A degradation study of cefepime hydrochloride in solutions under various stress conditions by TLC–densitometry

A rapid, accurate and sensitive thin‐layer chromatography (TLC) method with densitometric detection has been developed and validated for the determination of cefepime in pharmaceuticals. Chromatographic separation was achieved on a silica gel TLC F₂₅₄ plates with a mobile phase consisting of ethanol–2‐propanol–glacial acetic acid 99.5%–water (4:4:1:3, v/v). Densitometric detection was carried out at wavelength of 266 nm in reflectance/absorbance mode. The validation of the method was found to be satisfactory with high accuracy (from 99.24 to 101.37%) and precision (RSD from 0.06 to 0.36%). Additionally, the stability of cefepime in solution was investigated, including the effect of pH, temperature and incubation time. Favorable retention parameters (R_f, R_s, α) were obtained under the developed conditions, which guaranteed good separation of the studied components. The degradation process of cefepime hydrochloride was described by kinetic and thermodynamic parameters (k, t_0.1, t_0.5 and E_a). Moreover, the chemical properties of degradation products were characterized by the R_f values, absorption spectra, HPLC‐MS/MS and TLC‐densitometry analysis. As the method could effectively separate the active substance from its main degradation product (1‐methylpyrrolidine), it can be employed as a method to indicate the stability of this drug. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and specific biodegradation of novel polyesteramides containing amino acid residues

Novel polyesteramides were synthesized from p‐nitrophenyl esters of sebacic or adipic acids and diamines containing α‐amino acid ester groups. The optimal polymerization condition was 60 °C in N,N‐dimethylformamide. The structures of these polymers were confirmed by IR and NMR. The number‐average molecular weights of these polyesteramides ranged from 2280 to 23,600 (except for the polymers containing glycine residues), depending on the nature of the amino acid used. The biodegradability of the polyesteramides was investigated by in vitro hydrolysis with proteases and a lipase as catalysts in borate buffer solutions. The results indicated that the polymers containing L ‐phenylalanine were hydrolyzed most effectively by α‐chymotrypsin, subtilisin Carlsberg, and subtilisin BPN′. The polyesteramides containing other amino acid residues also underwent hydrolysis to different extents, reflecting the substrate specificity of the proteases. Lipase had almost no effect on the hydrolytic degradation of these polyesteramides. The polymers containing glycine residues were hardly decomposed by any of the enzymes used. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 1318–1328, 2001     

Large-scale recovery and purification ofl-asparaginase fromErwinia carotovora

A large-scale process was developed to purify gram quantities of a therapeutic enzyme,L-asparaginase, from submerged cultures ofErwinia carotovora. Cells were harvested from 150 L of fermentation broth and washed. A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer. After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover. The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column ofl-asparagine Sepharose CL-4B. After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product. the final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility. The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis,N-terminal sequencing, peptide mapping, and immunological comparison.     

High-Performance Liquid Chromatographic Determination of Cefepime and Cefazolin in Human Plasma and Dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of cefepime and cefazolin in human plasma and dialysate. For component separation, the method utilized a C₁₈ column with an aqueous mobile phase of dibasic potassium hydrogen phosphate (pH 7.0) and methanol gradient at a flow rate of 1 mL min⁻¹. The method demonstrated linearity from 2.0 to 100.0 μg mL⁻¹ (r > 0.999) with detection limit of 1 μg mL⁻¹ for both cefepime and cefazolin. The method was utilized for evaluation of plasma and dialysate samples in a clinical study evaluating the dialyzer clearance of cefepime and cefazolin using high-flux hemodialysis with varying blood flow rates in chronic kidney failure patients undergoing hemodialysis and peritoneal dialysis treatment.     

Study on the preparation and biological evaluation of <Superscript>99m</Superscript>Tc–gatifloxacin and <Superscript>99m</Superscript>Tc–cefepime complexes

The aim of this work is the development of new radiopharmaceuticals for imaging infection and inflammation in human. Gatifloxacin (fluoroquinolone derivative) and cefepime (cephalosporine derivative) are antibiotics used to treat bacterial infections were investigated to label with one of the most important radioactive isotopes (technetium-99m). The reaction parameters that affect the labeling yield such as substrate concentration, stannous chloride dihydrate concentration, pH of the reaction mixture, and reaction time were studied to optimize the labeling conditions. Maximum radiochemical yield of ^99mTc–gatifloxacin (90  ± 1.8%) complex was obtained by using 50 μg SnCl₂·2H₂O and 2.5 mg gatifloxacin at pH 10 while ^99mTc–cefepime was prepared at pH 8 with a maximum radiochemical yield of 98  ± 1.4% by adding ^99mTc to 5 mg cefepime in the presence of 50 μg SnCl₂·2H₂O. Biological distribution of ^99mTc–gatifloxacin and ^99mTc–cefepime was carried out in experimentally induced infection rats, in the left thigh, using Escherichia coli. Both thighs of the rats were dissected and counted and the ratio of bacterial infected thigh/contralateral thigh was then evaluated. T/NT for both ^99mTc–gatifloxacin and ^99mTc–cefepime was found to be 4.5  ± 0.3 and 8.4  ± 0.1, respectively, which was higher than that of the commercially available ^99mTc–ciprofloxacin. The abscess to normal muscle ratio indicated that ^99mTc–cefepime could be used for infection imaging. Besides, in vitro studies showed that ^99mTc–cefepime can differentiate between bacterial infection and sterile inflammation.     

A green capillary zone electrophoresis method for the simultaneous determination of piperacillin,tazobactam and cefepime in pharmaceutical formulations and human plasma

A green, novel, rapid, accurate and reliable capillary zone electrophoresis method was developed and validated for the simultaneous determination of piperacillin, tazobactam and cefepime in pharmaceutical preparations. Separation was carried out using fused silica capillary (50 µm i.d. × 48.6 cm and 40.2 cm detection length) and applied potential of 20 kV (positive polarity) and a running buffer containing 15 m m sodium borate buffer adjusted to pH 9.3 with UV detection at 215 nm. Amoxicillin was used as an internal standard. The method was suitably validated according to International Conference on Harmonization guidelines. The method showed good linearity in the ranges of 10–100, 20–400 and 10–400 µg/mL with limits of quantitation of 1.87, 3.17 and 6.97 µg/mL and limits of detection of 0.56, 0.95 and 2.09 µg/mL for tazobactam, piperacillin and cefepime, respectively. The proposed method was successfully applied for the analysis of these drugs in their synthetic mixtures and co‐formulated injection vials. The method was extended to the in vitro determination of the two drugs in spiked human plasma. It is considered a ‘green’ method as it consumes no organic solvents. Copyright © 2015 John Wiley & Sons, Ltd.     

Thermokinetic Studies on the Activation of Arginase by Glycine

The activation of bovine liver arginase, which catalyzes the hydrolysis of L‐arginine to L‐ornithine and urea, by glycine was studied by thermokinetic methods at 37°C in 40 mmol·L^?1 sodium barbiturate‐HCl buffer solution (pH 9.4). Results of this experiment indicate that an appropriate concentration of glycine can enhance the activity of arginase, and the relative activation rate reached its maximum value, 74%, when the concentration of glycine in reaction system was 1 mmol·L^?1 and the initial concentration of arginine was 5 mmol·L^?1. With the increase of substrate concentration, the relative activation rate decreased in a definite glycine concentration. Michealis constant K_m of reaction decreased from 5.53 to 3.31 mmol·L^?1 and inhibition constant of product L‐ornithine K_p increased from 1.18 to 3.73 mmol·L^?1 when glycine concentration was 1 mmol·L^?1. For these reasons one possible activation mechanism of arginase by glycine was suggested that the activation effect results from the competition of glycine and arginine to enzyme activity position. When one or two of the activity positions of arginase are occupied by glycine, it is propitious for the enzyme to complex with substrate and obstruct L‐ornithine from combining with enzyme, and when all of the activity positions are occupied by glycine, the activation effect vanishs and the inhibition effect appears.     

Boron equilibria with high molecular weight guar: An NMR study

Equilibrium constants have been derived from ¹¹B-NMR spectra for water-soluble complexes between borate anion and high molecular weight guar (e.g., 2 × 10⁶ Da). The validity of constants was confirmed by their ability to predict the pH changes measured on mixing various combinations of sodium borate, guar, and caustic into aqueous salt solutions, by correlation of computed crosslink concentrations with the dynamic storage modulii measured on the same fluids, as well as published G′ data for borate/guar/HPG gels. A smooth correlation was also found between the computed crosslink concentrations and the viscosities of borate/guar gels (measured at constant shear). These equilibrium constants were also of the same order as those published for simple sugars forming six-member rings with borate anion. The stoichiometries of the complexes were obtained directly from the NMR peak areas rather than by analogy with simple, single-ring sugars, and at the low concentrations of chemicals used to produce moderately viscous fluids [e.g., 5700 CP at 1.5 mM boron, 10 g/L guar, and 4% salt]. The required analytical sensitivity was achieved with a 600 MHz NMR spectrometer. © 1996 John Wiley & Sons, Inc.     

Boron heterocycles derived from 2-guanidinobenzimidazole

The syntheses and structure determinations of a series of boron heterocycles derived from 2-guanidinobenzimidazole 1 are reported. Structures of new compounds, 2-guanidino-1-methyl-benzimidazole 2, diphenyl-(2-guanidinobenzimidazole-N,N′)-borate 3, diphenyl-(2-guanidino-1-methyl-benzimidazole-N,N′)borate 4, hydroxy-phenyl-(2-guanidino-benzimidazole-N,N′)borate 5, hydroxy-phenyl-(2-guanidino-1-methyl-benzimidazole-N,N′)borate 6,methoxy-phenyl-(2-guanidinobenzimidazole-N,N′)borate 7, isopropoxy-phenyl-(2-guanidinobenzimidazole-N,N′)borate 8, acetoxy-phenyl-(2-guanidinobenzimidazole-N,N′)borate 9, methoxy-phenyl-(2-guanidino-1-methyl-benzimidazole-N,N′)borate 10, dihy-droxy-(2-guanidino-1-methyl-benzimidazole-N,N′)borate 16, difluoro-(2-guanidinobenzimidazole-N,N′)borate, 17, dihydroxy-(2-guanidino-1-benzimidazole-N,N′)borate potassium salt 19, diphenyl-(2-guanidinium-10H-benzimidazole-N,N′)borate hydro-chloride 20, methoxy-phenyl-(2-guanidinobenzimidazole-N,N′)borate hydrochloride 21, and N10-borane-(diphenyl-2-guanidinobenzimidazole-N,N′)borate 22, were determined based on ¹H, ¹³C, ¹⁵N, and ¹¹B spectroscopy. The X-ray diffraction structures of 3–7, 19, and 20 were obtained. The formation of N3-borane adducts 11 and 12 derived from compounds 1 and 2, respectively, and the dihydride-(2-guanidinobenzimidazole-N,N′)borate 13 and dihydride-(2-guanidino-1-methyl-benzimidazole-N,N′)borate 14 were observed by ¹¹B NMR. The results show that 2-guanidinobenzimidazole gives stable borate heterocycles with a delocalized π electronic system. A dynamic exchange of N–H protons was observed with preferred protonation at N-12. The new heterocycles are protonated at N-10 by acidic substances to give pyridinium-type heterocycles or can lose a proton to give iminium salts. © 1998 John Wiley & Sons, Inc. Heteroatom Chem 9:399–409, 1998     

Transition Metal Borate Complexes Containing κ0-κ6 Denticity of Scorpionate Borate Ligands

The recent developments in the field of transition metal (TM) borate complexes have been a landmark in modern coordination chemistry. The structural diversities of these complexes play an important role in several catalytic processes. Generally, polypyrazolyl borate ligands, [BH_n(pz)_4-n]⁻ (n=1, 2; pz=pyrazolyl), popularly known as scorpionates have been used extensively for the preparation of TM borate complexes. The presence of multiple donor atoms in the flexible borate proligands led to several coordination modes. Based on the electronic and steric properties of these ligands and the metals, the denticity of borate ligands in TM complexes varied from κ⁰ to κ⁶. The presence of different bonding modes of these borate ligands made them very interesting in main group organometallic chemistry. In addition, cooperative activation of boranes by TM complexes containing metal-nitrogen or metal-sulfur bonds has become an alternative to the utilization of borate proligands for the synthesis of TM borate complexes. This review summarizes the advancements of the chemistry of TM borate complexes focusing exclusively on the synthetic methods and various bonding scenarios.     

Hydrolysis kinetics of the sulfonylurea herbicide Sulfosulfuron

The kinetics of hydrolytic degradation of Sulfosulfuron was investigated to predict the fate of the herbicide in an aqueous environment. The study revealed that the hydrolytic degradation of Sulfosulfuron followed first-order kinetics. The degradation of the herbicide was dependent on pH and temperature. Hydrolysis rate was faster in acidic condition (t _1/2 = 9.24 d at pH 4.0) than alkaline environment (t _1/2 = 14.14 d at pH 9.2). Several fold increase in the degradation rate was found when temperature was increased from 10 ± 1°C (t _1/2 = 518 h) to 50 ± 1°C (t _1/2 = 10 h). Activation energy (E _a) was also calculated as 63.87 KJ mol^?1, which is required for the hydrolytic degradation of the molecule. Both media pH and temperature effects were coupled together and derived a complex equation to estimate the overall effect of these two abiotic factors. The major degradation mechanism of the compound was the breaking of the sulfonylurea bridge yielding corresponding sulfonamide and aminopyrimidine. The possible significance of the results to persistence of the herbicide in the field condition is discussed.     

Separation of Ribose-5-phosphate,Ribose-1-phosphate,Deoxyribose-1-phosphate by High Performance Liquid Chromatography and Spectrophotometric Determination Using 2-Cyanoacetamide

Abstract

A simple procedure for separation of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate is based on high performance liquid chromatography using reversed phase 4 × 300 mm “μ Bondapak/NH₂” column. The column is equilibrated with 0.13 M borate buffer (pH 7.5) followed by gradient elution of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate using water, 0.05 M borate buffer containing 0.1 M MgCl₂ (pH 9.6) and 0.05 M sodium acetate-acetic acid buffer containing 0.1 M MgCl₂ (pH 5.0) as eluants respectively. Eluates of borate complex “μ Bondapak/NH₂” column are brought to pH 9.6 by the addition of 1 N KOH and enzymatically hydrolysed with alkaline phosphatase (EC 3.1.3.1) to release the free pentoses. The free pentoses are mixed with a reagent solution prepared from aqueous solution of 2% cyanoacetamide and 0.6 M borate buffer (pH 9.6), and the mixture is boiled for 10 minutes and the absorbance of the product is measured at 276 nm using a spectrophotometer.     

Borate‐Driven Gatelike Scaffolding Using Mesoporous Materials Functionalised with Saccharides

We report the development of an MCM‐41 mesoporous support that is functionalised with saccharides at the pore outlets and contains the dye [Ru(bipy)₃]²⁺ in the pores (solid S1 ; bipy=2,2′‐bipyridyl). For this hybrid system, the inhibition of mass transport of the dye from the pore voids to the bulk solution in the presence of borate is demonstrated in water at neutral pH. The formation of the corresponding boroester derivative is related to the selective reaction of borate with the appended saccharides. This control is selective and only anion borate, among several anions and cations, can act as a molecular tap and inhibit the delivery of the entrapped guest. Additionally, the S1 –borate system behaves as pH‐controlled gatelike scaffolding. This pH‐responsive release can be achieved in an acidic pH (due to hydrolysis of the boroester), whereas the system remains closed at neutral pH. Molecular dynamic simulations using force‐field methods have been made to theoretically study the open/close borate‐driven mechanism. A mesoporous silica structure was constructed for this purpose, taking the plane (1?11) of the β‐cristobalite structure as a base on which hexagonal nanopores and anchored saccharide derivatives were included. The final model shows a highly flexible nanopore diameter of approximately 12.5 Å of similar size to the [Ru(bipy)₃]²⁺ complex (ca. 12 Å). However, the anchoring of borate to the appended saccharides results in a remarkable reduction of the pore size (down to ca. 6.4 Å) and a significant constraint in the flexibility and mobility of the saccharides. The theoretical calculations are in agreement with the experimental results and enable visualisation of the functional borate‐driven dye‐delivery‐inhibition outcome.     

The catalytic properties of alkaline phosphatases under various conditions

A comparative study was performed to examine the catalytic properties of alkaline phosphatases from bacteria Escherichia coli and bovine and chicken intestines. The activity of enzyme dimers and tetramers was determined. The activity of the dimer was three or four times higher than that of the tetramer. The maximum activity and affinity for 4-nitrophenylphosphate was observed for the bacterial alkaline phosphatase (K _M = 1.7 × 10^?5 M, V _max = 1800 μmol/(min mg of protein) for dimers and V _max = 420 μmol/(min mg of protein) for tetramers). The Michaelis constants were equal for two animal phosphatases in various buffer media (pH 8.5) ((3.5 ± 0.2) × 10^?4 M). Five buffer systems were investigated: tris, carbonate, hepes, borate, and glycine buffers, and the lowest catalytic activity of alkaline phosphatases at equal pH was observed in the borate buffer (for enzyme from bovine intestine, V _max = 80 μmol/(min mg of protein)). Cu²⁺ cations formed a complex with tris-(oxymethyl)-aminomethane (tris-HCl buffer) and inhibited the intestine alkaline phosphatases by a noncompetitive mechanism.     

Rapid analysis of N-methylpyrrolidine in cefepime with thermal desorption ion mobility spectrometry

N-methyl-pyrrolidine (NMP) a potential impurity in the cephalosporin antibiotic cefepime is analysed using a rapid thermal desorption – ion mobility spectrometry (TD-IMS) method. The thermal desorption approach is shown to be capable of rapidly extracting NMP from the cefepime at 80 °C without causing thermal degradation of the cefepime. The ion mobility method has an analysis time of 1 min and demonstrates good linearity over a range from 0.3–3.0 μg ml^?1 of NMP, with limits of detection and quantification of 0.056 and 0.1875 μg ml^?1 respectively. The developed method was applied to the analysis of a cefepime sample and determined that NMP was present in a cefepime sample at a level of 0.0376 % with a percentage relative standard deviation (n = 6) of 3.2 %. This was compared with a LC-UV method which was in close agreement measuring NMP at 0.0384 % in the cefepime sample with a percentage RSD (n = 6) of 5.7 %. These results show that the TD-IMS method gives comparable data to the established LC methods and demonstrates the potential of TD-IMS for rapid measurement of volatile compounds in pharmaceutical matrices.     

Investigation of cobalt sorption on polyethylene using an in situ radioactive tracer method

Experiments on sorption of cobalt onto polyethylene foil from buffered borate solutions are reported in this paper. Sorption isotherms and sorption kinetics of cobalt as a function of pH and the effect of borate species on cobalt sorption were investigated. The results show that the extent of cobalt sorption is lower in borate buffer than in Na ClO₄ solution. It is found that the adsorption isotherms obtained are dependent on the pH of the solution. Possible reasons of the influence of pH on the sorption isotherms are discussed.     

Equilibria and kinetics for pH-dependent axial ligation of ethylester and methylester(aquo)cobaloximes with aromatic and aliphatic N-donor ligands and a molecular mechanistic study of the Co-C bond

Equilibrium constants are determined for the reaction of ethylester and methyl ester (aquo) cobaloximes with histamine, histidine, glycine and ethyl glycine ester as a function of pH at 25°C, using spectrophotometric technique. The functional dependence of pK _a on the substitution rate of H₂O varies with the pK _a of the incoming ligand, establishing the existence of nucleophilic participation of the ligand in the transition state. This data is interpreted with the help of kinetic data where dissociation kinetic reactions were also studied as a function of pH. Binding and kinetic data were correlated based on the basicity, steric hindrance of the entering ligand and HSAB principle. To compare the rate constants of the entering ligands pH-independent second-order rate constants were calculated. The effect of incoming ligand on Co-C bond is studied using molecular mechanics     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.

     

Further studies on the reaction of amines and proteins with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole

The reaction of glycine with NBD-F (4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole) is investigated to establish conditions that provide a high formation rate of NBD-glycine and a low hydrolysis rate of the reagent. The reaction rate increases with higher temperature, larger contents of organic solvent and a sodium borate buffer. The use of borate buffer decreases ther ate of hydrolysis of the reagent. For low-molecular-weight amines, condition for a suitable liquid chromatographic postcolumn reactor include a high content of acetonitrile and 0.1 M sodium borate (pH 8.0–8.5). For proteins, separated by molecular-exclusion chromatography, water is needed for sensitive reactions. Suitable postcolumn reactor conditions include borate buffer (pH 7.9) containing 0.1 M potassium chloride, a 0.02% (w/v) NBD-F solution in acetonitrile with reaction at 50°C for about 45 s. The detection limits for human serum albumin, β-lactoglobulin and myoglobin are 6.6 pmol, 8.4 pmol and 11 pmol, respectively.     

Sulfur Deficiency Changes Mycosporine-like Amino Acid (MAA) Composition of Anabaena variabilis PCC 7937: A Possible Role of Sulfur in MAA Bioconversion

In the present investigation we show for the first time that bioconversion of a primary mycosporine-like amino acid (MAA) into a secondary MAA is regulated by sulfur deficiency in the cyanobacterium Anabaena variabilis PCC 7937. This cyanobacterium synthesizes the primary MAA shinorine (RT = 2.2 min, λ_max = 334 nm) under normal conditions (PAR + UV-A + UV-B); however, under sulfur deficiency, a secondary MAA palythine-serine (RT = 3.9 min, λ_max = 320 nm) appears. Addition of methionine to sulfur-deficient cultures resulted in the disappearance of palythine-serine, suggesting the role of primary MAAs under sulfur deficiency in recycling of methionine by donating the methyl group from the glycine subunit of shinorine to tetrahydrofolate to regenerate the methionine from homocysteine. This is also the first report for the synthesis of palythine-serine by cyanobacteria which has so far been reported only from corals. Addition of methionine also affected the conversion of mycosporine-glycine into shinorine, consequently, resulted in the appearance of mycosporine-glycine (RT = 3.6 min, λ_max = 310 nm). Our results also suggest that palythine-serine is synthesized from shinorine. Based on these results we propose that glycine decarboxylase is the potential enzyme that catalyzes the bioconversion of shinorine to palythine-serine by decarboxylation and demethylation of the glycine unit of shinorine.