Separation and Determination of Liposomal and Non-Liposomal (Free) Doxorubicin from Human Plasma by SPE and LC-MS/MS

Significance of the Topic

Accurate quantification of liposomal and free doxorubicin in human plasma is essential for assessing the safety and efficacy of pegylated liposomal formulations. Distinguishing encapsulated drug from its unencapsulated counterpart informs pharmacokinetic profiles, optimizes dosing regimens, and supports regulatory compliance in oncology bioanalysis.

Objectives and Study Overview

This study demonstrates a streamlined workflow combining Thermo Scientific SOLA HRP solid phase extraction with Accucore C18 UHPLC separation and TSQ Vantage tandem mass spectrometry to:

• Separate liposomal (encapsulated) and non-liposomal (free) doxorubicin in plasma
• Quantify each fraction with high sensitivity and accuracy over 1–500 ng/mL
• Validate the method against label claims for free drug content in liposomal injections

Methodology and Instrumentation

Sample pretreatment avoids vigorous mixing to preserve liposome integrity. The two-stage SPE protocol uses a hydrophobic reversed-phase cartridge: the free fraction is retained and eluted with acidified methanol while the encapsulated fraction passes through for subsequent liposome disruption by protein precipitation and a second SPE cleanup. Chromatographic separation employs a 2.6 μm Accucore C18 column with a water–methanol gradient containing 0.1 % formic acid at 0.4 mL/min and 30 °C. Detection uses positive-mode heated electrospray ionization in SRM mode, monitoring m/z 544.2→361.1 for doxorubicin and 528.2→321.1 for daunorubicin IS.

Main Results and Discussion

The method achieved linear calibration (r2=0.999) from 1 to 500 ng/mL with a limit of quantitation of 1 ng/mL. Intra- and inter-day precision at 3, 200, and 400 ng/mL showed %RSD below 10 %. Recovery ranged from 89.8 to 102.5 % with matrix interference under 4 %. Free doxorubicin measurements in spiked liposome injections aligned with the 1.5–2.5 % label claim, confirming effective separation and quantification.

Benefits and Practical Applications

The SOLA HRP SPE format offers high reproducibility, cleaner extracts, and reduced solvent consumption. Combined with Accucore C18 UHPLC and TSQ Vantage MS, the workflow delivers robust throughput suitable for clinical and bioanalytical laboratories, supporting therapeutic monitoring and formulation development.

Future Trends and Applications

Emerging opportunities include extending this approach to other nanocarrier systems, integrating automation for high-throughput screening, and miniaturizing sample handling. Continued advances in core shell LC columns and novel SPE chemistries will enhance sensitivity and selectivity for complex drug delivery platforms.

Conclusion

The integrated SPE-LC-MS/MS method provides reliable separation and quantification of liposomal versus free doxorubicin in plasma, combining simplicity with excellent analytical performance. It supports critical bioanalytical requirements for liposomal drug development and therapeutic monitoring.

References

U.S. Food and Drug Administration Guidance for Industry Bioanalytical Method Validation 2001