Accessible Requires Authentication Published by De Gruyter August 19, 2015

Comparing the effect of isotopically labeled or structural analog internal standards on the performance of a LC-MS/MS method to determine ciclosporin A, everolimus, sirolimus and tacrolimus in whole blood

Henar Valbuena, Maria Shipkova, Sophie-Maria Kliesch, Simon Müller and Eberhard Wieland


Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used for analysis of immunosuppressive drugs. This study investigated whether replacing analog internal standards (ANISs) with isotopically labeled internal standards (ILISs) has an impact on the performance of a LC-MS/MS method for the quantification of tacrolimus (TAC), sirolimus (SIR), ciclosporin A (CsA) and everolimus (EVE) in whole blood.

Methods: Following hemolysis, protein precipitation, and extraction with either ANISs (ascomycin, desmethoxy-rapamycin, CsD), or ILISs (TAC-13C,D2; SIR-13C,D3; CsA-D12; EVE-D4), samples were centrifuged and injected into a LC-MS/MS device equipped with a C18 reversed phase column. The effect of the two ISs on the linearity, precision, accuracy, trueness, matrix effects, and carryover was investigated by using the same patient-, proficiency testing-, and quality control samples. Statistical analysis of agreement between results includes a standard random effects model and Passing-Bablok regression.

Results: Within-day imprecision was <10%, between-day <8%, and trueness 91%–110% for all the analytes with both ISs. No carryover or matrix effects were observed. The median accuracy was −2.1% for CsA, 9.1% for EVE, 12.2% for SIR, and −1.2% for TAC with the ILISs; and −2% for CsA, 9.8% for EVE, 11.4% for SIR, and 0.2% for TAC with the ANISs. Results of patient and proficiency testing samples were not statistically different.

Conclusions: Although ILISs are generally considered superior to ANISs, they may not be always essential. When optimizing a LC-MS/MS method other factors must be also considered.

Corresponding author: Henar Valbuena, Department of Clinical Chemistry, Vall d’Hebron University Hospital. Barcelona, Spain, Fax: +34 933868198, E-mail:


Henar Valbuena was awarded with an Exchange Programme Fellowship from Fundación José Luis Castaño to complete her scientific education and be able to take part in this project.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.


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Supplemental Material:

The online version of this article (DOI: 10.1515/cclm-2015-0519) offers supplementary material, available to authorized users.

Received: 2015-6-4
Accepted: 2015-6-30
Published Online: 2015-8-19
Published in Print: 2016-3-1

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