Bioanalytical methods for breast milk and plasma

The ConcePTION project has developed standardized and validated bioanalytical methods to study medicine concentrations in human breast milk and plasma. The methods were developed as part of the clinical lactation studies, and the data has supported validation of models for medicine transfer to breast milk.

The developed methods are rapid, robust, and capable of quantifying compounds of interest in a range that is relevant to clinical concentrations, which is crucial for the determination of pharmacokinetic (PK) parameters as well as to perform the physiologically-based pharmacokinetics (PBPK) and population PK (popPK) modeling.

Milk and plasma samples were collected in the project’s clinical demonstration studies (cetirizine, venlafaxine, metformin, and prednisolone), and shipped to Uppsala Biobank where samples were aliquoted and stored at -80°C pending analysis. Analysis was performed using liquid-chromatography tandem mass spectrometry (LC-MS/MS) at the Uppsala Drug Optimization and Pharmaceutical (UDOPP/ADMEot) unit, SciLifeLab DDD, Department of Pharmacy, Uppsala University, Uppsala, Sweden.

The samples from the demonstration studies are now part of the ConcePTION breast milk collection for clinical lactation studies. The methods are standardized and validated according to the regulatory guidelines from the FDA (Bioanalytical Method Validation Guidance, 2020) and EMA (Guideline on bioanalytical method validation, 2012). The following parameters were validated: stability, dilution integrity, accuracy and precision of the method, calibration curve range, selectivity and determination of lower limit of quantification (LLOQ). All parameters were validated in the relevant matrix (human breast milk and/or plasma) based on the planned demonstration studies. Stability was also assessed in human whole blood.

The value of performing proper validation of the analytical methods according to regulatory guidelines increases confidence in the results from lactation studies, and increases the chance of the results being used either in clinic or on the label of the drug in question. By setting up these procedures for the bioanalysis of samples from lactation studies we provide a framework for future studies to build upon. This framework has been published Open Access: 

Using the ConcePTION biobanalysis framework

The procedures for bioanalysis of biobank samples from lactation studies can guide how to set up analytical processes for the rapid and accurate analysis of human breast milk and plasma samples. Of key importance is the validation of stability in both milk and plasma, as well as in human whole blood, as this will guide timelines for sample processing and handling in conjunction with the lactation studies. Validated stability will increase confidence in the bioanalytical results and any following conclusion. A short stability in whole blood increases demands on the capabilities on the clinical center performing the lactation study as they will need to have the capabilities to centrifuge the blood on site.

The standardized procedures for sample preparation and analysis, where all compounds are able to be treated in the same way, with the same sample preparation, analysis on the same type of 96-well plate and on the same instrument provide a robust method that is easily described and transferrable.

Contact persons

Pawel Baranczewski
pawel.baranczewski@uu.se

Aljona Saleh
aljona.saleh@uu.se

Department of Pharmacy, Uppsala Drug Optimization and Pharmaceutical Profiling (UDOPP/ADMEoT), SciLifeLab Drug Discovery and Development Platform, Uppsala University, Box 580, SE-75123 Uppsala, Sweden.