Estimation of affinities of ligands in mixtures via magnetic recovery of target-ligand complexes and chromatographic analyses: chemometrics and an experimental model

Table 1 Parameters for HPLC-MS-SIM analyses and approximation of the minimum quantity of SMPP with each mixture sample of a single candidate biotin derivative.

Compound	BCHA	BMPL	BDETA	BME
m/z for M+H⁺	326	314	332	259
Slope for linear response^a	862.1	279.8	116.9	49.2
Intercept for linear response^a	322.1	143.4	59.9	24.2
R² for linear response	> 0.998	> 0.998	>0.994	>0.992
Minimum quantity of the ligand in 5 μL solution to meet the prerequisites of Eq.(6) (pmol)^b	1.5	2.0	2.1	2.0
Minimum quantity of the ligand in 40 μL extract to meet the prerequisites of Eq.(6) (pmol)	12	16	16.8	16
Recovery ratio^c	0.47	1.00	0.57	0.82
Minimum quantity of the ligand bound to SMPP in competitive binding systems (pmol)^d	26	16	31.5	20
Minimal quantity of SMPP to bind the ligand (μL)^e	6.5	4.0	7.8	5.0
Summed minima of SMPP for a PMFS (μL)	11	-	12	9

^a both peak area and intercept were in μA.ms and the quantity of each pure biotin derivative was in pmol.
^b calculated as the quantity of the biotin derivative for peak areas five times of the absolute value of its intercept for linear response.
^c determined in triplicate with variation coefficient below 9%.
^d calculated considering recovery ratio to give the required extract of bound ligand.
^e calculated with the binding capacity of 4 nmol per mL SMPP for a extract of equal contents of one candidate ligand and BMPL as the reference ligand.

ISSN: 1472-6750