| Full text | |
| Author(s): Show less - |
Romano, Paschoalina
[1, 2]
;
Fernandes, Maria da Luz
[2]
;
Rezende Ebner, Persio de Almeida
[1]
;
de Oliveira, Nayara Duarte
[2]
;
Okuda, Larissa Mitsue
[2]
;
Agena, Fabiana
[2]
;
Mendes, Maria Elizabete
[1]
;
Sumita, Nairo Massakazu
[1]
;
Coelho, Veronica
[3]
;
David-Neto, Elias
[2]
;
Galante, Nelson Zocoler
[2]
Total Authors: 11
|
| Affiliation: | [1] Univ Sao Paulo, Sch Med, Cent Lab Div LIM 3, Hosp Clin, Sao Paulo, SP - Brazil
[2] Univ Sao Paulo, Sch Med, Hosp Clin, Kidney Transplant Serv, Sao Paulo, SP - Brazil
[3] Univ Sao Paulo, Sch Med, Lab Immunol LIM 19, Heart Inst, Sao Paulo, SP - Brazil
Total Affiliations: 3
|
| Document type: | Journal article |
| Source: | Journal of Pharmaceutical and Biomedical Analysis; v. 152, p. 306-314, APR 15 2018. |
| Web of Science Citations: | 3 |
| Abstract | |
Monitoring tacrolimus (Tac) exposure in cell matrices enriched with lymphocytes can improve Tac therapeutic drug monitoring (TDM) in solid organ transplant recipients. An UPLC-MS/MS based assay for Tac quantification in peripheral blood T CD4+ and B CD19+ lymphocytes was developed. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and highly purified (purity >90%) T CD4+ and B CD19+ cell suspensions were acquired by magnetic negative selection from whole blood of 6 healthy volunteers. The purity of lymphocyte suspensions was checked by flow cytometry. Tac extraction was performed in a liquid-liquid zinc sulfate, methanol and acetonitrile based medium. Ascomycin was used as internal standard. The equipment used was a Waters (R) Acquity (TM) UPLC system (Waters Corporation, Milford, MA, USA). The chromatographic run was performed on a Waters (R) MassTrak TDM C18 (2.1 x 10 mm) column (Waters Corporation, Milford, MA, USA). at a flow rate of 0.4 mL/min. The instrument was set in electrospray positive ionization mode. The method was validated according to Clinical Laboratory Standard Institute (CLSI) guidelines and showed a high sensitivity and specificity over a range of 0-5.2 ng/mL in PBMC, 0-5.0 ng/mL in T CD4+ Lymphocytes and 0-5.3 ng/mL in B CD19+ lymphocytes. Precision was appropriate with CV of intra-assay quantifications ranging from 4.9 to 7.4%, and of inter-assay quantifications from 7.2 to 13.9%. Limit of detection and quantification were 0.100 and 0.115 ng/mL in PBMC, 0.058 and 0.109 ng/mL in T CD4+ and 0.017 and 0.150 ng/mL in B CD19+ cells. Matrix effect was not significant among all the studied matrices. Samples showed stability for Tac quantification over a period of 90 days either at room temperature or at -30 degrees C storage conditions. The method was applied to clinical samples of 20 kidney transplant recipients. Concentrations ranged from 2.200 to 11.900 ng/mL in whole blood, from 0.005 to 0.570 ng/10(6) cells in PBMC, from 0.081 to 1.432 ng/10(6) cells in T CD4+, and from 0.197 to 1.564 ng/10(6) cells in B CD19+ cell matrices. The method has potential applicability for Tac TDM in solid organ transplant recipients. (C) 2018 Elsevier B.V. All rights reserved. (AU) | |
| FAPESP's process: | 16/10083-9 - Optimization of the magnetic negative selection purification technique of peripheral blood T CD4+ and B lymphocyte with analysis of cell viability, yield and purity with flow cytometry |
| Grantee: | Larissa Mitsue Okuda |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| FAPESP's process: | 16/10043-7 - Validation of an ultra high performance liquid chromatography - mass spectrometry assay for tacrolimus quantification in matrices of peripheral blood T (CD4+) and B lymphocytes |
| Grantee: | Nayara Duarte de Oliveira |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| FAPESP's process: | 15/11389-1 - Monitoring intracellular concentration and pharmacodynamic effects of tacrolimus in peripheral blood T CD4+ and B lymphocytes of kidney transplant recipients |
| Grantee: | Nelson Zocoler Galante |
| Support Opportunities: | Regular Research Grants |