Ultrafast Quantitation of Immunosuppressant Drugs in Whole Blood by Agilent 6475 LC/MS

Significance of the Topic

A precise and rapid measurement of immunosuppressant drugs in whole blood is critical for optimizing transplant patient outcomes and minimizing adverse effects such as nephrotoxicity and neurotoxicity. Therapeutic drug monitoring (TDM) ensures that drug concentrations remain within a narrow therapeutic window, reducing the risk of under- or overdosing.

Study Objectives and Overview

This application note describes the development and validation of a 2.5-minute liquid chromatography–mass spectrometry (LC/MS) method using an Agilent 6475 triple quadrupole system and a commercial RECIPE ClinMass TDM kit to quantify four key immunosuppressants (tacrolimus, sirolimus, everolimus, and cyclosporine A) in whole blood. The primary goals were to achieve outstanding sensitivity, linearity, and interday precision while significantly increasing sample throughput.

Instrumentation

The analytical setup combined:

• Agilent 1290 Infinity II binary pump, multisampler, and column thermostat
• RECIPE ClinMass prefilter (MS9032) and analytical column (MS9030)
• Agilent Jet Stream source and 6475 triple quadrupole mass spectrometer

Methodology

Sample preparation involved protein precipitation of 100 µL whole blood with internal standard and precipitant, agitation, centrifugation, and direct injection into the LC/MS.
The chromatographic gradient ran from 10% to 95% organic over 1.7 minutes at 0.9 mL/min, with column and source temperatures optimized at 70 °C and 250–350 °C, respectively. Multiple reaction monitoring (MRM) transitions for each analyte and corresponding isotopically labelled internal standard were optimized using MassHunter Optimizer. Data processing was performed with MassHunter Qualitative and Quantitative Analysis software.

Main Results and Discussion

– The total run time of 2.5 minutes allowed elution of all four drugs within a tight retention window.
– Lower limits of quantification were below 0.5 µg/L for tacrolimus, sirolimus, and everolimus, and 6.8 µg/L for cyclosporine A.
– Calibration curves over six concentration levels showed excellent linearity (R2 > 0.996) across three days.
– Interday precision (RSD) remained below 6.5% and accuracy ranged from 93% to 113% for quality control samples.
– No significant carryover was observed, supporting reliable high-throughput operation.

Benefits and Practical Applications

– Ultrafast analysis supports processing of over 500 whole blood samples per day.
– Simple sample preparation with no filtration or extensive cleanup required.
– High sensitivity and specificity of MRM transitions minimize matrix interferences.
– The ready-to-use RECIPE TDM kit provides a turnkey solution for clinical and research laboratories engaged in immunosuppressant monitoring.

Future Trends and Possibilities

As demand for TDM expands, further integration of automated sample preparation and data analysis workflows will be important. Emerging mass spectrometry platforms may enable multiplexed assays including additional drug classes. Machine learning–assisted peak detection and quantitation could further streamline data processing and quality control. Standardization of kits and protocols across laboratories will enhance comparability and support regulatory compliance.

Conclusion

The validated 2.5-minute LC/MS method on the Agilent 6475 platform combined with the RECIPE ClinMass TDM kit delivers highly sensitive, linear, and precise quantitation of four immunosuppressants in whole blood. Its ultrafast throughput and minimal sample preparation make it well suited for routine clinical and research TDM applications.

References

1. Yeung P. et al. Mass Spectrometry Quantitation of Immunosuppressive Drugs in Clinical Specimens Using Online Solid-Phase Extraction and Accurate-Mass Full Scan-Single Ion Monitoring. J. Mass Spectrom. Adv. Clin. Lab. 2023; 99–104.
2. Gunay G. et al. Simultaneous Analysis of Tacrolimus, Sirolimus, Everolimus, and Cyclosporine A in Whole Blood Using an Agilent Ultivo Triple Quadrupole LC/MS. Agilent Technologies Application Note 5994-2395EN.