MSACL 2025 Abstract

Sample Cleanup Approaches for the Analysis of A Multi-Class Drugs of Abuse Panel in Breast Milk

Kyle Dukes (1), Esraa AboJasser (1), Lee Williams (2), Sohel Rana (1)
(1) Biotage LLC, (2) Biotage GB

Esraa Abojasser (Presenter) Biotage

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) Other Potential Conflicts Biotage / Employee Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Breastfeeding offers significant health benefits for both infants and mothers, promoting optimal nutrition, immune protection, and developmental outcomes. However, maternal use of illicit drugs or misuse of prescription medications during lactation can result in the transfer of potentially harmful substances into breast milk, posing potentially harmful risks to the nursing infant. Consequently, the ability to accurately detect and quantify drugs of abuse in breast milk is critical for clinical toxicology, forensic investigations, and public health monitoring

OBJECTIVE:
This study demonstrates a reproducible and sensitive analytical workflow for the extraction and detection of commonly encountered drugs of abuse in human breast milk with good recoveries, low detection limits, and reliable quantification across the range of analytes.

METHODS:
A panel of 55 analytes with diverse chemical backbones and a wide range of LogP (-0.6-7.05) and pKa (0.13-10.47) was spiked into breast milk aliquots at a concentration range of 0.01-50ng/mL. Sample extraction was investigated by comparing protein precipitation, protein and phospholipid depletion (PLD), supported liquid extraction (SLE), and cSPE for QuEChERS. Extraction performance was compared by evaluating extract recovery, matrix effect, and reproducibility. The optimized protocol using cSPE for QuEChERS was transferred to the Extrahera® automated sample preparation workstation and evaluated for linearity and precision. LC-MS/MS analysis was performed using a Shimadzu Nexera X2 UHPLC system coupled to a SCIEX 5500 QTrap MS system.

RESULTS:
Breast milk can be a challenging biomatrix due to the high protein and lipid content. These matrix components can interfere with target drug analytes analysis causing ion suppression. We observed consistent extraction performance using the cSPE for QuEChERS workflow, with the majority of investigated analytes achieving 70%-90% recovery with excellent reproducibility (RSD=6-15%) with the exception of amphoteric analytes such as ritalinic acid, gabapentin, and pregabalin, with recoveries around 30-35%, and strongly and moderately polar benzodiazepines and opioids, which recoveries ranged between 55-70%. The cSPE QuEChERS extraction effectively removed over 99% of the phospholipids that co-extracted during protein precipitation, while maintaining good extraction recoveries for the target analytes. In comparison, lower recoveries and higher matrix effects were observed when analytes were extracted with the other techniques. The optimized cSPE QuEChERS extraction method was simpler and faster than traditional SPE.

CONCLUSION:
The cSPE for QuEChERS provided better recoveries for the target panel of drugs of abuse testing in breast milk while maintaining low matrix effects and RSDs below 3%. The final optimized protocol demonstrated excellent linearity and coefficients of determination, r2 > 0.99 for all analytes.