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 μm, 150 mm × 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 μmol 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 μmol L⁻¹ and 1.0 μmol 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.     

Simultaneous analysis of citrulline and arginine in serum and tissue

We developed a method for simultaneously determining l-citrulline and l-arginine levels in serum and tissue samples using RP-HPLC with ultraviolet (UV) detection. The serum was deproteinized by trichloroacetic acid and heat; the tissue was homogenized by trichloroacetic acid and deproteinized as the same as serum. Phenyl-isothiocyanate (PITC) solution was used as derivatization reagent and a gradient elution was carried out. The linearity for l-arginine and l-citrulline ranged from 0 to at least 1000 μmol/L. R² values were above 0.9999 for both. LODs for l-arginine and l-citrulline were 0.0462 μmol/L and 0.0195 μmol/L, respectively, while LOQs were 0.530 μmol/L and 0.417 μmol/L, respectively. Intra- and inter-day CVs were less than 3.5% and 7.5% in serum, respectively. The average recovery was from 85.5% to 116.5% in serum. Intra-assay CVs were 3.8% and 10.3%, and inter-assay CVs were 13.7% and 10.7% for l-arginine and l-citrulline respectively in tissue. The average recovery was from 92.8% to 113.5% in tissue. This is a reliable and convenient analytical method which is suitable for most clinical laboratories.     

Simultaneous determination of 11 drugs belonging to four different groups in human urine samples by reversed-phase high-performance liquid chromatography method

A new, accurate, sensitive and fast reversed-phase high-performance liquid chromatography (RP-HPLC) as an analytical method for the quantitative determination of 11 drugs in human urine was worked out, optimized and validated. The objects of analysis were imipenem (IMP), paracetamol (PAR), dipyrone (DPR), vancomycin (VCM), amikacin (AMK), fluconazole (FZ), cefazolin (CFZ), prednisolone (PRE), dexamethasone (DEX), furosemide (FUR) and ketoprofen (KET) belonging to four different groups (antibiotics, analgesic, demulcent and diuretic). For HPLC analysis, diode array (DAD) and fluorescence (FL) detectors were used. The separation of analyzed compounds was conducted by means of a LiChroCART^® Purospher^® C₁₈e (125 mm × 3 mm, particle size 5 μm) analytical column with LiChroCART^® LiChrospher^® C₁₈ (4 mm × 4 mm, particle size 5 μm) pre-column with gradient elution. Analyzed drugs were determined within 20 min. The mobile phase was comprised of various proportions of methanol, acetonitrile and 0.05% trifluoroacetic acid in water. AMK was separated and determined from human urine using ortho-phthaldialdehyde-3-mercaptopropionic acid (OPA-3-MPA) as a fluorescent reagent by RP-HPLC-FL. The following retention times for drugs IMP, PAR, DPR, VCM, AMK, FZ, CFZ, PRE, DEX, FUR and KET in human urine were found: 4.01 min, 4.86 min, 6.71 min, 8.14 min, 9.46 min, 10.01 min, 10.90 min, 13.34 min, 14.06 min, 16.03 min and 18.98 min, respectively. Excellent linearity was obtained for compounds in the range of concentration: 0.35-42 μg ml⁻¹, 0.5-45 μg ml⁻¹, 4.5-38 μg ml⁻¹, 0.25-25 μg ml⁻¹, 0.5-35 μg ml⁻¹, 0.25-22 μg ml⁻¹, 0.03-52 μg ml⁻¹, 0.15-25 μg ml⁻¹, 0.25-28 μg ml⁻¹, 0.05-18 μg ml⁻¹ and 0.15-35 μg ml⁻¹ for IMP, PAR, DPR, VCM, AMK, FZ, CFZ, PRE, DEX, FUR and KET, respectively. The limits of detection (LOD) and limits of quantification (LOQ) for analyzed drugs were calculated in all cases and recovery studies were also performed. Ten human urine samples obtained from patients treated in hospital have been tested. In analyzed samples, one or more drugs from the 11 examined drugs were detected. The concentrations of examined drugs in urine samples ranged between: 1.5-12 μg ml⁻¹ of PAR, 5.2-11.5 μg ml⁻¹ of DPR, 0.13-9.5 μg ml⁻¹ of CFZ and 0.1-8 μg ml⁻¹ of FUR. This method can be successfully applied to routine determination of all these drugs in human urine samples.     

Rapid analysis of L-dopa in urine samples using gold nanoelectrode ensembles

This work describes the application of a three-dimensional gold nanoelectrode ensembles (GNEE) for monitoring l-dopa in standards and human urine samples using flow injection analysis (FIA) with amperometric detection. Analytical results reveal that the GNEE exhibited better electrocatalytic activity than a gold disk or glassy carbon electrode. Under optimal conditions of l-dopa analysis at GNEE, the calibration plot has a linear range of 5-300 ng/mL with a coefficient of variation (CV) of 3.1% in pH 7.0 phosphate buffer saline (PBS, pH 7.0). The detection limit was 3.0 ng/mL for FIA. The high precision and sensitivity of GNEE provides a feasible means of directly determining l-dopa in urine samples.     

Determination of L- and D-enantiomers of methotrexate using amperometric biosensors

In order to determine the enantiopurity of methotrexate (Mtx), seven biosensors were proposed for the assay of l-Mtx and three biosensors for the assay of d-Mtx. The biosensors were designed using physical and chemical immobilization of glutamate oxidase and/or l-amino acid oxidase (l-AAOD) and/or horseradish peroxidase (HRP) for the assay of l-methotherexate, and d-amino acid oxidase (d-AAOD) and HRP for the assay of d-Mtx. Electrode characteristics were obtained and compared for the different carbon paste based biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol l⁻¹ to pmol l⁻¹, magnitude order with limits of detection in the fmol l⁻¹ to nmol l⁻¹ concentration range. All biosensors were successful for the determination of the enantiopurity of Mtx as raw material, and in its pharmaceutical formulations (tablets and injections).     

A highly efficient synthesis of unnatural l-sugars from d-ribose

A preparative and short synthesis of l-ribose and l-apiose was accomplished starting from d-ribose via stereoselective cis-dihydroxylation and C2-hydroxymethylation, respectively. These l-sugars can serve as versatile intermediates for the synthesis of l-nucleosides.     

Solid phase extraction of Co ions using l-tyrosine immobilized on multiwall carbon nanotubes

A study was performed to assess the performance of aminoacids immobilized on carbon nanotubes (CNTs) for their employment as a sorbent for solid phase extraction systems. An immobilization method is introduced and the aminoacid l-tyrosine was chosen as a case study. A spectrophotometric study revealed the amount of aminoacid immobilizated on CNTs surface, and it turned to be of 3174 μmol of l-tyr g⁻¹. The material was tested for Co retention using a minicolumn inserted in a flow system. At pH 7.0, the amount of Co retained by the column was of 37.58 ± 3.06 μmol Co g⁻¹ of CNTs. A 10% (v/v) HNO₃ solution was chosen as eluent. The pH study revealed that Co binding increased at elevated pH values. The calculation of the mol ratio (moles of Co bound at pH 9 to moles of l-tyr) turned to be 3:1. The retention capacity was compared to other bivalent cations and showed the following tendency: Cu²⁺ > Ni²⁺ > Zn²⁺ ? Co²⁺. The analytical performance was evaluated and an enrichment factor of 180 was obtained when 10 mL of 11.37 μg L⁻¹ Co solution was loaded onto the column at pH 9.0; reaching a limit of detection (LoD) of 50 ng L⁻¹. The proposed system was successfully applied to Co determination in QC-LL2 standard reference material (metals in natural water).     

Enthalpies of transfer of amino acids from water to aqueous solutions of formamide

Enthalpies of solution of glycine, l-alanine, l-serine in water and aqueous solutions of formamide were measured at 298.15 K. Transfer enthalpies of amino acids from water to aqueous solutions of formamide were derived and interpreted qualitatively with hydration co-sphere overlap model. The results show that the structure interaction between formamide and zwitterionic head-group and hydrophilic side chain of amino acids make a negative contribution to transfer enthalpy, while that with the hydrophobic side chain is positive. In the solvent composition range studied, transfer enthalpies decrease overall with the increasing concentration of formamide, with the relative order of l-serine < glycine < l-alanine.     

Electropolymerisation of L-arginine at carbon paste electrode and its application to the detection of dopamine, ascorbic and uric acid

L-arginine was electropolymerised on a carbon paste electrode (CPE) to form the biopolymer by free radical formation in the electro oxidation process of the amino and carboxylic group containing compound by cyclic voltammetric technique. The modified electrode shows an excellent electrocatalytic activity towards the oxidation of both dopamine (DA) and ascorbic acid (AA). It was demonstrated that the deposited biopolymer has positive charges over the bare carbon electrode surface, which leads to the formation of electrical double layer made the fast electron transfer process could leads to the diffusion of dopamine, ascorbic acid and uric acid on their charge gradient by cyclic voltammetric technique. The response of the sensor was tested towards the different dopamine concentration. The catalytic peak current obtained was linearly related to DA concentrations in the ranges of 5×10(-5) to 1×10(-4)M L(-1) with correlation co-efficient of 0.9924 which reveals the adsorption controlled process. The detection limit for dopamine was 5×10(-7)M L(-1). The interference studies showed that the modified electrode exhibits excellent selectivity in the presence of large excess of ascorbic acid (AA) and response is fast stable, reliable, resistant to biofouling and can be applied for the real sample analysis in medical, pharmaceutical and biotechnological sectors. The adsorption-controlled process and kinetic parameters of the poly(L-arginine) were determined using electrochemical approaches.     

An amperometric method for the determination of trace mercury(II) by formation of complexes with l-tyrosine

A selective and sensitive amperometric method of analysis has been developed for determination of the trace amounts of mercury in waters at a platinum electrode based on the effect of the presence of mercury ions on the current due to oxidation of l-tyrosine. A decrease of signal was observed due to the formation of a complex of tyrosine with the Hg(II) ion adsorbed on the electrode surface. Several parameters were varied, such as applied potential, pH and concentration of tyrosine. The calibration plot was linear in the range from 0.02 to 3 μmol l⁻¹ Hg(II) with r=0.997 and the detection limit (3σ) was 0.014 μmol l⁻¹; the relative standard deviation was 2.2%. The study of interferences from other metal ions revealed a good selectivity of this method towards mercury(II). The stoichiometry of the mercury-tyrosine complex was determined to be 1:2 and the formation constant 627±19. Formation of complexes with mercury ions was also demonstrated with several catechol compounds and other amino acids. The method was applied to the analysis of contaminated waters.     

A fully automated system for inorganic antimony preconcentration and speciation in urine

A study was undertaken to ascertain the analytical capabilities of l-methionine immobilized on controlled pore glass for Sb preconcentration and speciation. A fully automated on-line system, implemented with hydride generation (HG) and inductively coupled plasma optical emission spectrometry (ICP OES), was used. Sb(III), at pH 10 was selectively retained in the column containing the immobilized aminoacid, while Sb(V) was not retained at all. A 30% HCl solution was used as eluent agent. Prior to total Sb determination, a pre-reduction step with thiourea was necessary. An on-line pH adjusting and pre-reduction of Sb(V) was achieved in a fully automated system. The detection limit for the preconcentration of 10 mL of an aqueous solution was 70 ng L⁻¹ with a relative standard deviation of 2%. An enrichment factor of 20 was achieved when 10 mL of sample was passed through the system, reaching a throughput of 23 samples per hour. The method was successfully applied to the determination of Sb(III) and total Sb in urine.     

A novel capillary electrophoresis method for the determination of d-serine in neural samples

A capillary electrophoresis method has been developed for the determination of d-serine in neural samples. d/l-serine was tagged with naphthalene-2,3-dicarboxaldehyde (CBI-d/l-Ser), and the separation of CBI-d/l-Ser enantiomer was achieved by using a dual chiral selector system consisting of β-cyclodextrin (β-CD) and chiral micelles formed by sodium deoxycholate (SDC). No resolution was observed when either β-CD or SDC was used alone. Moreover, the combined use of β-CD with achiral micelles of sodium dodecylsulfate (SDS) exhibited no resolving effect. With laser induced fluorescence detection, the limit of detection was 3.0 × 10⁻⁸ M Ser. Under the separation conditions selected, no other amino acids co-eluted with l-/d-Ser enantiomers. Using the present method, d-Ser level in Aplysia ganglia homogenates was found to vary significantly from animal to animal. Interestingly, d-Ser was not detected in single neurons isolated from Aplysia ganglia.     

Enantioanalysis of l-2-hydroxyglutaric acid in urine samples using enantioselective, potentiometric membrane electrodes based on maltodextrins

Quantitative assay of l-2-hydroxyglutaric acid (l-2-HGA) is important for the diagnosis of l-2-hydroxyglutaric aciduria. Three enantioselective, potentiometric membrane electrodes (EPMEs) based on maltodextrins with different dextrose equivalent (DE) (DE: 4.0-7.0 (I), 13.0-17.0 (II), 16.5-19.5 (III)), were designed for the enantioanalysis of l-2-HGA. The enantioselective, potentiometric membrane electrodes can be used reliably for enantiopurity assay of l-2-HGA using the direct potentiometric method in the ranges of 10⁻⁹-10⁻⁵, 10⁻⁶-10⁻³ and 10⁻⁸-10⁻⁵ mol L⁻¹ for the enantioselective, potentiometric membrane electrodes based on maltodextrins I, II and III, respectively, with very low detection limits. A high reliability was obtained when the electrodes were used for the assay of l-2-HGA in urine samples.     

A d,l-proline catalyzed diastereoselective trimolecular condensation: an approach to the one-pot synthesis of perhydrofuro[3,2-b]pyran-5-ones

d,l-Proline was found to catalyze efficiently the one-pot trimolecular condensation of indoles, a sugar hydroxyaldehyde, and Meldrum’s acid followed by intramolecular cyclization with evolution of carbon dioxide and elimination of acetone to afford 7-(1H-3-indolyl)-2,3-dimethoxyperhydrofuro[3,2-b]pyran-5-ones. The reaction proceeded cleanly at ambient temperature to afford the products in good yields with high diastereoselectivity.     

A flexible approach for the synthesis of selectively labelled l-arginine

A simple and efficient synthesis of l-arginine has been achieved in 12 steps and 24% overall yield. Regioselective reduction and functional group manipulation of the β-side chain of aspartic acid allowed the preparation of an ornithine derivative, which was then guanylated with a bis-protected 1-guanyl-pyrazole and deprotected to give l-arginine. This approach allows the flexible incorporation of stable isotopes and this is demonstrated using potassium ¹³C-cyanide, which has resulted in the preparation of 5-¹³C-l-arginine.     

New building blocks for peptide and depsipeptide synthesis: hexafluoroacetone protected l-homoisoserine and d,l-homoisocysteine derivatives

Efficient syntheses of l-homoisoserine and d,l-homoisocysteine derivatives starting from l-malic and d,l-thiomalic acid using hexafluoroacetone as protecting and activating agent are described. The new compounds are interesting building blocks for the preparation of non-natural peptides and depsipeptides as well as for the construction of new GABA derivatives.     

An efficient synthesis of l-allono-1,4-lactone from 2,3:5,6-di-O-isopropylidene-d-mannono-1,4-lactone

We reported herein an efficient synthesis of l-allono-1,4-lactone from 2,3:5,6-di-O-isopropylidene-d-mannono-1,4-lactone in five steps. The key feature of this method involved a one-pot, ‘double inversion’ procedure at the stereocenters of C-4 and C-5 of d-mannono-1,4-lactone to afford the target molecule.     

l-Arginine bearing an anthrylmethyl group: fluorescent molecular NAND logic gate with H and ATP as inputs

A novel l-arginine derivative was synthesized and its interactions with ATP in aqueous solutions were demonstrated by fluorescence, UV-vis, and ¹H NMR spectroscopy. A two-input NAND logic gate was presented on the basis of this single molecule.     

Separation and determination of l-tyrosine and its metabolites by capillary zone electrophoresis with a wall-jet amperometric detection

A method of capillary electrophoresis with wall-jet amperometric detection (AD) has been developed for separation and determination of l-tyrosine (Tyr) and its metabolites, such as Tyramine (TA), p-hydroxyphenylpyruvic (pHPP), homogentisic acid (HGA) and some dipeptides containing Tyr, such as Tyr-Gly-Gly (YGG), Tyr-Arg (YR) and Tyr-d-Arg (Y-d-R). A carbon disk electrode was used as the working electrode and the optimal detection potential was 1.00 V (versus Ag/AgCl). At 18 kV of applied voltage, the seven compounds were completely separated within 20 min in 110 × 10⁻³ mol/L Na₂HPO₄-NaH₂PO₄ buffer (pH 7.10) containing 3 × 10⁻³ mol/L β-cyclodextrin (β-CD). Good linear relationship was obtained for all analytes and the detection limits of seven analytes were in the range of 0.95-4.25 ng/mL. The proposed method has been applied to examine the metabolic process of l-tyrosine in rabbit's urine.