Background: A bioalanytical method for the quantification of tacrolimus (TAC) and 3 metabolites, 13-O, 15-O, and 31-O-demethylated TAC (M-I, M-III, and M-II) in human whole blood using liquid chromatography, electrospray ionization, tandem mass spectrometry (LC–ESI–MS/MS) was developed and validated.
Method: The analytes were extracted from 85 μL of blood by protein precipitation followed by solid-phase extraction and a concentration step. The analytes and the internal standard (IS, ascomycin) were separated on a C18 column using a slow gradient mobile phase elution, with an analysis time of 3.3 minutes. The ammonium-adduct ions with transitions of m/z 821.5 > 768.7 (TAC), 807.5 > 754.7 (M-I, M-III, M-II), and 809.4 > 756.7 (IS) were measured in selected reaction monitoring mode using electrospray ionization.
Results: Measuring ranges were 0.1–50 ng/mL for M-II, M-III, and TAC and 0.15–39 ng/mL for M-I. Imprecision in quantification was <20% for all analytes, whereas accuracy was within ±20%. Recovery was calculated to be >50% for all analytes. The sample's stability was proven for 1 month at −20°C and 72 hours at room temperature. Three freeze–thaw cycles had no significant effect on the stability. The prepared samples were stable at least 16 hours at 8°C. Analysis of 53 patient samples resulted in average concentrations of 7.2 for TAC, 0.8 for M-I, 0.4 for M-III, and 0.2 ng/mL for M-II. The total metabolite concentration was 17% (4%–52%) of the TAC concentration. The TAC concentration measured by LC–MS/MS was 36.1% ± 27.1% lower than by immunochemical (enzyme multiplied immunoassay technique) analysis. When adding the metabolite crossreactivity in the presence of TAC, the difference between the 2 methods was still 29.8% ± 28.3%, indicating that the overestimation of TAC concentration of enzyme multiplied immunoassay technique compared with liquid chromatography–tandem mass spectrometry cannot only be ascribed to the demethylated metabolites.
Conclusions: An LC–ESI–MS/MS method for the quantitative analysis of TAC and 3 metabolites, using a 2-step sample preparation was successfully developed, validated, and applied on 53 patient samples.
*Laboratory for TDM and Clinical Toxicology, Department of Pharmacy, University Medical Center Groningen, Groningen, The Netherlands
†Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institutet, Karolinska University Hospital Huddinge
‡Department of Medicine, Division of Clinical Pharmacology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden.
The authors declare no conflicts of interest.
Correspondence: Anton Pohanka, PhD, Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden (e-mail: email@example.com).
Received September 23, 2011
Accepted January 13, 2012