Buffer partition coefficient measurement

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

Strichartz, G. R., Sanchez, V., Arthur, G.R., Chafetz, R., Martin, D. Fundamental properties off local anesthetics. II. Measured octanol buffer partition coefficients and pKa values of clinically used drugs, Anesth. Analg. 1990, 71, 158-170. 

In the traditional shake-flask method, the apparent partition coefficient, log D, is measured, usually at pH 7.4 (sometimes at pH 6.5). Different buffers are used to control each pH used in the determinations [70]. Usually, in a comprehensive study, several pH measurements are made, and values of log are plotted against the pH. This plot is often called the lipophilicity profile . One can determine the true partition coefficients (log P ) and the ionization constants from the features in such a curve. 

Ketoprofen, a weak-acid drug, with a pKa 4.12 (25°C, 0.01 M ionic strength), was selected to illustrate Eqs. (7.20) and (7.21) in a series of simulation calculations, as shown in Fig. 7.16. The membrane-buffer apparent partition coefficients /(piii were calculated at various pH values, using the measured constants from... 

Fig. 20.1. Correlation between the air/water partition coefficient, Kaw, determined from measurements of the surface pressure as a function of drug concentration (Gibbs adsorption isotherm) in buffer solution (50 mM Tris/HCI, containing 114 mM NaCI) at pH 8.0 and the inverse of the Michaelis Menten constant, Km obtained from phosphate release...

PARTITION COEFFICIENTS (P ), RETENTION FACTOR (K) AND MOBILITY DATA OF DYE INTERMEDIATES MEASURED WITH A PHOSPHATE-(TTAB) BUFFER AT PH 5.0 ... 

The recent work of Wang and Lien (29) illustrates that ion-pair partitioning occurs to a greater extent than previously realized. Partition coefficients calculated from measurements made on partially ionized compounds depend not only on the pH, but on the buffer used. They may vary by more than one log unit. The authors derived equations to correct log P to octanol/water values, but these can still be off by several tenths of a log unit. A preferable solution would be to know the log Pj and account for ion-pair partitioning. 

Two retention indices are then measured, log kgs (log k obtained with a mobile phase containing 95% of acetonitrile and 5% of buffer solution) and log ko (retention factor obtained with a pure aqueous mobile phase). The difference between these two values (log ko 9s) is correlated to the 1-octanol/water partition coefficient of the neutral form of tested compounds (Equation 5.13) ... 

A system similar to the one described above was used for determination of partition coefficients. A FIA system with segmented flow was devised so that the partitioning of a drug between aqueous buffer and chloroform could be measured. The aqueous and organic phases were separated using a phase separator. The system could be set up to measure the concentration of the drug in either the organic or the aqueous phase. Such a system enables rapid repeat determinations of partition coefficient at various pH values with minimal sample consumption." ... 

These measurements were conducted in ATP system consisting of 5.1% (w/w) PEG 8000 and 10% (w/w) dextran 70 in phosphate buffer at 4°C. Under these conditions, the volume ratio of PEG (light) to dextran (heavy) phases was 3 7. The composition (w/w) of PEG phase was 9.25% PEG and 0.52% dextran. The composition of the dextran phase was 0.54% PEG and 21.1% dextran. The phase composition was unaffected by NaCl concentration in the range of 0-5 M. The partition coefficient is defined as ratio of concentration in PEG phase to that in dextran phase. The triazine dye, Cibacron blue FGF, was ligated to PEG for affinity partitioning. 

Partition coefficients in the octanol-pH 7.4-phosphate-buffer system. c Nitrothiazole oxidation-reduction potentials (volts) as calculated from their half-wave potentials, as determined using a Polarecord E 261 polarograph (Metrohm AG, Herisau, Switzerland) and a saturated Ag/AgCl reference electrode. Measurements were performed at 20°C and a drop time of 1 drop/2.8 sec. The compounds were dissolved in 1 ml dimethyl formamide and added to 24 ml of a borax-potassium biphosphate buffer of pH 7.3 [prepared according to J. M. Kolthoff, J. Biol. Chem. (1925) 68, 135]. A pH of 7.4 resulted. Standard error of determination 3 mv. 

The classical measurement of LogP is the shake flask method [17]. A known amount of drug is dissolved in a flask containing both octanol phase and aqueous buffer at controlled pH to ensure the existence of only nonionic form (at least two units from the drug pA) ). The flask is shaken to equilibrate the sample between two phases. There must be no undissolved substance present in both phases. After the system reaches its equilibrium, which is time- and temperature-dependent, the concentration of drug is analyzed by HPLC in both phases. Partitioning coefficient is calculated as... 

Table 1 Selected true (corrected) partition coefficients of representative drug molecules measured in 1-octanol-water or 1-octanol-phosphate buffer...

The distribution of lomefloxacin between 1-octanol and aqueous buffers was studied at 25°C, pH 5,7 and 9 (17). The apparent partition coefficients of lomefloxacin as determined by the analysis of both the organic and aqueous phases using spectrophotometry were 0.118 0.001,0.139 0.004 and 0.112 0.002, respectively. The thermodynamic partition coefficient was determined to be 0.146 0.005. Takacs-Novac, et al. (18) measured the octanol-water partition coefficient spectrophotometrically by analysis of the aqueous phase only and obtained a value of 0.150. It was found that the presence of metal ions (Al3+, Ca2+, Mg2+, Bi3+ and Fe3+) decreased the partition coefficient of lomefloxacin. (15, 19). 

The pJCa 0 verapamil hydrochloride (I) was determined by measuring the partition coefficient of I between n-heptane and aqueous buffer solution at various pH values. The effect of ionic strength and temperature on the pJCa was also measured. The estimated pJCa hi human plasma was 8.75."... 

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