Application of simultaneous separation and derivatization for the determination of α‐lipoic acid in urine samples by high‐performance liquid chromatography with spectrofluorimetric detection

Abstract

To help to clarify therapeutic functions of lipoic acid (LA) in biochemical and clinical practice we have elaborated a fast, simple and accurate HPLC method enabling determination of LA in human urine. The proposed analytical approach includes reduction of LA with tris(2‐carboxyethyl)phosphine and simultaneous separation and derivatization of the analyte with butylamine and o‐phthaldialdehyde followed by spectrofluorimetric detection at λex = 340 nm and λem = 440 nm. The assay was performed using gradient elution and the mobile phase containing 0.0025 mol L−1 o‐phthaldialdehyde in 0.0025 mol L−1 NaOH and acetonitrile. Linearity of the detector response for LA was observed in the range of 0.3–8 μmol L−1. Limits of detection and quantification for LA in urine samples were 0.02 and 0.03 μmol L−1, respectively. The total analysis time, including sample work‐up, was <20 min. The analytical procedure was successfully applied to analysis of real urine samples delivered from six healthy volunteers who received a single 100 mg dose of LA.

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

“…It indicates the need to control the concentration of each of these compounds. Although many analytical protocols dedicated to the determination of LA or metabolically related amino thiols in human plasma have been presented in the literature, all of them require the determination of LA and GSH, Cys, Hcy in two different assays [21][22][23][24][25][26][27][28][29][30].…”

“…Taking under consideration protocols described in previously published papers, the simultaneous determination of antioxidants such as LA and GSH, and its metabolic relatives in human plasma samples seems to be challenging. The assays require four steps of sample preparation protocols, such as reduction of disulfide bonds, derivatization of the thiol group, deproteinization and centrifugation for protein removal [21][22][23][24][25][26][27][28][29][30]. All of these compounds occur in the human body mainly in the oxidative form not accessible for the derivatization reagent.…”

“…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.…”

“…To avoid these troubles, usually deproteinization with the use of The high separation capacity of high performance liquid chromatography (HPLC) makes it the preferred technique for biological samples analysis. For quantification of LA in biological fluids, mainly in human plasma, several HPLC methods which exploit spectrophotometric [21], spectrofluorometric [22][23][24], electrochemical [25,26], and mass spectrometry [27] detection have been developed and described in the literature. Although these assays allow quantification of LA in human plasma, they do not give the possibility to control levels of metabolically important endogenous amino thiols such as GSH, Cys, Hcy, and Cys-Gly.…”

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

“…The schemes of the reactions of GSH with OPA and NAC with OPA and B–NH 2 are shown in Figure 1. On-column derivatization was successfully exploited earlier for HCY and/or methionine determination in plasma, urine, and wine samples [27,28,29] and for the determination of LA in urine samples [30]. The selection of optimal conditions for sample preparation was performed using the urine sample spiked with NAC as well as the plasma and homogenization of brain tissues spiked with GSH and NAC.…”