Validation of an analytical method by high-performance liquid chromatography and microbiological assay, biological safety and in silico toxicity for danofloxacin

Danofloxacin is a veterinary fluoroquinolone used to treat respiratory and gastrointestinal diseases of birds, pigs and cattle. The literature reviewed shows some analytical methods to quantify this fluoroquinolone, but microbiological and biological safety studies are limited. The analytical methods were validated by the Official Codes. The LC-DAD method was developed and validated using an RP-18 column, mobile phase containing a mixture of 0.3% triethylamine (pH 3.0) and acetonitrile (85:15, v/v). The microbiological assay was performed by agar diffusion method (3 x 3) and Staphylococcus epidermidis as a microorganism test. Forced degradation studies were performed in both methods. The minimum inhibitory concentration (MIC) was performed by test microdilution and toxicity studies were evaluated using in silico study, cell proliferation, cell viability test, micronuclei and comet assay. LC and a microbiological assay proved linear, accurate, precise, and robust to quantify danofloxacin, but only the LC method showed selectivity to quantify the drug in the presence of its degradation products. These results demonstrate that the LC method is suitable for stability studies of danofloxacin, but a microbiological assay cannot be used to quantify the drug due to the biological activity of the photoproducts. Ex-vivo cytotoxicity and theoretical and experimental genotoxicity were also observed.

Consumer profile and problems associated with uninspected raw milk consumption in western Paraná / Perfil dos consumidores e problemas associados ao consumo de leite cru informal no oeste do Paraná

The objectives of this study were to analyze consumer profile; to identify the main reasons for raw milk consumption; and to analyze in laboratory samples of uninspected raw milk from five towns in the western region of the state of Paraná, Brazil. The types of milk most frequently consumed were: 42.3% ultra-high temperature (UHT), 38.3% pasteurized milk, 17.6% uninspected raw milk, and 1.7% powered milk. The frequencies of households that preferred uninspected raw milk were, according to the town, 32.7% in Iporã, 29.2% in Marechal Cândido Rondon, 18.9% in Assis Chateaubriand, 17.6% in Palotina, and 10% in Toledo. Flavor was the main reason for uninspected raw milk consumption, and the purchase of this product was more frequent in households whose income was between one to four minimum wages. It was observed that the sales of uninspected milk are more financially advantageous to the producer than sales of inspected milk. All samples analyzed showed lack of compliance with at least one parameter, 60.9% for mesophilic counts, 56.6% for non-fat dry matter, 52.1% for freezing point, 43.5% for acidity, 23.9% for density, 23.9% for the casein macropeptide, 17.4% for fat content, 8.7% were reactors in the milk ring test, and 2.2% were reactors in microbial growth inhibitor test. Fraud by addition of water was observed in 20% of the samples. Uninspected raw milk analyzed in this study involved a low-quality product that is a financial hazard as it may be adulterated, and that poses risk to consumer health.(AU)

Os objetivos desta pesquisa foram verificar o perfil, identificar os principais motivos para o consumo e avaliar laboratorialmente as amostras de leite cru informal de cinco municípios do oeste do estado do Paraná, Brasil. Os tipos de leite mais frequentemente consumidos foram: 42,3% ultra-high temperature (UHT), 38,3% pasteurizado, 17,6% leite cru informal e 1,7% em pó. As frequências de consumidores que preferiram leite cru informal, por cidade, foram: 17,6% em Palotina, 29,2% em Marechal Cândido Rondon, 18,9% em Assis Chateaubriand, 17,6% em Palotina e 10% em Toledo. Sabor foi o principal motivo que influenciou o consumo de leite cru informal, e famílias com renda familiar de um a quatro salários mínimos foram as que mais consumiram esse tipo de leite. A comercialização informal de leite foi mais vantajosa financeiramente para o produtor em relação à comercialização formal. Todas as amostras analisadas apresentaram-se em desacordo em pelo menos um parâmetro, sendo 60,9% para mesófilos, 56,6% para estrato seco desengordurado, 52,1% para índice crioscópico, 43,5% para acidez, 23,9% para densidade, 23,9% para índice de caseíno macropeptídeo, 17,4% para teor de gordura, 8,7% reagente ao teste do anel em leite (TAL), e 2,2% reagente para presença de inibidor de crescimento bacteriano. A adulteração encontrada foi a adição de água em 20% das amostras analisadas. O leite cru informal avaliado nesta pesquisa envolveu a comercialização de um produto sem qualidade e que constitui um perigo financeiro, em razão do consumo de produtos adulterados, além do risco à saúde do consumidor.(AU)

Study on laboratory information system setting of microbiology specimen types / 国际检验医学杂志

Objective The microbiology specimen types were various and complex ,the system of main specimen types in micro-biological detection was established under the laboratory information system (LIS) for realizing the management target of quality control before microbiological analysis .Methods A total of 3304 submitted microbiological samples were collected from January 1 to 31 in 2015 .After setting the microbiological item application procedure of main specimen types in LIS ,1532 submitted microbio-logical specimens from June 20 to 24 were performed the statistics .The error rates of specimen types were compared before and af-ter setting .Then 1635 and 1340 submitted microbiological specimens were re-collected form July 9 to 13 and August 10 to 15 ;the change of error rates was continuously observed for comparing whether the statistical difference of error rates existing between be-fore and after setting .Results The error rate of submitted microbiological specimens before setting was 4 .6% (152/3304) ,which after setting was 1 .3% (20/1532)(χ2 =31 .224 ,P<0 .001) ,which during the continuous observation period maintained the lower level of 1 .04% (17/1635) ,χ2 =39 .658 ,P<0 .001) and 0 .9% (13/1340 ,χ2 =34 .673 ,P<0 .001) .Conclusion Re-setting the LIS reduces the error rate of microbiological specimen type ,effectively increase the working efficiency and reaches the quality control in-dex before microbiological analysis .

Selection of appropriate analytical tools to determine the potency and bioactivity of antibiotics and antibiotic resistance$ / 药物分析学报

Antibiotics are the chemotherapeutic agents that kill or inhibit the pathogenic microorganisms. Re-sistance of microorganism to antibiotics is a growing problem around the world due to indiscriminate and irrational use of antibiotics. In order to overcome the resistance problem and to safely use antibiotics, the correct measurement of potency and bioactivity of antibiotics is essential. Microbiological assay and high performance liquid chromatography (HPLC) method are used to quantify the potency of antibiotics. HPLC method is commonly used for the quantification of potency of antibiotics, but unable to determine the bioactivity; whereas microbiological assay estimates both potency and bioactivity of antibiotics. Additionally, bioassay is used to estimate the effective dose against antibiotic resistant microbes. Simultaneously, microbiological assay addresses the several parameters such as minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), mutation prevention concentration (MPC) and critical concentration (Ccr) which are used to describe the potency in a more informative way. Microbiological assay is a simple, sensitive, precise and cost effective method which gives reproducible results similar to HPLC. However, the HPLC cannot be a complete substitute for microbiological assay and both methods have their own significance to obtain more realistic and precise results.

A fully validated microbiological assay for daptomycin injection and comparison to HPLC method

abstract Daptomycin (DPT) was the first lipopeptide antibiotic available for commercialization. It is active against gram-positive bacteria, including resistant strains. This work aimed to develop and validate a turbidimetric microbiologic assay to determine daptomycin in an injectable form. A 3x3 design was employed, at concentrations of 1, 2 and 4.0 µg/mL. The microorganism test used was Staphylococcus aureus ATCC 6538p, and Antibiotic Medium 3 was used as the culture medium. Method validation demonstrated that the bioassay was linear (r=0.9995), precise (RSD=2.58%), accurate (recovery 100.48± 2.11%), and robust. Degradation kinetics was also performed in an alkaline medium, indicating that daptomycin degradation follows first order kinetics under these conditions. The analyses of degraded solutions showed that daptomycin degradation products do not possess bactericidal activity. The bioassay was compared to HPLC method that was previously developed and no significant difference was found between them (p>0.05). The method proved to be appropriate for daptomycin injection quality control.

resumo A daptomicina (DPT) é o primeiro lipopeptídeo cíclico disponível para comercialização. Possui atividade frente a bactérias gram-positivas, incluindo cepas resistentes. O objetivo deste trabalho foi desenvolver e validar um ensaio microbiológico turbidimétrico para quantificar a daptomicina na forma injetável. Empregou-se delineamento 3x3, nas concentrações de 1,0; 2,0 e 4,0 µg/mL. Como micro-organismo teste foi usado Staphylococcus aureus ATCC 6538p, e Meio para Antibióticos nº 3 foi empregado como meio de cultura. A validação do método demonstrou que o ensaio foi linear (r=0,9995), preciso (RSD=2,55%), exato (recuperação de 100,48 ± 2,11%) e robusto. A cinética de degradação em meio alcalino foi avaliada, indicando que a daptomicina segue cinética de primeira ordem nessa condição. A análise das soluções degradadas mostrou que os produtos de degradação da daptomicina não possuem atividade antimicrobiana. O bioensaio foi comparado com o método por CLAE previamente desenvolvido e não houve diferença significativa entre ambos (p<0,05). O método mostrou-se apropriado para o controle de qualidade da daptomicina injetável.

Development and validation of a rapid turbidimetric assay to determine the potency of norfloxacin in tablets

Norfloxacin is one of the first commercially available (and most widely used) fluoroquinolone antibiotics. This paper reports the development and validation of a simple, sensitive, accurate and reproducible turbidimetric assay method to quantify norfloxacin in tablets formulations in only 4 hours. The bioassay is based on the inhibitory effect of norfloxacin upon the strain ofStaphylococcus epidermidis ATCC 12228 IAL 2150 used as test microorganism. The assay was performed 3x3 parallel lines like, three tubes for each concentration of reference substance and three tubes for each sample concentration. The results were treated statistically by analysis of variance and were found to be linear (r2 = 0.9999) in the selected range of 25-100 μg mL-1; precise (intra-assay: relative standard deviation (RSD) = 1.33%; inter-assay: RSD = 0.21%), accurate (100.74%) and robust with RSD lower than 4.5%. The student's t-test showed no statistically significant difference between the proposed turbidimetric method and an HPLC method previously validated. However the turbidimetric assay can be used as a valuable alternative methodology for the routine quality control of this medicine, complementary to other physical-chemical methods.

O norfloxacino foi a primeira fluorquinolona (e mais utilizada) disponível no mercado. Este trabalho divulga um novo desenvolvimento e validação de um método turbidimétrico simples, sensível, preciso e reprodutível para a quantificação de norfloxacino em comprimidos em apenas 4 horas. O bioensaio é baseado no efeito inibitório de norfloxacino sobre a cepa Staphylococcus epidermidis ATCC 12228 IAL 2150, utilizada como micro-organismo teste. O bioensaio foi efetuado através do delineamento de linhas paralelas 3x3, em que três tubos foram utilizados para a solução padrão e três tubos para as concentrações da amostra. Os resultados foram tratados estatisticamente pela análise de variância, apresentando coeficiente de correlação linear der2 = 0,9999, na faixa de 20 a 100 μg mL-1; precisão (intra-ensaio: desvio padrão relativo (RSD) 1,33%; inter-ensaio: RSD=0,21%), exatidão (100,74%) e robustez com RSD menor que 4,5%. O teste-tmostrou não haver diferença estatisticamente significativa entre o método turbidimétrico proposto e um método por HPLC previamente validado. No entanto, o ensaio turbidimétrico pode ser utilizado como método alternativo para o controle de qualidade de rotina para este antimicrobiano, como um complemento de outros métodos físico-químicos.

Synthesis of Silver Nanoparticles and Incorporation with Certain Antibiotic Using Gamma Irradiation.

Aims: The study was explaining that silver nanoparticles (AgNPs) were synthesized biologically by Bacillus megaterium culture supernatant (as reducing and stabilizing agents) by the optimization of medium components for nitrate reductase production to enhance the synthesis of AgNPs. And use of gamma irradiation for the synthesis and incorporation of AgNPs with selected antibiotics at distinct dose. Place and Duration of Study: The study was carried out in 2012 in the Department of Drug Radiation Research, Egyptian Atomic Energy Authority. Methodology: The optimized conditions for AgNPs formation by B. megaterium culture supernatant were as follows; media containing: (%) yeast extract: 0.15, peptone: 0.25, KNO3:0.1 temp: 30ºC and incubation period 24 h with maximum nitrate reductase activity of (680.89U/ml). Physical synthesis of AgNPs and incorporation with antibiotics such as (Sodium Cefotaxime, Gentamycin sulphate and Amoxicillin) by γ-rays doses such as (0.5, 1, 1.5, 2, 2.5 and 3 kGy) were studded. AgNPs were characterized by (UV-Vis.), (DLS), (FT-IR) and (TEM) analysis. Combined and individual antibacterial activities of Amoxicillin and AgNPs were investigated against different pathogenic bacterial species by measuring the (ZOI) and by determining the (MIC). Results: This method shows that Aqueous Ag+ ions were reduced to AgNPs when added to the cell-free supernatant of B. megaterium this is indicated by the color change from whitish yellow to brown and the control showed no color change. In physical method Amoxicillin was incorporated with AgNPs perfectly at 2.5kGy. The decreasing order of the average antibacterial activity against bacterial group was observed to be AgNPs > Amoxicillin > Amoxicillin + AgNPs. Conclusion: The radiation-induced AgNPs synthesis is a simple, clean which involves radiolysis of aqueous solution that provides an efficient method to reduce metal ions. B. megaterium was found to be an effective biological tool for the extracellular biosynthesis of AgNPs. The bactericidal activity have proved that AgNPs in combination with amoxicillin kill bacteria at such low concentrations (units of ppm), which do not reveal acute toxic effects on human cell, in addition to overcoming resistance, and lowering cost when compared to conventional antibiotics.

Herb drug interaction：effect of Manix? on pharmacokinetic parameters of pefloxacin in rat model

Objective: To evaluate the effect of Manix?, the commonly used polyherbal formulation on pefloxacin pharmacokinetic parameters.Methods:from hospitalized patients.Results:Microbiological assay was employed using clinical isolate of Escherichia coli samples Manix? altered the bioavailability parameters of pefloxacin as thus, maximal concentration (Cmax) of pefloxacin (0.91±0.31) μg/mL occurred at time to reach maximal concentration (tmax) 4.0 h while in the group that received Manix? alongside pefloxacin Cmax was (0.22±0.08) μg/mL at tmax 1.0 h respectively. The area under curve of pefloxacin alone was (7.83±5.14) μg/h/mL while with Manix? was (2.60±0.08) μg/h/mL. There was a significant difference between Cmax, tmax and area under curve between pefloxacin alone and pefloxacin after Manix? pre-treatment (P<0.05).Conclusions:The concurrent use of Manix? and pefloxacin has been found to compromise the therapeutic effectiveness of pefloxacin which could lead to poor clinical outcomes in patients.

Herb drug interaction: effect of Manix® on pharmacokinetic parameters of pefloxacin in rat model

<p><b>OBJECTIVE</b>To evaluate the effect of Manix®, the commonly used polyherbal formulation on pefloxacin pharmacokinetic parameters.</p><p><b>METHODS</b>Microbiological assay was employed using clinical isolate of Escherichia coli samples from hospitalized patients.</p><p><b>RESULTS</b>Manix® altered the bioavailability parameters of pefloxacin as thus, maximal concentration (Cmax) of pefloxacin (0.91±0.31) µg/mL occurred at time to reach maximal concentration (tmax) 4.0 h while in the group that received Manix® alongside pefloxacin Cmax was (0.22±0.08) µg/mL at tmax 1.0 h respectively. The area under curve of pefloxacin alone was (7.83±5.14) µg/h/mL while with Manix® was (2.60±0.08) µg/h/mL. There was a significant difference between Cmax, tmax and area under curve between pefloxacin alone and pefloxacin after Manix® pre-treatment (P<0.05).</p><p><b>CONCLUSIONS</b>The concurrent use of Manix® and pefloxacin has been found to compromise the therapeutic effectiveness of pefloxacin which could lead to poor clinical outcomes in patients.</p>

Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution

Chlorhexidine (CHX) is a broad-spectrum antiseptic that is used in many topical pharmaceutical formulations. Because there is no official microbiological assay reported in the literature that is used to quantify CHX, this paper reports the development and validation of a simple, sensitive, accurate and reproducible agar diffusion method for the dosage of chlorhexidine digluconate (CHX-D) in an aqueous solution. The assay is based on the inhibitory effect of CHX-D upon the strain of Staphylococcus aureus ATCC 25923, which is used as the test microorganism. The design 3x3 parallel-line model was used. The results were treated statistically by analysis of variance (ANOVA), and they were excellent in terms of linearity (r = 0.9999), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 0.5 to 4.5%. The results obtained were precise, having relative standard deviations (RSD) for intra-day and inter-day precision of 2.03% and 2.94%, respectively. The accuracy was 99.03%. The method proved to be very useful and appropriate for the microbiological dosage of CHX-D in pharmaceutical formulations; it might also be used for routine drug analysis during quality control in pharmaceutical industries.

Clorexidina (CHX) é um antisséptico com amplo espectro de ação utilizada em muitos tipos de preparações farmacêuticas para uso tópico. Uma vez que não há na literatura ensaio microbiológico oficial para quantificar a clorexidina, este trabalho objetivou o desenvolvimento e validação de um ensaio microbiológico simples, sensível, exato e reprodutível, por difusão em ágar, para doseamento de digliconato de clorexidina (CHX-D) em solução aquosa. O ensaio é baseado no efeito da inibição de Staphylococcus aureus ATCC 25923, utilizado como microorganismo teste, pela CHX-D. Utilizou-se o delineamento 3x3. Os resultados foram verificados estatisticamente pela análise de variância (ANOVA) e apresentaram excelente linearidade (r = 0,9999), demonstrando que o método segue o modelo linear com regressão significativa entre o diâmetro da zona de inibição e o lagaritmo da concentração no intervalo de 0,5 a 4,5%. Os resultados obtidos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 2,03% e DPR para precisão inter-dias de 2,94%. A exatidão foi 99,03%. O método provou ser muito útil e apropriado para doseamento microbiológico da CHX-D em formas farmacêuticas e pode ser empregado para análise desta substância no controle de qualidade em indústrias farmacêuticas.

Validated microbiological and HPLC methods for the determination of moxifloxacin in pharmaceutical preparations and human plasma

The article presents a comparison between microbiological and high performance liquid chromatographic (HPLC) assays for quantification of moxifloxacin in tablets, ophthalmic solutions and human plasma. The microbiological method employed a cylinder-plate agar diffusion assay using a strain of Esherichia coli ATCC 25922 as the test organism and phosphate buffer (pH8) as the diluent. The calibration curves were linear (R²> 0.98) over a concentration range of 0.125 to 16 µgml-1. The within day and between days precisions were < 4.47% and < 6.39% respectively. Recovery values were between 89.4 and 110.2%. The HPLC assay used Hypersil® BDS C18 reversed phase column (250×4.6 mm, 5µm) with a mobile phase comprising 20 mM ammonium dihydrogen orthophosphate (pH3) and acetonitrile (75:25) and flowing at 1.5 ml/min. The detection was at 295nm. The calibration curves were linear (R²> 0.999) over the range of 0.125 to 16 µg ml-1. The within day and between days precisions were < 4.07% and < 5.09% respectively. Recovery values were between 97.7 and 107.6%. Similar potencies were obtained after the analysis of moxifloxacin tablets and ophthalmic solutions by both methods. Also pharmacokinetic parameters were calculated after the analysis of plasma samples of six male healthhy volunteers by both validated methods.

Pharmacokinetics of teicoplanin in rabbit eyes / 中华实验眼科杂志

Background Endophthalmitis is a serious infectious eye disease.Efficient drug therapy plays a key role in the early stage.There have been few researches on teicoplanin treating endophthalmitis and pharmacokinetics in ocular tissue.Objective The present study was to investigate the intraocular pharmacokinetic course and feature of intravitreal administration of teicoplanin.Methods Thirty-three Japanese white rabbits were included in this study and randomized into 6 groups.The right eyes of the rabbits were used in experiment.5 g/L of teicoplanin was injected into the vitreous cavity,and vitreous and aqueous humor samples were extracted after 15 minutes,30 minutes,1,2,4,6,12,24,48,96 and 192 hours,and the concentration of teicoplanin was determined by bioassay.Results The logarithmic value of the concentration of teicoplanin was raised with the increase in the bacterial inhibition zone diameters,of which the equation of the regression curve was Y =0.174X-0.813(R2=0.999).A good linear relationship was presented within 1.0-80.0 mg/L.Single intravitreal injection of teicoplanin was compliant with the two-compartment model.Moreover,the distribution phase Tα1/2 and elimination phase Tβ1/2 of vitreous were 1.68 and 152.15 hours,separately.And Tα1/2 and Tβ1/2 of the aqueous humor were 2.83 hours and 70.56 hours,individually.The peak teicoplanin concentrations in the vitreous and aqueous humor were(358.47±21.53)mg/L and(102.17±9.54)mg/L at 1 hour,respectively and remained at(4.38±0.68)mg/L and(2.38±0.38)mg/L,respectively 192 hours later.Conclusions Intravitreal injection of 0.5 mg of teicoplanin can remain therapeutic concentration for a long time in the vitreous and aqueous humor.

Development of agar diffusion method for dosage of gramicidin / Desenvolvimento do método de difusão em agar para dosagem de gramicidina

Gramicidin, an antimicrobial peptide active against Gram positive bacteria, is commonly used in pharmaceutical preparations for topical use. Considering that only the turbidimetric method has been described in the literature, the present study sought to develop and validate an agar diffusion method for the dosage of gramicidin. The method was developed and validated using the Kocuria rhizophila ATCC 9341 as a test microorganism. Two designs were used: a 3x3 parallel-line model, and a 5x1 standard curve. The validation demonstrated that the method follows the linear model (r²= 0.994), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 5 to 25.3 µg/mL. The results obtained for both designs were precise, having a relative standard deviation (R.S.D.) for intra-day precision of 0.81 for the 3x3 assay and 1.90 for the 5x1 assay. For the inter-day precision, the R.S.D. was 1.35 for the 3x3 and 2.64 for the 5x1. The accuracy was verified and results confirmed to be accurate, having a tolerance interval of 95 percent, which lay within permitted limits and appropriate trueness. In addition, the method was considered selective, with limit of detection and upper and lower limits of quantification of 2.00, 5.00 and 25.3 µg/mL, respectively. No difference in precision between the designs used in the agar diffusion method was evident (p>0.05). The method proved to be appropriate for the microbiological dosage of the raw material gramicidin.

A gramicidina, um peptídeo antimicrobiano ativo contra bactérias Gram positivo, é utilizada em preparações farmacêuticas de uso tópico. Neste trabalho procurou-se desenvolver e validar outro método para o doseamento de gramicidina tendo em vista que somente o método turbidimétrico é descrito. O método de difusão em ágar foi desenvolvido e validado utilizando como microrganismo teste Kocuria rhizophila ATCC 9341. Foram utilizados dois delineamentos: retas paralelas 3x3 e curva padrão 5x1. A validação demonstrou que o método segue o modelo linear (r²= 0,994) havendo regressão significativa entre o diâmetro dos halos de inibição e o logaritmo da concentração na faixa de 5,00 a 25,3 µg/mL. Os resultados obtidos por ambos os delineamentos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 0,81 para ensaio 3x3 e de 1,90 para ensaio 5x1. Para a precisão inter-dias o DPR foi de 1,35 para 3x3 e de 2,64 para 5x1. A exatidão foi verificada e os resultados foram exatos apresentando intervalo de tolerância a 95 por cento dentro dos limites permitidos e veracidade adequada. O método foi seletivo com limites de detecção e quantificação inferior e superior iguais a 2,00, 5,00 e 25,3 µg/mL, respectivamente. Não foi observada diferença entre a precisão dos delineamentos empregados no método de difusão em ágar (p>0.05). O método se mostrou adequado para a dosagem microbiológica de gramicidina matéria-prima.

Determination of apramycin in oral soluble powder by a HPLC method using pre-column derivatization with o-phthalaldehyde and UV detection / Determinação da apramicina em pó solúvel oral por um método de HPLC utilizando pré-coluna de derivação com o de detecção de ftalaldeído e UV

A high-performance liquid chromatographic method employing pre-column derivatization with o-phthalaldehyde (OPA) and 2-mercaptoacetic acid was developed for the determination of apramycin, an aminoglycoside antibiotic used in veterinary medicine, in the oral soluble powder form. The chromatographic separation was done by ion-pair HPLC using a C18 reversed-phase column, Synergy Hydro (150 mm x 4.6 mm x 4 µm) and mobile phase composed of 0.005 mol/L sodium octanosulfonate in a mixture of acetonitrile: water: acetic acid (45:55:2) (v/v/v) with a flow rate of 1.0 mL/min; the UV detector was operated at 332 nm. The developed method was validated according to official compendia guidelines, having demonstrated robustness, selectivity and linearity for the concentration range of 0.02 to 0.05 mg/mL, precision (with RSD < 2.0 percent both for intra and inter-day precision) accuracy (average recuperation of 99.33 percent) and detectivity (quantification and detection limits of 0.08 and 0.02 µg/mL, respectively). Three batches of commercial apramycin oral soluble powder were analyzed by both the proposed method and the official microbiological method, where all the results obtained were in the acceptable range (95 percent to 105 percent of labeled value of apramycin). Both methods were statistically compared by the t test, which yielded no significant differences (α = 0.05) thereby confirming the equivalence of the methods.

Foi desenvolvido um método por cromatografia líquida alta eficiência empregando derivatização pré-coluna com o-ftalaldeído (OPA) e ácido mercaptoacético para determinação de apramicina, um antibiótico aminoglicosídeo de uso veterinário, em pó oral solúvel. A separação cromatográfica foi feita por fase reversa com pareamento iônico utilizando-se coluna Synergy Hydro C18 (150 x 4,6 mm x 4 µm) e fase móvel composta por octanossulfonato de sódio 0,005 mol/L em mistura de acetonitrila:água:ácido acético nas proporções 45:55:2 (v/v/v), numa vazão de 1.0 mL/min; efetuou-se detecção por UV a 332 nm. O método foi validado de acordo com os compêndios oficiais e demonstrou robustez, seletividade, linearidade na faixa de 0,02 a 0,05 mg/mL, precisão (com DPR < 2,0 por cento tanto para a precisão intra-dia quanto para a precisão inter-dia), exatidão (recuperação média de 99,33 por cento) e detectabilidade (limite de quantificação e de detecção iguais a 0,08 e 0,02 µg/mL, respectivamente). Analisaram-se 3 lotes de apramicina pó oral solúvel pelo método proposto e pelo método microbiológico oficial e todos os resultados obtidos estavam dentro do limite de aceitação (95 por cento-105 por cento valor rotulado de apramicina). Ambos os métodos foram comparados estatisticamente pelo teste t, não sendo encontradas diferenças significativas entre eles para α=0,05, sendo os dois equivalentes.

Folate content and retention in selected raw and processed foods / Contenido de folatos y retención en determinados alimentos crudos y procesados

Adequate intake of folate reduced the risk of abnormalities in early embryonic brain development such as the risk of malformations of the embryonic brain/spinal cord, collectively referred to as neural tube defects (NTDs). Folate is extremely sensitive to destruction by heat, oxidation and UV light. The purpose of this study was to evaluate the use of different extraction procedures and enzymatic treatment to determine folate concentrations in variety of foods using a microbiological assay (MA) with Lactobacillus rhamnosus as the test organism. This study also aimed to evaluate the retention of folate in foods after using different cooking processes. Nine of the most commonly consumed foods in Argentina and that contain folate were analyzed: broccoli, spinach, potato, lentil, soy (raw and boiled); hen whole egg and yolks (raw, boiled and fried); beef liver (raw and cooked); strawberry (raw) and white bread. For this study, rat plasma (RP) and human plasma (HP) conjugases together with acetate and phosphate buffers were tested. In extraction step for all analyses, RP conjugase was selected since it was easily available in our laboratory and small quantities were required. The acetate buffer was chosen since better growth and more reproducible results were obtained in the different conditions assayed. The results allowed the foods to be grouped into a) rich sources of folate: hen eggs, yolks, spinach, soybean (raw) and strawberry (100 and 350mg/100g fresh weight (FW); b) good sources of folate: broccoli (raw), soybean (boiled), lentils (raw) and potato (56 to 83mg/100g FW) and c) moderate sources of folate: broccoli, lentils (boiled), white breads, onions and beef liver (15 to 30mg/100g FW). The folate retention was in the range 14-99% according to both type of food and method of processing. Contents and losses of folate vary widely according to type of food and cooking method.

La ingesta adecuada de folatos reduce el riesgo de las anormalidades en el desarrollo temprano del cerebro embrionario, tales como el riego de malformaciones en el cerebro/médula espinal, conocidas en conjunto como defectos del tubo neural (NTDs). Los folatos son extremadamente sensibles al tratamiento con calor, la oxidación y la luz UV. El objetivo de este trabajo fue evaluar el uso de diferentes procedimientos de extracción y de tratamientos enzimáticos para determinar el contenido de folato en distintos alimentos empleando un método microbiológico que utiliza el microorganismo Lactobacillus rhamnosus. En este trabajo se evaluó también la retención de folatos en alimentos sometidos a diferentes procesos de cocción. Se analizaron 9 de los alimentos que contienen folatos y más comúnmente consumidos en Argentina: brócoli, espinaca, papa, lente ja, soja (crudos y cocidos): huevo entero de gallina y yema (crudo, hervido y frito). Bife de hígado vacuno (crudo y cocido); frutillas (crudas) y pan: blanco. Se probó para este estudio conjugasas de plasma de rata (PR) y de plasma humano (PH) conjuntamente con buffers fosfato y acetato. En la extracción para todos los análisis se escogió la conjugasa de PR por ser accesible para nuestro laboratorio y por que se utiliza en pequeñas cantidades. El buffer acetato fue elegido debido a que se obtuvo resultados más reproducibles y un mejor crecimiento en las diferentes condiciones ensayadas. Los resultados permitieron agrupar los alimentos en: a) fuente rica de folatos: huevo y su yema, espinaca, soja (cruda) y frutilla (100 a 350mg/100g peso fresco); b) fuente buena de folatos: brócoli (crudo), soja (hervidas), lentejas (cruda) y papa (cruda y hervida) (56 a 83mg/100g peso fresco) y c) fuente moderada de folatos: brócoli y lentejas (hervidos), bife de hígado, pan blanco y cebollas (15 a 30mg/100g peso fresco). La retención de folato estuvo en el rango de 14-99% de acuerdo al tipo de alimento y el método de procesado. El contenido de folato y sus perdidas fueron muy variables dependiendo del alimento y del método de cocción empleados.

Comparison of microbiological assay and HPLC-UV for determination of fluconazole in capsules / Comparação de ensaios microbiológicos e CLAE-UV para determinação de fluconazol em cápsulas

The development of a specific agar diffusion bioassay for the quantitative determination of fluconazole formulated in capsules was carried out using a strain of Candida albicans ATCC 18804 as the test organism. A prospective validation of the method showed adequate linearity (r²=0.9995), precision (R.S.D. = 4.0 percent for intra-day and 4.5 percent for inter-day precision) and accuracy (mean recovery = 102.9 percent). High performance liquid chromatography was chosen as a comparison method for the fluconazole determination. The contents of fluconazole determined by both methods, for four capsule samples, showed a strong correlation, confirmed by Pearson's correlation coefficient value (r = 0.9884). The bioassay is a suitable method for both research and pharmaceutical industry laboratories.

Este trabalho visou ao desenvolvimento e validação de um método microbiológico por difusão em ágar para quantificação de fluconazol em cápsulas utilizando o isolado Candida albicans ATCC 18804 como reagente biológico. O método foi validado e foi verificada linearidade (r²=0,9995), precisão (D.P.R. = 4.0 por cento para precisão intra-dia e 4,5 por cento para precisão inter-dia) e exatidão (recuperação média = 102,9 por cento). Concomitantemente, foi realizado o doseamento de fluconazol nas cápsulas por meio de cromatografia líquida de alta eficiência. Os teores encontrados por ambos os métodos demonstraram alta correlação, confirmada pelo Coeficiente de Correlação de Pearson (r = 0,9884). O ensaio microbiológico desenvolvido pode ser considerado ferramenta valiosa tanto para a pesquisa científica quanto para a rotina da indústria farmacêutica.

Bioequivalence of Cefdinir Dispersible Tablets in Healthy Volunteers / 中国药房

OBJECTIVE:To study the pharmacokinetics and the bioequivalence of cefdinir dispersible tablets in healthy volunteers.METHODS:Microbiological assay method was used to determine the plasma concentration at different time in 20 healthy volunteers after oral administration of single dose of 400mg of cefdinir dispersible tablets(test preparation) and cefdinir capsule(reference preparation) by cross-over way.RESUTLS:The concentration-time curves of test preparation and reference preparation of cefdinir fitted one compartment open model.The pharmacokinetic parameters of the test preparation vs.the reference preparation were as follows:tmax(3.48?0.53)h vs.(3.60?0.48)h,Cmax(2.10?0.32)mg?L-1 vs.(2.15?0.26)mg?L-1.t1/2ke(2.41?0.39)h vs.(2.33?0.41)h,AUC0～12(9.51?1.65)mg?h?L-1 vs.(10.05?1.72)mg?h?L-1,AUC0～∞(10.43?1.62)mg?h?L-1 vs.(11.01?1.81)mg?h?L-1,respectively.The relative bioavailability of cefdinir dispersible tablet as against its reference preparation was(96.03?14.89)%.CONCLUSION:The two preparations of cefdinir were proved to be bioequivalent.

Influence of endotoxin induced fever on the pharmacokinetics of intramuscularly administered cefepime in rabbits

This study examined the effect of experimentally induced fever on the pharmacokinetics of cefepime (75 mg/kg BW) administered intramuscularly to six rabbits. The study was carried out in two consecutive phases separated by a two-week washout period. An infection was induced by an intravenous inoculation of 5 x 10(8) colony-forming units of Escherichia coli 24 h before the pharmacokinetic investigation. A quantitative microbiological assay was employed to measure the plasma cefepime concentrations using an agar-gel diffusion method with Bacillus subtilis ATCC 6633 as the test organism. Twenty-four hour after the injection, the rectal temperature in the infected animals increased by 1degrees C. There was a significant reduction in the elimination halflife by 21.8% in the febrile rabbits compared to healthy animals. In addition, the infection significantly increased the peak plasma concentrations by 11.9%, the mean residence time by 19.9%, the area under the plasmaconcentration- time curve by 53.6% and the area under the moment curve by 62.3%. In conclusion, the endotoxin-induced febrile state produced significant changes in the plasma levels as well as some of the pharmacokinetic variables of cefepime in rabbits.

Development of Gentamicin Loaded PLGA Microspheres for the Treatment of Musculoskeletal Infection: In Vivo Evaluation / 대한정형외과연구학회지

PURPOSE: We evaluate in vitro and in vivo efficacy of newly developed gentamicin loaded PLGA microspheres for the treatment of musculoskeletal infection. MATERIALS AND METHODS: Controlled gentamicin sulfate releasing microspheres manufactured from biodegradable PLGA were prepared with an Oil/Oil solvent evaporation method for the treatment of musculoskeletal infection. The in vitro release amount of GS was analyzed by high performance liquid chromatography (HPLC) and the released GS activity was determined by microbiological assay using staphylococcus (S) aureus, respectively. The results of inhibition zone test agree well with the HPLC results obtained from the in vitro release test. RESULTS: The microspheres of different size were obtained with varying the experimental conditions, and the shape of microspheres was smooth and spherical. The PLGA microspheres release gentamicin for 67 days in in vitro test. There was significant inhibition around microphere PLGA from 1 day to 7th week in inhibition zone test The inhibition was reduced after 8th week. and there was no inhibition at 9th week PLGA microspheres. CONCLUSION: It can be suggested that GS/PLGA MSs implantable system that provided a prolonged delivery of GS was found to be effective against S. aureus infection for desired period.