ADME Studies

ADME Studies

ADME (Absorption, Distribution, Metabolism and Excretion) studies are conducted during any stage of drug development to characterize molecules before final testing in humans. Experts at Blirt are specialized in in vitro ADME-Tox studies. Our ADME in vitro services include physicochemical properties analysis, in vitro permeability, protein binding, metabolism and toxicology (non GLP) (genotoxicity, cytotoxicity and proliferation).


Determination of the solubility of the organic compound in selected solvents provides preliminary information on the nature of the tested compound. It is crucial information during the preclinical stage of development of the molecule – a potential drug. The use of selected set of solvents provides package of valuable information. It helps to make a preliminary assessment of the processes’ speed of absorption and excretion of potential drugs from the body.

Compound requirements2.5 mg solid (for solubility assessment)
1 mg solid (for standard preparation)
Solventswater, diethyl ether, 5% aqueous NaOH solution, 5% aqueous NaHCO3 solution, 5% aqueous HCl solution, concentrated sulfuric acid, 85% phosphoric acid, 5% glucose solution, 0.9% NaCl solution
Number of Replicatesn = 2 (aliquots from filtrate)
Incubation TimeOvernight
Incubation TemperatureAmbient temperature
Analysis methodHPLC-UV-VIS, Spectrophotometric determination
Output dataSolubility [mg/ml]


The chemical stability assay enables to determinate degradation of compound in aqueous buffer by non-enzymatic processes (for example hydrolysis, oxidation or light-catalyzed degradation). Chemical stability of potential drugs is crucial because compounds that are highly unstable may not be suitable as drug candidates due to difficulties in maintenance of a therapeutically effective formulation. In addition compounds designed for oral administration must exhibit chemical stability at the low pH. A range of different pH values are available.

Test Compound Concentration1 μM
DMSO Concentration0.33%
Time Points0, 5, 15, 30, 45, 120 min
Number of Replicates2
Incubation Plate MaterialPTFE (all time points) and polypropylene (2 h only)
Compound Requirements50 µL of 10 mM solution
Analysis MethodLC-MS/MS
Output data% Parent compound remaining at each time point
% Parent compound to polypropylene compared to PTFE


The partition coefficient between the immiscible organic and aqueous phase (typically n-octanol and water) for a pharmaceutical composition is determined by extraction technique.

Analytical methods for measuring the concentration of the solute in both phases are selected on the basis of the physicochemical properties of an investigated molecule (usually a UV-VIS spectrometry is used). Organic compounds with very low solubility in aqueous solutions have often biological activity. For those compounds we propose an innovative method for determining the value of the partition coefficient using chromatographic techniques (without prior extraction from the water to the organic phase).

MethodMiniaturised shake flask method
Partition Solventn-Octanol
Ratio of Buffer:Octanol50:1, 5:1, 1:2 (v/v)
Positive Control CompoundsAcebutolol, Ketoconazole
Compound Requirements1 mg solid compound
Analysis MethodUV-VIS or HPLC-UV-VIS
Output dataLogP, Log D7.4


The ability of the molecule to dissociate in physiological environment of living cell is very important for specificity of drug biological activity. An acid dissociation constant (also known as acidity constant, or acid-ionization constant) – Ka describes this ability of the compound. The ionization of the molecule affects the absorbance value and on the UV-VIS spectrum shape.

MethodpH-metric titration or spectrophotometric methods
Compound Requirements1 mg solid compound
Output datapKa

Drugs are metabolized by many sites in the body, including the gut wall, lungs, kidney and plasma. However, the majority of drug metabolism takes place in the liver which is the most metabolically active tissue per weight unit. We provide metabolic stability testing for small molecules including S9, microsomal and Hepatocytes stability assays.


S9 liver fraction is commonly used to support in vitro ADME studies, including phase I and phase II metabolism. S9 fraction is a mixture of microsomes and cytosol, which contains a wide variety of drug metabolizing enzymes and can be supplemented with cofactors such as NADPH and UDPGA. S9 fractions from different species are available to examine the differences in drug metabolism.

Standard S9 stability protocol:

Available speciesHuman, rat, mouse, rabbit, dog, monkey
Test Compound Concentration3 μM
S9 Concentration1 mg/mL
Time Points0, 5, 15, 30, 45 min
Temperature37°C
CofactorsNADPH, UDPGA
ControlsBlank sample
Sample without cofactor (45 min only)
Postive control (2 compounds)
Number of replicates3 samples/time-point for test compound
1 sample/time-point for controls
Analysis MethodLC-MS
Output dataPercent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life


Liver microsomes are commonly used to support in vitro ADME studies. Liver microsomes contain a wide variety of drug metabolizing enzymes and are commonly used to examine the potential for first-pass metabolism of orally administered drugs. Microsomes are pooled form multiple donors to minimize lot-to-lot variation caused by interindividual variability. Microsomes from different species are available to examine the differences in drug metabolism.

Standard microsomal stability protocol:

Available speciesHuman, rat, mouse, rabbit, dog, monkey
Test Compound Concentration3 μM
Microsome Concentration0.5 mg/mL
Time Points0, 5, 15, 30, 45 min
Temperature37°C
CofactorsNADPH
ControlsBlank sample
Sample without cofactor (45 min only)
Postive control (2 compounds)
Number of replicates3 samples/time-point for test compound
1 sample/time-point for controls
Analysis MethodLC-MS
Output dataPercent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life


Stability of compounds in biological fluids is important parameter because substances which undergo rapid degradation exhibit low efficacy in vivo. Information about plasma instability is useful for other in vitro studies (e.g., plasma protein binding where data can be difficult to interpret), in vivo studies (e.g., storage and handling pre-clinical and clinical samples may be challenging) and for screening of drugs, where rapid conversion in plasma is desirable (e.g., prodrugs and antedrugs).

Standard plasma stability protocol:

Available speciesHuman, rat, mouse, rabbit, dog, monkey, bovine
Test Compound Concentration1 μM
Time Points0, 15, 30, 60 and 120 min
Temperature37°C
ControlsBlank sample
Postive control (1 compounds)
Number of replicates3 samples/time-point for test compound
1 sample/time-point for controls
Analysis MethodLC-MS
Output dataPercent of the parent compound remaining after each incubation period


Hepatocytes are commonly used to support in vitro ADME studies requiring intact cellular systems. Intact hepatocytes contain the major hepatic drug-metabolizing enzymes required to study the four categories of xenobiotic biotransformation: hydrolysis, reduction, oxidation and conjugation. The assay is performed on human hepatic cells Hep2G. Primary cryopreserved hepatocytes from different species are also available up on request to examine the differences in drug metabolism.

Standard hepatocyte stability assay protocol:

Test SystemHuman Hep2G liver cells, Human, rat, mouse, dog, monkey, minipig (cryopreserved hepatocytes)
Test Compound Concentration3 µM
Time points0, 5, 10, 20, 40 and 60 min
Temperature37°C
ControlsBlank sample
Postive control (2 compounds)
Number of replicates3 samples/time-point for test compound
1 sample/time-point for controls
Analysis MethodLC-MS
Output dataPercent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life

In vitro permeability and transport studies are performed to assess the potential of oral absorption and distribution of drug candidates. We provide whole range of in vitro tests to characterize properties of small molecules subjected to passive transport – PAMPA.


The Parallel Artificial Membrane Permeation Assay (PAMPA) is used as an in vitro model of passive, transcellular permeation and absorption of drugs by a PVDF membrane filter pretreated with a solution of the lipid. Passive diffusion is an important factor in determining transport of the drug through the gastrointestinal tract, transport across cell membranes penetration of the blood brain barrier, as well as transport across cell membranes.

Standard PAMPA assay protocol:

Test Compound Concentration10 μM
Number of Replicates3
Membrane CompositionLecithin in dodecane (1% v/v)
Incubation Time16 hours
TemperatureRoom temperature
Compound Requirements100 µL of 10 mM DMSO solution
Integrity MarkerLucifer Yellow
Analysis MethodLC-MS/MS or HPLC-UV-VIS
Data DeliveryLog Pe


To understand the distribution potential of tested compound a fraction of unbound in plasma compound is determined with a usage of equilibrium dialysis. The measure of binding to plasma affects the way of potential drug distribution into tissues in the body. Extensive plasma protein binding also limits the amount of free compound available to access sites of action in the cell, this may cause slower metabolism and elimination. Equilibrium dialysis is the most widely accepted method for determination of plasma protein binding due to minimized non-specific binding effects, when compared to other methods, for example ultrafiltration.

Standard plasma protein binding assay protocol:

MethodEquilibrium Dialysis (at 10 %, 50 % or 100 % plasma)
Test Compound Concentration5 μM (different concentrations available)
Number of Replicates2
Compound Requirements150 μL of 10 mM solution
Analysis MethodLC-MS/MS quantification (both plasma and buffer standards prepared)
Data DeliveryFraction unbound in 100% plasma Recovery


Protein binding has a very important effect on drug dynamics since only the free (unbound) fraction of drug interacts with receptors and may cause pharmacological effects. It is more common to determine the concentration of a drug in plasma rather than whole blood. On the other hand it is crucial for the interpretation of pharmacokinetic data if differential binding to a specific component in the blood occurs.

Standard whole blood protein binding assay protocol:

Test methodRapid equilibrium dialysis (RED, Pierce)
(8K MWCO, distribution: 100% blood-buffer)
Available speciesHuman, rat, mouse, rabbit, dog, monkey
Test Compound Concentration5 µM
Incubation time4h
Temperature37°C
ControlsBlank sample
Positive control (2 compounds: Metoprolol; Chlorthalidone)
Number of replicates3 samples for test compound
1 sample for controls
Analysis MethodLC-MS
Output dataFraction unbound in blood
Recovery


Differences between plasma and brain composition are significant. Plasma contains twice as much protein and brain contains 20 fold more lipids.

Standard brain tissue binding assay protocol:

Test methodRapid equilibrium dialysis (RED, Pierce)
(8K MWCO, distribution: 100% brain tissue homogenate-buffer)
Available speciesHuman, rat, mouse, rabbit, dog, monkey
Test Compound Concentration5 µM
Incubation time4h
Temperature37°C
ControlsBlank sample
Positive control (2 compounds: Metoprolol; Chlorthalidone)
Number of replicates3 samples for test compound
1 sample for controls
Analysis MethodLC-MS
Output dataFraction unbound in brain tissue homogenate
Recovery


It is proved that observed kinetics of metabolism in the in vitro assays can be influenced by nonspecific liposomal binding causing problems with accurate prediction of clearance. Several examples proved that knowledge of microsomal binding leads to more precise understanding of connections between in vitro metabolism and in vivo pharmacokinetics.

Standard microsomal binding assay protocol:

Test methodRapid equilibrium dialysis (RED, Pierce)
(8K MWCO, distribution: 100% microsomes-buffer)
Available speciesHuman, rat, mouse, rabbit, dog, monkey
Test Compound Concentration5 µM
Incubation time4h
Temperature37°C
ControlsBlank sample
Positive control (2 compounds: Atenolol, Propranolol)
Number of replicates3 samples for test compound
1 sample for controls
Analysis MethodLC-MS
Output dataFraction unbound in microsome
Recovery


Blood to plasma distribution is calculated using plasma protein binding assay and whole blood protein binding assay.