Feb 11, 2003

Affinity extraction combined with stable isotope dilution LC/MS for the determination of 5-methyltetrahydrofolate in human plasma

Analytical Biochemistry
Bryant C NelsonChad P Nelson


The predominant circulating folate monoglutamate in human plasma (>90%), and thus the most significant folate for accurately diagnosing folate deficiency, is 5-methyltetrahydrofolic acid (5 MT). Folate deficiency is typically indicated when circulating folate levels are < or = 3 ng/mL. The quantitative determination of plasma folates in general, and of 5 MT in particular, is complicated by their naturally low levels (pg/mL to ng/mL), their instability, and their tendency to interconvert. Highly specific and sensitive analytical methods are needed to accurately quantify endogenous 5 MT in human plasma. A method that utilizes the specific high-affinity binding sites of bovine folate binding protein (FBP) and the selectivity and sensitivity of selected ion monitoring mode isotope-dilution liquid chromatography/mass spectrometry (LC/MS) to quantify plasma 5 MT has been developed. The method is based on the solid-phase affinity extraction (SPAE) of 5 MT and its stable isotopically labeled analogue ([13C(5)]5 MT) from plasma (1 mL) using FBP immobilized to polymeric beads. The excess high-affinity binding sites on the affinity columns enable quantitative extraction of 5 MT from plasma under optimized sample pH conditions. Additionall...Continue Reading

  • References16
  • Citations19


  • References16
  • Citations19


Mentioned in this Paper

Human plasma
Hormone Receptors, Cell Surface
Bos taurus
5-methyltetrahydrofolate, calcium salt (1: 1), (L-Glu)-isomer
Selected Ion Monitoring
Mass Spectrometry
Plasma Proteins

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