Validation of high-performance liquid chromatography–tandem mass spectrometry assays quantifying omacetaxine mepesuccinate and its 4′‑des-methyl and cephalotaxine metabolites in human plasma and urine

doi:10.1016/j.jchromb.2015.08.015

Journal of Chromatography B

Volume 1002, 1 October 2015, Pages 152-159

https://doi.org/10.1016/j.jchromb.2015.08.015 Get rights and content

Highlights

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These assays were fully validated according to the latest FDA and EMA guidelines.
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Deuterated isotopes were used as internal standards.
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The assays were considered very suitable to support clinical pharmacologic studies of omacetaxine mepesuccinate.

Abstract

Since omacetaxine is mainly metabolised by esterases, the plasma samples were immediately stabilised after collection with an esterase inhibitor and stored at a nominal temperature of −80 °C. Urine samples were stored at −80 °C immediately after collection. Protein precipitation was applied as the sample pretreatment method for the plasma samples, and urine samples were processed using solid-phase extraction (SPE). For both assays, the dried and reconstituted extracts were injected on a XBridge BEH Phenyl column for analysis of all analytes. Gradient elution was applied with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionised using a turbospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer.

The validated plasma assay quantifies all analytes in the concentration range of 0.1–100 ng/mL and the urine assay in the range of 0.1–50 ng/mL. At all concentrations, the accuracies were within ±15% of the nominal concentrations and precisions were ≤15%. The developed methods have successfully been applied in a human mass balance study of omacetaxine.

Introduction

Omacetaxine mepesuccinate (hereafter called omacetaxine) is a modified cephalotaxine and is registered (Synribo^®) for the treatment of adult patients with chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs). Omacetaxine is a semi-synthetic product from the leaves of the Cephalotaxus fortunei, which is identical to the natural product homoharringtonine [1]. Homoharringtonine was first extracted from the bark of this tree, and the alcoholic extract showed effects on various types of acute leukemia by inhibition of protein synthesis. Subsequently a semi-synthetic process was developed to produce large quantities of highly purified omacetaxine [2], [3]. Omacetaxine is mainly metabolised by esterases into its inactive 4′-des-methyl(4′-DMHHT) and cephalotaxine metabolites.

To support clinical pharmacological studies an assay for quantification of omacetaxine and its metabolites is needed. Since the activity of Cephalotaxus alkaloid extracts such as homoharringtonine and cephalotaxine have been under investigation for approximately 40 years, several assay procedures have already been described using different detection methods [4], [5], [6], [7]. Only one method describes the quantification of 4′-DMHHT, but without bioanalytical details [8]. However, methods for quantitation of the parent drug and the two circulating metabolites in human plasma and urine were needed for use in support of a human mass balance study. Full validation of the developed assays was performed in compliance with the OECD principles of good laboratory practice (GLP) [9] and according to the FDA and latest EMA guidelines on bioanalytical method validation [10], [11]. Additionally, the applicability of the assay in clinical sample analysis was demonstrated.

Section snippets

Chemicals

Omacetaxine, 4′-DMHHT and cephalotaxine (Fig. 1) were manufactured by Novasep (Pompey, France) and provided by Teva Pharmaceuticals (North Wales, PA, USA). Deuterated stable isotope-labeled (²H₅) analogues of all compounds were used as internal standards, and these were manufactured by Chemtos (Austin, TX, USA) and provided by Teva Pharmaceuticals. Sigma–Aldrich (St. Louis, MO, USA) supplied paraoxon which served as esterase inhibitor to stabilise the analytes in plasma. Methanol (UPLC grade

Calibration model

Calibration standards were prepared and analysed in duplicate during three analytical runs. The linear regression of peak area ratios versus the concentrations was weighted 1/x² to obtain the lowest total bias over the range. The calibration range of omacetaxine, 4′-DMHHT and cephalotaxine in plasma was 0.1–100 ng/mL and in urine was 0.1–50 ng/mL. Calibration curves were accepted if 75% of the non-zero calibration standards and 50% of each calibration level, including a LLOQ and an ULOQ, had a

Conclusion

A sensitive LC–MS/MS assay for the quantification of omacetaxine, 4′-DMHHT and cephalotaxine in human plasma and urine was developed and validated. The validated range for all analytes in plasma is from 0.1 to 100 ng/mL using 100-μL plasma aliquots and in urine from 0.1 to 50 ng/mL using 400-μL urine aliquots. Dilution integrity experiments show that samples can be diluted 25-fold in control matrix prior to analysis. The extended concentration range for plasma is therefore from 0.1 to 2000 ng/mL

Footnote

This work was supported by Teva Branded Pharmaceutical R&D, Inc.

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Pharmacokinetic (PK) study of anticancer drugs in cancer patients is highly crucial for dose selection and dosing intervals in clinical applications. Once an anticancer drug is administered, it undergoes various metabolic pathways; to determine these pathways, it is necessary to follow the administered drug in biological samples via different analytical methods. In addition, multi-drug quantification methods in patients undergoing multi-drug regimens of cancer therapy can have several benefits, such as reduced sampling time and analysis costs. In order to collect and categorize these studies, we conducted a systematic review of HPLC methods reported for the analysis of anticancer drugs in biological samples. A systematic search was performed on PubMed Medline, Scopus, and Web of Science databases, and 116 studies were included. In summary of included studies, when the objective of a method was to quantify a single drug, MS, or UV detectors were utilized equivalently. On the other hand, in methods with the aim of quantifying drug and metabolite(s) in a single run, MS detectors were the most utilized. This review can provide a comprehensive insight for researchers prior to developing a quantification method and selecting a detector.
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At present, HHT substance and injection are only official in Chinese Pharmacopoeia [8] which describes an HPLC method for related substance test, and no specified impurity is listed. In the literature, only a few HPLC and HPLC-MS methods are available for determination of HHT in plant extracts, biological samples, pharmaceutical ingredient and dosage form [9–13]. He et al. have reported the structure identification of three HHT congeners as well as five degradation products under hydrolytic (acidic, basic, neutral) and thermal stress condition in HHT substances [9].
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Cephalotaxus alkaloids represent a family of plant secondary metabolites known for 60 years. Significant activity against leukemia in mice was demonstrated for extracts of Cephalotaxus. Cephalotaxine (CET) (1), the major alkaloid of this series was isolated from Cephalotaxus drupacea species by Paudler in 1963. The subsequent discovery of promising antitumor activity among new Cephalotaxus derivatives reported by Chinese, Japanese, and American teams triggered extensive structure elucidation and biological studies in this family. The structural feature of this cephalotaxane family relies mainly on its tetracyclic alkaloid backbone, which comprises an azaspiranic 1-azaspiro[4.4]nonane unit (rings C and D) and a benzazepine ring system (rings A and B), which is linked by its C3 alcohol function to a chiral oxygenated side chain by a carboxylic function alpha to a tetrasubstituted carbon center. The botanical distribution of these alkaloids is limited to the Cephalotaxus genus (Cephalotaxaceae). The scope of biological activities of the Cephalotaxus alkaloids is mainly centered on the antileukemic activity of homoharringtonine (HHT) (2), which in particular demonstrated marked benefits in the treatment of orphan myeloid leukemia and was approved as soon as 2009 by European Medicine Agency and by US Food and Drug Administration in 2012. Its exact mechanism of action was partly elucidated and it was early recognized that HHT (2) inhibited protein synthesis at the level of the ribosome machinery. Interestingly, after a latency period of two decades, the topic of Cephalotaxus alkaloids reemerged as a prolific source of new natural structures. To date, more than 70 compounds have been identified and characterized. Synthetic studies also regained attention during the past two decades, and numerous methodologies were developed to access the first semisynthetic HHT (2) of high purity suitable for clinical studies, and then high grade enantiomerically pure CET (1), HHT (2), and analogs.
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View full text

Validation of high-performance liquid chromatography–tandem mass spectrometry assays quantifying omacetaxine mepesuccinate and its 4′‑des-methyl and cephalotaxine metabolites in human plasma and urine

Highlights

Abstract

Introduction

Section snippets

Chemicals

Calibration model

Conclusion

Footnote

J. Chromatogr.

J. Pharm. Biomed. Anal.

J. Chromatogr.

Homoharringtonine: history, current research, and future direction

Cancer

Homoharringtonine for the treatment of chronic myelogenous leukemia

Expert Opin. Pharmacother.