Micro-Determination of L-Carnitine and Its Relative Compounds in Human Plasma
L-Carnitineand its relative compounds are quaternary ammonium compounds which exist naturally in the human body. Due to L-carnitine is an important cofactor for fatty acid oxidation in the mitochondria, it is used as a drug for patients with fatty acid metabolism deficiency and also as an adjuvant for losing weight.
In this study, we established a rapid, sensitive, and selective method by using narrow-bore liquid chromatography with fluorescence detection (FLD) for determination of L-carnitine and its related compounds. Pre-column derivatization coupled with aqueous solvent-based dispersive liquid-liquid microextraction (AS-DLLME) method to improve biosample extraction efficiency. In this method, plasma (1 μL) was utilized and derivatized with 4-bromomethylbiphenyl in acetonitrile at 90℃ for 9 min. After adding 85 μL of toluene as remover, 5 μL of 1 M ammonium acetate aqueous solution was used as extractant for plasma. Then 5 μLextractant was taken and injected into reversed-phase narrow-bore liquid chromatography.
Because L-carnitine, betyrobetaine, and acetyl-L-carnitine are endogenous in human, we used standard addition method as a quantitative method and applied this method to human plasma successfully. The concentration of L-carnitine is in the range from 43.25 to 47.67 μM in health volunteer. In human plasma, butyrobetaine and acetyl-L-carnitine are in the range from 1.08 to 1.27 μM and 2.79 to 6.65 μM, respectively. The linearity (r2>0.996) and reproducibility of standard addition method were good enough for quantitation. All the relative compounds were identified by LTQ Orbitrap mass spectrometer. The results showed that the proposed method were successfully developed for analysis of hydrophiliccompounds, such as L-carnitineand its relative compounds.
Index Terms-Aqueous solvent-based dispersive liquid-liquid microextraction, L-carnitine, fluorescence derivatization, narrow-bore liquid chromatography.