Rapid Determination of Total Homocysteine in Blood Spots by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry,

[摘要] The determination of total homocysteine (tHcy) plays an important role in diagnosis and therapy of folate and cobalamin (vitamin B12) deficiencies. In addition, it is now widely accepted that increased tHcy is an independent risk factor for thromboembolism and cardiovascular disease, including coronary occlusive disease (1)(2)(3).Although the causal role of Hcy in the development of vascular occlusive disease has yet to be determined, prospective intervention trials that will effectively lower total plasma Hcy currently are in progress (4). As a result, various analytical methods have been established to measure tHcy in serum or plasma (5)(6)(7)(8)(9)(10). We have established a more rapid protocol suitable for accurately measuring tHcy in hemolysates by use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS/MS). The LC/MS/MS method circumvents the disadvantages of time-consuming derivatization and allows the processing of >400 samples per day.Blood was collected in hemolysate tubes that keep the tHcy concentration constant for at least 48 h at room temperature (11). For longer time periods, the hemolysate was stored at −20 °C. Hemolysate (20 μL) was pipetted onto filter paper (Neonatal Screening Card; Schleicher & Schuell). For each sample, two spots (equivalent to 4.7 μL) were punched out and incubated for 15 min at room temperature with a mixture of 20 μL of reducing agent (500 mmol/L dithiothreitol in doubly distilled water) and of 20 μL of internal standard {0.0025 mmol/L d,l …

[学科分类] 过敏症与临床免疫学