An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization.

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.

Development of a method of analysis for profiling of the impurities in phenoxymethylpenicillin potassium based on the analytical quality by design concept combined with the degradation mechanism of penicillins.

Accurate analysis of all of the impurities present in a substance is critical for controlling the impurity profiles of drugs. Penicillins can easily yield a formidable array of degradation-related impurities (DRIs) with significantly different polarities and charge properties, which renders identifying each one a complicated matter. In this work, phenoxymethylpenicillin potassium (Pen V) was selected to find a way to quickly establish a robust analysis method for the impurity profiling of penicillin. Based on the analytical quality by design (AQbD) concept and the degradation mechanism of the drug, structures of all of the DRIs were first proposed. Then Pen V and its detected DRIs were separated and identified by liquid chromatography-tandem mass spectrometry method (LC-MS). Characteristic fragment ions and mass fragmentation process of Pen V and its detected DRIs were summarized. In addition, a quantitative structure-retention relationship (QSRR) model was constructed to predict the retention times of undetected impurities and to evaluate whether the chromatographic system can separate them. Finally, a stability-indicating high-performance liquid chromatography (HPLC) method was developed that can separate all of the DRIs of Pen V.

Molecularly imprinted solid-phase extraction of cephalexin from water-based matrices.

In the present paper, we describe the synthesis of a cephalexin (CFX) molecularly imprinted polymer (MIP), the direct application of the MIP to SPE for the determination of CFX (which is a beta-lactam antibiotic) in human urine and the use of the MIP in a tandem SPE system to determine CFX in river water. The molecularly imprinted polymers (MIP) showed cross-selectivity for amoxicillin (AMX; also a beta-lactam antibiotic). This allowed both CFX and AMX to be quantified in acidified human urine, with recoveries of 78 and 60% for CFX and AMX, respectively, when the urine samples were spiked with CFX and AMX at 4 mg/L. These analyses were facile because the molecularly imprinted solid-phase extraction (MISPE) extracts were clear compared to the nonpurified samples. In order to increase the sample volume for river water analyses, a tandem SPE system incorporating a commercially available sorbent was implemented. With this set-up, CFX was determined with recoveries in excess of 50% when 200 mL of acidified river water samples spiked at 10 microg/L with CFX were percolated through the tandem system.

Quantitative determination of penicillin V and amoxicillin in feed samples by pressurised liquid extraction and liquid chromatography with ultraviolet detection.

A rapid and simple method is proposed for the routine determination of amoxicillin (AMOX) and penicillin V (PENV) in swine feedingstuffs. The method is based on pressurised liquid extraction (PLE) followed by high performance liquid chromatography with ultraviolet detection (PLE-HPLC-UV) for antibiotic analysis. Parameters affecting PLE procedure, such as temperature, solvent composition, number of extraction cycles and sample cell size, were evaluated in order to achieve the highest extraction efficiency. The optimised method employed 11mL extraction cells, acetonitrile-water mixtures (25:75, v/v) for AMOX and (50:50, v/v) for PENV, as extraction solvent, 102.07atm of extraction pressure, 50 degrees C of extraction temperature, 5min of static time and 60% flush volume of the cell size. Extracts were filtered and directly analysed by HPLC-DAD/UV without further clean-up. Mean recovery rates for feed samples fortified with 200-500mgkg(-1) of both antibiotics were 86% for AMOX (RSD< or =6%) and 95% for PENV (RSD< or =3%). The method was successfully applied to the analysis of a commercial medicated swine feedingstuff, and the results were in good agreement with those obtained using mechanical shaking or ultrasonic extraction combined with solid phase extraction (UE-SPE), previously applied in the literature for feed analysis. The extraction efficiencies were evaluated by statistical comparison (analysis of variance, ANOVA-single factor) of the results obtained using the different extraction methods. Compared to the alternative techniques, PLE offers several practical advantages: easy to perform, fast, savings in solvent volume and in time, all steps are fully automated and further clean-up is not necessary for penicillin analysis.

Development of a Minimally Invasive Microneedle-Based Sensor for Continuous Monitoring of ß-Lactam Antibiotic Concentrations in Vivo.

Antimicrobial resistance poses a global threat to patient health. Improving the use and effectiveness of antimicrobials is critical in addressing this issue. This includes optimizing the dose of antibiotic delivered to each individual. New sensing approaches that track antimicrobial concentration for each patient in real time could allow individualized drug dosing. This work presents a potentiometric microneedle-based biosensor to detect levels of ß-lactam antibiotics in vivo in a healthy human volunteer. The biosensor is coated with a pH-sensitive iridium oxide layer, which detects changes in local pH as a result of ß-lactam hydrolysis by ß-lactamase immobilized on the electrode surface. Development and optimization of the biosensor coatings are presented, giving a limit of detection of 6.8 µM in 10 mM PBS solution. Biosensors were found to be stable for up to 2 weeks at -20 °C and to withstand sterilization. Sensitivity was retained after application for 6 h in vivo. Proof-of-concept results are presented showing that penicillin concentrations measured using the microneedle-based biosensor track those measured using both discrete blood and microdialysis sampling in vivo. These preliminary results show the potential of this microneedle-based biosensor to provide a minimally invasive means to measure real-time ß-lactam concentrations in vivo, representing an important first step toward a closed-loop therapeutic drug monitoring system.

Antibiotic levels in pericardial fluid.

An experimental model was designed to study the ability of antibiotics to enter the pericardial compartment. Noninfected and infected pericardial fluid and serum antibiotic activities were determined in adult mongrel dogs before and at intervals after antibiotic administration. After the administration of penicillin G, methicillin, cephaloridine, streptomycin, or gentamicin, clinically adequate antibiotic levels in the noninfected pericardial fluid were obtained within 1 h, and these levels approached or exceeded the serum levels within 2-4 h. Antibiotic levels obtained from infected dog pericardial fluids were higher than those from noninfected animals. Patients' serum and pericardial fluid antibiotic levels were measured after penicillin G, penicillin V, cephalothin, and gentamicin administration. We have found, both in the canine and human studies, that pericardial antibiotic levels taken at least 2 h after antibiotic administration are almost identical to those in the blood.

Comparison of Fiber Optic and Conduit Attenuated Total Reflection (ATR) Fourier Transform Infrared (FT-IR) Setup for In-Line Fermentation Monitoring.

The performance of a fiber optic and an optical conduit in-line attenuated total reflection mid-infrared (IR) probe during in situ monitoring of Penicillium chrysogenum fermentation were compared. The fiber optic probe was connected to a sealed, portable, Fourier transform infrared (FT-IR) process spectrometer via a plug-and-play interface. The optical conduit, on the other hand, was connected to a FT-IR process spectrometer via a knuckled probe with mirrors that had to be adjusted prior to each fermentation, which were purged with dry air. Penicillin V (PenV) and its precursor phenoxyacetic acid (POX) concentrations were determined by online high-performance liquid chromatography and the obtained concentrations were used as reference to build partial least squares regression models. Cross-validated root-mean-square errors of prediction were found to be 0.2 g L-1 (POX) and 0.19 g L-1 (PenV) for the fiber optic setup and 0.17 g L-1 (both POX and PenV) for the conduit setup. Higher noise-levels and spectrum-to-spectrum variations of the fiber optic setup lead to higher noise of estimated (i.e., unknown) POX and PenV concentrations than was found for the conduit setup. It seems that trade-off has to be made between ease of handling (fiber optic setup) and measurement accuracy (optical conduit setup) when choosing one of these systems for bioprocess monitoring.

Analysis of phenoxymethylpenicillin potassium by capillary electrophoresis.

The application of capillary electrophoresis for separation of penicillin V and its impurities was investigated. The phosphate-borate buffer supplemented with sodium dodecyl sulfate (SDS) 20.0 g/L (69 mM) and pentanesulfonic acid sodium salt (PS) 2.2 g/L (12.5 mM) adjusted to pH 6.3, and current voltage 15 kV seem to provide optimal conditions for this aim. The resolution between penicillin V and each impurity was very good. The statistical analysis of phenoxymethylpenicillin V assay showed no significant differences between the results obtained by CE and HPLC methods.

Comparison of human serum, parotid and mixed saliva levels of phenoxymethylpenicillin, ampicillin, cloxacillin and cephalexin.

1. A study has been made of serum, mixed and parotid salivary levels attained in normal volunteers following oral dosage of 500 mg phenoxymethylpenicillin tablets, 500 mg crushed phenoxymethylpenicillin tablets in capsules, 500 mg ampicillin, 500 mg cloxacillin and 500 mg cephalexin.2. High mixed saliva levels were obtained with phenoxymethylpenicillin tablets and it is considered that these were due to rapid intra-oral dissolution of surface powder from friable tablets. No saliva levels were detected when tablets from the same batch were put into capsules.3. Low or no saliva levels were achieved with ampicillin, cloxacillin and cephalexin.4. The mode of action of antibiotics in oral infections is discussed.

The distribution of antibacterial agents between plasma and lymph in the dog.

1. Plasma, peripheral and thoracic lymph concentrations of penicillin V, phenethicillin, carbenicillin, ampicillin, cloxacillin, penicillin G, chloramphenicol and sulphadiazine were determined at various time intervals up to 6 h following intramuscular administration of 50 mg/kg to dogs.2. Peak plasma concentrations of the penicillins occurred within half an hour after administration with the peak lymphatic concentrations occurring 1.5 to 3 h afterwards. For the remaining period of the test the concentration in the lymph exceeded the corresponding concentration in the plasma. Sulphadiazine gave concentrations in thoracic lymph equal to the plasma concentration, but the peripheral lymph concentrations were lower while the concentrations of chloramphenicol in both peripheral and thoracic lymph were always lower than the plasma concentrations.3. After the peak concentrations were reached, the concentration curves for penicillins in lymph followed the same pattern as found in plasma, the penicillin concentrations declining exponentially. Sulphadiazine produced more persistent levels both in lymph and in plasma while the concentrations of chloramphenicol were still rising 6 h after administration.4. The free concentrations of penicillin in lymph were equal to the free concentrations in plasma, whereas the concentrations of free sulphadiazine and chloramphenicol in lymph were less than those in the plasma.

Implementation of a thermal biosensor in a process environment: on-line monitoring of penicillin V in production-scale fermentations.

The production of penicillin V was monitored in 0.5 m3 and 160 m3 bioreactors. The thermal biosensor was an enzyme thermistor modified for split-flow analysis. The heat signal generated in the enzyme column was corrected for any nonspecific heat with the use of an identical but inactive reference column. The on-line monitoring was performed in the fermentation pilot plant and in a fermentation plant of Novo Nordisk A/S. Immobilized beta-lactamase was used to monitor three consecutive 0.5 m3 penicillin fermentations. Broth samples were continuously filtered through a tangential flow filtration unit in a sterile external loop. The on-line penicillin V values were 10% higher than those obtained by off-line HPLC analysis. Alternatively a polypropylene filtration probe was inserted into a 160 m3 bioreactor and samples were withdrawn at 0.5 ml/min. The same experiments were repeated with purified and immobilized penicillin V acylase. The on-line penicillin V values obtained with this enzyme correlated very well with those from HPLC analysis. The on-line monitoring was controlled and analysed by a software program written in Labtech Notebook.

A novel microphysiometer based on MLAPS for drugs screening.

This paper presents a novel microphysiometer for simultaneous measurements of several extracellular ions concentrations in living cells based on MLAPS (multi-light addressable potentiometric sensor). In the microphysiometer, different sensitive membranes are illuminated in parallel with n light sources at different frequencies, the response amplitudes of each frequency component can be measured on-line by parallel processing algorithm. By the experiments, we can analyze the relations of the extracellular environmental H(+), Na(+), K(+), Ca(2+) under the effects of western medicines (dilantin, phenobarbital sodium, penicillin sodium) and Chinese drugs (scutellaria, medlar, hemlock parsley), and estimate the effects of several drugs. As the novel microphysiometer works under regular cell culture conditions, cells can be repeatedly simulated with drugs to complete dose-response curve within a few hours. With the detection of a general parameter (extruded protons and ions), the system can be used to monitor the real-time process of the cells' metabolism, observe the functional responses of different kinds of membrane-bound receptors, evaluate the drugs.

Two FIA-based biosensor systems studied for bioprocess monitoring.

In this paper, two different FIA-based biosensor systems are described for application to different biotechnologically relevant purposes. In the first system, single fiber optodes were used to determine the pH, urea and penicillin V concentrations. A two-channel system was developed for the simultaneous monitoring of different variables to increase the analysis accuracy. This system was used for monitoring the penicillin V concentration during a cultivation of Penicillium chrysogenum. The second system described is a calorimetric immunoassay based on the use of an enzyme thermistor. A sandwich assay with protein A immobilized on a solid support for the determination of various IgGs was established. A fusion protein of protein A and beta-galactosidase obtained from a recombinant E. coli strain was used in the labelling and detection reaction. This system is designed for future application in bioprocess monitoring.

Application of ion-exchange cartridge clean-up in food analysis. II. Determination of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in meat using liquid chromatography with ultraviolet detection.

A multiresidue analytical method was developed for the simultaneous determination of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in meat. The method involves the use of an ion-exchange cartridge for sample clean-up followed by ion-pair high-performance liquid chromatography with ultraviolet detection. The recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from pork muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg were in the range of 77-90, 73-95 and 80-93% with coefficients of variation of 0.5-1.7, 1.6-4.4 and 3.2-6.6%, respectively. For beef muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg, the recoveries of these compounds were 83-92, 71-86 and 77-90% with coefficients of variation of 1.7-4.4, 2.6-7.0 and 3.9-6.4%, respectively. The detection limits for each penicillin were 0.02 mg/kg in meat.

High-performance liquid chromatographic analysis of penicillin V benzathine oral suspensions.

A rapid high-performance liquid chromatographic assay for penicillin V content of penicillin V benzathine bulk drug and oral suspensions is described. Dilution of the oral suspension with methanol containing 1,3,5-trimethoxybenzene as the internal standard allowed for direct analysis on a reversed-phase column with a mobile phase of 53% methanol in 0.05 M aqueous pH 3.5 phosphate buffer. A relative standard deviation of less than 1% was obtained on commercial formulations, and the system was linear over a range 10-40 microgram injected.

Application of proton and 13C-NMR spectroscopy to estimation of diastereoisomer ratio in phenethicillin.

A procedure for the determination of the diastereoisomer ratio in phenethicillin potassium and its formulations is described. The method utilizes analyses of PMR and 13C-NMR data. Assignments of 13C-chemical shifts in D- and L-phenethicillin potassium also are included.

In-beam electron ionization mass spectra of penicillins.

The characteristics of in-beam electron ionization mass spectra of 6-aminopenicillanic acid and several penicillins, which yield no detectable molecular ion peaks using a conventional direct-insertion probe, have been established. The spectra of all compounds studied, with the exception of amoxicillin, exhibited molecular ion or (M+1) peaks with spectral features similar to the reported methyl ester or amide derivatives of the compounds. The fragmentation of penicillin G on electron impact under in-beam conditions can be described on the basis of data from mass analyzed ion kinetic energy spectrometry. A desorption technique utilizing polyethylene glycol 4000 was used as a means of obtaining satisfactory spectra of ampicillin and amoxicillin.

Evaluation of LC methods for the separation of phenoxymethylpenicillin and its related substances.

Five isocratic reversed-phase liquid chromatography (LC) methods have been examined for the separation of phenoxymethylpenicillin and its related substances. A method previously selected for the analysis of benzylpenicillin gave the best selectivity. The mobile phase of this method is composed of 0.5 M phosphate buffer (pH 3.5)-water-methanol (10:50:40), the amount of organic modifier was slightly adapted when it was used with different columns. Similar selectivity is obtained not only on different C18 materials but also on C8 packings. The selectivity on poly(styrenedivinylbenzene) is less good. As method performance test a resolution test with benzylpenicillin was used. The robustness of this method was examined by applying a full factorial design to test the influence of the content of organic modifier in the mobile phase, the pH of the mobile phase, the concentration of buffer in the mobile phase and the temperature of the column. The results show that the method is robust.

Association of 6-oxo-piperidine-2-carboxylic acid with penicillin V. Production on Penicillium chrysogenum fermentations.

Analysis of a 13C NMR spectrum of a concentrated broth from Penicillium chrysogenum fermentation revealed the presence of penicillin V and 6-oxo-piperidine-2-carboxylic acid(1) as the principal constituents. The latter lactam, identical to an authentic sample prepared by the cyclization of alpha-aminoadipic acid was present to the extent of 28 mol% of penicillin V. The lactam isolated form the broth was nearly racemic, having a slight excess of the L-isomer. This isolation provides further evidence regarding the biosynthetic precursors of the hydrophobic penicillins.