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Improved GC-MS methods for analysis of homocysteine, methionine and methylmalonate in cultured cells

Conference Paper


Abstract


  • Purpose: The aim of the present study is to establish a new gas

    chromatography mass spectrometry (GC-MS) method using a triple

    quadrupole which would allow for more sensitive quantification of

    changes in homocysteine, methionine and methylmalonate in small

    numbers of cultured cells and brain tissue. Homocysteine, methionine

    and methylmalonate are established markers of cobalamin deficiency.

    Methods: Cultured cells (1 x 106) and 5mg of human brain were incubated

    with 10 mg/ml dithiothreitol in methanol for one hour at room temperature

    to reduce homocysteine disulfide bonds. Lipids were removed using

    methyl-tert-butyl ether and the aqueous phase derivatized with N-Methyl-

    N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Samples were

    subsequently analysed with a 7000 Agilent triple-quadrupole GC-MS.

    Multiple reaction monitoring was performed on the compounds in

    electron ionisation mode at 70eV. Corresponding deuterated internal

    standards were used to quantify compounds. Results: We identified

    the following transitions: 420->392, 318 424 ->396, 322; 320->292, 218,

    323->295, 221; 289->189, 147 292->192, which allowed for sensitive

    quantification of homocysteine, methionine and methylmalonate and

    their deuterated standards respectively. The lowest limits of detection for

    homocysteine, methionine and methylmalonate were found to be 0.001

    pM in three different cell lines and brain tissue. Sample preparation and

    analysis was complete in 4 h. Conclusion: Homocysteine, methionine

    and methylmalonate were successfully quantified in low cell numbers.

    This method is widely applicable for studies of cobalamin function in

    cells and has the potential advantage of conserving precious human

    tissue samples.

Authors


  •   Spiro, Adena S. (external author)
  •   Jenner, Andrew M. (external author)
  •   Garner, Brett

Publication Date


  • 2013

Citation


  • Spiro, A. S., Jenner, A. M. & Garner, B. (2013). Improved GC-MS methods for analysis of homocysteine, methionine and methylmalonate in cultured cells. 33rd Meeting of the Australian Neuroscience Society: Program, Abstracts & List of Registrants (pp. 130-130). Australia: ANS.

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/312

Start Page


  • 130

End Page


  • 130

Abstract


  • Purpose: The aim of the present study is to establish a new gas

    chromatography mass spectrometry (GC-MS) method using a triple

    quadrupole which would allow for more sensitive quantification of

    changes in homocysteine, methionine and methylmalonate in small

    numbers of cultured cells and brain tissue. Homocysteine, methionine

    and methylmalonate are established markers of cobalamin deficiency.

    Methods: Cultured cells (1 x 106) and 5mg of human brain were incubated

    with 10 mg/ml dithiothreitol in methanol for one hour at room temperature

    to reduce homocysteine disulfide bonds. Lipids were removed using

    methyl-tert-butyl ether and the aqueous phase derivatized with N-Methyl-

    N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Samples were

    subsequently analysed with a 7000 Agilent triple-quadrupole GC-MS.

    Multiple reaction monitoring was performed on the compounds in

    electron ionisation mode at 70eV. Corresponding deuterated internal

    standards were used to quantify compounds. Results: We identified

    the following transitions: 420->392, 318 424 ->396, 322; 320->292, 218,

    323->295, 221; 289->189, 147 292->192, which allowed for sensitive

    quantification of homocysteine, methionine and methylmalonate and

    their deuterated standards respectively. The lowest limits of detection for

    homocysteine, methionine and methylmalonate were found to be 0.001

    pM in three different cell lines and brain tissue. Sample preparation and

    analysis was complete in 4 h. Conclusion: Homocysteine, methionine

    and methylmalonate were successfully quantified in low cell numbers.

    This method is widely applicable for studies of cobalamin function in

    cells and has the potential advantage of conserving precious human

    tissue samples.

Authors


  •   Spiro, Adena S. (external author)
  •   Jenner, Andrew M. (external author)
  •   Garner, Brett

Publication Date


  • 2013

Citation


  • Spiro, A. S., Jenner, A. M. & Garner, B. (2013). Improved GC-MS methods for analysis of homocysteine, methionine and methylmalonate in cultured cells. 33rd Meeting of the Australian Neuroscience Society: Program, Abstracts & List of Registrants (pp. 130-130). Australia: ANS.

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/312

Start Page


  • 130

End Page


  • 130