Highly sensitive and rapid determination of tacrolimus in peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry.
Biomed Chromatogr
; 33(1): e4416, 2019 Jan.
Article
in En
| MEDLINE
| ID: mdl-30362145
ABSTRACT
After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC-MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 µL containing 1 million PBMCs was used in combination with MagSiMUS-TDMPREP . To each sample we added 30 µL lysis buffer, 20 µL reconstitution buffer containing 13 C2 H4 -tacrolimus as internal standard, 40 µL MagSiMUS-TDMPREP Type I Particle Mix and 175 µL Organic Precipitation Reagent VI for methanol-based protein precipitation. A 10 µL aliquot of the supernatant was injected into the UPLC-MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r2 = 0.997) over the range 5.0-1250 pg/1 × 106 PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Leukocytes, Mononuclear
/
Chromatography, Liquid
/
Tacrolimus
/
Tandem Mass Spectrometry
Type of study:
Diagnostic_studies
/
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
Biomed Chromatogr
Year:
2019
Document type:
Article
Affiliation country:
Netherlands