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Quantitative Measurement of Plasma and Urinary Excretion of 3‐Hydroxyisovaleryl Carnitine by UPLC‐MS/MS
Author(s) -
Wang Zhu
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.917.5
Subject(s) - carnitine , chemistry , chromatography , high performance liquid chromatography , urine , analyte , acetylcarnitine , detection limit , electrospray ionization , excretion , mass spectrometry , biochemistry
Objective An increased plasma concentration of 3‐hydroxyisovaleryl carnitine (3HIA‐carnitine) results from impairment in the leucine catabolic pathway at the conversion of 3‐methyl‐crotonyl‐CoA to 3‐methylglutaconyl‐CoA which is caused by reduced activity of the biotin‐dependent enzyme 3‐methylcrotonyl‐CoA carboxylase. In order to validate plasma 3HIA‐carnitine as a novel biomarker of biotin deficiency rats models, an LC‐MS/MS method for the quantitation of 3HIA‐carnitine in plasma and urinary excretion has been established. Materials & Methods Plasma and urinary excretion samples were centrifugated with high speed at 3000×g for 10 minutes, the MCX solid phase extraction column was used to filter and purify the liquid samples, the following procedures were elution, wash‐out, redissolve, detection. The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (1.7μm, 2.1×50mm), with a mobile phase composed of 0.1% formic acid in water‐methanol with gradient elution. Analyte quantification was performed in the positive electrospray ionization mode and multiple monitoring. The quantification and qualification were performed using the internal standard of 3‐Hydroxyisovaleryl carnitine, then the evaluation for accuracy and precision were followed. Main Findings The RSD of six parallel determination for the 3HIA‐carnitine in plasma and urine were 4.8%, 5.5%, respectively. The linear range of 3‐Hydroxyisovaleryl carnitine was 0.5~20ng/ml in the plasma 5~200ng/ml in urine, with both the linear correlation coefficient was above 0.99. Moreover in three standard levels, rate of recovery in plasma and urine were more than 102.5%, 79%. The detection and quantification limits for urine and plasma were 20ng/L, 50ng/L and 10ng/L, 33ng/L, respectively. Conclusion & Recommondations The sample preparation method is simple and fast, and the method can be used to analyze 3‐Hydroxyisovaleryl carnitine in plasma and urinary excretion efficiently and sensitively. If confirmed in deeper studies, 3HIA‐carnitine in plasma and urinary is likely to be an important indicator of biotin status in a variety of clinical circumstances especially for the high‐risk population like pregnant women. Support or Funding Information Youth fund of national natural science fundation in China (81302428)