Determination of folic acid and its metabolites in human serum based on ultra-high performance liquid chromatography-tandem mass spectrometry
Objective To establish an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method to determine the concentrations of folic acid and its metabolites in human serum.Methods An Agilent EclipsePlus-C18(2.1 mm x 50 mm,1.8 μm)column was used with 0.1％formic acid-water(A)-0.1％formic acid-methanol solution(B)as mobile phase gradient eluted at a flow rate of 0.3 mL/min,40℃ column temperature,and electrospray ionization source(positive ion mode).Multiple reaction monitoring(MRM)scanning method was used for mo-nitoring.Internal standard method was used for quantification.Results The linear correlation coefficient of the standard curve for three metabolites of the folate cycle was 0.9956 to 0.9999.The accuracy was between 91.2％and 109.2％,and the precision was less than 10％.The minimum detection limit was 1 ng/mL and the matrix effects of the three metabolites in the folate cycle were negligible.The results of stability test were in accordance with the requirements.Blood samples from 150 healthy adults were measured.The folic acid concentration ranged from 0.00 to 3.99 ng/mL,with an average concentration of(2.29±0.31)ng/mL.The 5-methyl tetrahydrofolate concentration ranged from 1.22 to 29.07 ng/mL,with an average concentration of(6.10±4.75)ng/mL.The homocysteine concentration ranged from 4.57 ng/mL to 68.22 ng/mL,with an average concentration of(12.47±8.19)ng/mL.Conclusion The method was rapid,sensitive,accurate,specific,and reproducible.It can be applied to analyzing folic acid and its metabolites in human serum.
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