Measurement of reduced and total mercaptamine in urine using liquid chromatography with ultraviolet detection

Abstract

A simple liquid chromatographic method for the determination of reduced and total mercaptamine in human urine is described. The method is based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet-absorbance detection at 355 nm. Total mercaptamine was determined by reductive conversion of its oxidized fraction to the thiol form before the derivatization step. Baseline separation was achieved on an analytical Zorbax SB C(18) (5 microm, 150 x 4.6 mm) column with a mobile phase consisting of pH 2.0 0.05 mol L(-1) trichloroacetic acid buffer (component A) and acetonitrile (component B) pumped at 1.2 mL min(-1). Gradient elution was used: 0-3 min 12% B, 3-9 min 12-30% B, 9-12 min 30-12% B. The response of the detector was linear within the ranges studied, from 0.1 to 50 micromol L(-1) for reduced mercaptamine and from 0.4 to 400 micromol L(-1) for total mercaptamine. The imprecision ranges for reduced and total mercaptamine were within 1.45-11.71 and 0.73-10.61%, respectively. The analytical accuracy for determined compounds was from 98.79 to 109.77%. The lower limits of detection and quantitation were 0.05 and 0.1 micromol L(-1) of urine for reduced mercaptamine, and 0.2 and 0.4 micromol L(-1) of urine for total mercaptamine, respectively. This method can be used for routine clinical monitoring of the title thiol-drug and its reduced and oxidized fractions. Moreover, cysteine and cysteinylglycine can be measured concurrently, if needed.

Recent advances in separation and detection methods for thiol compounds in biological samples

“…The mild labeling conditions (i.e., room temperature for a few minutes in neutral medium) and the absorption property (around 355 nm) seem to be suitable for the determination of thiols. Of course, the method using CMQT is applicable not only for cysteamine, but also various thiol compounds such as HCySH and GSH [45][46][47][48][49][50]. Some sulfhydryl-containing drugs, i.e., Pen, TP and captopril, in human urine are also determined by LC with UV detection after derivatization with CMQT [51][52][53][54].…”

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis

“…7) at quaternary nitrogen atom (BCPB). This phenomenon allows the use of a large reagent excess in order to push reaction to the completion without excess reagent peak on the chromatogram [66]. Of different functionalities of aminothiols (e.g.…”

“…tration ranges in plasma, and particularly in urine, gradient elution is preferred [75][76][77][78][79][80][81][82][83][84][85], as is the case for cysteamine and endogenous thiols in plasma [80] or cysteine [70] and drugs tiopronin and Dpenicillamine in urine [83] shown in Fig. 12.…”

“…According to Dolan [145] ion-pairing reagents can never be washed fully from the column, even with extensive column flushing, and is advised to dedicate a particular column to ion-pairing application only. A satisfactory improvement of retention of the cationic thiols analytes can be achieved by chromatographing them with the mobile phase containing trichloroacetic acid [61][62][63][64][65][66][67][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85]89,91]. Trichloroacetic acid acts as pairing reagent and at the same time constitute the main component of the mobile phase buffer.…”

Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC

“…Over the years, several analytical methods have been developed for thiols determination such as liquid chromatography (LC) [20,21], gas chromatography [22,23], ion-exchange chromatography [24,25] and capillary electrophoresis [26,27]. High performance liquid chromatography (HPLC) with several detection techniques, such as ultraviolet [4,28,29], fluorescence (FL) [30][31][32], electrochemical [5,6,33] and mass spectrometry [34][35][36], is the most reported methodology. All the referred methods have basic limitations in terms of equipment, reagent costs, complexity, sample preparation, run time, number of thiols simultaneously quantified, and/or validation assessment, which delay their use for high-throughput routine clinical or research purposes [8].…”