Ratiometric fluorescence and colorimetric dual-mode sensing platform based on carbon dots for detecting copper(II) ions and D-penicillamine.

A sensing platform with both ratiometric fluorescence and colorimetric responses towards copper(II) ions (Cu2+) and D-penicillamine (D-pen) was constructed based on carbon dots (CDs). o-Phenylenediamine (OPD) was employed as a chromogenic development reagent for reaction with Cu2+ to generate the oxidation product 2,3-diaminophenazine (oxOPD), which not only emits green fluorescence at 555 nm, but also quenches the blue fluorescence of CDs at 443 nm via the inner filter effect (IFE) and Förster resonance energy transfer (FRET). Additionally, oxOPD exhibits obvious absorption at 420 nm. Since the intense chelation affinity of D-pen to Cu2+ greatly inhibits the oxidation of OPD, the intensity ratio of fluorescence at 443 nm to that at 555 nm (F443/F555) and the absorbance at 420 nm (A420) were conveniently employed as spectral response signals to represent the amount of D-pen introduced into the testing system. This dual-signal sensing platform exhibits excellent selectivity and sensitivity towards both Cu2+ and D-pen, with low detection limits of 0.019 µM and 0.092 µM, respectively. In addition, the low cytotoxicity of the testing reagents involved in the proposed sensing platform facilitates its application for live cell imaging.

Fluorescent MUA-stabilized Au nanoclusters for sensitive and selective detection of penicillamine.

In this study, we have developed a facile method for preparation of highly fluorescent Au nanoclusters (AuNCs) using 11-mercaptoundecanoic acid (MUA) as both the reducing and stabilizing agent. The as-prepared MUA functionalized AuNCs (MUA-AuNCs) have good water solubility, excellent photostability, and strong fluorescence emission at 610 nm with a quantum yield of 7.28% in water. The fluorescence of MUA-AuNCs was first quenched by copper ions through electron transfer, subsequently caused obvious restoration by competitive effect after adding penicillamine, making it a potential fluorescent sensor for penicillamine with a detection limit of 0.08 µM. Furthermore, the newly designed fluorescence "on-off-on" assay was explored for the measurement of penicillamine in complex real water and urine samples with satisfactory results.

A graphene quantum dots-Pb2+ based fluorescent switch for selective and sensitive determination of D-penicillamine.

Taking consideration of metal-induced fluorescence quenching and excellent coordination effect of D-penicillamine (D-PA), a graphene quantum dots (GQDs)-based fluorescent switch for D-PA detection was designed and established firstly with the help of lead ions. GQDs obtained from citric acids made them rich in carboxyl and hydroxyl groups, giving GQDs the ability to combine with lead ions. As anticipated, the fluorescence intensity was quenched by Pb2+ through electron transfer process. Further, the addition of D-PA effectively recovered the fluorescence due to the departure of Pb2+ from GQDs aroused by the strong coordination between D-PA and Pb2+. Thus, a fluorescent switch was activated for D-PA detection. The fluorescence recovery efficiencies were found to be proportional to the concentration of D-PA in the range of 0.6-50 µmol L-1 and the detection limit was 0.47 µmol L-1. The real sample detection was performed in human urea sample and satisfactory recoveries of 96.84%-102.13% were obtained. The GQDs-Pb2+ based fluorescent switch sensing method was firstly established with low detection limit and wide linear range, making it a supplement and improvement for D-PA detection.

The renal clearance of amino acids in cystinuria.

The renal clearance of cystine, lysine, ornithine, arginine, and glycine has been compared with the simultaneously determined glomerular filtration rate in nine cystinuric patients. Five were studied before and after stabilization on penicillamine therapy, two were studied only while taking penicillamine, and two were studied only in the absence of penicillamine administration. The renal clearances of cysteine-penicillamine and of penicillamine disulfide were also determined in the patients who were taking the drug. Amino acids were determined by quantitative ion exchange chromatography, and the reliability of the method has been evaluated in terms of its reproducibility and of the recovery of known amounts of amino acids added to plasma and to urine. The plasma levels of cystine and of the basic amino acids were less than normal in all the patients. Cysteine-penicillamine and penicillamine disulfide were cleared by the kidney at rates similar to that of cystine. Two of the patients had glycine clearances that were considerably above the normal value. The renal clearance of cystine exceeded the glomerular filtration rate in six of the nine patients. The results form a continuum from values approximately equal to the glomerular filtration rate to values about twice this amount. The renal clearances of cystine and of the basic amino acids vary independently of one another in the disease. The significance of these results is discussed in terms of the nature of the renal tubular transport defect that underlies the disorder.

Penicillamine kinetics in normal subjects.

The kinetic characteristics of penicillamine are reported in four fasting subjects after four oral doses each. On late test days, tow of the subjects received an additional single dose 30 min after a large breakfast. On subject originally included in the study had to drop out because of gastrointestinal disturbances following each of two single doses of penicillamine. The fasting plasma levels of penicillamine observed in this study displayed an unusual double peak in the plasma levels after single doses. Individual subjects had consistent plasma level patterns for each of the four single doses but there was marked intersubject variability in patterns and kinetic parameters. The half-life of unchanged penicillamine ranged from 1.66 to 3.15 hr and the apparent plasma clearance ranged from 530 to 2300 ml/min. The administration of penicillamine following a large breakfast caused a reduction in the area under the penicillamine plasma concentration-time curve corresponding to a decrease in the extent of absorption of unchanged penicillamine.

Determination of urinary cystine for monitoring anticystinuric therapy.

Cystine is assayed in urine by a method adapted from the procedure of Haux and Natelson (Clin Chem 1970;16:366-369) and modifications of the method, which improved its specificity, are presented. Assay imprecision (CV) at four different cystine concentrations (range: 0.042-1.658 mmol/l) varied from 23.1% to 3.7%. Analytical recovery was 94.6 +/- 6.0%. D-Penicillamine, alpha-mercaptopropionylglycine, L-glutamine and metabolites, added to urine, had negligible effect on the results of the cystine assay. Samples were stable for 3 wk when stored at 4 degrees C or frozen. Cystine excretion (mmol/24 h) in apparently healthy persons ranged from 0.041 to 0.170 (mean +/- SD: 0.099 +/- 0.039). Cystine excretions are presented in patients treated with anticystinuric drugs.

Determination of D-penicillamine in serum and urine of patients with rheumatoid arthritis.

A chromatographic auto-analyser method is described for the determination of total D-penicillamine in biological fluids. After oxidation with performic acid, D-penicillaminic acid is separated from other ninhydrin-positive acidic amino acids by anion-exchange chromatography. Total cysteine/cystine is simultaneously determined as cysteic acid, which makes the method equally suitable for patients with cystinuria. The detection limit in serum and urine is 2 microM D-penicillamine, which is more than sufficient for clinical application. The metabolite S-methyl-D-penicillamine is oxidized by performic acid to a sulphone, which can be determined after separation by cation-exchange chromatography.

Characterization of protein-bound gold in rat urine following aurothiomalate administration and of rat and human albumin-gold-thiomalate.

The metabolites of gold in the urine of rats given the antiarthritic drug aurothiomalate were investigated by gel permeation chromatography, electrophoresis, and chemical studies. Following a single dose of aurtothiomalate, the excreted gold was protein-bound in the high-molecular-weight (greater than or equal to 150,000 dalton) and serum albumin fractions. Electrophoresis confirmed the presence of albumin, but showed that the other proteins present differ from those in normal or in vitro aurothiomalate-incubated rat sera. The pattern of the proteins establishes that the proteinuria was of the glomerular type. The alterations in the gold distribution produced by incubation of the urine with the low-molecular-weight thiol penicillamine and with exogenously added aurothiomalate indicated the existence of a labile equilibrium of gold among protein binding sites in the urine. Incubation of rat and human sera and commercially prepared serum albumins with aurothiomalate increased the electrophoretic mobility of the albumin. The significance of this change in electrophoretic mobility with respect to two models of gold binding by serum albumin is discussed.

An overview of assay methods for D-penicillamine.

The stability and biotransformation of D-penicillamine as it relates to assay development is discussed. A review of published literature on assays is presented with respect to blood/plasma levels of D-penicillamine in man and to statistical evaluation of assays. Also, the paper describes gas chromatographic assay for D-penicillamine disulfide and L-cysteine D-penicillamine disulfide in urine and plasma, as well as a new high performance liquid chromatography assay for D-penicillamine in plasma.

The pharmacokinetics of D-penicillamine in man.

High performance liquid chromatography (HPLC), coupled with an amalgamated gold electrochemical detector, to measure plasma and urine concentrations of D-penicillamine, was used to determine the pharmacokinetics of this drug following both its intravenous and oral administration in doses of 800 mg. After iv administration, plasma elimination half-life (T 1/2 beta) was 62.7 +/- 5.3 min; plasma clearance (CI) 560.7 +/- 42.8 ml/min; volume of distribution (Vd), 57.0 +/- 9.3 l; and % D-penicillamine excreted within 24 h, 42.1% +/- 6.2%. Following oral administration, T 1/2 beta was 60.7 +/- 8.2 min, % D-penicillamine excreted within 24 h, 21.2 +/- 2.3%; and fraction of absorption (f) 41.2 +/- 5.5%. Urinary copper excretion paralleled urine and plasma D-penicillamine concentrations.

A survey of analytical approaches to the measurement of D-penicillamine and penicillamine disulfides.

This paper evaluates the usefulness of different methods for measuring aminothiols and disulfides in biological fluids. The major approaches usually involve liquid or gas-liquid chromatography. Techniques tested and currently used by the authors are discussed.

A simple method for determination of D-penicillamine on the carbon paste electrode using cupric ions.

The interaction of D-penicillamine (PA) with copper at the carbon paste electrode (CPE) in the presence of cupric ions (Cu(2+)) was used for the determination of PA at very low potential (0.1 V vs. Ag/AgCl) without applying of any modifier. The electrochemical response of copper is changed considerably in the presence of negligible amount of PA. In this report some important parameters, such as pH effect, Cu(2+) concentration and scan rate are studied, which the selected conditions were acetate buffer (pH=6) and 1 mM Cu(2+). The linear range for PA was from 1.0×10(-6) to 1.0×10(-4) M with an experimental detection limit of 1.0×10(-7) M. The relative standard deviation for 6 measurements was 3.8%. The interfering effects of some important inorganic ions were investigated, which there was no significant effect on the PA measurements. Also three organic interferences including ascorbic acid (AA), uric acid (UA) and l-cysteine (Cys) were examined, which the effect of AA was not notable, the interference of UA was moderate and for Cys was significant, but moderate at the levels which fined in the urine samples. This method was applied successfully for the determination of PA in urine sample.

Simultaneous determination of tiopronin and d-penicillamine in human urine by liquid chromatography with ultraviolet detection.

d-Penicillamine and tiopronin are drugs widely used for the treatment of many diseases. Because of the relatively high frequency of side effects to these compounds, some of which are dose-related, drug monitoring in urine samples during treatment is advisable. In this paper, we describe a simple method for the determination of tiopronin and d-penicillamine in human urine. The method was based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet-absorbance detection. 2-S-quinolinium derivatives of thiols were detected at 355 nm. The derivatization was optimized in terms of pH and time of the reaction. Baseline separation was achieved on an analytical Zorbax SB C-18 (5 microm, 150 mm x 4.6 mm) column with a mobile phase consisting of pH 2.0 0.09 mol L(-1) trichloroacetic acid buffer (component A) and acetonitrile (component B) pumped at 1.0 mL min(-1). Gradient elution was used: 0-4 min, 12% B; 4-8 min, 12-40% B; 8-12 min, 40-12% B. The d-penicillamine and tiopronin standards added to the urine show that the response of the detector is linear within the range studied, from 1 to 200 micromol L(-1) urine. The imprecision ranges for tiopronin and d-penicillamine were within 1.61-8.24% and 2.92-10.60%, respectively. The analytical accuracy for determined compounds was from 97.24 to 109.39%. The lower limits of detection and quantitation were 0.5 micromol L(-1) and 1.0 micromol L(-1) urine, respectively. This method can be used for routine clinical monitoring of the title thiol-drugs. Cysteine can be measured concurrently, if needed.

Rapid automated determination of D-penicillamine in plasma and urine by ion-exchange high-performance liquid chromatography with electrochemical detection using a gold electrode.

A high-performance liquid chromatographic method for the determination of D-penicillamine in plasma and urine has been developed, based on separation on a cation-exchange resin followed by an amperometric detection of the SH group of D-penicillamine oxidized on a gold electrode. The method has been automated and separations from plasma and urine take 7 and 9 min, respectively. The detection limits are 0.05 micrograms/ml in plasma and 0.2 micrograms/ml in urine, with a coefficient of variation of 2.9% (n = 10).

High-performance liquid chromatographic determination of penicillamine in whole blood, plasma, and urine.

A high-performance liquid chromatographic method for the determination of penicillamine in plasma, whole blood, and urine samples is described. The method uses a commercially available electrochemical detector at a potential of +0.1 V versus the Ag/AgCl reference electrode. This method is selective and sensitive for sulfhydryl compounds. The chromatography separates penicillamine from other endogenous sulfhydryl compounds with a limit of detection for penicillamine in biological samples of ca. 10(07) M.