Frontal and Zonal Affinity Chromatography Coupled to Mass Spectrometry

Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

“…The employed detector depends on the complexity and concentration of the infused sample and could include a radioflow, UV or fluorescence detector, and a mass spectrometer. The detection of the analyte requires either a selective labeling of a binder and the use of a corresponding detector,for example radiolabeled marker with a radioflow detector or a fluorescently labeled binder with a fluorescent detector; or a detector capable of discriminating between coeluting compounds, for example a mass spectrometer (MS), as long as each compound has a unique m/z or a characteristic transition (MS/MS) for tandem systems, thus allowing a label-free assay, making it the most flexible and generalized strategy [24–26]. …”

“…V 0 can be calculated from the retention volume of a compound with no affinity for the immobilized target, from the retention volume of the binders using a similar chromatographic column without the immobilized target or the retention volume of a saturating concentration of the ligand. The chromatographic profile can be analyzed with a polynomial equation to derive the inflection point corresponding to the breakthrough volume (V) [4, 25, 26]. $$false(\mathrm{normalV}-{\mathrm{V}}_{0}false)={\mathrm{B}}_{\mathrm{t}}\times {false(false[\mathrm{normalA}false]+K_{\mathrm{d}}false)}^{-1}$$…”

“…Equation 2 is used to calculate K d and B t . $${false[Afalse]}_0+y=B_{t}true(\frac{1}{V-V_0}true)-K_d$$ Where V – V 0 is the corrected breakthrough volume for the binder, B t is the number of available active binding sites in mol, A 0 refers to the binder infusion concentration and the summed concentrations ([A] 0 +y) refer to the initial concentration of the binder for the first step of the staircase; while for the subsequent steps will be the sum of that step and all its predecessors [4, 26]. The slope of a plot of [A] 0 +y versus reciprocal (V-V 0 ) provides the column capacity B t and the negative intercept provides K d .…”