Simultaneous Determination of Contents of Flavonol Glycosides and Terpene Lactones in Ginkgo Biloba Tablets by Ultra High Performance Liquid Chromatography Tandem Single Quadrupole Mass Spectrometry Detector

Ginkgo biloba leaf tablets is an effective ingredient in the treatment of cardiovascular and cerebrovascular diseases. In the process of drug production, the quality of ginkgo preparations is often controlled by measuring the content of seven ingredients in ginkgo leaves. To establish UPLC-MS multicomponent analysis method for ginkgo biloba tablets and to simultaneously determine the contents of quercetin (QUE), isorhamnetin(ISO), kaempferol(KAE) and GinkgolideA (GA),ginkgolideB(GB),ginkgolideC(GC) and bilobalide (BB) in ginkgo tablets. Waters Xbridge C18(4.6×150mm,3.5um) column was used, mobile phase A was acetonitrile and mobile phase B was water (containing 0.10% formic acid). The injection volume was 10μL.Negative ion mode monitoring was conducted with ESI. Scanning range:m/z100∼1400.The detection ions of the seven tested components includem/z301.0(QUE),m/z284.9(KAE),m/z315.1(ISO),m/z453.1(GA),m/z423.1(G B),m/z439.0(GC)and m/z325.0(BB), respectively. Within a space of 10min, flavonoids and terpene lactones in ginkgo biloba tablets were completely separated. The peak area exhibited an excellent linear relationship with the concentration. The sample recovery rate ranged from 91.74% to 109.77%.Precision RSDs of within-day and between-day were lower than 2.879% and 3.928% respectively. The method for determination of seven components in ginkgo biloba tablets displays good repeatability, recovery rate and precision, for which it can be applied to quality control of ginkgo biloba tablets.


72
As specified in the first part of the 2015 edition of the Chinese Pharmacopoeia [17], the terpene 73 lactones in specification A shall be 4.8mg·tablet -1 as a minimum and the terpene lactones content in 74 specification B shall be 2.4mg·tablet -1 as a minimum(n=3). The results indicated that the content of 75 terpene lactones in ten batches of ginkgo bilobo tablets produced by five manufacturers was 76 compliant, as shown in Table 2.

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A precise measurement was taken of the series of standard solutions and the determination was 82 performed according to the "chromatographic conditions"(n=3). The concentrations of QUE, KAE, 83 ISO, GA, GB, GC, BB were taken as abscissa, the peak areas were taken as ordinate and a linear 84 regression equation was derived. The results indicated that the linear relationship of each standard 85 was excellent within a certain range. The mixed reference substance solution was diluted 86 incrementally. The concentration of each reference substance when S/N=10:1 and S/N=3:1 was 87 regarded as the quantitative limit and detection limit [18]. The quantitative limit range was 5.0*10 -6 88 to 1.0*10 -5 , mg·mL -1 and the detection limit range was 1.0*10 -5 to 2.5*10 -5 mg·mL -1 , as shown in Table  89 3.

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After one tablet of sample A1 was taken and re-extracted.1000μL of filtered extract was taken.
93 According to the ratio of 1:0.5(low),1:1.0 (medium) and 1:1.5(high) of the content of each measured 94 chemical substance, standard solution with various substance concentrations of 1mg·mL -1 was added 95 respectively. In addition, the recovery rates of high, medium and low concentration substances were 96 tested after the standard was added. Based on the determination of "chromatographic 97 conditions"(n=3), the recovery rate of this method was 91.74%~109.77%. The results confirmed that 98 the recovery rate of this method could satisfy the requirements, as shown in Table 4. 102 standard solution of low, medium and high concentration was prepared and determined based on 103 "chromatographic conditions" (n=6). Intra-day precision refers to six parallel tests within one day. 104 Daytime precision refers to two parallel tests within one day for three consecutive days. The results 105 confirmed that the intra-day and inter-day precision of the method could satisfy the requirements, as 106 shown in Table 5. 118 revealed that the sample solution was capable of remarkable stability within forty-eight hours, as 119 shown in Table 7.

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Totally ten batches of ginkgo leaf samples were taken and determined based on the terms of 123 "preparation of test solution" and "chromatographic conditions"(n=3). The established analysis 124 method was effective in separating the components in ginkgo biloba tablets and a quantitative 125 analysis was performed of seven components. After calculation, the results revealed that QUE, KAE, 126 ISO and BB are higher than other components in ten batches of ginkgo biloba tablets, as shown in 127 Table 8. The LC-MS spectra of the 7 components reference substance were observed to be consistent 128 with the corresponding component spectra of the sample, as shown in Figures 1 and 2. And the 129 primary mass spectra of the reference substance and the sample are presented in Figures 3, 4, 5, 6, 7, 130 8 and 9.

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High performance liquid chromatography-mass spectrometry (HPLC-MS) has been extensively 144 applied to the quality research of drugs due to its impressive specificity and higher sensitivity [19-145 24]. In this experiment, the greatest advantage displayed by the UPLC-MS was simple operation. It 146 took as little as five minutes of mass spectrometry equilibrium time to complete qualitative analysis 147 of samples. Moreover, it can be used for qualitative and quantitative analysis of compounds that is 148 incapable of ultraviolet absorption.Negativeion mode monitoring was conducted by Electron Spray 149 Ionization and the results demonstrated that the relative molecular mass parameters were 150 respectively QUE301.0,KAE284.9, ISO315.1,GA453.1,GB423.1,GC439.0 and BB325.0, which was 151 discovered to be consistent with literature reports [22].Not only does this method provide reference 152 and basis for the quality control of ginkgo biloba tablets, it also lays a solid foundation for further 153 study on the pharmacodynamical characteristics of in vivo index components of ginkgo biloba after 154 oral administration in rats. However, this method remains subjected to various limitations and some 155 drawbacks are exposed. In this sense, further research is deemed necessary to improve the quality 156 control of ginkgo biloba tablets and to figure out their efficacy.