Analysis of free amino acids in islets of Langerhans by high-performance liquid chromatography using pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

• Published in: Journal of separation science 2007-12, Vol.30 (18), p.3154-3163
• Main Authors: Yang, Cailing, Jiang, Xiaoman, Guo, Linyuan, Zhang, Haixia, Liu, Mancang
• Format: Article
• Language: English
• Subjects: 3-diazole (NBD-Cl); 4-Chloro-7-nitrobenzo-2-oxa-1; 4-Chloro-7-nitrobenzofurazan; 4‐Chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl); Amino Acids - analysis; Amino Acids - chemistry; Amino Acids - metabolism; Animals; Biological and medical sciences; Buffers; Chromatography, High Pressure Liquid - methods; Fluorescence detection; Free amino acids; Fundamental and applied biological sciences. Psychology; General aspects; HPLC; Hydrogen-Ion Concentration; Islets of Langerhans; Islets of Langerhans - chemistry; Islets of Langerhans - metabolism; Rats; Temperature; Time Factors; Vertebrates: anatomy and physiology, studies on body, several organs or systems
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.200700160

A simple high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was developed and used for the analysis of free amino acids in islets of Langerhans; 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) served as pre-column derivatization reagent. Islets of Langerhans were separated from the pancreas of normal and obese rats, treated with pre-cooling methanol-water (80:20, v/v), and ultrasonicated to fragmentize the islets and effect deproteination. Several parameters influencing the derivatization reaction and chromatographic separation were optimized. Amino acid derivatives obtained under optimal conditions were separated on a C18 column with acetonitrile-acetate buffer as mobile phase and detected at 470 nm/540 nm (Ex/Em). Matrix effects were investigated and good linearities with correlation coefficients better than 0.9972 were obtained over a wide range of 0.42-42.11 μM for most of the amino acids. The detection limits (S/N = 3) were within the range of 6.1-51 nM. The precision of the method and recoveries were in the ranges of 1.43-10.76% (RSD%) and 85.07-108.82%, respectively. The analytical results showed that the serine content was markedly higher in normal rats than in obese rats, whereas methionine was of relatively lower content in both normal and obese rats.