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.
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