Online acid barrage stacking anti-salt injection for capillary electrophoresis of 9-fluorenylmethylchloroformate-derivatized amino acids in high ionic strength solutions by UV detection

Electrophoresis. 2006 Nov;27(21):4240-8. doi: 10.1002/elps.200600149.

Abstract

An acid barrage stacking (ABS) method has been shown to be feasible for online anti-salt injection in CE of 9-fluorenylmethyl chloroformate (FMOC)-labeled amino acids (AAs) detected by common UV absorption. The operation was performed on normal polar CE by sucking in an extra plug of acid following a sample zone, serving as a selective acid barrage to block the backward migration of weak anionic analytes due to a sudden mobility reduction via acid-base reaction which does not affect strong co-ions such as Cl(-) to penetrate the barrage freely. By CE-UV of FMOC-AAs in various NaCl solutions, the effectiveness of ABS was firmly validated, able to stand up to 500 mM NaCl and to stack analytes by 10(3)-fold calculated from the UV detection limits, that is 0.01 microM for ABS and 10 microM for non-stacking injection. The method was also validated by determining trace Glu and Asp in real samples of rat brain microdialysate, rat serum and human saliva. The intraday RSDs were 0.33-4.9% for migration time and 1.8-9.6% for peak area. The recoveries measured by spiking technique were 82-115% for Glu and 86-116% for Asp. Working equations were obtained by plotting peak height vs. concentration at 0.1-50 microM, with correlation coefficients of >0.999. The contents of Glu and Asp were thus found at 0.26-0.83 microM and 0.24-0.64 microM respectively, in rat brain microdialyste; 37-40 microM and 8.4-10 microM, respectively, in rat serum; and 3.5-5.8 microM and 1.0-4.1 microM, respectively in human saliva. They were consistent with the data from other methods.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Amino Acids / analysis*
  • Amino Acids / blood
  • Animals
  • Brain Chemistry
  • Electrophoresis, Capillary / methods*
  • Flow Injection Analysis
  • Fluorenes / chemistry*
  • Humans
  • Hydrogen-Ion Concentration
  • Online Systems*
  • Osmolar Concentration
  • Rats
  • Saliva / chemistry
  • Sodium Chloride / chemistry
  • Solutions
  • Spectrophotometry, Ultraviolet

Substances

  • 2,7-di-tert-butyl-9-fluorenylmethoxycarbonyl chloride
  • Amino Acids
  • Fluorenes
  • Solutions
  • Sodium Chloride
  • 1-(9-fluorenyl)methyl chloroformate