Determination of oxalate in urine and plasma using reversed-phase ion-pair high-performance liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II)-electrogenerated chemiluminescence detection

Oxalate is quantitated in both urine and plasma samples using reversed-phase ion-pair high-performance liquid chromatography (HPLC) with tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺]-electrogenerated chemiluminescent (ECL) detection. Underivatized oxalate was separated on a reversed-phase column (Zorbax ODS) using a mobile phase of 10% methanol in 100 mM phsophate buffer at pH 7.0. The eluted compounds were combined with a stream of 2 mM Ru(bpy)₃²⁺ at a mixing tee before the ECL flow-cell. In the flow-cell, Ru(bpy)₃²⁺ is oxidized to Ru(bpy)₃²⁺ at a platinum electrode, and reacts with oxalate to produce chemiluminescence (CL). Urine samples were filtered and diluted prior to injection. Plasma samples were deproteinized before injection. A 25-μl aliquot of sample was injected for analysis. Possible interferants, including amino acids and indole-based compounds, present in biological samples were investigated. Without the separation, amino acids interfere by increasing the total observed CL intensity; this is expected because they give rise to CL emission on their own in reaction with Ru(bpy)₃³⁺. Indole compounds exhibit a unique interference by decreasing the CL signal when present with oxalate. Indoles inhibit their own CL emission at high concentration. By use of the indicated HPLC separation, oxalate was adequately separated from both types of interferants, which thus had no effect on the oxalate signal. Urine samples were assayed by both HPLC and enzymatic tests, the two techniques giving similar results, differing only by 1%. Detection limits were determined to be below 1 μM (1 nmol/ml) or 25 pmol injected. The working curve for oxalate was linear throughout the entire clinical range in both urine and plasma.     

A dimeric tris(2,2′-bipyridine)ruthenium(II) system: Emission spectrum and cyclic voltammogram

A dimeric ruthenium(II) compound in which two Ru(bpy)₃ groups are linked by an amide bonding has been prepared as a model compound to study an energy transfer between Ru(bpy)₃ chelates. The nature of the solution luminescence spectrum varied with concentration: the emission maximum appeared at 650 nm for dilute solutions and at 670 nm for concentrated solutions. This concentration dependence has been interpreted in terms of excimers that are formed due to an energy transfer between two Ru(bpy)₃ groups in a dimer molecule. The cyclic voltammogram for the Ru³⁺/Ru²⁺ reaction is quasireversible: the reaction is governed by a sluggish electron transfer which may be due to an intradimer electronic interaction.     

Determination of lansoprazole in pharmaceuticals using flow injection with rhodamine 6G–diperiodatoargentate(III) chemiluminescence detection

A chemiluminescence (CL) method based on rhodamine 6G (R6G)–diperiodatoargentate(III) (silver(III) complex) reaction in acid solution is reported for the determination of lansoprazole (LNP) combined with a flow injection (FI) technique. The most likely mechanism for CL reaction was elucidated considering reported data, spectrophotometric and spectrofluorimetric studies. The weak CL reaction between R6G and silver(III) complex could be magnanimously increased in the presence of LNP with a limit of detection (LOD) of 0.002 mg L⁻¹ (S/N = 3), a linear range of 0.01 to 10 mg L⁻¹ (R² = 0.9997, n = 7), a relative standard deviation (RSD) of 1.2 to 3.2% (n = 4) and an injection throughput of 140 h⁻¹. No interference activity of commonly found excipients in LNP was detected. After LNP extraction from pharmaceutical samples, the recovery rate ranging from 93 to 110% (RSD, 1.4–3.3%, n = 4) was calculated. The results of the proposed flow CL method were assessed with a spectrophotometric approach applying paired Student's t-test and the calculated value (0.178) was lower than the distributed value (2.20) at a 95% confidence limit.     

Specific cation effect on quenching reactions of excited tris(α,α-diimine)ruthenium(II) and tris(2,2-bipyridine)chromium(III) by tris(oxalato)- and tris(malnato)chromates(III) in aqueous solutions

Specific salt effects were studied on the quenching reaction of excited [Ru(N-N)₃]²⁺ (N-N=2,2^′-bipyridine (bpy), 1,10-phenanthrorine (phen)) and [Cr(bpy)₃]³⁺ by [Cr(ox)₃]³⁻ (ox=oxalate ion) and [Cr(mal)₃]³⁻ (mal=malonate ion) in aqueous solutions as a function of alkali metal ions which were added for adjustment of ionic strength. The value of k_q, quenching rate constants, and k₁, energy transfer rate constant in encounter complex, is changed by the cations as the order of Li⁺ > Na⁺ > K⁺ ≈ Rb⁺ ? Cs⁺, although diffusion rate constants are not changed by the co-existing cations. Among the quenching reactions investigated in this work, a ratio of k₁ values in the aqueous solutions whose ionic strength was adjusted with LiCl and KCl, k₁^LiCl/k₁^KCl, is larger for quenching systems of closely approached donor-acceptor pair than loosely bounded pair. These results indicate that co-existing alkali cation tunes the distance between donor and acceptor in encounter complex where energy transfer occurs.     

Determination of organophosphate flame retardant tris(2-chloroethyl)phosphine based on the luminol–H2O2 chemiluminescence system

Organophosphorus flame retardants (OPFRs) are new types of environmental pollutants, therefore the rapid and sensitive detection of OPFRs is a very important objective. A new experimental phenomenon was found in which tris(2-chloroethyl)phosphine (TCEP), a type of OPFR, could effectively enhance the signal of the luminol–H₂O₂ chemiluminescence (CL) system. Combined with the controllability of flow injection analysis, a rapid, stable, and sensitive CL method was established. The CL intensity responded linearly to the concentration of TCEP in the range 0.5–100 μg/L (R² = 0.999) with a low detection limit of 33 ng/L. Relative standard deviation (RSD) was 2.2% (n = 7, c = 100 μg/L). Water samples were labelled and recycled with RSDs of 1.1–5.7% and recoveries of 88.7–116.1%. Based on these results, this study established a new CL detection method for the environmental pollutant TCEP.     

Multispectroscopic DNA-binding studies of a terbium(III) complex containing 2,2′-bipyridine ligand

Agarose gel electrophoresis, absorption, fluorescence, viscosity, and circular dichroism (CD) have been used in exploring the interaction of terbium(III) complex, [Tb(bpy)₂Cl₃(OH₂)] where bipy is 2,2′-bipyridine, with Fish salmon DNA. Agarose gel electrophoresis assay, along with absorption and fluorescence studies, reveal interaction between the corresponding complex and FS-DNA. Also, the binding constants (K_b) and the Stern–Volmer quenching constants (K_sv) of Tb(III) complex with FS-DNA were determined. The calculated thermodynamic parameters suggested that the binding of mentioned complex to FS-DNA was driven mainly by hydrophobic interactions. A comparative study of this complex with respect to the effect of iodide-induced quenching, ionic strength effect, and ethidium bromide exclusion assay reflects binding of explicit to the FS-DNA primarily in a groove fashion. CD and viscosity data also support the groove binding mode. Furthermore, Tb(III) complex have been simultaneously screened for their antibacterial and antifungal activities.     

DNA interaction of europium(III) complex containing 2,2′-bipyridine and its antimicrobial activity

The interaction of native fish salmon DNA (FS-DNA) with [Eu(bpy)₃Cl₂(H₂O)]Cl, where bpy is 2,2′-bipyridine, is studied at physiological pH in Tris-HCl buffer by spectroscopic methods, viscometric techniques as well as circular dichroism (CD). These experiments reveal that Eu(III) complex has interaction with FS-DNA. Moreover, binding constant and binding site size have been determined. The value of K_b has been defined 2.46 ± .02 × 10⁵ M^?1. The thermodynamic parameters are calculated by Van’t Hoff equation, the results show that the interaction of the complex with FS-DNA is an entropically driven phenomenon. CD spectroscopy followed by viscosity as well as fluorescence and UV––Vis measurements indicate that the complex interacts with FS-DNA via groove binding mode. Also, the synthesized Eu(III) complex has been screened for antimicrobial activities.     

Flow injection assays for NADH and ethanol using photosensitized rose bengal and luminol–copper (II) chemiluminescence system

The online photoreaction of the rose bengal photosensitized luminol–copper (II) chemiluminescence (CL) system was used for the determination of β-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10⁻⁸ and 2.17 × 10⁻⁵ M, and linear range 1.3 × 10⁻⁷ to 2.5 × 10⁻⁵ M (R² = 0.9998, n = 6) and 0.11–2.17 × 10⁻³ M (R² = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h⁻¹ with a relative standard deviation (n = 3) of 1.5–4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly.     

Complexes of platinum(II) containing 2,2′-bithiazoline and 2,2′-bipyrimidine as binucleating ligands

Dimethyl and diphenyl platinum(II) complexes containing binucleating α-diimine ligands BN (BN = 2,2′-bithiazoline and 2,2′-bipyrimidine) have been isolated and characterized. Electrophilic attack of mercuric chloride on the mononuclear compounds leads to binuclear systems of C_2v symmetry, with the two chelating moieties of the ligands occupied by platinum and mercury, respectively. ¹H NMR spectroscopy suggests a large transmission of electronic effects between the metals through the ligands.     

Crystal structure of a new form of copper(II)—inosine 5′-monophosphate—2,2′-dipyridylamine ternary complex

Two crystalline forms of the [Cu(II) (IMP) (DPA) (H₂O)]₂·nH₂O (IMP=inosine 5′-monophosphate, DPA=2,2′-dipyridylamine) complex were obtained from aqueous solution at pH=6.2. The crystals of the two forms belong to the monoclinic system, space group P2₁. The cell parameters are: a=9.445(2), b=33.902(4), c=7.802(2) Å, β=90.48(2)°, Z= 2, D_c=1.69g cm⁻³ and μ(Mo Kα) = 10.49cm⁻¹ (form α, n=4), and a=7.828(2), b=18.552(3), c=17.378(3) Å, β=91.16(2)°, Z=2, D_c=1.66 g cm⁻³, μ(Mo Kα) = 10.40 cm⁻¹ (form β, n=3.62). Bau and coworkers reported the preparation of form α by vapor diffusion of CH₃CN into aqueous solution containing Cu(NO₃)₂, Na₂IMP and DPA in a 1:1:1 molar ratio and the analysis of the compound by single crystal X-ray diffraction [1].Intensities for 3412 reflections were collected from a crystal of form β in the present work. Graphite-monochromatized Mo Kα radiation was employed. The structure was refined to final R and R_w values of 0.1000 and 0.1115 respectively. The dimeric units contain two copper ions in square-pyramidal coordination polyhedra. Each polyhedron consists of two nitrogen atoms of DPA, two oxygen atoms from two phosphate groups and a water molecule in the axial position. A statistical disorder was found in a nucleotide moiety of the dimer. Two sets of atomic positions corresponding to the purine system were refined with site occupation factors of 0.62(1) and 0.38(1) respectively. Also the ribose ring shows a disorder with two possible conformations. The puckering mode of the prevailing conformation is C(3′)-endo. In the other nucleotide molecule of the dimer the furanose puckering mode is C(3′)-endo. The rotation around the glycosyl linkages can be described as ‘anti’ in the structure of form β. The C(4)N(9)C(1′)O(4′) torsion angle values are −97(2) and −94(3)° for the disordered nucleotide molecule and +91(2)^o for the other nucleotide moiety. Strong intermolecular DPADPA and purine-purine stacking interactions stabilize the crystal lattice. The differences on the nucleotide conformation between the structure of form α and form β can probably be ascribed to differences in the hydrogen bonds and stacking interactions.     

The mechanism of antimalarial action of the ruthenium(II)–chloroquine complex [RuCl2(CQ)]2

The mechanism of antimalarial action of the ruthenium-chloroquine complex [RuCl(2)(CQ)](2) (1), previously shown by us to be active in vitro against CQ-resistant strains of Plasmodium falciparum and in vivo against P. berghei, has been investigated. The complex is rapidly hydrolyzed in aqueous solution to [RuCl(OH(2))(3)(CQ)](2)[Cl](2), which is probably the active species. This compound binds to hematin in solution and inhibits aggregation to beta-hematin at pH approximately 5 to a slightly lower extent than chloroquine diphosphate; more importantly, the heme aggregation inhibition activity of complex 1 is significantly higher than that of CQ when measured at the interface of n-octanol-aqueous acetate buffer mixtures under acidic conditions modeling the food vacuole of the parasite. Partition coefficient measurements confirmed that complex 1 is considerably more lipophilic than CQ in n-octanol-water mixtures at pH approximately 5. This suggests that the principal target of complex 1 is the heme aggregation process, which has recently been reported to be fast and spontaneous at or near water-lipid interfaces. The enhanced antimalarial activity of complex 1 is thus probably due to a higher effective concentration of the drug at or near the interface compared with that of CQ, which accumulates strongly in the aqueous regions of the vacuole under those conditions. Furthermore, the activity of complex 1 against CQ-resistant strains of P. falciparum is probably related to its greater lipophilicity, in line with previous reports indicating a lowered ability of the mutated transmembrane transporter PfCRT to promote the efflux of highly lipophilic drugs. The metal complex also interacts with DNA by intercalation, to a comparable extent and in a similar manner to uncomplexed CQ and therefore DNA binding does not appear to be an important part of the mechanism of antimalarial action in this case.     

Photophysical studies of hetero-bischelated complexes of rhodium(III) containing 2,2′-dipyridylamine

Four mixed-ligand complexes, cis-Rh[(bipy)(HDPA)Cl₂]Cl (1), cis-[Rh(phen)(HDPA)Cl₂]Cl (2), cis-[Rh(bipy)(DPA)Cl₂] (3), and cis-[Rh(phen)(DPA)Cl₂] (4) (where bipy = 2,2′-bipyridine, phen = 1,10-phenantroline, HDPA = 2,2′-dipyridylamine, and DPA = the deprotonated form of 2,2′-dipyridylamine) have been synthesized and characterized. In slightly acidic solution and at low temperature (77 K), both complexes 1 and 2 show a broad, symmetric and structureless red emission with microsecond lifetime identified as dd* phosphorescence. In slightly basic solution, the deprotonated complexes (3 and 4) exhibit a broad and asymmetric blue emission, showing no vibrational structure with a lifetime in the order of microseconds. Emission of complex 3 reveals a blue shift of 0.81 μm⁻¹ compared to the emission of complex 1 and that of complex 4 shows a blue shift of 0.77 μm⁻¹ with respect to complex 2. Electrochemical data have also been obtained for the four complexes in CH₃CN. There are two reduction peaks observed for both complexes 1 and 2. Each peak is followed by a one-electron reduction at the metal, with an elimination of chloride during each reduction step, which is in consistent with the dd* phosphorescence assignment for the two complexes. For complexes 3 and 4, only a one-electron reduction process occurs at the metal with an elimination of chloride. Based on the luminescence and electrochemical data, the emission of complexes 3 and 4 are assigned as πd* phosphorescence. Results from density functional theory (DFT) calculations provide theoretical evidence in support of this πd* assignments.     

Stabilization of a hydrated ketone by metal complexation. The crystal and molecular structures of bis-2,2′,N,N′-bipyridyl ketone-hydrate nickel(II) sulfate,copper(II) chloride and copper(II) nitrate

The crystal structure of the complexes (I)Ni[C₁₁N₈N₂(OH)₂]₂SO₄, (II) Cu[C₁₁H₈N₂(OH)₂]₂Cl₂· 4H₂O and (III) Cu[C₁₁H₈N₂(OH)₂]₂(NO₃)₂·2H₂O have been determined by three-dimensional X-ray analysis methods. Crystal data are: (I), monoclinic, space group C2/c, Z = 4, a = 19.666(4), b = 7.994(2), c = 16.045(6) /rA, /gb = 111.231(9)°, (II), monoclinic, space group C2/c, Z = 4, a = 14.504(4), b = 12.333(8), c = 14.630(3) Å, /gb = 90.92°; and (IIl), monoclinic, space group P2₁/n, Z = 2, a = 7.601(5), b = 11.977(4), c = 14.463(6) Å, β = 93.10(8)°. These structural investigations clearly demonstrate that in each case hydration occurs across the ketone double bond in the ligand and that the resulting hydroxyl group coordinates to the metal. Two di-2-pyridyl ketone ligands are thus bonded to the metal atom in a tridentate fashion. In the nickel complex (I), all six coordination interactions appear to have approximately the same strength. However, in the copper complexes (II) and (III), the pyridyl nitrogens are strongly coordinating to the metal in the equatorial plane, while the hydroxyl groups are more weakly coordinating in the axial direction. The metal to ligand bond distances are: (I) d_Ni−O = 2.098(4), d_NiN = 2.062(4), 2.087(4) Å, (II) d_CuO = 2.465(5), d_CuN = 1.994(5), 2.006(5) Å, (III) d_CuO = 2.464(5), d_CuN = 1.990(5), 2.036(5) Å. The neutral diol that results from hydrolysis of di-2-pyridyl ketone is stabilized by coordination to the metal and such coordination is little affected by changes in the metal, the anion or the extent of hydration.     

Specific cation effect on quenching reactions of excited tris(α,α′-diimine) ruthenium(II) and chromium(III) complexes by cyanide complexes in aqueous solutions

Specific salt effects were studied on the quenching reaction of excited [Ru(NN)₃]²⁺ (NN=2,2′-bipyridine(bpy), 1,10-phenanthrorine(phen)) and [Cr(bpy)₃]³⁺ by [Cr(CN)₆]³⁻, [Fe(CN)₆]³⁻ and [Ni(CN)₄]²⁻ in aqueous solutions as a function of alkali metal ions which were added for adjustment of ionic strength. The quenching rate constants in [Ru(NN)₃]²⁺-[Cr(CN)₆]³⁻ and [Cr(bpy)₃]³⁺-[Cr(CN)₆]³⁻ systems are changed by the cations as Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺. On the other hand, the rate constants in [Ru(NN)₃]²⁺-[Fe(CN)₆]³⁻ and [Ru(NN)₃]²⁺-[Ni(CN)₄]²⁻ systems, which are diffusion-controlled reactions, are not varied by the alkali metal cations. The obtained order (Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺) of the quenching rate constant is quite different from salt effects, Li⁺<Na⁺<K⁺<Rb⁺<Cs⁺, which have been obtained in the electron transfer reactions between complex anions.     

Synthesis, electrochemistry and mixed-valence state of dinuclear tris(acetylacetonato)ruthenium(III) complexes bridged by sulfur atoms at the γ-position of acetylacetonate

Dinuclear tris(acetylacetonato)ruthenium(III) complexes bridged by one sulfur atom (1) or two sulfur atoms (2) at the γ-position of the acetylacetonate have been synthesized by the reactions of tris(acetylacetonato)ruthenium(III) with SCl₂ or S₂Cl₂. The molecular structure of 2 has been determined by single crystal X-ray diffraction study. The cyclic voltammograms of both the dinuclear complexes exhibit two one-electron reduction waves in acetonitrile (AN), dichloromethane (DM), benzonitrile (BN), and N,N-dimethylformamide (DMF). While the complex 1 exhibited two one-electron oxidation waves in AN, DM, and BN, complex 2 showed only irreversible waves in all the solvents. The comproportionation constants (K_c) for mixed-valence states of both Ru^II/Ru^III and Ru^III/Ru^IV were calculated from the redox potentials of dinuclear complexes. The values of log₁₀K_c (Ru^II/Ru^III) (for complexes 1 and 2) and log₁₀K_c (Ru^III/Ru^IV) (for complex 1) were between 1.35 and 3.55. These values are not so large and hence, the complexes may be classified as class II in the Robin and Day classification. Although no relationship could be found between K_c and the dielectric constant of the solvent, there exists a correlation between donor number (DN) of the solvent and log₁₀K_c values.     

Kinetics of the oxidation of bromide ions by bis(2,2′-bipyridine)manganese(III) ions in aqueous perchlorate media

The kinetics of the oxidation of bromide ions by the bis(2,2′-bipyridine)manganese(III) ion have been investigated over a range of acid concentrations for several temperatures. Initial rates of reaction are measured and from their variation with [Mn^III] and [Br−] it is concluded that the observed order in [Mn(bipy)₂³⁺_aq] is one and that in [Br⁻] is intermediate between zero and one. It is shown kinetically that intermediate complexes between Mn^III and Br⁻ ions are involved and from the variation of the rate constant with acidity it is concluded that the decomposition of Mn(bipy)₂Br²⁺_aq is definitely involved as a rate controlling step and that the decomposition of the protonated complex Mn(bipy)(bipyH⁺)Br³⁺_aq is also probably involved as a rate controlling step. The kinetics and mechanism for this oxidation are compared with those found for other cations complexed with bipyridine, for aqua-cations and other complexes of Co^III.     

Inhibition of Streptomyces chromofuscus Phospholipase D Activity by Dichloro-(2,2′:6′,2′-terpyridine)-platinum (II) Dihydrate

To determine the catalytic site of Streptomyces chromofuscus phospholipase D (PLD), which lacks an HKD motif, we examined the effects of inhibitors on the hydrolytic activity of the PLD by comparing it with cabbage and Streptomyces PLDs, which have two HKD motifs. We showed that dichloro-(2,2′:6′,2"-terpyridine)-platinum (II) dihydrate, a His- and Cys-directed chemical modifier, had inhibitory effects on the activities of all types of PLD examined. On the other hand, N -ethylmaleimide, a thiol-directed modifier had no such effects on PLD activity. These results suggest that the His residue plays an important role in the activity of Streptomyces chromofuscus PLD.     

Protein recognition of hetero-/homoleptic ruthenium(II) tris(bipyridine)s for α-chymotrypsin and cytochrome c

We examined the relationship between the structures of hetero-/homoleptic ruthenium(II) tris(bipyridine) metal complexes (Ru(II)(bpy)(3)) and their binding properties for α-chymotrypsin (ChT) and cytochrome c (cyt c). Heteroleptic compound 1a binds to both ChT and cyt c in 1:1 ratio, whereas homoleptic 2 forms 1:2 protein complex with ChT but 1:1 complex with cyt c. These results suggest that the structure of the recognition cavity in Ru(II)(bpy)(3) can be designed for shape complementarity to the targeted proteins. In addition, Ru(II)(bpy)(3) complexes were found to be potent inhibitors of cyt c reduction and to permeate A549 cells.