Two Original Experimental Setups for Staircase Frontal Affinity Chromatography at the Miniaturized Scale.

Frontal affinity chromatography is a powerful, underappreciated technique for the qualitative (screening) and quantitative (Kd determination) evaluation of biological interactions. Its development has been previously hampered by its sample consumption, limited throughput, and lack of dedicated instrumentation especially at a miniaturized scale. This work describes two original experimental devices allowing nano-frontal affinity chromatography titrations (nano-FAC) to be automatically implemented in the time-saving staircase mode. The first nano-FAC system utilizes a capillary electrophoresis device (7100 CE Agilent system) in the pressurization mode with in situ UV detection. The second nano-FAC experimental setup implements a nano-LC device (Ultimate 3000 Thermo) modified with a 10-port valve equipped with two superloops (loop volume, 5 µL) operating alternatively and automatically in a single run. The benefits and drawbacks of each approach are exemplified using two model protein-ligand interactions (concanavalin A-mannose and concanavalin A-glucose). The two methods result in concordant dissociation constants (Kd) and number of active site (Bact) values, obtained in a fully automated manner, with low sample consumption and good throughput.

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma.

Here we describe a new HPLC-MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds. Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.

Quantitative analysis of the tumor suppressor dendrogenin A using liquid chromatography tandem mass spectrometry.

Dendrogenin A (DDA) was recently identified as a mammalian cholesterol metabolite that displays tumor suppressor and neurostimulating properties at low doses. In breast tumors, DDA levels were found to be decreased compared to normal tissues, evidencing a metabolic deregulation of DDA production in cancers. DDA is an amino-oxysterol that contains three protonatable nitrogen atoms. This makes it physico-chemically different from other oxysterols and it therefore requires specific analytical methods We have previously used a two-step method for the quantification of DDA in biological samples: 1) DDA purification from a Bligh and Dyer extract by RP-HPLC using a 250×4.6mm column, followed by 2) nano-electrospray ionization mass spectrometry (MS) fragmentation to analyze the HPLC fraction of interest. We report here the development a liquid chromatography tandem mass spectrometry method for the analysis of DDA and its analogues. This new method is fast (10min), resolving (peak width <4s) and has a weak carryover (<0.01%). We show that this technique efficiently separates DDA from its C17 isomer and other steroidal alkaloids from the same family establishing a proof of concept for the analysis of this family of amino-oxysterols.