Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacifica algae: a chemometric approach

An experimental design procedure was used to investigate the effects of some operating parameters on the supercritical fluid extraction of carotenoids beta-carotene, beta-cryptoxanthin and zeaxanthin from Spirulina Pacifica algae, a carotenoid-rich dietary product. Variables tested were temperature and pressure of the supercritical fluid, dynamic extraction time and percentage of ethanol added as the modifier. Each variable was tested at three levels; 31 experiments were performed in random order. Analyses of the extracts were performed by high-performance liquid chromatography with UV-Vis photodiode array detection. Analytical responses (chromatographic peak areas) were processed by using a stepwise multiple regression analysis, in order to find polynomial functions describing the relationships between variables and responses. For all the analytes the experimental conditions providing the highest extraction yield inside the experimental domain considered were found. Supercritical fluid extraction results obtained in these conditions were compared with those obtained by performing solvent extraction in order to evaluate the effectiveness of the supercritical fluid extraction procedure.     

Determination of phylloquinone (vitamin K1) by high performance liquid chromatography with UV detection and with particle beam-mass spectrometry

A reversed-phase PB LC-MS method was used for the determination and unequivocal identification of vitamin K(1) in some vegetable samples. The method was also applied to the identification of other constituents in the food analyzed. LC-MS experiments were carried out in both EI and negative-ion CI modes. Preliminary sensitivity studies on vitamin K(1) showed a detection limit of 2 ng (signal-to-noise ratio=3) in negative-ion operation and in single-ion monitoring at m/z 450. Quantitative results obtained with LC-MS for the extracts of four vegetables were compared with those of LC-UV analyses performed at 247 nm.     

Development and validation of a method using on-line solid-phase extraction and liquid chromatography with ultraviolet detection for the determination of bisphenol A, octylphenol, and nonylphenol in groundwater

Alkylphenols are nonbiodegradable metabolites arising from various pathways of aerobic and anaerobic degradation of the nonionic surfactants alkylphenol ethoxylates. A method based on the use of on-line solid-phase extraction (SPE) and liquid chromatography (LC) with UV detection was developed to determine bisphenol A, octylphenol, and nonylphenol at trace levels in water. The on-line coupled SPE procedure automatically enables an approximately 300-fold preconcentration of analytes, which can be further enhanced by an increase in applied sample volume. By using C18 cartridges, recoveries of >90% were obtained for all the analytes. A validation procedure was carried out with a groundwater sample to ensure the quality of the results; performance criteria such as detection limits (LODs), quantitation limits (LOQs), linearity, and precision were evaluated. LODs and LOQs in the range of 0.17-0.36 and 0.35-1.88 microg/L, respectively, were found; for all the analytes, linearity was established over 2 orders of magnitude (r2 >0.997, n = 54). The intraday repeatability values expressed as relative standard deviations were <5.3%; a nested analysis of variance was performed to verify the influence of 3 different factors, i.e., different days, extraction procedure, and LC replicate injection, on data precision.     

Development and in-house validation of a liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli products

An accurate method based on the use of reversed-phase (RP) liquid chromatography-tandem mass spectrometry interfaced with electrospray (LC-ESI-MS/MS) was devised for the determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli food samples. A simple sample treatment procedure entailing the use of an extraction step with acetone without clean-up was developed. A C18 column with an aqueous formic acid/methanol mixture as the mobile phase was used under isocratic conditions. Mass spectral acquisition was done in positive ion mode by applying selected reaction monitoring of three fragmentation transitions per compound to provide a high degree of selectivity. The method was in-house validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, sensitivity, accuracy, recovery, and selectivity on two kinds of hot chilli sauces. Good results in the low ng/g level were obtained for LOD and LOQ of all analytes in matrices. Under both intra-day repeatability (R.S.D. between 1 and 13%) and intermediate precision (about 5-15% R.S.D. for both chilli sauce matrices) conditions, precision proved to be typical of determinations based on electrospray LC-MS and acceptable for routine monitoring purposes. Extraction recoveries for all four azo-dyes in chilli tomato sauce ranged from 92 to 103% at a spiking level of 5 microg/kg, whereas values between 72 and 97% were calculated in chilli tomato and cheese sauce at the same concentration level. The applicability of the method to the determination of Sudan azo-dyes in hot chilli products was demonstrated.     

Liquid—liquid extraction and determination of uranium(IV) with 2,6-diacetylpyridine bis(benzoylhydrazone)

2,6-Diacetylpyridine bis(benzoylhydrazone) (H₂DPBH) is proposed as a ligand for the extraction of uranium(VI). Complete extraction from aqueous solutions into dichloromethane is achieved with a ligand/metal mole ratio of < for 10^?5?10^?4 M uranyl ion. Potentiometric measurements indicate that the extracted species is UO₂ (DPBH). Uranium can be determined in the extract by spectrophotometric measurements at 420 nm and by differentail pulse polarography (E_p = ?0.67 V) with tetraethylammonium bromide as supporting electrolyte. For both methods, the detection limit is about 2 × 10^?6 M in the extract.     

Magnetic particles functionalized with PAMAM-dendrimers and antibodies: a new system for an ELISA method able to detect Ara h3/4 peanut allergen in foods

An innovative enzyme-linked immunosorbent assay (ELISA) format based on antibody-coated magnetic micro-particles (MPs) for the sensitive detection of Ara h3/4 allergen in food is described. The immunosupport is suspended in the incubation solutions and the MPs with the captured allergen can be easily harvested on a magnet, separated from the solutions, and washed using an easy-to-use, fast and selective approach that allows its detection and quantification. Two differently coated MPs, ProteinA-Pn-b and MP-NH₂-PAMAM G 1.5 -Pn-b immunosupports, were tested. The functionalization of the MPs with PAMAM-sodium carboxylate dendrimers elicits a major stability on the immunoglobulin activity resulting in a threefold enhancement of the analytical sensitivity for the assay with respect to a ProteinA immobilization. Validation was carried out on two different matrices: corn flakes and biscuits. In the case of MP-NH₂-PAMAM G 1.5 -Pn-b immunosupport, limit of detection was found to be 0.2 mg peanuts/kg matrix in both matrices; the linear response range was demonstrated from 2.5 to 15 mg peanuts/kg matrix by performing statistical tests (homoscedasticity and Mandel fitting tests). Good accuracy and recovery (>80 ± 2%) were obtained. Different food samples were tested and the results were compared with those obtained with a commercially available ELISA kit. The results obtained in this work demonstrated the applicability of the immunomagnetic ELISA methods on real samples and the possibility to perform the assay with significantly reduced reagent and sample consumption.     

Identification of in vivo nitrosylated phytochelatins in Arabidopsis thaliana cells by liquid chromatography-direct electrospray-linear ion trap-mass spectrometry

Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the –NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PC_s was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC₂, PC₃ and PC₄ resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS² experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule.     

Particle-packed column versus silica-based monolithic column for liquid chromatography-electrospray-linear ion trap-tandem mass spectrometry multiallergen trace analysis in foods

A bicarbonate buffer-based extraction method for the simultaneous analysis of five nut allergens (Ana o 2, cashew-nut; Cor a 9, hazelnut; Pru 1, almond; Ara h3/4, peanut; Jug r 4, walnut) in cereals and biscuits using liquid chromatography-electrospray-linear ion trap-tandem mass spectrometry (LC-ESI-LIT-MS(2)) was developed and validated. The method was based on our earlier published LC-MS(2)-based method in a research frame aimed at the identification and determination of hidden allergens in foods by using selective biomarker peptides. A C18 particle-packed column and a silica-based C18 monolithic column were compared in terms of chromatographic performances, such as peak shape, resolution, analysis time and selectivity. The C18 particle-packed column exhibited better performances and was further used for method development and validation. By operating under MS(2) selected reaction monitoring (SRM) acquisition mode, linearity, limits of detection (LOD) and quantitation, trueness and precision were evaluated on breakfast samples enriched with a mix of the five nuts. Good linearity of the matrix matched-calibration curves was obtained and detection limit values generally varied from 14 to 55 mg nut/kg matrix. Recoveries were in the 76±4% to 94±3% range with RSD <15%. The capabilities of LIT to perform MS(n) fragmentation was exploited to improve selectivity of the analysis, and the LC-(SRM) MS(2) method was compared in terms of LOD, linearity, precision and accuracy with a LC-(SRM) MS(3) method. Finally, both the LC-MS(2) and LC-MS(3) methods were successfully applied to the analysis of nut traces in commercially available breakfast cereals and biscuits.     

Trends in analytical atomic and molecular mass spectrometry in biology and the life sciences

Trends in analytical atomic and molecular mass spectrometry (MS) are reviewed here with an emphasis on problems related to biology and the life sciences. The move towards novel desorption/ionization techniques including matrix-free laser desorption-ionization MS and recent technological developments in MS imaging of elements, small molecules, and proteins are discussed. Recent developments in the interfacing of microfluidics with MS are also reviewed. Metabolic profiling, evaluation of biomarkers, pharmaceutical metabolite identification, and many related topics are addressed.