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Simultaneous quantification of homocysteine, methionine, cysteine in human plasma using LC/MS/MS: a combination which may help to explain disorders in


V. Ducros1

1D廧artement de Biologie Int嶲r嶪, Centre Hospitalier Universitaire, Grenoble, France

First, we developed an LC/MS/MS method for total homocysteine (tHcy) determination in order to be able to measure this amino acid as a biochemical marker in vascular disease [1] but sometimes we were aware that the knowledge of the other sulphur amino-acids could help us to interpret a high value of homocysteinemia. Therefore we decided to add methionine (Met) and total cysteine (tCys) in the LC/MS/MS method.
Homocysteine, cysteine, methionine stock solutions were prepared from homocystine, cystine and methionine. Deuterated homocysteine (Hcy-d4) and deuterated cysteine (Cys-d2) stock solutions were prepared from homocystine-d8 and cystine-d4 (Cambridge Isotope Laboratory). Standards (included in each batch of samples) at concentrations of 0, 3.73, 7.45, 14.9, 29.8, 49.7, 74.5, 99.3 and 149 痠ol/L for tHcy and 0, 8.25, 16.5, 33, 66, 110, 165, 220 and 330 痠ol/L for tCys and 0, 3.35, 6.7, 13.4, 26.8, 44.7, 67, 89.3, and 134 for Met were prepared in acidic aqueous solutions. Sample preparation was carried out by adding 100 無 of diluted internal standards (~50 痠ol/L of homocysteine-d4 and ~ 80 痠ol/L of cysteine-d2) to 100 無 of plasma, or quality control, or standard. Complete reduction of plasma disulfides was accomplished by the addition of 30 無 of a freshly prepared solution of dithiothreitol (DTT) at 500 mmol/L. After mixing, the reduction step was performed at room temperature for 15 min. Proteins were precipitated by the addition of 200 無 of a solution of 1 ml/L formic acid and 0.5 mL/L trifluoroacetic acid in acetonitrile and incubated 10 minutes on ice to improve precipitation. After centrifugation at 13000 g for 5 min, 200 無 of the clear supernatant was transferred to an autosampler vial.
An Applied Biosystems/MDS SCIEX API 3000 Triple-Quadrupole Mass Spectrometer equipped with a TurboIonSpray source was used. The source operated in positive ion mode at an ion spray voltage of +5500 V. The turbo gas (nitrogen) temperature was set to 250 蚓 at a flow rate of 7 L/min.
Maximum sensitivity for tHcy, tCys, and Met was obtained by measuring product ions from the fragmentation of the protonated [M+H]+molecule in the positive ion mode. The multiple reaction monitoring (MRM) mode was used with the following transitions 136.1>90 and 140>94 for homocysteine and homocysteine-d4, respectively; 122>76 and 124>78 for cysteine and cysteine-d2, respectively; and 150>104 for methionine. Aqueous calibration curves were established to calculate the amount of homocysteine, cysteine and methionine in the sample on the basis of the integrated peak area ratio of homocysteine to homocysteine-d4, of cysteine to cysteine-d2, and methionine to homocysteine-d4.
The chromatography was performed on an Agilent HP 1100 system using a short Supelcosil CN column, 3 痠 (33 x 4.6 mm) (Supelco, reference 58979). The column temperature was set to 20蚓. Autosampler injections of 5 無 were made using isocratic elution in 30:70 with 0.1% formic acid in water/acetonitrile at 1 ml//min. A split ratio 1:5 was used in the source. Total acquisition cycle was 4 min per sample.
Reference values of plasma tHcy, tCys and Met concentrations were determined in 31 healthy adults. The simultaneous assay of Met and tCys helps to distinguish between transulfuration and remethylation pathway deficiency when an hyperhomocysteinemia is found. A few cases of this help will be detailled during the workshop.

References
[1] Ducros V, Belva-Besnet H, Casetta B, Favier A. A robust LC/MS/MS method for total plasma homocysteine determination in clinical practice. Clin. Chem. Lab Medecine 2006 44: 987-90.

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