Application of CE, HPLC and LC-MS-MS for the analysis and quality control of Ginkgo biloba dosage forms
- Authors: Dubber, Mary-Jean
- Date: 2006
- Subjects: Pharmaceutical chemistry -- Quality control Ginkgo Micelles Capillary electrophoresis High performance liquid chromatography Drugs -- Dosage forms Flavonoids Terpenes Herbals
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3757 , http://hdl.handle.net/10962/d1003235
- Description: Natural products are complex mixtures of compounds with therapeutic effects which are often reported to be due to the synergistic action of multiple and sometimes unknown components. Consequently, standardization of these products is complex and a lack of effective quality control (QC) criteria in most countries has led to marketing of commercial products with questionable quality, safety and efficacy (QSE). The aim of this study was therefore to develop qualitative and quantitative analytical methods for use in the QC of Ginkgo biloba solid oral dosage forms. Initially, a micellar electrokinetic chromatography (MEKC) method was developed for the identification of the flavonol glycosides, rutin and quercitrin as well as 3 flavonol aglycones, quercetin, kaempferol and isorhamnetin in crude extracts of 4 Ginkgo biloba solid oral dosage forms using ultraviolet (UV) detection. A reversed-flow cyclodextrin-modified MEKC method was subsequently developed for the simultaneous determination of the aforementioned flavonols as well as ginkgolide A, B, C, J and bilobalide (all positive markers) in Ginkgo commercial products. A non-aqueous capillary electrophoresis (CE) method was also developed for fingerprinting the presence of ginkgolic acids (negative markers) in Ginkgo biloba leaf extracts, which are purported to be associated with toxic properties. This method was also applied to 2 Ginkgo biloba commercial products. Since the flavonols have strong UV absorbing chromophores, a reversed phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated using photo-diode-array (PDA) detection which was then successfully applied to fingerprint commercially available Ginkgo biloba solid oral dosage forms as well as quantify the relevant flavonol markers present in these extracts. Sample preparation was simple, rapid and cost efficient with minimal clean-up and the employment of a minibore column which requires low mobile phase flow rates contributed to the economy of the method. Unlike the conventional QC approach, samples were not hydrolyzed and direct determination of 2 intact flavonol glycosides, together with the usual aglycone markers was facilitated which provided maximal content information for fingerprint comparisons. On the other hand, terpene trilactones possess poor chromophores and an alternative detection method to UV was required in order to obtain suitable sensitivity. RP-HPLC with evaporative light scattering detection (ELSD) was selected for quantification of these non-volatile constituents in Ginkgo dosage forms and this method was deemed suitable for the routine QC analysis of these positive markers in commercial products. Since approximately 33 flavonoids have been identified in Ginkgo biloba leaf extracts, baseline separation using UV/PDA detection normally requires complex gradient programs and long analysis times. In addition, unequivocal identification of the flavonoids with similar UV spectra and elution times cannot be guaranteed. A liquid chromatographic tandem mass spectrometric (LC-MS-MS) method was therefore developed and validated in order to ensure accurate quantification of the selected flavonol marker compounds in Ginkgo commercial products. LC-MS-MS analysis of Ginkgo extracts revealed, in addition to rutin, the possible presence of other quercetin analogues, quercetin-3-Orhamnoside-7-O-glucoside or quercetin-3-O-glucoside-7-O-rhamnoside, previously unreported in Ginkgo biloba leaf extracts or dosage forms. In terms of evaluating the most suitable analytical method for QC, CE shows exceptional potential in the future analysis of Ginkgo biloba dosage forms while HPLC-PDA and HPLC-ELSD are currently the most affordable and practical instruments for the routine analysis of the flavonols and terpenoids, respectively. LC-MS-MS proved to be pivotal for the accurate identification and quantification of the flavonols due to interference by other flavonoid compounds with similar retention times and UV spectra to the peaks of interest. All quantitative and qualitative results revealed large discrepancies in the marker content between the products regardless of which batch was analysed and product labels disclosed little relevant information. Although currently not required by most regulatory agencies, some of the usual quality criteria applied to orthodox medicines was evaluated. In particular, dissolution analysis, disintegration, tablet hardness and weight uniformity were assessed and revealed similar inconsistencies. This thesis emphasises that implementation of effective QC criteria is long overdue and is essential to ensure consistent product QSE of commercially available Ginkgo biloba solid oral dosage forms.
- Full Text:
- Date Issued: 2006
Application of reverse-flow micellar electrokinetic chromatography for the simultaneous determination of flavonols and terpene trilactones in Ginkgo biloba dosage forms
- Authors: Dubber, Mary-Jean , Kanfer, Isadore
- Date: 2006
- Language: English
- Type: text , Article
- Identifier: vital:6357 , http://hdl.handle.net/10962/d1006049
- Description:
A reverse-flow micellar electrokinetic chromatographic (RF-MEKC) method was developed for the simultaneous qualitative determination of 10 components consisting of the flavonol glycosides, rutin and quercitrin, the flavonol aglycones, isorhamnetin, kaempferol and quercetin, the terpene trilactones, ginkgolides A, B, C and J and the sesquiterpene, bilobalide. This method was used to fingerprint Ginkgo biloba solid oral dosage forms and validated for the quantitation of the marker compounds, rutin and quercetin in some commercial products. In addition to the usual variables, the influence of some essential background electrolyte (BGE) components such as sodium dodecyl sulphate (SDS) and
-cyclodextrin concentrations were investigated. A polyimide fused-silica square capillary column (75 μm I.D. × 360 μm O.D.) with a total length of 60.0 cm and effective length of 45.0 cm was used for the separation. The final BGE consisted of 20 mM phosphoric acid, 40 mM SDS and 12 mM -cyclodextrin (pH 2.2) using reverse polarity with a voltage of −17.5 kV. Samples were injected electrokinetically at −5 kV for 3 s for the qualitative analysis and hydrodynamically at 20 mbar for 0.6 s for the quantitative assay. The total run time was 22 min and the limits of detection were 3.13 μg/ml and 1.88 μg/ml for rutin and quercetin, respectively. Fingerprint profiles of the solid oral dosage forms and the results of the quantitative analysis indicated that there were major discrepancies in the marker content between products and illustrates the value of this method for use as a procedure to assess product quality of commercially available Ginkgo biloba products. - Full Text:
- Date Issued: 2006