Bioanalytical method for quantitative determination of mithramycin analogs in mouse plasma by HPLC–QTOF

Joseph M. Eckenrode; Prithiba Mitra; Jürgen Rohr; Markos Leggas

doi:10.1002/bmc.4544

Bioanalytical method for quantitative determination of mithramycin analogs in mouse plasma by HPLC–QTOF

Joseph M. Eckenrode, Prithiba Mitra, Jürgen Rohr, Markos Leggas

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of <20%. MTMSA-Trp was stable for 30 days at −80°C and for at least three freeze–thaw cycles. Methanol (−80°C) extraction afforded 92% of MTMSA-Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.

Original language	English
Article number	e4544
Journal	Biomedical Chromatography
Volume	33
Issue number	8
DOIs	https://doi.org/10.1002/bmc.4544
State	Published - Aug 2019

Bibliographical note

Funding Information:
This work was supported by the United States Department of Defense grant number PC150300P1 (W81XWH-16-1-0477) to M.L. and J.R. We acknowledge the Markey Cancer Center, Dance Blue Foundation, and UK Center for Pharmaceutical Research Innovation for their resources and financial support. The authors acknowledge University of Kentucky staff member, Dr Jamie Horn PhD, for her assistance with LC/QTOF training and Reiya Hayden for assistance with plasma protein binding experiments.

Funding Information:
This work was supported by the United States Department of Defense grant number PC150300P1 (W81XWH‐16‐1‐0477) to M.L. and J.R. We acknowledge the Markey Cancer Center, Dance Blue Foundation, and UK Center for Pharmaceutical Research Innovation for their resources and financial support.

Publisher Copyright:
© 2019 John Wiley & Sons, Ltd.

Keywords

Ewing sarcoma
bioanalytical
mass spectrometry
method validation
mithramycin

ASJC Scopus subject areas

Analytical Chemistry
Biochemistry
Molecular Biology
Pharmacology
Drug Discovery
Clinical Biochemistry

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title = "Bioanalytical method for quantitative determination of mithramycin analogs in mouse plasma by HPLC–QTOF",

abstract = "Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of <20%. MTMSA-Trp was stable for 30 days at −80°C and for at least three freeze–thaw cycles. Methanol (−80°C) extraction afforded 92% of MTMSA-Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.",

keywords = "Ewing sarcoma, bioanalytical, mass spectrometry, method validation, mithramycin",

author = "Eckenrode, {Joseph M.} and Prithiba Mitra and J{\"u}rgen Rohr and Markos Leggas",

note = "Funding Information: This work was supported by the United States Department of Defense grant number PC150300P1 (W81XWH-16-1-0477) to M.L. and J.R. We acknowledge the Markey Cancer Center, Dance Blue Foundation, and UK Center for Pharmaceutical Research Innovation for their resources and financial support. The authors acknowledge University of Kentucky staff member, Dr Jamie Horn PhD, for her assistance with LC/QTOF training and Reiya Hayden for assistance with plasma protein binding experiments. Funding Information: This work was supported by the United States Department of Defense grant number PC150300P1 (W81XWH‐16‐1‐0477) to M.L. and J.R. We acknowledge the Markey Cancer Center, Dance Blue Foundation, and UK Center for Pharmaceutical Research Innovation for their resources and financial support. Publisher Copyright: {\textcopyright} 2019 John Wiley & Sons, Ltd.",

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N2 - Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of <20%. MTMSA-Trp was stable for 30 days at −80°C and for at least three freeze–thaw cycles. Methanol (−80°C) extraction afforded 92% of MTMSA-Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.

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Bioanalytical method for quantitative determination of mithramycin analogs in mouse plasma by HPLC–QTOF

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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