On-column preconcentration of glutathione and glutathione disulfide using pH-mediated base stacking for the analysis of microdialysis samples by capillary electrophoresis

Abstract

Capillary electrophoresis (CE) has become a useful analytical tool for the analysis of microdialysis samples. However, CE with UV detection (CE-UV) is not sensitive enough to quantify glutathione (GSH) and glutathione disulfide (GSSG) in high ionic strength sample matrices such as microdialysates, because of the small optical path length in the capillary. To overcome this limitation, an on-column preconcentration technique, pH-mediated base stacking, was used in this study to improve the sensitivity of CE-UV. In pH-mediated base stacking, sample injection is followed by the injection of sodium hydroxide to achieve on-column preconcentration of analytes. This stacking technique allowed large volumes of high ionic strength samples to be injected without deterioration of the separation efficiency and resolution. The limit of detection for GSH and GSSG was found to be 0.75 µM (S/N = 6) and 0.25 µM (S/N = 6), respectively. A 26-fold increase in sensitivity was achieved for both GSH and GSSG using the pH-mediated base stacking, relative to normal injection without stacking. The developed method was used to analyze GSH and GSSG in liver microdialysates of anesthetized Sprague Dawley male rats. The basal concentrations of GSH and GSSG in the liver microdialysates of male rats were found to be 4.73 ± 2.08 µM (n = 7) and 5.52 ± 3.66 µM (n = 7), respectively.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips

“…For bases, an electrolyte with a weak acid and injection with a strong acid must be used. This approach has been shown to be exceptionally simple and powerful for the analysis of highly saline samples such as ringer's solution and microdialysates and can generally provide up to a 100-fold increase in sensitivity as demonstrated by Lunte and co-workers [10,[48][49][50][51][52]. However, care must be taken to optimise the base (acid) injection times to maintain efficiency and resolution.…”

Simultaneous Determination of Glutathione and Glutathione Disulfide in an Acid Extract of Plant Shoot and Root by Capillary Electrophoresis

“…Values are also similar to those found through chromatographic techniques 8 , although not as low as those found through CE coupled to the use of more sensitive detectors such as laser-induced fluorescence 18 and mass spectrometry 19 . EC methods have been described based on other modalities such as sample stacking 32 or MECK 13 with lower detection limits than those found in the present study, but they have not been successfully applied to highly acid matrices such as those formed by MPA. The method reproducibility, both intra-and interday, was good, with RSD values below 4% for the area under the curve and below 1% for migration time.…”

DNA Concentration Effect of Various Hydroxide Compounds on Stacking in Capillary Electrophoresis

“…For example, CE has low detection sensitivity due to the small sample volume. [8][9][10][11] Recently, various on-column pre-concentration techniques such as pH-mediated base stacking, 4,5 micellar electrokinetic chromatography (MEKC), 6 field amplification or enhancement, [9][10][11] moving reaction boundary (MRB), 12 micelle to solvent stacking (MSS) 13 and base stacking (BS) 14,15 have been developed and applied to eliminate these constraints. Some methods combine two concentration techniques, such as field-enhanced sample injection (FESI) and sweeping for the on-line concentration of analyte.…”

“…However, previous BS studies were considered and performed with only sodium hydroxide (NaOH), 4,5,14,15 although there are many other hydroxide compounds. In this study, the pre-concentration effect of DNA molecules on the BS was investigated using various hydroxide compounds such as lithium hydroxide (LiOH), potassium hydroxide (KOH), ammonium hydroxide (NH 4 OH), magnesium hydroxide (Mg(OH) 2 ), calcium hydroxide (Ca(OH) 2 ), strontium hydroxide (Sr(OH) 2 ) and barium hydroxide (Ba(OH) 2 ).…”

“…Each hydroxide compound with different cations showed different BS effects such as sensitivity enhancement of DNA molecules (i.e., increasing peak height) at the same conditions ( Table 2). The sensitivity enhancement of hydroxide compounds in descending order is as following: NaOH > NH 4 …”