Liquid Chromatography with Amperometric Detection at a Silver Based Detector for the Determination of Thiocompounds: Application to the Assay of Thiopurine Antimetabolites in Urine

Abstract

A silver amperometric detector coupled to liquid chromatography (LC) was used for the determination of 6-thioguanine (6-TG) and two of its metabolites, thiouric acid (TU) and 2-amino-6-mercaptopurine riboside (6-TGR). The silver detector coupled to LC operated at a low applied potential (0.08 V vs Ag/AgCl) and offered a chromatogram with peak responses corresponding to molecules interacting with silver, namely, chloride ions and small soluble biothiols in addition to the organothiol drug compounds investigated. Online electrochemical surface cleaning permitted the improvement of the repeatability and peak shape of the recorded signal compared to direct current amperometric detection (AD) when operating in chloride containing media. The studied molecules were eluted isocratically within 5 min on a reversed-phase C18 column without interference from endogenous biothiols present in urine samples. Diluted urine samples (1:1) were directly injected in the LC setup; a linear calibration curve was obtained between peak area and analyte concentration between 0.1 and 10 μM for all the studied molecules. Limits of detection (LODs) were 0.03, 0.008, and 0.01 μM, and the limits of quantification (LOQs) were 0.1, 0.02, and 0.03 μM for TU, 6-TG, and 6-TGR, respectively. Within-day RSDs were 2%, 0.8%, and 1% and between-day RSDs were 2%, 0.9%, 2% for TU, 6-TG, and 6-TGR, respectively. Recoveries in spiked urine were 99.8%, 99.9%, and 99.0% for TU, 6-TG, and 6-TGR, respectively.

Determination of Thiols and Free Thiol Content in a Protein with Liquid Chromatography Coupled to Amperometric Detection at a Silver Based Carbon Paste Electrode

“…The derivative started to form within 5 min and the reaction was completed after 20 min. Amperometric experiments were performed at +0.080 V vs. Ag/AgCl based on the CV profile (Figure ) and in agreement with previous LC‐AD results at a bare silver electrode . The flow rate was varied between 0.5 and 1.0 mL/min and best results were achieved at a flow rate of 0.7 mL/min.…”

“…C and D, respectively). DTT provided a peak twice as high as Cys and GSH likely because of its two functional groups reacting with Ag and silver ions at the electrode solution interface (Figure E) . The chromatogram of a reaction mixture between Cys and 2‐DPDS gave the product 2‐TP (t R 4.51 min) and the peak of Cys had completely disappeared indicating completion of the derivatization reaction (Figure F).…”

“…Thereaction can also be monitored by electrochemistry at as ilver based electrode thanks to the reactive thiol group.T his was confirmed by cyclic voltammetry of as olution of cysteine at the AgCPE (Figure 2). Thei nteraction of the silver composite electrode,p olarizeda t0 .08 V vs.A g/Ag + ,w ith Cys produced an oxidation wave close to 0.00 Volt correspondingt ot he catalytic oxidation of silver (particles) at the AgCPE [27][28][29].N oo xidation wave was obtained by CV of 2-DPDS in the potential range investigated other than an increase of the AgCPE capacitive current.…”

“…Several electrochemical methods have also been reported for the determination of free sulfhydryl groups [23] with good selectivity and sensitivity achieved when combined to liquid chromatography.T he LC detector may operate by amperometry or pulsed amperometry using gold, gold amalgamatedw ith mercury,s ilver or boron doped diamond as workinge lectrode [24][25][26][27][28][29].T o the best of our knowledge,a mperometric detection coupled to LC for the assay of biomoleculesb earing thiol groups following previous derivatizationh as not been reported.S ilverw as shown to be ag ood electrode substrate for the selective determination, at low appliedp otential, of thio-compounds in the submicromolar concentration range [22][23][24][25][26][27][28][29].I ta ppeared, however, to be not sensitive to large molecules such as proteins bearing free thiol groups such ash uman serum albumin (HSA). It was thus of interest to exploitt he DPDS derivatizationr eaction for subsequent LC-amperometric detection of the liberated thiol product namely 2-TP.T he detector used in the presentw ork consistedo fasilver modified solid carbon paste electrodea si to ffered the selective reactivityo f silver towards thiols,alow baseline currenta nd it was readily prepareda nd renewed.…”

Antineoplastic drugs and their analysis: a state of the art review

“…120,121,125 6-Methylmercaptopurine produced by TPMT was frequently sought in globules of patients undergoing treatment to define their phenotyping, since TPMT deficiency led to severe toxicity. 121 In addition to UV detection (which was the most widely used), amperometric detection (AD), 129 fluorescence detection (FD) 130 and MS detection 121,126,127,131 were also coupled to LC for the determination of purine analogues. LOQ of 1 ng mL −1 were usually obtained for purine analogues determination by LC-MS. 127,126 Concentrations of 6-mercaptopurine at 0.0615 ng mL −1 in urine samples were detected by a LC-FD method using metal palladium to form coordination complexes and enhance the detection signal.…”