A CLINICAL RESEARCH METHOD FOR THE ANALYSIS OF IMMUNOSUPPRESSANT DRUGS IN WHOLE BLOOD USING CAPITAINER™ B DEVICES

Importance of the Topic

Monitoring immunosuppressant drugs such as cyclosporine, everolimus, sirolimus, and tacrolimus is central to transplant patient care. Traditional methods require venous sampling of large blood volumes under clinical supervision, leading to patient discomfort, logistical challenges, and increased resource use. Microliter-scale remote sampling using dried blood spot devices offers a less invasive, more convenient alternative, potentially enhancing patient compliance and reducing sampling costs.

Goals and Study Overview

This work aims to develop and validate a clinical research workflow for quantifying key immunosuppressants in whole blood via microsampling. By employing Capitainer™ B devices to collect 10 µL dried blood spots (DBS) from a 30 µL blood sample, the study evaluates linearity, precision, accuracy, and throughput using UPLC-MS/MS technology.

Methodology and Sample Preparation

• Calibrators and controls: MassTrak™ immunosuppressant sets.
• Sampling: 30 µL whole blood applied to Capitainer™ B device to form a 10 µL DBS; dried overnight.
• Extraction: DBS punched into 2 mL tube; add 200 µL internal standard solution (labeled cyclosporine, ascomycin, everolimus, sirolimus); mix and sonicate.
• Acidification and liquid–liquid extraction: Add 10 µL 0.05 M HCl and 1 mL tert-methyl butyl ether; vortex, centrifuge, transfer 850 µL organic layer.
• Evaporation and reconstitution: Dry under nitrogen at 40 °C; reconstitute in 200 µL mobile phase (water/acetonitrile with ammonium fluoride).

Used Instrumentation

• Waters ACQUITY™ UPLC I-Class FL system with HSS C18 SB column (1.8 µm, 2.1×30 mm).
• Xevo™ TQ Absolute triple quadrupole mass spectrometer in ESI+ mode.
• Gradient elution: water/acetonitrile/ammonium fluoride; total run time ~1.5 min.

Main Results and Discussion

• Linearity: r2 > 0.99 across calibration ranges (1–30 ng/mL for everolimus, sirolimus, tacrolimus; 25–1500 ng/mL for cyclosporine) over five runs.
• Precision and repeatability: ≤ 7.6% CV at QC levels (low, mid, high) across analytes.
• Accuracy: Mean bias within ±6% compared to external quality assurance trimmed mean.
• Throughput: Fast analysis supports high-volume clinical research sampling.

Benefits and Practical Applications

The microsampling approach reduces blood volume requirements and simplifies logistics for decentralized clinical research. The validated method affords high sensitivity, precision, and accuracy comparable to conventional venous sampling, while saving time and resources in sample collection and handling.

Future Trends and Opportunities

Ultrafast UPLC-MS/MS workflows combined with microsampling devices are poised to expand remote therapeutic drug monitoring. Integration with telemedicine platforms and automated sample processing could further streamline patient-centric studies and support real-world pharmacokinetic investigations.

Conclusion

A robust clinical research method for immunosuppressant quantification using Capitainer™ B microsampling and UPLC-MS/MS has been demonstrated. The technique achieves validation criteria for linearity, sensitivity, precision, and accuracy, offering a practical alternative to conventional sampling in transplant patient monitoring and clinical research.