p-iodophenol-enhanced luminol chemiluminescent assay applied to discrimination between acute lymphoblastic and minimally differentiated acute myeloid (FAB-M0) or acute megakaryoblastic (FAB-M7) leukemias

Introduction: In this report, we propose the application of the p-iodophenol-enhanced luminol chemiluminescent technique to the determination of peroxidase (myeloperoxidase and/or platelet peroxidase) activity in blasts of minimally differentiated acute myeloblastic leukemia (AML-M0) and acute megakaryoblastic leukemia (AML-M7).Methods: the frozen blast cells from 29 patients were thawed and submitted to the optimized protocol.Results: All cases of AML-M7 and AML-M0 exhibited integrated light emission greater than 73 (10(2) mV x s), which was the arbitrary cutoff point set for the discrimination between AML and acute lymphoblastic leukemia (ALL) (mean + 3 x s.d. of ALL samples, n = 10). In addition, five out of seven cases of AML-M0 showed results above the Cutoff point.Conclusion: This highly sensitive enhanced chemiluminescent technique may be applied to discriminate between ALL and AML-M7 or AML-M1 cases, and most AML-M0 cases. It is very simple, cheap and easy to perform compared to other procedures used to measure MPO activity in AML-leukemias including AML-M7 and AML-M0.

Microbiological assay for ceftazidime injection

A simple, sensitive, and specific biodiffusion assay for the! antibacterial ceftazidime was developed using a strain of Staphylococcus epidermidis (ATCC 12228) as the test organism. Ceftazidime was measured in powder for injection at concentrations ranging from 100 to 400 mu g/mL. The calibration graph for ceftazidime was linear (r(2) = 1), and the method validation showed that it was precise (relative standard deviation = 0.415) and accurate. The results obtained by biodiffusion assay were statistically calculated by linear parallel model and by means of regression analysis and were verified using analysis of variance. It was concluded that the microbiological assay is satisfactory for in vitro quantification of the antibacterial activity of ceftazidime in pharmaceuticals.

Microbiological assay for gatifloxacin in pharmaceutical formulations

A simple, sensitive and specific agar diffusion bioassay for the antibacterial gatifloxacin was developed using a strain of Bacillus subtilis ATCC 9372 as the test organism. Gatifloxacin could be measured in tablets and raw material at concentration ranging 4-16 mu g ml(-1). The calibration graph for gatifloxacin was linear from 4.0 to 16.0 mu g ml(-1). A prospective validation of the method demonstrated that the method was linear (r(2) = 0.9993), precise (R.S.D. = 1.14%) and accurate. The results confirmed its precision and did not differ significantly from others methods described in the literature. The validated method yielded good results in terms of the range, linearity, precision, accuracy, specificity and recovery. We concluded that the microbiological assay is satisfactory for in vitro quantification of the antibacterial activity of gatifloxacin. (c) 2005 Elsevier B.V. All rights reserved.

Development and validation of a stability-indicative agar diffusion assay to determine the potency of linezolid in tablets in the presence of photodegradation products

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

LC Assay for Ciprofloxacin Hydrochloride Ophthalmic Solution

The objective of the current study was to develop and subsequently validate a simple, sensitive and precise reversed-phase LC method for the determination of ciprofloxacin hydrochloride in ophthalmic solution form. The chromatographic separation of ciprofloxacin hydrochloride was achieved on a Symmetry Waters C(18) column using UV detection at 275 nm. The optimized mobile phase consisted of 2.5% acetic acid solution: methanol:acetonitrile (70:15:15, v/v/v). The proposed method provided linear responses within the concentration range 1.0-6.0 mu g mL(-1) for ciprofloxacin hydrochloride. Correlation coefficient (r) for the ciprofloxacin hydrochloride was 0.9994. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 5% in all instances. No interference from any components of pharmaceutical dosage forms was observed.

Assessment of genetic damage induced by dental bleaching agents on mouse lymphoma cells by single cell gel (comet) assay

Dental bleaching is a simple and conservative procedure for aesthetic restoration of vital discoloured teeth. However, dental bleaching agents may represent a hazard to human health, especially by causing DNA strand breaks. Genotoxicity tests form an important part of cancer research and risk assessment of potential carcinogens. In the current study, the genotoxic potential associated with exposure to dental bleaching agents was assessed by the single cell gel (comet) assay in vitro. Six commercial dental bleaching agents (Clarigel Gold - Dentsply; Whitespeed - Discus Dental; Nite White - Discus Dental; Magic Bleaching - Vigodent; Whiteness HP - FGM and Lase Peroxide - DMC) were exposed to mouse lymphoma cells in vitro. The results pointed out that all dental bleaching agents tested contributed to the DNA damage as depicted by the mean tail moment. Clear concentration-related effects were obtained for DNA damaging, being the strongest effect observed at the highest dose of the hydrogen peroxide (Whiteness HP and Lase Peroxide, at 35% concentration). on the contrary, Whitespeed (Discus Dental) induced the lowest level of DNA breakage. Taken together, these results suggest that dental bleaching agents may be a factor that increases the level of DNA damage as detected by the single cell gel (comet) assay.

Lack of genotoxicity induced by endogenous and synthetic female sex hormones in peripheral blood cells detected by alkaline Comet assay

The etiology of hormone-induced cancers has been considered to be a combination of genotoxic and epigenetic events. Currently, the Comet assay is widely used for detecting genotoxicity because it is relatively simple, sensitive, and capable of detecting various kinds of DNA damage. The present study evaluates the genotoxic potential of endogenous and synthetic sex hormones, as detected by the Comet assay. Blood cells were obtained from 12 nonsmoking and 12 smoking women with regular menstrual cycles and from 12 nonsmoking women taking low-dose oral contraceptives (OC). Peripheral blood samples were collected at three phases of the menstrual cycle (early follicular, mean follicular, and luteal phases), or at three different moments of oral contraceptive intake. Three blood samples were also collected from 12 healthy nonsmoking men, at the same time as oral contraceptive users. Results showed no significant difference in the level of DNA damage among the three moments of the menstrual cycle either in nonsmoking and smoking women, or between them. No significant difference in DNA damage was also observed among oral contraceptive users, nonusers, and men. Together, these data indicate lack of genotoxicity induced by the physiological level of the female sex hormones and OC as assessed by the alkaline Comet assay. In conclusion, normal fluctuation in endogenous sex hormones and use of low-doses of oral contraceptive should not interfere with Comet assay data when this technique is used for human biomonitoring.

Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.

Colorimetric enzymatic assay of L-malic acid using dehydrogenase from baker's yeast

A colorimetric method has been developed and optimized to measure L-malic acid in samples of fruit juices and wine. This method is based on oxidation of the analyte, catalyzed by malate dehydrogenase (MDH) from dry baker's yeast, and in combination with the reduction of a tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). In the present study, the method exhibited sensitivity in the range of 500-4000 mu M of L-malic acid in the reaction cuvette, with the lower detection limit of 6.7-10(-2) g/L, the upper limit of 53.6.10(-2) g/L and a maximum standard deviation of only 2.5 % for the analyzed samples. The MDH activity from baker's yeast was also optimized, the enzyme showed a high stability at pH=8.0-9.0 and the activity was maintained completely at temperatures up to 40 degrees C for 1 hour. The results show that the colorimetric method using enzymatic preparations from dry baker's yeast is a simple and low-cost method with possibility of wide application.

Voltammetric assay of albendazole in pharmaceutical dosage forms

A simple, rapid inexpensive voltammetric method have been developed for the quantitative determination of albendazole (ABZ) as the pure assay, by direct dissolution of commercial tablets in HCl solutions. Studies with linear sweep (LSV), square-wave (SWV) and differential pulse voltammetry (DPV) were carried out ABZ in aqueous medium at a glassy carbon electrode. A well defined irreversible oxidation peak current was obtained at 1,00V vs. SCE. The method permits a precise quantitative determination of ABZ using the standard addition method. The detection limits for the three voltammetric techniques were found to be 3.0 x 10(-5) M (LSV), 6.2 x 10(-5) M (SWV) and 4.0 x 10(-5) M (DPV).

A novel PCR-RFLP assay for the detection of the single nucleotide polymorphism at position+1440 in the human CXCR2 gene

We designed a novel PCR-RFLP assay to genotype for the CXCR2 +1440 (G/A) single nucleotide polymorphism, which provides a simple, low-cost, practical, and reproducible method. Allele frequencies in healthy Brazilian individuals were found to be 0.65% for allele A and 0.35% for allele G.

Characterization of myeloid or lymphoid acute leukemia by a chemiluminescence assay: Comparison with immunocytochemistry using an antimyeloperoxidase antibody

A simple and sensitive chemiluminescence assay for the demonstration of the activity of intracellular myeloperoxidase (MPO) is described, which is useful for the distinction between myeloid and lymphoid commitment in blasts from acute leukemia patients. When the cut-off point was settled at 13 mV of chemiluminescence all cases of acute myeloid leukemia (AML) were distinguished from those of acute lymphoid leukemia. In addition, this technique was able to demonstrate MPO activity in AML poorly differentiated (FAB-M0) which usually does not stain for MPO in classical cytochemistry preparations and could be negative also by immunocytochemistry with anti-MPO monoclonal antibody. Therefore the method here described presented a higher sensitivity than the immunocytochemistry procedure with anti-MPO.

Microbiological assay for the determination of azithromycin in ophthalmic solutions

The validation of a simple, sensitive and specific agar diffusion bioassay, applying cylinder-plate method, for the determination of the antibiotic azithromycin in ophthalmic solutions is described. Using a strain of Bacillus subtilis ATCC 9372 as the test organism, azithromycin at concentrations ranging from 50.0 to 200.0 μg·mL-1 could be measured in 1.666 7 mg·mL-1 ophthalmic solutions. A prospective validation of the method showed that the method was linear (r = 0.999 9) and precise (RSD = 0.70) and accurate (it measured the added quantities). The results obtained by bioassay method could be statistically calculated by linear parallel model and by means of regression analysis and verified using analysis of variance (ANOVA). We conclude that the microbiological assay is satisfactory for quantification of azithromycin in ophthalmic solutions.

Development and validation of a microbiological agar assay for determination of orbifloxacin in pharmaceutical preparations

Orbifloxacin is a fluoroquinolone with broad-spectrum antimicrobial activity, and belongs to the third generation of quinolones. Regarding the quality control of medicines, a validated microbiological assay for determination of orbifloxacin in pharmaceutical formulations has not as yet been reported. For this purpose, this paper reports the development and validation of a simple, sensitive, accurate and reproducible agar diffusion method to quantify orbifloxacin in tablet formulations. The assay is based on the inhibitory effect of orbifloxacin upon the strain of Staphylococcus aureus ATCC 25923 used as test microorganism. The results were treated statistically by analysis of variance and were found to be linear (r = 0.9992) in the selected range of 16.0-64.0 μg/mL, precise with relative standard deviation (RSD) of repeatability intraday = 2.88%, intermediate precision RSD = 3.33%, and accurate (100.31%). The results demonstrated the validity of the proposed bioassay, which allows reliable orbifloxacin quantitation in pharmaceutical samples and therefore can be used as a useful alternative methodology for the routine quality control of this medicine. © 2011 by the authors; licensee MDPI, Basel, Switzerland.