Article pubs.acs.org/JAFC
High-Resolution Gas Chromatography/Mass Spectrometry Method for Characterization and Quantitative Analysis of Ginkgolic Acids in Ginkgo biloba Plants, Extracts, and Dietary Supplements Mei Wang,† Jianping Zhao,† Bharathi Avula,† Yan-Hong Wang,† Cristina Avonto,† Amar G. Chittiboyina,† Philip L. Wylie,§ Jon F. Parcher,† and Ikhlas A. Khan*,†,‡,∥ †
National Center for Natural Products Research, and ‡Division of Pharmacognosy, Department of BioMolecular Science, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States § Agilent Technologies, 2850 Centerville Road, Wilmington, Delaware 19808-1610, United States ∥ Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia ABSTRACT: A high-resolution gas chromatography/mass spectrometry (GC/MS) with selected ion monitor method focusing on the characterization and quantitative analysis of ginkgolic acids (GAs) in Ginkgo biloba L. plant materials, extracts, and commercial products was developed and validated. The method involved sample extraction with (1:1) methanol and 10% formic acid, liquid−liquid extraction with n-hexane, and derivatization with trimethylsulfonium hydroxide (TMSH). Separation of two saturated (C13:0 and C15:0) and six unsaturated ginkgolic acid methyl esters with different positional double bonds (C15:1 Δ8 and Δ10, C17:1 Δ8, Δ10, and Δ12, and C17:2) was achieved on a very polar (88% cyanopropyl) aryl-polysiloxane HP-88 capillary GC column. The double bond positions in the GAs were determined by ozonolysis. The developed GC/MS method was validated according to ICH guidelines, and the quantitation results were verified by comparison with a standard highperformance liquid chromatography method. Nineteen G. biloba authenticated and commercial plant samples and 21 dietary supplements purported to contain G. biloba leaf extracts were analyzed. Finally, the presence of the marker compounds, terpene trilactones and flavonol glycosides for Ginkgo biloba in the dietary supplements was determined by UHPLC/MS and used to confirm the presence of G. biloba leaf extracts in all of the botanical dietary supplements. KEYWORDS: Ginkgo biloba, ginkgolic acids, gas chromatography/mass spectrometry, TMSH derivatization, ozonolysis, dietary supplements
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INTRODUCTION Ginkgo biloba L. (Ginkgoaceae), also known as Maidenhair tree, products and extracts constitutes one of the most widely used classes of herbal medicines, dietary supplements, or phytopharmaceuticals in the world.1 The plant is the sole surviving species of the Ginkgoaceae family and is often referred to as a living fossil tree.2,3 Purported medicinal benefits mostly center on improved cognitive functions (memory and concentration), deceleration of aging, treatment of cardiovascular and neurodegenerative disorders, as well as improvement of blood circulation.4−6 Other beneficial properties include antitumor, antidepressant, and antistress activities.7 The positive medicinal effects are most commonly attributed to the plant’s major components, viz., terpene trilactones (TTLs) and flavonol glycosides.1 It is often observed that herbal medicines and dietary supplements may exhibit concomitant, adverse, as well as beneficial pharmaceutical effects. Such is the case with G. biloba. The detrimental medicinal effects attributed to G. biloba extracts and commercial products include contact allergic dermatitis, possible herb−drug interactions, as well as potential cytotoxic, mutagenic, carcinogenic, neurotoxic, or tumorpromoting properties.8−11 The medical validity of many of these claims is not well established; however, there is general agreement that most adverse effects are due to the presence of bioactive alkylphenols, such as ginkgolic acids (GAs), © 2014 American Chemical Society
Figure 1. Chemical structures of the main components of alkylphenols in Ginkgo biloba.
cardanols (ginkgols), and cardols.12−15 The structures of these compounds are given in Figure 1. The ginkgolic acids have also been designated as 2-hydroxy-6-alkylbenzoic acids, 6-alkylsalicyclic acids, and anacardic acids. These potentially hazardous components are chemically similar to urushiols found to be responsible for the allergenic properties of poison ivy and sumac.16 Received: Revised: Accepted: Published: 12103
August 18, 2014 October 30, 2014 November 10, 2014 November 10, 2014 dx.doi.org/10.1021/jf503980f | J. Agric. Food Chem. 2014, 62, 12103−12111
Journal of Agricultural and Food Chemistry
Article
pretreatment schemes required for other chromatographic methods. This method was used to resolve seven GAs and three cardanols including C13:0, C15:0 as well as double bond positional isomers C15:1 Δ8 and Δ10, C17:1 Δ8 and Δ12 along with one GA with two unsaturation sites, viz., C17:2. No C17:0 or C17:1 Δ10 isomers were detected. Possible reaction schemes for the electron impact fragmentation of the methylated GAs were discussed. In earlier work, Schotz16 developed a GC/MS method for the analysis of the silyl derivatives of a standardized extract EGb 761. Five GAs, viz., C13:0, C15:0, C15:1 (Δ8 and Δ10), and C17:1 (Δ12), were identified. In 2004, Choi et al.27 reported an NMR method for the analysis of GAs. This spectroscopic method did not require derivatization of the GAs or calibration standards. The peaks associated with the aromatic protons in GAs were individually integrated to give the total GA concentration rather than individual components. This method, however, was not sufficiently sensitive and suffered interference problems when applied for the analysis of commercial samples. Very recently, van Beek’s group addressed the problem of the lack of pure standards of ginkgolic acids.28 Magnetic nanoparticles were used to selectively adsorb GAs from petroleum ether extracts of ginkgo leaves. The mixed acids were subsequently desorbed with strong acid or base and isolated by prep-scale LC on a C8 column. Significant quantities of eight GAs were isolated by this novel procedure. The objectives of the present study were to develop and validate a high-resolution gas chromatographic method for the analysis of the individual isomers of C13, C15, and C17 GAs in ginkgo leaves, extracts, and commercial products. In addition, the double bond position and the concentration of each isomer will be determined. This information may improve our understanding of the GA isomer distribution in various plant parts. The accuracy of the GC method will be assessed by comparison with the accepted HPLC methods. Additionally, the veracity of the extracts in dietary supplements will be established by detection of the marker compounds for G. biloba, TTLs and flavonol glycosides, by UHPLC/MS analysis.
There are two types of leaf extracts: full extracts and standardized extracts. The full extracts are usually prepared with alcohol and thus contain all constituents soluble in alcohol. The standardized extracts constitute the major commercial ginkgo products and contain 6% TTLs, 24% flavonol glycosides, and trace amount of ginkgolic acid. Such extracts are prepared in a multistep process, in which some compounds are enriched (TTLs and flavonoids) while others (biflavones and ginkgolic acids) are removed. Because of the significant risk factors of the ginkgolic acids, the content of these undesirable components in standardized extracts of G. biloba are currently limited to 5 ppm GAs ranging from 5 to 56 ppm. The two samples (15913 and 15925) containing the highest concentrations of GAs (56 and 30 ppm, respectively) were both leaf extracts, not standardized extracts. The major active components of G. biloba leaf extracts are the flavonoids and sesquiterpenes lactones.33,34 Standardized commercial extracts usually contain >6% ginkgolides (A, B, C, and J) and bilobalide and 24% flavonoids (quercetin, kaempferol, and isorhamnetin). In order to confirm the presence of G. biloba leaf extracts in the botanical dietary supplements, all of the dietary supplements shown in Table 2 were analyzed by a previously published method29 involving the use of [M + H]+ ions of five sesquiterpene lactones and three flavonoids which were observed at m/z 425.2 (ginkgolide J and ginkgolide B), m/z 441.2 (ginkgolide C), m/z 327.3 (bilobalide), m/z 409.2 (ginkgolide A), m/z 303.1 (quercetin), m/z 287.1 (kaempferol), and m/z 317.1 (isorhamnetin). Based on the retention times and mass spectra in comparison with standard compounds, all commercial ginkgo preparations analyzed were found to contain all eight compounds indicating that all of the analyzed dietary supplements contained ginkgo leaf extracts. Confirmation with Standard HPLC Technique. HPLC with a C8 column, acetonitrile eluent and UV detection at 310 nm is a commonly accepted method for the analysis of GAs without distinction between the double bond positional isomers.3,20,23,28,35 One leaf sample, 1095, was analyzed in a derivatized form by GC/MS and in the acid form by HPLC. The underivatized 1095 sample was analyzed by LC using three detection schemes, viz., UV at 310 nm, MS scan and MS SIM at m/z 319, 347, 345, and 373 for C13:0, C15:0, C15:1 and C17:1, respectively. The results for the quantitation of GAs in the ginkgo leaf sample (1095) are given in Table 4.
Quantitative analysis results obtained with the GC method were comparable with results achieved by a commonly accepted HPLC method. The ratios of the concentrations of C15:1 Δ10 to Δ8 observed for leaf samples of ginkgo were in the range of 1−3, whereas the same ratio in the seed samples was always
