Penicilloic Acid Research Articles

Activation of laccase by penicillin and derivatives

Capable of oxidizing a wide range of reducing substances, laccase has a great potential for various biocatalytic applications. Extensive research effort is being made for improving laccase's activity, specificity, and stability. We observed that pre-incubating with penicillin led to a significant activation of a Rhizoctonia solani laccase, although penicillin was not a redox substrate of the enzyme. The activation impacted mainly the k cat of the laccase, and the effect was optimal at pH 5. Ampicillin and penicilloic acid were also active on the laccase (although their effect were weaker than that of penicillin), but 6-aminopenicillanic acid, phenylacetic acid, and cephalexin were not active. The apparent effect from penicillin was attributed to a non-redox interaction with the polypeptide backbone of the laccase. The effect was significantly weaker on Myceliophthora thermophila and Trametes villosa laccases.

Insights into the reaction of β-lactam antibiotics with copper(II) ions in aqueous and micellar media: Kinetic and spectrometric studies

Degradation of β-lactam antibiotics by means of metallic cations seems to have a very complex chemistry, involving not only the catalytic effect of the metal ion but also complex formation. Many different compounds, such as methylpyrazines, oxazolones, penicilloic, penicillenic, and penicillonic acids, have been reported as degradation products of such antibiotics, although not many details about the progress of the reaction can be found in the literature. Two novel fluorimetric and spectrophotometric methods previously published by the authors, as well as kinetic studies, have been used to propose a possible reaction mechanism for the ampicillin degradation in the presence of copper(II) ions. Likewise, we have proposed the chemical structure required by the β-lactam antibiotics to develop absorption or fluorescence properties. Kinetics in micellar and aqueous media shows that the copper–ampicillin reaction proceeds through different pathways depending on the reaction medium.

Electrochemical synthesis and impedance characterization of nano-patterned biosensor substrate

The nano-porous anodic aluminum oxide has been used as a substrate material for enzymatic biosensor operating in aqueous solutions. Nano-scale porous structure was formed by electrical anodization in an acid solution. By changing anodization conditions, such as electrolyte concentration, temperature, and anodization time, the ordered hexagonal porous structure with well-controlled pore size and depth can be obtained. Nano-porous alumina substrate with adsorbed enzymes was used as an enzyme electrode and pH sensor. The pH changes are driven by the enzymatic reactions, e.g. penicillin G hydrolysis to form penicilloic acid in the presence of penicillinaze. The advantage of physical adsorption used to bound penicillinaze, the model enzyme in this work, to the porous structure, is that usually no reagents are required and only a minimum of “activation” or clean-up steps. Adsorption tends to be less disruptive to enzyme proteins than chemical attachment. Due to the increased active sensor area, the immobilization of enzymes has been enhanced, which in turn improved the electrode’s sensitivity. To characterize the interactions of enzymes with nano-porous alumina oxide, electrochemical impedance spectroscopy (EIS) was used.

Food processing effects on residues: penicillins in milk and yoghurt

Milk samples spiked with benzylpenicillin, cloxacillin, oxacillin, dicloxacillin, ampicillin or nafcillin at and above maximum residue limit concentrations were used to produce yoghurt according to the typical industrial process. As expected, coagulation was negatively influenced by increasing concentrations of residual penicillins. On the other hand, the concentration of the penicillins was reduced due to various parameters of the production process. The temperature during heat treatment (90 °C, 15 min) of the milk before the yoghurt cultures were added, the binding of the penicillins to milk proteins, the fermentation time and temperature, and the influence of the starter cultures proved to be main factors leading to the decrease of residual penicillins. Main degradation products of penicillin G as identified by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) were penillic, penicilloic, and penilloic acids.

Monitoring of ampicillin and its related substances by NMR

A 1H NMR procedure for the monitoring of ampicillin (Amp) and its main related substance in different media, has been developed. The characteristics peak of Amp, 6-aminopenicillanic acid (6-APA), phenylglycine (PhG), and penicilloic acid in the range of 0.5–0.9 and 3.0–4.5 ppm were used for their identification in drugs and serum samples. The quantitative works were performed based on the intensity of protons of the methyl group link to the β-lactam cyclic of Amp and 6-APA and the aromatic protons of PhG relative to the protons of methylene group of maleic acid, as internal standard, at constant temperature. The resulting data are compared with those obtained with an HPLC method proposed by British Pharmacopoeia. Statistical studies show that, at a confidence limit of %95, there is no significant difference between the two methods. In comparison with the HPLC method, the proposed NMR method does not require any sample pretreatment, standard solution preparation, long analysis time and use of any carcinogenic solvent. The method was applied to the determination of Amp and its related substances in synthetic mixtures, drug powders and serum samples.

Influence of temperature on the kinetics of adsorption and desorption of clavulanic acid by ionic exchange

Beta-lactamases are enzymes that deactivate penicillins and cephalosporins by hydrolyzing their beta-lactam rings, producing penicilloic acid derivatives and, in the case of cephalosporins, analogous degradation products. Clavulanic acid (CA) is a beta-lactam antibiotic produced by Streptomyces clavuligerus, which has a potent beta-lactamase inhibiting activity. This antibiotic displays only weak antibacterial activity, therefore rendering it unsuitable for use by itself. However, its use in combination with certain penicillins (amoxycillin and ticarcillin) is effective in clinical use. The recovery process of CA involves steps such as adsorption, ion exchange chromatography or liquid–liquid extraction, but with low yields. Thus, in the case of CA, an investigation of the influence of temperature on the adsorption process is very important because of the unstable structure of this antibiotic, which degrades rapidly under normal processing conditions. In the work reported on herein, the influence of temperature on the CA adsorption process was investigated, involving equilibrium and kinetic studies and carrying out batch shake experiments with the ion exchanger resin Amberlite IRA 400 at four different temperatures. A model of the CA adsorption process, taking into account mass transfer limitation, is proposed. An estimate of the thermodynamics of the adsorption process was made based on van’t Hoff’s equation.

Depolarization of the Membrane Potential by β-Lactams as a Signal to Induce Autolysis

The effect of β-lactam antibiotics that are known to inhibit cell wall biosynthesis and induce cell wall autolysis on the electrophysiological state of the plasma membrane in Streptomyces griseus was studied. Addition of various β-lactam antibiotics induced a dose- and growth-stage-dependent depolarization of the membrane potential of Streptomyces griseus. The hydrolyzed biologically inactive derivative penicilloic acid had no depolarizing effect on the membrane potential. The ionophore gramicidin D, while depolarizing the membrane potential, also induced a dose-dependent increase in cell wall lysis. These observations suggest that alteration of the transmembrane potential could be an important signal in triggering cell wall autolysis of S. griseus.

A validated stability-indicating high performance liquid chromatographic assay of amoxicillin

The purpose of this work is to develop a selective, simple, rapid and accurate liq. chromatog. method for the anal. of amoxicillin and its possible degrdn. products in bulk drug. Amoxicillin and its degrdn. products were analyzed on Ultrasphere C18 column, using a mobile system contg. 4​% methanol in 0.01 M potassium dihydrogen phosphate soln., 2 mM triethylamine, and adjusted to pH 3.6 with 0.1 M phosphoric acid. The samples were monitored at wavelength 242 nm. Amoxicillin was forcedly degraded in powder and soln. forms under all possible stress conditions. The forced degrdn. conditions were including the effect of daylight, heat, moisture, acid-​base hydrolysis, sonication, and oxidn. Amoxicillin penicilloic acid (AMP) was the major degrdn. product formed under all stress conditions. However, penicillamine (PM) and 6-​aminopenicillanic acid (6-​APA) were generated under severe stress conditions. The detection and quantification limit of 6-​APA, PM and clavulanic acid were enhanced by the use of triethylamine as ion-​pair. This method was also applicable for the detn. of both, amoxicillin and clavulanic acid in Augmentin parenteral.

1021 Preparation and analysis of penicilloic acid and penilloic acid using high resolution nuclear magnetic resonance

1021 Preparation and analysis of penicilloic acid and penilloic acid using high resolution nuclear magnetic resonance

Pharmacodynamic and pharmacokinetic rationales for the development of an oral controlled-release amoxicillin dosage form

The goal of this investigation was to develop an oral sustained-release formulation for amoxicillin that would maximize the duration of active drug concentration in the extracellular fluid, thus increasing the dosing interval while assuring antimicrobial activity. This rationale is based on the pharmacodynamic properties of the drug which is non- concentration dependent on the one hand, while requiring long exposure of the pathogen to the drug with minimal post-antibiotic effect on the other. Due to pharmacokinetic constraints, including short biological half-life and limited `absorption window' (confined to the small intestine) with poor colonic absorption, the new matrix tablet formulation, composed of hydrophilic (hydroxypropyl methyl-cellulose) polymer, was designed to release 50% of its contents within the first 3 h and to complete the drug release process over 8 h (under in vitro conditions). The pharmacokinetics of the new formulation was evaluated in 12 healthy volunteers and compared to a conventional gelatin capsule with both formulations containing 500 mg amoxicillin. The plasma concentrations of active amoxicillin and penicilloic acid were determined by an HPLC method with a fluorometric detector. It was found that the area under the concentration–time curve and maximal serum amoxicillin concentrations following the sustained release preparation were lower than the immediate release formulation. However, the time over the required threshold concentrations, i.e. the minimal inhibitory concentration (MIC) as well as the more clinically relevant parameter — four times MIC of the drug against susceptible pathogens, was found to be maintained for significantly longer periods. The results suggest that in order to achieve a twice daily dosing regimen that will provide therapeutic concentrations for the whole 12 h dosing intervals, a larger dose of the new formulation should be given (e.g. 750 mg or even 1g twice daily). This recommendation is based on the large interindividual differences of the extent of amoxicillin absorption found in this investigation, and is intended to assure that the `poor' absorbers will also benefit from full antibiotic efficacy. This dosing regimen will lead to increased patient compliance and improved therapeutic outcome.

Spectroscopic and polarographic investigations: Copper(II)-penicillin derivatives

Copper(II) derivatives of penicillins (benzypenicillin, phenoxymethylenepenicillin, ampicillin, amoxycillin and carbenicillin) have been prepared. Copper(II) ions promote hydrolysis of penicillins (L) to corresponding penicilloic acids (L∗) owing to β-lactam group. Coordination compounds isolated under equilibrium conditions showed stoichiometries of the type CuL∗, CuL∗ 2, and CuL∗ 3. Two compounds with molar metal to ligand ratio 1:1 and 1:3 are aqueous soluble and interactions at molar ratio 1:2 result in precipitate formation. The solubility of the precipitates undergoes no changes in a broad pH region. It takes place only in highly acidic or alkaline media. The infrared spectra suggest that penicillins behave as monoanionic bidentate ligands coordinating the Cu(II) ion through the carboxylic group.

The interference of penicilloic acids with Karl Fischer titration of penicillins

Phenoxymethylpenicilloic acid monohydrate was titrated with 12 different brands of Karl Fischer reagent. It was shown that only the four pyridine-containing reagents gave correct results for water content, providing that the sample solution was cooled in an ice bath or that the delay time of the amperometric end-point determination was 10 s instead of 30 s, as prescribed by the European Pharmacopoeia. The other reagents always gave results, which were too high. When these reagents were used to titrate the water in sodium amoxicillin, sodium carbenicillin or sodium ticarcillin the same conclusions could be drawn with regard to the accuracy of the results, the usefulness of the reagents and the reaction conditions. These penicillins may contain up to 9% penicilloic acids. The maximum relative error obtained with pyridine-free reagents was about 50% for ticarcillin. It has been confirmed that penicilloic acids interfere with Karl Fischer titration of water and that this interference is greatly dependent on the brand of reagent and on the reaction conditions.

Investigation of the reaction mechanism of the mercurimetric determination of benzylpenicillin

The assay of different penicillins in the European Pharmacopoeia was carried out by mercurimetric titration with potentiometric determination of the end-point. The consecutive formation of reaction products during titration was followed by reversed-phase liquid chromatography (LC) and ultraviolet detection. In one experiment the titration was carried out with 14C-labeled benzylpenicillin and the reaction was followed with LC coupled to radiochemical detection. The identity of the intermediates and final reaction products was deduced from their retention times in comparison with reference products obtained by independent chemical transformation of benzylpenicillin. This allowed one to define for the first time the complete reaction scheme. This involves the isomerization of the natural penicilloic acid followed by decarboxylation, which has not been reported previously. At the end of the titration, only benzylpenilloaldehyde and a 1:1 complex of mercury and penicillamine were present in the solution.

On-line monitoring of glucose and penicillin by sequential injection analysis

A sequential injection analysis (SIA) system has been developed for on-line monitoring of glucose and penicillin during cultivations of the filamentous fungus Penicillium chrysogenum. The SIA system consists of a peristaltic pump, an injection valve, two piston pumps, two multi-position valves and a detector. The glucose analyzer is based on an enzymatic reaction using glucose oxidase, which converts glucose to glucono-lactone with formation of hydrogen peroxide and subsequent detection of H 2O 2 by a chemiluminescence reaction involving luminol. The penicillin analysis is based on formation of penicilloic acid by penicillinase. Penicilloic acid is detected either by a chemiluminescence method or by a decolorization method. In the chemiluminescence method the penicilloic acid is quantified by its quenching effect on the chemiluminescence signal obtained when luminol reacts with iodine. In the decolorization method the penicilloic acid is detected spectrophotometrically by the decrease in the absorbance of an iodine-starch complex.

Amperometric Penicillin Biosensor and Its Application in Flow Injection Analysis System for Determination of Penicillin in Broth

An amperometric biosensor for determination of penicillin was developed. Penicillinase was immobilized by cross-linking with glutaraldehyde and bovine serum albumin, which was dropped on a platinum disc electrode. The principle of this electrode is based on the classical iodometric penicillin assay. The required hydrolysis is performed by immobilized penicillinase. Enzymatic hydrolysis of the β-lactam ring results in the formation of the corresponding penicilloic acid, which consumes iodine. The excess of iodine is determined amperometrically. The electrode was used to perform more than 1000 assays in 2 weeks and successfully used to determine penicillin in broth. The linearity, pH, interferences, and buffer strength dependence of the electrode were examined and discussed.

Simultaneous monitoring of glucose, lactic acid and penicillin by sequential injection analysis

A sequential injection analysis (SIA) system for measurement of glucose, lactic acid and penicillin has been developed. The system is built around a 10 port multiposition valve, a piston pump, and a chemiluminescence detector. The SIA-system may measure all three components using the same hardware, and it is therefore well suited for monitoring of penicillin cultivations, where glucose and lactic acid are typical substrates and penicillin is the major product. The analysis of glucose is based on an enzymatic conversion - using glucose oxidase - to glucono-δ-lactone with parallel formation of hydrogen peroxide. The formed hydrogen peroxide is detected by a chemiluminescence reaction involving luminol. The analysis of lactic acid using lactate oxidase is similar to that of glucose. The analysis of penicillin is based on formation of penicilloic acid by pumping the sample through a column of immobilized penicillinase upstream of the multiposition valve. When thereafter penicilloic acid reacts with iodine it prevents a simultaneous chemiluminescence generating reaction between iodine and luminol.

Determination of penicillin by triangle programmed coulometric acid–base flow titration in a system incorporating an enzyme reactor

AbstractA flow‐through coulometric titration technique is described which can be applied for the determination of a wide variety of compounds. If necessary, the sample can be transformed in the measuring system into a form which can be titrated. Thus an enzyme‐catalyzed reaction can also be used as a sample pretreatment step. In this article a measurement technique and apparatus are described in which an enzyme‐catalyzed reaction is used for the fast and reliable determination of penicillin compounds. The principle of the technique is that an enzyme reactor is built into the sample line of the measurement system used for performing coulometric triangle programmed titrations. One of the components of the reaction mixture leaving the reactor (in this case penicilloic acid) is determined by coulometric acid–base titration. This technique is especially useful for the analysis of large sample series.

High resolution 1H NMR spectroscopic studies of the metabolism and excretion of ampicillin in rats and amoxycillin in rats and man.

High resolution proton nuclear magnetic resonance (1H NMR) spectroscopy has been used to investigate the metabolism and urinary excretion of the aminopenicillins, ampicillin and amoxycillin, in rats and of amoxycillin in man. 1H NMR resonances of the aminopenicillins, together with those for their 5R, 6R and 5S, 6R penicilloic acids and diketopiperazine metabolites were detected, assigned and quantified in urine samples with the aid of spin-echo NMR techniques. The dimer of amoxycillin was detected in rat urine for the first time together with novel drug-related resonances assigned to amoxycillin carbamate. Quantitative 1H NMR spectroscopic results were consistent with HPLC and microbiological data considering that only single measurements were recorded. Due to the short analysis time and simple sample preparation, NMR was particularly useful for studying the metabolism of the aminopenicillins for which sample degradation poses analytical problems. The non-invasive character of 1H NMR spectroscopic analysis of urine also provided unique information on a reversible reaction between amoxycillin and bicarbonate, an endogenous urinary metabolite.

Industrial on-line monitoring of penicillin V, glucose and ethanol using a split-flow modified thermal biosensor

Penicillin V, glucose and ethanol were monitored in 0.5 and 2.5 m 3 bioreactors using an enzyme thermistor, modified for split-flow analysis. Penicillin V was also monitored in a 160 m 3 bioreactor. The samples were split immediately before similtaneously entering the enzyme column and an identical reference column, (without enzyme). By using a reference column the non-specific heat arising from mixing and solvation effects caused by high salt and metabolite concentrations in the fermentation broth can be eliminated. Immobolized β-lactamase in the enzyme column was used to monitor three consecutive fermentations, but the penicillin V values were 10% higher than the values obtained by off-line liquid chromatographic(LC) analysis of the same samples. Another enzyme was therefore used with the same set-up. After purification from a broth supernatant, penicillin V acylase was used for on-line monitoring of penicillin production in a 160 m 3 bioreactor. The concentrations of penicilloic acid, p-hydroxypenicillin V and penicillin V were determined by off-line LC. The sum of these concentrations matched the on-line biosensor values very well. Glucose was monitored from the start in a Saccharomyces fermentation but after 5 h the analysis was switched to ethanol monitoring. It took 60 min to switch from a glucose oxidase column to a column with alcohol oxidase and obtain the first on-line ethanol value.

On-line monitoring of penicillin V during penicillin fermentations: a comparison of two different methods based on flow-injection analysis

Two different flow-injection analysis (FIA) methods used for on-line monitoring of penicillin V during fed-batch penicillin fermentations are compared. The fermentations are carried out using a high yielding strain of Penicillium chrysogenum and both analyzers are used continuously for on-line monitoring during 200 h. Both methods are based on a reaction catalyzed by β-lactamase, which converts penicillin V to penicilloic acid. In analyzer 1 the sample is injected into a buffer stream and carried to a stirred measurement cell containing an enzyme electrode. The enzyme electrode consists of β-lactamase immobilized onto a sensitive pH glass electrode with a very low response time. No predilution of the sample is required in this analyzer. In analyzer 2 the sample is injected into an enzyme reactor containing immobilized β-lactamase. The formed penicilloic acid is detected by decolorization of an iodine-starch complex. By means of a second injection valve it is possible to have the sample by-pass the enzyme reactor and instead pass a dummy reactor in which no β-lactamase is immobilized. Thereby both the concentration of penicillin and penicilloic acid can be measured. On-line dilution of the sample is ensured by a gradient technique. Experimental results from on-line monitoring of penicillin V using the two methods are presented. The large amount of precise measurements enables a very good resolution of the penicillin production; something of extreme value for verification of mathematical models describing the fermentation kinetics.