Development and validation of a simple HPLC-MS/MS method for the quantification of methylmalonic acid in human serum without a derivatization step.

A simple and rapid HPLC-MS/MS analytical method was developed and validated for the determination of methylmalonic acid (MMA) in human serum without a derivatization step. Serum samples (200 µl) were pretreated using a simple method based on ultrafiltration using a VIVASPIN 500 ultrafiltration column. Chromatographic separation was achieved on a Luna Omega C18 column with a PS C18 precolumn guard by gradient elution using 0.1% (v/v) formic acid in water (mobile phase A) and 0.5% (v/v) formic acid in acetonitrile (mobile phase B) at a flow rate of 0.2 ml min-1 . The total run time of the analysis was 4.5 min. Negative electrospray ionization and multiple reaction monitoring mode were used. The lower limit of detection and lower limit of quantification for MMA were determined to be 13.6 and 42.3 nmol L-1 , respectively. The developed method enabled the quantification of MMA in a wide linear range of 42.3-4230 nmol L-1 with a correlation coefficient of 0.9991.

Bacillus amyloliquefaciens-Derived Lipopeptide Biosurfactants Inhibit Biofilm Formation and Expression of Biofilm-Related Genes of Staphylococcus aureus.

Biosurfactants (BSs) are surface-active compounds produced by diverse microorganisms, including the genus Bacillus. These bioactive compounds possess biological activities such as antiadhesive, antimicrobial and antibiofilm effects that can lead to important applications in combating many infections. Based on these findings, we decided to investigate the antibiofilm activity of BSs from the marine Bacillus amyloliquefaciens against Staphylococcus aureus CCM 4223. Expression of biofilm-related genes was also evaluated using qRT-PCR. Isolated and partially purified BSs were identified and characterized by molecular tools and by UHPLC-DAD and MALDI-TOF/MS. Bacillus amyloliquefaciens 3/22, that exhibited surfactant activity evaluated by oil spreading assay, was characterized using the 16S rRNA sequencing method. Screening by PCR detected the presence of the sfp, srfAA, fenD and ituD genes, suggesting production of the lipopeptides (LPs) surfactin, fengycin and iturin. The above findings were further supported by the results of UHPLC-DAD and MALDI-TOF/MS. As quantified by the crystal violet method, the LPs significantly (p < 0.001) reduced biofilm formation of S. aureus in a dose-dependent manner and decreased expression of biofilm-related genes fnbA, fnbB, sortaseA and icaADBC operon. Data from our investigation indicate a promising therapeutic application for LPs isolated from B. amyloliquefaciens toward prevention of S. aureus biofilm infections.

A new HPLC-MS/MS analytical method for quantification of tazobactam, piperacillin, and meropenem in human plasma.

A simple and fast high-performance liquid chromatography with tandem mass spectrometry method for quantification of tazobactam, piperacillin, and meropenem in human plasma has been developed and validated. Simple sample preparation with a volume of 10 µL was done by protein precipitation with a mixture of methanol-acetonitrile-water (6:2:2, v/v/v). Chromatographic separation was achieved on a Luna column with a precolumn security guard by gradient elution using a mobile phase consisting of water with the addition of 0.1% formic acid (component A) and mixture methanol-acetonitrile (8:2, v/v) with the addition of 0.1% formic acid (component B). The run time was 2.7 min. The lower limits of detection and lower limits of quantification were for piperacillin 0.03 and 0.1 mg/L, for meropenem 0.04 and 0.2 mg/L and for tazobactam 0.16 and 0.5 mg/L. The validated method was used for therapeutic monitoring of tazobactam, piperacillin, and meropenem in samples of patients treated in the intensive care unit.

A new HPLC-MS/MS method for simultaneous determination of Cyclosporine A, Tacrolimus, Sirolimus and Everolimus for routine therapeutic drug monitoring.

A rapid, simple and robust HPLC-MS/MS method for simultaneous determination of immunosuppressants Cyclosporine A, Tacrolimus, Sirolimus and Everolimus has been developed and validated. Sample of whole blood with volume of 50 µL was prepared by a protein precipitation with methanol and 0.5 mol. L-1 ZnSO4. Chromatographic separation was achieved on a Phenyl-Hexyl column by a gradient elution using 20 mmol.L-1 ammonium formate/0.1% (v/v) formic acid in water (mobile phase A) and 20 mmol.L-1 ammonium formate/0.1% (v/v) formic acid in methanol (mobile phase B) with flow rate 1 mL.min-1. The run time was 3.5 min. Electrospray ionization and multiple reaction monitoring was used. The lower limit of quantification (LLOQ) was set at 0.5 µg.L-1 for Tacrolimus, Sirolimus and Everolimus and 5 µg.L-1 for Cyclosporine A. The method demonstrated adequate accuracy and precision with sufficient linearity range.

Thin-Layer Chromatography: an Efficient Technique for the Optimization of Dispersive Liquid-Liquid Microextraction.

Thin-layer chromatography (TLC) is an often omitted analytical technique due to its lower sensitivity and separation capacity. Even in the era of high-performance liquid chromatography (HPLC), thin-layer chromatography still offers many advantages, such as simplicity, rapidity, and cost-effectiveness, which predict TLC to be the first-choice method for the laborious optimization process requiring analysis of numerous samples. In this work, a thin-layer chromatography method with chemical and densitometric detection was used to optimize a dispersive liquid-liquid microextraction (DLLME) process for the extraction and preconcentration of estradiol in human urine. The chromatographic system consisted of silica gel plates as the stationary phase and toluene-ethanol (9:1; v/v) mixture as the developing solvent. The plates were dyed with 10% phosphomolybdic acid reagent and sequentially evaluated densitometrically at λ = 430 nm. In the context of DLLME optimization, parameters including the type and volume of extraction and dispersive solvents, centrifugation, salt addition and extraction time, were studied. The proposed DLLME-TLC method was successfully applied to the determination of estradiol in real human urine samples.

Dispersive liquid-liquid microextraction as an effective preanalytical step for the determination of estradiol in human urine.

In this work, a fast and effective dispersive liquid-liquid microextraction was developed for the isolation and preconcentration of free 17 ß-estradiol, the main human estrogen, from real human urine samples. To optimize the extraction technique, few important parameters such as type and volume of extraction and dispersive solvents, centrifugation conditions, effect of salt addition, and extraction time were studied. Optimal conditions were obtained when injecting 600 µL mixture of tetrachloromethane as extraction solvent and ethanol as dispersive solvent (1:5, v/v) into 2 mL of urine containing 8% NaCl and following centrifugation at 10 000 rpm, thus reaching enrichment factor 28 and extraction recovery 98% for estradiol. Procedure was evaluated by means of high-performance liquid chromatography with UV detection (λ = 280 nm) using a C-18 column and methanol/water (60:40, v/v) as the mobile phase. The method was linear within the concentration range 1.0-250.0 mg/L (r = 0.9997) and provided a limit of detection of 0.25 mg/L. The proposed method was applied to the determination of free estradiol in real human pregnancy urine.

Thin-layer chromatography analysis of fructooligosaccharides in biological samples.

This study presents the application of a rapid, simple and inexpensive thin-layer chromatography (TLC) for the analysis of fructooligosaccharides (FOSs) as feed additives (prebiotics) in complicated biological samples with minimal pre-treatment. Prebiotics have been monitored in different parts of the intestinal tract (jejunum, ileum and colon) of monogastric animals to which commercially available dietetic products containing fructooligosaccharides Raftifeed IPX, Raftilose and polysaccharide maltodextrin have been added into the feed. Thereby it contributes to a clarification of fructooligosaccharides and polysaccharides transformation in the digestive system. Chromatographic separation has been studied on different chromatographic systems (stationary and mobile phases). Optimal separation of fructooligosaccharides in dietetic products as well as in the samples from intestinal tract of monogastric animals has been achieved on glass-backed precoated silica gel layers impregnated with sodium acetate. The layers were developed with butanol-ethanol-water (5:3:2, v/v) in a vertical trough glass chamber with mobile phase vapour saturation. The visualisation of FOSs on chromatograms was performed with mixture of diphenylamine-aniline-phosphoric acid in acetone as primary detection reagent. Coloured spots of FOSs (blue-pink spots) on chromatograms have also been detected by reflectance densitometry at wavelength lambda=370nm. Simultaneously, the concentration of acetic acid, which is one of FOSs fermentation product, was monitored in the intestinal contents from jejunum, ileum and colon by capillary isotachophoresis.