Detection of tiopronin in body fluids and pharmaceutical products using red-emissive DNA-stabilized silver nanoclusters as a fluorescent probe.

Tiopronin is a widely used drug for treatment of cystinuria, rheumatoid arthritis and hepatic disorders. It is also an antidote to heavy metal poisoning and a radioprotective agent. A method is described for rapid and sensitive determination of tiopronin using DNA-stabilized silver nanoclusters (DNA-AgNCs) as a fluorescent probe. Tiopronin can selectively bind to DNA-AgNCs to form a stable Ag-S bond upon which the red photoluminescence (best measured at excitation/emission wavelengths of 590/640 nm) is quenched. The finding is used to design an assay that has a linear response in the 1-150 nM tiopronin concentration range and a 270 pM limit of detection. Compared with previously reported methods, the present approach is more rapid, highly sensitive and selective. It has been successfully applied in the detection of tiopronin in spiked urine and serum, and in pharmaceutical products (tablets and injections). Graphical abstract An ultrasensitive and reliable method for tiopronin assay is developed using red-emissive silver nanoclusters as a fluorescent probe. It has been successfully applied in the determination of tiopronin in biological fluids and pharmaceutical products.

Simultaneous determination of tiopronin and its primary metabolite in plasma and ocular tissues by HPLC.

Tiopronin, formally 2-mercaptopropionylglycine (MPG), is currently prescribed to treat cystinuria and rheumatoid arthritis, and its antioxidant properties have led to its investigation as a treatment for cataracts, a condition in which oxidative stress is strongly implicated. To study its accumulation in the eye, a reliable, isocratic HPLC method was developed for the determination of MPG and its primary metabolite 2-mercaptopropionic acid (MPA) in plasma and relevant ocular tissues. This method utilizes pre-column derivatization and fluorescence detection. The 3.5 min separation enables high-throughput analysis, and validation experiments demonstrated that this method is suitable for evaluating ocular accumulation of MPG and MPA at concentrations as low as 66 and 33 nm, respectively. Excellent linearity was achieved over the working concentration range with R2 > 0.997. Extraction recovery was reproducible within each matrix and exceeded 97%. Accuracy was within 13.3% relative error, and intra- and inter-day precisions were within 6% CV and 7% CV, respectively. Sample stability was demonstrated under various storage conditions, and the use of an internal standard conferred exceptional ruggedness. This method has been successfully applied for the determination of MPG and MPA in plasma, cornea, lens and retina following intraperitoneal administration of the drug in Wistar rats.

Label-free fluorescence turn on detection of tiopronin with tunable dynamic range based on the ensemble of Alizarin Red S/copper ion.

In this study, a new type of rapid, label-free fluorescence turn-on assay for detection of tiopronin using Alizarin Red S (ARS)/copper ion ensemble is developed. ARS is high fluorescence in BR buffer solution. But, the fluorescence of ARS can be significantly quenched by copper ions due to ground-state complexation. However, in the presence of tiopronin, copper ions were released from the ARS and thus restored the fluorescence of ARS. The assay has several important features. First, the system is simple in design, fast in operation and is more convenient and promising than other methods. Second, the proposed assay eliminated the separation process and sophisticated instrumentations. Third, the detection process can be seen with the naked eye and can be easily adapted to automated high-throughput screening. At last, the assay has high sensitivity and selectivity for tiopronin and the detection limit is 0.8 ng/mL which is lower than or at least comparable to the previous methods. Moreover, the dynamic range of the sensor can be tuned simply by adjusting the concentration of copper ions. Importantly, the protocol offers high selectivity for the determination of tiopronin in pharmaceutical tablets, injection and biological samples with satisfactory results. Thus, the assay shows great potential applications in the fields of pharmaceuticals and clinical analysis.

Quantitative determination of tiopronin in human plasma by LC-MS/MS without derivatization.

Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC-MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4-dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post-treatment samples were analyzed on a C8 column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol-5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0-5000 ng/mL. The intra- and inter-day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC-MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP.

Sensitive LC-MS-MS method for the determination of tiopronin in human plasma.

A highly selective and sensitive LC-MS-MS method was developed and validated to quantify tiopronin in human plasma, using fudosteine as the internal standard (IS). L-Cysteine and 1,4-dithiothreitol (DTT) were used as the reducer and the stabilizer to release and stabilify tiopronin from a dimmer and mix forms with endogenous thiols in the treatment of plasma samples. After a simple liquid-liquid extraction with ethyl acetate in acidic condition, the post-treatment samples were analyzed on a C(18) column interfaced with a triple-quadruple tandem mass spectrometer using negative electrospray ionization. Methanol and water (40:60, v/v) were used as the isocratic mobile phase, with 0.2% formic acid and 1.0 mM tris (hydroxymethyl) aminomethane (Tris) in water. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy and precision of measurements. The assay was linear over the concentration range 0.078-10 microg/mL. The correlation coefficients for the calibration curves ranged from 0.9980 to 0.9990. The intra- and inter-day precisions, calculated from quality control samples, were not more than 10.49%. The method was employed in a pharmacokinetic study after oral administration of 200 mg tiopronin tablets to 24 healthy volunteers.

Stereoselective analysis of tiopronin enantiomers in rat plasma using high-performance liquid chromatography-electrospray ionization mass spectrometry after chiral derivatization.

A specific and relatively sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) was developed for the quantitative analysis of tiopronin enantiomers in rat plasma. The method is based on the derivatization of (+)-tiopronin and (-)-tiopronin with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate (GITC) in acetonitrile. The separation of resulting diastereomic derivatives was performed on C18 column (150 mm x 2.0 mm ID, packed with 5.0 mum C(18) silica RP particle), using a mobile phase of methanol/water (containing 5.3 mM formic acid) with gradient elution. LC-MS was performed in the selected ion monitoring and positive ion mode using target ions at m/z: 575 for the diastereomic derivatives of tiopronin and m/z: 603 for the derivative of N-isobutyryl-D-cysteine (internal standard). The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The calibration curves were linear over the concentration range of 0.025-5 microg/ml for both enantiomers of tiopronin. For both enantiomers of tiopronin, the interbatch and intrabatch variability values were less than 15%, and the accuracy was within +/-17% in terms of relative error. The method was successfully applied to a pharmacokinetic study of rac-tiopronin in rat.

[Determination of tiopronin in rat plasma by HPLC following fluorescent derivatization].

A sensitive, rapid method for determining reduced tiopronin concentration in rat plasma has been developed by using a high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl) maleimide (NPM). The analytes were separated on a Kromasil C18 column (250 mm x 4.6 mm, 5 microm) using 0.2% glacial acetic acid aqueous solution including 0.015 mol x L(-1) KH2PO4 and acetonitrile (56:44) as a mobile phase at a flow-rate of 0.8 mL x min(-1), and fluorescence detection wavelength were set at lamda(e x) = 340 nm and lamda(e m) = 375 nm, the column temperature was 30 degrees C. The calibration curve was found to be linear over a range of 0.1 - 10.0 microg x mL(-1), the limit of quantitation was 0. 1 mg x L(-1). The coefficients of the variation for the within-run and between-run precisions ranged from 5.3% to 10.8% and 7.0% to 10.8%, respectively. The percentage of absolute recovery ranged from 73.7% to 79.7%. The method was used to determine the concentration of tiopronin in rat plasma after a single intragastric administration of 25 mg x kg(-1) tiopronin to 6 healthy male Wistar rats. The pharmacokinetic process was fitted to a two-compartment model. The method has been successfully applied to the determination of tiopronin in rat plasma.

Determination of total tiopronin in human plasma by LC-ESI-MS using tris (2-carboxy-ethyl) phosphine as reducing reagent and methyl acrylate as derivatization reagent for the thiol group.

A quantitative method for the determination of total tiopronin (TP) in human plasma was developed by liquid chromatography with electrospray ionisation (ESI) mass spectrometric detection. After reduction with tris (2-carboxy-ethyl) phosphine (TCEP) and derivatization with methyl acrylate (MA) for the thiol group of TP, plasma samples were processed successively by deproteinization and solid phase extraction. N-acetyl-l-cysteine (NAC) was selected as internal standard undergoing the same treatment as TP. The method was validated that it could meet the need of biological analysis. The lower limit of quantitation (LLOQ) of TP in plasma was 0.02microg/mL. Finally, the method was successfully applied to a pharmacokinetic study in 20 healthy Chinese male volunteers after an oral dose of 200mg TP tablets.

High-performance liquid chromatography-electronspray ionization mass spectrometry for determination of tiopronin in human plasma.

A simple and sensitive HPLC/ESI-MS method for the determination of tiopronin in human plasma was described. Vitamin C and 2-mercaptoethanol (2-Me) were used as the reducer and the stabilitizer to release and stabilify tiopronin from a dimmer and mix forms with endogenous thiols in the treatment of plasma samples. The analytes were separated on a Johnson Spherigel analytical column packed with 5 microm C8 silica, using the formic acid aqueous solution (pH 4.5) including tris(hydroxymethyl) aminomethane (Tris) and 2-Me (0.5 and 1 mM, respectively) as a mobile phase. Cyclamate was used as the internal standard (I.S.) for the quantification of tiopronin. The correlation coefficient of the calibration curve were better than 0.998 in the range of 0.107-5.35 microg/ml in human plasma. The limit of quantification (LOQ) was 0.107 microg/ml (S/N 10:1, RSD 7.1%). The inter-day and intra-day accuracy was below 7.1 and 6.8%, respectively. As a preliminary application, this method has been successfully applied to the determination of tiopronin in the human plasma.

Liquid chromatography analysis of N-(2-mercaptopropionyl)-glycine in biological samples by ThioGlo 3 derivatization.

N-(2-Mercaptopropionyl)-glycine (MPG) is a synthetic aminothiol antioxidant that is used in the treatment of cystinuria, rheumatoid arthritis, liver and skin disorders. Recent studies have shown that MPG can function as a chelating, cardioprotecting and a radioprotecting agent. Several other studies have shown that it may also act as a free radical scavenger because of its thiol group. Thiol-containing compounds have been detected in biological samples by various analytical methods such as spectrophotometric and colorimetric methods. However, these methods require several milliliters of a sample, time-consuming procedures and complicated derivatization steps, as well as having high detection limits. The present study describes a rapid, sensitive and relatively simple method for detecting MPG in biological tissues by using reverse-phase HPLC. With ThioGlo 3 [3H-Naphto[2,1-b] pyran, 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) phenyl-3-oxo-)] as the reagent, highly fluorescent derivatives of thiols can be obtained that are suitable for HPLC. MPG is derivatized with ThioGlo 3 and is then detected flourimetrically by reverse phase HPLC using a C18 column as the stationary phase. Acetonitrile: Water (75:25) with acetic acid and phosphoric acid (1 mL/L) is used as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for MPG is linear over a range of 10-2500 nM (r=0.999) and the coefficients of the variation of within-run and between-run precision were found to be 0.3 and 2.1%, respectively. The detection limit was 5.07 nM per 20 microL injection volume. Quantitative relative recovery of MPG in the biological samples (plasma, lung, liver, kidney and brain) ranged from 90+/-5.3 to 106.7+/-9.3 %. Based on these results, we have concluded that this method is suitable for determining MPG in biological samples.

Simultaneous determination of tiopronin and its metabolites in rat blood by LC-ESI-MS-MS using methyl acrylate for stabilization of thiol group.

A sensitive and reproducible HPLC-electrospray tandem mass spectrometric method has been developed for the analysis of tiopronin (TP) and its metabolites, 2-mercaptopropionic acid (2-mpa) and S-methylated TP (SA13), in rat blood using methyl acrylate (MA) for the stabilization of a thiol group. The thiol groups of TP and 2-mpa in rat blood were immediately derivatized by the addition of MA-acetonitrile solution in 0.1 M Tris HCl (pH 9.1). The purification of the derivatives was accomplished by a simple liquid-liquid extraction procedure involving protein precipitation step. The analysis was performed on a Zorbax SB-C18 analytical column by a gradient elution with methanol-0.05 M acetic acid (15:85 and 7:3, v/v). Negative ion electrospray ionization with selected reaction monitoring was employed for the detection of analytes. Linearity of calibration was observed over the range of 0.5-1000 ng/ml for TP and 2-mpa, and 2-1000 ng/ml for SA13. The intra- and inter-assay variability for all analytes at the limit of quantitation (LOQ) level ranged from 5.47 to 16.75% and 4.95 to 7.23%, respectively. The LOQs estimated for TP, 2-mpa and SA13 were 0.5, 0.5 and 2 ng/ml, respectively. This assay method was successively applied to a pharmacokinetics study after an oral administration of TP (10 mg/kg) to rats.

Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine.

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine--cysteine, homocysteine, cysteinylglycine, and glutathione--as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97-107%, with little difference between plasma and urine samples. The detection limit was approximately 50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 micromol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.

Evidence for an essential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in conscious pigs.

Conscious pigs underwent a sequence of 10 2-min coronary occlusions, each separated by 2 min of reperfusion, for three consecutive days (days 1, 2, and 3). On day 1, pigs received an i.v. infusion of a combination of antioxidants (superoxide dismutase, catalase, and N-2 mercaptopropionyl glycine; group II, n = 9), nisoldipine (group III, n = 6), or vehicle (group I [controls], n = 9). In the control group, systolic wall thickening (WTh) in the ischemic-reperfused region on day 1 remained significantly depressed for 4 h after the 10th reperfusion, indicating myocardial "stunning." On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 53% on day 2 and 56% on day 3 compared with day 1 (P < 0.01), indicating the development of a powerful cardioprotective response (late preconditioning against stunning). In the anti-oxidant-treated group, the total deficit of WTh on day 1 was 54% less than in the control group (P < 0.01). On day 2, the total deficit of WTh was 85% greater than that observed on day 1 and similar to that observed on day 1 in the control group. On day 3, the total deficit of WTh was 58% less than that noted on day 2 (P < 0.01). In the nisoldipine-treated group, the total deficit of WTh on day 1 was 53% less than that noted in controls (P < 0.01). On days 2 and 3, the total deficit of WTh was similar to the corresponding values in the control group. These results demonstrate that: (a) in the conscious pig, antioxidant therapy completely blocks the development of late preconditioning against stunning, indicating that the production of reactive oxygen species (ROS) on day 1 is the mechanism whereby ischemia induces the protective response observed on day 2; (b) antioxidant therapy markedly attenuates myocardial stunning on day 1, indicating that ROS play an important pathogenetic role in postischemic dysfunction in the porcine heart despite the lack of xanthine oxidase; (c) although the administration of a calcium-channel antagonist (nisoldipine) is as effective as antioxidant therapy in attenuating myocardial stunning on day 1, it has no effect on late preconditioning on day 2, indicating that the ability of antioxidants to block late preconditioning is not a nonspecific result of the mitigation of postischemic dysfunction on day 1. Generation of ROS during reperfusion is generally viewed as a deleterious process. Our finding that ROS contribute to the genesis of myocardial stunning but, at the same time, trigger the development of late preconditioning against stunning supports a complex pathophysiological paradigm, in which ROS play an immediate injurious role (as mediators of stunning) followed by a useful function (as mediators of subsequent preconditioning).

Gas chromatographic-mass spectrometric determination of tiopronin in human blood using acrylic acid esters as a derivatization reagent for the thiol group.

A gas chromatographic-mass spectrometric method for determining tiopronin, which has a thiol group, in human blood has been described. To prevent the oxidative degradation of tiopronin in the blood, its thiol group was immediately protected by treatment with isobutyl acrylate, which reacted readily with tiopronin in a 0.1 M Na2HPO4 solution (pH 9.1). The reaction was quantitative within 30 min. The blood sample was deproteinized and purified by a combination of liquid-liquid extraction and solid-phase extraction. Finally, the carboxyl moiety of the ester adduct was derivatized to the pentafluorobenzyl ester. The derivatives of tiopronin and the internal standard were analysed with gas chromatography-mass spectrometry. The precision of the method was satisfactory, and the calibration curve had good linearity in the concentration range investigated. The limit of determination of tiopronin in blood was estimated to be ca. 1 ng/ml.

Pharmacokinetics of oral tiopronin.

Ten healthy subjects were given 500 mg (3064 mumol) tiopronin, or 2-mercaptopropionylglycine (2-MPG) by mouth. Cmax was reached after 3-6 h, and after a shorter beta-phase a long terminal half-life of 53 h of total tiopronin was found. Tiopronin measured as unbound (non-protein-bound) drug disappeared more rapidly from plasma, with a calculated t1/2 of 1.8 h. Mean residence time was higher (58 h) when calculated as total tiopronin than as unbound tiopronin (6 h), and this was also the case for the volume of distribution (V lambda = 455 l vs V lambda,u = 41 l). The results indicate extensive protein binding in plasma and a deep pool of tissue bound tiopronin after the first absorption and distribution phases. Absolute bioavailability (f) was 63%, and bioavailability calculated from urinary excretion was 47%, which are well correlated with each other. Urinary excretion was mainly confined to the first 6 h (74%) and was almost complete (98%) within 12 h. We conclude that the maximal absorption of the tiopronin was late, protein and tissue binding of the drug were high and its bioavailability varied. The renal excretion of low molecular weight tiopronin occurred early, which implies that the drug should be given in divided doses, at least twice daily, for optimal efficiency in the treatment of cystinuria.

Pharmacokinetics of 2-(alpha-thenoylthio)-propionylglycine (TTPG) in healthy volunteers--an oral dose-proportionality investigation.

The dose linearity of 2-(alpha-thenoylthio)-propionylglycine (TTPG) pharmacokinetics after a single oral administration at three different TTPG doses (180, 540 and 1080 mg) was evaluated in 12 healthy volunteers according to an open, randomized, cross-over study with a 1-week wash-out period between each administration. The duration of the study, for each subject, was 4 weeks. Plasma concentration and urinary excretion of TTPG and its two systemic metabolites, namely propionylglycine (tiopronin) and thiophenecarboxylic acid (TCA) were assayed by a previously well validated HPLC method. Due to differences in the physical and chemical properties of these compounds, two assays were needed, one to measure TTPG and TCA as such, and one to measure derivatized tiopronin. Both used UV detection. TTPG, tiopronin and TCA were quickly detected in plasma, suggesting that the drug administered is rapidly absorbed and biotransformed, in part, in the systemic circulation into the two metabolites noted above. Time-to-peak for all three analytes showed a trend to increase with increasing doses of TTPG, being: 0.42, 0.40 and 0.67 h (P < 0.01) with TTPG; 0.53, 0.47 and 0.73 h (P < 0.05) with TCA; and 1.33, 2.13 and 2.58 h (P < 0.01) with tiopronin. Cmax showed the opposite behaviour with values (ng ml-1) normalized to the dose of 540 mg: 1235, 905 and 513 (P < 0.001) with TTPG; 888, 547 and 383 (P < 0.001) with TCA; and 7290, 6950 and 5170 (P < 0.01) with tiopronin.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacokinetics of tiopronin and its principal metabolite (2-mercaptopropionic acid) after oral administration to healthy volunteers.

We have studied the pharmacokinetics of tiopronin and its principal metabolite, 2-mercaptopropionic acid (2-MPA) in healthy volunteers after the oral administration of 500 mg (2 Acadione tablets), followed by simultaneous assay of the two compounds in plasma over a period of 48 h using a new method (emission of fluorescence after HPLC and post-column derivatization by pyrene-maleimide). The absorption of tiopronin was slow (tmax between 4 and 6 h) and the plasma concentrations subsequently fell biexponentially. The principal metabolite 2-MPA appeared later in the plasma (tmax between 10 and 12 h after a lag-time of 3 h) then disappeared monoexponentially. About 15% of the tiopronin was metabolized to 2-MPA.

Determination of 2-mercaptopropionylglycine and its metabolite, 2-mercaptopropionic acid, in plasma by ion-pair reversed-phase high-performance liquid chromatography with post-column derivatization.

A simple and fast high-performance liquid chromatographic method was developed for the simultaneous measurement of 2-mercaptopropionylglycine (Tiopronine) and its metabolite (2-mercaptopropionic acid) in human plasma after the administration of a pharmaceutical dosage form (Acadione). The sample treatment before high-performance liquid chromatographic analysis consisted of the reduction of the corresponding disulphides by tri-n-butylphosphine and protein precipitation with ethanol. Separation was achieved by ion-pair high-performance liquid chromatography on a reversed-phase column (LiChrospher RP 18e) with cetrimonium bromide as counter ion and detection by fluorimetry after post-column derivatization with a selective thiol reagent, i.e. pyrenemaleimide. The high frequency of the analyzed samples and validation results make the method suitable for pharmacokinetic studies, and this was demonstrated by the first results obtained after the administration of an oral dose of 500 mg of Tiopronine to two healthy subjects.

Determination of 2-mercaptopropionylglycine in plasma and urine by high-performance liquid chromatography.

Methods for quantitative analysis of total and non-protein-bound 2-mercaptopropionylglycine (2-MPG) in plasma, and total 2-MPG in urine, have been developed. By reduction of urine, plasma or deproteinized plasma samples with tributylphosphine, 2-MPG is liberated from its disulphides, and after clean-up of the sample, 2-MPG is derivatized with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The 2-MPG-DACM derivative is then quantified by high-performance liquid chromatography (HPLC) with fluorimetric detection. Both ion-suppression and ion-pair HPLC gave satisfactory chromatograms. The precision of the methods was satisfactory (coefficient of variation 3.1-5.8%), analytical recovery was quantitative (85-99%) and the two HPLC techniques were well correlated (r = 0.99). Five healthy subjects receiving 500 mg of 2-MPG showed maximal total plasma concentration of 13.8-26.9 mumol/l at 3-5 h after intake, and their non-protein-bound 2-MPG was, at the same time, 62-77% of the total 2-MPG. The urinary excretion was 27.8 +/- 3.8% (mean +/- S.D.) of the given dose, most of it excreted within 12 h after intake.