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www.rsc.org/methods Page 1 of 20 Analytical Methods

1 2 3 4 5 6 7 DETERMINATION OF ELEVEN IN CHAMOMILE AND LINDEN 8 9 EXTRACTS BY CAPILLARY ELECTROPHORESIS 10 11 12 13 1 2* 14 Senem ŞANLI and Craig LUNTE 15 16 17 1Department of Chemistry, Faculty of Science and Arts, Usak University, Usak, 64100, 18 19 TURKEY 20 2 21 Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Manuscript 22 23 Chemistry, University of Kansas, Lawrence, KS, 66045, USA 24 25 26 27 Abstract 28 29 A sensitive, dependable, simple and rapid method based on capillary electrophoresis with diode 30 array detection (CE–DAD) was developed for identification and determination of eleven

31 Accepted 32 flavonoids (apigetrin, naringin, , catechin, , , , , 33 34 , , and ) in Chamomile and Linden flower extracts. Several 35 36 parameters which influence the separation were investigated to determine the optimum 37 conditions. At room temperature, the eleven flavonoids could be well separated within 11 min. in 38 39 a 55cm length capillary at a separation voltage of 24 kV with 40 mM borate buffer (pH 8.9).

40 Methods 41 Under optimum conditions, linearity was achieved within the concentration ranges of 2.5-100.0 42 43 M for all analytes except myricetin and luteolin (5.0-100.0 M) with correlation coefficients ≥ 44 45 0.999. The method was successfully applied to the determination of flavonoids in plant samples 46 such as Chamomile and Linden flowers with satisfactory recoveries. 47 48 49 50 Keywords: Flavonoids, Chamomile, Linden Flower, Capillary Electrophoresis 51 Analytical 52 53 54 55 56 57 58 59 60 Analytical Methods Page 2 of 20

1 2 3 4 1 Introduction 5 6 7 Flavonoids are benzo-γ-pyrone derivatives consisting of phenolic and pyrane rings and are 8 9 classified according to substitutions. Flavonoids are further divided into sub-classes: , 10 1 11 flavanones, , isoflavanones, anthocyanidins and flavan-3-ol derivatives: catechins. 12 13 Flavonoids have been widely used as therapeutics due to their spasmolytic, antiphlogistic, 14 antiallergic, and diuretic properties.2 It has been proposed that these activities are a result of the 15 16 flavonoids oxygen scavenging capacity.3 Flavonols and flavones are of particular interest 17 18 as they provide both and free radical scavenging activity to foods.4 In addition, 19 20 epidemiological studies indicate that their consumption result in a reduced risk of cancer and 21 5-7 Manuscript 22 cardiovascular diseases. 23 The structures of the eleven flavonoids used in this study and frequently found in plants, are 24 25 listed in Table 1. Most of the compounds possess similar structure and chemical characteristics 26 27 in addition with naringin and apigetrin which are flavanone and flavone glycosides, respectively. 28 29 Extracts from linden flowers (Tilia cordata), have been used in folk medicine to induce 30 sweating with colds and influenza, as well as to treat nervous tension, anxiety, insomnia, high

31 Accepted 32 blood pressure, arteriosclerosis, migraines and digestive upsets.8 Chamomile extracts (Matricaria 33 34 chamomilla) are used as anti-inflammatory, anti-emetic, bactericide, fungicide and spasmolytic.9- 35 10 36 In this work, we have successively developed a sensitive, dependable, simple and rapid 37 38 method for the determination of eleven flavonoids (apigetrin, naringin, naringenin, catechin, 39 galangin, apigenin, luteolin, quercetin, myricetin, kaempferol, and kaempferide, Table 1) in plant

40 Methods 41 extracts. 42 43 So far, analysis of flavonoids has been accomplished predominately by high performance 44 11-14 15,16 45 liquid chromatography (HPLC) and gas chromatography (GC) . There have been capillary 46 electrophoretic and micellar electrokinetic chromatography methods for the separation of 47 48 selected flavonoids reported17-30. Dadakova et al.18 studied quercetin in plant samples by micellar 49 19 50 electrokinetic capillary chromatograph. Fonseca et al. determined ten flavonoids in Analytical 51 20 52 Chamomilla recutita using capillary electrochromatography. Zhang et al. developed a micellar 53 54 electrokinetic capillary method for determination of several flavonoids in traditional Tibetan 55 herbal tea. Tonin et al.27 determined flavonoids in neem sample by solvent-modified micellar 56 57 electrokinetic chromatography. Kocevar et al.28 developed both a CE and an HPLC method for 58 59 60 Page 3 of 20 Analytical Methods

1 2 3 determination of five flavonoids in medicinal plants. The main advantages of CE over HPLC are 4 5 that run times are relatively short and only minimal reagents, mostly of a non-toxic nature, are 6 7 consumed during analysis, resulting in a more cost effective and environment-friendly technique 8 9 that both rivals and complements HPLC. 10 11 In this paper, we describe a simple capillary electrophoresis method for the simultaneous 12 determination of several flanonoids. The main advantages of this method are a shorter analysis 13 14 time (11 min) and lower limits of detection relative to previously reported methods.. Analytical 15 16 conditions established in this study were applied to determine the content of flavonoids in 17 18 chamomile and linden samples by CE coupled with diode array detection (DAD). 19 20 21 Manuscript 22 2. Experimental 23 24 2.1 Chemicals 25 26 27 Catechin, apigenin, luteolin, quercetin, kaempferol, naringenin, and galangin were obtained 28 from Sigma (St. Louis MO, USA). Kaempferide, myricetin, naringin, and apigetrin (apigenin-7- 29 30 glucoside) were purchased from Fluka (Switzerland). All flavonoids were used as received

31 Accepted 32 without further purification. Methanol (MeOH, HPLC grade) was purchased from Fisher 33 34 Scientific (Pittsburgh, PA, USA). Sodium tetraborate (Na2B4O7), boric acid (H3BO3) sodium 35 36 hydroxide (NaOH) and butylated hydroxytoluen (BHT) were obtained from Sigma (St. Louis 37 MO, USA). Ultrapure water was obtained with a Milli-Q system (Millipore, Bedford, MA, 38 39 USA).

40 Methods 41 42 Stock solutions of flavonoids were prepared by dissolving enough of the studied compound 43 in MeOH (50 mL) to give a 1.00 mM solution. Borate buffer (40 mM) was prepared by mixing 44 45 Na2B4O7 solution with H3BO3 (500 mM) in ultrapure water. All reagents were protected from 46 47 light. Prepared solutions were stored at 4o C for several days and at -20o C for long-term storage. 48 49 50 2.2 Apparatus 51 Analytical 52 All separations were performed on a Beckman Coulter P/ACE MDQ Capillary 53 54 Electrophoresis System with a diode array detector (Palo Alto, CA). A 55 cm × 50 µm i.d. fused 55 56 silica capillary (Polymicro Technologies, Tucson, AZ) with an effective length of 45 cm was 57 58 used for the separation. 59 60 Analytical Methods Page 4 of 20

1 2 3 A Fisher Scientific AB 15 pH/ion analyser with Fisher Scientific Acument combination pH 4 5 electrode was used. The pH measurements were performed in triplicate to ensure stability and 6 7 reproducibility of the potentiometric system. 8 9 10 2.3 Procedures 11 12 The separation capillary was activated in the first use by flushing sequentially with MeOH 13 (5 min), 1.0 M HCl (2 min), water (2 min), 1.0 M NaOH (20 min), water (2 min), and then with 14 15 buffer (20 min). The capillary was conditioned daily by washing with 1.0 M NaOH (20 min), 16 17 water (2 min) and finally with the running buffer (20 min). Between each analysis, the capillary 18 19 was flushed with 1.0 M sodium hydroxide (3 min), water (2 min) and then the running buffer (3 20 min). Prior to use, buffer solutions were filtered through 0.25 μm membrane filters (Millipore, 21 Manuscript 22 Bedford, MA) and degassed by ultrasonication for approximately 10 min. Buffer, containing 40 23 24 mM Na2B4O7, was adjusted to pH 8.9 with 0.5 M H3BO3. The Applied voltage was 24 kV, 25 26 pressure injection was at 10 psi for 5 s, and the detection was performed at 210 nm. 27 28 2.4 Sample preparation 29 30

31 Chamomile and Linden samples were collected from the Aegean part of Turkey. They were Accepted 32 33 dried in the air. Approximately 0.5 g of each sample was ground into powder in a mortar and 34 31 35 accurately weighed. The extraction and hydrolysis procedure of Proestos et al. was used with 36 some modifications. To each weighed sample were added 25 mL methanol, 15 mL water and 37 38 0.04 g BHT. For hydrolysis, 10 mL 6 M HCl was added. The extraction mixture was refluxed in 39 0

40 a water bath at 80 C for 1.5 hours in the dark. After cooling, 5 mL of this solution was put in a Methods 41 42 tube and vacuumed until dryness. The residue was dissolved with buffer. Before the injection, 43 samples were filtered through 0.25 µm membrane filter. All sample solutions were stored in the 44 45 dark. 46 47 48 3 Result and Discussion 49 50 51 3.1 Effects of pH value and the Buffer Concentration Analytical 52 53 The mechanism of separation in CE is based on differences between the charge-to-size ratios 54 55 of the analytes. The molecules of the flavonoids investigated have at least one ionizing phenolic 56 57 hydroxyl group; ionization of this group is one of the main factors determining the 58 59 60 Page 5 of 20 Analytical Methods

1 2 3 electrophoretic mobility of these flavonoids.32 pK values reported in the literature for water 4 a 5 solutions are shown in Table 1. 33-39 6 7 8 Borate buffer was employed as the buffer in this work because borate can chelate with the 9 40 10 analytes to form more soluble complex anions. The pH dependence of the migration time was 11 investigated over the pH range of 8.7–9.1. The migration times of all analytes increase with 12 13 increasing pH except mycretin. At pH 8.7, the myricetin peak was split into two smaller peaks, 14 15 while at pH 9.1, myricetin and apigenin were not separated. The Rs values for myricetin and 16 17 apigenin are given in Table 2. The optimum pH was determined to be pH 8.9 based on the good 18 resolution, selectivity, and peak shapes of the studied compounds within a relatively short 19 20 analysis time (11 min). Flavonoids are also more susceptible to oxidative degradation at higher 21 Manuscript 22 pH and longer separation times. 23 24 25 In addition to pH, the concentration of borate buffer is also an important parameter for the 26 separation. The effect of the borate buffer concentration on migration time was also studied. 27 28 When the borate buffer concentration increased, migration times increased. Good resolution was 29 30 achieved with the shortest separation time using 40 mM borate.

31 Accepted 32 33 3.2 Effects of Separation Voltage 34 35 The influence of the separation voltage on the migration time of the analyte was also 36 37 studied. The results indicated that increasing the separation voltage gave shorter migration time 38 39 for all studied compounds, but also increased the baseline noise and decreased the resolution of

40 Methods 41 the analytes. However, too low of a separation voltage increased the analysis time considerably 42 and caused peak broadening. The optimal voltage was determined by performing runs at 43 44 increasing voltages (20, 24 and 26 kV) until deterioration in resolution was noted. Separation of 45 46 galangin and kaempferol cannot be achieved when the separation voltage was 26 kV or greater. 47 48 The Rs values for galangin and kaempferol are given in Table 2. The optimum separation voltage 49 was found to be 24 kV, at which good separation can be obtained for all of the analytes. 50 51 Analytical 52 From the above experiments the optimum conditions for separation of all eleven flavonoids 53 54 was determined to be; 40 mM borate, pH 8.9 with an applied voltage of 24 kV. A typical 55 56 electropherogram for a standard solution of the eleven analytes is shown in Fig. 1; it is apparent 57 that satisfactory separation can be achieved within 11 min. 58 59 60 Analytical Methods Page 6 of 20

1 2 3 As shown in Fig. 1, the type and position of substituents can strongly effect migration times 4 5 of the flavonoids. Naringin has a disaccharide substituent, apigetrin has a monosaccharide 6 7 substituent, while the rest of the flavonoids studied are aglycones. Under the separation 8 9 conditions utilized, the flavonoids are negatively charged and their electrophoretic mobility is 10 11 against the electroosmotic flow and away from the detector end of the capillary. Therefore 12 naringin elutes first because of a slower electrophorectic mobility due to the large size of the 13 14 disaccharide group. Likewise, apigetrin elutes next because of the size of its monosaccharide 15 16 substituent. The aglycones are similar in size but vary in pKa values. Catechin is only 17 18 partially charged at the separation pH and therefore has the slowest electrophoretic mobility of 19 the flavonoid aglycones and is thus the first of this group to elute. Luteolin and quercetin are 20 21 dianions at the separation pH and therefore elute last as they have the fastest electrophoretic Manuscript 22 23 mobility. The rest of the flavonoid aglycones studied elute between catechin and luteolin based 24 25 on a combination of their pKa values and sizes. 26 27 3.3 Linearity, Repeatability and Detection Limits 28 29 30 A series of the standard mixture solutions of the eleven analytes were tested to determine

31 41 Accepted 32 the validation parameters. The linearity was calculated by plotting the peak area versus 33 34 concentration of the compounds. The calibration curves were obtained by linear least squares 35 regression. The validation data are reported in Table 3. The method exhibited good linearity 36 37 based on a correlation coefficient > 0.999 for all compounds studied The LOD and LOQ were 38 39 calculated as LOD = 3.3s/m and LOQ = 10s/m, where s is the standard deviation of response and

40 42,43 Methods 41 m is the slope of the corresponding calibration curve. 42 43 In the literature, there is no method for the simultaneous determination of these eleven 44 45 compounds by capillary electrophoresis for plant samples. The MEKC method of Dadakova et 46 18 -1 19 47 al. provided an LOC of 0.5 µg mL with an analysis time of 20 minutes. Fonseca et al. 48 -1 28 49 reported an LOD of 35 µg mL . The CE method of Kocevar et al. required an analysis time of 50 18 minutes. The method described above provides a combination of faster analysis time and Analytical 51 52 improved limits of detection. 53 54 55 3.4 Sample Analysis and Recovery 56 57 58 59 60 Page 7 of 20 Analytical Methods

1 2 3 The validated methodology was applied to quantification of the flavonoids in Chamomile 4 5 and Linden samples. Typical electropherogram of Chamomile and Linden extracts are shown in 6 7 Fig 2 and Fig 3. Peaks were identified by standard addition and comparison of absorbance 8 9 spectra. As an example the absorbance spectra of a quercetin standard and from the peak 10 11 identified as quercetin in a chamomile extract are shown in Fig 3. All peak identities were 12 determined by co-elution with standards and comparision of absorbance spectra with standard 13 14 spectra. The contents of the two plant extracts were quite different. In Chamomile extracts, three 15 16 of the studied compounds were detected (catechin kaempferol and quercetin) under the optimum 17 18 conditions. The contents of quercetin were highest in Chamomile extracts. In Linden extracts, 19 catechin, kaempferol, apigenin and myricetin were detected with catechin occurring in the 20 21 highest concentration. The concentration of the analytes in the samples were calculated using Manuscript 22 23 their peak areas from the calibration curve obtained under the same conditions. 24 25 26 Accurate amounts of analytes were added to sample and recovery values were obtained 27 using their peak area from the calibration curve. Each sample was analyzed in triplicate. Content 28 29 and recovery analysis were given in Table 4 for chamomile and linden extracts, respectively. The 30

31 recoveries for these compounds were between 91.0 % and 100.2 %. The recovery results showed Accepted 32 33 that this method is suitable for real sample analysis. 34 35 36 37 38 4 Conclusions 39

40 Methods 41 Although several methods have been reported for determination of the different types of 42 43 flavonoids, to the best of our knowledge, there are no methods yet for simultaneous 44 determination of the eleven flavonoids in this study from the extracts of the herbal plants. In this 45 46 work, simple, fast and effective CE-DAD method has been applied to simultaneously determine 47 48 flavonoids in Chamomile and Linden extracts without using organic modifier. Also, this method 49 50 has been found to be reproducible and highly sensitive. The proposed method promises to be 51 Analytical 52 applicable to the identification and determination of the eleven flavonoids in other plant extracts 53 as well. 54 55 56 5 Acknowledgments 57 58 59 60 Analytical Methods Page 8 of 20

1 2 3 This work was supported by the Higher Education Council of Turkey (YOK). 4 5 6 7 8 9 10 References 11 12 1 C. A. Rice-Evans, N. Miller, Structure-antioxidant activity relationships of flavonoids and 13 14 isoflavonoids. In C. A. Rice-Evans, L. Packer, Flavonoids in health and disease, New York: 15 16 Marcel Dekker Inc. 1997, p. 199 – 219. 17 18 2 G. Cao, E. Sofic, R. L. Prior, Free Radic. Biol. Med., 1997, 5, 749-760. 19 3 H. M. Merken, G. R. Beecher, J. Chromatogr. A., 2000, 897, 177-184. 20 21 4 F. Shahidi, P. K. Wanasundara, Crit. Rev. Food Sci. Nutr., 1992, 32, 67-103. Manuscript 22 23 5 L. Wattenburg, Cancer Res., 1985, 45, 1-8. 24 25 6 L. Wattenburg, Proc. Nutr. Soc., 1990, 49, 173-180. 26 7 H. Wei, L. E. Tye, D. F. Bresnick, Birt Cancer Res., 1990, 50, p. 499-502. 27 28 8 M. Coleta, M. G. Campos, M. D. Cotrim, et al. Pharmacopsychiatry 34 (suppl 1): S20–1, 29 30 2001.

31 Accepted 32 9 N. G. Bisset, Herbal Drugs and Phytopharmaceuticals: A Handbook for Practice on a 33 34 Scientific Basis, CRC Press, Boca Raton, FL, 1994, p. 332. 35 10 World Health Organization. Monographs on Selected Medicinal Plants, vol. 1: Flos 36 37 Chamomillae. WHO, Geneva, 1999, p. 86. 38 39 11 A. Obmann, S. Purevsuren, M. Zehl, C. Kletter, G. Reznicek, S. Narantuya, S. Glasl,

40 Methods 41 Phytochem. Anal., 2012, 23, 254–259. 42 12 T. Z. Niu, Y. W. Zhang, Y. L. Bao, Y. Wu, C. L. Yu , L. G. Sun, J. W. Yi, Y. X. Huang, Y. 43 44 X. Li, J. Pharm. Biomed Anal., 2013, 76, 44–48. 45 46 13 M. Francisco, D. A. Moreno, M. E. Cartea, F. Ferreres, C. G. Viguera, P. Velasco. J. 47 48 Chromatogr. A., 2009, 1216, 6611–6619. 49 50 14 G. H. Shui, L. P. Leong, J. Chromatogr. A 2002, 977, 89–96. Analytical 51 15 C. S. Creaser, M. R. Koupai-byzani, G. R. Stephenson, J. Chromatogr., 1989, 478, 415-420. 52 53 16 R. Christov, V. Bankova, J. Chromatogr., 1992, 623, 182-185. 54 55 17 Z. K. Shihabi, T. Kute, H. Y. Garcia, M. Hinsdale, J. Chromatogr. A., 1994, 680, 181-185. 56 57 18 E. Dadakova, E. Prochazkova, M. Krizek, Electrophoresis, 2001, 22, 1573-1578. 58 59 60 Page 9 of 20 Analytical Methods

1 2 3 19 F. N. Fonseca, M. F. M. Tavares, C. Horváth, J. Chromatogr. A., 2007, 1154, 390-399. 4 5 20 Y. Zhang, J. Chen, X. M. Ma, Y. P. Shi, J. Pharm. Biomed. Anal. 2007, 45, 742-746. 6 7 21 X. J Chen, H. Ji, Y. T. Wang, S. P. Li, J. Sep. Sci. 2008, 31, 881-887. 8 9 22 M. N. Luo, H. W. Lu, H. Q. Ma, J. Pharm. Biomed. Anal. 2007, 44, 881-886. 10 11 23 Y. Lua, C. Jiaa, Q Yao, H. Zhong, M. C. Breadmore, J. Chromatogr. A., 2013, 1319, 160– 12 165. 13 14 24 M. E. Yue, T. F. Jiang, Y. P. Shi, Talanta 2004, 62, 695–699. 15 16 25 S. Zhang, S. Dong, L. Chi, P. He, Q. Wang, Y. Fang, Talanta 2008, 76, 780-784. 17 18 26 S. P. Wang, K. J. Huang, J. Chromatogr. A., 2004, 1032, 273– 279. 19 27 F. G. Tonin, A. V. Jager, G. A. Micke, J. P. S. Farah, M. F. M. Tavares, Electrophoresis 20 21 2005, 26, 3387–3396. Manuscript 22 23 28 N. Kocevar, I. Glavac, R. Injac, S. Kreft, J. Pharm. Biomed. Anal. 2008, 46, 609–614. 24 25 29 T. F. Jiang, X. Du, Y. P. Shi, Chromatographia, 2004, 59, 255-258. 26 30 G. Chen, H. Zhang, J. Ye, Anal Chim Acta, 2004, 20, 69-76. 27 28 31 C. Proestos, N. Chorianopoulos, G. J. E. Nychas, M. Komaitis, J. Agric. Food Chem., 2005, 29 30 53, 1190-1195.

31 Accepted 32 32 P. H. , J.C. Archambault, P. Andre, M. Dreux, E. Gaydou, J Chromatogr A., 1997 33 34 791, 289–297. 35 33 H. Serra, T. Mendes, M. R. Bronzea, A. L. Simplicio, Bioorg. Med. Chem., 2008, 16, 4009– 36 37 4018. 38 39 34 E. A. González, M. A. Nazareno, C. D. Borsarelli, J Chem Soc Perkin Trans., 2002, 2, 2052-

40 Methods 41 2056. 42 35 I. Kron, Z. Pudychová-Chovanová, B. Veliká, J. Guzy, P. Perjési, Monatsh. Chem., 2012, 43 44 143, 13-17. 45 46 36 M. Tungjai, W. Poompimon, C. Loetchutinat, S. Kothan, N. Dechsupa, S. Mankhetkorn, The 47 48 Open Drug Delivery Journal, 2008, 2, 10-19. 49 50 37 S. Ramešová, R. Sokolová, I. Degano, J. Bulíčková, J. Zabka, M. Gál, Anal Bioanal Chem., Analytical 51 2012, 402, 975-82. 52 53 38 M. E. Arrington, Inhibitor Screening and Characterization Using 19F 54 55 NMR. Thesis, Department of Chemistry and Physics, Western Carolina University, 2010. 56 57 http://libres.uncg.edu/ir/wcu/f/Arrington2010.pdf. 58 59 60 Analytical Methods Page 10 of 20

1 2 3 39 http://www.hmdb.ca/metabolites/HMDB37441 4 5 40 S. Hoffstetter-Kuhn, A. Paulus, E. Gassmann, H. Michael, Anal Chem., 1991, 63, 1541– 6 7 1546. 8 9 41 CH, Topic Q2A validation of analytical procedures methodology PMP/ICH/281/95. 10 11 42 C. M. Riley, T. W. Rosanske, Development and Validation of Analytical Methods, Elsevier, 12 New York, 1996. 13 14 43 M. E. Swartz, I. S. Krull, Analytical Method Development and Validation, Marcel Dekker 15 16 Inc., New York, 1997. 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 Page 11 of 20 Analytical Methods

1 2 3 Figure Captions 4 5 6 Fig 1. Electropherogram of eleven flavonoids under optimum conditions. Running buffer 7 40 mM sodium tetraborate solution (pH 8.9); uncoated fused silica capillary 55 cm (45 8 cm to detector) x 50 µm i.d.; applied voltage 24 kV; detection UV absorbance at 210 nm; 9 pressure injection, 10 psi for 5 s; ambient temperature. Peaks represent 1: Naringin, 2: 10 11 Apigetrin, 3: Catechin, 4: Naringenin, 5: Kaempferide, 6: Galangin, 7: Kaempferol, 8: 12 Apigenin, 9: Mycretin, 10: Luteolin, 11: Quercetin (Each compounds are 0.1 mM) 13 14 Fig 2. Typical electropherogram of a chamomile extract. 1: Catechin; 2: Kaempferol; 3: 15 Quercetin 16 17 18 Fig 3. Typical electropherogram of a linden extract. 1: Catechin; 2: Kaempferol; 3: 19 Apigenin; 4: Myricetin 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 Analytical Methods Page 12 of 20

1 2 3 Fig 1. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 Page 13 of 20 Analytical Methods

1 2 3 Fig 2. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37

38 39

40 Methods 41 42

43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 Analytical Methods Page 14 of 20

1 2 3 Fig 3. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 Page 15 of 20 Analytical Methods

1 2 3 Table 1. 4 5 6 Compounds Chemical Structure 7 8 9 Apigetrin 10 11 (Apigenin-7-glucoside) 12 13 CAS No : 578-74-5 14 15 -1 16 C21H20O10 MW: 432.38 g mol 17 18 19 Naringin 20 21 (4',5,7-Trihydroxyflavanone-7- Manuscript 22 rutinoside) 23 24 25 pKa1 = 10.2 ± 0.1 [33]; 9.04 [34] 26 27 pKa2 > 11.50 [33] −1 28 C27H32O14 MW: 580.53 g mol 29 CAS No : 10236-47-2 30

31 Accepted 32 Naringenin 33 34 35 pKa1 = 6.8 ± 0.1 [33]; 7.08 ± 0.12 36 [35] 37 pKa2 = 10.4 ± 0.2 [33] 38 pKa2 > 11.50 [33] 39

40 Methods 41 CAS No : 480-41-1 C H O MW: 272.26 g mol−1 42 15 12 5 43 44 Catechin 45 46 pKa = 8.6 [36] 47 48 CAS No : 154-23-4 49 50 Analytical 51 52 −1 53 C15H14O6 MW: 290.27 g mol 54 55 56 57 58 59 60

Analytical Methods Page 16 of 20

1 2 3 Galangin 4 5 6 pKa1 = 7.19 7 pKa2 = 9.44 [36] 8 9 10 11 CAS No : 548-83-4 −1 12 C15H10O5 MW: 270.24 g mol 13 14 15 Apigenin 16 17 pKa = 7.86 [36] 18 19 CAS No : 520-36-5 20 21 Manuscript 22 −1 23 C15H10O5 MW: 270.24 g mol 24 25 Luteolin 26 27 pK = 5.99 ± 0.32 28 a1 29 pKa2 = 8.40 ± 0.42 [37] 30

31 Accepted 32 CAS No : 491-70-3 33 34 −1 C15H10O6 MW: 286.24 g mol 35 36 37 Kaempferol 38 39 pKa = 7.89 [36]

40 Methods 41 42 43 CAS No : 520-18-3 44 −1 45 C15H10O6 MW: 286.24 g mol 46 47 Ouercetin 48 49 50 pKa1 = 5.87 ± 0.14 Analytical 51 pKa2 = 8.48 ± 0.09 [37]; 7.76 [36] 52 53 54 CAS No : 117-39-5 55 56 −1 57 C15H10O7 MW: 302.24 g mol 58 59 60

Page 17 of 20 Analytical Methods

1 2 3 Myricetin 4 5 6 pKa = 6.5 [38] 7 8 CAS No : 529-44-2 9 10 11 −1 12 C15H10O8 MW: 318.24 g mol 13 14 Kaempferide 15 16 pKa = 6.44 [39] 17 18 CAS No : 491-54-3 19 20

21 Manuscript 22 −1 23 C16H12O6 MW: 300.26 g mol 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60

Analytical Methods Page 18 of 20

1 2 3 Table 2. R values for kaempferol –galangin and apigenin-myricetin 4 s 5 6 7 8 Voltage pH 9 10 Compounds 20 24 26 8.7 8.9 9.1 11 12 13 14 15 16 Kaempferol-galangin 2.844 3.195 2.835 1.821 3.981 5.862 17 18 19 Apigenin-Myricetin 3.520 3.523 3.832 4.950 3.596 1.917 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60

Page 19 of 20 Analytical Methods

1 2 3 Table 3. Statistical evaluation of the calibration data of studied flavonoids by CE–DAD. 4 5 6 Naringin Apigetrin Catechin Naringenin Kaemp- Galangin Kaemp- Apigenin Myricetin Luteolin Quercetin 7 Compounds 8 feride ferol 9 10 Linear Range (µM) 2.5-100 2.5-100 5-100 2.5-100 2.5-100 2.5-100 2.5-100 2.5-100 5-100 5-100 2.5-100 11 12 Correlation Coefficient 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 13 14 LOD (µM) 0.143 0.298 0.181 0.098 0.298 0.346 0.5640 0.611 2.019 0.966 1.434 15 16 LOQ (µM) 0.434 0.904 0.550 0.297 0.904 1.049 1.712 1.851 6.731 2.929 4.346 Manuscript 17 18 6.578 7.109 7.785 8.360 8.819 9.351 9.806 10.097 19 Migration Time 4.770 5.226 6.226 20 0.072 1.031 0.287 0.414 0.707 0.315 0.467 0.307 21 %RSD of Migration 0.334 1.011 0.557 22

23 Accepted 24 25 26 27 28 29

30 Methods 31 32 33 34 35 36 37 38 Analytical 39 40 41 42 43 44 45

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Methods Page 20 of 20

1 2 3 4 5 a b 6 Table 4. Determination results of recovery in this method with dried Chamomile and Linden 7 8 extracts (n = 3) 9 10 11 12 13 Compounds Original RSD Added Found Recovery RSD 14 content of (%) amount (µg/g) (%) (%) 15 sample (µg/g) (µg/g) 16 17 18 a Catechin 43.36 0.6 34.80 77.19 97.2 2.0 19

20 b 21 44.69 1.2 58.00 101.69 98.3 0.7 Manuscript 22 23 a 24 Kaempferol 28.44 0.5 22.90 51.39 100.2 1.8 25 26 26.75b 0.3 74.42 100.80 99.5 1.2 27 28 29 Quercetin 25.05a 1.2 12.09 36.68 96.2 1.8 30

31 Accepted 32 Apigenin 36.90b 0.2 87.07 116.16 91.0 0.4 33 34 35 b 36 Mycretin 35.68 0.6 50.92 83.62 94.2 1.6 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60