High-Performance Liquid Chromatography-Mass Spectrometry for the Determination of Flavonoids in G.biloba Leaves

Food Science and Technology Research, 22 (2), 199 _ 204, 2016 Copyright 2016, Japanese Society for Food Science and Technology doi: 10.3136/fstr.22.199 http://www.jsfst.or.jp Technical paper High-Performance Liquid Chromatography-Mass Spectrometry for the Determination of Flavonoids in G.biloba Leaves Yan-Ling Su College of Bioengineering, Jinzhong University, Jinzhong, Shanxi, PR China Received December 13, 2013 ; Accepted November 27, 2015 The aim of the present work was to develop a multimethod for the analysis of five flavonoids in G.biloba leaves. The selected flavonoids were rutin, quercetin 3-D-galactoside, baicalin, isorhamnetin and rhamnetin. Leaf extraction was sonicated by using ethanol. The extract was passed through solid-phase extraction columns. Then, the extract was analyzed by high-performance liquid chromatography coupled in line with mass spectrometry. Calibration was carried out over the concentration range of 0.5 to 50 mg/l. The calibration data presented high correlation coefficients (>0.99). The limits of detection were 4.7, 5.3, 9.7, 6.9 and 12.9 μg/l for rutin, quercetin 3-D-galactoside, baicalin, isorhamnetin and rhamnetin, respectively. Mean recoveries ranged from 66% to 72% for the HPLC MS analysis. Reproducibility was estimated by the relative standard deviation (RSD) which ranged between 2.21 and 3.93%. Five flavonoids were found to be present in G.biloba leaves. The developed method was readily applied to quantify the flavonoids in some commercially available G.biloba leaves. Keywords: G.biloba leaves, flavonoids, relative standard deviation, capillary Introduction A variety of molecules with potent biological activities derived from the Ginkgo leaf extract have been shown to play different physiological roles in the organism. Among these, flavonoids are of particular interest because of their capacity to act mainly as antioxidants/free radical scavengers, enzyme inhibitors, and cation chelators (DeFeudis and Drieu, 2000; DeFeudis et al., 2003). Extracts of G.biloba leaves are being marketed as therapeutic dietary supplements to counteract a variety of disorders including vertigo, depression, short-term memory loss, hearing loss, lack of attention or vigilance. Although the putative therapeutic benefits of G.biloba leaf extract may reside in the synergistic effect of all of its components, isolated constituents have been found to be active in a variety of assays. For example, flavonoids are a group of low molecular weight substances that account for around 24% of the total Ginkgo leaf extract (Smith and Luo, 2003, 2004). In general, the bioavailability of flavonoids is relatively low due to limited absorption and rapid elimination (Goh and Barlow, 2004). Flavonoids in the glycosidic form are poorly absorbed in the intestine; only in the aglycone form can they be absorbed directly (Goh and Barlow, 2004). Unabsorbed flavonoids that reach the colon may be subject to metabolism by bacterial enzymes, and then absorbed (DeFeudis and Drieu, 2000, 2004). Once absorbed, flavonoids reach the liver where they are metabolized to conjugated derivatives (DeFeudis and Drieu, 2000). The flavonoids have been shown to be have antioxidant activity including free radical scavenging activity (Pietri et al., 1997). The flavonoids are known to exert their effects through inhibition of the cyclooxygenase-2 enzyme, which is a part of prostaglandin synthesis,and its inhibition is known to reduce colon carcinogenesis. The flavonoids (quercitin, kaempferol, and isorhamnetin) are also known to improve coronary blood flow by improving contractile functions which are due to increased release of catecholamines from *To whom correspondence should be addressed. E-mail: jzxysyl@163.com

200 endogenous liver tissue reserves (Mahady, 2002). Therefore, isolation and quantification of the flavonoids in G.biloba leaf extract are essential to ensure the activity of the flavonoids. Various analytical techniques have been employed to isolate and quantify the flavonoids in G.biloba leaf extract. Analysis of flavonoids in G.biloba with a binary HPLC gradient system was reviewed by Pietta and co-workers as early as 1991. Li and Fitzloff (2002) used HPLC with photodiode array detector to compare to flavonole aglycone content in pharmaceutical products. Recently, a new method to evaluate Ginkgo leaf extract using HPLCfingerprinting has been suggested as a better method to monitor various flavonoids (Sun and Liu, 2007). The aim of this study is to develop a robust analytical method for rapid determination of the flavonoids, with minimal sample treatment. We evaluated the use of HPLC MS to quantify the flavonoids, and the method was validated by determining its reproducibility, linearity, detection limit and recovery. Finally, this method was applied to G.biloba leaf extract. Materials and Methods Preparation of standard solutions Rutin (95%), quercetin 3-D-galactoside (97%), baicalin (95%), isorhamnetin (95%) and rhamnetin (99%) were purchased from Sigma Chemical, St. Louis, MO. Stock solution of 100 mg/l for five flavonoids were prepared. Y.-L. Su Five-point calibration curves (0.5, 1, 5, 10, and 50 mg/l) of the reference compounds were prepared in Milli-Q deionized water (Millipore, Bedford, MA). Extraction methods Two kinds of G.biloba leaves were bought in different pharmacies. They were grated and were then passed through a 50 mesh sieve. Five grams of each was weighed and extracted with 50 ml of 70% ethanol for 20 min at 60 using a ultrasonic extraction system (Kun Shan Ultrasonic Instruments Co., Ltd, Jiangsu, China). The primary extracts were filtered to remove suspended particulate matter and then were passed through solid-phase extraction columns containing octadecyl-bonded porous silica to extract the flavonoid at a flow rate of 10 ml/min. To prepare the samples for HPLC-MS analysis, the extract was filtered through 0.22 μm filters. HPLC MS Analysis The HPLC-MS analyses were performed on an LCQ Advantage MAX HPLC-MS system (Thermo Electron Corporation Inc, USA). The HPLC system was equipped with a quaternary gradient pumping system, an on line degasser, a photodiode array detector (PDA), and a mass spectrometer (MS). Chromatographic separations were performed with a Thermo ODS- 2 HYPERSIL C 18 column (100 mm 4.6 mm, 3 μm particle size, Thermo Electron Corporation Inc, USA). Solvent A was water and solvent B was acetonitrile. The flow rate was 0.3 ml/min and the solvent gradient was 20% B at 0 min; 30% B at 4 min; 43% B at Table 1. Linear equation, correlation coefficient of calibration curves. constituents Linear equation Regression (R 2 ) Rutin Y = 94.8 X + 6269.7 0.9983 Quercetin 3-D-galactoside Y = 90.9 X + 2393.5 0.9976 Baicalin. Y = 74.6 X 2593.3 0.9979 Isorhamnetin Y = 281.1 X + 263.4 0.9991 Rhamnetin Y = 138.7 X 14088 0.9982 Note: Y: peak area (μv.s) X: concentration (μg/l) Table 2. The RSD and the detection limits of the five flavonoids constituents RSD% limits of detection (μg/l) Rutin 3.43 4.7 Quercetin 3-D-galactoside 2.48 5.3 Baicalin. 2.21 9.7 Isorhamnetin 3.93 6.9 Rhamnetin 3.89 12.9 Table 3. The recoveries of five flavones spiked in G.biloba leaves (mean ±RSD, %, n = 3) constituents Amount added (5 μg/g) Amount added (10 μg/g) Amount added (50 μg/g) Rutin 68.74 ± 3.11 72.45 ± 2.18 71.54 ± 3.63 Quercetin 3-D-galactoside 69.65 ± 3.94 71.75 ± 1.81 70.32 ± 3.42 Baicalin. 65.94 ± 2.01 67.94 ± 2.21 70.53 ± 3.14 Isorhamnetin 69.350 ± 3.23 69.95 ± 2.97 72.11 ± 2.14 Rhamnetin 71.32 ± 3.73 70.89 ± 5.17 71.34 ± 2.55

High-Performance Liquid Chromatography-Mass Spectrometry for the Determination of Flavonoids in G.biloba Leaves 201 Fig. 1. High-performance liquid chromatogram of G.biloba leaf extract. Fig. 2. LC-MS total ion current chromatogram of G.biloba leaf extract. 16 min; 45% B at 25 min; 60% B at 29 min; 70% B at 36 min; 50% B at 41 min; 20% B at 50 min The analytical column was equilibrated for 10 min between samples to obtain a stable baseline for subsequent analysis. The effluents were monitored by measuring the absorbance at 255 nm and directed into the mass spectrometer via the electrospray interface. Nitrogen was used as the nebulizing and drying gas. The detector (MS) operated under the following conditions: electrospray ionization interface operating in negative mode; source: 4.5 kv, sheath gas flow: 60 (arbitrary units), auxiliary gas flow: 5 (arbitrary units). The capillary was held at _ 10 kv and kept at 275. For HPLC MS, spectra were recorded over the mass-tocharge (m/z) range of 100 to 1,300 with a target mass of 609 m/z, 445 m/z, 463 m/z, 315 m/z and an average of about 5 spectra. All the analyses were carried out using an injection volume of 10 µl; triplicate injections were done for each point on the calibration curves and duplicate injections were done for samples. Flavonoids in these samples were quantified by mass spectrometry using calibration curves prepared in the same system with reference standards at least 95% purity. The quantifications for HPLC MS were performed using the Xcalibur software Version 1.4 (Thermo Electron Corp., Waltham, MA). Plots were made of the HPLC MS peak areas for the deprotonated molecular ion at m/z 609, 445, 463 and 315. The determination by HPLC MS was based on the peak areas of the most abundant product ions from fragmentation of the molecular ion with m/z 609, 445, 463 and 315. Statistical methods A minimum of three replicate extractions were performed for plant sample, and extract was analyzed at least three times by HPLC-MS analysis. Statistical analyses were

202 Y.-L. Su Fig. 3. High-performance liquid chromatogram of 10 µl of five Flavonoids standards at 5 mg/l. Fig. 4. LC-MS total ion current chromatogram of 10 µl of five Flavonoids standards at 5 mg/l conducted on treatment means using Student s t-test. Results and Discussion Method Validation Linearity A linear regression analysis of the peak area vs. concentration of each reference standard flavonoid dissolved in water was calculated by using 3 replicates at 5 different concentrations of HPLC-MS analysis. Calibration curves were constructed at the relevant wavelength of maximum absorption of each reference flavonoid. The HPLC peaks were monitored at the UV range from 200 _ 400 nm. Calibration curve was constructed using the peak area of deprotonated molecular ion on MS chromatogram detected at HPLC retention time of each reference flavonoid, which was confirmed by UV peaks of HPLC at 255 nm for rutin, quercetin 3-D-galactoside, isorhamnetin and rhamnetin, and 275 nm for baicalin. Assessment of peak purity showed peak homogeneity excluding the possibility of the presence of interfering components and rendering the method specific. Linear equation and correlation coefficients were depicted in Table 1. The results obtained for all HPLC MS analyses corresponded to linear equations. The values for the coefficient of determination (R 2 in Table 1) were higher than 0.99, which indicated that the fits were acceptable. Detection Limits The calibration curve obtained by HPLC MS was used for quantification. The detection limit was estimated as the flavonoids concentration giving a signal equal to the blank signal plus 3 standard deviations of the blank (Miller and Miller, 2000). The limits of detection were 4.7, 5.3, 9.7, 6.9 and 12.9 μg/l for rutin, quercetin 3-D-galactoside, baicalin, isorhamnetin and rhamnetin, respectively. This detection limit can easily be

High-Performance Liquid Chromatography-Mass Spectrometry for the Determination of Flavonoids in G.biloba Leaves 203 Fig. 5. Mass spectra in negative mode of 10 µl of the five flavonoids. standards at 1 mg/l. A. Rutin; B. Quercetin 3-D-galactoside; C. Baicalin; D. Isorhamnetin; E. Rhamnetin. improved by increasing the flow rate that was passed to the electrospray source (from 20 to 300 L/min), but higher flow rates also resulted in the formation of a deposit on the cone of the mass spectrometer that should be removed periodically to avoid deterioration of the response. Precision and accuracy The reproducibility of the assay was assessed by high, medium, low concentrations of the reference standard. Each concentration was prepared in triplicates to

204 determine precision. Reproducibility was estimated by the relative standard deviation (RSD) of the peak areas for 9 consecutive analyses of the reference standard. The values obtained for this parameter were shown in Table 2 and ranged between 2.21 and 3.93%. Recovery has been estimated as (the amount found in the spiked sample the amount found in the sample) 100/the amount added (Massart et al., 1990). The mean values were calculated from the recovery experiments by using 3 different concentrations of five reference standards (5 μg/g, 10 μg/g, and 50 μg/g) added to the extract of G.biloba leaves, blank extract were prepared to determine the original concentration of the reference standard, and triplicate additions of each point were performed. The recovery values in Table 3 were achieved when using the calibration curves. Recovery data obtained ranged from 66% to 72% for the HPLC MS analysis. Therefore, the linear regressions can be used to calculate the concentration of flavonoids. Analysis of the extract of G.biloba leaves The extract of G. biloba leaves was selected to optimize the analytical conditions for determination of the flavonoids. The HPLC analysis of this extract resulted in a very complex profile (Figure 1).The flavonoids of interest, rutin, quercetin 3-D-galactoside, baicalin, isorhamnetin and rhamnetin, eluted together with other fragments. The total ion chromatogram of HPLC-MS of this zone also revealed a complex mixture of flavonoids (Figure 2). A significantly better specificity was obtained by extracting the molecular ion with m/z 609, m/z 445, m/z 463 and m/z 315, where some high-intensity peaks with different retention times were obtained. The total chromatogram was separated into fractions labeled 1, 2, 3, 4 and 5, which corresponded to rutin, quercetin 3-D-galactoside, baicalin, isorhamnetin and rhamnetin, respectively (Figure 1; Figure 2). Although the selectivity and intensity of the HPLC signal were sufficient to perform a quantitative determination of the flavonoids, the responses of five standard flavonoids obtained by performing HPLC-MS were also evaluated. HPLC chromatogram at λ=255 nm of 10 µl of reference standards at 5 mg/l, with corresponding retention times was shown in Figure 3. LC-MS total ion current chromatogram was shown in Figure 4. As can be observed, good separation was achieved. Analysis by HPLC-MS Analysis by HPLC-MS was used to confirm the HPLC results. First, a standard flavonoids solution was injected under conditions described in Materials and Methods. Figure 5 shows the full scan mass spectra of the 5 selected flavonoids. For all the analyzed flavonoids, the peak area of the deprotonated molecular ion [M H] _, for ruin at m/z 609, for 3-D-galactoside at m/z 463, for baicalin at m/z 445, for isorhamnetin and rhamnetin at m/z 315, was used for quantification. The sample was injected by HPLC-MS and the result was confirmed by comparison of their retention time, UV spectra and mass spectra of standards and sample using the same HPLC-MS conditions. Y.-L. Su Conclusion The method described is a good alternative analytical tool for the routine determination of these flavonoids in G.biloba leaves. The use of MS produced very clean chromatograms for five flavonoids. The method had variability with RSD lower than 4% and good linearity in the range from 0.5 to 50 mg/l required for the direct determination of these flavonoids in G.biloba leaves. The analysis was performed without any sample pretreatment or concentration.analysis by HPLC-electrospray ionization mass spectrometer is an appropriate and useful method for identification of the five flavonoids. Acknowledgements The work was supported by academic innovation team of Jinzhong university. References DeFeudis, F. V. and Drieu, K. (2000). G.bilobaextract (EGb 761) and CNS functions: basic studies and clinical applications. Curr. Drug. Targets, 1, 25-58. DeFeudis, F. V. and Drieu K. (2004). Stress-alleviating and vigilanceenhancing actions of G.biloba extract (EGb 761), Drug. Dev. Res., 62, 1-25. DeFeudis, F. V., Papadopoulos,V., and Drieu, K. (2003). G.biloba extracts and cancer: a research area in its infancy. Fundam. Clin. Pharmacol., 17, 405-17. Goh, L.M.L. and Barlow, P. J. (2004). Flavonoid recovery and stability from G.biloba subjected to a simulated digestion process. Food Chem., 86, 195-202. Li, W. and Fitzloff, J.F. (2002). HPLC determination of flavonoids and terpene lactones in commercial G.biloba products. J. Liq. Chrom. Rel. Technol., 25, 2501-2514. Mahady, G. B. (2001). Ginkgo biloba: a review of quality, safety, and efficacy, Nutr. Clin. Care, 4, 140-7. Miller, J. N. and Miller, J. C. (2000). Statistics and Chemometrics for Analytical Chemistry. Pearson Education. Harlow, UK. Pietri, S., Maurelli, E., Drieu, K., and Culcasi, M. (1997). Cardioprotective and antioxidant effects of the terpenoid constiuents of G.biloba extract (EG761). J Mol. Cell. Cardiol, 29, 733-742. Pietta, P., G, Mauri, P. L., and Gardana, C. (1991). Identification of flavonoids from Ginkgo biloba L, Anthemis nobilis L and Equisetum arvense L, by high-performance liquid. chromatography with diode-array UV detector. J Chromatography, 553, 223-231. Smith, J.V. and Luo, Y. (2003). Elevation of oxidative free radicals in Alzheimer s disease model scan be attenuated by G.biloba extract EGb 761. J Alzheimers Dis., 5, 287-300. Smith, J.V. and Luo, Y. (2004). Studies on molecular mechanisms of G.biloba extract. Appl. Microbiol. Biotechnol., 64, 465-72. Sun,G. and Liu, J. (2007). Qualitative and quantitative assessment of the HPLC fingerprints of G.biloba extract by the involution similarity method. Anal. Sci., 23, 955-962.