Simplified high-performance liquid chromatography-mass spectrometry assay for measurement of tacrolimus and its metabolites and cross-validation with microparticle enzyme immunoassay.
In this study, a modified, specific assay for measurement of tacrolimus and its metabolites in blood and urine from transplant patients using high-performance liquid chromatography (HPLC) linked to mass spectrometry (MS) is described. Samples were prepared for HPLC-MS by modified solid-liquid extraction. The original two-step washing procedure was replaced by a single washing step, and samples were eluted with acetonitrile/water instead of dichloromethane, thus avoiding an evaporation step. Samples were injected automatically every 3 min into the HPLC-MS system. Time-consuming gradient elution was replaced by isocratic elution. This procedure resulted in a lower limit of quantitation of 0.2 microgram/L. The interassay variability was 14.5% for 5 micrograms/L and 15.8% for 25 micrograms/L. The intrassay variability was 11.2% for 5 micrograms/L and 4% for 25 micrograms/L. The recovery for tacrolimus in blood was 90.4% for 1 microgram/L, 78.9% for 10 micrograms/L, and 81.3% for 25 micrograms/L. Measurement of tacrolimus and its metabolites in samples from various transplant patients showed that the main metabolites found in blood and urine are demethyl-tacrolimus, di-demethyl-tacrolimus and demethyl-hydroxy-tacrolimus. Cross validation of the modified HPLC-MS assay with a microparticle enzyme immunoassay showed a significant correlation between the two assays, with r = 0.915.
Gonschior
A K
AK
Institüt für Allgemeine Pharmakologie, Medizinische Hochschule Hannover, Germany.
Christians
U
U
Winkler
M
M
Schiebel
H M
HM
Linck
A
A
Sewing
K F
KF
D000097:Acetonitriles / Q000737:chemistry; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D007166:Immunosuppressive Agents / Q000097:blood* / Q000378:metabolism / Q000652:urine; D016030:Kidney Transplantation / Q000276:immunology; D016031:Liver Transplantation / Q000276:immunology; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000097:blood* / Q000378:metabolism / Q000652:urine; D014867:Water / Q000737:chemistry
C098104:32-O-acetyltacrolimus; D000097:Acetonitriles; D007166:Immunosuppressive Agents; D014867:Water; D016559:Tacrolimus; C032159:acetonitrile