Simultaneous Analysis of Tacrolimus, Sirolimus, Everolimus, and Cyclosporin A in Whole Blood Using the Agilent RapidFire High-Throughput Mass Spectrometry System

Significance of the Topic

Therapeutic monitoring of immunosuppressive drugs is critical for patient management in organ transplantation and autoimmune disorders. Traditional LC MS platforms provide excellent sensitivity and selectivity but are limited by longer analysis times. The Agilent RapidFire high throughput MS system integrates ultrafast solid phase extraction with tandem mass spectrometry to reduce cycle times to under 13 seconds per sample addressing the growing demand for rapid and reliable clinical testing.

Objectives and Study Overview

This study aimed to evaluate performance of the Agilent RapidFire 360 system coupled to a 6460 triple quadrupole mass spectrometer for simultaneous quantification of tacrolimus, sirolimus, everolimus, and cyclosporin A in whole blood. Key goals included assessing throughput, linearity, precision, accuracy, and sensitivity compared to conventional LC MS workflows.

Methodology and Instrumentation

Sample preparation involved protein precipitation of whole blood with zinc sulfate and methanol spiked with stable isotope labeled internal standards. Extracts were loaded onto a reversed phase C18 RapidFire cartridge. Three mobile phases were utilized: aqueous ammonium acetate with formic and trifluoroacetic acids; methanol; and ammonium acetate in methanol. The SPE cycle was automated with timed sensor states enabling a sample cycle of less than 13 seconds. Mass spectrometric detection was performed on an Agilent 6460 triple quadrupole MS operating in multiple reaction monitoring mode. Source conditions included a gas temperature of 225 C, nebulizer pressure of 40 psi and capillary voltage of 4000 V. MRM transitions for each analyte and corresponding internal standard were optimized for quantifier and qualifier ions, dwell times, and collision energies.

Results and Discussion

Calibration curves demonstrated strong linearity with correlation coefficients above 0.995 across all analytes. The quantification range spanned 0.8 to 50 ng/mL for tacrolimus, sirolimus and everolimus and 7.8 to 1000 ng/mL for cyclosporin A. Limits of quantification were established at 0.78 ng/mL for the three mTOR inhibitors and 7.8 ng/mL for cyclosporin A with signal to noise ratios exceeding 40:1 and 100:1 respectively. Intra and inter day precision values were below 10% for all concentrations and accuracies remained within ±15%. No significant carryover was observed confirming robustness for high throughput analysis.

Benefits and Practical Applications

The RapidFire MS MS workflow delivers sample throughput exceeding 270 samples per hour without compromising analytical performance. This capability is particularly advantageous for clinical laboratories conducting large scale therapeutic drug monitoring, pharmacokinetic studies or high volume screening of small molecule analytes in whole blood.

Future Trends and Applications

Emerging developments may include expansion to broader panels of immunosuppressants, enhanced automation through integration with robotic platforms and adaptation for additional drug classes. Ongoing improvements in SPE cartridge design and MS instrumentation could further shorten cycle times and increase sensitivity advancing the feasibility of near real time therapeutic monitoring.

Conclusion

The Agilent RapidFire high throughput mass spectrometry system successfully achieved rapid, accurate and precise simultaneous quantification of four key immunosuppressant drugs in whole blood. By dramatically reducing analysis times while maintaining stringent analytical criteria this approach supports the growing demands of clinical research and diagnostic laboratories.

Used Instrumentation

• Agilent RapidFire 360 high throughput SPE system
• Agilent 6460 triple quadrupole mass spectrometer
• Agilent MassHunter Qualitative Analysis and RapidFire Integrator software

References

1. Schlicht KE, Korman EW, Miller VP, et al High throughput analysis of tacrolimus in whole blood using ultrafast SPE MS MS Poster 160 59th ASMS Conference June 7 2011 Denver CO