Challenges of Developing a Bioanalytical Method for a Macrolide Immunosuppressant Compound by LC–MS/MS

Abstract

The quantification of tacrolimus in human whole blood was developed by LC-MS/MS for a range of 50.0 to 50,000.0 pg/ml. Different challenges were faced during method development due to ion-suppression, lack of sensitivity and low recovery. The optimization of the extraction procedure played a crucial role as tacrolimus had to be isolated from red blood cells, to which it is strongly bound. Another particular challenge arose from the freeze-thaw stability where the extracted samples from fresh blood always showed a lower recovery. Finally, matrix effect was observed in some matrices over time, which resulted in a failed long-term stability in whole blood. In order to resolve the matrix effect issue, the sample procedure had to be improved. The final assay showed good recovery, low matrix effect, linearity, blood stability and good precision and accuracy.

Quantitative Analysis of Peptides with Mass Spectrometry: Selected Reaction Monitoring or High‐Resolution Full Scan?

“…In HPlc-MS/MS method development for a macrolide immunosuppressant, matrix effects prevented successful validation and introduction of HrMS/ MS considerably reduced chemical noise [20]. A first example of a full validation of a bioanalytical method using HrMS was published by Fung et al [46] for the quantification of prednisone and prednisolone in human plasma.…”

“…However, the product ion spectrum is scanned at high resolution instead of unit resolution. For small molecules, it has been reported that selection of product ions at high resolution can improve selectivity and specificity [20]. However, the challenges of the scan time paradigm discussed for SrM workflows are also relevant for HrMS SrM.…”

“…In particular, when multiple analytes are to be quantified in a single injection, the SrM workflow has limitations and full scan HrMS has been proposed as a better alternative. Examples in food safety, forensic and environmental research, as well as toxicology and therapeutic drug monitoring have been described in the literature [16][17][18][19][20]. In HrMS experiments, selectivity is achieved through narrow width extracted ion chromatograms (EIc) achievable through a combination of high resolution and good mass accuracy.…”

A Robust Procedure for Determination of Immunosuppressants Cyclosporine A and Tacrolimus in Blood Samples with Detection of LC–MS/MS

“…3 And other LC-MS/MS methods commonly used zinc sulfate (ZnSO 4 ) and methanol or acetonitrile as protein precipitant. [20][21][22] Zinc sulfate is a nonvolatile inorganic salt which is di cult to remove after entering the mass spectrum. The instrument is easy to get dirty, which increases the frequency of maintenance and cleaning.…”

Clinical and genetic determinants of tacrolimus pharmacokinetics and pharmacodynamics in the transplant population

“…Accuracy and precision data shows that intra-and interassay bias and coefficient of variation (CV) were <20% for LLOQ and <15% for quality control samples ( 123 . The addition of a washout step with isopropyl alcohol: tetrahydrofuran (90:10) in between runs has helped minimize build-up of ion-suppressing matrix components which seemed to have a more pronounced effect at lower analyte concentrations.…”