A Fast, Simple and Sensitive Method for Estimation of Leuprolide in Human Plasma using Shimadzu LCMS-8045

Significance of the topic

Leuprolide acetate is a potent synthetic nonapeptide analog of natural gonadotropin releasing hormone employed in treatment of hormone dependent cancers and reproductive disorders. Precise quantification of low picogram levels in human plasma is essential for pharmacokinetic profiling, therapeutic monitoring and formulation evaluation.

Objectives and Study Overview

The aim was to develop and partially validate a rapid, simple and sensitive LC MS MS assay for measuring leuprolide in human plasma using the Shimadzu LCMS 8045. Key goals included achieving a lower limit of quantification of 25 pg/mL, minimizing matrix effects, streamlining sample preparation, and enabling high throughput analysis for pharmacokinetic investigations.

Used Instrumentation

• Shimadzu Nexera X2 UHPLC system coupled to LCMS 8045 triple quadrupole mass spectrometer
• Shim pack GIST C18 column 2.1 × 50 mm with 3 µm particle size
• Electrospray ionization in positive mode with UF Qarray ion guide technology

Methodology and Sample Preparation

Samples of 200 µL human plasma spiked with leuprolide and deuterated internal standard undergo single step protein precipitation. After vortexing and centrifugation, 25 µL of supernatant is injected onto the UHPLC. Chromatography uses a gradient of 0.1 percent formic acid in water and acetonitrile at 0.6 mL per minute and 40 °C, yielding a total run time of four minutes. MRM transitions monitor doubly charged precursors to specific product ions under optimized collision energies.

Main Results and Discussion

• Calibration curve linear over 25 to 30000 pg/mL with correlation coefficient above 0.997
• Lower limit of quantification at 25 pg/mL with accuracy 117 percent and precision 15.5 percent at LLOQ
• Intraday and interday precision and accuracy for quality control levels met acceptance criteria with RSD below 6.4 percent and accuracy within 85 to 115 percent
• Extraction recovery ranged from 69.6 to 77 percent across QC levels with consistent reproducibility
• Matrix effect evaluation yielded factors between 0.87 and 0.98, indicating minimal ion suppression
• No significant carryover observed in blank injections following the highest calibrator

Benefits and Practical Applications

• Four minute analysis time supports high throughput sample screening
• Low plasma volume and simple precipitation reduce matrix effects and prolong column lifetime
• Sub picogram sensitivity enables pharmacokinetic monitoring of depot formulations and low dose therapies
• Robust performance suitable for clinical pharmacokinetic studies, bioequivalence trials and therapeutic drug monitoring

Future Trends and Potential Applications

Advancements could include microflow and nanoflow LC MS MS to further reduce sample consumption, integration of automated sample handling for increased throughput, and use of high resolution mass analyzers for multiplexed peptide profiling. Emerging extraction technologies and bioanalytical platforms may expand capability for simultaneous quantification of multiple peptide drugs in complex matrices.

Conclusion

A rapid, reliable and highly sensitive LC MS MS method for quantifying leuprolide in human plasma was established and partially validated on the Shimadzu LCMS 8045. The assay meets rigorous criteria for sensitivity, precision and accuracy, providing a valuable tool for pharmacokinetic and clinical studies requiring low level peptide analysis.

References

1. https://www.kegg.jp/entry/D00989 accessed Jun 14 2024
2. https://en.wikipedia.org/wiki/Leuprorelin accessed Jul 08 2019
3. Periti P Mazzei T Mini E Clinical pharmacokinetics of depot leuprorelin Clin Pharmacokinet 2002 41 7 485 504