Anal. Chem. 2000, 72, 3396
Comments on “An Extraction Method for Determination of Ginkgolides and Bilobalide in Ginkgo Leaf Extracts” SIR: Recently Lang and Wai1 published an interesting new quantitative procedure for the analysis of terpene trilactones in Ginkgo leaves and extracts. However, there might be some problems associated with this procedure. To avoid possible wrong results by those wanting to apply this method, in the following the potential pitfalls are brought to the attention of your readership. Lang and Wai extracted their samples first by boiling for 2 min in water and then by boiling with a 0.1% Na2HPO4 solution (pH ) 8) for 15 min. It has been reported that bilobalide is unstable under neutral or basic conditions,2,3 and this was confirmed by Lang and Wai themselves.1 This means that any bilobalide which was not extracted during the first 2 min will be partially degraded during the second alkaline extraction step. This could lead to lower values for bilobalide. Of all samples except number 1, the bilobalide content reported is indeed low in comparison with the combined ginkgolide content. Normally the bilobalide content is 40-50% of the total terpene trilactone content.4-6 During their sample cleanup, Lang and Wai used dichloromethane to extract the terpene trilactones from the acidified aqueous phase. However, all terpene trilactones are poorly soluble in this organic solvent. Dichloromethane has been used by others to separate apolar impurities from terpene trilactones.7 In particular, the most polar ginkgolides, C and J (further abbreviated as G-C and G-J), will not partition from the aqueous phase into dichloromethane. Unfortunately, the authors had no G-C and G-J at their disposal and were thus unable to determine this by recovery experiments. The poor extraction of these two ginkgolides is confirmed by the absence of a peak for G-J in their chromatogram (should appear between G-A and G-B) and the low values reported for G-C relative to those of G-A and G-B. (1) Lang, Q.; Wai, C. M. Anal. Chem. 1999, 71, 2929-2933. (2) Fourtillan, J. B.; Brisson, A. M.; Girault, J.; Ingrand, I.; Decourt, J. P.; Drieu, K.; Jouenne, P.; Biber, A. The´ rapie 1995, 50, 137-144. (3) Chen, Z.; Ying, M.; Mao, X.; Hu, L. In Proceedings of ′97 International Seminar on Ginkgo; The State Science and Technology Commission: Beijing, China, 1997; pp 154-157. (4) Hasler, A.; Meier, B. Pharm. Pharmacol. Lett. 1992, 2, 187-190. (5) Lolla, E.; Paletti, A.; Peterlongo, F. Fitoterapia 1998, 69, 513-519. (6) van Beek, T. A.; Taylor, L. T. Phytochem. Anal. 1996, 7, 185-191. (7) Wada, K.; Sakaki, K.; Miura, K.; Yagi, M.; Kubota, Y.; Matsumoto, T.; Haga, M. Biol. Pharm. Bull. 1993, 16, 210-212. (8) van Beek, T. A.; Scheeren, H. A.; Rantio, T.; Melger, W. C.; Lelyveld, G. P. J. Chromatogr. 1991, 543, 375-387.
3396 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
Lang and Wai used BSA at 100 °C for silylation of the terpene trilactones prior to gas chromatography. However, the tertiary OH is difficult to derivatize. Hasler and Meier have studied the derivatization in detail and have reported that BSA alone did not give good results and that BSTFA with 1% TMCS was a better choice.4 A derivatization with BSA alone could possibly lead to less-reproducible results. The extraction and sample cleanup parts of the method of Lang and Wai1 were compared with those of an existing method (twice extraction with refluxing 10% methanol in water8 and sample cleanup by SPE5) by the same experienced phytochemical analyst with the same Ginkgo leaf sample on the same day. The same detection method (HPLC-ELSD) was used for both extracts. In the chromatograms obtained by the Lang and Wai method, the peaks for G-A and G-B were somewhat lower, the peak for bilobalide was a factor of 10 lower, while no peaks for G-C and G-J were detectable in comparison with the 10% methanol extraction and SPE cleanup. When the aqueous fraction remaining after the dichloromethane partitioning was analyzed, a trace of G-A, about as much bilobalide and G-B as was found in the dichloromethane layer, and large amounts of G-C and probably G-J could be detected. These results are in clear contradiction with the distribution coefficient experiments carried out by Lang and Wai. Assuming an equal practical expertise, two possible explanations can be suggested. In the comparative experiment, no optional THF was added to the dichloromethane, because no slow phase separations occurred.1 Adding 1 mL of THF to 5 mL of dichloromethane could significantly influence the distribution coefficient in favor of the dichloromethane layer. Furthermore, Lang and Wai used the pure substances in water for their determination of the distribution coefficients. However, in real leaf samples and extracts, significant amounts of natural surfactants occur which will alter the distribution coefficient in favor of the aqueous layer. The use of an alkaline solution in the second extraction step will make this worse in the case of leaves and crude extracts, as the normally insoluble ginkgolic acids, which are surfactants, will become more soluble. In conclusion, this method should be used with some caution. Possible improvements might be the replacement of the 0.1% Na2HPO4 solution by distilled water during the second extraction step and the standard addition of 1 mL of THF to 4 mL of dichloromethane. Further validation should be considered, in particular, recovery experiments for bilobalide and G-C and G-J.
Teris A. van Beek
Phytochemical Section, Laboratory of Organic Chemistry Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands November 17, 1999. Accepted April 27, 2000. AC991324C 10.1021/ac991324c CCC: $19.00
© 2000 American Chemical Society Published on Web 06/10/2000
