Stability of Drugs, Drug Candidates, and Metabolites in Blood and Plasma

Gregory A. Reed1

1 Department of Pharmacology, Toxicology, and Therapeutics and the University of Kansas Cancer Center, Kansas City, Kansas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.6
DOI:  10.1002/cpph.16
Online Posting Date:  December, 2016
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Abstract

Determination of drug or drug metabolite concentrations in biological samples, particularly in serum or plasma, is fundamental to describing the relationships between administered dose, route of administration, and time after dose for achieving the optimal clinical response. While a well‐characterized, accurate analytical method is needed to define these parameters, it must also be established that the analyte concentration in the sample at the time of analysis is identical to the concentration at sample acquisition. This is necessitated by the fact that drugs and their metabolites are susceptible to degradation in samples due to metabolism or to physical and chemical processes, resulting in a lower measured concentration than was in the original sample. Careful examination of analyte stability during processing and storage and, if necessary, adjustment of procedures and conditions to maximize stability, are a critical component of method validation to ensure the accuracy of the data. The protocols provided in this unit address the stability of the analytes in whole blood and blood‐derived samples prior to sample preparation for analysis. Issues addressed include sample acquisition, processing of whole blood, and storage of blood‐derived samples. © 2016 by John Wiley & Sons, Inc.

Keywords: drug stability; bioanalytical method validation; plasma stability

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Drug and Drug‐Candidate Metabolite Stability in Whole Blood
  • Basic Protocol 2: Long‐Term Drug and Drug Candidate Metabolite Stability in Frozen Plasma
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Drug and Drug‐Candidate Metabolite Stability in Whole Blood

  Materials
  • 10 mg/ml tetrahydrouridine (THU; Sigma‐Aldrich, cat. no. 584222‐10MG) in H 2O
  • 25 ml whole blood with sodium heparin (drug‐free, heparinized, blood is available from several commercial sources, or expired donated blood may be obtained locally through blood banks)
  • Spiking solutions drug and drug‐candidate metabolite stability in whole blood, low (25 μg/ml) and high (500 μg/ml) analyte concentration (see recipe)
  • 42 2‐ml microcentrifuge tubes labeled for whole blood samples
  • 42 2‐ml microcentrifuge tubes labeled for plasma samples
  • Timer
  • Ice bath for 0°C samples
  • Racks or 25°C heating block for microcentrifuge tubes with ambient temperature samples
  • Refrigerated microcentrifuge
  • Freezer for plasma storage (preferably −70°C or below, but at least −20°C)
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Figures

Videos

Literature Cited

  Aimone, L.D. and de Lannoy, I.A. 2014. Overview of pharmacokinetics. Curr. Protoc. Pharmacol 66:7.1.1‐7.1.31. doi: 10.1002/0471141755.ph0701s66.
  Briscoe C.J. and Hage, D.S. 2009. Factors affecting the stability of drugs and drug metabolites in biological matrices. Bioanalysis 1:205‐220. doi: 10.4155/bio.09.20.
  Garofolo, F., Rocci, M.L., Dumont, I., Martinez, S., Lowes, S., Woolf, E., van Amsterdam, P., Bansai, S., Barra, A.C.G., Bauer, R., Booth, B.P., Carrasco‐Triguero, M., DeSilva, B., Gallicano, K., Gouty, D., Ho, S., Hucker, R., Jemal, M., Katoril, N., Le Blaye, O., Lee, J., Li, W., Michael, S., Nehls, C., Nicholson, R., Ormsby, E., Tang, D., Viswanathan, C.T., Weiner, R., and Young, G. 2011. 2011 White paper on recent issues in bioanalysis and regulatory findings from audits and inspections. Bioanalysis 3:2081‐2096. doi: 10.4155/bio.11.192.
  Marangon, E., Sala, F., Caffo, O., Galligioni, E., D'Incalci, M., and Zucchetti, M. 2008. Simultaneous determination of gemcitabine and its main metabolite, dFdU, in plasma of patients with advanced non‐small‐cell lung cancer by high‐performance liquid chromatography‐tandem mass spectrometry. J. Mass. Spectrom. 43:216‐223. doi: 10.1002/jms.1293.
  Shord, S.S., Faucette, S.R., Gillenwater, H.H., Pescatore, S.L., Hawke, R.L., Socinski, M.A., and Lindley, C. 2003. Gemcitabine pharmacokinetics and interaction with paclitaxel in patients with advanced non‐small‐cell lung cancer. Cancer Chemother. Pharmacol. 51:328‐336. doi: 10.1007/s00280‐002‐0560‐1.
  Tirona, R.G. and Pang, K.S. 1999. Bimolecular glutathione conjugation kinetics of ethacrynic acid in rat liver: In vitro and perfusion studies. J. Pharmacol. Exp. Therap. 290:1230‐1241.
Internet Resources
  http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm064964.htm
  FDA, 2013. Bioanalytical method validation: Draft guidance.
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