6-aminoquinolyl-n-hydroxysuccinimidyl-carbamate and acetonitrile

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.

Topics: Acetonitriles; Amino Acids; Aminoquinolines; Buffers; Carbamates; Chromatography, High Pressure Liquid; Ethylamines; Humans; Hydrogen-Ion Concentration; Reproducibility of Results; Temperature; Ultrafiltration

A homogeneously labeled insulin sample was prepared using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as the fluorescent-labeling reagent, and this was successfully applied to a chromatographic immunoassay. This labeled insulin was prepared by tagging all the three amino groups with AQC. Both CE and chromatographic immunoassay experiments indicated that the prepared insulin still kept its immunoaffinity to its antibody. It was observed that appropriate concentrations of acetonitrile (ACN) were efficient in lowering the quenching of the fluorescent signal of tagged insulin, in keeping the dilute, tagged insulin in solution, and in improving its peak shape during a chromatographic immunoassay. The tagged insulin was found to be 20-400 times more sensitive than native insulin detected under ultraviolet detection conditions. A competitive chromatographic immunoassay system was set up and calibrated. The system was used for analyses of an insulin-spiked urine sample, with a 96% recovery obtained.

Topics: Acetonitriles; Aminoquinolines; Antibodies; Carbamates; Chromatography, High Pressure Liquid; Electrophoresis, Capillary; Fluorescent Dyes; Humans; Immunoassay; Insulin; Staining and Labeling