Determination of biothiols by a novel on-line HPLC-DTNB assay with post-column detection

Abstract

A novel on-line HPLC-DTNB method was developed for the selective determination of biologically important thiols (biothiols) such as l-cysteine (Cys), glutathione (GSH), homocysteine (HCys), N-acetylcysteine (NAC), and 1,4-dithioerythritol (DTE) in pharmaceuticals and tissue homogenates. The biothiols were separated on C18 column using gradient elution, reacted with the postcolumn reagent, DTNB in 0.5% M-β-CD (w/v) solution at pH 8, to form yellow-colored 5-thio-2-nitrobenzoic acid (TNB), and monitored with a PDA detector (λ = 410 nm). With the optimized conditions for chromatography and the post-column derivatization, 40 nM of NAC, 40 nM of Cys, and 50 nM of GSH can be determined. The relative standard deviations of the recommended method were in the range of 3.2–5.4% for 50 μM biothiols. The negative peaks of biothiol constituents were monitored by measuring the increase in absorbance due to TNB chromophore. The detection limits of biothiols at 410 nm (in the range of 0.04–0.58 μM) after post-column derivatization with DTNB + M-β-CD were much lower than those at 205 nm UV-detection without derivatization, and were distinctly lower than those with post-column DTNB alone. The method is rapid, inexpensive, versatile, nonlaborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of biothiol constituents of biological fluids and pharmaceuticals.

Introduction

Endogenous low molecular weight biothiols such as cysteine (Cys), N-acetylcysteine (NAC), homocysteine (HCys), reduced (GSH) and oxidized glutathione (GSSG) play a central role in a variety of physicological and pathological processes in the human body (i.e., protection against reactive oxygen and nitrogen species, control of gene expression, heavy metal detoxification, markers of various health disorders and signal transduction) [1], [2], [3]. The concentrations of biothiols and the change in their molar ratio are key indicators for monitoring cell functionality and oxidative stress.

The main challenges directed to individual identification and quantification of biothiols in complex matrices such as biological fluids lie in their unfavorable physicochemical properties (lack of strong chromophore or fluorophore). The high polarity and water solubility of these compounds make their analysis difficult in complex matrices [4]. The choice of derivatizing reagent – used in pre- or post-column mode – is important not only for the sensitive detection but also for stabilization of biothiols and improvement of chromatographic properties [5]. Moreover, the chemical instability, especially the liability of the thiol group to oxidation, often causes inaccurate analytical results [4]. Therefore, various thiol-reactive probes (i.e., 2-chloro-1-methylquinolinium tetrafluoroborate, 1-benzyl-2-chloropyridinium bromide) [5] for derivatizing low molecular weight thiols in complex biological matrices for different analytical assays (i.e., HPLC [6], [7], GC–MS [8], capillary electrophoresis (CE) [9]) have been reported that employ chromatographic and electrophoretic separation. However, most of these assays in conjunction with pre- or post-column derivatization of thiols have basic limitations in terms of complexity, sample processing and run times, and number of thiols simultaneously quantitated. On the other hand, a potential disadvantage of pre-column derivatization is that the derivatization process is administered to all matrix simultaneously and there is the possibility of non-uniform conversion and changes in the relative concentration of analyte derivatives differing from their precursors in the untreated sample. Post-column technique allows automation of the derivatization step and has several advantages over pre-column method (i.e., minimized sample treatment, lack of necessity to remove the excess of the reagent) [10]. Therefore, post-column techniques are more suitable for standardization of biothiol assays.

The determination of biothiols by using HPLC with post-column derivatization has been studied intensively over past several decades, and there is a great interest in developing new post-column detection systems for this purpose. Traditional post-column reactions generally produce spectroscopically measurable compounds by labeling the biothiols with molecular chromophores or fluorophores (i.e., labeling with o-phthalaldehyde (OPA), ninhydrin via thiazolidine reaction, maleimide, and sulfhydryl/disulfide exchange reaction) [10].

Recently, several papers have been published on the usage of Ellman's reagent (5,5′-dithio-bis(2-nitrobenzoic acid), DTNB) (with UV–vis detection) as a post-column reagent in the determination of biothiols [6], [11]. Nozal et al. [12] also used DTNB in the presence of cationic micelles of hexadecyltrimethylammonium bromide that enhanced the sensitivity. In this paper, it is aimed to develop and validate a new post-column derivatization technique using the well-known DTNB reagent in the presence of 0.5% (w/v) methyl-β-cyclodextrin (M-β-CD) for individual biothiols comprising Cys, HCys, GSH, NAC, and DTE, for synthetic thiol-containing mixtures, biological fluids and pharmaceutical samples. Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides known for their ability to form essentially non-covalent inclusion complexes with a variety of poorly soluble compounds contained in drugs or biological fluids. The host–guest inclusion complexes with CDs usually exhibit increased solubility in aqueous solutions, as well as improved stability and bioavailability of the guest molecule [13], [14], [15]. It is expected that the inclusion complexation of biothiols with M-β-CD may have an enhancing effect on the stability of these compounds in complex matrices. By similar reasoning, Suliman et al. developed a spectrofluorometric method for the determination of penicillamine (PA) in pharmaceutical formulations by measuring the fluorescence intensity of the PA-fluorescamine derivative in the presence of M-β-CD [16] as sensitivity enhancer. Male and Luong also used M-β-CD to stabilize OPA derivatives of biogenic amines by forming inclusion complexes, and developed CD-modified capillary electrophoresis coupled with laser-induced fluorescence detection for the analysis of such OPA derivatives [17]. Özyürek et al. [18] reported that CUPRAC (cupric reducing antioxidant capacity) absorbance values of some antioxidant compounds in acetonated aqueous solution were slightly increased with an increase of M-β-CD concentration and reached a plateau after a critical concentration of 2% M-β-CD.

The instrumental set-up of the designed on-line system is given in Fig. 1. In this system, the HPLC-separated biothiols react with the DTNB reagent along a reaction coil, and the yellow colored TNB (5-thio-2-nitrobenzoic acid) chromophore having an absorption maximum at 412 nm is produced. Thus, separation and quantification of biothiols can be performed simultaneously. It was observed that the increase in the area of negative peaks as a function of increased available concentration of analytes (especially from CD-solubilized and stabilized complex matrices) are reflected in their increased Cys-equivalent thiol content (CETC).