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.
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.
Henar
H
Valbuena
Maria
M
Shipkova
Sophie-Maria
SM
Kliesch
Simon
S
Müller
Eberhard
E
Wieland
D001774:Blood Chemical Analysis / Q000592:standards*; D002853:Chromatography, Liquid / Q000592:standards*; D016572:Cyclosporine / Q000097:blood; D000068338:Everolimus / Q000097:blood; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D007553:Isotope Labeling / Q000592:standards*; D020123:Sirolimus / Q000097:blood; D016559:Tacrolimus / Q000097:blood; D053719:Tandem Mass Spectrometry / Q000592:standards*
D007166:Immunosuppressive Agents; D016572:Cyclosporine; D000068338:Everolimus; D020123:Sirolimus; D016559:Tacrolimus