Aug 03, 2025
In-Vivo Mouse and Rat PK Bioanalysis
- Nick Lynch1,2
- 1Curlew Research;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nick Lynch 2025. In-Vivo Mouse and Rat PK Bioanalysis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkrqdwv5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 29, 2025
Last Modified: August 03, 2025
Protocol Integer ID: 219101
Keywords: ADME, DMPK, drug discovery, PK, pharmacokinetic, bioanalysis, rat pk bioanalysis in vivo pk bioanalysi, rat pk bioanalysi, vivo pk bioanalysi, drug concentrations in biological sample, measuring drug concentration, crucial for drug discovery, other pharmacokinetic parameter, drug discovery, biological sample, bioanalysi, studying drug behaviour, drug, vivo mouse, drug behaviour, clinical trial
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Abstract
In vivo PK bioanalysis involves studying drug behaviour within a living organism to understand how it is absorbed, distributed, metabolized, and excreted (ADME). This is crucial for drug discovery and development, providing insights into a drug's efficacy and safety before clinical trials. Bioanalysis plays a key role in accurately measuring drug concentrations in biological samples, like plasma, to assess ADME and other pharmacokinetic parameters.
Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Summary
In vivo PK bioanalysis involves studying drug behaviour within a living organism to understand how it is absorbed, distributed, metabolized, and excreted (ADME). This is crucial for drug discovery and development, providing insights into a drug's efficacy and safety before clinical trials. Bioanalysis plays a key role in accurately measuring drug concentrations in biological samples, like plasma, to assess ADME and other pharmacokinetic parameters
| Options Run | |
| in-vivo mouse single dose PK IV | |
| in-vivo mouse single dose PK PO | |
| in-vivo rat single dose PK IV | |
| in-vivo rat single dose PK PO |
Sample Preparation
Bioanalysis begins with the preparation of a primary stock solution at 1,000,000 ng/mL using DMSO or a suitable alternative, which is then serially diluted to generate standard and quality control (QC) working solutions from 2000ng/ml to 0.5ng/ml.
These solutions are stored at 4°C for up to one week. Calibration standards and QCs are prepared in 96-well plates, ensuring that the volume of organic solvent does not exceed 10% of the plasma to prevent protein precipitation.
For sample preparation, thawed biological samples are added to the plates, mixed thoroughly, and diluted if necessary. An internal standard (IS) in acetonitrile is then added before centrifugation.
Assay
Tissue samples are homogenized in phosphate buffer, with dilution ratios tailored to the tissue type, and analysed using either a tissue-specific standard curve or surrogate matrix when required.
During LC-MS/MS analysis, 100 µL of supernatant is transferred to a new plate, diluted with 0.1% formic acid, and analysed following the generated run list.
Data Analysis
Data processing involves assessing calibration curve fit, where at least 75% of calibration points must fall within ±25% of their nominal values. QC sample acceptance requires two-thirds to meet ±25% criteria, including half at each concentration level.
Carryover should not exceed 1% of the top standard, and any interference in blanks must remain below 20% of the lowest standard. Internal standard response variation over 30% must be reviewed by senior staff, though early run variation is typically acceptable. For unknown samples, if neat and diluted values differ by more than 20%, the diluted value is reported to account for potential matrix effects.
- AUC (Area Under the Curve): Represents the total drug exposure over a specific time period.
- Cmax (Maximum Concentration): The highest drug concentration in the blood stream.
- t½ (Half-life): The time it takes for the drug concentration to halve.
- Vss (Volume of Distribution): The apparent volume of the body in which the drug is distributed.
- Clearance: The rate at which the drug is eliminated from the body.
Acknowledgements
Grateful to Concept Life Sciences for supplying the original protocol steps