Krzysztof Kuśmierek

Determination of endogenous thiols and thiol drugs in urine by HPLC with ultraviolet detection

Analysis of urine for endogenous thiols and thiol drugs content by HPLC with ultraviolet detection is addressed. Other methodologies for detection and determination of thiols in urine are only mentioned. Outline of metabolism, role of main biological thiols in physiological and pathological processes and their reference concentrations in urine are presented. In particular, urine sample preparation procedures, including reduction of thiol disulfides, chemical derivatization and reversed-phase HPLC separation steps are discussed. Some experimental details of analytical procedures for determination of endogenous thiols cysteine, cysteinylglycine, homocysteine, N-acetylcysteine, thioglycolic acid; and thiol drugs cysteamine, tiopronin, D-penicillamine, captopril, mesna, methimazole, propylthiouracil and thioguanine are reviewed.

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

Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.

Determination of methimazole in urine by liquid chromatography.

A liquid chromatography methodology is developed and validated for detection and quantification of methimazole in urine. The approach is based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate, reversed-phase high-performance liquid chromatography (HPLC) separation of so formed methimazole 2-S-quinolinium derivative from other urine matrix components, followed by detection and quantification with the use of ultraviolet-visible detector. Neither extraction, nor preconcentration of the sample are necessary. The methimazole standards added to normal urine before derivatization step show that the response of the detector, set at 345nm, is linear within the concentration range studied, that is, from 0.25 to 50mg/l urine. The relative standard deviation values for precision and recovery within the calibration range were from 1.8 to 5.0% and from 95.7 to 103.3%, respectively. Lower limits of detection and quantitation were 0.15 and 0.25mg/l urine, respectively.

Reversed-phase liquid chromatography method for the determination of total plasma thiols after derivatization with 1-benzyl-2-chloropyridinium bromide.

A high-performance liquid chromatography method was developed for simultaneous detection and quantitation of total cysteine, glutathione, homocysteine and cysteinylglycine in human plasma. The two key steps in the analysis are reduction of disulfides and treatment with 1-benzyl-2-chloropyridinium bromide, which rapidly and quantitatively reacts with thiol groups to form stable S-pyridinium derivatives with intense UV absorption. The derivatives are well separated on a Zorbax SB C(18) column using reversed-phase high-performance liquid chromatography and monitored at 315 nm. The calibration graphs were linear over concentration ranges covering most experimental and clinical cases with a regression coefficients better than 0.999. The detection and quantitation limits for all analytes were 0.2 and 0.5 micromol/L, respectively. The recoveries were 99.25-101.68%. The intra- and interassay imprecisions were 0.88-4.24 and 1.68-5.14%, respectively. The method was applied for plasma samples donated by apparently healthy volunteers.

Urinary mesna and total mesna measurement by high performance liquid chromatography with ultraviolet detection

We describe in this report a method for determination of mesna and total mesna in urine by high performance liquid chromatography with ultraviolet detection. The method involves a treatment of the urine sample with tri-n-butylphosphine in order to convert mesna disulfides to its reduced counterpart mesna, ultraviolet labelling with 2-chloro-1-methylquinoluinium tetrafluoroborate, reversed-phase HPLC separation, and detection and quantitation at 350nm. The result corresponds to total mesna that is sum of mesna, dimesna and its mixed disulfides with endogenous thiols. For determination of mesna the reduction step is omitted. Content of disulfide forms of mesna can be calculated by subtracting the concentration of mesna from the total mesna concentration. The separation of 2-S-quinolinium derivatives of mesna from those of endogenous urinary thiols and internal standard was achieved on an analytical Waters Nova-Pak C18 (150mmx3.9mm, 5mum) column. A mixture of an aqueous solution of pH 2.3, 0.05M trichloroacetic acid and acetonitrile (88:12, v/v) was used as a mobile phase at flow rate of 1.2ml/min and ambient temperature. The assay for mesna and total mesna in urine was proved to be linear over the studied ranges of 0.2-30 and 0.2-800nmol/ml urine, respectively. The mean recoveries over the calibration ranges were 95.4% for mesna and 99.7% for total mesna. The lower limits of detection and quantitation were 0.1 and 0.2nmol/ml for both the procedures, respectively. The imprecision did not exceed 8.5%. No interference from endogenous substances was observed.

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

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.