Analysis of cyanobacterial toxins by hydrophilic interaction liquid chromatography-mass spectrometry

The combination of hydrophilic interaction liquid chromatography with electrospray mass spectrometry (HILIC-MS) has been investigated as a tool for the analysis of assorted toxins produced by cyanobacteria. Toxins examined included saxitoxin and its various analogues (1-18), anatoxin-a (ATX-a, 19), cylindrospermopsin (CYN, 20), deoxycylindrospermopsin (doCYN, 21), and microcystins-LR (22) and -RR (23). The saxitoxins could be unequivocally detected in one isocratic analysis using a TSK gel Amide-80 column eluted with 65% B, where eluent A is water and B is a 95% acetonitrile/water solution, both containing 2.0 mM ammonium formate and 3.6 mM formic acid. The analysis of ATX-a, CYN and doCYN required 75% B isocratic. Simultaneous determination of 1-21 was also possible by using gradient elution. HILIC proved to be suitable for the analysis of microcystins, but peak shape was not symmetric and it was concluded that these compounds are best analysed using existing reversed-phase methods. The HILIC-MS method was applied to the analysis of field and cultured samples of Anabaena circinalis and Cylindrospermopsis raciborskii. In general, the method proved quite robust with similar results obtained in two different laboratories using different instrumentation.     

Comparison of combustion sources for sulfur chemiluminescence detection

Three different types of SCD combustion source have been evaluated for use in the chromatographic analysis of atmospheric sulfur compounds. The conventional FID source and the newer inverted burner source were found to be less sensitive and less stable than the flameless design. Overall, the flameless source was superior for use with HRGC-SCD.     

Analysis of Chloramphenicol Residues in Honey by Liquid Chromatography–Tandem Mass Spectrometry

Antibiotics are often used in bee-keeping to control European and American foulbrood. The broad spectrum antibiotic chloramphenicol (CAP) was used for curative purposes in veterinary medicine, but is now forbidden in numerous countries, although still used in south-east Asia. A liquid-chromatographic method with tandem mass spectrometric detection (LC–MS–MS) has been developed for analysis of sub-g kg^–1 residues of chloramphenicol in honey. Results from full validation of the procedure and analysis of 75 honey samples obtained commercially in Switzerland are presented. These show the method is satisfactory and useful for monitoring chloramphenicol residues in honey.     

Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry

In contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine beta-casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine beta-casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1-1.0%) to the sample solution; a 10-fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C(18)-bonded stationary phase silica surface.     

Effect of nature of support and impregnating agent on lipophilicity determination for nonionic surfactants by reversed-phase thin-layer chromatography

Summary The retention of 9 nonylphenyl ethylene oxide oligomers was determined in 15 reversed-phase chromatographic systems using silicone oils of various molecular mass as impregnating agents and silica, cellulose and alumina supports. The data were evaluated with principal component analysis carried out on the covariance and the correlation matrices. The R_M values did not follow the additivity rule and they did not change linearly with increasing length of the ethylene oxide chain. This is probably due to the folded state of the chain in the eluent. The molecular mass of the silicone oils had a negligible effect on the retention whereas the retention increased with increasing level of impregnation. The support considerably influenced the lipophilicity values of these nonionic surfactants and cellulose seemed to be the most appropriate support. Calculations proved that the application of a correlation matrix may cause data distortion; therefore, the use of a covariance matrix is strongly proposed.     

Concurrent adsorption and complex formation in reversed-phase liquid-liquid chromatography with tri-n-octylphosphine oxide

Summary Reversed-phase liquid-liquid chromatographic systems consisting of an aqueous mobile phase and an organic liquid stationary phase of the proton acceptor tri-n-octylphosphine oxide (TOPO) inn-decane, coated on LiChrosorb RP-8, have been studied. The solutes were hydrophilic aromatic carboxylic acids and phenol. The retention of the carboxylic acids shows a minimum at 10 mM of TOPO, whereas increasingly tailing peaks have been obtained with decreasing concentrations of TOPO. This behaviour is due to a concurrent complex formation by hydrogen bonding with TOPO in the liquid stationary phase and adsorption at the interface between the support and the liquid stationary phase. The adsorption of TOPO, ketones and aromatic acids from hexane on Li-Chrosorb RP-8 has been studied, and seems to be due to residual silanol groups. The adsorption isotherm of TOPO has been determined and can be described by a two-site Langmuir adsorption model. Non polar solutes are not adsorbed. The influence of TOPO on the retention and the peak symmetry of carboxylic acids in the liquid-liquid chromatographic system appears to be due to a competition between TOPO and the acids for the same adsorption sites. No competition was found for phenol.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Determination of the enantiomeric composition of chiral carboxylic acids using chiral derivatization and HPLC

Summary The enantiomers of chiral carboxylic acids were separated as their diastereomeric amides with (1R,2R)-(−)-1-(4-nitrophenyl)-2-amino-1,3-propanediol (“levobase”) and with “dextrobase” (the enantiomer of levobase) by high-performance liquid chromatography using a conventional C-18 column and various solvent systems containing acetonitrile, methanol, water, and phosphoric acid.     

LC Determination of 4-Bromoaniline in Green Algae Chlamydomonas reinhardtii After Continuous-Flow Microextraction

In this study, the determination of 4-Bromoaniline (4-BA) in green algae Chlamydomonas reinhardtii (C. reinhardtii) was investigated by applying continuous-flow microextraction (CFME) combined with high-performance liquid chromatography (HPLC). Continuous-flow microextraction was conducted in a homemade glass chamber, i.e. the sample solution flowed through a constant volume drop of solvent in the chamber at a constant flow rate. The effects of different factors on extraction efficiencies were also investigated and these factors included the kind of extraction solvent, solvent drop volume, sample flow rate, extraction time and addition amount of salt. Under the optimum extraction conditions (extraction solvent, carbon tetrachloride; solvent drop volume, 3.5 μL; sample flow rate, 1.0 mL min⁻¹; extraction time, 10 min; no addition of salt), the calibration plot was set up by plotting peak area against a series of 4-Bromoaniline concentrations (0.01–10 μg mL⁻¹) in aqueous solution. The correlation coefficient (r) was 0.9990. The limit of detection (LOD) was 0.6 ng mL⁻¹. The precision of this method was obtained by successive five time analyses of 100-ng mL⁻¹ standard solution of 4-Bromoaniline, and the relative standard deviation (RSD) was 3.5%. The concentration factor was calculated by the ratio of peak area of the analyte obtained after and before extraction and found to be 10.6. 4-Bromoaniline residues in Chlamydomonas. reinhardtii cells and tap water samples were satisfactorily analyzed according to the method described above.     

Dominant Factors Affecting the Chromatographic Behaviour of Bile Acids

With the aim of optimizing the chromatographic process by avoiding any preliminary derivatizing step, we examined the chromatographic behaviour of a selected set of unconjugated bile acids looking at the dominant factors that affect the performances of three different stationary phases: RP-8, RP-18 and RP-18 Base Deactivated (RP-18-BD). Accordingly to its structural peculiarity, the RP-18-BD column combined with a specific mobile phase has proved to be the most suitable one, in enhancing both separation factor α and resolution R _S within the selected set of analytes. Pronounced changes in the chromatographic profiles by only slightly changing the mobile phase composition (pH, buffer concentration, percentage and kind of organic modifier) prompted us to achieve satisfactory results in the separation and resolution of the selected set of bile acids.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th symposium on the practical applications for the analysis of proteins, nucleotides and small molecules, Montreal, Canada, August 12–16, 2005.An erratum to this article can be found at     

Concentration and characterization of dilute colloidal samples by potential-barrier field-flow fractionation

Summary Potential-barrier field-flow fractionation, which is a combination of potential-barrier chromatography and sedimentation field-flow fractionation, is shown to be a convenient and accurate method for the concentration and analysis (separation and characterization) ofdilute colloidal samples. Two sizes (0.158 and 0.271 μm) of haematite (α-Fe₂O₃) monodisperse colloidal samples diluted in volumes of up to 20 cm³ are used as model colloids. The particle diameters found by the present concentration procedure under various experimental conditions are in good agreement with those determined by conventional sedimentation field-flow fractionation, in which a small concentrated sample volume was injected directly into the column.