Simultaneous determination of amoxicillin and potassium clavulanate in combined medicinal forms: procedure transfer from HPLC to UPLC.

This article presents the results of transferring a high-performance liquid chromatography (HPLC) procedure for the assay of amoxicillin and potassium clavulanate in tablets to the ultra-performed liquid chromatography (UPLC) conditions. Since the State Pharmacopoeia of Ukraine (SPhU) does not contain the monograph for the simultaneous analysis of amoxicillin and clavulanic acid, the British Pharmacopoeia procedure was used. Parameters of the procedure were optimized to fit the UPLC and to make a better performance. Transfer of the method to the UPLC conditions allowed to shorten the run time from 15 min to 7.5 min, which makes the process less time-consuming and more cost-effective. The upgraded procedure was further validated. Validation of both methods was performed in terms of linearity, precision, accuracy, specificity and stability. HPLC method was verified to later implementation into the SPhUs monograph. Afterwards, the methods were compared in terms of their impact on the environment using the eco-scale that included hazards of the solution, the amount of produced wastes, the impact on environmental and laboratory staff, etc. Both methods appeared to be eco-friendly with a moderate advantage of UPLC method. Moreover, the statistical comparison was performed using Passing Bablok regression method. It showed that both methods are statistically comparable.

Suitability of Amoxicillin-Clavulanic Acid for Administration via Prolonged Infusion.

RATIONALE: Previously, we have been able to outpace bacterial mutation by replacing increasingly ineffective antibiotics with new agents. However, with the discovery of new antibiotics diminishing, optimising the administration of existing broad-spectrum antibiotics such as co-amoxiclav has become a necessity. METHODS: A stability indicating HPLC method was developed and validated in compliance with International Council for Harmonisation (ICH) guidelines. Stability of co-amoxiclav at clinical concentration was evaluated at three temperatures (4°C, ambient (23-25°C) and 37°C) in three diluents (water for injection (WFI), 0.9% w/v NaCl and Ringer's solution). To establish whether there were significant differences at the level of both diluent and temperature, results were analysed using analysis of covariance (ANCOVA) to assess differences between the attained slopes of regression. RESULTS: Data obtained indicated co-amoxiclav stability superior to that previously proposed making it suitable for extended infusion therapy. The degradation of amoxicillin appeared to follow a linear trend, with the rate of degradation elevated at higher temperatures, demonstrated by the magnitude of the regression slopes in these conditions. Analysis of regression slopes via ANCOVA demonstrated that diluent and temperature both significantly affected co-amoxiclav stability. Amoxicillin retained 90% of its initial concentration for 7.8 to 10 hrs when stored at 4°C, 5.9 to 8.8 hrs at ambient and 3.5 to 4.5 hrs when incubated at 37°C. CONCLUSION: Co-amoxiclav is suitable for administration via prolonged infusion. Findings from this study aid in ameliorating current dosing regimens to optimise antibiotic efficacy. Other valuable applications conferred from these findings include the ability to pre-prepare solutions for use in bolus administration, minimising preparation time and workload.

Are antibiotics substandard in Lebanon? Quantification of active pharmaceutical ingredients between brand and generics of selected antibiotics.

BACKGROUND: In developing countries, brand-generic substitution is not based on validated scientific evidence that confirm the therapeutic equivalence of the generic to the originator. Rather, decisions are made based on the availability of generic medications. Substitution by inappropriate preparations applies to antibiotics, which may increase the risk of resistance in case of underdosing. This analytical study aims to dose and assess for the accuracy of labeling three oral antibiotic preparations, namely ciprofloxacin hydrochloride, amoxicillin trihydrate and amoxicillin trihydrate-clavulanate potassium, the active pharmaceutical ingredients (APIs) found in brand and generic tablets available on the Lebanese market. METHODS: One brand and 4 generics of ciprofloxacin tablets, 3 generic amoxicillin tablets, and 1 brand and 4 generics of amoxicillin-clavulanic acid medications, were quantified, taking 2 batches of each. According to the United States Pharmacopeia (USP) guidelines, ultra-high pressure liquid chromatography was used to measure the APIs content within tablets. The USP required assay limit of the API was taken as the main comparison criteria. RESULTS: Out of the 5 ciprofloxacin medications tested, all 5 were out of the 2% required range, thus being substandard. For amoxicillin, all 3 medications were within the 20% range. As for amoxicillin-clavulanic acid medications, 4 out of 5 medications met the 30% required range of clavulanic acid and one exceeded the claimed amount of clavulanic acid, while all 5 met the assay limit for amoxicillin. CONCLUSION: These findings raise safety and efficacy concerns, providing solid grounds for potential correlations of antibiotic resistance/substandard antibiotics.

Single-run capillary electrophoresis method for the fast simultaneous determination of amoxicillin, clavulanate, and potassium.

We report a new fast method for the simultaneous determination of amoxicillin, clavulanate, and potassium by capillary electrophoresis with capacitively coupled contactless conductivity detection. Samples containing potassium as the cation, and both amoxicillin and clavulanate as anions were determined simultaneously in a single run (in less than 45 s) using 10 mmol/L of both 2-amino-2-hydroxymethyl-propane-1,3-diol and 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid (pH 8.4) as the background electrolyte. Limits of detection were 25.0, 5.0, and 4.0 µmol/L for amoxicillin, clavulanate, and potassium, respectively. The proposed method is inexpensive, simple, fast (75 injections h-1 ), environment friendly (minimal waste generation), and accurate (recovery values between 98 and 103%). The results obtained with the proposed method were statistically similar (95% confidence level) to those obtained by using high-performance liquid chromatography (amoxicillin and clavulanate) and flame photometry (potassium).

Simultaneous determination of clavulanic acid and tazobactam in bovine milk by HPLC.

A rapid and specific high-performance liquid chromatographic method was developed and validated for the simultaneous determination of the ß-lactamase inhibitors clavulanic acid and tazobactam in bovine milk. The HPLC system was equipped with ultraviolet absorption detection using a C18 column with a detection wavelength of 215 nm. The mobile phase (pH 4) was 0.02% phosphoric acid and methanol in the ratio of 90:10 v/v. The retention times were 5.67 min for clavulanic acid and 7.22 min for tazobactam. The method exhibited good linearity for clavulanic acid and tazobactam, with R2 > 0.9988. Recovery ranged from 81.953% to 87.688% for clavulanic acid and from 85.007% to 92.991% for tazobactam. The precision expressed as RSD ranged from 0.975% to 1.248% for clavulanic acid and from 0.872% to 1.650% for tazobactam. A simple and precise HPLC method was developed for the determination of clavulanic acid and tazobactam in bovine milk. This method is intended for use in surveys of milk adulteration by the relevant inspection departments.

Atividade antimicrobiana de moléculas bioativas produzidas por Streptomyces parvulus DPUA 1573 frente a Staphylococcus spp. multirresistentes de mastite bubalina / Antimicrobial activity of bioactive molecules produced by Streptomyces parvulus DPUA 1573 against Staphylococcus spp. multi-drug resistant isolates from mastitis buffalo

A mastite é uma inflamação na glândula mamária que pode acarretar perdas na produção e na qualidade do leite, gerando prejuízos econômicos para a pecuária leiteira. O tratamento é baseado na utilização de antibióticos, sendo, em muitos casos, ineficazes devido à resistência bacteriana já conhecida para esta doença. O objetivo deste trabalho foi selecionar linhagens de Streptomyces spp. produtoras de biocompostos com atividade antimicrobiana frente a isolados do gênero Staphylococcus multirresistentes de búfalas com mastite. Bem como, determinar os melhores parâmetros de produção, e avaliar a produção simultânea de ácido clavulânico. A seleção de Streptomyces spp. com capacidade de produzir compostos com atividade antimicrobiana foi realizada através da técnica bloco de gelose. Dentre as 30 espécies de Streptomyces spp. testadas, o micro-organismo Streptomyces parvulus DPUA 1573 apresentou melhores resultados, sendo capaz de inibir o crescimento de 7 isolados Staphylococcus spp. multirresistentes. Posteriormente, a espécie selecionada Streptomyces parvulus DPUA 1573 foi cultivada em diferentes condições pré-determinadas pelo planejamento fatorial 24, onde as variáveis independentes foram: concentração de soja (0,5; 1,0; 1,5%), glicose (0; 0,5; 1g/L), agitação (150; 200; 250rpm) e temperatura (28; 32; 37°C) e todos os ensaios do planejamento foram monitorados até 120 horas de cultivo. Todas as variáveis independentes influenciaram positivamente no crescimento celular, enquanto que para atividade antimicrobiana apenas as variáveis temperatura e agitação apresentaram efeitos significativos positivos. O líquido metabólito produzido por Streptomyces parvulus DPUA 1573 foi capaz de inibir o crescimento de sete Staphylococcus spp. multirresistentes. As melhores condições de cultivo para a produção de moléculas bioativas por este micro-organismo foi a 37?C, com 250rpm de agitação por período de 72 horas. Nos ensaios que apresentaram atividade antimicrobiana, foi avaliada a produção de ácido clavulânico ao longo do cultivo. A maior concentração de ácido clavulânico foi de 269,84g/L obtidas nas condições de 1,5% de farinha de soja em ausência de glicose no tempo de 96 horas. A linhagem Streptomyces parvulus DPUA 1573 foi eficiente contra Staphylococcus spp. multirresistentes isolados de mastite em búfalas, ainda apresentando concomitantemente produção de ácido clavulânico com o potencial uso farmacêutico.(AU)

Mastitis is an inflammation in one or more mammary glands which can lead to reduction in production and quality of milk causing economic losses to dairy farming. The use of antibiotics is the key for the treatment of this disease, but in many cases ineffective due to bacterial resistance already known for this condition. The aim of this study was to select strains of Streptomyces spp. producing biomolecules with antimicrobial activity against multidrug-resistant Staphylococcus isolated from buffaloes with mastitis, as well as to determine the best production parameters to the evaluation of simultaneous production of clavulanic acid. Thirty species of Streptomyces spp. were used to selecting the greatest producer spectrum of antimicrobial activity (agar block technique), with selection of Streptomyces parvulus DPUA 1573, and 7 multidrug-resistant Staphylococcus spp. sensitive to its biocompounds. The selected strain of Streptomyces parvulus DPUA 1573 was cultured in different conditions predetermined by the factorial design 24, where the independent variables were: soybean concentration (0.5, 1.0, 1.5%), glucose (0, 0.5, 1g/L), agitation (150, 200, 250rpm) and temperature (28, 32, 37°C); all the tests were monitored up to 120 hours of cultivation. All independent variables influenced positively the cell growth, while for antimicrobial activity only the variables temperature and agitation showed positive effects. The antimicrobial bio compounds showed activity against seven multidrug-resistant Staphylococcus spp under the conditions: temperature 37°C, agitation 250rpm, with 72 hours of production process. In the tests which showed antimicrobial activity, was also assessed the production of clavulanic acid along with the cultivation. The highest concentration of clavulanic acid was 269.84g/L obtained under the conditions of 1.5% of soybean flour and absence of glucose in 96 hours. The strain Streptomyces parvulus DPUA 1573 was effective against multidrug-resistant strains of Staphylococcus spp. of mastitis from buffaloes, still showing concomitantly production of clavulanic acid for pharmaceutical use.(AU)

Label-Free Photonic Crystal-Based ß-Lactamase Biosensor for ß-Lactam Antibiotic and ß-Lactamase Inhibitor.

A simple, label-free, and visual photonic crystal-based ß-lactamase biosensor was developed for ß-lactam antibiotic and ß-lactamase inhibitor in which the penicillinase (a ß-lactamase) was immobilized on the pH-sensitive colloidal crystal hydrogel (CCH) film to form penicillinase colloidal crystal hydrogel (PCCH) biosensing film. The hydrolysis of penicillin G (a ß-lactam antibiotic) can be catalyzed by penicillinase to produce penicilloic acid, leading to a pH decrease in the microenvironment of PCCH film, which causes the shrink of pH-sensitive CCH film and triggers a blue-shift of the diffraction wavelength. Upon the addition of ß-lactamase inhibitor, the hydrolysis reaction is suppressed and no clear blue-shift is observed. The concentrations of ß-lactam antibiotic and ß-lactamase inhibitor can be sensitively evaluated by measuring the diffraction shifts. The minimum detectable concentrations for penicillin G and clavulanate potassium (a ß-lactamase inhibitor) can reach 1 and 0.1 µM, respectively. Furthermore, the proposed method is highly reversible and selective, and it allows determination of penicillin G in fish pond water samples.

An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus.

Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial ß-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate-imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400mg/L. The detection and quantification limits were 0.01 and 0.02mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate-imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.

Development and validation of stability-indicating HPLC method for simultaneous determination of meropenem and potassium clavulanate.

A stability-indicating LC assay method was developed and validated for a simultaneous determination of meropenem and potassium clavulanate in the presence of degradation products formed during acid-base hydrolysis, oxidation and thermolysis. The isocratic RP-HPLC method was developed with a LiChrospher RP-18 (250 mm x 4.6 mm, 5 microm) column and gradient elution of 12 mmol/L ammonium acetate and acetonitrile. The flow rate of the mobile phase was 1.0 mL/min, the detection wavelength 220 nm and the temperature 303 K. The method was validated with regard to linearity, accuracy, precision, selectivity and robustness, and was applied successfully for the determination of meropenem and potassium clavulanate separately as well as jointly in pharmaceutical formulations.

Studies on the formation and forming mechanism of the related substance E in potassium clavulanate production by HPLC-MS/MS

The objective of this study was to investigate the formation and forming mechanism of the related substance E in potassium clavulanate production. The impurity with retention time of 11.1 min in potassium clavulanate final product was confirmed as the related substance E by high performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS).The related substance E analysis during the production of clavulanic acid showed that this impurity could be formed during both the fermentation and purification processes, especially in the later fermentation stage, filtration concentration and back-extraction procedure. Clavulanic acid was the precursor of the related substance E. Studies on its forming mechanism showed that the related substance E was formed by the combination of the imino group of one molecule of clavulanic acid with the carboxyl group of another molecule of clavulanic acid with the opening of β-lactam ring. Results of a multi-factor orthogonal test confirmed that the concentration of clavulanic acid was the dominant factor to accelerate the reaction, while the temperature was another contributing factor. The pH 5.0-6.5 had little impact on the generation of the related substance E.

O objetivo deste estudo foi investigar a formação da substância E e o respectivo mecanismo na produção de clavulanato de potássio. Confirmou-se a impureza com tempo de retenção de 11,1 min no produto final, clavulanato de potássio, como substância E, por meio de cromatografia líquida de alta eficiência, em conjunto com detecção por espectrometria de massas (CLAE-MS-MS). A análise da substância relacionada E durante a produção do ácido clavulânico mostrou que essa impureza pode ser formada tanto durante a fermentação quanto durante os processos de purificação, especialmente no estágio final de fermentação, filtração, concentração e procedimento de extração. O ácido clavulãnico foi o precursor da substância E. Estudos no mecanismo de sua formação mostraram que a substância E formou-se pela combinação do grupo imina da molécula do ácido clavulânico com o grupo carboxílico de outra molécula de ácido clavulânico, com a abertura do anel β-lactâmico. Resultados do teste ortogonal multifatorial confirmaram que a concentração do ácido clavulânico foi o fator dominante para acelerar a reação, enquanto a temperatura foi outro fator que contribuiu. O pH de 5,0 a 6,5 teve pouco impacto na geração da substância E.

Utilização de sistemas poliméricos de duas fases aquosas (SPDFA) compostos por polietileno glicol/ácido poliacrílico (PEG/APA) para extração de ácido clavulânico / Utilization of aqueous two phase systems (ATPS) composed of polyethylene glycol/polyacrylic acid (PEG/APA) in the extraction of clavulanic acid

A viabilidade da produção em escala industrial de produtos biotecnológicos de interesse comercial e terapêutico, como os fármacos, depende significativamente das técnicas de separação e purificação utilizadas. A aplicação do sistema de duas fases aquosas (SDFA) é proposta como alternativa para a purificação, pois permite a separação e análise de biomoléculas, de modo que estas não percam sua atividade ou propriedades desejadas. Esta técnica é interessante para a purificação em larga escala, pois permite partição seletiva, com potencial de obtenção de altos rendimentos, além de apresentar boa relação custo-benefício. O presente trabalho estudou a purificação por extração líquido-líquido do ácido clavulânico em SDFA utilizando um novo sistema polimérico aquoso, formado pelos polímeros polietileno glicol (PEG) e ácido poliacrílico (APA). Foram estudadas diferentes composições do sistema polimérico aquoso PEG/APA, empregando diferentes massas molares e concentrações para o PEG e utilizando a massa molar 8000g/mol para o APA. Com base nas informações obtidas o melhor ponto de extração para o ácido clavulânico na presença de Na2SO4 foi definido como MPEG=400 g/mol, CPEG=17,5% (m/m) e CNaPA=22,5% (m/m) com K= 19,14, ηT=91,21%, BM=101,69 e R=0,45. Enquanto que na presença de NaCl, o melhor ponto encontrado foi: MPEG=400 g/mol, CPEG=35% (m/m) e CNaPA=10% (m/m) com K=11,96 ηT=80,04%, BM=90,18 e R=0,66. No trabalho será avaliada, também, a influência da temperatura, pH e força iônica nesse sistema. Estabeleceram-se os melhores parâmetros de separação do ácido clavulânico presente em meio fermentado produzido por Streptomyces clavuligerus utilizando a metodologia de fermentação extrativa com SDFA PEG/APA. O efeito do ácido clavulânico no diagrama de fases do sistema PEG-APA, bem como sua partição na forma pura e na presença de homogeneizado celular, foi estudado principalmente através da determinação do coeficiente de partição e recuperação do respectivo fármaco

The viability of industrial scale production of commercial and therapeutical biotechnological products, such as drugs, is significantly dependent on the separation and purification techniques applied. The use of two-aqueous phase systems (ATPS) is proposed as an alternative to purification because it allows the separation and analysis of biomolecules, so that they do not lose their activities or desired properties. This technique is interesting for large scale purification because it allows selective partition with high potential yield and good cost/benefit ratio. The present work studied the purification of clavulanic acid (CA) by liquid-liquid extraction in ATPS applying a new aqueous polymeric system composed of two polymers, namely polyethylene-glicol (PEG) and sodium polyacrylate (NaPA). Different compositions of the aqueous polymeric system (PEG/PAA) were utilized, employing different PEG molar masses (MPEG) and concentrations (CPEG) and a molar mass of PAA of 8000 g/mol. In the light of the results obtained, the best conditions for clavulanic acid extraction, in the presence of Na2SO4, were MPEG = 400 g/mol, CPEG = 17.5% (m/m) and CNaPA = 22.5% (m/m), which allowed obtaining a partition coefficient (K) of 19.14, a yield in the top phase (ηT) of 91.21%, a mass balance (MB) of 101.69 and a volume ratio (R) of 0.45. On the other hand, in the presence of NaCl, the best results (K = 11.96, ηT = 80.04%, MB = 90.18 and R = 0.66) were found at: MPEG = 400 g/mol, CPEG = 35% m/m and CNaPA = 10% m/m. The effect of clavulanic acid in the PEG-PAA system phase diagram and its partition either in its pure form or in the cell homogenate were studied mainly through both the determination of the partition coefficient and the recovery of the drug selected for this study

[Determination of clavulanic acid residue in milk by high performance liquid chromatography-tandem mass spectrometry].

An analytical method was developed for the determination of clavulanic acid (CLAV) in milk by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A 2 g milk sample was deproteinized by ethanol. The supernatant was transferred into a pear-shaped bottle to be evaporated to about 0.5 mL, and the residue was dissolved with ammonium acetate solution. The sample was determined by HPLC-MS/MS after the purification. The chromatographic separation was achieved on a Luna 5u C8 column using 0.1% formic acid in water and acetonitrile as mobile phases with gradient elution. The identification of CLAV was carried out by MS/MS equipped with electrospray ionization in negative scanning and multiple reaction monitoring (MRM) modes. Matrix-matched calibration standard was used for the quantification. The calibration curve showed perfect linear in the range of 10 - 400 microg/kg with the correlation coefficient of 0.999. The limit of detection (LOD, S/N > or = 3) was 10 microg/kg in milk, and the limit of quantification (LOQ, S/N > or = 10) was 20 microg/kg. The mean recoveries varied from 80.00% to 91.25% at the four spiked levels of LOQ, 1/2MRL (the maximum residue limit), MRL, and 2MRL with the relative standard deviations of 5.60% -8.77%. In conclusion, the established method can be applied for the determination of CLAV residues in milk.

Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples.

We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a ß-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ (max)=390 nm) is converted to a red product (λ (max)=486 nm). Since CA can irreversibly inhibit ß-lactamase activity, the level of CA in a sample can be measured as a function of the A (390)/A (486) ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 µg L(-1) and 50 µg L(-1) to 10 mg L(-1), respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.

AmpC beta lactamases among Gram negative clinical isolates from a tertiary hospital, South India

AmpC â-lactamases are cephalosporinases that hydrolyze cephamycins as well as other extended-spectrum cephalosporins and are poorly inhibited by clavulanic acid. Although reported with increasing frequency, the true rate of occurrence of AmpC â-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Pondicherry state, South India. A total of 235 Gram negative clinical isolates were tested for resistance to cefoxitin, third generation cephalosporin (3GC) antibiotics, ampicillin, amikacin, co-trimoxazole, gentamicin, meropenem and tetracycline by disc diffusion method. Isolates found resistant to 3GC and cefoxitin were tested for the production of AmpC â -lactamases by three dimensional extraction method and AmpC disc method. Isolates found to sensitive to 3GC were subjected to disc antagonism test for inducible AmpC production. One hundred and thirty four (57 percent) strains were resistant to 3GC, among which 63(47 percent) were positive for plasmid-mediated AmpC beta lactamases production. Among the 101 strains sensitive to 3GC, 23 (22.7 percent) revealed the presence of inducible AmpC beta lactamases by disc approximation test. A total of 80.9 percent (51/63) of screen positive isolates were detected by Amp C disc test and 93.6 percent (59/63) by three dimensional extraction method. Out of the 86 AmpC producers, 67 (77.9 percent) were cefoxitin resistant .Inducible AmpC was not found in Esch.coli and Klebsiella spp. The AmpC producers also concurrently showed multidrug resistance pattern. AmpC producers were found to be prevalent in our hospital and though three dimensional extraction test detects AmpC better, the disk test is easier to perform routinely and is user- friendly.

Simultaneous chemiluminescence determination of amoxicillin and clavulanic acid using least squares support vector regression.

In this work, both the batch and flow injection chemiluminescence (CL) methods have been proposed for the simultaneous determination of two structurally similar beta-lactams including amoxicillin and clavulanic acid (CLAV). Chemiluminescence spectral overlap of these two structurally similar beta-lactams is the main limitation for the simultaneous analysis of the two compounds. Least squares support vector regression (LS-SVR) was applied to relate concentration of both the compounds to their CL profiles. The parameters of the model, consisting of kernel parameter, sigma(2), and the regularization parameter, gamma, were optimized by constructing different LS-SVR models and the model with the minimum root mean squared error of cross-validation (RMSECV) for the calibration set was selected as the best model and its parameters were chosen as the optimized values. The performance of LS-SVR model was compared with Partial Least Squares (PLS) and the results revealed the superiority of the LS-SVR over PLS model. Under the optimized experimental conditions for both the compounds, when LS-SVR was applied, the detection limits obtained were 0.2 and 0.60 micromol L(-1) in the batch mode and 0.3 and 0.5 micromol L(-1) in the flow injection mode for CLAV and amoxicillin, respectively. The proposed method was utilized for the simultaneous determination of the compounds in pharmaceutical formulations and spiked plasma samples.

Development of a new HPLC method for the simultaneous determination of ticarcillin and clavulanic acid in pharmaceutical formulations.

A new HPLC method has been developed and validated for the simultaneous determination of ticarcillin (TIC) and clavulanic acid (CA) in pharmaceutical formulations. The HPLC separation was achieved on a beta-cyclodextrin column (Cyclobond I, 250 x 4.6 mm, 5 microm) with methanol-16 mM pH 6.0 ammonium acetate buffer (50 + 50, v/v) mobile phase at a flow rate of 0.8 mL/min. Detection was at 220 nm. Validation of the method was performed by evaluating specificity, robustness, accuracy, and precision. The calibration curves were linear in the range of 1-100 microg/mL for CA and 2-200 microg/mL for TIC. The LOQs based on the standard regression lines were 0.42 and 1.42 microg/mL for CA and TIC, respectively, and the LOD were 0.14 and 0.47 microg/mL, respectively. Total recoveries of synthetic mixtures (CA:TIC = 1:10, 1:15, and 1:30) were 99.25-100.99% for CA and 99.54-100.82% for TIC. Compared with the U.S. Pharmacopeia method, the proposed method has the advantage of a relatively low flow rate and short analysis time. The proposed method was successfully applied for the simultaneous determination of these two drugs in sterilized H20 and 5% dextrose injection solutions.

Simultaneous spectrophotometric determination of Cefpodoxime proxetil and Potassium clavulanate.

Two accurate, precise, sensitive and economical procedures for simultaneous estimation of Cefpodoxime proxetil and Potassium clavulanate in tablet dosage form have been developed. The methods employed were absorbance correction method (I) and first order derivative spectroscopic method (II). The first method employs wavelength 288 nm for direct estimation of Cefpodoxime proxetil where Potassium clavulanate shows nil absorbance. Estimation of Potassium clavulanate is carried out after correction for absorbance of Cefpodoxime proxetil at 218 nm. The second method is based on first order derivative spectroscopy. Wavelengths 235.6 nm and 308.2 nm were selected for the estimation of the Potassium clavulanate and Cefpodoxime proxetil, respectively. Both the drugs obey Beer's law in the concentration range 5-50 microg/ml. The results of analysis have been validated statistically and by recovery studies. The percentage assay was found to be 99.54 +/- 0.285 for Cefpodoxime proxetil and 98.53 +/- 0.760 for Potassium clavulanate (Mean +/- SD) by method I and 99.93 +/- 0.270 for Cefpodoxime proxetil and 99.40 +/- 0.723 for Potassium clavulanate (Mean +/- S.D) by method II respectively.

Simultaneous determination of potassium clavulanate and cefixime in synthetic mixtures by high-performance liquid chromatography.

A simple, precise, and sensitive high-performance liquid chromatographic method was developed and validated for the simultaneous determination of potassium clavulanate and cefixime in synthetic mixture form. The analytes were separated on a C18 column by using 0.03 M disodium hydrogen phosphate buffer (pH 6.5)-methanol (84 + 16, v/v) as the mobile phase with detection at 220 nm. The method exhibited high sensitivity and good linearity in the concentration ranges of 12.5-62.5 and 20-100 microg/mL for potassium clavulanate and cefixime, respectively. The total run time for the 2 components was <8 min, and the average recovery was >101.5% with a relative standard deviation of <1.0%. The proposed method was validated according to guidelines of the International Conference on Harmonization by evaluation of linearity, recovery, selectivity, robustness, limits of detection and quantitation, and within- and between-day precision. The results obtained for the synthetic mixture show that the method is highly precise and accurate for the simultaneous determination of potassium clavulanate and cefixime.

Applying near-infrared spectroscopy in downstream processing: one calibration for multiple clarification processes of fermentation media.

The use of near-infrared spectroscopy (NIRS) is demonstrated in the first downstream processing (DSP) steps of an active pharmaceutical ingredient (API) manufacturing process. The first method developed was designed to assess the API content in the filtrate stream (aqueous) of a rotary drum vacuum filter. The PLS method, built after spectral preprocessing and variable selection, had an accuracy of 0.01% (w/w) for an API operational range between 0.20 and 0.45% (w/w). The robustness and extrapolation ability of the calibration was proved when samples from ultrafiltration and nanofiltration processes, ranging from 0 to 2% (w/w), were linearly predicted (R2=0.99). The development of a robust calibration model is generally a very time-consuming task, and once established it is imperative that it can be useful for a long period of time. This work demonstrates that NIR procedures, when carefully developed, can be used in different process conditions and even in different process steps of similar unit operations.

Amoxicillin-clavulanic acid and trimethoprim- sulfamethoxazole in rodent feed and water: effects of compounding on antibiotic stability.

We assessed the concentrations of 2 antibiotic combinations, amoxicillin-clavulanic acid and trimethoprim-sulfamethoxazole when compounded in reverse osmosis [RO] (pH 6.0), tap (pH 6.7), and acidified water (pH 2.6) over 7 d, and pre- and post-pelleting, post-gamma irradiation and shipping, and monthly until 180 d post-milling in feed. Amoxicillin concentrations in RO and tap water varied between 1.18 and 1.29 mg/ml, and 1.09 and 1.22 mg/ml, respectively. The concentration of amoxicillin declined immediately and remained between 0.43 and 0.50 mg/ml in acidified water. Clavulanic acid exhibited a slow time-dependent decrease in concentration to 0.05 mg/ml at day 7 in RO water, immediately declined and varied from 0.02 to 0.05 mg/ml in tap water, and was undetectable in acidified water. Trimethoprim and sulfamethoxazole concentrations were near expected in RO, tap, and acidified water. In food, amoxicillin, trimethoprim, and sulfamethoxazole concentrations were each reduced to approximately 60% of expected after pelleting, but remained stable thereafter for 180 d. The initial clavulanic acid concentration in feed was less than 10% of expected and was undetectable after 1 mo. Plasma drug concentrations were determined in C57BL/6NCrl mice at 4 h after commencement of the dark and light cycles following administration of antibiotic food for at least 72 h. Plasma amoxicillin and sulfamethoxazole concentrations were 3- and 10-fold greater, respectively, during the dark period. Plasma levels of clavulanic acid and trimethoprim were consistent at both time points. These results indicate that the antibiotic concentration can be influenced by compounding in feed and water, and differs in plasma during the light and dark phases of the photoperiod.