Separation and assay of 14C-labeled glyceryl trinitrate and its metabolites by OPLC coupled with on-line of off-line radioactivity detection

I. Klebovich, György Morovján, István Hazai, E. Mincsovics

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12 Citations (Scopus)

Abstract

Glyceryl trinitrate (nitroglycerin), useful in the prevention and treatment of acute anginal attacks characteristic of ischemic heart disease, has been used as a model compound to evaluate the applicability of overpressured layer chromatography (OPLC) coupled with several radioactivity detection methods in metabolism research. Radioactivity detection methods studied included digital autoradiography (DAR), flow-cell solid scintillation radioactivity detection (RD), and the phosphor-imaging technique. After treatment of rats with 14C-labeled glyceryl trinitrate, glyceryl trinitrate and its metabolites 1,2- and 1,3-glyceryl dinitrate and 1- and 2-glyceryl mononitrate were isolated from plasma by liquid-liquid extraction. OPLC separation of metabolites and glyceryl trinitrate was performed on fine-particle silica gel-coated TLC plates with acetonitrile-dibutyl ether mixtures as mobile phases and stepwise gradient elution. Detection of radiocarbon-labeled parent drug and its metabolites was performed off-line by digital autoradiography and the phosphor-imaging technique. On-line detection was performed by flow-cell solid scintillation radioactivity detection. OPLC coupled with RD combines the selectivity of radiochemical detection with the separation efficiency of overpressured liquid chromatography (OPLC); this is of great importance during metabolite screening and isolation. After sample extraction and application to the plate the selectivity of the system could be optimized. The optimization could be monitored by DAR, which could be chosen as detection method for the off-line analysis of multiple samples simultaneously separated on a single plate. Isolation of separated sample constituents could be performed after applying the sample as a single band to the plate and elution of metabolites of interest followed by their detection by use of on-line RD. Strongly retained compounds could be detected by DAR and removed mechanically, with the adsorbent, and extracted with a strong solvent. The recently developed on-line OPLC-RD method and the previously reported OPLC-DAR and HPLC-RD techniques are rapid, economic, and effective separation systems for application in metabolism research.

Original languageEnglish
Pages (from-to)404-409
Number of pages6
JournalJournal of Planar Chromatography - Modern TLC
Volume15
Issue number6
DOIs
Publication statusPublished - Nov 2002

Fingerprint

Radioactivity
Nitroglycerin
Metabolites
Chromatography
Assays
Autoradiography
Scintillation
Metabolism
Phosphors
Imaging techniques
Liquid-Liquid Extraction
Silica Gel
Liquid chromatography
Liquids
Research
Liquid Chromatography
Adsorbents
Myocardial Ischemia
Rats
Screening

Keywords

  • Digital autoradiography (DAR)
  • Glyceryl trinitrate
  • HPLC-radioactivity detection (HPLC-RD)
  • Hyphenated techniques
  • Metabolism study
  • On-line OPLC-radioactivity detection (OPLC-RD)
  • Phosphor imaging/bioimaging

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

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title = "Separation and assay of 14C-labeled glyceryl trinitrate and its metabolites by OPLC coupled with on-line of off-line radioactivity detection",
abstract = "Glyceryl trinitrate (nitroglycerin), useful in the prevention and treatment of acute anginal attacks characteristic of ischemic heart disease, has been used as a model compound to evaluate the applicability of overpressured layer chromatography (OPLC) coupled with several radioactivity detection methods in metabolism research. Radioactivity detection methods studied included digital autoradiography (DAR), flow-cell solid scintillation radioactivity detection (RD), and the phosphor-imaging technique. After treatment of rats with 14C-labeled glyceryl trinitrate, glyceryl trinitrate and its metabolites 1,2- and 1,3-glyceryl dinitrate and 1- and 2-glyceryl mononitrate were isolated from plasma by liquid-liquid extraction. OPLC separation of metabolites and glyceryl trinitrate was performed on fine-particle silica gel-coated TLC plates with acetonitrile-dibutyl ether mixtures as mobile phases and stepwise gradient elution. Detection of radiocarbon-labeled parent drug and its metabolites was performed off-line by digital autoradiography and the phosphor-imaging technique. On-line detection was performed by flow-cell solid scintillation radioactivity detection. OPLC coupled with RD combines the selectivity of radiochemical detection with the separation efficiency of overpressured liquid chromatography (OPLC); this is of great importance during metabolite screening and isolation. After sample extraction and application to the plate the selectivity of the system could be optimized. The optimization could be monitored by DAR, which could be chosen as detection method for the off-line analysis of multiple samples simultaneously separated on a single plate. Isolation of separated sample constituents could be performed after applying the sample as a single band to the plate and elution of metabolites of interest followed by their detection by use of on-line RD. Strongly retained compounds could be detected by DAR and removed mechanically, with the adsorbent, and extracted with a strong solvent. The recently developed on-line OPLC-RD method and the previously reported OPLC-DAR and HPLC-RD techniques are rapid, economic, and effective separation systems for application in metabolism research.",
keywords = "Digital autoradiography (DAR), Glyceryl trinitrate, HPLC-radioactivity detection (HPLC-RD), Hyphenated techniques, Metabolism study, On-line OPLC-radioactivity detection (OPLC-RD), Phosphor imaging/bioimaging",
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T1 - Separation and assay of 14C-labeled glyceryl trinitrate and its metabolites by OPLC coupled with on-line of off-line radioactivity detection

AU - Klebovich, I.

AU - Morovján, György

AU - Hazai, István

AU - Mincsovics, E.

PY - 2002/11

Y1 - 2002/11

N2 - Glyceryl trinitrate (nitroglycerin), useful in the prevention and treatment of acute anginal attacks characteristic of ischemic heart disease, has been used as a model compound to evaluate the applicability of overpressured layer chromatography (OPLC) coupled with several radioactivity detection methods in metabolism research. Radioactivity detection methods studied included digital autoradiography (DAR), flow-cell solid scintillation radioactivity detection (RD), and the phosphor-imaging technique. After treatment of rats with 14C-labeled glyceryl trinitrate, glyceryl trinitrate and its metabolites 1,2- and 1,3-glyceryl dinitrate and 1- and 2-glyceryl mononitrate were isolated from plasma by liquid-liquid extraction. OPLC separation of metabolites and glyceryl trinitrate was performed on fine-particle silica gel-coated TLC plates with acetonitrile-dibutyl ether mixtures as mobile phases and stepwise gradient elution. Detection of radiocarbon-labeled parent drug and its metabolites was performed off-line by digital autoradiography and the phosphor-imaging technique. On-line detection was performed by flow-cell solid scintillation radioactivity detection. OPLC coupled with RD combines the selectivity of radiochemical detection with the separation efficiency of overpressured liquid chromatography (OPLC); this is of great importance during metabolite screening and isolation. After sample extraction and application to the plate the selectivity of the system could be optimized. The optimization could be monitored by DAR, which could be chosen as detection method for the off-line analysis of multiple samples simultaneously separated on a single plate. Isolation of separated sample constituents could be performed after applying the sample as a single band to the plate and elution of metabolites of interest followed by their detection by use of on-line RD. Strongly retained compounds could be detected by DAR and removed mechanically, with the adsorbent, and extracted with a strong solvent. The recently developed on-line OPLC-RD method and the previously reported OPLC-DAR and HPLC-RD techniques are rapid, economic, and effective separation systems for application in metabolism research.

AB - Glyceryl trinitrate (nitroglycerin), useful in the prevention and treatment of acute anginal attacks characteristic of ischemic heart disease, has been used as a model compound to evaluate the applicability of overpressured layer chromatography (OPLC) coupled with several radioactivity detection methods in metabolism research. Radioactivity detection methods studied included digital autoradiography (DAR), flow-cell solid scintillation radioactivity detection (RD), and the phosphor-imaging technique. After treatment of rats with 14C-labeled glyceryl trinitrate, glyceryl trinitrate and its metabolites 1,2- and 1,3-glyceryl dinitrate and 1- and 2-glyceryl mononitrate were isolated from plasma by liquid-liquid extraction. OPLC separation of metabolites and glyceryl trinitrate was performed on fine-particle silica gel-coated TLC plates with acetonitrile-dibutyl ether mixtures as mobile phases and stepwise gradient elution. Detection of radiocarbon-labeled parent drug and its metabolites was performed off-line by digital autoradiography and the phosphor-imaging technique. On-line detection was performed by flow-cell solid scintillation radioactivity detection. OPLC coupled with RD combines the selectivity of radiochemical detection with the separation efficiency of overpressured liquid chromatography (OPLC); this is of great importance during metabolite screening and isolation. After sample extraction and application to the plate the selectivity of the system could be optimized. The optimization could be monitored by DAR, which could be chosen as detection method for the off-line analysis of multiple samples simultaneously separated on a single plate. Isolation of separated sample constituents could be performed after applying the sample as a single band to the plate and elution of metabolites of interest followed by their detection by use of on-line RD. Strongly retained compounds could be detected by DAR and removed mechanically, with the adsorbent, and extracted with a strong solvent. The recently developed on-line OPLC-RD method and the previously reported OPLC-DAR and HPLC-RD techniques are rapid, economic, and effective separation systems for application in metabolism research.

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KW - HPLC-radioactivity detection (HPLC-RD)

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KW - Metabolism study

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KW - Phosphor imaging/bioimaging

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