Short communicationDetermination of the cyclic depsipeptide FK228 in human and mouse plasma by liquid chromatography with mass-spectrometric detection
Introduction
FK228 (romidepsin, formerly FR 901228, NSC 630176; (E)-(1S, 4S, 10S, 21R)-7-[(Z)-ethylidene]4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]tricos-16-ene-3,6,9,19,22-pentanone;) (Fig. 1) is a naturally occurring bicyclic tetrapeptide that was first isolated from Chromobacterium violaceum by Fujisawa Company [1], [2]. This agent is a novel histone deacetylase inhibitor that has demonstrated potent cytotoxic activity against human tumor cell lines and in vivo efficacy against both human tumor xenografts and murine tumors [3], [4]. Multiple phase I and phase II clinical trials of FK228 have been initiated at the National Cancer Institute [5], [6], [7].
An analytical method was previously developed and validated in our laboratory for the quantitation of FK228 in human plasma, in support of multiple clinical trials [8]. However, the lack of internal standard became a major concern in daily practice, since the assay was being used long-term to analyze large numbers of samples. Furthermore, the original assay was not suitable for analysis of preclinical mouse samples. These animal experiments require a reliable analytical method that only uses 100 μL plasma or less, as compared to the 500 μL that was required in the previous method. In addition, the original assay was not suitable for the analysis of samples following higher doses of the drug. As exposure increased, due to the rapid dose escalation design of the study, the original upper limit of quantitation (ULOQ), 100 ng/mL, was no longer sufficient. Attempts at extending the range failed due to signal fluctuation without the application of an internal standard. Due to these major limitations, we report here an improved analytical method for determination of FK228 concentration in human and mouse plasma based on liquid chromatography coupled with single-quadrupole mass-spectrometric detection.
Section snippets
Chemicals
FK228 was supplied by Pharmaceutical Management Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, NCI (Bethesda, MD, USA). Internal standard, harmine, was purchased from Sigma–Aldrich (St. Louis, MO, USA). Formic acid (98%) was obtained from Fluka (through Sigma–Aldrich, St. Louis, MO, USA). Ethyl acetate (Fisher Scientific, Fairlawn, NJ, USA) and methanol (J.T. Baker, Phillipsburg, NJ, USA) are of HPLC grade. Deionized water was generated with a
Specificity
Due to the previously reported difficulty in finding a suitable internal standard with similar chemical structure and properties [8], a wider variety of different compounds were tested, including Boc-Met-Leu-Phe-OH and 6-methylprednisolone. Finally, harmine (Fig. 1) was chosen. Fig. 2 displays typical chromatograms of an extract of a blank human plasma sample (A), and an extract of a plasma sample spiked with IS and FK228 at a concentration of 2 ng/mL (B). The selectivity of the analysis is
Application
The validated method was then applied to study the pharmacokinetics of FK228 in patients on NCI initiated phase I and phase II clinical trials, as well as on additional preclinical mouse studies. Fig. 3 shows the observed plasma concentration time profile of FVB mice that received 3.6 mg/kg of FK228 as an I.V. bolus. Each data point was the average from three individual mice. A typical chromatogram of a human patient sample is presented in Fig. 2(C) with a calculated concentration of 777.72 ng/mL.
Conclusion
In conclusion, a novel chromatographic method with mass-spectrometric detection has been developed and validated for the quantitative determination of FK228 in human and mouse plasma. This method is specific, accurate and precise, and can be easily implemented into routine practice. Compared to our previously published LC/MS method [8], the addition of IS greatly reduced assay variability, allowing for the reduction of plasma volume from 500 to 100 μL, which made it possible to apply this method
Acknowledgements
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400 (ER Gardner). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
This research was supported by the Intramural Research Program of the NIH,
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