Highly sensitive quantitative analysis of Leuprolide from rat plasma using LC-MS/MS

Significance of the Topic

The quantitative measurement of leuprolide in rat plasma is crucial for pharmacokinetic profiling during drug discovery. It informs dosage optimization, safety assessments, and supports regulatory bioanalysis.

Objectives and Article Overview

This work aimed to establish a highly sensitive and selective LC-MS/MS method capable of detecting leuprolide at picogram-per-milliliter levels in rat plasma. The method was validated across a broad concentration range to support preclinical and translational studies.

Methodology and Instrumentation

Sample preparation employed solid phase extraction with conditioning in methanol and water, plasma loading, sequential washing with water and 20% acetonitrile, and elution in methanol. The eluate was evaporated and reconstituted prior to analysis.

Chromatographic separation used a Shimadzu Nexera X2 UHPLC system equipped with a Shim-pack C18 column (100 mm × 2.1 mm, 2.7 µm) under a buffer–acetonitrile gradient at 0.5 mL/min and 40 °C.

Detection was performed on a Shimadzu LCMS-8060 triple quadrupole mass spectrometer with heated electrospray ionization. Multiple reaction monitoring transitions were m/z 605→249 for leuprolide and m/z 635→221 for the goserelin internal standard.

Key Results and Discussion

The assay achieved a lower limit of quantitation of 10 pg/mL, with a linear dynamic range from 10 to 300 000 pg/mL and a correlation coefficient of 0.9952. Extraction recovery exceeded 70% for leuprolide and internal standard. Matrix factor evaluation yielded an average normalized factor of 1.07, indicating minimal ion suppression or enhancement. Intra- and inter-batch precision and accuracy met acceptance criteria (<15% for QC levels and <20% at LLOQ), and no interfering peaks were observed at the retention time of leuprolide.

Benefits and Practical Applications

This validated LC-MS/MS protocol delivers ultra-sensitive quantitation with low plasma volume requirements, making it ideal for pharmacokinetic, toxicokinetic, and bioequivalence studies of peptide therapeutics. Its wide dynamic range supports analysis of both early time-point low concentrations and later high concentrations within a single run.

Future Trends and Potential Applications

Ongoing innovations in ionization sources, high-speed scanning, and microflow UHPLC will further enhance sensitivity and throughput. The methodology can be adapted to other peptide and small-molecule drugs in various biological matrices, facilitating next-generation bioanalysis and personalized medicine studies.

Conclusion

A robust and ultrasensitive LC-MS/MS method for leuprolide quantitation in rat plasma has been developed and validated. It employs solid phase extraction, UHPLC separation, and triple quadrupole detection to meet stringent regulatory standards, supporting comprehensive preclinical pharmacokinetic investigations.

References

1. Pradeep K Vuppala Journal of Bioequivalence & Bioavailability Volume 5 Issue 4 2013
2. EMA Guideline on Bioanalytical Method Validation Rev.1 Corr.2 EMEA/CHMP/EWP/192217/2009
3. US FDA Guidance for Industry Bioanalytical Method Validation Rev.1 Draft September 2013
4. Japan MHLW Guideline on Bioanalytical Method Validation in Pharmaceutical Development 25 July 2013