On-line solid-phase extraction capillary electrophoresis for enhanced detection sensitivity and selectivity: application to the analysis of metallothionein isoforms in sheep fetal liver

C B Knudsen, J H Beattie

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    50 Citations (Scopus)

    Abstract

    A simple and rapid capillary electrophoresis (CE) method for analysis of metallothionein (MT) isoforms is described. A modified two-step solvent extraction procedure was used in combination with CE and an on-line solid-phase preconcentration device for sensitive and reproducible detection of MT isoforms in sheep fetal liver. Preparation of twenty samples was practicable within a working day with subsequent automated overnight analysis by solid-phase extraction (SPE)-CE. A commercially available divinylbenzene-based reversed-phase resin was found to be most suitable for the SPE device because it is resistant to extremes of pH and can be adequately regenerated between analyses. Each SPE device was readily constructed from commonly available materials and was used for the reproducible separation of over 100 biological samples before replacement. Precision of analysis within or between sample batches was <10% and usually <5% while detection limits were at least 28 ng/ml for standards and 272 ng/ml for biological samples. This would indicate a detection limit of about 0.5 mu g/g wet weight of tissue. Recovery of MT from tissue cytosols by solvent extraction was measured using radiolabeled MT and was found to be just over 50% increasing to almost 70% by addition of NaCl to the homogenisation buffer. The combined solvent extraction and SPE-CE methodology was applied to the analysis of MT in sheep fetal liver and the results compared favorably with those obtained by high-resolution chromatography. MT-1 levels were 2 to 4-times higher than those of MT-2 and both isoforms decreased from day 89 to day 136 of gestation. These results were compared with MT levels in fetal liver from sheep embryos that had been perturbed by temporary transfer to an advanced uterine environment. Hepatic MT levels at day 136 of gestation were 3 to 8-times higher than in normal fetal liver and significant differences were observed with both isoforms. (C) 1997 Elsevier Science B.V.

    Original languageEnglish
    Pages (from-to)463-473
    Number of pages11
    JournalJournal of Chromatography A
    Volume792
    Issue number1-2
    Publication statusPublished - 19 Dec 1997

    Keywords

    • selectivity
    • solid-phase extraction
    • metallothioneins
    • PERFORMANCE LIQUID-CHROMATOGRAPHY
    • ZONE ELECTROPHORESIS
    • ONLINE PRECONCENTRATION
    • MASS-SPECTROMETRY
    • SEPARATION
    • ZINC
    • RAT
    • ISOTACHOPHORESIS
    • INDUCTION
    • ISOMETALLOTHIONEINS

    Cite this

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    title = "On-line solid-phase extraction capillary electrophoresis for enhanced detection sensitivity and selectivity: application to the analysis of metallothionein isoforms in sheep fetal liver",
    abstract = "A simple and rapid capillary electrophoresis (CE) method for analysis of metallothionein (MT) isoforms is described. A modified two-step solvent extraction procedure was used in combination with CE and an on-line solid-phase preconcentration device for sensitive and reproducible detection of MT isoforms in sheep fetal liver. Preparation of twenty samples was practicable within a working day with subsequent automated overnight analysis by solid-phase extraction (SPE)-CE. A commercially available divinylbenzene-based reversed-phase resin was found to be most suitable for the SPE device because it is resistant to extremes of pH and can be adequately regenerated between analyses. Each SPE device was readily constructed from commonly available materials and was used for the reproducible separation of over 100 biological samples before replacement. Precision of analysis within or between sample batches was <10{\%} and usually <5{\%} while detection limits were at least 28 ng/ml for standards and 272 ng/ml for biological samples. This would indicate a detection limit of about 0.5 mu g/g wet weight of tissue. Recovery of MT from tissue cytosols by solvent extraction was measured using radiolabeled MT and was found to be just over 50{\%} increasing to almost 70{\%} by addition of NaCl to the homogenisation buffer. The combined solvent extraction and SPE-CE methodology was applied to the analysis of MT in sheep fetal liver and the results compared favorably with those obtained by high-resolution chromatography. MT-1 levels were 2 to 4-times higher than those of MT-2 and both isoforms decreased from day 89 to day 136 of gestation. These results were compared with MT levels in fetal liver from sheep embryos that had been perturbed by temporary transfer to an advanced uterine environment. Hepatic MT levels at day 136 of gestation were 3 to 8-times higher than in normal fetal liver and significant differences were observed with both isoforms. (C) 1997 Elsevier Science B.V.",
    keywords = "selectivity, solid-phase extraction, metallothioneins, PERFORMANCE LIQUID-CHROMATOGRAPHY, ZONE ELECTROPHORESIS, ONLINE PRECONCENTRATION, MASS-SPECTROMETRY, SEPARATION, ZINC, RAT, ISOTACHOPHORESIS, INDUCTION, ISOMETALLOTHIONEINS",
    author = "Knudsen, {C B} and Beattie, {J H}",
    year = "1997",
    month = "12",
    day = "19",
    language = "English",
    volume = "792",
    pages = "463--473",
    journal = "Journal of Chromatography A",
    issn = "0021-9673",
    publisher = "Elsevier",
    number = "1-2",

    }

    TY - JOUR

    T1 - On-line solid-phase extraction capillary electrophoresis for enhanced detection sensitivity and selectivity: application to the analysis of metallothionein isoforms in sheep fetal liver

    AU - Knudsen, C B

    AU - Beattie, J H

    PY - 1997/12/19

    Y1 - 1997/12/19

    N2 - A simple and rapid capillary electrophoresis (CE) method for analysis of metallothionein (MT) isoforms is described. A modified two-step solvent extraction procedure was used in combination with CE and an on-line solid-phase preconcentration device for sensitive and reproducible detection of MT isoforms in sheep fetal liver. Preparation of twenty samples was practicable within a working day with subsequent automated overnight analysis by solid-phase extraction (SPE)-CE. A commercially available divinylbenzene-based reversed-phase resin was found to be most suitable for the SPE device because it is resistant to extremes of pH and can be adequately regenerated between analyses. Each SPE device was readily constructed from commonly available materials and was used for the reproducible separation of over 100 biological samples before replacement. Precision of analysis within or between sample batches was <10% and usually <5% while detection limits were at least 28 ng/ml for standards and 272 ng/ml for biological samples. This would indicate a detection limit of about 0.5 mu g/g wet weight of tissue. Recovery of MT from tissue cytosols by solvent extraction was measured using radiolabeled MT and was found to be just over 50% increasing to almost 70% by addition of NaCl to the homogenisation buffer. The combined solvent extraction and SPE-CE methodology was applied to the analysis of MT in sheep fetal liver and the results compared favorably with those obtained by high-resolution chromatography. MT-1 levels were 2 to 4-times higher than those of MT-2 and both isoforms decreased from day 89 to day 136 of gestation. These results were compared with MT levels in fetal liver from sheep embryos that had been perturbed by temporary transfer to an advanced uterine environment. Hepatic MT levels at day 136 of gestation were 3 to 8-times higher than in normal fetal liver and significant differences were observed with both isoforms. (C) 1997 Elsevier Science B.V.

    AB - A simple and rapid capillary electrophoresis (CE) method for analysis of metallothionein (MT) isoforms is described. A modified two-step solvent extraction procedure was used in combination with CE and an on-line solid-phase preconcentration device for sensitive and reproducible detection of MT isoforms in sheep fetal liver. Preparation of twenty samples was practicable within a working day with subsequent automated overnight analysis by solid-phase extraction (SPE)-CE. A commercially available divinylbenzene-based reversed-phase resin was found to be most suitable for the SPE device because it is resistant to extremes of pH and can be adequately regenerated between analyses. Each SPE device was readily constructed from commonly available materials and was used for the reproducible separation of over 100 biological samples before replacement. Precision of analysis within or between sample batches was <10% and usually <5% while detection limits were at least 28 ng/ml for standards and 272 ng/ml for biological samples. This would indicate a detection limit of about 0.5 mu g/g wet weight of tissue. Recovery of MT from tissue cytosols by solvent extraction was measured using radiolabeled MT and was found to be just over 50% increasing to almost 70% by addition of NaCl to the homogenisation buffer. The combined solvent extraction and SPE-CE methodology was applied to the analysis of MT in sheep fetal liver and the results compared favorably with those obtained by high-resolution chromatography. MT-1 levels were 2 to 4-times higher than those of MT-2 and both isoforms decreased from day 89 to day 136 of gestation. These results were compared with MT levels in fetal liver from sheep embryos that had been perturbed by temporary transfer to an advanced uterine environment. Hepatic MT levels at day 136 of gestation were 3 to 8-times higher than in normal fetal liver and significant differences were observed with both isoforms. (C) 1997 Elsevier Science B.V.

    KW - selectivity

    KW - solid-phase extraction

    KW - metallothioneins

    KW - PERFORMANCE LIQUID-CHROMATOGRAPHY

    KW - ZONE ELECTROPHORESIS

    KW - ONLINE PRECONCENTRATION

    KW - MASS-SPECTROMETRY

    KW - SEPARATION

    KW - ZINC

    KW - RAT

    KW - ISOTACHOPHORESIS

    KW - INDUCTION

    KW - ISOMETALLOTHIONEINS

    M3 - Article

    VL - 792

    SP - 463

    EP - 473

    JO - Journal of Chromatography A

    JF - Journal of Chromatography A

    SN - 0021-9673

    IS - 1-2

    ER -