Quantitative profiling of bile acids in blood, adipose tissue, intestine, and gall bladder samples using ultra high performance liquid chromatography-tandem mass spectrometry.

An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed for the determination of 33 target and 28 unknown bile acids (BAs) in biological samples. Sixty-one BAs could be measured in 20 min using only a small amount of sample and with a simple sample preparation. The method proved to be very sensitive (limit of detection 5-350 pg/mL, lower limit of quantitation 0.1-2.6 ng/mL), linear (R(2) > 0.99) and reproducible (typically CV <15 % in biological matrixes). The method was used to analyze human adipose tissue, plasma, and serum (from same subjects) and mouse serum, gall bladder, small intestine, and colon samples (from same animals). Cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, deoxycholic acid, and their conjugates (mainly glycine, but also taurine conjugates) were the main metabolites in human samples, and cholic acid, glycine cholic acid, and several taurine conjugates in mouse samples. Using the method, 28 unknown BAs could also be detected. UHPLC-MS/MS spectra, accurate mass, and tissue distribution suggested that nine of the unknown bile acids were taurine conjugates, 13 were glycine conjugates, and six were intact BAs, respectively. To our knowledge, this was the first time BAs were detected in adipose tissue. Results showed that 17 targeted BAs were found at ng/g level in human adipose tissue. Our findings give a novel insight of the endogenous role of BAs in adipose tissue and their role as biomarkers (e.g., in metabolic diseases).

On-line coupled dynamic sonication-assisted extraction-liquid chromatography for the determination of phenolic acids in Lamiaceae herbs.

A fast and efficient on-line coupled dynamic sonication-assisted extraction-liquid chromatography (DSAE-LC) method was developed for the determination of phenolic acids in basil, oregano, rosemary, sage, spearmint and thyme. The extraction and chromatography were coupled via a solid-phase trap filled with strong anion exchange material. The relative standard deviations (RSDs) for the retention times were less than 0.4% and those for the peak heights less than 3% except for gallic acid (RSD 1.2% for the retention times and 11% for the peak heights). Limits of detection were below approximately 3ng.

Comprehensive two-dimensional liquid chromatography in the analysis of antioxidant phenolic compounds in wines and juices.

A novel comprehensive two-dimensional liquid chromatographic (LCxLC) system was developed for the quantification of antioxidant phenolic compounds in wine and juice. The system allows parts of the sample that are well separated in the first column to pass directly to the detector after the first column, while the rest of the sample proceeds to the second column. The optimised LCxLC system employed a combination of two C18 columns, the latter column with an ion-pair reagent (tetrapentylammonium bromide). The relative standard deviations (RSD) for the retention times were better than 0.01% in the first dimension and on average 6.3% in the second. The RSD values of the peak volumes varied from 3% (protocatechuic acid) to 13% (caffeic acid, n = 3, 10 microg/ml).

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages.

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.

Comprehensive two-dimensional liquid chromatography in analysis of Lamiaceae herbs: characterisation and quantification of antioxidant phenolic acids.

A fast and effective dynamic sonication assisted ethanol extraction method was developed for extracting phenolic acids from basil, oregano, rosemary, sage, spearmint and thyme of the Lamiaceae family. The results were compared with results obtained by conventional solvent extraction techniques. A comprehensive two-dimensional liquid chromatography (LC x LC) system interfaced to electrospray ionisation time-of-flight (TOF) mass spectrometry was then optimised for analysis and quantification of the herb extracts. The optimised LC x LC system employed a combination of C18 and cyano columns. The relative standard deviations for the retention times were better than 0.05% (rosmarinic acid 0.1%) and those for the peak areas 2-14% (2 mg/l, n=3). Limits of detection were 18-90 ng/ml. The LC x LC-MS method was applied to the quantitative analysis of phenolic acids, and the results were compared with those obtained with conventional LC-MS.

Rapid quantitative analysis of carnitine and acylcarnitines by ultra-high performance-hydrophilic interaction liquid chromatography-tandem mass spectrometry.

l-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance-hydrophilic interaction liquid chromatography-tandem mass spectrometry (UHP-HILIC-MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600ng/ml for carnitine and acetylcarnitine and 5-200ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5ng/ml for carnitine and acetylcarnitine and approximately 0.5ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.

Polyethylenimine-modified metal oxides for fabrication of packed capillary columns for capillary electrochromatography and capillary liquid chromatography.

The need for novel packing materials in both capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) is apparent and the development towards more selective, application-oriented chromatographic phases is under progress world-wide. In this study we have synthesized new polyethyleneimine (PEI) functionalized Mn(2)O(3), SiO(2), SnO(2), and ZrO(2) particles for the fabrication of packed capillary columns for CEC and CLC. The nanocasting approach was successful for the preparation of functionalized metal oxide materials with a controlled porosity and morphology. PEI functionalization was done using ethyleneimine monomers to create particles which are positively charged in aqueous solution below pH 9. This functionalization allowed the possibility to have both hydrophobic (due to its alkyl chain) and ionic interactions (due to positively charged amino groups) with selected compounds. For comparison aminopropyl-functionalized silica was also synthesized and tested. Both slurry pressure and electrokinetic packing procedures used gave similar results, but fast sedimentation of the material caused some problems during the packing. The high stability and wide pH range of PEI-functionalized SiO(2) material, with potential for hydrophobic and electrostatic interactions, proved to be useful for the CEC and CLC separation of some model acidic and neutral compounds.

Particle-into-liquid sampler on-line coupled with solid-phase extraction-liquid chromatography-mass spectrometry for the determination of organic acids in atmospheric aerosols.

In the present study, sample collection and preparation were directly integrated with a chromatographic system by coupling a particle-into-liquid sampler for the first time on-line with solid-phase extraction-liquid chromatography-tandem mass spectrometry. Several organic acids, such as adipic, hydroxyglutaric, mandelic, vanillic, cis-pinonic, pinic, azelaic and sebacic, were used in the research. For sample pretreatment and concentration, strong anion exchange material was used in the extraction. Sampling, extraction and analysis conditions were optimized to obtain reliable information about aerosol chemical composition. To evaluate the performance of the on-line coupled system, half of each sample was analysed on-line and the other half was derivatized and analysed off-line by gas chromatography-mass spectrometry. Comparison of the two techniques with use of t-test showed the results to be in an excellent agreement. Limits of detection of studied acids in on-line system were between 0.1 and 0.9 ng. The on-line coupled system is fast and reliable and a promising tool for the real time analysis of organic acids in atmospheric aerosols.

Data analysis programs for comprehensive two-dimensional chromatography.

User-friendly and easy-to-use laboratory-written programs for visualisation and interpretation of comprehensive two-dimensional chromatographic data were developed. The programs that are not tied to any particular commercial instrument, and data obtained either by comprehensive two-dimensional liquid (LC x LC) or gas (GC x GC) chromatography can be analysed. Operations of the programs allow visualisation of 2D and 3D plots, comparison of two 2D plots at a time, as well as determination of retention times and peak heights and volumes.

Comparison of GC-MS and LC-MS methods for the analysis of antioxidant phenolic acids in herbs.

Two methods were developed for the quantitative analysis of phenolic acids in herb extracts. The methods were based on liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) and gas chromatography-mass spectrometry (GC-MS). The methods were compared in terms of their linearity, repeatability, selectivity, sensitivity and the speed of the analysis. The sensitivity was good for both methods, with limits of detection of <80 ng/ml for most of the compounds. The relative standard deviations (RSD) of the peak areas were on average 7.2% for the LC-TOFMS method and 1.4% for the GC-MS method. Both methods were found to be suitable for the determination of the target analytes, although GC-MS was better suited to the quantitative determination of compounds present at low concentrations.