Interference of raw milk autochthonous microbiota on the performance of conventional methodologies for Listeria monocytogenes and Salmonella spp. detection

Pathogen detection in foods by reliable methodologies is very important to guarantee microbilogical safety. However, peculiar characteristics of certain foods, such as autochthonous microbiota, can directly influence pathogen development and detection. With the objective of verifying the performance of the official analytical methodologies for the isolation of Listeria monocytogenes and Salmonella in milk, different concentrations of these pathogens were inoculated in raw milk treatments with different levels of mesophilic aerobes, and then submitted to the traditional isolation procedures for the inoculated pathogens. Listeria monocytogenes was inoculated at the range of 0.2-5.2 log CFU/mL in treatments with 1.8-8.2 log CFU/mL. Salmonella Enteritidis was inoculated at 0.9-3.9 log CFU/mL in treatments with 3.0-8.2 log CFU/mL. The results indicated that recovery was not possible or was more difficult in the treatments with high counts of mesophilic aerobes and low levels of the pathogens, indicating interference of raw milk autochthonous microbiota. This interference was more evident for L. monocytogenes, once the pathogen recovery was not possible in treatments with mesophilic aerobes up to 4.0 log CFU/mL and inoculum under 2.0 log CFU/mL. For S. Enteritidis the interference appeared to be more non-specific. (C) 2007 Elsevier GmbH. All rights reserved.

Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay

Objectives: The resazurin microtitre plate assay (REMA) was evaluated to determine the susceptibility of Mycobacterium tuberculosis to pyrazinamide, and was compared with the broth microdilution method (BMM), the absolute concentration method (ACM) and pyrazinamidase (PZase) determination. Methods: Thirty-four M. tuberculosis clinical isolates (26 susceptible and 8 resistant to pyrazinamide) and reference strains M. tuberculosis H37Rv ATCC 27294 and Mycobacterium bovis AN5 were tested. Results: REMA and BMM showed 100% specificity and sensitivity when compared with ACM; BMM, however, demanded more reading time. The PZase determination assay showed 87.50% and 100% sensitivity and specificity, respectively. Conclusions: All tested methods in this preliminary study showed excellent sensitivity and specificity for the determination of pyrazinamide susceptibility of M. tuberculosis, but REMA was faster, low-cost and easy to perform and interpret. Additional studies evaluating REMA for differentiating pyrazinamide-resistant and-susceptible M. tuberculosis should be conducted on an extended panel of clinical isolates.

Methods for detection of anatoxin-a(s) by liquid chromatography coupled to electrospray ionization-tandem mass spectrometry

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253 > 58, 253 > 159, 235 > 98 and 235 > 96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS. (C) 2009 Elsevier Ltd. All rights reserved.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Detection of the TLR4 1196C > T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

Background: Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. Results: Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C> T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK-plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 mu L PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 mL PCR product. In standardized conditions, TLR4 1196C> T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C> T.

Determination of Atropine Enantiomers in Ophthalmic Solutions by Liquid Chromatography Using a Chiral AGP (R) Column

Many therapeutic agents are commercialized under their racemic form. The enantiomers can show differences in the pharmacokinetic and pharmacodynamic profile. The use of a pure enantiomer in pharmaceutical formulations may result in a better therapeutic index and fewer adverse effects. Atropine, an alkaloid of Atropa belladonna, is a racemic mixture of l-hyoscyamine and d-hyoscyamine. It is widely used to dilate the pupil. To quantify these enantiomers in ophthalmic solutions, an HPLC method was developed and validated using a Chiral AGP (R) column at 20 degrees C. The mobile phase consisted of a buffered phosphate solution (containing 10 mM 1-octanesulfonic acid sodium salt and 7.5 mM triethylamine, adjusted to pH 7.0 with orthophosphoric acid) and acetonitrile (99 + 1, v/v). The flow rate was 0.6 mL/min, with UV detection at 205 nm. In the concentration range of 14.0-26.0 mu g/mL, the method was found to be linear (r > 0.9999), accurate (with recovery of 100.1-100.5%), and precise (RSD system: <= 0.6%; RSD intraday: <= 1.1%; RSD interday: <= 0.9%). The method was specific, and the standard and sample solutions were stable for up to 72 h. The factorial design assures robustness with a variation of +/-10% in the mobile phase components and 2 degrees C of column temperature. The complete validation, including stress testing and factorial design, was studied and is presented in this research.

Simultaneous Determination of Choline Citrate and Acetylmethionine in Injectable Solutions by High Performance Liquid Chromatography

Choline citrate (CC) and acetylmethionine (AM) are lipotropic drugs used in several pharmaceutical formulations. The objective of this research was to develop and validate a high performance liquid chromatographic (HPLC) method for simultaneous determination of CC and AM in injectable solutions, aiming its application in routine analysis for quality control of these pharmaceutical formulations. The method was validated using a Shim-Pack (R) C18 (250 x 4.6 mm, 5 mu m) column. The mobile phase was constituted of 25 mM potassium phosphate buffer solution, pH 5.7, adjusted with 10 % orthophosphoric acid, acetonitrile and methanol (88:10:2, v/v/v). The flow rate was 1.1 mL.min(-1) and the UV detection was made at 210 nm. The analyses were made at room temperature (25 +/- 1 degrees C). The method is precise, selective, accurate and robust, and was successfully applied for simultaneous quantitative determination of CC and AM in injectables.

Simultaneous determination of econazole nitrate, main impurities and preservatives in cream formulation by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of econazole nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-chlorobenzl alcohol and alpha-(2,4-dicholorophenyl)-1H-imidazole-1-ethanol) in cream formulations, has been developed and validated. Separation was achieved on a column Bondclone (R) C18 (300 mm x 3.9 mm i.d., 10 mu m) using a gradient method with mobile phase composed of methanol and water. The flow rate was 1.4 mL min(-1), temperature of the column was 25 C and the detection was made at 220 nm. Miconazole nitrate was used as an internal standard. The total run time was less than 15 min, The analytical curves presented coefficient of correlation upper to 0.99 and detection and quantitation limits were calculated for all molecules. Excellent accuracy and precision were obtained for econazole nitrate. Recoveries varied from 97.9 to 102.3% and intra- and inter-day precisions, calculated as relative standard deviation (R.S.D), were lower than 2.2%. Specificity, robustness and assay for econazole nitrate were also determined. The method allowed the quantitative determination of econazole nitrate, its impurities and preservatives and could be applied as a stability-indicating method for econazole nitrate in cream formulations. (C) 2008 Elsevier B.V. All rights reserved.

HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies

In this study, a simple, rapid and sensitive HPLC method with UV detection is described for determination of metformin in plasma samples from bioequivalence assays. Sample preparation was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed on a reversed-phase phenyl column at 40 degrees C. Mobile phase consisted of a mixture of phosphate buffer and acetonitrile at flow rate of 1.0 ml/min. Wavelength was set at 236 nm. The method was applied to a bioequivalence study of two drug products containing metformin, and allowed determination of metformin at low concentrations with a higher throughput than previously described methods. (c) 2007 Elsevier B.V. All rights reserved.

Validation of an HPLC analytical method for determination of pancuronium bromide in pharmaceutical injections

Pancuronium bromide is used with general anesthesia in surgery for muscle relaxation and as an aid to intubation. A high performance liquid chromatographic method was fully validated for the quantitative determination of pancuronium bromide in pharmaceutical injectable solutions. The analytical method was performed on an amino column (Luna 150mm4.6mm, 5m). The mobile phase was composed of acetonitrile:water containing 50mmol L-1 of 1-octane sulfonic acid sodium salt (20:80v/v) with a flow rate of 1.0mL min-1 and ultraviolet (UV) detection at 210nm. The proposed analytical method was compared with that described in the British Pharmacopoeia.

Determination of Vecuronium Bromide in Pharmaceuticals: Development, Validation and Comparative Study of HPLC and CZE Analytical Methods

Vecuronium bromide is a neuromuscular blocking agent used for anesthesia to induce skeletal muscle relaxation. HPLC and CZE analytical methods were developed and validated for the quantitative determination of vecuronium bromide. The HPLC method was achieved on an amino column (Luna 150 x 4.6 mm, 5 mu m) using UV detection at 205 nm. The mobile phase was composed of acetonitrile:water containing 25.0 mmol L(-1) of sodium phosphate monobasic (50:50 v/v), pH 4.6 and flow rate of 1.0 mL min(-1). The CZE method was achieved on an uncoated fused-silica capillary (40.0 cm total length, 31.5 cm effective length and 50 mu m i.d.) using indirect UV detection at 230 nm. The electrolyte comprised 1.0 mmol L(-1) of quinine sulfate dihydrate at pH 3.3 and 8.0% of acetonitrile. The results were used to compare both techniques. No significant differences were observed (p > 0.05).

Effects of light intensity and dilution rate on the semicontinuous cultivation of Arthrospira (Spirulina) platensis. A kinetic Monod-type approach

Semicontinuous cultures were carried out at different dilution rates (D) and light intensities (I) to determine the maximum productivity of Arthrospira platensis cultivated in helicoidal photobioreactor up to the achievement of pseudo-steady-state conditions. At I = 108 mu mol photons m(-2) s(-1), the semicontinuous regime ensured the highest values of maximum cell concentration (X(m) = 5772 +/- 113 mg L(-1)) and productivity (P(XS) = 1319 +/- 25 mg L(-1) d(-1)) at the lowest (D = 0.1 day(-1)) and the highest (D = 0.3 day(-1)) dilution rates, respectively. A kinetic model derived from that of Monod was proposed to determine the relationship between the product of light intensity to dilution rate (ID) and the cell productivity, which were shown to exert a combined influence on this parameter. This result put into evidence that pseudosteady-state conditions could be modified according to circumstances, conveniently varying one or other of the two independent variables. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of carbon dioxide feeding rate and light intensity on the fed-batch pulse-feeding cultivation of Spirulina platensis in helical photobioreactor

The behavior of S. platensis was investigated in this study through fed-batch pulse-feeding cultures performed at different carbon dioxide feeding rates (F = 0.44-1.03 g L-1 d(-1)) and photosynthetic photon flux density (PPFD = 80-250 mu mol photons m(-2) s(-1)) in a bench-scale helical photobioreactor. To achieve this purpose, an inorganic medium lacking the carbon source was enriched by gaseous carbon dioxide from a cylinder. The maximum cell concentration achieved was 12.8 g L-1 at PPFD = 166 mu mol photons m(-2) s(-1) and F= 0.44 g L-1 d(-1) of CO2. At PPFD = 80 and 125 mu mol photons m(-2) s(-1), the carbon utilization efficiency (CUE) reached maximum values of 50 and 69%, respectively, after about 20 days, and then it decreased, thus highlighting a photolimitation effect. At PPFD = 166 mu mol photons m(-2) s(-1), CUE was >= 90% between 20 and 50 days. The photosynthetic efficiency reached its maximum value (9.4%) at PPFD = 125 mu mol photons m(-2) s(-1). The photoinhibition threshold appeared to strongly depend on the feeding rate: at high PPFD, an increase in the amount of fed CO2 delayed the inhibitory effect on biomass growth, whereas at low PPFD, excess CO2 addition caused the microalga to stop growing. (c) 2007 Elsevier B.V. All rights reserved.

UV-Derivative Spectrophotometric and Stability-Indicating High-Performance Liquid Chromatographic Methods for Determination of Simvastatin in Tablets

A stability-indicating high-performance liquid chromatographic (HPLC) and a second-order derivative spectrophotometric (UVDS) analytical methods were validated and compared for determination of simvastatin in tablets. The HPLC method was performed with isocratic elution using a C18 column and a mobile phase composed of methanol:acetonitrile:water (60:20:20, v/v/v) at a flow rate of 1.0 ml/min. The detection was made at 239 nm. In UVDS method, methanol and water were used in first dilution and distilled water was used in consecutive dilutions and as background. The second-order derivative signal measurement was taken at 255 nm. Analytical curves showed correlation coefficients > 0.999 for both methods. The quantitation limits (QL) were 2.41 mu g/ml for HPLC and 0.45 mu g/ml for UVDS, respectively. Intra and inter-day relative standard deviations were < 2.0 %. Statistical analysis with t- and F-tests are not exceeding their critical values demonstrating that there is no significant difference between the two methods at 95 % confidence level.

Development and Validation of Stability-Indicating HPLC Methods for Quantitative Determination of Pravastatin, Fluvastatin, Atorvastatin, and Rosuvastatin in Pharmaceuticals

High-performance liquid-chromatographic (HPLC) methods were validated for determination of pravastatin sodium (PS), fluvastatin sodium (FVS), atorvastatin calcium (ATC), and rosuvastatin calcium (RC) in pharmaceuticals. Two stability-indicating HPLC methods were developed with a small change (10%) in the composition of the organic modifier in the mobile phase. The HPLC method for each statin was validated using isocratic elution. An RP-18 column was used with mobile phases consisting of methanol-water (60:40, v/v, for PS and RC and 70:30, v/v, for FVS and ATC). The pH of each mobile phase was adjusted to 3.0 with orthophosphoric acid, and the flow rate was 1.0mL/min. Calibration plots showed correlation coefficients (r)0.999, which were calculated by the least square method. The detection limit (DL) and quantitation limit (QL) were 1.22 and 3.08 mu g/mL for PS, 2.02 and 6.12 mu g/mL for FVS, 0.44 and 1.34 mu g/mL for ATC, and 1.55 and 4.70 mu g/mL for RC. Intraday and interday relative standard deviations (RSDs) were 2.0%. The methods were applied successfully for quantitative determination of statins in pharmaceuticals.