Rapid Determination of Total Homocysteine in Human Plasma by Using N(O,S)-Ethoxycarbonyl Ethyl Ester Derivatives and Gas Chromatography–Mass Spectrometry,

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The measurement of total homocysteine (tHcy), i.e., the sum of free and protein-bound homocysteine, and the homocysteinyl moieties of the disulfides homocystine and cysteinylhomocysteine in human serum or plasma is of significant value for the diagnosis and follow-up of folate and cobalamin (vitamin B12) deficiencies (1)(2). These deficiency states are the most abundant causes of marked increases of plasma homocysteine. In addition, a moderately increased concentration of homocysteine is of growing interest for its claimed link with the development of thromboembolism and vascular disease, including coronary atherosclerosis (3)(4)(5)(6). Previous studies have dealt with several of different protocols to measure tHcy concentrations in human serum or plasma by using HPLC and gas chromatography–mass spectrometry (GC-MS) techniques (7)(8). The derivatization protocols most widely used are laborious, time consuming, and require expensive reagents. Here we present a more rapid methodology suitable for sensitive tHcy determination in human plasma by using a simple aqueous, room-temperature, one-step derivatization protocol with ethyl chloroformate (ECF) that provides volatile N ( O , S )-ethoxycarbonyl amino acid ethyl esters (9)(10) and GC-MS analysis. Blood was collected by venipuncture into Vacutainer Tubes (Sarstedt) containing EDTA at a final concentration of 2.7 mmol/L. The study was in accordance with ethical standards of the local committee. Plasma was recovered without delay after centrifugation at 5000 g for 3 min at 0–2 °C. Plasma can be stored at −20 °C until analysis. Plasma aliquots (100 μL) were supplemented with 20 μL of reducing agent (tri- n -butylphosphine, 360 mmol/L in dimethylformamide) and dl-[3,3,3′,3′,4,4,4′,4′-2H8] homocystine (isotopic purity 98%; CIL; final concentration 40 μmol/L) as an internal standard (IS). The samples were incubated for 30 min at 4 °C to reduce and release (endogeneous) homocysteine from …