Bioavailability of wilforlide A in mice and its concentration determination using an HPLC-APCI-MS/MS method
Introduction
Wilforlide A (WA) and triptolide are two active components in Tripterygium wilfordii Hook F (TW, also called Thunder God Vine), a traditional Chinese medicine (named Lei Gong Teng in Chinese) known for treatment of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and psoriasis for years (Fig. 1) [[1], [2], [3]]. Currently, a product called Tripterygium wilfordii glycosides tablet or Lei Gong Teng Duo Gan Tablet is readily commercially available in China. In our previous study, the TW extract has also been found to sensitize prostate cancer resistant to docetaxel [4]. However, the use of TW extract has been associated with serious side effects such as immunosuppression and hepatotoxicity as well as quality control issues of herbal extracts [[5], [6], [7]]. Thus its active chemical markers, such as WA (content of 0.056 mg/g in root of TW) and triptolide (content of 0.067 mg/g to 0.8 mg/g in root of TW) would be attractive for further potential development [8,9]. Of the two chemical markers, triptolide has been reported to cause more side effects, such as immune suppression and anti-fertility than WA [10,11]. Its content is restricted to be no more than 10 μg per 10 mg (one tablet) in the commercial tablets [12]. In comparison, the recommended content of WA in the TW tablet is greater than 10 μg per 10 mg, consistent with a safer projection of this maker. In previous studies, WA has shown efficacious anti-inflammatory activities in carrageenan-induced rat pedal swelling and in tampon-induced rat granulation models with no significant immune suppressive effect [13]. Furthermore, we have observed an enhanced anticancer effect when combined with a conventional chemotherapeutic drug [14]. Thus, WA seems to be a good drug candidate to be further developed due to its various favorable activities, but less toxicity. However, there is no absorption and pharmacokinetic information available for WA. Such information will be useful for planning pre-clinical efficacy studies as well as future clinical trials in human subjects. In addition, the oral pharmacokinetic profile of WA will be relevant to its designation as a quality control marker for the TW product when given orally [15]. Therefore, in this study the bioavailability of WA following its oral and intraperitoneal administration is investigated using a newly developed high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometric assay (HPLC-APCI-MS/MS).
Section snippets
Materials and instruments
WA and celastrol were purchased from the National Institutes for Food and Drug Control (Beijing, China) with purities of greater than 95%. The analytical grade formic acid was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) with a purity of more than 99%. HPLC-grade methanol and acetonitrile were obtained from Fisher Scientific (Pittsburgh, PA, USA). Methyl tert-butyl ether (MTBE) was purchased from VWR International, LLC. (Brisbane, CA, USA). The blank mouse plasma (CD-1) was got from
Specificity and sensitivity
WA and celastrol were well separated under the chromatographic conditions without significant interference. The retention time of WA and celastrol was 10.3 and 9.2 min respectively (Fig. 3). This method was linear over the concentration range from 10 to 5000 ng/ml with R2 greater than 0.99. A1/x2 weighted linear regression was used for all calibration curves (x is the nominal concentration). The LOD and LLOQ of WA were identified to be 5 ng/ml and 10 ng/ml respectively, which indicated that
Discussion
In the present study, an assay method of WA was developed and applied to a bioavailability study in mice. Prior to our assay development, there was one previously published assay of WA which used a single quadrupole mass to detect WA using the Selected Ion Monitoring (SIM) mode by detecting the m/z of its molecular ion [17]. Since more than 300 components from Tripterygium wilfordii have been reported and many share similar chemical properties [1], a significant interference of the signal could
Conclusion
We have successfully developed and validated a simple, sensitive and accurate HPLC-APCI-MS/MS method suitable for the bioavailability/pharmacokinetic study of WA following IV, IP and PO dosing. The bioavailability of oral WA is extremely low (about 0.58%), which suggests that WA is not likely to be effective when given orally in human subjects. Thus, it may not be suitable to be a relevant active marker for quality control of Tripterygium wilfordii for oral administration.
Acknowledgements
This work was supported by a bridge fund of Western University of Health Sciences (M. Chow). We thank Dr. Yilong Zhang (Ph.D) for his input in the pharmacokinetic model analysis.
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