Equilibrium Dialysis Method Research Articles

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes

It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65–70 percent ammonium sulfate fraction (190ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A–I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60–65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A–I in the plasma of tiger pufferfish in in vivo and in vitro studies.

Equilibrium studies of binding of cationic surfactants to sodium humate

The binding of the cetylpyridinium chloride surfactant (CPC) to sodium humate was studied by the method of equilibrium dialysis without the use of a background electrolyte, at pH=7, at 30°C. The binding isotherm was obtained in a wide concentration range of the CPC: 6·10-6 to 4.96·10-4 mol/L. By increasing the concentration, the isotherm first tends to a saturation step which is followed by a cooperative type of behaviour. The first step was evaluated by an equation derived from the principles similar to those used in the treatment of complexation equilibria. The calculated value of the binding constant (KX=68 900) and the maximum amount of the bounded CPC (qT=8.36 mmol/g C) are in a good agreement with literature data obtained by different experimental method and a different model equation.

Bio-accessibility of bioactive compounds (ACE inhibitors and antioxidants) from extruded maize products added with a red seaweed Porphyra columbina

An expanded maize product added with red seaweeds Porphyra columbina (3.5 g 100 g−1) was developed and bio-accessibility of minerals and bioactive compounds such as ACE inhibitors and antioxidants provide by algae were evaluated using a pepsin/pancreatin digestion and equilibrium dialysis method. Extruded maize added with red seaweed showed higher dialyzability of ACE inhibitor compounds (41.0% ACE inhibition), total phenolic content (0.83 mg gallic acid/g dialysate) and antioxidant capacity (36.6% DPPH inhibition, 2.4 mM TEAC, power reduction and 99.4% copper-chelating activity) than extruded maize. Results about bio-accessibility of bioactive compounds provided by red edible seaweeds may help food technologists to tailor new bio-functional foods, such as functional snacks.

Effect of Ca (II) ion on the in vitro availability and protein binding of Amlodipine besylate

BackgroundThe present study explicates the effect of metals ion (Ca2+) on the in vitro availability of Amlodipine besylate owing to drug–metal interaction. MethodsSpectral studies were performed in an aqueous system at a fixed temperature (37 ± 0.5)°C and under different pH by UV spectrophotometric method at various concentrations of drug and metal. A Job plot was used to determine the stoichiometry of a binding event and The Ardon's method confirmed the complexation. An in vitro study of protein binding of Amlodipine besylate and their 1:1 mixture with Ca2+ ion had been conducted by equilibrium dialysis method at (37 ± 0.5)°C and at pH 7.4 by using Bovine Serum Albumin (BSA). ResultsSpectral studies detected the initial complexation. By Job's plot it was found that the interaction of Amlodipine besylate with metal ion (Ca2+) form one complex with metal at composition of 1:1. The Ardon's spectrophotometric method confirmed the 1:1 complexation and the value of stability constant was higher at pH 7.4 (0.11). The percentage of protein binding of Amlodipine besylate with BSA was found to be 86% and 42% at high and low concentration range respectively. In presence of Ca2+ the percentage of protein binding of drug increased 46% at lower concentration range and 94% at higher concentration zone. The results were statistically significant (p < 0.05).The Scatchard plots showed that in class I binding sites, the value of affinity constant and number of binding sites of 1:1 complexes with Ca2+ was 1.04 and 20.8 respectively. ConclusionDrug–metal complex might, therefore, decrease the free drug in plasma and tissue systems. This may change the pharmacokinetic properties of the drug and may affect the pharmacological effects. It is thus inferred that care and monitoring must be taken during combination therapy of Amlodipine besylate and Ca2+.

Study on pharmacokinetics of asperosaponin VI and its active metabolite in rats

To study the pharmacokinetics, excretion characteristics and plasma protein binding rate of asperosaponin VI (A-VI) and its active metabolite hederagenin (M1). A-VI and M1 concentrations in plasma, bile, urine and feces were determined by established LC-MS/MS to calculate the pharmacokinetic parameters. The plasma protein binding rate of A-VI was determined by equilibrium dialysis method. the double peaks was observed in the A-VI plasma concentration-time curve, after rats were orally administered with low, medium and high doses of A-VI (0.03, 0.09, 0.27 g x kg(-1)). The Cmax1 and Cmax2 of A-VI were (28.88 +/- 49.78) and (4.480 +/- 1.872) microg x L(-1), (35.19 +/- 23.53) and (22.11 +/- 16.15) microg x L(-1), (73.37 +/- 37.28) and (132.2 +/- 160.7) microg x L(-1), respectively. The AUC0-t, of A-VI were (43.21 +/- 37.32), (133.9 +/- 102.5) and (779.6 +/- 876.9) microg x h x L(-1), respectively. The t1/2 of A-VI were (3.3 +/- 0.8), (3.2 +/- 2.3) and (4.5 +/- 1.2) h, respectively. The Cmax of M1 were (16.03 +/- 9.336), (26.41 +/- 11.95) and (28.71 +/- 5.874) microg x L(-1), respectively. The AUC0-t, of M1 were (105.6 +/- 73.60), (260.0 +/-153.9) and (323.1 +/- 107.9) microg x h x L(-1), respectively. The t1/2 of M1 were (4.1 +/- 3.4), (4.4 +/- 2.3), (3.9 +/- 0.9) h, respectively. No significant gender difference was found in the in vivo pharmacokinetics of A-VI and M1. There was no accumulation of A-VI and M1 after multiple administrations of A-VI (0.09 g x kg(-1)). After oral administration of A-VI, the double peaks were also observed in biliary and urinary excretion rate-time curves for A-VI. M1 was detected in the feces samples at 6 h after oral administration. The average plasma protein binding rate of A-VI was 92. 9% in rats.

Why mammals more susceptible to the hepatotoxic microcystins than fish: evidences from plasma and albumin protein binding through equilibrium dialysis

To elucidate the interspecies variation of susceptibility to microcystins (MCs), fresh plasma and purified albumin from six kinds of mammals and fish were used in toxins-substances binding test. Protein contents in the test plasma were analyzed and the binding characteristics to MCs were compared. Two kinds of widely observed MCs, microcystin-LR (MC-LR) and microcystin-RR (MC-RR) were tested and data were collected through the method of equilibrium dialysis. It was found that total plasma protein and albumin content in mammals were nearly two times and four times higher than that in fish, respectively. In the test range of 0-100 μg/mL, binding rates of fish plasma to MCs were considered significant lower (p < 0.01) than that of mammals. And human plasma demonstrated the highest binding rate in mammals. In all the test species, plasma protein binding rates of MC-RR were significantly higher than MC-LR (p < 0.01). Besides, binding profiles of albumin were acquired under the protein content of 0.67 mg/mL. Human serum albumin demonstrated the highest affinity to MCs throughout the six species and differences among the other five species were considered not significant (p > 0.05). From the view of protein binding, it is concluded that both the variation of plasma protein composition and albumin binding characteristic could influence the existing form of MCs in circulation, change MCs utilization, alter MCs half-life and further contribute to the difference of susceptibility between mammals and fish.

Measurement of associations of pharmaceuticals with dissolved humic substances using solid phase extraction

An innovative method was developed to determine association of carbadox, lincomycin and tetracycline with dissolved humic acids using solid phase extraction (SPE). Dissolved organic matter (DOM) and DOM-bound pharmaceuticals passed through the SPE cartridge while the cartridge retained freely dissolved pharmaceuticals from water. This method was validated by comparison with the results measured using the common equilibrium dialysis technique. For the SPE method pharmaceutical interaction with DOM required ∼30h to approach the equilibration, whereas 50–120h was needed for the equilibrium dialysis technique. The uneven distributions of freely membrane-penetrating pharmaceuticals and protons inside vs. outside of the dialysis cell due to the Donnan effect resulted in overestimates of pharmaceutical affinity with DOM for the equilibrium dialysis method. The SPE technique eliminates the Donnan effect, and demonstrates itself as a more efficient, less laborious and more accurate method. The measured binding coefficients with DOM followed the order of carbadox<lincomycin<tetracycline. Pharmaceutical bindings with Leonardite humic acid were greater than those with Aldrich humic acid due to more interaction sites, i.e. carboxylic and phenolic functional moieties, present in the Leonardite humic acid. The results obtained suggest that many pharmaceuticals could be significantly bound to DOM, which alters their fate and mobility in the environment.

PRT064445 but Not Recombinant Fviia Reverses Rivaroxaban Induced Anticoagulation As Measured by Reduction in Blood Loss in a Rabbit Liver Laceration Model

AbstractAbstract 3414We used a modified rabbit liver laceration model to demonstrate the effects of PRT064445, a recombinant fXa derivative, to reverse rivaroxaban induced anticoagulation as measured by reduction of blood loss, decrease of unbound fraction of rivaroxaban in plasma and relevant pharmacodynamic markers: anti-fXa activity, prothrombin (PT) and activated thromboplastin (aPTT) times. Recombinant fVIIa (rfVIIa) was tested in the same model for comparison.Anesthetized rabbits were administered vehicle or 1 mg/kg rivaroxaban via IV bolus injection. After 30 minutes to allow the rivaroxaban to biodistribute, vehicle, PRT064445 or rfVIIa was administered as a bolus injection followed by laceration of two liver lobes (5× each lobe: 1-cm long and 3-mm deep incisions with scalpel blade) and blood loss collected on pre-weighed gauze over 15 minutes. Blood loss due to rivaroxaban anticoagulation represented approximately 10% of the animal’s total blood volume. Total rivaroxaban concentration in plasma was measured by LC-MS/MS. Free fraction of rivaroxaban (non-protein, non-PRT064445 bound) was assessed using equilibrium dialysis method followed by LC-MS/MS quantitation. Anti-fXa activity was measured using a modified chromogenic LMW heparin kit. PT and aPTT was measured using commercial reagents (HemosIL). Anticoagulation by rivaroxaban (1.65 μM average plasma concentration at 30 min time point) resulted in 2.3-fold and 1.9-fold prolongation of PT and aPTT, respectively, and increased blood loss by 3.2-fold over vehicle. PRT064445 (75 mg/rabbit) administration reduced blood loss due to rivaroxaban anticoagulation by >85% and decreased peak anti-fXa activity by 98%, PT by 74%, aPTT by 66% and the free fraction of rivaroxaban in plasma from 26% to <0.5%. In contrast, rfVIIa (150 μg/kg) had no effect on blood loss but reversed PT by 85% and aPTT by 54%. PRT064445 and rfVIIa alone had no effect on blood loss, but rfVIIa decreased PT by 35%.These data demonstrate that PRT064445 can reduce blood loss due to rivaroxaban induced anticoagulation using a single bolus administration. Reduction of blood loss with PRT06445 correlated to the decrease in the free fraction of rivaroxaban in plasma as well as PD markers anti-fXa activity and PT, while rfVIIa decreased PT but had no effect on blood loss. These and our previous data with reversal of enoxaparin-induced anticoagulation in a rat tail transection model indicate that PRT064445 can be used as a universal antidote for both direct and indirect fXa inhibitors. Disclosures:Hollenbach:Portola Pharmaceuticals: Employment. Lu:Portola Pharmaceuticals: Employment. Tan:Portola Pharmaceuticals: Employment. Lee:Portola Pharmaceuticals: Employment. Athiwat:Portola Pharmaceuticals: Employment. Inagaki:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment.

The Possibility of Predicting Prolongation of the Pharmacological Action of Biologically Active Polymeric Substances

The physicochemical properties and the formation, stability, and dissociation constants of complexes of poly acids with organic bases possessing physiological activity were determined by methods of potentiometric titration, conductometry, and equilibrium dialysis, and by biological methods. The relationship between the level of physicochemical characteristics of complexes and the possibility of predicting the prolonged pharmacological (antimicrobial, anaesthetising) action of physiologically active substances in complexes with polymers was established.

Effect of arsenic on paracetamol binding to bovine serum albumin using site specific probes

Arsenic contamination in groundwater is a global health challenge. A large number of people worldwide are affected by arsenic poisoning. Paracetamol is a widely used analgesic-antipyretic drug. Effect of arsenic on paracetamol binding to protein has been investigated using two site specific probes and equilibrium dialysis method was used for the experiment. In absence of any site specific probes free concentration of paracetamol bound to bovine serum albumin increased from 3.95 ± 1.164% to 25.36 ± 1.164%. In presence of site-I specific probe warfarin sodium the % release of drug was steady at around 14%. But in presence of site-II specific probe an increment of free drug concentration was observed from 14.38 ± 1.164% to 54.72 ± 1.552%. Thus it can be assumed that the free concentration of paracetamol was increased to a greater extent in presence of arsenic and probably arsenic bound to site-II of BSA. Thus arsenic may displace paracetamol by binding with high affinity binding site, site-II in the BSA and probably arsenic has little effect to site-I.DOI: http://dx.doi.org/10.3329/icpj.v1i11.12061 International Current Pharmaceutical Journal 2012, 1(11): 361-365

Determination of plasma protein binding rate of vitexin

To establish an analytical method on vitexin concentration in plasma to determine the plasma protein binding rate of vitexin. The equilibrium dialysis method and HPLC were adopted to determine vitexin concentration in plasma, calculate human plasma protein binding rate of vitexin and compare rat and human plasma protein binding rates of vitexin. At 2-16 mg x L(-1), there was no significant difference in the plasma protein binding rate. But the human plasma protein binding rate of vitexin was higher than its rat plasma protein binding rate, indicating a significant difference in rat and human plasma protein binding rates of vitexin. Vitexin has a higher protein binding rate with both rat plasma and human plasma.

P10—Comparisons between free thyroxine levels measured by analogue and equilibrium dialysis methods, in the presence of PFOS and PFOA

P10—Comparisons between free thyroxine levels measured by analogue and equilibrium dialysis methods, in the presence of PFOS and PFOA

Influence of decortication, popping and malting on bioaccessibility of calcium, iron and zinc in finger millet

Finger millet is a rich source of calcium and other minerals. The presence of high amounts of phytates, polyphenols and dietary fibre, may lower the bioaccessibility of minerals. The present study was aimed to determine the influence of decortication, popping and malting on bioaccessibility of calcium, iron and zinc in finger millet. The seed coat fraction of the millet was also included in the studies. Bioaccessibility of minerals was estimated by equilibrium dialysis method while phytic acid by colorimetric method. Polyphenols and dietary fibre were estimated by Folin–Ciocalteu and enzymatic methods respectively. Decortication decreased the total mineral contents, but increased the bioaccessibility of calcium, iron and zinc by 15, 26 and 24 g/100 g respectively. Popping decreased the bioaccessible calcium by about 10 g/100 g and increased that of iron by 5 and 14 g/100 g respectively in native and decorticated millet. No significant increase in bioaccessibility of zinc was observed on popping the decorticated millet but native millet showed an increase of 18 g/100 g. Malted millet showed higher values of bioaccessibility for all the minerals while seed coat fractions exhibited comparatively lower values, because of high proportion of inhibitory factors. Total phytic acid, polyphenols and dietary fibre decreased significantly on decortication and malting.

Rapid Equilibrium Dialysis (RED): an In-vitro High-Throughput Screening Technique for Plasma Protein Binding using Human and Rat Plasma

Determining the extent to which a molecule binds to plasma proteins is a critical phase of drug development because the amount of plasma-bound drug influences compound dosing, efficacy, clearance rate and potential for drug interactions. Determination of the free (%Fu) and bound (%Bound) fractions of a test article in plasma is therefore a critical parameter, which is routinely determined in the process of drug discovery and development. This determination is enabled by equilibrium dialysis, an accepted and standard method for reliable estimation of the non-bound drug fraction in plasma. Although it is the preferred method, equilibrium dialysis has historically been labor-intensive, time-consuming, cost-prohibitive and difficult to automate. A Rapid Equilibrium Dialysis (RED) drug-protein binding assay using LC-MS/MS was developed using a novel technique that resulted in significantly improved assay precision and offers a speed advantage. A panel of compounds covering a range of expected protein binding was tested in plasma of human and rat species. A sensitive and selective method using quadruple tandem mass spectrophotometer interfaced with electro spray ionization was developed for the quantification of unbound drug in pretreated plasma. The mobile phases used were 0.1% Formic acid in Acetonitrile: 0.1% Formic acid in Water with gradient HPLC method. The unbound fraction of drugs was detected by mass spectrometer operated in ESI mode. In addition, data for a set of ten compounds was compared with literature values. With the described method, it is possible to screen a relatively large number of compounds for PPB in a drug discovery environment.

Plasma protein binding of sorafenib, a multi kinase inhibitor: in vitro and in cancer patients

Sorafenib is an orally administered multikinase inhibitor that exhibits antiangiogenic and antitumor activity. Few investigators have been able to correlate cumulative sorafenib dose or total exposure to pharmacodynamic effects. This discrepancy may be in part due to poorly understood protein binding characteristics. Since unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug, an equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (F(u)) sorafenib in human plasma and in isolated protein solutions. Unbound sorafenib concentrations were determined in cancer patients receiving the drug orally at a dose of 400 mg and 600 mg twice daily. Sorafenib was extensively bound with mean F(u) value of 0.3% in both non-cancer and cancer patient's plasma. The binding in plasma was concentration independent, indicating a low-affinity, possibly nonspecific and nonsaturable process. In isolated protein solutions, 99.8% and 79.3% of sorafenib was bound to human serum albumin (HSA) (4 g/dL) and α(1)-acid glycoprotein (AAG) (0.1 g/dL) with binding constants of 1.24 × 10(6) M(-1) and 1.40 × 10(5) M(-1), respectively. In cancer patients receiving sorafenib, unbound sorafenib was not correlated with patient characteristics or laboratory values. In conclusion, sorafenib is highly protein bound in human plasma with a higher affinity towards albumin and limited free drug may be partly responsible for its borderline clinical activity.

Abstract 5466: Clinically relevant plasma protein binding of RO4929097, a gamma-secretase inhibitor targeting notch signaling

Abstract Purpose. Aberrant activation of the Notch pathway contributes to tumor initiation and/or progression. RO4929097 (RO) is an orally bioavailable, small-molecule inhibitor of γ-secretase, which inhibits Notch signaling in tumor cells. RO is being evaluated in phase I/II clinical trials as monotherapy or in combination with other anticancer agents (e.g., Hedgehog inhibitor GDC-0449). RO exhibits large inter-individual pharmacokinetic (PK) variability, which may contribute to difference in treatment outcome. Unbound drug plasma concentration is believed to be more relevant to pharmacological and toxicological effects than total drug. Thus, it is desirable to determine RO binding in plasma and factors influencing this process. The objective of this study was to investigate RO binding to plasma proteins and blood cells, and to examine the influence of GDC-0449 on RO plasma protein binding and PK in cancer patients. Method. An equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining RO fraction unbound (fu) in human plasma. RO binding in plasma from four different species and in isolated plasma protein solutions, as well as drug partitioning in human blood cells were investigated. The PK of total and unbound RO was evaluated in 5 cancer patients who received daily oral RO 20 mg alone or in combination with daily oral GDC-0449 150 mg. Results. RO4929097 was extensively bound in human, dog, rat, and mouse plasma, with a mean fu value of 4.7%, 2.1%, 10.6%, and 1.5%, respectively. In human whole blood, the blood-to-plasma concentration ratio was 0.73. In isolated protein solutions, ∼ 88% and 95% of RO4929097 was bound to human serum albumin (HSA, 40 mg/ml) and alpha1-acid glycoprotein (AAG, 1.4 mg/ml), with a binding constant of 1.8 × 104 M−1 and 1.0 × 106 M−1, respectively. The presence of 25 μM of GDC-0449 increased RO fu by 8.4 and 1.1 folds in AAG (1.4 mg/ml) and HSA (40 mg/ml) solution, respectively. In cancer patients, RO fu exhibited a large intra- and inter-individual variability (range, 0.2 – 3.0%; mean, 1.0 ± 0.7%); co-administration of GDC-0449 increased RO fu by 3.7 to 8.5 folds. Co-administration of GDC-0449 significantly decreased the plasma exposure to total RO, but did not have apparent influence on the unbound RO exposure. Conclusion. RO is highly bound to plasma proteins (mainly to AAG), with a large inter-species and inter-individual variability. Consideration of protein binding is important in the scale-up of RO PK and pharmacodynamic parameters from animal models to humans. Changes in protein binding caused by drug-drug interactions (such as by GDC-0449) or disease states (e.g., increased AAG concentration in cancer patients) can influence the plasma exposure to the total but not unbound RO. Measurement of unbound RO plasma concentration is recommended to avoid misleading conclusions or unadvisedly adjusting RO dose based on total plasma levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5466. doi:10.1158/1538-7445.AM2011-5466

The determination of the binding of salicylate to serum proteins.

Abstract The ratios of protein-bound to unbound salicylate in bovine, calf, horse and human sera were measured by equilibrium dialysis, ultrafiltration and frontal elution techniques. The equilibrium dialysis method gave the best measure of binding capacity, but several factors were found to affect the results. These factors include adsorption of the drug onto the dialysing membrane, the composition of the dialysing fluid, the necessity to allow sufficient time for equilibrium to be attained in the presence of serum, and the occurrence of dialysable substances in sera which may either compete with or displace salicylate from its binding sites.

In vitro interaction study of retinoic acid isomers with telmisartan and amlodipine by equilibrium dialysis method using UV spectroscopy

The in vitro protein binding of retinoic acid isomers (isotretinoin and tretinoin) and the antihypertensive drugs (amlodipine and telmisartan) was studied by equilibrium dialysis method. In this study, free fraction of drugs and the % of binding of drugs in the mixture to bovine serum albumin (BSA) were calculated. The influence of retinoic acid isomers on the % of protein binding of telmisartan and amlodipine at physiological pH (7.4) and temperature (37 ± 0.5 °C) was also evaluated. The in vitro displacement interaction study of drugs telmisartan and amlodipine on retinoic acid isomers and also interaction of retinoic acid isomers on telmisartan and amlodipine were carried out.

Interaction between PAMAM-NH2 G4 dendrimer and 5-fluorouracil in aqueous solution

The formation equilibrium of poly(amidoamine) dendrimer (PAMAM-NH2 G4) complex with an oncologic drug such as 5-fluorouracil (5-FU) in water at room temperature was examined. Using the results of the drug solubility in dendrimer solutions and the method of equilibrium dialysis, the maximal number of drug molecules in the dendrimer–drug complex and its equilibrium constant were evaluated. Solubility results show that PAMAM-NH2 G4 dendrimer can transfer tens 5-fluorouracil molecules in aqueous solution. The number of active sites in a dendrimer macromolecule being capable of combining the drug, determined by the separation method, amounts to n=30±4. The calculated equilibrium constant of the 5-FU-active site bonding is equal to K=(400±120).

Development and Validation of a Higher-Throughput Equilibrium Dialysis Assay for Plasma Protein Binding

We describe the practical aspects of developing a semiautomated, higher-throughput plasma protein binding (PPB) assay. The assay has a capacity of 32 PPB measurements per screen using triplicate incubations per measurement, and it is flexible with respect to the number of compounds and the number of plasma types used. The described method is based on the 48-well format rapid equilibrium dialysis (RED) device in combination with a robotic liquid handling platform and quantitative bioanalysis. The RED device method was optimized with respect to equilibration time. Method validation was performed by comparison of results from the semiautomated RED PPB assay with both of those obtained using an alternative, manual equilibrium dialysis method and with literature values. Propranolol and warfarin were used as control compounds. We have modeled the effect of dialysis membrane leakage on the measured unbound fraction and implemented a test for measuring protein content in the buffer compartment to confirm the integrity of each insert of the RED device. With the described method, it is possible to screen a relatively large number of compounds for PPB in a drug discovery environment.