Comparing the ESA HPLC total homocysteine assay with electrochemical detection to the CDC in-house HPLC assay with fluorescence detection

Abstract

Background: Environmental science has developed a simple high performance liquid chromatography (HPLC) assay with electrochemical detection for total homocysteine (tHcy) measurement that does not require derivatization of free thiols. We evaluated this method and compared it with the CDC HPLC assay with fluorescence detection (FD). Methods: tHcy is measured after reduction of disulfides/protein-bound thiols and protein precipitation using four channels of an ESA CoulArray detector. l-homocystine is used as calibrator, penicillamine as internal standard. Results: Aqueous calibration of the ESA assay resulted in overestimation of tHcy by ∼30% compared to the HPLC-FD method. Calibration in plasma alleviated the matrix effect. The within- ( n=3) and between-run ( n=20) imprecision was <6%, the linearity up to 100 μmol/l was excellent, and the recovery of tHcy added to plasma was nearly complete (98.7%±2.3%). Good correlation was observed between both methods for 266 plasma samples. The ESA assay showed a minimal negative bias of 0.28 μmol/l (3.3%). Conclusion: The ESA tHcy assay performed well in terms of accuracy and precision, and showed good agreement with the CDC HPLC-FD assay when calibrated in plasma. The major advantage of this assay is that it does not require sample derivatization. Disadvantages include instability of the prepared samples for prolonged storage and matrix effects.