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Sirolimus measurement by LC-MS/MS without solid phase extraction – overcoming the obstacles

N. W. Brown, P. Arampatzis, C. E. Gonde, M. Pires, J. Tracey, M. Tredger

Ann Transplant 2008; 13(1): 36-36 :: ID: 880193

Abstract

Background: Local clinical practice dictated the need for a 3-4 hour turn-around time for sirolimus (SRL) analysis. Of the many reported methods using LC-MS/MS, most proved unsuitable because they involved time consuming sold-phase extraction (SPE) or markedly over-estimated SRL in patient samples (by 1.5 to 2-fold) and spiked quality assurance samples (by 15-20%), showed poor (>15%) interassay precision or low sensitivity when evaluated in-house.
Material/Methods: Using stepwise optimisation of the pre-treatment, chromatography and tandem-mass spectrometry stages of the method we were able to successfully validate a method to FDA standards. Pretreatment with methanolic NH[sub]4[/sub] CO[sub]3[/sub]/ZnSO[sub]4[/sub] improved precipitation and optimised sirolimus extraction. Chromatography was enhanced using a C8 column and a column purge with 100% methanol reduced apparent ion-enhancement. Mass spectrometry with a low desolvation temperature removed a co-eluting peak.
Results: We identified a lower limit of quantification of 1.5 μg/L, inter-assay precision <12% from 2.9 to 40.0 μg/L and linearity to at least 50 μg/L. Recovery of spiked samples gave a median recovery of 107% and analysis of 27 stored quality assurance samples (patient pools and spiked samples) a median z value of +0.3 compared to the LC-MS/MS group mean. Comparison of results in 186 samples from liver, renal, pancreatic islet and bone marrow transplant recipients showed good agreement with a reference laboratory using LC-MS/MS and SPE: median (interquartile range) bias of +4.30% (-0.10 to 9.03%).
Conclusions: Timely analysis of SRL in patient samples is possible with satisfactory sensitivity and precision using LC-MS/MS without prior SPE.

Keywords: Sirolimus, Chromatography, analysis

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Annals of Transplantation eISSN: 2329-0358
Annals of Transplantation eISSN: 2329-0358