Kinetic resolution of both 1-phenylethanol enantiomers produced by hydrolysis of 1-phenylethyl acetate with <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B in different solvent systems

The enzymatic resolution of (R, S)-1-phenylethanol produced by hydrolysis of (R, S)-1-phenylethyl acetate catalyzed by immobilized Candida antarctica lipase B (CALB) was successfully carried out in different solvent systems. A systematic screening and optimization of the reaction parameters such as enzyme amount, the nature and the content of organic solvent, pressure and temperature in supercritical carbon dioxide (SC-CO₂) and phosphate buffer, with respect to the conversion rate, were performed. CALB exhibits high enantioselectivity in both tert-butanol with 0.025 mol/l phosphate buffer (pH 7.5) and SC-CO₂ with 0.025 mol/l phosphate buffer (pH 7.5) systems. The conversion rate was 41.2% higher in SC-CO₂ with 0.025 mol/l phosphate buffer (pH 7.5) then in tert-butanol with 0.025 mol/l phosphate buffer (pH 7.5) and the reaction time decreased from 8 h to 90 min.     

Aspects of Degradation Kinetics of Azithromycin in Aqueous Solution

The chemical stability of azithromycin (AZM) in aqueous solution has been investigated utilizing a stability-indicating LC assay with ultraviolet detection. The degradation kinetics were studied as functions of pH (4–7.2), buffer composition (phosphate, acetate, and citrate), buffer concentration, ionic strength, drug concentration and temperature. The observed rate obtained by measuring the remaining intact AZM was shown to follow pseudo-first-order kinetics. The maximum stability of AZM occured at an approximate pH 6.3 in 0.05 M potassium phosphate. The observed degradation rate increased with ionic strength, buffer concentration and obeyed the Arrhenius equation over the temperature range investigated (70–100 °C). The apparent energy of activation (E _a) for AZM in solution was found to be 96.8 kJ mol^?1 and by application of the Arrhenius equation the stability at 25 °C (k ₂₅) and 40 °C (k ₄₀) had been predicted. Moreover, the degradation rate of AZM was independent on its initial concentration. Trace metal ions are unlikely to be involved in the degradation of AZM in aqueous solution. The major degradation product of AZM in aqueous solution was isolated and identified by LC–MS–MS and ¹H and ¹³C NMR spectra.     

The determination of phenobarbital and diphenylhydantoin in blood by differential pulse polarography

A sensitive differential pulse polarographic assay was developed for the determination of phenobarbital or diphenylhydantoin in blood. The assay involves the selective extraction of the compound into chloroform from whole blood buffered to pH 7.0. After suitable “clean-up” of the sample, each compound is nitrated in 10% potassium nitrate in sulfuric acid at 25° for 1 h. The nitro-derivatives are extracted into ethyl acetate, and the residues are dissolved in 1 M phosphate buffer (pH 7.0) or 0.1 M sodium hydroxide for phenobarbital and diphenylhydantoin, respectively; the solutions are deoxygenated, and analyzed by differential pulse polarography. The overall recovery of phenobarbital and diphenylhydantoin from blood was 72.3% ±6.5 (s_r) and 76.7 ±2.3 (s_r) respectively. The sensitivity limit is 1–2 μg ml^-1 of blood for both compounds. A modified assay for the determination of both compounds in blood with t.l.c. separation was also developed.     

Photochemical Reaction Mechanisms of 2‐Nitrobenzyl Compounds in Solution,I. 2‐Nitrotoluene: Thermodynamic and Kinetic Parameters of the aci‐Nitro Tautomer

The largely reversible, light‐induced tautomerization of 2‐nitrotoluene ( 1 ) to the quinonoid aci‐nitro tautomer aci‐ 1 was studied by flash photolysis as a benchmark for comparison with the widely used nitrobenzyl phototriggers (`caged compounds'). The pH‐rate profile for the decay of aci‐ 1 in aqueous solution exhibits downward curvature at pH 3 – 4, which is attributed to pre‐equilibrium ionization of the nitronic acid aci‐ 1 to its anion 1 ⁻ (pK_a=3.57). Two regions of upward curvature, at pH ca. 6 and <0 (H₀≈−1), each indicate a change in the reaction mechanism. The elementary reactions that dominate between the curved regions are assigned on the basis of kinetic isotope effects and the observation of general acid catalysis: Hydronium ions regenerate 2‐nitrotoluene by C‐protonation of 1 ⁻ in the pH range of 0 – 6, and H₂O is the proton source at pH>6. A hird, irreversible Nef‐type isomerization of aci‐ 1 prevails in highly acidic solutions (pH<0). The equilibrium constant for the thermal tautomerization of 1 to aci‐ 1 is estimated as pK_T=17.0±0.2 based on kinetic data.     

Equations for calculation of the pH of buffer solutions containing sodium or potassium dihydrogen phosphate, sodium hydrogen phosphate, and sodium chloride at 25°C

Published thermodynamic data measured in aqueous mixtures of sodium or potassium dihydrogen phosphate with hydrogen phosphate and chloride at 25°C were used to test recently developed methods for calculation of the pH of phosphate buffer solutions. Equations for ionic activity coefficients are used in these methods. It is shown that all data used in the tests up to an ionic strength of about 0.5 mol-kg^-1 can be accurately predicted by the two methods recommended. In one of these methods, equations of the Hückel type are used for ionic activity coefficients and in the other equations of the Pitzer type. Several sets of phosphate buffer solutions are recommended,e.g., for calibrations of glass electrode cells. In the recommended sets, the pH of the buffer solutions can be calculated either by the Hückel or Pitzer method, and the pH predictions of these methods agree in most cases within 0.005 at least up to ionic strengths of about 0.2 mol-kg^-1. The pH values of the two primary pH standards endorsed by IUPAC based on aqueous mixtures of KH₂PO₄ and Na₂HPO₄,i.e., pH values of 6.865 and 7.413, can also be accurately predicted by the equations recommended in this study.     

Glucose oxidase electrodes of a terpolymer poly(aniline-co-o-anisidine-co-o-toluidine) as biosensors

A simple technique is described for constructing a glucose sensor by the entrapment of glucose oxidase (GOD) in a poly (aniline-co-o-anisidine-co-o-toluidine) [P(A-co-o-A-co-o-T)] thin films, which were electrochemically deposited on a platinum plate in phosphate and acetate buffer. The maximum current response was observed for the said electrodes at pH 5.5 and potential 0.60 V (versus Ag/AgCl). The phosphate buffer gives high stability and fast response as compared to acetate buffer in amperometric measurements.     

901 — Electrode process of glutamate dehydrogenase in differential pulse polarography

The anodic and cathodic responses of sulfhydryl groups in glutamate dehydrogenase (GDH) were studied using the d.p.p. method. The current peaks were detected at the potentials U_p,anod and U_p,cath of approx. −1.45 V and −1.50 V, respectively, versus s.c.e. in phosphate buffer, pH 7. Both the cathodic and the anodic responses of GDH showed autoinhibiting diffusion-controlled adsorption effects. Both peak potentials U_p,anod and U_p,cath are shifted positively with increasing pH up to pH 8.5 (U_p,anod by 40 mV/pH, U_p,cath by 60 mV/pH) and then become pH-independent. The preceding chemical reaction (field-induced homolysis) with pH-dependent rate yields the electroactive form, the thiyl radical, through abstraction of the hydrogen atom from the -SH group. The radical could be reduced, yielding the thiol anion after the uptake of one electron. The two-electron anodic oxidation in d.p.p. involves the product of this reduction process and another thiol anion in an adjacent position. The dissociation rate constant of the thiol group near that undergoing homolysis was estimated from the d.p.p. data (log k_d = 2.9 ± 0.2).     

Investigation of the interaction mechanism of the recombinant human antibody MDJ8 and its fragments with chromatographic apatite phases

The chromatographic behaviour of a recombinant human antibody (IgG₁-subtype, κ-light chain, MW: 149.5 kD, pI: 9.3) was investigated as a function of the buffer pH and buffer type (HEPES, phosphate, borate) on fluoroapatite and hydroxyapatite stationary phases. HEPES buffer was used at pH 7.0, phosphate buffer at pH 8.2 and borate buffer between pH 8.5 and 11. Elution was by a double gradient method of first a salt gradient from 0 to 1 M NaCl in the corresponding buffer, followed by a step gradient to 0.4 M sodium phosphate. Regardless of the pH and buffer type, the antibody eluted in the NaCl gradient; capacity factors decreased with increasing pH. At pH 11 the antibody eluted in the flow-through. Retention was thus dominated by electrostatic interaction throughout the investigated pH-range. Investigation of antibody fragments obtained by papain digestion (fc- and fab-fragments) and deglycosylated fc-fragments showed that the sugar structures had no influence on the chromatographic behaviour. Instead the chromatographic behaviour was dominated by that of the fab-fragment. ζ-Potential measurements verified that the apatite surface bore a negative surface charge in the investigated pH range, while the antibody net surface charge switched from positive to negative as the pH increased. The corresponding isoionic point was a function of both the buffer concentration and the buffer species. However, above a pH of 8.3 the ζ-potential of the antibody generally was negative. Simulations of the molecular electrostatic potential of the antibody and the two fragments revealed the presence of a positively charged patch within the fab-fragment, which only disappeared above a pH of 10. Most likely this patch was responsible for the observed behaviour.     

Reaction mechanisms between methylamine and a few Schiff bases: Ab initio potential energy surfaces of a catalytic step in semicarbazide sensitive amino oxidases (SSAO)

The potential energy surfaces for the transamination reaction catalyzed by SSAO were explored for some of the possible reactants considered in a preliminary investigation (Comput Chem 2000, 24 , 311). The proton transfer to methylamine (as a model of the catalytic base belonging to the enzyme active site)—either from the keto or enol form of the reactant Schiff bases with one of the possible cofactors, pyridoxal phosphate, PLP (using as a model the pyridoxal ring protonated at N)—was investigated. The enol form seems to be preferred in the region of the neutral intermediate, because even the keto form undergoes a spontaneous rearrangement to the enol form once the C_α proton is delivered to methylamine, producing methylammonium. When the proton is returned back to the Schiff base (on C₁), the adduct is about 1.4 kcal/mol more stable than the reactants, while a canonical electron distribution is obtainable only for the enol form. The proton transfer to methylamine was also studied in the presence of the other possible cofactor (para or ortho) topaquinone, TQ. A steep uphill pathway, similar to the keto‐pyridoxal Schiff base one, is obtained using the Schiff base with pTQ, which requires a rearrangement to the final intermediate. On the contrary, using the oTQ structures with the quinonoid O on the same side of methylamine, the proton abstracted from the Schiff base goes spontaneously onto the other quinonoid oxygen. The effect on the barrier heights produced by the presence of a variety of functional groups in the vicinity of the pyridoxal ring nitrogen was also examined. © 2001 John Wiley & Sons, Inc. Int J Quant Chem, 2001     

New Cocrystals of Antipsychotic Drug Aripiprazole: Decreasing the Dissolution through Cocrystallization

Cocrystallization is an important route to tuning the solubility in drugs development, including improving and reducing. Five cocrystals of aripiprazole (ARI) with resveratrol (RSV) and kaempferol (KAE), ARI-RSV, ARI₂-RSV₁·MeOH, ARI-KAE, ARI-KAE·EtOH, ARI-KAE·IPA, were synthesized and characterized. The single crystal of ARI₂-RSV₁·MeOH, ARI-KAE·EtOH, and ARI-KAE·IPA were analyzed by single crystal X-ray diffraction (SCXRD). The SCXRD showed multiple intermolecular interactions between API and the coformers, including hydrogen bond, halogen bond, and π-π interactions. Dissolution rate of the two nonsolvate ARI-RSV and ARI-KAE cocrystals were investigated through powder dissolution experiment in pH = 4.0 acetate buffer and pH = 6.8 phosphate buffer. The result showed that RSV could reduce the dissolution rate and solubility of ARI in both medium through cocrystallization. However, KAE improved the dissolution rate and solubility of ARI in pH = 4.0 medium, on the contrary, the two solubility indicators of ARI were both reduced for ARI-KAE cocrystal.     

The lon-chromatographic behavior of arsenite,arsenate, methylarsonic acid and dimethylarsinic acid on the hamilton PRP-X100 anion-exchange column

The HPLC separation of arsenite, arsenate, methylarsonic acid and dimethylarsinic acid has been studied in the past but not in a systematic manner. The dependence of the retention times of these arsenic compounds on the pH of the mobile phase, on the concentration and the chemical composition of buffer solutions (phosphate, acetate, potassium hydrogen phthalate) and on the presence of sodium sulfate or nickel sulfate in the mobile phase was investigated using a Hamilton PRP-X100 anion-exchange column. With a flame atomic absorption detector and arsenic concentrations of at least 10 mg dm^?3 all investigated mobile phases will separate the four arsenic compounds at appropriate pH values in the range 4–8. The shortest analysis time (?3 min) was achieved with a 0.006 mol dm^?3 potassium hydrogen phthalate mobile phase at pH 4, the longest (?10 min) with 0.006 mol dm^?3 sodium sulfate at pH 5.9 at a flow rate of 1.5 cm³ min^?1. With a graphite furnace atomic absorption detector at the required, much lower, flow rate of ?0.2 cm³ min^?1 acceptable separations were achievable only with the pH 6 phosphate buffer (0.03 mol dm^?3) and the nickel sulfate solution (0.005 mol dm^?3) as the mobile phase. To become detectable approximately 100 ng arsenic from each arsenic compound (100 μl injection) must be chromatographed with the phosphate buffer, and approximately 10 ng with the nickel sulfate solution.     

A method for studying swelling kinetics based on measurement of electrical conductivity

A novel method was applied to the study of swelling kinetics of pH-responsive hydrogels. This technique is based on the pH-dependent electrical conductivity of these materials, which is measured by coating planar interdigitated electrode arrays with thin hydrogel membranes. To demonstrate the utility of the method, the swelling kinetics of a well-characterized pH-responsive hydrogel were studied. Cross–linked copolymers of 2-hydroxyethyl methacrylate (HEMA) with up to 20 mol% dimethylaminoethyl methacrylate (DMA) were studied as a function of copolymer composition in phosphate or triethanolamine buffer at buffer concentrations from 1 to 100 mM. The experiments consisted of measuring the change in electrical resistance of a hydrogel-coated electrode array following a small pH change in the external buffer medium. The characteristic response time to reach a new equilibrium following a pH change was proportional to the concentration of DMA within the polymer and was inversely proportional to the buffer concentration. The characteristic response times for devices tested in phosphate buffer were a function of the magnitude of the pH step, increasing from 2.6 to 5.6 min as the step size increased from 0.2 to 0.57 pH units. However, the response times for devices tested in triethanolamine were independent of step size. The observed dependences upon the values of the dissociation constant (pK_a) of the buffering ion, the apparent pK_a of DMA, and the pH of the external bath agreed with buffer-mediated diffusion–reaction theory, and as such this conductimetric method represents a powerful tool for the study of swelling kinetics of responsive hydrogels.     

The Determination of Folic Acid (Pteroylmonoglutamic Acid) in Fortified Products by Reversed Phase High Pressure Liquid Chromatography

Abstract

A reversed phase HPLC method was developed for the separation and determination of pteroylglutamic acid (PGA) in fortified foods. Extraction was carried out by heating with phosphate-citrate buffer, pH 8.0 containing ascorbate, and incubation with papain at 40°C for 4 hrs. The extracts were purified and concentrated on a short DEAE column which was rinsed with phosphate buffer, pH 7.0, of increasing molarity. PGA was eluted with 0.1M phosphate buffer, pH 7.0, containing 0.5M NaCl. The eluants were chromatographed on a Spherisorb ODS 10 μm column (250 × 4.6 mm) using a 30 min linear gradient of 2% to 30% acetonitrile in 0.1M acetate buffer, pH 4.0, at 1 ml/min and an absorbance detector at 280 nm. The coefficients of variation on analysis of 8 replicate samples of a milk and soy protein based infant formulas were 5.9% (at 4.6 ng/50 μl inject) and 6.8% (at 1.8 ng/50 μl inject) respectively.     

蝶呤类化合物的荧光性能研究

研究了蝶呤类化合物的天然荧光特性。着重考察了新蝶呤、生物蝶呤、黄蝶呤和蝶呤在 p H7.7磷酸盐缓冲溶液条件下的荧光光谱及各种因素对其荧光强度的影响。在最佳实验条件下 ,四种蝶呤类化合物的线性范围为 :蝶呤 0 .6～ 2 .8μg/m L,新蝶呤 0～ 2 .6μg/m L,生物蝶呤 0～ 2 .4μg/m L,黄蝶呤 0～ 6.0 μg/m L,检出限依次为 :4.2 9× 1 0 - 7g/m L,6.71× 1 0 - 8g/m L,5.79× 1 0 - 9g/m L和 1 .75× 1 0 - 8g/m L     

Water-soluble supramolecular porphyrin dimer: self-organization of mono(imidazolyl)-substituted Zn porphyrin to a special-pair type dimer in water

Tris(4-carboxylphenyl)-mono(N-methylimidazolyl)-substituted Zn porphyrin was synthesized as a precursor for a water-soluble supramolecular porphyrin dimer. The dimer formation was performed in a NaHCO₃ aq solution (pH 8.4) and phosphate buffer solutions (pH 7.4-9.0). The split Soret bands of Zn porphyrin observed in the absorption spectra clearly showed self-organization to a special-pair type slipped cofacial dimer via metal coordination of imidazole even in water.     

Simultaneous determination of two active ingredients in Flos daturae by capillary electrophoresis with electrochemiluminescence detection

The coupling of Ru(bpy)₃²⁺ based electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) was developed for the simultaneous determination of the two major active ingredients (atropine and scopolamine) in Flos daturae. Parameters related to the separation and detection were discussed and optimized. It was proved that 20 mM phosphate buffer at pH 8.48 could achieve the most favorable resolution, and the high sensitivity of detection was obtained by maintaining the detection potential at 1.2 V. Under the optimized conditions: ECL detection at 1.2 V, 20 mM phosphate buffer at pH 8.48, 5 mM Ru(bpy)₃²⁺ and 50 mM phosphate buffer at pH 7.48 in the detection reservoir, detection limits of 5 × 10⁻⁸ mol/l for atropine and 1 × 10⁻⁶ mol/l for scopolamine were obtained. Relative standard derivations of the ECL intensity and the migration time were 5.16 and 0.71% for atropine and 5.07 and 1.22% for scopolamine, respectively. Developed method was successfully applied to determine the amounts of both alkaloids in Flos daturae. A baseline separation for atropine and scopolamine was achieved within 11 min.     

Rapid Purification of the Unstable Enzyme Carbamoyl Phosphate Synthase by High Pressure Liquid Chromatography

Abstract

Extracts of soluble proteins obtained from rat liver mitochondria by freeze-thawing and subsequent diafiltration were fractionated by HPLC on a I 250 protein column. The column was eluted either with 0.05 M phosphate buffer pH 6.85 or 0.1 M acetate buffer pH 7.15. Specific fractions obtained by elution with either phosphate or acetate buffer showed a 6.1-fold or 5.5-fold increase in the specific activity of Carbamoyl phosphate synthase when compared with that of crude mitochondrial preparations. The purification and the molecular weight of carbamoyl phosphate synthase were verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.     

Development of efficient SPE–TLC method and evaluation of biological interactions of contraceptives with progesterone receptors

TLC–SPE methodologies were developed to ascertain biological interactions of norethindrone acetate and dydrogesterone contraceptives with plasma progesterone receptor proteins. TLC solvent system for plain and Cu(II) impregnated silica gel plates was n-hexane-n-butanol (90:10, v/v), which took 20 min to run up to 10.0 cm. The best separation was on Cu(II) impregnated plates due to maximum difference in R_f values and compact spots. The optimized SPE conditions were pH 2.0 and 3.0 of phosphate buffer (50 mM) for norethindrone acetate and dydrogesterone, respectively. The flow rate of plasma and eluting solvent (methanol) through C₁₈ cartridge was 0.10 mL/min. The interactions of these contraceptives with progesterone receptor proteins were analysed by TLC–SPE results, which were supported by modelling using PyMOL and Autodoc4 softwares. The dydrogesterone was found to be bound strongly than norethindrone acetate. Attempts have been made to discuss the drugs’ interactions at chemo-supramolecular level.     

Novel applications of chloroperoxidase: enantioselective oxidation of racemic epoxyalcohols

The CPO-catalysed enantioselective oxidations of racemic glycidol and cis-2,3-epoxyhexanol to the corresponding aldehydes were studied using tert-butyl hydroperoxide in the 9:9:1 and 18:1 mixtures of hexane:ethyl acetate:buffer and hexane:buffer, respectively. Temperature and pH (100 mM citrate buffer, pH 4.0–7.0) effects on enantioselectivity in the terms of ee for the less reactive alcohol enantiomers with conversion were studied.     

Micellar electrokinetic capillary chromatography for the separation of cefalexin and its related substances

Micellar electrokinetic capillary chromatography (MEKC) was examined for analysis of cefalexin and its related substances. Good selectivity was obtained with two different buffer solutions: a sodium acetate buffer (50 mM, pH 5.25) containing sodium dodecyl sulfate (50 mM SDS) or sodium phosphate buffer (40 mM, pH 7.0) containing 100 mM SDS. Both methods permit cefalexin to be completely separated from its ten related substances within 20 min. The robustness of the method, using pH 5.25 acetate buffer, was examined by means of a full-fraction factorial design to test the influence of buffer pH, concentration of SDS and buffer concentration. The parameters for validation such as linearity, precision, limit of detection and limit of quantitation are also reported. The results show that method 1 is suitable for the analysis of cefalexin.