Standardization (External and Internal) of HPLC Assay for Plasma Homocysteine

Abstract

Measurement of plasma homocysteine may be of value in several clinical conditions including homocystinuria, atherosclerosis, thrombophilia, and folate/vitamin B12 deficiency. The increasing interest in measuring total homocysteine in plasma has led to the development of several different methods (1). A widely used technique for measuring total plasma homocysteine is reversed-phase HPLC with fluorescence detection after derivatization of plasma thiols with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) (2)(3). Most published methods use external calibration alone for quantitation of homocysteine because of the difficulty in selecting an internal standard. We have modified this method by adding cysteamine hydrochloride as an internal standard to the plasma or homocysteine calibrator to compensate for variations in thiol derivatization and sample injection procedures. HPLC was carried out by an isocratic system with fluorescence detection (SFM 25 spectrofluorometer), autosampler (SA 360), and HPLC pump (325) supplied by Kontron Instruments. Chemicals were obtained from Sigma. The method has been adapted from that of Ubbink et al. (2) on the basis of the chemical description provided by Araki and Sako (3). The plasma or homocysteine calibrator (150 μL) was incubated with 100 mL/L tri- n -butylphosphine in dimethylformamide (15 μL) for 30 min at 4 °C to reduce and release protein-bound thiols. Deproteinization was achieved by the addition of 100 g/L trichloroacetic acid (150 μL) and centrifugation. An aliquot of the supernatant (50 μL) was mixed with sodium hydroxide (10 μL, 1.55 mol/L), borate buffer (125 μL, 0.125 mol/L, pH 9.5, containing 4 mmol/L EDTA), and SBD-F (50 μL, 1 g/L) and incubated for 60 min at 60 °C. The SBD-F derivative from the supernatant (20-μL aliquot) was eluted isocratically from the Spherisorb ODS2 [4.6 mm (i.d.), 5-μm particles] analytical column (Jones Chromatography). The mobile phase was 0.1 mol/L KH2PO4, pH 2.0, containing 40 …