Determination of beta-lactam antibiotics in water by fluorescence quenching of mercurochrome, and application for simple investigation of potency

The fluorescence quenching reaction between fluorescein mercury or halogeno-fluorescein mercury compounds (fl. Hg, 2,7- or 2,4-dichloro-fl.Hg, 3',4',5',6'-tetrachloro-fl.Hg, mercurochrome) and beta-lactam antibiotics (ampicillin (AB-PC) and cephalexin (CEX] was investigated, and mercurochrome was selected for the detection of beta-lactam antibiotics; the detection limit was about 0.8 micrograms/ml. A fluorimetric assay of beta-lactam antibiotics was established by measuring the fluorescence of mercurochrome and mercurochrome-beta-lactam antibiotics solutions in weakly basic media to determine the degree of fluorescence quenching. The maximum emission wavelength of mercurochrome solution was at 544 nm with excitation at 470 nm. The calibration graphs were linear over the ranges of about 0-6 micrograms/ml beta-lactam antibiotics penicillins (AB-PC, penicillin G, sulbenicillin, amoxicillin, cyclacillin, oxacillin, hetacillin and piperacillin) and cepham antibiotics (CEX, cefazolin, cephaloglycin, cephaloridine and cefpyramide), and the relative standard deviation was 2.7% for 1.4 micrograms/ml of AB-PC (n = 5). This fluorescence quenching reaction between mercurochrome and beta-lactam antibiotics was applied in a survey of decomposition and remaining potency of beta-lactam antibiotics.     

An approach for decontamination of beta-lactam antibiotic residues or contaminants in the pharmaceutical manufacturing environment

An effective procedure for decontamination of beta-lactam antibiotic residues or contaminants in the pharmaceutical manufacturing environment was investigated. Decontamination with solutions of hydrochloric acid, sodium hydroxide, hydrogen peroxide and hydroxylamine as agents for degradation was assessed. According to the results, the beta-lactam antibiotics were significantly degraded with sodium hydroxide and hydroxylamine. From the structural analysis of the degradation products of a cephem antibiotic, cefpodoxime proxetil, it was found that hydroxylamine degraded the beta-lactam structure under mild conditions, while sodium hydroxide did not. Therefore, hydroxylamine was considered an appropriate decontamination agent for beta-lactam antibiotics.     

Catalysis of hydrolysis and aminolysis of non-classical beta-lactam antibiotics by metal ions and metal chelates.

The Zn(2+)-tris (hydroxymethyl)aminomethane (Tris) system has a great catalytic effect on the hydrolysis and aminolysis of some beta-lactam antibiotics. In order to ascertain the mechanism of this catalysis we have analysed the effects of the beta-lactam antibiotic structure. First we studied the kinetics of the decomposition of imipenem, SCH 29482, aztreonam and nocardicin A in aqueous solution of Tris at 35.0 degrees C, 0.5 mol.dm-3 ionic strength and in the presence of metal ions (Zn2+, Cd2+, Co2+, Cu2+, Ni2+ and Mn2+). From these studies, we conclude that Tris and metal ions (in separate solutions) exert a great catalytic effect on the hydrolysis of imipenem and SCH 29482. We suggest that in metal ion solutions a 1:1 complex is formed between the metal ion and beta-lactam antibiotic, which is attacked by hydroxide ions. Studies of the degradation of the antibiotics studied in solutions of Tris and metal ions together indicate that the systems Cd(2+)-Tris and Zn(2+)-Tris have a great catalytic effect on the hydrolysis and aminolysis of imipenem and SCH 29482. We suggest that this catalysis takes place via a ternary complex in which the metal ion plays a double role by (a) placing the antibiotic and the Tris in the right position for the reaction and (b) lowering the pKa of the hydroxide group of Tris, which is coordinated with the metal ion, generating a strong nucleophile.     

Free-radical destruction of beta-lactam antibiotics in aqueous solution

Many pharmaceutical compounds and metabolites are being found in surface and ground waters, indicating their ineffective removal by conventional wastewater treatment technologies. Advanced oxidation/reduction processes (AO/RPs), which utilize free-radical reactions to directly degrade chemical contaminants, are alternatives to traditional water treatment. This study reports the absolute rate constants for reaction of three beta-lactam antibiotics (penicillin G, penicillin V, amoxicillin) and a model compound (+)-6-aminopenicillanic acid with the two major AO/RP reactive species: hydroxyl radical ((*)OH) and hydrated electron (e(-)aq). The bimolecular reaction rate constants (M(-1) s(-1)) for penicillin G, penicillin V, amoxicillin, and (+)-6-aminopenicillanic acid for (*)OH were (7.97 +/- 0.11) x 10(9), (8.76 +/- 0.28) x 10(9), (6.94 +/- 0.44) x 10(9), and (2.40 +/- 0.05) x 10(9) and for e(-)aq were (3.92 +/- 0.10) x 10(9), (5.76 +/- 0.24) x 10(9), (3.47 +/- 0.07) x 10(9), and (3.35 +/- 0.06) x 10(9), respectively. To provide a better understanding of the decomposition of the intermediate radicals produced by hydroxyl radical reactions, transient absorption spectra were observed from 1 to 100 micros. In addition, preliminary degradation mechanisms and major products were elucidated using (137)Cs gamma irradiation and LC-MS. These data are required for both evaluating the potential use of AO/RPs for the destruction of these compounds and studies of their fate and transport in surface waters where radical chemistry may be important in assessing their lifetime.     

Rational design of a novel fluorescent biosensor for beta-lactam antibiotics from a class A beta-lactamase

A rational design strategy was used to construct a sensitive "turn-on" biosensor for beta-lactam antibiotics and beta-lactamase inhibitors from a class A beta-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Omega-loop of the E166C mutant close to the active site of the beta-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Omega-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent. This process is accompanied by an increase in the fluorescence of the labeled enzyme. The fluorescence intensity of the biosensor increases with the concentration of antibiotics or inhibitors, which can detect penicillin G at concentrations as low as 50 nM in water. This approach opens a possibility for converting highly active and nonallosteric enzymes into substrate-binding proteins for biosensing purposes.     

Stability in aqueous solution of two monocyclic beta-lactam antibiotics: aztreonam and nocardicin A.

The catalytic effect of various buffer systems (citrates, acetates, phosphates, borates and carbonates) on the degradation of aztreonam and nocardicin A in aqueous solution was studied at 35 degrees C and a constant ionic strength of 0.5 mol.dm-3 over a pH range of 3.50 to 10.50. The observed degradation rates, obtained by measuring the remaining intact antibiotic, were shown to follow pseudo-first-order kinetics with regard to antibiotic concentrations and to be influenced by general acid and general base catalysis. The changes in the concentration of intact beta-lactam antibiotic in the solutions were established by reverse-phase HPLC with UV-detection. In general the buffer systems employed in the kinetic studies showed a very weak catalytic effect on the degradation of aztreonam and nocardicin A. The pH-rate profiles for these antibiotics showed degradation minimums at pH 5.38 and 6.13, respectively. Aztreonam is slightly more reactive with hydrogen ions than nocardicin A and is much more reactive with hydroxide ions. In comparison with other beta-lactamic antibiotics, aztreonam and nocardicin A are much more stable in aqueous solution, except for aztreonam in a base solution, which is just as unstable as penicillins and cephalosporins. The Arrhenius activation energies were determined for aztreonam and nocardicin A at pH's 4.23, 6.59 and 8.60.     

Fluorescence enhancing by alkaline degradation of tetracycline antibiotics and its application

Tetracycline antibiotics could be degraded after being beated in boiling-water bath in alkaline solution (in KOH medium).The fluorescence excitation and emission wavelengths of the degradation products changed and the fluorescence intensity increased by 5-190 folds as compared with that of antibiotics themselves It was considered that there occurred a nucleophilic ring rupture reaction to form the degradation products with lactome structure The mteraction of the lone pair electrons of oxygen on the degradation products with the conjugated π electrons induced the in crease of the delocalization for the conjugated electron system in the molecule and thus enhanced the fluorescence intensity.     

Use of SDS micelles for improving sensitivity, resolution, and speed in the analysis of beta-lactam antibiotics in environmental waters by SPE and CE

This study dealt with the potential of MEKC with LIF detection involving derivatization with sulfoindocyanine succinimidyl ester (Cy5) for the separation and determination of beta-lactam antibiotics (ampicillin, amoxicillin, cephradine, and cephalexin) in environmental water samples. Water samples of 50 mL were enriched by SPE by passage through a weak base-cation Amberlite(R) IRA-93 exchange column. SDS micelles play important roles in the whole analytical process by improving the yield (sensitivity) and the kinetics of the labeling reaction, the elution of the retained antibiotics from the SPE preconcentration system and the electrophoretic resolution of their Cy5-derivatives. The optimum procedure includes a derivatization step of the antibiotics at 25 degrees C for 10 min and direct injection for MEKC analysis, which is conducted within about 15 min using 15 mM SDS in the running buffer (35 mM sodium borate at pH 9.3). LODs from 30 to 45 ng/L and RSDs (within-day precision) from 3.5 to 5.9% were obtained for the antibiotics in water samples with average recoveries ranging from 96.4 to 99.4%. These results indicate that the method proposed is a straightforward and sensitive tool for the determination of these antibiotics in environmental water samples providing similar quantitative results to those using more expensive equipment like LC-electrospray MS/MS.     

Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices

The penicillin-binding protein PBP 2x* from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the detection and determination of penicillins and cephalosporins with intact beta-lactam structure in milk, bovine and porcine muscle juice, honey and egg. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and digoxigenin as functional groups (DIG-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses anti-digoxigenin F(ab) fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more antibody fragments are bound via the digoxigenin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present. A fractional factorial design was applied to detect chemometrically effects and interactions of the assay parameters. For optimisation of the significant parameters a Box-Behnken design was used. The assay has been developed for various food matrices as screening test with the option for a quantitative assay, when the identity of the residual beta-lactam is known (e.g. elimination studies). Cefoperazon, cefquinome, cefazolin, cloxacillin, ampicillin and benzylpenicillin could be detected at levels corresponding to 1/2 EU maximum residue limit (MRL) in milk, meat juice from muscle tissue of different species, egg and honey (where applicable) without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.     

Trace determination of beta-lactam antibiotics in environmental aqueous samples using off-line and on-line preconcentration in capillary electrophoresis

A sensitive and reliable method using capillary zone electrophoresis with UV-diode array detection (CZE-DAD) has been developed and validated for trace determination of beta-lactam antibiotics in waste, well and river water matrices. Due to the lack of sensitivity of the UV-vis detection, a solvent extraction/solid-phase extraction (SPE) method applied for off-line preconcentration and cleanup of water samples, in combination with an on-line preconcentration methodology named large volume sample stacking (LVSS) have been applied. The analytes included nafcillin, dicloxacillin, cloxacillin, oxacillin, ampicillin, penicillin G and amoxicillin. Average recoveries for water samples fortified with the studied beta-lactams at different concentration levels (1.0, 2.0 and 4.0 microg/L) were ranging between 94 and 99%, with relative standard deviations (RSDs) lower than 10%. The precision, calculated as intra-day and inter-day standard deviations fell within acceptable ranges (3.3-7.2%). The limits of detection were estimated to range between 0.08 and 0.80 microgL(-1) for the studied compounds. All the samples analyzed were negative for all the analytes at these levels of concentration and the method showed its usefulness for the detection of these widely applied beta-lactam antibiotics in different kinds of waters.     

Hydrophobicity of beta-lactam antibiotics. Explanation and prediction of their behaviour in various partitioning solvent systems and reversed-phase chromatography.

beta-Lactam antibiotics tend to undergo self-association in hydrophilic organic solvents, which leads to a strong dependence of their experimentally observable log P values on the partitioning conditions. As a result, most of the earlier obtained log P values for beta-lactam antibiotics cannot be applied as a common hydrophobicity measure, but they proved to be linearly related to each other and to a large body of reversed-phase chromatographic data. The retention of cephalosporins on reversed-phase liquid chromatographic columns is complicated by silanophilic interactions. However, under elution conditions that eliminate these silanophilic interactions, good correlations with log P data are observed, and a unified hydrophobicity scale for 90 penicillin and cephalosporin compounds could be evaluated. The Hansch and Leo additive scheme was shown to be valid for the calculation of hydrophobicities for penicillin and cephalosporin C-6(7) substituents, but it failed when applied to the prediction of cephalosporin C-3-substituent hydrophobicities. The hydrophobic increments for the sixteen most common cephalosporin C-3-substituents were empirically evaluated from literature data, and a simple equation was derived for an overall beta-lactam antibiotic hydrophobicity calculation. The proposed scale is valid for predicting the partitioning of most beta-lactam antibiotics in both hydrophilic and lipophilic organic-water systems, although it should be used with caution when applied to antibiotics containing additionally charged side-chains.     

Catalytic mechanism of class A beta-lactamase. I. The role of Glu166 and Serl30 in the deacylation reaction

The tetrahedral intermediate formation process, which is the first step in the deacylation reaction by class A beta-lactamase, was investigated by the ab initio molecular orbital method. In this study, benzyl penicillin was used as the substrate. From the results of our molecular dynamics study of the structure of beta-lactam antibiotics-beta-lactamase complex, the substrate, Ser70, Lys73, Ser130, Glu166 and a water molecule for the deacylation reaction were considered for construction of a model for calculation. The calculation results indicated that Glu166 plays a role in holding a water molecule, which is necessary for the deacylation reaction, and that the hydrogen bond network among Lys73Nzeta, Ser130Ogamma, and the carboxyl group of the beta-lactam antibiotics was formed by the uptake of beta-lactam antibiotics by beta-lactamase. The activation energy for this reaction was 33.3 kcal/mol, and it is very likely that the reaction occurred at body temperature. Subsequent calculation results obtained by using the model excluding Ser130 and the carboxyl group of the substrate indicated that the activation energy for this reaction was 40.8 kcal/mol, which is 7.5 kcal/mol higher than that of the previous reaction. It was found that the hydrogen bond network plays an important role in decreasing the activation energy for the tetrahedral intermediate formation reaction. Lys73Nzeta, which is located at the edge of the hydrogen bond network, played a role in forming a hydrogen bond with Glu166Oepsilon in order to help the deacylation reaction. The role of amino acid residues around the active site of class A beta-lactamase was also discussed.     

The biosynthetic gene cluster for the beta-lactam carbapenem thienamycin in Streptomyces cattleya

beta-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic beta-lactam antibiotics. This involves the participation of a beta-lactam synthetase. Using available information from beta-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces cattleya. Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of thienamycin biosynthesis leading to the formation of the beta-lactam ring. Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.     

Multidimensional ion-pair HPLC for the purification of aminoglycoside antibiotics with refractive index detection

Summary Gradient chromatographic conditions with refractive index detection are defined for the ion-pair separation of the non-UV-absorbing aminoglycoside antibiotics. The method was applied with an off-line ion-pair prepurification step to the separation and characterization of unknown aminoglycoside starting from filtered broths. An on-line prepurification process has also be designed.     

Cold-temperature stability of five beta-lactam antibiotics in bovine milk and milk extracts prepared for liquid chromatography-electrospray ionization tandem mass spectrometry analysis

The stability of five major beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, oxacillin, and penicillin G) in fortified milk and in milk extracts prepared for LC-ESIMS/MS analysis was studied at varying cold temperatures (4, -20, and -76 degrees C). Storage of milk samples at 4 degrees C resulted in measurable losses of all beta-lactams after 6 days (>50% in most cases). Slow degradation of penicillin G, cloxacillin, and oxacillin was observed in milk stored at -20 degrees C, but no losses were recorded at -76 degrees C over 4 weeks. All antibiotics showed good stability at all temperature tested in milk extracts prepared for LC-ESIMS/MS analysis. The results of this study emphasize adherence to adequate sample handling and storage protocols as to reflect residue levels at the time of sample submission.     

Separation of beta-lactam antibiotics by micellar electrokinetic chromatography

The retention behaviour of beta-lactam antibiotics in micellar electrokinetic chromatography (EKC) was investigated. Sodium dodecyl sulphate (SDS) and sodium N-lauroyl-N-methyltaurate were used an anionic surfactants at concentrations of 0.05-0.3 M. It was found that the retention of ionic substances in micellar EKC is determined by the following three factors: the electrophoretic migration of the ionic substances, the interaction between the ionic substances and ionic surfactants and solubilization of the solute by the micellar phase. A difference in the retention behaviours of cationic substances was observed between the two anionic surfactants, which have different groups neighbouring the charge-bearing groups. The effect of an ion-pairing reagent was also investigated to make the effect of the micelle clearer. All test solutes were successfully separated by micellar EKC at SDS concentrations above 0.1 M, with theoretical plate numbers ranging from 70,000 to 260,000.     

Reversed-phase liquid chromatographic method for the analysis of aminoglycoside antibiotics using pre-column derivatization with phenylisocyanate

A pre-column derivatization liquid chromatographic method has been developed for the analysis of aminoglycoside antibiotics using phenylisocyanate as a derivatization reagent. Derivatives including kanamycin, neomycin and gentamicin were formed by reaction of the analytes with phenylisocyanate in the presence of triethylamine. Phenylisocyanato groups were attached to corresponding amino groups of aminoglycoside and their molecular mass was confirmed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The experimental conditions for derivatization and separation of aminoglycoside derivatives were optimized and validated. A simple liquid chromatographic method for the determination of aminoglycoside antibiotics was demonstrated.     

Trace determination of beta-lactam antibiotics in surface water and urban wastewater using liquid chromatography combined with electrospray tandem mass spectrometry

A sensitive and reliable method using liquid chromatography-electrospray tandem mass spectrometry has been developed and validated for the trace determination of beta-lactam antibiotics in natural and wastewater matrices. Water samples were enriched by solid-phase extraction. The analytes included amoxicillin (AMOX), ampicillin (AMP), oxacillin (OXA), cloxacillin (CLOX) and cephapirin (CEP). Average recoveries of beta-lactams (BLs) in fortified samples were generally above 75% (except amoxicillin) with the standard deviations lower than 10% in water matrices. Amoxicillin was not quantified due to poor recovery (less than 40%) in the investigated water matrices. Matrix effects were found to be minimal when measuring these compounds in water matrices. The accuracy, within- and between-run precision of the assay fell within acceptable ranges of 15% absolute. The method detection limit (MDL) was estimated to range between 8 and 10 ng/L in surface water, 13 and 18 ng/L in the influent and 8 and 15 ng/L in the effluent from a wastewater treatment plant. A large number of actual water samples were analyzed using this method in order to evaluate the occurrence of the beta-lactams in a river and a wastewater treatment plant in northern Colorado. Most of the samples were negative for all analytes. These compounds were found at 15-17 ng/L in the three influent samples and at 9-11 ng/L in three surface water samples out of a total of 200 samples. This indicates that contamination by beta-lactam antibiotics is of minor importance to the small mixed-watershed.     

Co-opting the cell wall in fighting methicillin-resistant Staphylococcus aureus: potent inhibition of PBP 2a by two anti-MRSA beta-lactam antibiotics

Methicillin-resistant Staphylococcus aureus (MRSA) is a global bacterial scourge that has become resistant to many classes of antibiotics, and treatment options for MRSA infections are limited. The cause of MRSA resistance to all commercially available beta-lactam antibiotics is the acquisition of the gene mecA, which encodes penicillin-binding protein 2a (PBP 2a). PBP 2a is a transpeptidase, which in contrast to the other transpeptidases of S. aureus does not experience inhibition by beta-lactam antibiotics. The lack of inhibition is due to a closed conformation for the active site for PBP 2a, which opens up only in the course of the catalytic function of the protein. Here we show that two new anti-MRSA antibiotics now undergoing clinical trials, ceftaroline and ME1036, are able to inhibit PBP 2a effectively, a process that is enhanced in the presence of a cell wall structural surrogate. It is likely that in the course of bacterial growth the occupancy of the allosteric site for the cell wall is co-opted by these antibiotics, and under these conditions the second-order rate constant for the encounter of the antibiotic and PBP 2a approaches the clinically useful value of 10(4)-10(5) M-1 s-1. These compounds are potent inhibitors of PBP 2a as well as PBPs from other species, and have potential as therapeutic agents for treatment of serious infections by MRSA and other resistant bacterial pathogens.     

Validation of the Charm MRL-3 for fast screening of beta-lactam antibiotics in raw milk

The biochemicals utilized in the Charm MRL beta-Lactam test (8 min test) were applied to faster flowing lateral components to create a new 3 min, one-step beta-lactam test called Charm MRL-3 (Charm Sciences Inc., Lawrence, MA). This new test was validated at T&V-ILVO according to Commission Decision 2002/657/EC. The following analytical parameters were checked: test specificity, detection capability, and test robustness (impact of deviation of the test protocol, and impact of the milk composition, batch differences of reagents). Further, the suitability of the Charm MRL-3 to screen heat-treated milk or milk from animal species other than the cow was also tested. Finally, the test was integrated in the monitoring of dairy samples to check the occurrence of false-negative or false-positive results, and the test was also included in a national ring trial and an international proficiency study. The results proved that the Charm MRL-3 is a fast, simple, and reliable cows' milk test that can be used at the farm level in order to prevent tanker milk contamination, or at the entrance of the dairy plant to screen tanker milk for the presence of beta-lactam antibiotics.