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.
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 microM 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.
Yang
Cailing
C
Department of Chemistry, Lanzhou University, Lanzhou, China.
Jiang
Xiaoman
X
Guo
Linyuan
L
Zhang
Haixia
H
Liu
Mancang
M
D009327:4-Chloro-7-nitrobenzofurazan; D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007515:Islets of Langerhans / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013696:Temperature; D013997:Time Factors
D000596:Amino Acids; D002021:Buffers; D009327:4-Chloro-7-nitrobenzofurazan