Use of a Mixed Cationic-Reverse Phase Column for Analyzing Small Highly Polar Metabolic Markers in Biological Fluids for Multiclass LC-HRMS Method

The determination of small highly polar metabolites at low concentrations is challenging when reverse-phase (RP) chromatography is used for multiclass analysis. A mixed cationic-RP column coupled to high-resolution tandem MS (HR-MS/MS) was tested for highly polar compounds in biological fluids, i.e., trimethylamine N-oxide (TMAO) and the isobaric molecules beta-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). The efficient retention and separation of the above compounds were obtained with common and MS-friendly RP conditions, reaching high selectivity and sensitivity. The method was firstly assessed in plasma and urine, showing good linearity in the range 50–1000 µg/L and 500–10,000 µg/L for TMAO and both BMAA and DAB, respectively. Excellent precision (RDS < 3%) and good accuracies (71–85%) were observed except for BMAA in plasma, whose experimental conditions should be specifically optimized. Preliminary tests performed on compounds with biological relevance and a wider range of polarities proved the effectiveness of this chromatographic solution, allowing the simultaneous analysis of a larger panel of metabolites, from very small and polar compounds, like trimethylamine, to quite lipophilic molecules, such as corticosterone. The proposed LC-HRMS protocol is an excellent alternative to hydrophilic interaction liquid chromatography and ion-pairing RP chromatography, thus providing another friendly analytical tool for metabolomics.

Electromembrane Extraction of Highly Polar Compounds: Analysis of Cardiovascular Biomarkers in Plasma

Cardiovascular diseases (CVDs) represent a major concern in today’s society, with more than 17.5 million deaths reported annually worldwide. Recently, five metabolites related to the gut metabolism of phospholipids were identified as promising predictive biomarker candidates for CVD. Validation of those biomarker candidates is crucial for applications to the clinic, showing the need for high-throughput analysis of large numbers of samples. These five compounds, trimethylamine N-oxide (TMAO), choline, betaine, l-carnitine, and deoxy-l-carnitine (4-trimethylammoniobutanoic acid), are highly polar compounds and show poor retention on conventional reversed phase chromatography, which can lead to strong matrix effects when using mass spectrometry detection, especially when high-throughput analysis approaches are used with limited separation of analytes from interferences. In order to reduce the potential matrix effects, we propose a novel fast parallel electromembrane extraction (Pa-EME) method for the analysis of these metabolites in plasma samples. The evaluation of Pa-EME parameters was performed using multi segment injection–capillary electrophoresis–mass spectrometry (MSI-CE-MS). Recoveries up to 100% were achieved, with variability as low as 2%. Overall, this study highlights the necessity of protein precipitation prior to EME for the extraction of highly polar compounds. The developed Pa-EME method was evaluated in terms of concentration range and response function, as well as matrix effects using fast-LC-MS/MS. Finally, the developed workflow was compared to conventional sample pre-treatment, i.e., protein precipitation using methanol, and fast-LC-MS/MS. Data show very strong correlations between both workflows, highlighting the great potential of Pa-EME for high-throughput biological applications.

Polar Compounds in Oils and Their Aquatic Toxicity

Polar compounds as found in oils are hydrocarbon compounds containing nitrogen, sulphur or oxygen. Measurement of the presence of these compounds in oils can be carried out using sophisticated analysis techniques, however quantification and separation of compounds is very difficult and will remain a problem for many years to come. Characterization of polar compounds in oils is at a state of infancy and little polar analysis for many oils has been carried out to date. In order to measure the toxicity of a specific compound or class of compounds, separation is needed. Separation is very difficult and in many cases, beyond the scope of today's technology. An alternative has been to synthesize the compound of concern and then test its toxicity. This approach ignores the matrix in which the compound is usually present and the compound of interest may be not bioavailable when present in the actual oil, due to its solubility in oil. Highly polar compounds are likely not present in produced oils due to the polar compound's high water solubility. Compounds with moderate or less polarity are typically more soluble in oil than water. Similarly, highly polar compounds produced by biodegradation or photo oxidation would be diluted in water during a spill. The aquatic toxicity of polar compounds compared to aromatic compounds has been tested by using evaporative weathering. Aromatic compounds, particularly that of the 2 to 5 ring polyaromatic hydrocarbons (PAHs), are fairly well-established as the primary toxic component of oils. Polar compounds are soluble in water and thus may pose another source of toxicity. Evaporative weathering tests where photo oxidation is not involved, in which some of the low molecular weight compounds and PAHs are lost from the oil, are thought to be one test of the comparison of polar compound toxicity compared to that of the PAHs. These tests show that polar compounds are generally less-aquatically-toxic than the 2 to 5-ring PAHs. Another test that has been performed is that of physical separation of oil components. In these type of tests, polar compounds have again been shown to have less aquatic toxicity that the PAHs in the same oil. Both tests have obvious limitations in that there are many compounds involved.

Multi-residue Determination of Polar Veterinary Drugs in Livestock and Fishery Products by Liquid Chromatography/Tandem Mass Spectrometry

Residues of 37 polar veterinary drugs belonging to six families (quinolones, tetracyclines, macrolides, lincosamides, sulfonamides, and others) in livestock and fishery products were determined using a validated LC-MS/MS method. There were two key points insample preparation. First, extraction was performed with two solutions of different polarity. Highly polar compounds were initiallyextracted with Na2EDTA-McIlvaine's buffer (pH 7.0). Medium polar compounds were then extracted from the same samples with acetonitrile containing 0.1% formic acid. Secondly, cleanup was performed using a single SPE polymercartridge. The first extracted solution was applied to the cartridge. Highly polar compounds were retained on the cartridge. Then, the second extracted solution was applied to the same cartridge. Both highly and medium polar compounds were eluted from the cartridge. This method satisfied the guideline criteria for 37 out of 37 drugs in swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, milk, and honey; 35 out of 37 in egg; and 34 out of 37 in flounder. The LOQ ranged from 0.1 to 5 μg/kg. Residues were detected in 24 out of 110 samples and analyzed using the validated method.

Molecular characterization of hot spring cyanobacteria and evaluation of their photoprotective compounds

Phylogenetic analysis of 4 cyanobacterial strains isolated from hot springs in Rajgir, India, was carried out using the 16S rRNA gene (1400 bp). These strains were identified as members of Chroococcales ( Cyanothece sp. strain HKAR-1) and Nostocales ( Nostoc sp. strain HKAR-2, Scytonema sp. strain HKAR-3, and Rivularia sp. strain HKAR-4). Furthermore, we evaluated the presence of ultraviolet-screening and (or) photoprotective compounds, such as mycosporine-like amino acids (MAAs) and scytonemin, in these cyanobacteria by using high-performance liquid chromatography. Well-characterized MAAs, including the critical and highly polar compounds shinorine, porphyra-334, and mycosporine-glycine, as well as several unknown MAAs, were found in these hot-spring-inhabiting microorganisms. The presence of scytonemin was detected only in Scytonema sp. strain HKAR-3 and Rivularia sp. strain HKAR-4. The results indicate that hot spring cyanobacteria, namely Cyanothece, Nostoc, Scytonema, and Rivularia, belonging to different groups possess various photoprotective compounds to cope up with the negative impacts of damaging radiations.