Biotransformation / Metabolism

In vitro biotransformation studies are a cornerstone of drug discovery and screening, providing critical insights into how drug candidates are metabolized in the body. These studies help identify potential metabolites, assess metabolic stability, and predict pharmacokinetic behavior—all essential for selecting safe and effective compounds for further development.

Metabolic Stability

Liver microsomes

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Species Liver Microsomes	As pe client request
NADPH Concentration	1 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	45 min @ 37 ± 1 °C
Time points	0 and 45 (60 min) (OR) 0, 5, 10, 15, 30, 45(60 min) min (+NADPH) and 45 (60 min) min (-NADPH)
Test compound Incubation concentration	1 µM
Final protein concentration	0.5 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	<0.1%
Assay Controls	Verapamil and Quinidine
Test compound requirement	~1mg of powder compound or 20 µL of 10 mM DMSO stock

Species

Liver S9 fractions

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Species S9 fraction	As pe client request
NADPH Concentration	1 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	45 min @ 37 ± 1 °C
Time points	0 and 45 (60 min) (OR) 0, 5, 10, 15, 30, 45(60 min) min (+NADPH) and 45 (60 min) min (-NADPH)
Test compound Incubation concentration	1 µM
Final protein concentration	0.5 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	<0.1%
Assay Controls	Verapamil and Quinidine
Test compound requirement	~1mg of powder compound or 20 µL of 10 mM DMSO stock

Species

Intestinal microsomes

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Species Intestinal Microsomes	As pe client request
NADPH Concentration	1 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	45 min @ 37 ± 1 °C
Time points	0 and 45 (60 min) (OR) 0, 5, 10, 15, 30, 45(60 min) min (+NADPH) and 45 (60 min) min (-NADPH)
Test compound Incubation concentration	1 µM
Final protein concentration	0.5 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	<0.1%
Assay Controls	Verapamil and Quinidine
Test compound requirement	~1mg of powder compound or 20 µL of 10 mM DMSO stock

Species

Intestinal S9 fraction

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Species Intestinal S9 fractions	As pe client request
NADPH Concentration	1 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	45 min @ 37 ± 1 °C
Time points	0 and 45 (60 min) (OR) 0, 5, 10, 15, 30, 45(60 min) min (+NADPH) and 45 (60 min) min (-NADPH)
Test compound Incubation concentration	1 µM
Final protein concentration	0.5 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	<0.1%
Assay Controls	Verapamil and Quinidine
Test compound requirement	~1mg of powder compound or 20 µL of 10 mM DMSO stock

Species

Hepatocytes

Protocol
Incubation matrix	Hepatocytes
Species	As per client request
Replicates	n=2
Test compound incubation concentration	1 µM or 0.5 µM
Incubation time	120 min at 37 °C
% DMSO content	<0.1%
Cells	0.5 or 1 *106 Cells/mL
Time points	0, 5, 10, 15, 30, 60, 90, and 120 min
Assay controls	Umbelliferone, Quinidine and Warfarin (Mouse, Rat,Dog and Monkey); Umbelliferone, Quinidine and Propranolol (Human)
Analysis	LC-MS/MS
Test compound requirement	~1mg of powder or 10 µL of 10 mM DMSO stock

Species

Plasma stability

Protocol
Incubation matrix	Plasma
Species	As per the client request
Replicates	2
Concentration	1 µM or as specified
Incubation temperature	37 °C
% DMSO content	≤ 0.1%
Time points	0, 15, 30, 60, 120 and 300 min
Assay controls	Propantheline Bromide
Analysis	LC-MS/MS
Compound requirement	10 mM DMSO stock 10 µL

Species

Blood stability

Protocol
Incubation matrix	Fresh blood pH 7.4
Species	Mice, Rat and Human
Replicates	2
Concentration	1 µM
Incubation temperature	37 °C
% DMSO content	≤ 0.1%
Time points	0, 15, 30, 60, and 120 min
Assay controls	Aclidinium Bromide
Analysis	LC-MS/MS
Compound requirement	10 mM DMSO stock 10 µL

Species

Conjugative metabolism (Phase II)

Glucuronidation

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Species Liver Microsomes / Liver S9 fractions	As per client request
UDPGA Concentration	5 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	60 min @ 37 ± 1 °C
Time points	0, 5, 10, 15, 30, 45 and 60 min (+UDPGA) and 60 min (-UDPGA)
Test compound Incubation concentration	1 µM
Final protein concentration	0.5 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	<0.1%
Assay Controls	Verapamil and Umbelliferone
Test compound requirement	~1mg of powder compound or 20 µL of 10 mM DMSO stock

Species

Sulfation

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Species Liver S9 fraction	As per client request
PAPS Concentration	0.1 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	45 min @ 37 ± 1 °C
Time points	0, 5, 10, 15, 30, 45 or 60 min (+PAPS) and 45 or 60 min (-PAPS)
Test compound Incubation concentration	1 µM
Final protein concentration	1 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	<0.1%
Assay Control	Daphnetin (3 µM)
Test compound requirement	~1mg of powder compound or 10 µL of 10 mM DMSO stock

Species

Identification of Phase I and Phase II metabolism

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Species Liver Microsomes	As per client request
NADPH Concentration	1 mM
UDPGA Concentration	5 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	60 min @ 37 ± 1 °C
Time points	0, 5, 10, 15, 30, 45 and 60 min (+NADPH, +UDPGA) and 60 min (-NADPH, -UDPGA)
Test compound Incubation concentration	1 µM
Final protein concentration	0.5 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	< 0.1%
Assay Controls	Verapamil and Umbelliferone
Test compound requirement	~1mg of powder compound or 20 µL of 10 mM DMSO stock

Species

Aldehyde oxidase

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Liver Cytosol	Human liver Cytosol
Preincubation time	10 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	60 min @ 37 ± 1 °C
Time points	0, 5, 10, 15, 30, and 60 min
Test compound Incubation concentration	1 µM
Final protein concentration	1 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final organic concentration	≤0.5%
Assay Control	Zoniporide
Aldehyde oxidase Inhibitor	Raloxifene Hydrochloride (10 µM)
Test compound requirement	~1mg of powder compound or 10 µL of 10 mM DMSO stock

Metabolite profiling and structure elucidation

Protocol
Buffer pH	100 mM potassium phosphate buffer, pH 7.4
Matrix	As per client request
Species	As per client request
NADPH Concentration	1 mM
Preincubation time	15 min (To ensure all reagents reaches 37 °C, before assay initiation)
Incubation time	45 min @ 37 ± 1 °C
Time points	0, 15 and 45 (60 min) (+NADPH) and 45 (60 min) min (-NADPH)
Test compound Incubation concentration	1 µM and 10 µM (As per client request)
Final protein concentration	0.5 mg/mL
Replicates	n=2
Analysis	LC-MS/MS
Final DMSO concentration	<0.1%
Test compound requirement	~1mg of powder compound or 20 µL of 10 mM DMSO stock

Species

Matrix

Biotransformation / Metabolism