A rapid LC-MS/MS method for the simultaneous quantification of 10 key folate cycle intermediates in human plasma

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• The folate cycle is vital for metabolic regulation, and its intermediates are crucial biomarkers for various diseases.
• Current LC-MS/MS methods struggle with polar folate metabolites, leading to long analysis times and poor retention.
• Accurate and efficient measurement of these metabolites is essential for clinical diagnostics and disease management.

Purpose of the Study:

• To develop and validate a novel HILIC-MS/MS method for the simultaneous quantification of 10 key folate cycle metabolites in human plasma.
• To improve chromatographic retention and reduce analytical run time compared to existing methods.
• To apply the developed method for assessing metabolic perturbations in critically ill patients, particularly those with acute kidney injury (AKI).

Main Methods:

• Development of a HILIC-LC-MS/MS method for simultaneous measurement of 5 folate intermediates, 4 related amino acids, and vitamin B12 in human plasma.
• Method validation including linearity, precision, recovery, and matrix effect assessment.
• Application of the validated method to analyze plasma samples from AKI patients and healthy controls.

Main Results:

• The novel HILIC-LC-MS/MS method achieved simultaneous measurement of 10 folate cycle metabolites in 8.5 minutes without derivatization.
• The method demonstrated excellent linearity (R² > 0.989), acceptable precision (intra-day: 1.3-11.3%, inter-day: 3.4-14.6%), recovery (89.5-113.8%), and matrix effect (81.6-115.8%).
• Analysis of clinical samples revealed significantly elevated levels of 5-methyltetrahydrofolate (5-MTHF) and glycine (Gly) in AKI patients, with positive correlations between serum creatinine (SCr) and 5-MTHF/homocysteine (hCys) levels.

Conclusions:

• The developed HILIC-LC-MS/MS method offers a rapid, sensitive, and reliable approach for quantifying key folate cycle metabolites in human plasma.
• This method facilitates the evaluation of folate nutritional status and the identification of metabolic disruptions associated with diseases like AKI.
• The findings highlight the potential of this assay in mitigating risks associated with folate-related disorders such as megaloblastic anemia and neural tube defects.