Mark Marzinke (Presenter)
Johns Hopkins University School of Medicine
Bio: Dr. Marzinke is an Assistant Professor of Pathology and Medicine in the Johns Hopkins University School of Medicine. He is the Director of Preanalytics and General Chemistry areas of the Core Laboratories of the Johns Hopkins Hospital and the Director of the Clinical Pharmacology Analytical Laboratory in the Division of Clinical Pharmacology. Dr. Marzinke received his B.A. degree from the College of the Holy Cross with a major in Biology, minor in Chemistry and a focused concentration in Biochemistry. He received his Ph.D. in Biochemistry from the University of Wisconsin-Madison in 2010, followed by a post-doctoral fellowship in Clinical Chemistry at the Johns Hopkins University School of Medicine, which he completed in 2012. He subsequently joined the faculty in the Departments of Pathology and Medicine. Dr. Marzinke is board certified in Clinical Chemistry by the American Board of
Authorship: Mark Marzinke and Pamela Hummert
Johns Hopkins University School of Medicine, 600 N. Wolfe St., Osler 501, Baltimore MD 21287
In order to better understand the pharmacokinetic-pharmacodynamic (PK-PD) relationships of anti-tuberculosis drugs in disease management, PK studies are required to assess drug concentrations. Thus, liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods for the multiplexed quantification of the common anti-TB medications rifampin (RIF), pyrazinamide (PZA), ethambutol (EMB), as well as the fourth-generation antibacterial agent moxifloxacin (MOX) and the experimental drug PA-824 in plasma have been developed and validation according to the recommendations of the FDA, Guidance for Industry: Bioanalytical Method Validation document. Further, the described work illustrates the bioanalytical considerations in working with these compounds.
Introduction: Tuberculosis, an infection caused by Mycobacterium tuberculosis, is associated with significant disease burden. In 2013, it was estimated that there were 9 million new cases of TB and 1.5 million TB-related deaths worldwide . Current treatment modalities include combinatorial therapies that are associated with 6-month treatment regimens and significant drug-related toxicities, including hepatotoxicity, neuropathy, ophthalmic toxicity, gastrointestinal abnormalities and rash. In an effort to better characterize traditional TB regimens in different dosing regimens, as well as assess experimental anti-TB drugs with improved toxicity profiles, analytical methods are required for drug quantification. This work describes the multiplexed quantification of the common anti-TB medications rifampin (RIF), pyrazinamide (PZA), ethambutol (EMB), as well as the fourth-generation antibacterial agent moxifloxacin (MOX) and the experimental drug PA-824, in plasma, via liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Methods: Blank human K2EDTA plasma was obtained from Biology Specialty Corporation (Colmar, PA). EMB, PZA, MOX, RIF, and the RIF structural analog rifamixin-d6, were all obtained from Toronto Research Chemicals (Toronto, Ontario). PA-824 was supplied via a collaboration with Dr. Eric Neuremberger at Johns Hopkins University. The antiprotozoal drug carnidazole was used an internal standard for PA-824 and was also acquired from Toronto Research Chemicals. Drugs of interest were isolated via protein precipitation. For sample analysis, 0.1 mL of plasma was combined with 0.05 mL of an internal standard mixture and 1 mL of methanol (MeOH) containing 0.1% formic acid. Following vortexing and centrifugation, supernatants were evaporated to dryness and reconstituted in a water:methanol mixture containing ascorbic acid. Samples were separated under a gradient elution at room temperature on a Kinetic C18 column, 2.1 x 50 mm, 2.6 micron column (Phenomenex Technologies, Torrance, CA). Analytes were detected on an API 4000 triple quadrupole mass spectrometer (AB SCIEX, CA, USA) with an ESI source operated in positive ionization mode. Analytes were monitored in selective reaction monitoring (SRM) mode. The assays were validated in accordance with the Food and Drug Administration (FDA) Guidance for Industry, Bioanalytical Method Validation guidelines .
Results: The analytical measuring ranges for this assay were as follows: 50 – 7500 ng/mL (EMB), 500 – 50000 ng/mL (PZA), 50 – 5000 ng/mL (MFX), 50 – 20000 ng/mL (RIF), and 50 – 5000 ng/mL (PA-824). The total analytical run time was 3.0 minutes. Analytical metrics assessed included both intra- and inter-assay precision and accuracy, dilutional analysis studies, stability challenges in response to freeze-thaw cycles, post-extraction and in sample matrix, as well as matrix effects analysis and selectivity studies. Quality control (QC) samples prepared at the lower limit of quantitation, as well as low, mid and high QC levels yielded intra- and inter-assay coefficients of variation (%CVs) and percent deviations (%DEV) were within acceptable limits for all compounds (≤ 20% at the lower limit of quantification, ≤ 15% for other QC levels). Dilutional analysis studies demonstrate that for drug concentrations above the limit of quantification, samples can be diluted and quantified, and observed concentrations are within 15% of theoretical concentrations in both specimen sources for both analytes. All analytes are stable following three freeze thaw cycles on the polyester-tipped swab, for three days in sample matrix maintained at room temperature, and for 3 days post-extraction in water containing 0.5% acetic acid. Acceptability criteria include ≤ 15% deviation from QC samples immediately prepared and analyzed. Finally to assess the potential influence of endogenous compounds on analyte suppression or enhancement, matrix effects studies were performed following the guidelines of Matuszewski and colleagues . Both PZA and EMB exhibited substantial ion suppression for both the analyte of interest as well as the internal standard. RIF showed slight ion enhancement, while there were negligible matrix effects for PA-824 and MOX.
Conclusions: The described work illustrates the development and validation of a multiplexed method for the quantification of several current and experimental anti-TB drugs used in the management of a Mycobacterium tuberculosis infection. The validated LC-MS/MS method described herein have been used in completed and ongoing clinical trials aimed at better understanding the pharmacokinetics of these drug regimens.
References & Acknowledgements:
1. World Health Organization. 2014. Global tuberculosis report 2014. Report WHO/HTM/TB/2014.08. World Health Organization, Geneva, Switzerland.
2. US Department of Health and Human Services UF, Center for Drug Evaluation and Research, Center for Veterinary Medicine, Guidance for industry: bioanalytical method validation. FDA, Rockville, MD, USA (2001)
3. B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng. Matrix effect in quantitative LC/MS/MS analyses of biological fluids: a method for determination of finasteride in human plasma at pictogram per milliliter concentrations. Anal Chem, 70 (1998), pp. 882–889
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