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A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid

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Abstract

A liquid chromatography(LC)/electrospray ionization tandem mass spectrometry (MS) method for the quantitative determination of total homocysteine and methylmalonic acid and the monitoring of methionine, homocystine and succinic acid in plasma has been developed. The analytes are determined under the presence of the deuterated internal standards methylmalonic acid-d 3 and homocystine-d 8. Although methylmalonic acid can be determined directly, a reduction step has to be carried out to ensure the measurement of total homocysteine. Ultrafiltration was applied afterwards to deproteinize the samples prior to LC/MS injection. LC/MS analysis is carried out isocratically using a mobile phase consisting of 5% methanol and 95% of a 0.06 M formic acid solution on a reversed-phase C18 column at a flow rate of 0.5 mL/min. The MS measurement was separated into several periods: homocysteine, homocystine and methionine were determined in the positive-ion mode, whereas the determinations of methylmalonic acid and succinic acid were carried out in the negative-ion mode. The intraday coefficients of variation (CVs) were 2.9% or less and 3.2% or less for homocysteine and methylmalonic acid, respectively. Interday CVs ranged from 3.8 to 5.9% for homocysteine and from 3.5 to 6.3% for methylmalonic acid. Analyte concentrations could reliably be determined, also far below the reference values. Furthermore, the linearity was determined and a correlation study with respect to the existing homocysteine and methylmalonic acid methods at Medisch Spectrum Twente Hospital was carried out.

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Authors and Affiliations

  1. Laboratory, Medisch Spectrum Twente Hospital, P.O. Box 50000, 7500 KA, Enschede, The Netherlands

    Christel Hempen & Gertjan van der Sluijs Veer

  2. Laboratory, Twenteborg Hospital, Hospital Group Twente, P.O. Box 7600, 7600 SZ, Almelo, The Netherlands

    Harry Wanschers

Authors
  1. Christel Hempen
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  2. Harry Wanschers
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  3. Gertjan van der Sluijs Veer
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Correspondence to Christel Hempen.

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Hempen, C., Wanschers, H. & van der Sluijs Veer, G. A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid. Anal Bioanal Chem 391, 263–270 (2008). https://doi.org/10.1007/s00216-008-1953-8

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  • DOI: https://doi.org/10.1007/s00216-008-1953-8

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