Standardization (external and internal) of HPLC assay for plasma homocysteine.

Measurement of plasma homocysteine may be of value in several clinical conditions including homocystinuria, ath-erosclerosis, thrombophilia, and folate/vitamin B 12 deficiency. The increasing interest in measuring total homo-cysteine 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 fluores-cence 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 hydrochlo-ride as an internal standard to the plasma or homocys-teine 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), au-tosampler (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 ␮L) was incubated with 100 mL/L tri-n-butylphosphine in dimethylformamide (15 ␮L) for 30 min at 4 °C to reduce and release protein-bound thiols. Depro-teinization was achieved by the addition of 100 g/L trichloroacetic acid (150 ␮L) and centrifugation. An ali-quot of the supernatant (50 ␮L) was mixed with sodium hydroxide (10 ␮L, 1.55 mol/L), borate buffer (125 ␮L, 0.125 mol/L, pH 9.5, containing 4 mmol/L EDTA), and SBD-F (50 ␮L, 1 g/L) and incubated for 60 min at 60 °C. The SBD-F derivative from the supernatant (20-␮L ali-quot) was eluted isocratically from the Spherisorb ODS2 [4.6 mm (i.d.), 5-␮m particles] analytical column (Jones Chromatography). The mobile phase was 0.1 mol/L KH 2 PO 4 , pH 2.0, containing 40 mL/L acetonitrile at a flow rate of 0.8 mL/min. Homocysteine calibrators (dl form) were prepared in the following matrices: deionized water, pooled plasma (anticoagulated with EDTA), phosphate buffer (0.1 mol/L, adjusted to pH 9.5), and borate buffers (0.1 mol/L, pH 9.5, with and without 2 mmol/L EDTA). Cysteamine hydrochloride (2-mercaptoethylamine) was used as an internal standard, which was added to the plasma or homocysteine calibrator to achieve a final concentration of 10.0 ␮mol/L (30 ␮L of 50.0 ␮mol/L cysteamine plus 120-␮L sample). Plasma samples from healthy adults (volunteer blood donors) were obtained from whole blood collected into evacuated EDTA tubes (Vacutainer Tubes, Becton Dick-inson) …