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Determination of total homocysteine (tHcy) in plasma is becoming an important diagnostic procedure in clinical chemistry because a slightly increased concentration of tHcy in plasma has been discussed as an important independent risk factor for atherosclerotic diseases (1). Many methods, mostly by HPLC, have been reported for measuring tHcy (2). However, these methods are relatively complex and require highly specialized equipment. Drew Scientific, Inc. (company named for identification purposes only; this evaluation does not constitute an endorsement by CDC) has developed the DS30 tHcy system for measuring tHcy in plasma. We evaluated this new system and compared it with our CDC reference HPLC method (3).The DS30 tHcy system comprises a small HPLC system using a 5-cm reversed-phase column, a tHcy assay reagent set that contains the necessary reagents and calibrators [5 and 20 μmol/L homocystine (concentration was equivalent to the free thiol)] and a quality-control (QC) set containing two concentrations of Hcy. The sample preparation requires 200 μL of plasma. A batch of 30 samples can be processed within 90 min. After the addition of 10 μL of the internal standard (IS; 2-mercaptoethylamine) to 200 μL plasma, the mixture of disulfides, mixed disulfides, and protein-bound thiols is reduced using 20 μL of tris(2-carboxyethyl)phosphine (TCEP). Protein is precipitated from this solution with trichloroacetic acid, and 100 μL of the supernatant is then derivatized with a fluorescent thiol-specific dye [ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBDF) in EDTA/borate buffer] at 60 °C for 50 min. The thiol derivatives are separated in a subsequent step by HPLC and detected by their fluorescence. …

Evaluation of the Drew Scientific DS30 Homocysteine Assay in Comparison with the Centers for Disease Control and Prevention Reference HPLC Method