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

Abstract

α-Lipoic acid, glutathione, cysteine, and cysteinylglycine can be applied as therapeutic agents in civilization diseases such as diabetes mellitus, cardiovascular diseases, and cancers. On the other hand, a higher concentration of homocysteine can result in health problems and has been indicated as an independent risk factor for cardiovascular disease and accelerated atherosclerosis. Here, the first simplified HPLC-UV assay that enables simultaneous determination of α-lipoic acid and low-molecular-mass thiols in plasma, reduces the number of steps, shortens the total time of sample preparation, and limits the amount of single-use polypropylene laboratory materials is described. The assay is based on reversed-phase high performance liquid chromatography with UV detection and simultaneous reduction of disulfide bound with tris(2-carboxyethyl)phosphine and the selective pre-column derivatization of the thiol group with 1-benzyl-2-chloropyridinium bromide. Linearity in the detector responses for plasma samples were observed in ranges: 0.12–5.0 nmol mL−1 for α-lipoic acid; 2.0–20.0 nmol mL−1 for glutathione, cysteinylglycine, and homocysteine; and 40.0–400.0 for cysteine. The LODs for α-lipoic acid and low-molecular-mass thiols were 0.08 and 0.12 nmol mL−1, respectively, while LOQs were 0.12 and 0.16 nmol mL−1, respectively. The usefulness of the proposed method has been proven by its application to real samples.

Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane

“…Detailed analytical data for total Cys, Hcy, GSH, Cys-Gly, and HSA are shown in Table 3 . These results are mostly in agreement with those previously reported [ 5 , 11 , 30 , 39 , 56 , 57 ]. The elaborated method can be successfully applied in large populations to monitor changes in thiol concentration in different physiological conditions.…”

“…In biological systems, thiols occur mainly in oxidized or protein-bound forms, and therefore the reduction of disulfide bonds to thiol groups must be performed prior to the derivatization reaction. NaBH 4 [ 5 , 41 , 54 ], dithiothreitol (DTT) [ 42 , 43 , 52 ], tris(2-carboxyethyl)phosphine (TCEP) [ 4 , 30 , 39 , 57 ], or 2-mercaptoethanol (2-ME) [ 6 , 52 ] are the most commonly used substances to reduce disulfide bonds. The choice of an appropriate reductant depends on the analytical procedure to be followed, especially on the type of the derivatizing reagent used and the detection method.…”

“…Despite these difficulties, numerous methods have been described for the determination of total thiols in biological fluids. The most commonly used techniques are high-performance liquid chromatography (HPLC) with different detection modes, mainly ultraviolet [ 4 , 5 , 6 , 30 , 39 , 40 ] and fluorescence [ 41 , 42 , 43 , 44 , 45 , 46 , 47 ], capillary electrophoresis [ 48 , 49 ], or spectrophotometry [ 12 , 50 ]. Although several techniques have been used to determine thiols, there are still some problems caused by the need for complicated instrumentation, complexity of the procedure, the time-consuming nature of the methods, and the number of thiols quantified simultaneously [ 14 , 30 , 51 , 52 ].…”

Quantification of Intracellular Thiols by HPLC-Fluorescence Detection

“…Currently available separation-based analytical methods for thiols include HPLCfluorescence detection, LC-mass spectrometry, and capillary electrophoresis [10][11][12][13]. We have previously developed analytical methods for thiol detection based on fluorescence derivatization using ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) (Figure 1b) and hydrophilic interaction chromatography (HILIC) [14][15][16][17].…”

HPLC/Elsd Determination and Validation of Alpha Lipoic Acid and Sucralose in Bulk and in Their Pharmaceutical Dosage Forms

“…In addition, ALA exerts metalchelating capacities and functions as a cofactor in various mitochondrial enzyme complexes involved in the decarboxylation of alpha-keto acids. 4,5 ALA possesses two sulfur atoms (at C6 and C8) connected by disulfide, as shown in Figure (1). IUPAC nomenclature of ALA is (R)-5-(1,2-Dithiolan-3-yl) pentanoic acid or 6,8-dithiooctanoic acid with a molecular formula of C8H14O2S2 and a molecular weight of 206.32 g/mol.…”