Application of Butylamine as a Conjugative Reagent to On-Column Derivatization for the Determination of Antioxidant Amino Acids in Brain Tissue, Plasma, and Urine Samples

Abstract

(1) Antioxidants are involved in body protection mechanisms against reactive oxygen species. Amino acids such as glutathione (GSH) and N-acetylcysteine (NAC) are known to be involved in providing protection against oxidative lethality. A quick and simple method for the determination of NAC and GSH in various biological matrices such as urine, plasma, and homogenates of brain tissues has been developed and described in this work. (2) The assay is based on reversed phase high performance liquid chromatography with spectrofluorimetric detection and on-column derivatization. Butylamine and o-phthaldialdehyde have been used as derivatization reagents. Since o-phthaldialdehyde constitutes a part of the mobile phase, the derivatization reaction and chromatographic separation occur simultaneously. (3) Linearity in the detector response for NAC in human urine was observed in the range of 5–200 nmol mL−1, and NAC and GSH in the brain tissue homogenates were observed in the range of 0.5–5 nmol mL−1 and 0.5–15 nmol mL−1, respectively. Human plasma linearity ranges covered 0.25–5.00 nmol mL−1 and 0.5–15 nmol mL−1 for NAC and GSH, respectively. The LODs for NAC and GSH were 0.01 and 0.02 nmol mL−1 while the LOQs were 0.02 and 0.05 nmol mL−1, respectively. The usefulness of the proposed method was proven through its application to real samples.

A Simplified Method for Simultaneous Determination of α-Lipoic Acid and Low-Molecular-Mass Thiols in Human Plasma

“…The assay is based on the simultaneous reduction with TCEP and derivatization with BCBP and elimination of the deproteinization step from the sample preparation protocol. Although in the literature, some assays dedicated to LA or GSH, or other amino thiols can be found, they do not allow the simultaneous determination of biologically important aminothiol antioxidants such as LA [24] and GSH [45] and other metabolically related low-molecular thiols [30]. The presented methodology exhibits some advantages when compared to other previously published reversed phase HPLC based methods.…”