5. Deuterium Labelled Sirolimus: Synthesis and Assessment as Internal Standard in a New LC/MS/MS Method
Tue 12:12 PM - PosterSplash Track 1
Kun-Young Sohn
McMaster University
Kun-Young Sohn and Johannes Zeidler.

Department of Pathology and Molecular Medicine, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S4L8, Canada
We report 1) a new method to quantitate the immunosuppressant drug Sirolimus, 2) the feasibility of preparing deuterated Sirolimus (Sirolimus-d3) in a clinical lab and 3) improved accuracy and precision with Sirolimus-d3 vs Ascomycin as internal standard in LC/MS/MS.

1) Sirolimus was enriched from whole blood by solid phase extraction after protein precipitation with ZnSO4 and acetonitrile and quantified with a Varian 1200L triple quadrupole LC/MS/MS system as ammonium adduct by a step gradient elution from a C18 guard column.

2) Sirolimus-d3 was prepared from unlabeled Sirolimus in one overnight step. The reaction can easily be performed by non-chemists in analytical labs. The product costs much less than the now commercially available deuterated drug and can be used without purification as internal standard.

3) The performance of Sirolimus-d3 as internal standard was compared to that of Ascomycin. All patient, quality control and proficiency samples submitted to analysis during 2008 were extracted after addition of Ascomycin and Sirolimus-d3. Data were collected for both internal standards, but only Sirolimus-d3 was used for reporting patient results. The comparison showed three main outcomes: A) Patient results were not significantly different on average with Ascomycin vs Sirolimus-d3. B) Precision and accuracy of quality control samples at three concentrations were much better with Sirolimus-d3 than with Ascomycin. A combined proportional and constant bias of opposite signs were found when comparing ascomycin with Sirolimus-d3. C) International proficiency testing results calculated with Sirolimus-d3 were consistently higher and gave an accuracy of 87% of the method mean compared to 66% with Ascomycin.

Summary: We show for the first time that the deuterated internal standard Sirolimus-d3 can easily and inexpensively be prepared without a synthetic chemistry laboratory according to a published procedure. Our in-house LC/MS/MS method for the quantitation of Sirolimus in whole blood showed better precision and accuracy with Sirolimus-d3 as internal standard than with ascomycin in quality control and proficiency samples. For patient samples no difference in accuracy was apparent between the two internal standards. Matrix effects in LC/MS/MS can be different for patient samples, quality control samples and proficiency testing samples.