Elsevier

Journal of Chromatography B

Volume 1053, 15 May 2017, Pages 20-26
Journal of Chromatography B

Determination of brusatol in plasma and tissues by LC–MS method and its application to a pharmacokinetic and distribution study in mice

https://doi.org/10.1016/j.jchromb.2017.04.012Get rights and content

Highlights

  • We developed a LC–MS method for simultaneous quantification of brusatol in plasma and tissues homogenate.

  • The plasma concentration of brusatol in mice decreased rapidly with the half-life time in 10 min.

  • Concentration of brusatol in plasma and tissues was both increased proportionally to doses.

  • A high concentration of brusatol was found in lung tissue.

Abstract

Objectives

The quassinoid brusatol, which can be isolated from Brucea javanica (L.) Merr., becomes popularly studied because of its anti-tumor activity. In order to further investigate brusatol and extend its applications, a sensitive analytical method for determination of brusatol in biological samples is essential. However, few methods had been reported until now. In this study, a highly sensitive and reproducible LC–MS method for simultaneous quantification of brusatol in mouse plasma and tissues was developed and validated.

Method

Plasma samples and tissue homogenate were extracted with diethyl ether after addition of the internal standard solution(IS). The supernatant was blown to dryness with nitrogen and residual was reconstituted with 100 μl of methanol. The separation was performed on an Intersil ODS-3 column and gradient elution was conducted with the mobile phase of water and methanol (0–5 min 47:53, 5–5.5 min 47:53–10:90, 5.5–9 min 10:90, posttime 4 min 47:53) at a flow rate of 0.8 mL/min. Quantification was performed in the selected ion monitoring (SIM) mode at m/z 543.2 for brusatol and 220.0 for IS (ornidazole). The method was validated by analyzing quality control plasma and tissue homogenate samples, and was applied to analyze samples obtained from mice after injections of brusatol via the tail vein.

Results

With ornidazole as the internal standard, calibration curve of the method ranged from 10 to 320 ng/ml for plasma and 10–240 ng/ml for tissues. Recovery rate of brusatol from plasma and tissues were between 71.09%–94.91%. Relative standard deviation (RSD) for inter- and intra-day precision was less than 15%, and the accuracy was between 96.1%–111.8%. The pharmacokinetics and distribution study of brusatol in mice after three single doses via the tail vein were carried out based on this method. The concentration of brusatol in plasma decreased rapidly and a more than 10 fold concentration of brusatol was found as compared to that in other tissues.

Conclusions

This is the first reported LC–MS method for detecting brusatol in tissues and can accurately determine the concentrations of these compounds in plasma and different tissues. Further research on the metabolism of brusatol in vivo is still needed.

Introduction

Brucea javanica (L.) Merr. belongs to the Simaroubaceae specie and is widely distributed in Southeast Asia and northern Australia [1]. Brucea javanica oil is extracted from the seed of this herb and has been used for treating various diseases including cancer, amoebic dysentery and malaria [2], [3]. In recent decades, phytochemical and biological activities of B.javanica were studied and many classes of constituents were separated, including quassinoids, triterpenoids, alkaloids, lignans and flavonoids. Quassinoids, include bruceine A, B, C, D, E, F, G, H, bruceantin, brusatol, bruceoside, brucamarin, brucedic acid, and etc., are the main anti-tumor ingredients of Brucea javanica [3].

The quassinoid brusatol, first isolated from the seeds of B.javanica in 1968[4], was confirmed to have various pharmacological effects of anticancer, antiprotozoal, anti-inflammatory, antiphytovirus, and antifedant [5], [6], [7], [8], [9]. The mechanism of anticancer effect was well studied, which include inhibition of protein synthesis, activation of NF-kB pathway, down-regulation of C-myc protein level, and inhibition of Nrf2 pathway by promoting ubiquitination [10], [11], [12], [13]. Nrf2 (Nuclear factor-E2-related factor 2) is an important factor in antioxidant response. It is controlled by Keap1 and regulates the expression of antioxidant protein and phase II detoxification enzyme through the antioxidant response element (ARE) [14], [15]. Recently, Nrf2 has been confirmed as a double-edged sword: it can suppress tumorigenesis in normal tissues while also can promotes tumor growth and enhances chemoresistance in tumor tissues [16]. Large numbers of cell researches and animal experiments have revealed that the pro-tumorigenic effect of accumulated Nrf2 in elevating the level of detoxifying enzyme in tumor cells, which enhance their resistance to chemotherapy [17], [18], [19]. Moreover, the latest study has identified brusatol as a unique inhibitor of Nrf2 pathway, which can selectively reduce the protein level of Nrf2 through stimulated ubiquitination and protelysis. Therefore, brusatol shows the activity of reducing tumor burden and ameliorating chemoresistance in both in vitro and in vivo models. Combination of brusatol and cisplatin in lung cancer cell line can significantly reduce cell proliferation and inhibit tumor growth when compared with cisplatin treatment alone. However, no cytotoxicity of brusatol was observed [13], [20]. This result indicates that brusatol has great potential to be developed into novel chemotherapy drug, and makes brusatol a hot topic of research.

To improve the development of brusatol, it is essential to develop a sensitive and reproducible analytical method that can be used in the pharmacokinetics and metabolism study. Early reported methods were rarely and companied with sever disadvantages like long run time, insufficient sensitivity, and not well validated [21], [22]. Moreover, no method for the quantification of brusatol in tissues has been described at present. In current study, a sensitive LC–MS method was established and validated for the quantification of brusatol in both plasma and tissues, and successfully used in pharmacokinetics and distribution studies in mice after three different single doses via the tail vein.

Section snippets

Chemicals and reagents

The reference standard of brusatol (purity >97%) was purchased from Shanghai Tauto Biotech Co., Ltd; ornidazole was purchased from Nanjing Sanhome Pharmaceutical Co., Ltd; methanol and ethyl acetate (HPLC grade) were obtained from J.T.Baker (USA); pure water was from Hangzhou Wahaha Group Co., Ltd.

Animals and drug administration

180 Kunming mice (18–22 g), half male and half female, were supplied by the Center of Laboratory Animal Service, Shandong University. The animal experiment protocols were in accordance with the

Method development

For the separation of brusatol and IS, the mobile phase was optimized. At the beginning, we chose methanol and water at a constant ratio of 53:47. Interferences was not found during the runtime, but appeared in the followed sample. Therefore gradient elution was used for washing out the interference. The mass spectrometric parameters were tuned in both positive and negative ion mode, better responses of brusatol and ornidazole were found in the positive mode. The fragment electric voltage for

Conclusion

Brusatol was identified as a unique inhibitor of the Nrf2 pathway and has great potential to be a novel adjuvant chemotherapeutic drug. In this study, a highly sensitive, accurate and reproducible LC–MS method for simultaneous quantification of brusatol in plasma and tissues was developed and validated. Pharmacokinetic and distribution studies were carried out by using this method. The plasma concentration of brusatol decreased rapidly with the half-life time in 10 min; the distribution of

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgement

This study was financially supported by Scientific Development Plan of Shandong Province (2013GSF11864).

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