A semi-automatization magnetic solid-phase extraction method of LC-MS/MS for the quantification of homocysteine and its related metabolites in serum

Clinical mass spectrometry is recognized for its high specificity and sensitivity in quantifying small-molecule biomarkers in serum. However, its broad adoption in clinical settings has been limited by challenges such as low automation and time-consuming workflows. This study aimed to develop a rapid, sensitive, and automated magnetic solid-phase extraction(MSPE) method using Hydrophilic-Lipophilic Balance (HLB) magnetic bead-based sample preparation coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous quantification of homocysteine(Hcy) and its related nine metabolites. The method was comprehensively validated for specificity, linearity, sensitivity, accuracy, precision, matrix effects, carry-over; and compared it with solid-phase extraction (SPE) methods. Results showed excellent linearity (r 2 > 0.995) for all nine biomarkers associated with the homocysteine metabolic cycle. Both the limits of detection and quantification met the clinical requirements. Recoveries at low, medium, and high spiking levels ranged from 85.46% to 114.48%. Intra-day precision (CV) was between 0.82% and 7.63%, and inter-day precision (CV) ranged from 1.62% to 11.43%. Matrix effects were acceptable, with internal standard–normalized matrix factors ranging from 0.83 to 1.19. Carry-over rates were between –7.43% and 5.21%. Method comparison with protein precipitation sample preparation showed correlation coefficients from 0.9462 to 0.9957, indicating no systematic bias. In conclusion, the developed semi-automatization method is rapid, highly sensitive, and reproducible, making it suitable for quantitative analysis of these nine homocysteine cycle–related biomarkers in clinical serum samples. It provides a reliable analytical tool for the diagnosis, treatment, and prevention of associated diseases. • This method enables semi-automatization with cost-effectiveness, greatly facilitating its clinical implementation. • A high-throughput LC-MS/MS method has been developed for the simultaneous quantification of homocysteine (Hcy) and nine of its metabolism-related biomarkers in a single run, which facilitates the systematic monitoring and diagnosis of diseases. • Comparison with the LC-MS/MS method employing protein precipitation pretreatment demonstrates strong correlation with minimal differences