A centrifuge-free, high-throughput liquid-liquid extraction phase separation filter column and its application in the detection of dihydrotestosterone in plasma

Liquid-liquid extraction (LLE) remains a foundational technique in clinical analysis for sample preparation. Traditional approaches typically require centrifugation to separate the aqueous and organic phases, followed by manual pipetting of the supernatant from individual samples. This process presents challenges for high-throughput applications. In this study, a phase separation filter column compatible with both 2.0 mL centrifuge tubes and 24-well plates was designed and optimized. This column effectively filters the organic phase into the column body, while its internal packing material facilitates the disruption and purification of the emulsion layer. As a result, the organic phase can be easily collected without the need for external equipment or centrifugation. Performance validation was performed by comparing the centrifugation method with the phase separation filter column approach using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of plasma dihydrotestosterone (DHT) via derivatization. The detection of DHT is routinely used in therapeutic drug monitoring (TDM) and drug efficacy assessment. Furthermore, no significant differences were observed between the two methods in terms of linearity, accuracy, limit of quantification, intra- and inter-day precisions, matrix effects, and actual sample analysis. These findings indicate that the phase separation filter column can be seamlessly integrated into clinical applications without altering existing LLE protocols and applied in pharmaceutical analysis, offering a promising solution for high-throughput LLE processes. • A derivatization method for the determination of dihydrotestosterone (DHT) has been established, characterized by its simplicity, high sensitivity, and a rapid 15 min derivatization process. • A phase separation filter column has been designed and developed to facilitate the demulsification and purification of samples for the extraction of DHT, thereby obviating the need for centrifugation. • The aqueous and organic phases are effectively separated by the filter column, enabling the effortless removal of the upper clear liquid without the risk of aspirating the lower aqueous phase, ensuring the accuracy of DHT extraction. • A filter column in a 24-well plate format has been developed to ensure compatibility with adjustable-spacing pipettes, thus supporting high-throughput operations for the extraction of DHT in serum.