High-performance liquid chromatographic method for measuring total plasma homocysteine levels

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Accurate measurement of total plasma homocysteine (tHcy) is crucial for clinical diagnostics.
• Existing methods for tHcy analysis can be complex and time-consuming.
• Optimization of analytical procedures is needed for routine clinical laboratory use.

Purpose of the Study:

• To develop and validate a modified high-performance liquid chromatographic (HPLC) method for routine clinical analysis of total plasma homocysteine (tHcy).
• To enhance the assay's efficiency, reproducibility, and safety through reagent and procedural modifications.

Main Methods:

• Modification of an existing HPLC procedure using SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization.
• Replacement of tri-n-butylphosphine with sodium borohydride as the reductant.
• Inclusion of mercaptopropionylglycine as an internal standard.
• Utilized basic isocratic HPLC equipment with a 14-minute run time.

Main Results:

• The modified HPLC assay demonstrated high reproducibility with within- and between-run coefficients of variation (C.V.) of 1.9% and 4%, respectively.
• Established reference values for fasting tHcy: 7.65±2.3 µmol/l (women) and 8.9±2.4 µmol/l (men).
• Determined post-methionine load tHcy levels: 19.9±5.5 µmol/l (women) and 26.8±5.5 µmol/l (men).

Conclusions:

• The modified HPLC method is suitable for routine clinical total plasma homocysteine analysis.
• The use of sodium borohydride and mercaptopropionylglycine improves assay safety and reproducibility.
• The optimized 14-minute run time enhances analytical efficiency for clinical laboratories.