RESUMEN
Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is <â¯10â¯min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.
Asunto(s)
Disulfuros/aislamiento & purificación , Metaboloma , Estrés Oxidativo , Compuestos de Sulfhidrilo/aislamiento & purificación , Cromatografía Liquida , Disulfuros/sangre , Disulfuros/orina , Glutatión/sangre , Glutatión/aislamiento & purificación , Glutatión/orina , Humanos , Espectrometría de Masas , Oxidación-Reducción , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/orinaRESUMEN
In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4 nM and the linear range in a log-log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Disulfuros/análisis , Mediciones Luminiscentes , Compuestos de Sulfhidrilo/análisis , Automatización , Cromatografía Líquida de Alta Presión/instrumentación , Complejos de Coordinación/química , Disulfuros/sangre , Disulfuros/orina , Análisis de Inyección de Flujo , Humanos , Oxidación-Reducción , Fosfinas/química , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/orina , Zinc/químicaRESUMEN
Carboplatin elicits minor side effects with respect to its first generation analogue cisplatin. Nevertheless, a dose-dependent nephrotoxicity of the drug has been reported to occur both in patients and in rats and a possible pathogenic role have been attributed to oxidative stress. We have studied the effect of carboplatin administration on the thiol/disulfide balance, on other biomarkers of oxidative stress and on antioxidant enzymes in the isolated perfused rat kidney. A 5-500microM carboplatin dose range did not alter renal function but significantly decreased levels of cysteine, glutathione and exposed protein sulfhydryl groups. Only a minimal increment in disulfides was observed, whereas malonyldialdehyde and protein carbonyls did not increase significantly. Among the antioxidant enzymes studied, only thioltransferase was inhibited by the treatment. Our data suggest that a minimal oxidative stress is present under our experimental conditions, thus indicating that platinum-based drugs do not produce significant amount of reactive oxygen species.
Asunto(s)
Carboplatino/farmacología , Homeostasis/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Antioxidantes/metabolismo , Carboplatino/administración & dosificación , Disulfuros/metabolismo , Disulfuros/orina , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Riñón/citología , Riñón/fisiología , Estrés Oxidativo/efectos de los fármacos , Perfusión , Ratas , Ratas Sprague-Dawley , Compuestos de Sulfhidrilo/orinaRESUMEN
A method for the simultaneous determination of a series of thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound thiols by tris(2-carboxyethyl)phosphine, the newly formed thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further thiols. The method was successfully applied to the analysis of free and disulfide-bound thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of thiols in urine samples. The thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.
Asunto(s)
Cromatografía Liquida/métodos , Disulfuros/orina , Compuestos Ferrosos/química , Maleimidas/química , Espectrometría de Masas/métodos , Coloración y Etiquetado/métodos , Compuestos de Sulfhidrilo/orina , Femenino , Humanos , Masculino , Metalocenos , Estructura Molecular , Sensibilidad y EspecificidadRESUMEN
Radiolabelled [UL-14C]-diphenyl sulphide, [UL-14C]-diphenyl sulphoxide and [UL-14C]-diphenyl sulphone were administered by gavage (1.0 mmol/kg body weight) to adult male Wistar rats following an overnight fast. For all compounds, faeces were the major route of excretion of radioactivity (50%). Urinary elimination (40%) was similar during the first (19%) and second (16%) days and a small amount of radioactivity (6%) was found within the carcass after four days. From urinary and faecal data, metabolism occurred via ring hydroxylation with subsequent conjugate formation. Oxidation of the sulphur to form the sulphoxide and sulphone also took place; a small amount of sulphoxide reduction was apparent but no sulphone reduction was found. No evidence for exclusion of the sulphur was obtained, and it appeared unlikely that extensive cleavage of the ring structures occurred.
Asunto(s)
Compuestos de Azufre/metabolismo , Animales , Derivados del Benceno/metabolismo , Derivados del Benceno/orina , Radioisótopos de Carbono , Cromatografía en Capa Delgada , Disulfuros/metabolismo , Disulfuros/orina , Heces/química , Cromatografía de Gases y Espectrometría de Masas , Masculino , Oxidación-Reducción , Ratas , Ratas Wistar , Compuestos de Azufre/orinaRESUMEN
Healthy male and female human volunteers were exposed to 50 ppm or 100 ppm trichloroethylene (Tri) by inhalation for 4 h. Blood and urine samples were taken at various times before, during, and after the exposure period for analysis of glutathione (GSH), related thiols and disulfides, and GSH-derived metabolites of Tri. The GSH conjugate of Tri, S-(1,2-dichlorovinyl)glutathione (DCVG), was found in the blood of all subjects from 30 min after the start of the 4-h exposure to Tri to 1 to 8 h after the end of the exposure period, depending on the dose of Tri and the sex of the subject. Male subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 2 h after the start of the exposure of 46.1 +/- 14.2 nmol/ml (n = 8), whereas female subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 4 h after the start of the exposure of only 13.4 /- 6.6 nmol/ml (n = 8). Pharmacokinetic analysis of blood DCVG concentrations showed that the area under the curve value was 3.4-fold greater in males than in females, while the t1/2 values for systemic clearance of DCVG were similar in the two sexes. Analysis of the distribution of individual values indicated a possible sorting, irrespective of gender, into a high- and a low-activity population, which suggests the possibility of a polymorphism. The mercapturates N-acetyl-1,2-DCVC and N-acetyl-2,2-DCVC were only observed in the urine of 1 male subject exposed to 100 ppm Tri. Higher contents of glutamate were generally found in the blood of females, but no marked differences between sexes were observed in contents of cyst(e)ine or GSH or in GSH redox status in the blood. Urinary GSH output exhibited a diurnal variation with no apparent sex- or Tri exposure-dependent differences. These results provide direct, in vivo evidence of GSH conjugation of Tri in humans exposed to Tri and demonstrate markedly higher amounts of DCVG formation in males, suggesting that their potential risk to Tri-induced renal toxicity may be greater than that of females.
Asunto(s)
Glutatión/análogos & derivados , Solventes/farmacocinética , Tricloroetileno/farmacocinética , Adulto , Cromatografía Líquida de Alta Presión , Disulfuros/sangre , Disulfuros/orina , Femenino , Cromatografía de Gases y Espectrometría de Masas , Glutatión/sangre , Glutatión/orina , Humanos , Masculino , Factores Sexuales , Solventes/toxicidad , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/orina , Tricloroetileno/toxicidadRESUMEN
Two isozymes of mammalian methionine adenosyltransferase, MAT I and MAT III, are expressed solely in adult liver. They are, respectively, tetramers and dimers of a single subunit encoded by the gene MAT1A. A third isozyme, MAT II, contains a catalytic subunit encoded by a separate gene, MAT2A, and is expressed in a variety of tissues, including (to a slight extent) adult liver. Based on a recent finding that 2 children with isolated hypermethioninemia and brain demyelination were homozygous for MAT1A mutations predicted to produce severely truncated proteins, and devoid of activity when expressed, it was concluded that complete lack of MAT I/III activity may be associated with neurological symptoms and demyelination. We now report that a 43-year-old man with persistent isolated hypermethioninemia, previously demonstrated to have deficient MAT activity in his liver, has normal brain myelination on MRI and normal neurological function, despite being homozygous for a 539 TG insertion in exon V of MAT1A, so that the gene is predicted to encode a protein of only 184 rather than the normal 395 amino acids. This patient's exon V mutation was demonstrated by SSCP analysis and verified by sequencing. Both parents are heterozygous for the same insertion. This suggests that MAT1A mutations producing severely truncated proteins do not necessarily produce brain demyelination. This finding has relevance to a previously reported 4-year-old girl who was also homozygous for the 539insTG mutation. Finally, our patient's 7% residual hepatic MAT activity, measured at 1 mM methionine, may reflect the hepatic activity of the more ubiquitous enzyme form, MAT II.
Asunto(s)
Encéfalo/metabolismo , Hígado/enzimología , Metionina Adenosiltransferasa/deficiencia , Vaina de Mielina/metabolismo , Adulto , Pruebas Respiratorias , Preescolar , Análisis Mutacional de ADN , Disulfuros/orina , Femenino , Homocigoto , Humanos , Isoenzimas/metabolismo , Masculino , Metionina/metabolismo , Metionina Adenosiltransferasa/genética , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , Sulfuros/análisisRESUMEN
We report the results of a biochemical evaluation of long-term treatment of cystinuria with the SH compound tiopronin (2-mercaptopropionylglycine). The effects of tiopronin were studied by monitoring the urinary excretion of free cysteine and the mixed disulfide between tiopronin and cysteine. Thirty-one patients with homozygous cystinuria were treated with tiopronin for 0.4-12 years (mean 7.8 years). The urinary concentration of free cysteine was used to adjust the tiopronin dose. In 28 of the 31 patients a mean urinary cystine concentration of less than 1,200 mumol/1(288 mg/l) was achieved with the final dose. The final daily doses of tiopronin ranged from 250 mg (1.5 mmol) to 3,000 mg (18.4 mmol; mean 1,540 mg; 9.4 mmol). In a majority of the patients the treatment reduced the 24-hour urinary free cystine excretion effectively, on average by 0.61 mumol (0.15 mg)/mg of tiopronin administered. No changes in the efficacy of tiopronin over time were observed, and the frequency of adverse effects was acceptable. To evaluate the effects of tiopronin on the metabolism of cystine we calculated the total urinary excretion of cystine as the sum of free cystine and the amount of cystine corresponding to the cysteine content of the tiopronin-cysteine disulfide. At low doses of tiopronin there was an increase in urinary excretion of the mixed disulfide as well as of total cystine. Monitoring urinary cystine concentration is necessary to achieve adequate individualized doses of tiopronin. Assessment of the mixed tiopronin-cysteine disulfide and the urinary excretion of total cystine shows that tiopronin may interfere with cystine metabolism in a more complex way than through a simple disulfide exchange reaction with urinary cystine.
Asunto(s)
Cistina/metabolismo , Cistinuria/tratamiento farmacológico , Cistinuria/orina , Tiopronina/uso terapéutico , Adolescente , Adulto , Anciano , Cistina/análisis , Disulfuros/orina , Relación Dosis-Respuesta a Droga , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Tiopronina/orinaRESUMEN
A method for the determination of N-acetylcysteine, cysteine and their disulfides in urine is described. The thiols and disulfides are separated by reversed-phase ion-pair chromatography with octyl sodium sulfate as the ion-pairing reagent and detected with a dual-electrode amperometric detector using Au/Hg amalgam electrodes. Both the thiols and disulfides are detected with this system. In addition, dimers and mixed disulfides can be detected individually.
Asunto(s)
Acetilcisteína/orina , Cisteína/orina , Disulfuros/orina , Cromatografía Liquida , Electroquímica , HumanosRESUMEN
The urinary excretion of meso-2,3-dimercaptosuccinic acid (DMSA), which is an effective chelating agent for lead, was determined after the oral administration of 10 mg DMSA/kg to six normal young men. The DMSA that was absorbed was extensively biotransformed. After 14 hours only 2.53% of the administered DMSA was excreted in the urine as unaltered DMSA and 18.1% as altered forms. The unaltered DMSA was 12% of the total DMSA found in the urine. The altered form(s) of DMSA was 88% of the total urinary DMSA. The altered DMSA can be converted to unaltered DMSA by electrolytic reduction, which indicates that the altered forms of DMSA are disulfides. The excretion of altered DMSA reached a peak between 2 and 4 hours after DMSA administration. There were small but statistically significant increases in the excretion of zinc, copper, and lead after DMSA administration. DMSA did not influence the urinary excretion of 27 other metals and elements.
Asunto(s)
Succímero/farmacocinética , Compuestos de Sulfhidrilo/farmacocinética , Adulto , Análisis de Varianza , Animales , Cromatografía Líquida de Alta Presión , Disulfuros/orina , Humanos , Masculino , Metales/orina , Minerales/orina , Ratas , Ratas Endogámicas , Manejo de Especímenes , Espectrofotometría Atómica , Succímero/orinaRESUMEN
We developed a method to measure disulfides which is applicable to biological fluids. It consisted of two parts. First, certain thiols and disulfides were separated by HPLC. Second, the eluted materials were submitted to postcolumn reaction with 2-nitro-5-thiosulfobenzoate in the presence of sulfite. The resultant yellow product, 2-nitro-5-thiobenzoate, was measured by its absorbance at 412 nm. We determined the elution characteristics of the thiols and disulfides derived from cysteine, glutathione, alpha-mercaptopropionylglycine (Thiola), and cysteamine. Penicillamine and its disulfide did not react. Cystine in the urine of 22 cystinuric patients, measured by this method, was compared with results obtained by automatic amino acid analysis.
Asunto(s)
Cromatografía Líquida de Alta Presión , Disulfuros/análisis , Compuestos de Sulfhidrilo/análisis , Cistinuria/orina , Disulfuros/orina , Humanos , Nitrobenzoatos , Compuestos de Sulfhidrilo/orinaRESUMEN
Dimesna was given to volunteers (n = 6) and levels of free thiols, mesna, cysteine and disulphides measured in urine. Mesna is excreted in the urine following oral dimesna administration. Peak urinary free thiol levels occur between 10 and 20 hr. Cysteine and mixed disulphides are also excreted. Mesna might be useful in prolonged bladder protection during oxazaphosphorine cancer chemotherapy.
Asunto(s)
Mercaptoetanol/análogos & derivados , Mesna/análogos & derivados , Administración Oral , Adulto , Cromatografía Líquida de Alta Presión , Disulfuros/orina , Femenino , Humanos , Masculino , Mesna/administración & dosificación , Mesna/metabolismo , Mesna/orina , Compuestos de Sulfhidrilo/orinaRESUMEN
Acivicin (AT-125; 6.25-200 mumol/kg i.v.) inhibited hepatic, biliary and renal gamma-glutamyltranspeptidase (GGT) activity up to 88, 99 and 97%, respectively, in 4-week-old rats. This inhibition of GGT by acivicin resulted in a 10- to 12-fold increase in the biliary excretion of reduced (GSH) and oxidized glutathione. Because the biliary excretion of cysteinylglycine (Cys-Gly), Cys-Gly disulfide, cysteine (Cys) and cystine concomitantly decreased (63-99%), the biliary excretion rate of total glutathione-derived thiols and disulfides did not change. In contrast, acivicin treatment dramatically elevated the urinary excretion rate of glutathione-derived thiols in a dose-dependent fashion, resulting in a 390-fold increase at the highest dosage. This mainly originated from enhancement of urinary excretion of GSH (up to 7200-fold), although the excretion of Cys and Cys-Gly into urine was also increased. Acivicin treatment did not affect hepatic and renal levels of GSH but, at high dosages, reduced the concentration of Cys in these organs. GSH and oxidized glutathione concentrations in serum were increased, whereas cystine was diminished in acivicin-treated rats. Inhibition of GGT by acivicin (100 mumol/kg i.v.) failed to influence the biliary excretion of methylmercury but increased urinary excretion 34-fold. Even though the urinary thiol excretion was much higher than the biliary thiol excretion in the acivicin-treated rats, methylmercury was preferentially excreted into bile rather than urine, indicating the importance of the liver as an excretory organ for methylmercury.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Bilis/metabolismo , Glutatión/metabolismo , Riñón/enzimología , Hígado/enzimología , Compuestos de Metilmercurio/metabolismo , gamma-Glutamiltransferasa/antagonistas & inhibidores , Animales , Disulfuros/metabolismo , Disulfuros/orina , Glutatión/orina , Isoxazoles/farmacología , Masculino , Compuestos de Metilmercurio/orina , Ratas , Ratas Endogámicas , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/orinaRESUMEN
Quantitative methodology was developed for the analysis of disulfide-containing peptide analogues of vasopressin in biologic media. The procedure employs sample clean-up by an ion-exchange solid-phase extraction cartridge, followed by high-performance liquid chromatography with electrochemical detection. The detector is a dual Hg/Au system operated in series in which the disulfide-containing peptides are first reduced at the upstream electrode and then detected as free thiols at the downstream electrode. The assay is linear in the range 2-100 ng/ml (approximately 2-100 pmol/ml) of urine with a lower limit of detection of 1 ng (approximately 1 pmol) on column. The method displayed general utility for a number of structural analogues of vasopressin.
Asunto(s)
Disulfuros/análisis , Péptidos/análisis , Vasopresinas/análisis , Cromatografía Liquida , Disulfuros/orina , Electroquímica , Electrodos , Humanos , Péptidos/orina , Vasopresinas/orinaRESUMEN
Sodium 2-mercaptoethanesulphonate (MESNA) is a uroprotective agent generally given i.v. to prevent haemorrhagic cystitis during oxazaphosphorine cancer chemotherapy. Oral administration of the drug is described since this might be an important route during long-term oxazaphosphorine treatment. MESNA is absorbed from the GI tract and excreted in the urine (about 41.5% of the dose), peak excretion being 2-3 hr after administration. A proportion of the excreted dose is as free thiols (about 24.2%) and the remainder is as disulphides. MESNA is shown to enhance excretion of cysteine in urine.
Asunto(s)
Mercaptoetanol/análogos & derivados , Mesna/orina , Administración Oral , Adulto , Cisteína/orina , Disulfuros/orina , Humanos , Masculino , Mesna/administración & dosificación , Mesna/uso terapéutico , Compuestos de Sulfhidrilo/orina , Enfermedades de la Vejiga Urinaria/prevención & controlRESUMEN
Ultratrace levels of organotin species and an organosulfur compound were detected in a National Bureau of Standards (NBS) human urine Standard Reference Material, SRM 2670, and a previously certified urine SRM 2672, using a purge and trap system coupled to a gas chromatograph equipped with a flame photometric detector. samples of the SRM were treated with sodium borohydride to form volatile tin hydrides. Species detected included dimethyltin (1.04 ng/ml), butyltin (0.03 ng/ml), and dimethyl-disulfide (2.73 ng/ml) in the new stock of freeze dried human urine SRM 2670 being prepared for issue by NBS and methyltin (1.0 ng/ml), butyltin (1.5 ng/ml), and inorganic tin (28.1 ng/ml) in the old stock of SRM 2672. This analytical technique should have useful applications in studies that are needed to develop a toxicological data base and monitoring programs for human organotin exposure.
Asunto(s)
Compuestos Orgánicos de Estaño/orina , Azufre/orina , Cromatografía de Gases/métodos , Disulfuros/orina , Humanos , Fotometría/métodos , Estándares de ReferenciaRESUMEN
Analysis of urinary captopril was necessary for dosage form bioavailability and dose titration studies. The necessity for long-term storage of samples prior to analysis and the presence of an oxidation-prone thiol of captopril required development of an acid-chelate stabilization method for urinary captopril. An electrochemical reduction released disulfide-conjugated captopril for thiol colorimetry. Of several rugged reduction cells evaluated, one with a porous glass disk separating the anode and the mercury pool cathode was preferred. Methylene chloride partitions from acidified salt-saturated urines, before and after reduction, allowed the measurement of free and disulfide-conjugated captopril. The drug partitioned into the solvent, whereas the aqueous phase retained acid protonated, amino group-bearing thiols like cystine. Subsequent solvent evaporation volatilized other potential colorimetric interferences. An automated thiol colorimetry of 25 samples/hr was developed for analysis of the aqueous reconstitutes. Results were confirmed by a subsequently developed HPLC method with electrochemical detection.
Asunto(s)
Captopril/orina , Prolina/análogos & derivados , Colorimetría/métodos , Disulfuros/orina , Electroquímica , Humanos , Oxidación-Reducción , Solubilidad , Compuestos de SulfhidriloRESUMEN
A gas chromatographic-mass spectrometric method for the simultaneous determination of captopril (SQ 14,255) and its disulphide (SQ 14,551) in biological fluids by means of selected ion monitoring is described. In order to prevent oxidative degradation, captopril was treated with N-ethylmaleimide (NEM). The captopril-NEM adduct and the disulphide were converted into the hexafluoroisopropyl esters, which were separated on a 10% Dexsil 300 GC column and determined by employing the captopril-N-butylmaleimide adducts as an internal standard. The blood and urine levels of captopril and its disulphide in dogs to which captopril had been administered orally were measured by the proposed method. The urinary excretion of these two substances in rats was also determined in a similar manner.