Determination of the Concentration of Orotic Acid in Human Urine by LC-MS/MS and Its Clinical Application
Objective:To establish a method to determine concentrations of orotic acid in human urine,and to apply this method in clinic.Methods:Urine sample was diluted 100 times with water and filtered by 0.22 μm filter membrane,then 400 μL methanol was added to 100 μL of the filtrate to precipitate pro-tein,carbmazepine(0.50 μg·mL-1,2 μL)was added as internal standard.After whirled for 1 min and cen-trifuged for 5 min at 13000 r·min-1,the supernatant(100 μL)was determined by liquid chromatography-tan-dem mass spectrometry(LC-MS/MS).The chromatographic column was Waters Atlantis dC18(4.6 mm×250 mm,5 μm),the mobile phase was 5 mmol·L-1 ammonium formate solution-acetonitrile(gradient elution),the flow rate was 0.50 mL·min-1,the column temperature was 4℃,and the injection volume was 3 μL.The positive and negative ion modes of the electric spray ion source were used.The detection mode was Full MS,the resolution of Full MS was 70000,and the mass scanning range was m/z 130-360.The detected ions of orotic acid and internal standard carbamazepine were m/z(-)155.00982 and(+)237.10224,re-spectively.Results:The concentration of orotic acid in urine ranged linearly from 0.016 to 1.0 μg·mL-1(r2= 0.9967),and the lower limit of quantification was 0.016 μg·mL-1;Intra-day and intra-day RSD<15%,RE<15%.Matrix effect was 88.70%-98.62%.The recovery rate was 95.93%-103.08%.The orotic acid urine sample was kept at room temperature for 24 h,and the treated sample was kept at 4℃for 24 h with good stability(RE<15%).The content of urinary orotic acid in normal people was(3.10±1.23)μg·mL-1,and that in ornithine carbamyltransferase deficiency(OTCD)patients was 354.82-2566.57 μg·mL-1.Conclusion:The research method is simple,specific and sensitive,and can be used for the auxiliary diagnosis of OTCD patients.
Liquid chromatography-tandem mass spectrometryOrotic acidOrnithine transcarbamylase deficiency
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