Organ transplantationplantation

Importance of the Topic

Effective therapeutic drug monitoring of immunosuppressants is essential for organ transplant patients due to narrow therapeutic windows and significant interindividual variability. Simultaneous quantification of sirolimus and tacrolimus in whole blood ensures accurate dose adjustment and improves patient safety.

Objectives and Study Overview

The study aimed to develop a semi-automated assay using HPLC coupled to a single quadrupole mass spectrometer for simultaneous determination of sirolimus and tacrolimus in EDTA whole blood. The method was validated over a clinical concentration range and assessed through proficiency testing.

Methodology and Instrumentation

• Sample preparation: Whole blood deproteinized with 0.1 M zinc sulphate/methanol (70:30 v/v) containing internal standards 32-desmethoxysirolimus and ascomycin
• Automated solid-phase extraction using C18 cartridges conditioned with methanol and 0.1% formic acid
• Elution with 2-propanol/0.1% formic acid, evaporation under nitrogen, and reconstitution in acetonitrile
• Chromatography: HPLC with C18 guard and analytical columns at 40 °C, mobile phase optimized for SRL and TRL separation
• Detection: Single quadrupole mass spectrometer (Shimadzu LCMS-2010) operated in selected ion monitoring

Results and Discussion

The calibration curves for both analytes were linear from 0 to 40 µg/L (r2>0.998). The lower limits of quantification were 1 µg/L for sirolimus and 0.5 µg/L for tacrolimus. Intra-day precision (CV) ranged from 5 to 12.5% and accuracy met ±15% criteria. Automated extraction enhanced reproducibility and eliminated carry-over. Column stability allowed analysis of over 1000 samples without sensitivity loss. Routine analysis of more than 1000 patient samples demonstrated the method’s robustness.

Benefits and Practical Applications

• High throughput with semi-automated sample handling
• Simplified workflow suitable for clinical laboratories
• Reliable quantification supporting therapeutic drug monitoring
• Cost and time savings through simultaneous drug analysis

Future Trends and Potential Applications

Further integration of fully automated sample preparation and tandem mass spectrometry is expected to improve sensitivity and selectivity. The method may be adapted for additional immunosuppressants or small molecules and for alternative sample matrices such as dried blood spots, enhancing remote patient monitoring.

Conclusion

The presented HPLC-MS assay offers a rapid, sensitive, and reproducible approach for monitoring sirolimus and tacrolimus in whole blood. Its semi-automated design meets clinical demands and supports high-volume therapeutic drug monitoring in transplant patient care.

References

1. Holt DW, Marwaha G, Jones K, Johnston A. Ther Drug Monit. 1993;15:472-7.
2. ASL International. International Proficiency Testing Scheme 2002.