Semi-empirical relationships between effective mobility, charge, and molecular weight of pharmaceuticals by pressure-assisted capillary electrophoresis: Applications in drug discovery

Abstract

Relationships between effective mobility (m(eff)), calculated charge (Z(c)), and molecular weight (MW) are semi-empirically derived for pharmaceuticals using pressure-assisted capillary electrophoresis (PACE). We determined the m(eff) at 12 different pH points (2.0-11.4) of 66 pharmaceutical-like compounds ranging in MW from 79 to 825 g/mol. Plots of the observed m(eff) values versus Z(c)/MW(x ) (where x is a fractional coefficient) gave linear relationships. For anions, it was found that the best correlation (R(2) = 0.9666) exists when the fractional coefficient is equal to 0.4920, resulting in the equation m(eff) = 0.1853 (Z(c)/MW (0.4920)). For cations, the best linear relationship (R(2) = 0.9861) gave the equation m(eff) = 0.3888 (Z(c)/MW (0.6330)). The m(eff), Z(c)/MW(x) relationships were then applied to: (i) developing a technique for selecting an appropriate pH to achieve optimal separation of pharmaceuticals and (ii) determining the maximum charge of a molecule in the pH range of determination of negative log of the dissociation constants (pK(a)) by PACE, thus enabling the correct choice of model equation to be automated without structure analysis.

Measurement of Dissociation Constants (pKa Values) of Organic Compounds by Multiplexed Capillary Electrophoresis Using Aqueous and Cosolvent Buffers**Advanced Analytical Technologies, Inc. (Formerly CombiSep), 2711 South Loop Drive, Suite 4200, Ames, IA 50010.

“…The molecular weight (MW) of the compound was entered into the software and the compound charge (Z c ) was estimated by the software using a previously described empirical relationship between MW, maximum m eff and Z c . 28 From the calculated compound charge, a suitable equation from a set of nine different model equations representing up to three ionizable groups 28,22 was selected by the software to perform a nonlinear regression analysis on the data. When performing a cosolvent experiment, the final result is the apparent s w pK a (I ¼ 50 mM) at the particular % methanol concentration.…”

“…28 In the case of methotrexate, a predicted charge of þ0.95 and À1.76 indicated that the compound was a monobase/diacid with three pK a values. Similarly, three pK a values were reported by Miller et al 28 using the aforementioned empirical relationship and by Bergström et al 62 using UV spectrophotometry. However, only two pK a values were reported by Wan et al 43 using CE-MS and 10 widely spaced pH buffers.…”

“…There have been a number of methods employed for pK a measurements based on solubility, potentiometric titration, 7-10 spectrophotometry, [11][12][13][14] HPLC [15][16][17] and, most recently, on capillary electrophoresis (CE). [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] The solubility method is of limited accuracy and potentiometric titrations typically require mg amounts of pure sample with a throughput of 20-40 min per compound. 34 In addition, the sample concentrations required for potentiometry often result in precipitation of poorly soluble compounds, necessitating mixed solvent extrapolation methods and further impacting sample throughput.…”

“…Equations relating the measured m eff and pH values to the pK a value via nonlinear regression analysis have been previously well described in the literature for up to three ionizable groups, which covers the majority of pharmaceutical compounds. 22,28 Theoretically, there is no restriction on the number of ionization equilibria that can be considered. 34 In Pharmaceutical Discovery operations it is often of interest to discriminate differences in pK a values among structural analogs and it is not unusual to encounter molecules with two or more very close pK a s. For the method to be suitable for the simultaneous measurement of the variety of possible pK a combinations, in a high throughput mode, it is necessary to have buffers spaced with relatively small increments and also covering as wide a pH range as possible.…”

Determination of Octanol-Water Partition Coefficient of Novel Coumarin Based Anticancer Compounds by Reversed-Phase Ultra-Fast Liquid Chromatography

“…A detailed literature survey showed that, in recent years many papers have been reported, that have showed the development of many higher throughput methods to determine lipophilicity in industries as there are many novel compounds synthesized [9][10][11]. Also, a large number of data base is available for the determination of log P values by different calculation methods [12].…”