Exometabolomic analysis of susceptible and multi-drug resistant Pseudomonas aeruginosa.

Multidrug resistant (MDR) Pseudomonas aeruginosa strains have recently become one of the major public health concerns worldwide leading to difficulties in selecting appropriate antibiotic treatment. Thus, it is important to elucidate the characteristics of MDR isolates. Herein, we aimed to determine the unique exometabolome profile of P. aeruginosa clinical isolates in monocultures that comprise high resistance to multiple antibiotics, and compare the differential metabolite profiles obtained from susceptible isolates by using GC/MS. Our results showed that partial least square-discriminant analysis (PLS-DA) score plot clearly discriminated the MDR and susceptible isolates indicating the altered exometabolite profiles, and highlighted the significantly enriched levels of trehalose and glutamic acid in MDR isolates. Expression of trehalose synthase (treS) was also 1·5-fold higher in MDR isolates, relatively to susceptible isolates. Overall, our study provides insights into the distinct footprints of MDR P. aeruginosa isolates in mono-culture.

Profile of Circulatory Metabolites in a Relapsing-remitting Animal Model of Multiple Sclerosis using Global Metabolomics.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Recently, metabolic aberrations have been defined in various disease processes either as contributing to the disease, as potential biomarkers, or as therapeutic targets. Thus in an attempt to define the metabolic alterations that may be associated with MS disease progression, we profiled the plasma metabolites at the chronic phase of disease utilizing relapsing remitting-experimental autoimmune encephalomyelitis (RR-EAE) model in SJL mice. At the chronic phase of the disease (day 45), untargeted global metabolomic profiling of plasma collected from EAE diseased SJL and healthy mice was performed, using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry. A total of 282 metabolites were identified, with significant changes observed in 44 metabolites (32 up-regulated and 12 down-regulated), that mapped to lipid, amino acid, nucleotide and xenobiotic metabolism and distinguished EAE from healthy group (p<0.05, false discovery rate (FDR)<0.23). Mapping the differential metabolite signature to their respective biochemical pathways using the Kyoto Encyclopedia of Genes and Genomics (KEGG) database, we found six major pathways that were significantly altered (containing concerted alterations) or impacted (containing alteration in key junctions). These included bile acid biosynthesis, taurine metabolism, tryptophan and histidine metabolism, linoleic acid and D-arginine metabolism pathways. Overall, this study identified a 44 metabolite signature drawn from various metabolic pathways which correlated well with severity of the EAE disease, suggesting that these metabolic changes could be exploited as (1) biomarkers for EAE/MS progression and (2) to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment of MS.

Multiresidue determination of quinolones regulated by the European Union in bovine and porcine plasma. Application of chromatographic and capillary electrophoretic methodologies.

This paper presents the multiresidue determination of the series of quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine) in bovine and porcine plasma using capillary electrophoresis and liquid chromatography with ultraviolet detection (CE-UV, LC-UV), liquid chromatography-mass spectrometry and -tandem mass spectrometry (LC-MS, LC-MS/MS) methods. These procedures involve a sample preparation by solid-phase extraction for clean-up and preconcentration of the analytes before their injection into the separation system. All methods give satisfactory results in terms of linearity, precision, accuracy and limits of quantification. The suitability of the methods to determine quinolones was evaluated by determining the concentration of enrofloxacin and ciprofloxacin in real samples from pig plasma and cow plasma.

Improved determination of quinolones in milk at their MRL levels using LC-UV, LC-FD, LC-MS and LC-MS/MS and validation in line with regulation 2002/657/EC.

This paper reports the multi-residue determination of quinolones included in the European Union regulations (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin and flumequine) in bovine milk, using liquid chromatography with ultraviolet (LC-UV), fluorescence (LC-FD), mass spectrometry (LC-MS) and tandem mass spectrometry detection (LC-MS/MS). The methods were validated, in line with EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit (CCalpha), detection capability (CCbeta), detection limit (LOD), quantification limits (LOQ), recoveries, precision, selectivity and stability were determined and adequate results were obtained. Recoveries higher than 80% were obtained for all quinolones. The methods developed were used to quantify enrofloxacin and its main metabolite, ciprofloxacin, in milk from animals treated with enrofloxacin.

Validation of a rapid micellar electrokinetic capillary chromatographic method for the simultaneous determination of isoniazid and pyridoxine hydrochloride in pharmaceutical formulation.

An efficient and reliable micellar electrokinetic capillary chromatography (MEKC) method has been developed for the simultaneous determination of isoniazid (ISO) and pyridoxine hydrochloride (PYR) in pharmaceutical formulations. A chemometric two level full factorial design approach was used to search for the optimum conditions of separation. Three parameters were selected for this study: the buffer pH, the buffer concentration and sodium dodecyl sulphate (SDS) concentrations. Resolution, peak symmetry and analysis time were established as response. The two analytes were separated within 6 min with the optimized conditions: 50 mM borate buffer, 25 mM SDS pH 7.8, 35 degrees C, at 50 mbar 4s injection and 30 kV by using a fused silica capillary (72 cm effective length, 50 microm i.d.). The detection wavelength was set to 205 nm. Meloxicam was used as internal standard. The method was validated with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity and robustness. The detection limits of the method were 1.0 microg mL(-1) for ISO and 0.40 microg mL(-1) for PYR and the method was linear at least in the range of 3.0-100 microg mL(-1) for ISO and 1.0-100 microg mL(-1) for PYR with excellent correlation coefficients (0.9995 for ISO and 0.9998 for PYR). Relative standard deviations (R.S.D.s) of the described method ranged between 0.54 and 2.27% for intra-day precision and between 0.65 and 2.69% for inter-day precision. The developed method was applied to the tablet form of ISO and PYR-containing the pharmaceutical preparations and the data were compared with obtained from the standard addition method. No statistically significant difference was found.

Determination of lornoxicam in pharmaceutical preparations by zero and first order derivative UV spectrophotometric methods.

Zero and first order derivative UV spectrophotometric methods were developed for the analysis of lornoxicam (LOR). The solutions of the standards and pharmaceutical samples were prepared in 0.05 N NaOH. Absorbances of LOR were measured at 376 nm for the zero order by measuring height of peak from zero and at 281 and 302 nm for the first order derivative spectrophotometric method by measuring peak to peak height. The linearity ranges were found to be 0.5-35 microg/mL for the zero order and 0.2-75 microg/mL for the first order derivative UV spectrophotometric method. The methods were validated and applied to the determination of LOR in pharmaceutical preparations (tablet and injectable, both containing 8 mg LOR). It was concluded that the methods developed were accurate, sensitive, precise, robust, rugged and useful for the quality control of LOR in pharmaceutical preparations.