Mass cytometry (CyTOF), a mass spectrometry-based single cell phenotyping technology, allows utilization of over 35 antibodies in a single sample and is a promising tool for translational human immunology studies. Although several analysis tools are available to interpret the complex data sets generated, a robust method for standardization and quality control within and across studies is needed. Here we report an efficient and easily adaptable method to monitor quality of individual samples in human immunology studies and to facilitate reproducible data analysis. Samples to be assessed are spiked with a defined amount of reference peripheral blood mononuclear cells from a healthy donor, derived from a single large blood draw. The presence of known standardized numbers and phenotypic profiles of these reference cells greatly facilitates sample analysis by allowing for: 1) quality control for consistent staining of each antibody in the panel, 2) identification of potential batch effects, and 3) implementation of a robust gating strategy. We demonstrate the utility of this method using peripheral blood and bronchoalveolar lavage samples from HIV+ patients by characterizing their CD8+ T-cell phenotypes and cytokine expression, respectively. Our results indicate that this method allows quality control of experimental conditions and results in highly reproducible population frequencies through a robust gating strategy.

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Mobile phase variables have a deep influence on the chromatographic behavior with polysaccharide-based chiral stationary phases. Basic additives are generally used to minimize peak broadening arising from unwanted interactions between polar solutes and underivatized silanols. However, basic additives can improve enantioselectivity through disruption of hydrogen bonds and modification of the polymer morphology. Acidic additives are incorporated into the mobile phase during the analysis of acidic compounds as efficiency enhancers. Acidic additives can also improve enantioselectivity by minimizing within the chiral recognition site nonenantioselective retention. Peak shape without acidic additive in the eluent could be severely distorted during the analysis of salified compounds. Concentration and type of alcohol modifier can have an effect on the morphology of the polymer. The different winding of the chiral selector, caused by alcohol modifiers of different size/shape, ultimately results in different stereo environment of the chiral cavities in the polymer chain. Trace amounts of water in normal-phase eluents can affect retention time, tailing, and resolution. Deliberate addition of water to the eluent can improve peak resolution and save analysis time and solvent needs. Immobilized-type polysaccharide-derived chiral stationary phases offer new selectivity profiles and often improved enantioselectivity.

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The presence of D-amino acids (D-AAs) as a consequence of natural or artificial interventions such as ageing, microorganism action, preservative and conservative processes (alkali or heat treatment), is a scarcely treated aspect from the scientific community. It is also fully documented that even a minor degree of racemisation on the proteins' AAs is the cause of a reduced digestion of such proteins. Besides interfering with the regular metabolism of L-AAs, D-AAs can also contribute to the development of pathological conditions in humans. So far, nearly all the most important chromatographic techniques were applied to quantify D-AAs in foodstuffs. However, most of them rely upon pre-or post-column derivatization procedures, often combined with sophisticated analytical equipments. Differently, in this paper we propose an easy-to-set up combination of monodimensional chromatographic methods to monitor the variation of the D-Ala, D-Asp and D-Glu content in two commercially available Spanish cheese samples prepared from the same milk mixture and characterized by a different maturity time: no ripening and six months ripening. After the free amino acid mixture was extracted from the two cheese samples, an ion-pairing RP-HPLC achiral protocol was firstly optimized with the objective to avail of a method enabling the complete distinction of Ala, Asp, and Glu from all the other aminoacidic species in the two extracts. An ionexchange-based chromatographic method was also optimized, thus allowing a profitable fractionation of the two aminoacidic mixtures. With such a procedure, less complex samples to be analyzed with a chiral ligand-exchange chromatography (CLEC) stationary phase based on S-trityl-L-cysteine (L-STC) units were obtained. The optimized CLEC conditions were then applied to the previously identified Ala, Asp and Glu containing fractions as well as to those including all the remaining species. For all the three compounds the enantiomeric excess (ee) was found to decrease passing from the ripened to the fresh cheese. As expected, the largest difference was found for Ala (ee value from 83.0% down to 20.5%), followed progressively by Asp (ee value from 90.5 to 75.0%) and Glu (ee value from 99.0 to 91.8%).

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In this study, we were interested in comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: the Cannara (Umbria region) and Imola (Emilia Romagna region) sites. Onions were cultivated in a comparable manner, mostly in terms of the mineral fertilization, seeding, and harvesting stages, as well as good weed control. Furthermore, in both regions, the plants were irrigated by the water sprinkler method and subjected to similar temperature and weather conditions. A further group of Cannara onions that were grown by micro-irrigation was also evaluated. After the extraction of the free amino acid mixture, an ion-pairing reversed-phase high performance liquid chromatography-evaporative light scattering detector (IP-RP HPLC-ELSD) method allowed for the separation and detection of almost all the standard proteinogenic amino acids. However, only the peaks corresponding to leucine (Leu), phenylalanine (Phe), and tryptophan (Trp), were present in all the investigated samples and they were unaffected from the matrix interfering peaks. The use of the beeswarm/box plots revealed that the content of Leu and Phe were markedly influenced by the geographical origin of the onions (with *** p << 0.001 for Phe), but not by the irrigation procedure. The applied HPLC method was validated in terms of the specificity, the linearity (a logarithm transformation was applied for the method linearization), the limit of detection (LOD) and limit of quantification (LOQ), the accuracy (≥90% for inter-day Recovery percentage), and the precision (≤10.51 for the inter-day RSD percentage), before the quantitative assay of Leu, Phe, and Trp in the onion samples. These preliminary findings are a good starting point for considering the quantity of the specific amino acids in the Rossa da inverno sel. Rojo Duro variety as a fingerprint of its geographical origin.

With the present contribution, we demonstrate that the baseline separation of ketoprofen enantiomers can be successfully achieved (α = 1.09; R(S) = 1.60) in the reversed-phase mode of elution with a commercially available anion-exchange-based chiral stationary phase, incorporating the quinine 2,6-diisopropylphenyl carbamate derivative as the enantioresolving unit. Focused modification of the eluent composition indicated a stereoselective role of hydrophobic and π-π interactions between the selector and selectand units, besides the prime ionic intermolecular interaction. The mechanistic hypotheses based on the chromatographic data were confirmed by in silico molecular dynamic simulations, which allowed us to establish the network of selector-selectand interactions underlying the stereorecognition process at a molecular level. The validated method was successfully used to evaluate the drug content and release profile of ketoprofen-loaded polymeric film, showing drug homogeneous distribution into the film and no preferential interactions between the polymer and one of the enantiomers, with the racemate released at each time point.