DEVELOPMENT AND VALIDATION OF QUANTIFICATION METHOD FOR HOMOCYSTEINE IN HUMAN PLASMA USING ULTRA-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY.

Abstract

We developed and validated a sensitive and robust method for quantifying homocysteine in human plasma using ultra-high-performance liquid chromatography-tandem mass spectrometry. Isocratic elution of the analyte and internal standard were achieved within 2 minutes using Zorbax Eclipse Plus C18 column, with 5% of mobile phase A (0.1% formic acid in water) and 95% of mobile phase B ( 0.1% formic acid in acetonitrile). Quantification was performed using multiple reaction monitoring (MRM) mode, based on parent and product ion transitions for L-Homocysteine (136 > 90.1, 56.2) and d8-DL-Homocysteine (140 > 94.1, 59.3) as the internal standard. The method was validated for linearity, sensitivity, accuracy, precision, recovery, and stability. Good linearity was observed within a range of 250-3000 ng-1 , with correlation coefficients (r2 = 0.98). The extraction recovery ranged between 83% and 92%. Homocysteine remained stable under all five tested conditions, including 2 and 8 hours on the benchtop at room temperature, overnight storage in the autosampler (10 ºC), three freeze-thaw cycles (-20 ºC), and long-term storage (1 month) at -20 ºC. Based on these findings, the developed method met the criteria as stipulated by European Medicines Agency (EMA), Food and Drug Administration (FDA), and International Council of Harmonisation (ICH). In conclusion, this validated method can be used for quantitating homocysteine levels in human plasma.