid extraction (twice);		 3500 ± 70
Chen et al. [26] Gallic acid; Chlorogenic acid; Caffeic acid;
Syringic acid; p-coumaric acid; Ferulic acid; Benzoic acid; Salicylic acid; Catechin;
Epicatechin; Rutin; Naringin; Hesperidin;
Quercetin; Resveratrol; Nobiletin; Tangeritin;		 Chinese
citrus and grape		 Fruit (Plant) Percolation;
Liquid–liquid extraction (twice);
Filtration (0.45 µm);		 394,800 ± 527,900 (citrus)
129,700 ± 146,600 (grape)
Khan et al. [27] 6‴-feruloylspinosin; Apigenin;
Apigenin-7-O-glucoside; Catechin;
Jujuboside A; Jujuboside B; Luteolin; Quercetin;		 Ziziphus
jujuba
and
Ziziphus
nummularia		 Fruits (Plants) Grinding;
Ultra-sonication assisted extraction;
Filtration 0.22 µm;		 15.5 ± 12.0
Jia et al. [28] Phloretin; Gallic acid; Protocatechuat E;
Catechin; 2,4-dihydroxybenzoic acid;
Chlorogenic acid; Proanthocyanidins-B2;
Vanillic acid; O-hydroxybenzene acetic acid; Coffeic acid; Syringate; p-coumaric acid;
Proanthocyanidins-A2; Veratronic acid; Ferulic acid; Benzoic acid; Salicylic acid; Naringin;
Hesperidin; Rutin; Ellagic acid; Myricetin; Naringenin; Quercetin; Kaempferol;		 Berries Fruit (Plant) Grinding;
Ultra-sonication assisted extraction;
Filtration;
Lyophilization;
Solvent resuspension;
Filtration (0.22 µm);		 11.5 ± 15.5
Sharma et al. [29] Rutin; Quercetin; Kaempherol; 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)chroman-4-one; 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)8-methylchroman-4-one; 5,7-dihydroxy-3-(4-methoxybenzyl)8-methylchroman-4-one; Polygonatum
verticillatum		 Rhizomes (Plant) Pulverization;
Percolation (fivefold);
Liquid–liquid extraction;
Filtration (0.25 µm);		 0.0243 ± 0.0044
Sharma et al. [30] Quercetin; Ferulic acid; Chlorogenic acid; Myristic
fragrans,
Hemidesmus
indicus,
and
Inula
racemosa		 Undisclosed Maceration;
Filtration (11 µm);
Lyophilization;
Solvent resuspension;
Filtration (undisclosed
diameter);		 0.0062
Ramaswamy et al. [31] Curcumin; Piperine; Quercetin; Rutin; Camellia sinensis L. (1);
Glycyrrhiza glabra L. (2);
Thymus
vulgaris L. (3);
Citrus
aurantium L. (4);		 Leaves (1, 3), rhizomes (2), tuberous roots (2), and rind (4) (Plants) Ultra-sonication assisted extraction;
Filtration 0.22 µm;		 C. s: 0.0036
C. a: 0.0011
G. g: 0.00095
T. v: 0.00087
Ali et al. [32] Rutin; Taxifolin; Quercetin; Apigenin; Kaempferol; Betulinic acid; Oleanolic acid;
Betulin; Lupeol; Stigmasterol; β-sitosterol;
Ursolic acid;		 Caesalpinia
pulcherrima (1);
Citrus lemon (2);
Opuntia
dellenii (3);
Bauhinia
variegata (4);
Polyalthia longifolia var. pendula (5);
Bombax ceiba (6);
Phlox drummondii (7);
Olea europea (8);
Tagetes
patula (9);
Melia
azedarach (10);		 Flower (1, 9, 10), fresh pods (1), seeds (2), cladodes (3), pod (4), root bark (5), wood (6), aerial part (7), leaves (8), and stem bark (6) (Plants) Ultra-sonication assisted extraction;
Filtration 0.22 µm;		 C. p (flowers): 234.56 µg/mL
C. p (fresh pods): 315.07 µg/mL
C. l: < LOQ
O. d: < LOQ
B. v: < LOQ
P. l: 579.51 µg/mL
B. c: < LOQ
P. d: < LOQ
O. e: 94.50 µg/mL
T. p: < LOQ
Macêdo et al. [33] Quercetin Triplaris
gardneriana Wedd		 Leaves (Plant) Pulverization;
Percolation (threefold);
Vacuum Liquid
Chromatography;		 9967 ± 1010
Urbstaite et al. [34] Chlorogenic acid; Myricetin-3-galactoside; Quercetin-3-galactoside; Quercetin-3-glucoside; Quercetin-3-α-Larabinopyranoside;
Quercetin-3-α-L-arabinofuranoside;
Quercetin-3-rhamnoside; Myricetin; Quercetin;		 Vaccinium
macrocarpon Aiton		 Fruit (Plant) Pulverization;
Ultra-sonication assisted extraction;
Filtration (0.22 µm);		 89.76 ± 1.58
Jan et al. [35] Rutin and Quercetin Buckwheat
(Fagopyrum spp.)		 Seeds and Leaves (Plant) Pulverization;
Percolation;
Filtration (0.22 µm);		 0.00011 ± 0.00014
B. c: Bombax ceiba; B. v: Bauhinia variegata; C. a: Citrus aurantium L.; C. l: Citrus lemon; C. s: Camellia sinensis L.; C. p: Caesalpinia pulcherrima; G. g: Glycyrrhiza glabra L.; O. d: Opuntia dellenii; O. e: Olea europea; P. d: Phlox drummondii; P. l: Polyalthia longifolia; T. p: Tagetes patula; T. v: Thymus vulgaris L.

2.2. Sample Treatment Prior Chromatographic Analysis

Most of the included studies promoted the fragmentation of samples by pulverizing [19][23][24][25][29][33][34][35] or grinding [20][27][28]. This procedure increases the effectiveness of the extractive methods subsequently applied, namely by increasing the area of contact with extractive solvents. Different extractive methods were adopted to ensure the recovery and concentration of QUE, generally combining multiple methodologies amongst the most conventional (such as maceration [30], percolation [20][25][26][29][33][35], reflux extraction [21][22], and liquid–liquid extraction [21][22][25][26][29]) and/or the most recent (namely, ultrasound-assisted extraction [19][23][24][27][28][31][32][35], solid-phase extraction [20], and chromatography techniques for sample preparation [22][33]). The preference for three of these techniques was quite evident: percolation, liquid–liquid extraction, and ultrasound-assisted extraction. Briefly, during percolation, the plant materials are soaked with a selected solvent and left to stand in a well-closed container, after which the whole materials are covered with enough amount of the selected solvent for extraction [36]. This exhaustive process in which soluble constituents are removed by extracting the crude drug with fresh solvent was preferred over its main competitor, maceration, for QUE extraction. Although simpler and less expensive, maceration is also less effective [37]. As to other similar alternatives, reflux extraction was only used in two studies [21][22]. Reflux extraction is more efficient than percolation, requiring less extraction time and solvent, but cannot be used to extract thermolabile compounds, which might be relevant in multiple-analyte studies [38]. Still, in most studies using percolation, subsequent combination with other extractive procedure(s) was performed. As for liquid–liquid extraction, this technique is based on the partitioning of organic compounds between an immiscible organic solvent and the aqueous sample [39]. Finally, in ultrasound-assisted extraction, the ultrasounds passing through the samples create compression and expansion, ultimately forming cavitation and accelerating the dissolution of the solute and heat transfer, further improving extraction efficiency [36][38]. Since ultrasound-assisted extraction is best suited for solid plant samples, it is not surprising that all the methods described it for the extraction of QUE in solid samples, usually after pulverization or grinding [36]. Being an effective technique with low solvent and energy consumption that is applicable for the extraction of thermolabile and unstable compounds [38], ultrasound-assisted extraction was the most frequently mentioned extraction technique in the included studies.

2.3. Chromatographic Conditions

Liquid chromatography methods were preferred for QUE detection and quantification over gas chromatography (GC), being reported in 100% of the included studies. GC has several advantages (e.g., easy to apply, inexpensive, requires less solvent, allows the analysis of volatile compounds, and there is no interaction of the mobile phase with the analyte), and in the case of QUE, its high operating temperatures are not significantly destructive since QUE is one of the most thermally stable flavonoids [16][40][41]. However, as previously mentioned, all but one of the studies carried out multi-analyte analyses, including compounds that are less thermally stable than QUE and that could be destroyed in the GC analysis. In addition, GC generally involves laborious derivatization procedures that increase the likelihood of making a mistake in sample preparation. Previous studies that determined QUE by GC described derivatization procedures that may have discouraged more recent studies from using this technique [42][43].

Failing to present all the main characteristics of the analytical method deemed as relevant for data synthesis and/or not identifying the specific natural product in which the analytical method was used (mixtures of compounds were excluded), thin layer-chromatography (TLC) methods were not fully depicted. However, there are also recent TLC methods that determined QUE in Itrifal formulations of Unani medicine [44], polyherbal formulations containing Terminalia species [45] and Myristica fragrans, Hemidesmus indicus, and Inula racemosa herbs [30].
The mobile phase can be a single solvent or a mixture [16][40]. All the analyzed studies employed mobile phases composed of a mixture of solvents, and water was present in most of the described methods. In chromatographic methods using reverse-phase HPLC, such as those herein included, it is frequent to use a moderately polar aqueous mobile phase and a nonpolar stationary phase [46]. Since QUE is a polar compound, and in reverse-phase HPLC, there is a stronger attraction of the polar molecules to polar solvents than to the stationary phase, a faster elution is ensured when water-containing mobile phases are used [46][47]. It can also be seen that most of the studies use acidified water, which may be related to the advantages that acidification of the mobile phase brings, such as increased chromatographic resolution, allowing more defined peaks to be obtained, and better separation of the peaks of all the compounds present in complex mixtures, and possibly a reduction in the time needed for the chromatographic run [15][16][17][40][48][49].
Different types of acids can be included in the mobile phase for the chromatographic analysis of samples [50][51][52][53]. Formic acid was the most widely used chemical for acidifying the mobile phase (75%), followed by acetic acid (19%), making them the most widely used in recently developed chromatography methods for QUE quantification. Orthophosphoric acid was employed in 6% of the articles. These results are not surprising since formic acid (first) and acetic acid (second) are described as two of the most used acids in chromatography [54]. The importance of adding acidic solutions to the mobile phases for analyzing QUE is further emphasized by its chemical characteristics. Since QUE is a weak acid, it is degraded by hydrolysis in alkaline solutions and is, therefore, more stable in acidic conditions [55][56].
The organic solvent acetonitrile (ACN) was included in 76% of the described mobile phases, followed by methanol. This is a solvent with a high affinity for a great variety of compounds and which is capable of enhancing chromatographic resolution when used in higher proportions. However, this solvent is more expensive compared with methanol, therefore increasing the costs associated with the method. It is usually recommended to start with ACN when optimizing multi-analyte chromatographic methods, further increasing the probability of ACN being the chosen organic solvent [40][57][58]. ACN is also associated with a decrease in retention time due to its strong elution capacity [40].
Elution can occur in two modes: isocratic (constant proportion over the analysis time) or gradient (different proportions of each solvent over time), as the mobile phase may require adjustment over time depending on the polarity of the analyte, and the number of analytes present in the sample [16][40][53][59][60][61].
Different types of detectors (UV/visible, MS, infrared, fluorescence, or electrochemical) can be used in liquid chromatography [40]. Different authors state that spectrophotometric methods are the most widely used in HPLC analysis, which was in line with the results since spectrophotometry is used in 70% of studies. [15][16][40].

2.4. Validation Parameters

To guarantee the reliability of the results obtained and the reproducibility of the method, all analytical methods must undergo a validation process after the analysis of certain compounds [62][63][64].
Different parameters should be evaluated to ensure methods are properly validated. Among these parameters, the most important to guarantee the reproducibility of the method are sensitivity, precision, and accuracy [62][63][64][65][66].
Regarding sensitivity, this is given by the LOD (lowest concentration of analyte that the method can detect) and the LOQ (lowest concentration of analyte that the method can quantify) [62][63][64][65][66].
The methods reported in the 17 studies analyzed have shown very variable sensitivity values, ranging over different orders of magnitude.
For the agreement of successive measurements of the same method, carried out under the same conditions, expressed by the intra-day precision (also called repeatibility), the obtained values ranged from 92.2% to 99.73%. Regarding the degree of agreement between measurements made after promoting variations of different factors such as different days, different analysts, or different equipment, which is expressed by inter-day precision (also known as intermediate precision), the values reported in the analyzed studies ranged from 92.1% to 99.47%.
The guidelines state that the RSD must be less than 15% for each standard concentration tested, meaning that the precision must be greater than 85%. This aspect is verified in all the studies analyzed, which indicates that the methods reported have good repeatability and intermediate precision, which allows us to conclude that the methods developed make it possible to carry out precise quantification, even if small variations may occur [63][64][65].

2.5. Bias Assessment

After analyzing the studies according to the criteria defined for their evaluation, most of the studies addressed the same bias evaluation parameters defined by the Johnson [67] criteria, and all the included studies mentioned/presented, totally or partially, at least four of the eight parameters.
One of the parameters is the consideration of the difference plot and the statistics of the differences. In other words, a comparison should be made of the results obtained with the calibration line and its equation in samples of known concentration, with a subsequent comparison of the values and statistical presentation of the difference [67]. The difference plots and difference statistics were not addressed in any of the studies. The usually considered guidelines to perform the method’s validation do not mention the need to consider the difference plot and statistics of difference [65][68], but the disagreement in linear analysis is easily seen in difference plots (in which differences between the comparison estimates are plotted against the mean of their values), are hard to detect in x–y plots, that might wrongly suggest agreement [67][69].
Another parameter is the performance and interpretation of the interference test, which involves constructing two straight-line equations: one with just the standard in the injection solvent and another equation with the standard extracted into the matrix and then evaluating the difference [67]. Despite the importance of assessing the potential contribution of any interferent in an analytical method, the interference test was rarely presented [33][35]. Interferents can be endogenous (e.g., coming from matrix components) or exogenous (e.g., resulting from the carryover effect) [70].
Another criterion is the performance and interpretation of the linearity test, in which the different parameters must be evaluated to ensure that the regression equation shows linearity within the defined range [67]. As to the linearity test, most studies interpreted the correlation coefficient obtained in the calibration curve to conclude about the linearity of the developed model and the suitability of the obtained equation [19][21][22][25][26][28][30][31][32][34][35]. Nonetheless, some authors consider that the correlation coefficient is not the most appropriate parameter for linearity determination and recommend that other evaluations should be performed, such as a relative error of the curve of less than 5% and zero contained in the ordinate of the origin [63][64][65][71].
Regarding the last parameter, carrying out and interpreting the recovery test, the samples must be spiked, and the accuracy assessed according to the recovery method [67]. Only two studies did not assess recovery [32][33]. The guidelines generally used to validate analytical methods mention that accuracy can be determined using different methods and do not mention the need to carry out a recovery test [65][68].

2.6. Assessment of the Methods

One of the main advantages of methods dedicated to a single analyte is the possibility of optimizing the method for detection with greater efficiency and lower solvent consumption. In the case of chromatographic analyses, efficiency is mainly reflected in retention time. Only one of the studies was exclusively dedicated to the detection of QUE but had a retention time of 32.9 min, much longer than other studies, ultimately resulting in higher solvent consumption [33]. If a quadrupole time-of-flight (QTOF) coupled to MS equipment is available, the method described by Sharma et al. [29] can be an interesting starting point for method development and/or adaptation, given the fact that it was the most sensible described method (LOD of 0.4 ng/mL) and presented a retention time of around 6 min. Furthermore, none of the MS methods completely satisfied more bias assessment parameters. Admitting that not all the laboratories possess MS equipment, the UHPLC-PDA method described by Srivastava et al. [23] might represent an alternative given the fact that it was the most sensitive spectrophotometric method with a retention time inferior to 10 min (LOD = 0.33 µg/mL; retention time = 6.4 min).

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Daniel Carvalho
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