Quantitative analysis of free/total carnitine in plasma by Liquid Chromatography/tandem mass spectrometry assay

• Main Authors: Sung-Fa Huang, 黃嵩發
• Other Authors: 楊沂淵
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/19827509910163010980

碩士 === 臺北醫學大學 === 醫學檢驗暨生物技術學系所 === 103 === Carnitine is a key substance for transportation of long-chain acyl-CoA esters across mitochondria membrane for subsequent fatty acid oxidation. Carnitine can be obtained from exogenous meat supply, and endogenous synthesis in liver. Defects of carnitine transportation and synthesis may eventually result in carnitine deficiency. The conventional method for quantitative analysis of plasma free/total carnitine is carnitine acetyltransferase (CAT) spectrophotometric assay. However, the CAT method is highly affected by hemolysis and any compound in physiological fluids containing–SH functional group, which may result in an analytic error of measurement. In addition, a larger volume of blood sample is required which makes the measurement more unfeasible. In this study, we intended to develop and to establish a liquid chromatography/tandem mass spectrometry method for clinical diagnostic purpose. By using multiple reaction monitoring (MRM) of tandem mass analysis, the mass-to-charge (m/z) of the precursor and the daughter ions of carnitine (Q1/Q3) was 162.2/85.0. The internal standard, a stable radio-isotope D3-carnitine, was used for the calibration and basis of quantitative measurement. The m/z of D3-carnitine was 165.2/103.0. According to the preliminary results, the within-run and between-run precisions of the method were excellent, in which the CV% were 4.1% and 5.5%, respectively. The recovery of this tandem mass assay was 94.5%, and the subsequent regression analysis showed good correction with the CAT method (r2=0.957). The reference values of free and total carnitine in normal control were 42.3 and 52.4 μmol/L, respectively, with a mean ± standard deviation of 42.3 ± 8.01(Free carnitine, n=130)；52.4±8.80(Total carnitine, n=130), and the samples showed a normal distribution. The developed LC-MS/MS method for plasma free/total carnitine determination is specific, sensitive, validated, accurate, and high throughput. The method is applicable and a great benefit particularly to the patients from pediatric department when a small volume of plasma sample is avaiable. This method is feasible and can be applied for clinical diagnostic services.