Studies On Determination Of Alkylphenols In Ginkgo Biloba Leaves By Gas Chromatography With On-line Pyrolytic Methylation

Ginkgo biloba extracts(EGb)are mainly used for the improvement of the blood circulation,both peripherally and centrally.However, alkylphenols including ginkgolic acids and ginkgols in Ginkgo leaves and extracts are claimed to be allergenic,mutagenic and slight neurotoxic. Accordingly,a requirement for a maximum concentration of ginkgolic acids in EGb have been included in the draft monographs of US and European pharmacopoeias by establishing a limit value of 5μg/g. Therefore,it is requested to develop a practical method to determine the alkylphenols in Ginkgo biloba leaves or extracts rapidly and precisely.In this thesis,the methods to determine alkylphenols in G.biloba leaves,G. biloba leaf tea and phytopharmaceuticals were developed on the basis of solvent extraction-pyrolytic methylation-gas chromatography/mass spectrometry(GC/MS)and direct pyrolytic methylation-GC.The volatile oil from G.biloba leaves was also analyzed by steam distillation-GC/MS.At first,a method of solvent extraction-pyrolytic methylation -GC/MS was applied to determine alkylphenols in Ginkgo biloba leaves ethanol extraction.Pyrolytic methylation of G.biloba leaves ethanol extract in the GC injector at 300℃in the presence of an organic alkali, tetramethylammonium hydroxide(TMAH)allowed quantitative analysis of alkylphenols including ginkgolic acids and ginkgols as their corresponding methyl derivatives.On the basis of the relative areas of these peaks,chemical composition of the alkylphenols in G.biloba leaves extract was determined with less than 3.4%RSD.Furthermore,trace amount of ginkgolic acids in phytopharmaceuticals was determined by pyrolytic methylation-GC/MS using selective ion monitoring.The results showed the linearity was satisfactory over a concentration range 0.50-100μg/ml for GA_13:0,GA_15:1-△8and GA_17:1-△12with the correlation coefficients higher than 0.998.The limits of detection ranged from 0.2 to 0.6μg/ml. The recoveries(＞91%)and precisions(R.S.D.s＜5%(n = 5))of the method were examined by analyzing EGb samples spiked at levels between 1.0 and 5.0μg/g.This simple,accurate and highly sensitive method may be used for chemical analysis and quality control of Ginkgo biloba leaves and extracs in various commercial ginkgol preparation processes.Secondly,a method of direct pyrolytic methylation-GC using a vertical microfurnace pyrolyzer was applied to determine alkylphenols in Ginkgo biloba leaves without using any tedious and time-consuming pretreatment such as solvent extraction and purification.Similarly,on the pyrograms of Ginkgo biloba leaves obtained at 300℃in the presence of TMAH,a series of sharp peaks of alkylphenols including ginkgolic acids and ginkgols as their corresponding methyl derivatives could be observed clearly.On the basis of the relative intensity of these peaks on the pyrogram obtained,chemical composition of the alkylphenols in G biloba leaves was determined rapidly and precisely with R.S.D less than 5.2%.The results showed the linearity was.satisfactory over a concentration range 50-1000 mg/l for GA₁₅:1-△8 with the correlation coefficients higher than 0.997.The limit of detection was 10μg/g.The recovery(＞92%)and precisions(R.S.D.s＜4.3%(n=5))of the method were examined by analyzing Ginkgo biloba leaf samples spiked GA₍15:1-△8 at 0.7 mg/g.Furthermore,the alkylphenols in Ginkgo biloba leaves collected at different growing stages and Ginkgo biloba leaf tea samples were also determined using this method with satisfactory results.Lastly,the volatile oil from G.biloba leaves was obtained by steam distillation and analyzed by GC/MS.The chemical components were separated and identified by GC/MS,their contents were quantitatively determined using a normalization method.78 compounds in the volatile oil from G.biloba leaves were identified,and amounted to 90.0%of their total compounds.The result showed that the volatile oil mainly consisted of 12.58%hydrocarbons,17.46%alcohol,8.56%aldehydes,31.73% ketones,2.79%esters and 0.73%hydroxybenzene.In these chemical constituents,the content ofβ-ionone was found highest(10.55%), followed by hexahydrofarnesyl acetone and farnesyl acetone,amounting to 5.07%,4.69%,respectively.