Article. Shan Lin 1,JiYe 1, Wei-dong Zhang 1, Bang-jing Cao 2, Xi-ke Xu 1, Lei Shan 1, and Juan Su 1, * Abstract. Introduction

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1 Journal of Chromatographic Science, 2016, Vol. 54, No. 6, doi: /chromsci/bmw045 Advance Access Publication Date: 10 April 2016 Article Article Development and Validation of an Analytical Method for the Determination of Flavonol Glycosides in Ginkgo Leaves and ShuXueNing Injections by a Single Marker Shan Lin 1,JiYe 1, Wei-dong Zhang 1, Bang-jing Cao 2, Xi-ke Xu 1, Lei Shan 1, and Juan Su 1, * 1 School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai , People s Republic of China, and 2 Department of Pharmacy, Jiangxi Traditional Chinese Medicine University, No. 18 Yunwan Road, Nanchang , People s Republic of China *Author to whom correspondence should be addressed. juansu_2008@126.com Received 28 December 2013; Revised 4 January 2016 Abstract The qualitative and quantitative analysis of the major bioactive components in traditional Chinese medicines (TCM) and their preparations is essential to evaluate their quality. However, the scarcity and high cost of chemical reference standards are common obstacles for quantitative analysis, especially for determining multicomponents. In this study, an effective and sensitive qualitative method to identify flavonol glycosides in Ginkgo leaves (Ginkgo Folium), and their preparations have been developed. Meanwhile, a simple, convenient and reproducible method for the quantitative analysis of multicomponents by a single marker (QAMS) has been established to simultaneously determine the major flavonol glycosides in Ginkgo leaves and their preparations. Among the 15 favonol glycosides that were found, 7 major flavonol glycosides with high contents were simultaneously determined by the QAMS and traditional external standard method (TES). Rutin was selected as the single marker, and the quantitative analysis was performed on a TSK gel ODS-100V C18 column using a gradient system of acetonitrile and water, with a variable wavelength detector (265 nm) within 50 min. The method validation was conducted, and the linearity was excellent (r 2 > ) with accuracy and precision within the required limits. The F-test (P > 0.05) indicated that the QAMS and TES method have no statistically significant difference. Introduction Traditional Chinese medicines (TCM) have been widely used to prevent and treat various diseases with complementary therapeutic effects due to active multicomponents (1). Characteristic components, especially active multicomponents, have been the focus of quality control of TCMs (2). Ginkgo biloba is the sole species of Ginkgophyta, which is widespread in the world. Its leaves (Ginkgo Folium) have been used to treat or prevent many diseases for centuries, such as asthma and circulatory diseases (3). Ginkgolides and flavonol glycosides are the major active ingredients of Ginkgo leaves (Ginkgo Folium). In China, ShuXueNing injection (SXNI), which is one of the pharmaceutical preparations of Ginkgo leaves, is applied to treat ischemic cardiovascular and cerebral vascular diseases. Ginkgolides are mainly measured by HPLC-ELSD (4 6) and reference standards including ginkgolide A, B, C, J and bilobalide, which are easily obtained and are chemically stable. However, in the case of flavonol glycosides that have been identified as glycosides derivatives of the aglycones quercetin, kaempferol and isorhamnetin (7), most reference standards are unavailable in commercial supply except rutin. The quantitative methods of flavonol glycosides can be divided into two types: direct assays and assays after acid hydrolysis (8, 9). The direct assays are time consuming, have high cost and are often affected by a The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oup.com 1041

2 1042 Lin et al. shortage of reference standards. Although assays after hydrolysis only measure flavonol aglycones, they cannot preclude the possibility of adulteration with the pure flavonol aglycones of other plant material containing flavonol aglycones. Therefore, quantitative analysis of flavonol glycosides in Ginkgo leaves and their preparations have to overcome more challenges due to the lack of reference standards and the complex separation conditions (10). In recent years, several reports have described the quantitative analysis of multicomponents by the single marker (QAMS) method, which applies a conversion factor (CF) to assay constituents that have similar characteristics, such as polarity, UV spectral and chromatographic behaviors. For example, the United States Pharmacopoeia used CF to analyze red clover (Trifolium pratente L.) (11) and St. John s Wort(Hypericum perforatum L.) (12). At the same time, Chinese groups also adopted CF to determine three lipophilic parts in Salvia Miltiorrhizae (Salvia miltiorrhiza Bunge) and multiple sesquiterpenes in Curcuma wenyujin (Curcuma aromatica Salisb. cv. Wenyujin) (13, 14). Rhubarb (Rheum palmatum Linn), one of most commonly used TCM, was also studied by this method, and emodin was used as a single reference standard to simultaneously determine six other anthraquinones (15). This method used a single marker facilitated quality control of multicomponents in TCM. To date, the European Pharmacopoeia (16) has employed the QAMS method to calculate the amount of Ginkgolides in the Ginkgo dry extract, rather than flavonol glycoside. Moreover, no quantitative procedure for major flavonol glycosides has yet been published by this method. In this work, flavonol glycosides were identified in 20 batches of Ginkgo leaves and 20 batches of SXNI. A simple, convenient and effective QAMS method was used to determine seven major flavonol glycosides using CFs. Finally, the potential factors of CFs including the variation of the environmental and operational conditions were investigated. Table I. Detail Information of the Ginkgo Leaves and SXNI Ginkgo biloba leaves ShuXueNing injections No. Origin Time of collection No. Origin Batch number 1 Jiangxi July 1 Hebei Hubei August 2 Hebei Guangxi October 3 Hebei Sicuan September 4 Hebei Jiangsu September 5 Hebei Shaanxi October 6 Hebei Hebei August 7 Hebei Anhui October 8 Hebei Shandong September 9 Hebei Liaoning July 10 Hebei Hunan August 11 Shanxi Henan October 12 Shanxi Shanghai September 13 Shanxi Fujian August 14 Shanxi Jiangsu July 15 Shanxi Shanghai September 16 Shanxi Beijing October 17 Shanxi Yunnan July 18 Shanxi Zhejiang August 19 Shanxi Guangdong October 20 Shanxi Experimental Plant material and chemicals Twenty batches of dry Ginkgo leaves were collected from different Chinese provinces from July to October in Twenty batches of SXNI were purchased from two manufacturers. The detailed information of all tested samples is listed in Table I. Rutin (quercetin- 3-O-rutinoside) (2) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) ( ), and narcissoside (isorhamnetin-3-o-rutinoside) (5) was purchased from the Shanghai Sunny Biotech Corporation (Shanghai, China). Kaempferol-3-O-2,6 -dirhamnosylglycoside (1), kaempferol-3-o-rutinoside (3), kaempferol-3-o-2 -glucosylrhamnoside (4), quercetin-3-o-2 -(6 -p-coumaroyl)glucosylrhamnoside (6) and kaempferol-3-o-2 -(6 -p-coumaroyl) glucosylrhamnoside (7) were isolated previously from Ginkgo leaves in our laboratory. The seven compounds were confirmed by LC MS and NMR spectroscopy and assayed by the peak areas normalization method on an HPLC-DAD. The purities of Compounds 2, 3, 4, 5 and 6 were greater than 98%; Compounds 7 and 1 were 95.1 and 92.2%, respectively. The structures of the seven compounds are given in Figure 1. HPLC grade acetonitrile was purchased from J.T. Baker (Phillipsburg, USA). Preparation of sample solutions Twenty batches of Ginkgo leaves were dried in the sun over 7 days and then ground to a powder that could pass through a 40-mesh sieve. One gram of Ginkgo leaves was accurately weighed, and the powder was Figure 1. Structures of the major flavonol glycosides in Ginkgo leaves. transferred to Soxhlet equipment with 150 ml chloroform and refluxed for 2 h at 70 C. After discarding the chloroform solution, the residues were removed to a 250-mL flask with 100 ml methanol and were refluxed for 4 h at 70 C (17). The dissolved extract solution was transferred to a 10-mL volumetric flask and diluted with methanol to

3 Flavonol Glycosides in Ginkgo Leaves and ShuXueNing Injections by a Single Marker 1043 volume, filtered through a 0.45-µm nylon membrane filter and stored at 4 C until use. Twenty batches of the SXNI injections were filtered through a 0.45-µm nylon membrane filter, then injected 10 µl of each into the column. Preparation of standard solutions The stock solution containing seven reference standards was dissolved in methanol in a 5-mL volumetric flask and then used to prepare working standards for calibration curves and recovery experiments. The concentrations of Compounds 1 7 in the stock solution were , , , , 68.82, and µg/ml, respectively. Instrumentation and reagents The quantitative analysis was performed on an Agilent-1100 HPLC system (Agilent Technologies, USA), equipped with a quaternary pump, a vacuum degasser, an autosampler, a column compartment and a variable wavelength detector (VWD). Samples were separated on a TSK gel ODS-100V C18 column ( mm i.d., 3 µm) with a guard column ( mm i.d., 5 µm). The mobile phase consisted of acetonitrile (Channel A) and water containing 0.1% formic acid (Channel B), and the eluted gradient was as follows: 16 21% (A) in 0 35 min, 21 25% (A) in min, 25 30% (A) in min, isocratic with 30% (A) in min. The flow rate was 0.8 ml/min, the column temperature was maintained at 30 C and the VWD was set to monitor at 265 nm. The water used for the HPLC was purified with a Milli-Q plus system (Millipore, Bedford, MA, USA). The other reagents used in the experiment were of analytical grade and were purchased from Shanghai Chemical Reagent (Shanghai, China). An Agilent HPLC-DAD-ESI/MS system was employed to identify the flavonol glycosides in samples with a TSK gel ODS-100V C18 column ( mm i.d., 3 µm) and the same chromatographic conditions mentioned above. The injection volume was 10 µl. After solvent splitting, 38% of the column effluent from the DAD was directed to an LC/MSD Ion Trap XCT mass spectrometer equipped with an ESI source (Agilent Technologies). The acquisition parameters were as follows: collision gas: ultrahigh-purity helium (He); nebulizer gas: high purity nitrogen (N 2 ); nebulizer gas (N 2 ): 35 psi; dry gas (N 2 ): 10 L/ min; drying temperature: 350 C; HV voltage: 3,500 V; mass range: m/z 200 1,600, target mass: m/z 500; compound stability: 100%; trap drive level: 100%; and collision energy: V. Data acquisition was performed by Chemstation software (Agilent Technologies). to solve this problem. The ratio of the factor of the single marker and the other compound was calculated as the CF, and the equation is described as follows: F K=M ¼ f K f M ¼ c k A M c M A k The f K and f M are the factors of the single marker used in the QAMS method (K) and the other reference standard (M). A k and A M are the peak areas of the single marker used in the QAMS method (K) and the other reference standard (M). Accordingly, C k and C M are the concentrations of the single marker used in the QAMS method (K) and the other reference standard (M). The relative retention time of the reference standard (RRT m/k )is another parameter that can be calculated as the ratio of the other standard reference (M) and single marker (K). RRT m=k ¼ Rt m Rt k Rutin was selected (peak 2 in Figure 2A) as the single marker because of its availability, stability and good separation from the other compounds in the chromatogram. Ruggedness and robustness tests In view of the diversity of the environmental and operational conditions, the value of the CF may fluctuate under different factors. The ruggedness test focused on the environmental factors; it was performed using three different reversed-phase C18 columns, including a TSK gel ODS-100V C18 column ( mm i.d., 3 µm), an Inertsil ODS-3 ( mm i.d., 5 µm) and an Agilent Extend-C18 ( mm i.d., 5 µm) and three instruments, namely, an Agilent 1100-VWD, an Agilent 1100-DAD and a Shimadzu LC2010A-VWD. The robustness test was performed by measuring the effects of the operational factors that embodied the analytical parameters in the analytical procedure, such as the proportion of formic acid aqueous solution (PH), the proportion of organic phase in the mobile phase (MP), the time program of the mobile phase (TP), the wavelength of the UV detector (WL), the flow rate (FR), and the column temperature (CT). Two peak measurement parameters comprising the peak width (PW) and slope sensitivity (SS) were also studied as well. The fluctuations of CFs were assayed by changing the parameters in a narrow range at three levels, and only one of these parameters was changed slightly at a time while the others were kept constant. Methods Method validation The developed QAMS method with rutin as the single marker was validated on parameters including linearity, limit of detection (LOD), limit of quantification (LOQ), precision (intra-day and inter-day variations), accuracy, ruggedness and robustness. The F-test (ANOVA) (P > 95%) was used to statistically analyze the results of the calculated accuracy for the QAMS method and the TES method. Calculation of the CF and the relative retention time The principle of the QAMS method is similar to the TES method, which is based on the Lambert Beer law. The absorption of an analyte is linearly proportional to the sample concentration over a certain linear range (18). However, the molar absorptivity of analytes is often different. When quantitative analysis is performed by using a single marker to determine the multicomponents, the CF must be calculated Results Qualitative analysis of flavonol glycosides A survey of the available literature showed that the flavonol glycosides of Ginkgo leaves were distinguished into four groups, corresponding to quercetin, kaempferol, isorhamnetin derivatives and others (19). Characterization experiments were performed in positive and negative ion mode, which showed [M + H] + and [M H] ions, as well as other fragment ions. Fragment ions at m/z 303, 287, 317, 319, 333 and 347 Da were attributed to quercetin, kaempferol, isorhamnetin, myricetin, patuletin and syringetin in the positive mode. The fragment ions representing the loss of 162 and 308 Da were thought to occur via the loss of a glucosyl unit and a glucosyl-rhamnosyl moiety (rutinoside unit), respectively. The fragment ions representing loss of 146 Da were considered to be due to a loss of a rhamnosyl group or a coumaroyl group (20 22). Using the principles described above and

4 1044 Lin et al. Figure 2. The HPLC-DAD chromatograms at 265 nm of (A) mixed standards, (B) SXNI and (C) Ginkgo leaves. Conditions: column: TSKgel ODS-100 V C18 column ( mm i.d., 3 µm) with a guard column ( mm i.d., 5 µm); mobile phase: acetonitrile (Channel A) water containing 0.1% formic acid aqueous solution (Channel B) as follows: 16 21% (A) in 0 35 min, 21 25% (A) in min, 25 30% (A) in min, isocratic with 30% (A) in min; flow rate: 0.8 ml/min; column temperature: 30 C; injection volume: 10 µl; wavelength of UV detector: 265 nm. The peak numbers of the compounds in (A) correspond to those given in Figure 1, and the peak numbers of compounds in (B) and (C) correspond to those given in Table II. comparing with reference standards, 15 flavonol glycosides were characterized in Ginkgo leaves and SXNI (Table II), seven compounds (peak 5, 7, 10, 11, 13, 14 and 15 in Table II) of which were unambiguously identified by comparing their retention times, the masses and fragment ions with those of reference compounds. Furthermore, the other compounds were tentatively assigned by matching the empirical fragmentation patterns with that of the known compounds previously reported in the literature (20 22). Finally, seven major flavonol glycosides with high content both in Ginkgo leaves and SXNI (peaks in Figure 2A) were selected for quantitative analysis. Validation of the QAMS method Specificity The specificity was performed by comparing the consistency of the retention time and the UV spectrum between each analyte and the corresponding reference standard after identification of all analytes by HPLC DAD ESI-MS analysis. Figure 2 shows that the retention times of the seven compounds in the samples and mixed standard solution were consistent. Linearity and sensitivities Calibration curves were established by injecting six samples of mixed reference standards at different concentration levels. Table III summarizes the excellent calibration curves obtained. The limits of detection (LOD) and limits of quantification (LOQ) under the present chromatographic conditions were determined on the basis of the response and slope of each regression equation at signal-to-noise ratios (S/N) of 3 and 10, respectively (Table III). Precision and accuracy The evaluation of the precision consisted of intra- and inter-day precision determinations. The intra-day precision was determined by analyzing the same mixed standard solution six times within one day. For inter-day precision test, the solution was examined in triplicate on three consecutive days. For the seven compounds, the relative standard deviations (RSDs) for the intra-day precision of the peak areas and retention times were <1.97 and 2.69%, and the RSDs for the inter-day precision of the peak areas and retention time were <0.89 and 1.99%, respectively (Table IV). For the recovery test, three different concentrations (high, medium and low) of the mixed reference standards were added to a certain amount of the sample. Three replicates were performed for each test level, including 50, 100 and 150% ranges of a known amount of mixed reference standards in the sample. The QAMS method was reproducible with good accuracy in the range of % and RSD % (Table IV). The results of the recovery test obtained by both methods (QAMS and TES) are not different by the F-test (P > 95%, n = 3).

5 Flavonol Glycosides in Ginkgo Leaves and ShuXueNing Injections by a Single Marker 1045 Table II. Mass Spectral Identification of the Flavonol Glycosides Analyzed by HPLC DAD MS Peak Rt a (min) [M + H] + (m/z) MS 2 fragment ion (m/z) Identification [M + H] [M + H-Glucoside] + Kaempferol-3-O-2 -glucosyl-6 -rhamnosylglucoside 449 [M + H-Rhamnosylglucoside] [M + H-Glucosyl-rhamnosylglucoside] [M + H] [M + H-Rhamnose] + Quercetin-3-O-2,6 -dirhamnosylglucoside 465 [M + H-Dirhamnose] [M + H-Dirhamnosylglucoside] [M + H] [M + H-Rhamnose] + Myricetin-3-O-rutinoside 319 [M + H-Rutinoside] [M + H] [M + H-Glucoside] + Quercetin-3-O-6 -rhamnosyl-2 -(6 -p-coumaroylglucosyl) 611 [M + H-Coumaroylglucosyl] [M + H-(Coumaroylglucosyl)glucoside] + glucoside [M + H] [M + H-Rhamnose] + Kaempferol-3-O-2,6 -dirhamnosylglucoside 449 [M + H-Dirhamnose] [M + H-Dirhamnosylglucoside] [M + H] [M + H-Rhamnose] + Isorhamnetin-3-O-2,6 -dirhamnosylglucoside 479 [M + H-Dirhamnose] [M + H- Dirhamnosylglucoside] [M + H] [M + H-Rhamnose] + Rutin (Quercetin-3-O-rutinoside) 303 [M + H-Rutinoside] [M + H] [M + H-Rhamnose] + Patuletin-3-O-rutinoside 333 [M + H-Rutinoside] [M + H] [M + H-Glucoside] + Quercetin-3-O-2 -glucosylrhamnoside 303 [M + H-Glucosylrhamnoside] [M + H] [M + H-Rhamnose] + Kaempferol-3-O-rutinoside 287 [M + H-Rutinoside] [M + H] [M + H-Rhamnose] + Isorhamnetin-3-O-rutinoside 317 [M + H-Rutinoside] [M + H] [M + H-Rhamnose] + Syringetin-3-O-2 -glucosylrhamnoside 347 [M + H-Glucosylrhamnoside] [M + H] [M + H-Glucoside] + Kaempferol-3-O-2 -glucosylrhamnoside 287 [M + H-Glucosylrhamnoside] [M + H] [M + H-Glucosylrhamnoside] + Quercetin-3-O-2 -(6 -p-coumaroyl)glucosylrhamnoside 303 [M + H-Coumaroyl-glucosylrhamnoside] [M + H] [M + H-Glucosylrhamnoside] + Kaempferol-3-O-2 -(6 -p-coumaroyl)glucosylrhamnoside 287 [M + H-Coumaroyl-glucosylrhamnoside] + a Retention time. Table III. Statistical Analysis of the Linear Regression Equation Employed in the Determination of the Seven Flavonol Glycosides No Calibration curve γ Test range (µg/ml) Conversion factors RRT a LOQ (ng) LOD (ng) F RSD 1 y = x y = x y = x y = x y = x y = x y = x a The relative retention time of the reference standard. Ruggedness and robustness tests Because to concerns with the variation of the response between different instruments and the resolution between different columns, the CF of each compound was measured in three columns and three instruments in a ruggedness test. The value of the CFs of Compounds 1 and 7 were not stable when changing columns and instruments, whereas the others had good stability (RSD < 4.73%) (Table V). We also observed from Table V that the CFs of Compounds 1 and 7 detected by a DAD were higher than the other two instruments whose detectors were the VWD. Therefore, it is necessary to investigate the influence of the wavelength of the UV detector. The wavelength of the UV detector was the most significant factor among the eight factors, as shown in Figures 3A F. Therefore, the wavelength should be strictly controlled to reduce the variation of CFs. In addition to the wavelength, the column temperature also influenced the CF value of Compound 5, possibly due to decreased column efficiency,

6 1046 Lin et al. Table IV. Recoveries and Precision of the Analytical Method for the Seven Flavonol Glycosides in SXNI No. Amount added (µg) Amount found (µg) 1 a (n =3) 2 b (n = 3) Intra-day precision (n = 6) Inter-day precision (n =3) Recovery RSD Recovery RSD RSD of PA c RSD of Rt d RSD of PA RSD of Rt P = 0.769* (n = 18) a Traditional external standard method. b Single standard to determine multicomponents. c Peak area. d Retention time. *P values were measured by the F-test (The P value is more than 0.05 means that there is no significant difference between the results of two methods). Table V. The CFs in the Ruggedness Tests (n =5) Compound Agilent 1100-VWD Agilent 1100-DAD Shimadzu LC2010A-VWD RSD Column 1 Column 2 Column 3 Column 1 Column 2 Column 3 Column 1 Column 2 Column which can be verified by the plates of Compound 5 decreasing notably from 47,952 to 7,958 when the column temperatures increased from 25 to 35 C. For Compound 1, the proportion of organic phase in the mobile phase had some influence on the CF; the result may be related to the purity of Compound 1 (92.2%). The increased proportion of organic phase in the mobile phase allowed the peaks of the compound and its impurity to overlap so that higher peak areas resulted in a lower value of CF. For Compound 7, the proportion of formic acid aqueous solution led to a decrease of the CF value due to improved column efficiency. However, a higher proportion of formic acid reduced column life and led to baseline drift. Application of the QAMS method The established QAMS and TES methods were used for simultaneously determining 20 batches of Ginkgo leaves from different origins and 20 batches of SXNI from the two manufacturers (Table I). The contents of the seven compounds were calculated as shown in Tables VI and VII. The results obtained from the two methods were compared using an F-test (P > 95%, n = 20), and there was no significant difference. Figure 4 indicates that the diversity of origins and the collection time influenced the contents of the flavonol glycosides in the Ginkgo leaves samples. In addition, the average of the total flavonol glycosides in the SXNI that was purchased from manufacturer B was higher than that from manufacturer A. Discussion The established quantitative method using a single marker (rutin) to simultaneously determine seven flavonol glycosides in Ginkgo leaves, and their preparations had excellent linearity, precision, and accuracy. The ruggedness and robustness tests demonstrated that the wavelength of the UV detector affected the value of the CFs, so it must be strictly controlled. Compared with the TES method by using an F-test (P >

7 Flavonol Glycosides in Ginkgo Leaves and ShuXueNing Injections by a Single Marker 1047 Figure 3. Results of the robustness test. (A F) The CFs of the six compounds at each level of eight factors, the line indicates the average value. The eight factors were as follows: the proportion of formic acid aqueous solution (PH), for investigation, was 0, 0.1 and 0.2%; the proportion of the organic phase in the mobile phase (MP), for investigation, was 15/20/24/29/29, 16/21/25/30/30 and 17/22/26/31/31 ; the time program of mobile phase (TP) for investigation was 0/33/34/46/48 (min), 0/35/36/48/50 (min) and 0/37/38/50/52 (min); the wavelength of the UV detector (WL) for investigation was 263, 265, and 267 nm; the flow rate (FR) and the column temperature (CT) for investigation were 25, 30 and 35 C; the peak width (PW) for investigation was 0.01, 0.04 and 0.1; the slope sensitivity (SS) for investigation was 5, 10 and 15. Table VI. Contents (mg/g) of the Seven Flavonol Glycosides in 20 Batches of Ginkgo Leaves No. Compound 2 Compound 1 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Sum 1 1 a 2 b *P (n = 20) a Traditional external standard method. b Single standard to determine multicomponents method. *P values were measured by F-test (The P value is more than 0.05 means that there is no significant difference between the results of two methods).

8 1048 Lin et al. Table VII. Contents (µg/ml) of the Seven Flavonol Glycosides in 20 Batches of SXNI No. Compound 2 Compound 1 Compound3 Compound 4 Compound 5 Compound 6 Compound 7 Sum 1 1 a 2 b *P (n = 20) a Traditional external standard method. b Single standard to determine multicomponents method. *P values were measured by F-test (The P value is more than 0.05 means there is no significant difference between the results of two methods). Figure 4. Contents (mg/g) of the total flavonol glycosides in 20 batches of Ginkgo leaves (A) and the contents (µg/ml) of the total flavonol glycosides in 20 batches of SXNI (B). The number of X title corresponds to those given in Table I.

9 Flavonol Glycosides in Ginkgo Leaves and ShuXueNing Injections by a Single Marker %, n = 20), there was no significant difference. It indicated that the established QAMS method was acceptable as a practical and an alternative technique for the quantitative analysis of flavonol glycosides in Ginkgo leaves and its preparations. Conclusion The qualitative and quantitative analysis of flavonol glycosides is important to the quality control of Ginkgo leaves and their preparations. The high cost and lack of reference standards have hindered the implementation of routine quality control. The use of a single reference standard to determine multicomponents with similar chemical structures can be regarded as an alternative approach. Among the seven identified high contents of flavonol glycosides in Ginkgo leaves ( peaks in Figure 2A), rutin is easily available, chemically stable and cheap. 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