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Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins
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A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects
Remco A Koster1,2, Pascal Niemeijer1,2, Herman Veenhof1
1Department of Clinical Pharmacy & Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Bioanalysis
|March 21, 2019
Summary
A new LC-MS/MS assay using volumetric absorptive microsampling tips was developed and validated for five immunosuppressants. The method showed stability and acceptable bias across various hematocrit levels, enabling reliable therapeutic drug monitoring.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Biotechnology
Background:
- Therapeutic drug monitoring (TDM) is crucial for immunosuppressants like tacrolimus, sirolimus, everolimus, cyclosporin A, and mycophenolic acid.
- Volumetric absorptive microsampling (VAMS) offers a minimally invasive alternative to traditional blood sampling for TDM.
- Investigating the impact of hematocrit (HT) on VAMS assays is essential for accurate drug quantification.
Purpose of the Study:
- To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for five immunosuppressants using VAMS.
- To evaluate the stability of these immunosuppressants in VAMS tips.
- To assess the influence of hematocrit variations on analyte recovery and assay accuracy.
Main Methods:
- Development and validation of a LC-MS/MS assay for tacrolimus, sirolimus, everolimus, cyclosporin A, and mycophenolic acid.
- Utilized volumetric absorptive microsampling tips for sample collection.
- Investigated analyte stability in VAMS tips for 14 days at 25°C.
- Assessed hematocrit effects on analyte recovery across a range of HT levels (0.20–0.60 L/L).
Main Results:
- The LC-MS/MS assay demonstrated robustness with maximum overall bias of 9.6% (sirolimus LLOQ) and coefficient of variation of 8.3% (everolimus LLOQ).
- All five immunosuppressants were stable in VAMS tips for at least 14 days at 25°C.
- Hematocrit-induced biases were within 15% for most analytes between HT levels of 0.20–0.60 L/L, with exceptions for cyclosporin A.
- Reduced recoveries were noted for sirolimus, everolimus, and temsirolimus at high analyte concentrations combined with low HT values.
Conclusions:
- A robust and validated LC-MS/MS method for analyzing immunosuppressants in VAMS tips was successfully developed.
- Hematocrit and concentration-dependent recovery effects were observed but remained within acceptable limits for the assay's intended purpose.
- This VAMS-based assay provides a reliable tool for therapeutic drug monitoring of immunosuppressants.
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