Abstract
A new HPLC quantification method for allergenic ginkgolic acids (GA) in Ginkgo biloba leaf extracts (GBE) was developed, using an acetonitrile-water gradient elution system with an Agilent-SB C18 column. With ginkgoneolic acid (13:0 GA) as a marker, the relative correlation factors of the four other GAs – 15:0, 15:1, 17:1, and 17:2 GA – to 13:0 GA were determined by HPLC and subsequently used for calculating their contents in 10 G. biloba leaf samples from Shandong, which were extracted according to the Chin.Ph. monograph for TCM GBE (not identical to the corresponding Ph.Eur. monograph). In other words, the content of 13:0 GA in the extracts was determined using the pure compound as an external standard. Subsequently the now known concentration of this compound functioned as an internal standard for the quantification of the other four GA derivatives via the relative correlation factors. The new HPLC method was validated by control measurements with external standards for each individual GA. The results did not differ significantly for any of the 10 leaf samples. Additionally, 10 commercial GBE (9 Chin.Ph., 1 Ph.Eur.) preparations were tested with the new method, in comparison to the current Chin.Ph. protocol. Although all 9 TCM extracts were in accordance with the regulation of a maximal content of 10 ppm GA when tested with the Chin.Ph. HPLC method, only two of these were below this concentration when tested with the new protocol (2.41 vs. 9.76 ppm/2.49 vs. 9.52 ppm), demonstrating that in many cases TCM GBE preparations on the Chinese market contain considerable amounts of GA that are not detected by the present Chin.Ph. HPLC. For the Ph.Eur. extract, 0.81 vs. 4.87 ppm were measured, both in agreement with the maximal content of 5 ppm GA according to Ph.Eur.. The newly developed protocol is therefore simple, reproducible, and can be used to determine the total contents of GA derivatives in G. biloba leaf extracts.
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