Validation of an LC-MS/MS method to measure tacrolimus in rat kidney and liver tissue and its application to human kidney biopsies.
BACKGROUND
Tacrolimus (TAC) has a narrow therapeutic index and high interindividual and intraindividual pharmacokinetic variability, necessitating therapeutic drug monitoring to individualize dosage. Recent evidence suggests that intragraft TAC concentrations may better predict transplant outcomes. This study aimed to develop a method for the quantification of TAC in small biopsy-sized samples of rat kidney and liver tissue, which could be applied to clinical biopsy samples from kidney transplant recipients.


METHODS
Kidneys and livers were harvested from Mrp2-deficient TR- Wistar rats administered TAC (4 mg·kg·d for 14 days, n = 8) or vehicle (n = 10). Tissue samples (0.20-1.00 mg of dry weight) were solubilized enzymatically and underwent liquid-liquid extraction before analysis by liquid chromatography tandem mass spectrometry method. TAC-free tissue was used in the calibrator and quality control samples. Analyte detection was accomplished using positive electrospray ionization (TAC: m/z 821.5 → 768.6; internal standard ascomycin m/z 809.3 → 756.4).


RESULTS
Calibration curves (0.04-2.6 μg/L) were linear (R > 0.99, n = 10), with interday and intraday calibrator coefficients of variation and bias <17% at the lower limit of quantification and <15% at all other concentrations (n = 6-10). Extraction efficiencies for TAC and ascomycin were approximately 70%, and matrix effects were minimal. Rat kidney TAC concentrations were higher (range 109-190 pg/mg tissue) than those in the liver (range 22-53 pg/mg of tissue), with median tissue/blood concentrations ratios of 72.0 and 17.6, respectively. In 2 transplant patients, kidney TAC concentrations ranged from 119 to 285 pg/mg of tissue and were approximately 20 times higher than whole blood trough TAC concentrations.


CONCLUSIONS
The method displayed precision and accuracy suitable for application to TAC measurement in human kidney biopsy tissue.
*Department of Clinical Pharmacology, The Queen Elizabeth Hospital, Woodville; †Discipline of Pharmacology, The University of Adelaide, Adelaide; ‡Central Northern Adelaide Renal Transplant Service, The Royal Adelaide Hospital, Adelaide, Australia; Departments of §Internal Medicine and ¶Hospital Pharmacy, Erasmus University Medical Centre, Rotterdam, the Netherlands.
Benjamin D
BD
Noll
Janet K
JK
Coller
Andrew A
AA
Somogyi
Raymond G
RG
Morris
Graeme R
GR
Russ
Dennis A
DA
Hesselink
Teun
T
Van Gelder
Benedetta C
BC
Sallustio
D000818:Animals; D001706:Biopsy; D002853:Chromatography, Liquid / Q000379:methods; D016903:Drug Monitoring; D006084:Graft Rejection / Q000378:metabolism / Q000517:prevention & control*; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry* / Q000493:pharmacokinetics*; D007668:Kidney / Q000737:chemistry* / Q000378:metabolism; D016030:Kidney Transplantation; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D016559:Tacrolimus / Q000737:chemistry* / Q000493:pharmacokinetics*; D053719:Tandem Mass Spectrometry / Q000379:methods
D007166:Immunosuppressive Agents; D016559:Tacrolimus