A sensitive, robust and high-throughput isotope dilution LC-MS/MS method for quantifying three folate forms in serum.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Clinical Chemistry

Background:

• Accurate quantification of folate forms in human serum is crucial for assessing nutritional status and guiding supplementation.
• Existing methods may lack the sensitivity, throughput, or robustness required for routine clinical monitoring.

Purpose of the Study:

• To establish and validate a sensitive isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining folic acid, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate in human serum.
• To apply this method for quantifying these folate forms in healthy adults and supplement users within the Chinese population.

Main Methods:

• Development of a stable 96-well solid-phase extraction (SPE) system for serum sample preparation.
• Optimization and validation of an isotope dilution LC-MS/MS method using a Shimadzu LCMS-8060NX.
• Assessment of method linearity, accuracy, precision, sensitivity, robustness, and throughput.

Main Results:

• The method demonstrated good linearity across specified ranges: 0.1-10 nmol/l for folic acid and 5-formyltetrahydrofolate, and 1.0-100 nmol/l for 5-methyltetrahydrofolate.
• High accuracy and precision were achieved, indicating reliable quantification.
• The method proved to be sensitive, robust, and high-throughput.

Conclusions:

• The developed isotope dilution LC-MS/MS method is highly suitable for the accurate and efficient determination of key folate forms in human serum.
• This validated method can be implemented for routine clinical monitoring of folate status, particularly in the Chinese population.