Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC-MS assay for delayed analyses of pharmacokinetic samples in rats

Jessica M. Jones, Michael D. Raleigh, Paul R Pentel, Theresa M. Harmon, Daniel E. Keyler, Rory P Remmel, Angela K Birnbaum

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain homogenate, bovine serum, and human plasma under various conditions. Analyte concentrations were measured by LC-MS. The goal was to identify conditions that allow maximum flexibility in scheduling sample collection and analysis, as well as gain more information on the stability of heroin in blood and tissue samples. A solid-phase extraction method with ice-cold solvents, sodium fluoride (NaF) and a low pH (3.0) maintained sample stability. Quality controls were within 94.0-105% of the target value. Variability was 4.0-8.9% for all analytes within the range of 5-200. ng/mL for heroin, 5-1000. ng/mL for 6-MAM, and 10-200. ng/mL for morphine. Heroin degradation to 6-MAM was faster in rat whole blood than in plasma, and faster in rat plasma than in rat brain homogenate. Maintaining NaF at 4. mg/mL throughout processing enhanced stability; higher NaF concentrations added to whole blood caused hemolysis. Samples processed through solid phase extraction and stored as dried pellets at 80. °C constituted the most stable environment for heroin, and was superior to the storing of samples in solution prior to or after extraction. Nevertheless, post-extraction heroin and 6-MAM levels declined by 6.7-8.3% over one week in rat plasma under these conditions, and by <1-4.7% in bovine serum or human plasma.

Original languageEnglish (US)
Pages (from-to)291-297
Number of pages7
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume74
DOIs
StatePublished - Feb 3 2013

Fingerprint

Pharmacokinetics
Heroin
Morphine
Rats
Assays
Blood
Plasma (human)
Degradation
Solid Phase Extraction
Plasmas
Brain
6-O-monoacetylmorphine
Sodium Fluoride
Ice
Metabolites
Hemolysis
Serum
Quality Control
Quality control
Scheduling

Keywords

  • Assay validation
  • Heroin
  • Morphine
  • Rat
  • Stability

Cite this

Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC-MS assay for delayed analyses of pharmacokinetic samples in rats. / Jones, Jessica M.; Raleigh, Michael D.; Pentel, Paul R; Harmon, Theresa M.; Keyler, Daniel E.; Remmel, Rory P; Birnbaum, Angela K.

In: Journal of Pharmaceutical and Biomedical Analysis, Vol. 74, 03.02.2013, p. 291-297.

Research output: Contribution to journalArticle

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abstract = "Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain homogenate, bovine serum, and human plasma under various conditions. Analyte concentrations were measured by LC-MS. The goal was to identify conditions that allow maximum flexibility in scheduling sample collection and analysis, as well as gain more information on the stability of heroin in blood and tissue samples. A solid-phase extraction method with ice-cold solvents, sodium fluoride (NaF) and a low pH (3.0) maintained sample stability. Quality controls were within 94.0-105{\%} of the target value. Variability was 4.0-8.9{\%} for all analytes within the range of 5-200. ng/mL for heroin, 5-1000. ng/mL for 6-MAM, and 10-200. ng/mL for morphine. Heroin degradation to 6-MAM was faster in rat whole blood than in plasma, and faster in rat plasma than in rat brain homogenate. Maintaining NaF at 4. mg/mL throughout processing enhanced stability; higher NaF concentrations added to whole blood caused hemolysis. Samples processed through solid phase extraction and stored as dried pellets at 80. °C constituted the most stable environment for heroin, and was superior to the storing of samples in solution prior to or after extraction. Nevertheless, post-extraction heroin and 6-MAM levels declined by 6.7-8.3{\%} over one week in rat plasma under these conditions, and by <1-4.7{\%} in bovine serum or human plasma.",
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