A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid
A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid
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DOI：
10.1007/s00216-008-1953-8
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发表时间：
2008-05-01
影响因子：
4.3
通讯作者：
Veer, Gertjan van der Sluijs
中科院分区：
文献类型：
--
作者：
Hempen, Christel;Wanschers, Harry;Veer, Gertjan van der Sluijs
建立了同时测定血浆中总同型半胱氨酸、甲基丙二酸和蛋氨酸、同型半胱氨酸、琥珀酸的高效液相色谱-电喷雾串联质谱法。分析物是在氚内标甲基丙二酸-d(3)和同型半胱氨酸-d(8)存在下测定的。虽然甲基丙二酸可以直接测定，但为了保证总同型半胱氨酸的测定，必须进行还原步骤。在LC/MS进样前对样品进行超滤去蛋白处理。LC/MS分析采用反相C18色谱柱，流动相为5%甲醇和95%0.06M甲酸溶液，流速为0.5mL/min。MS测定分为几个阶段：同型半胱氨酸、同型半胱氨酸和蛋氨酸在正离子模式下测定，甲基丙二酸和丁二酸在负离子模式下测定。同型半胱氨酸和甲基丙二酸的日内变异系数(CV)分别为2.9%和3.2%。同型半胱氨酸的日间变异系数为3.8%~5.9%，甲基丙二酸的日间变异系数为3.5%~6.3%。分析物浓度可以可靠地测定，也远远低于参考值。此外，测定了线性，并与Medisch Spectrum Twente医院现有的同型半胱氨酸和甲基丙二酸法进行了相关性研究。
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.