UPLC-MS/MS assay validation for tacrolimus quantitative determination in peripheral blood T CD4+and B CD19+lymphocytes

Autor(es):Mostrar menos -

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]

Afiliação do(s) autor(es)

^[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

• Tipo de documento: Artigo Científico
• Fonte: Journal of Pharmaceutical and Biomedical Analysis; v. 152, p. 306-314, APR 15 2018.
• Citações Web of Science: 3

Resumo

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)

• Processo FAPESP: 16/10083-9 - Otimização da técnica de purificação de populações de Linfócitos T CD4+ e b do sangue periférico por seleção magnética negativa com análise da viabilidade celular, recuperação, e pureza por citometria de fluxo
• Beneficiário: Larissa Mitsue Okuda
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 16/10043-7 - Validação do ensaio de cromatografia líquida de ultra alta eficiência acoplada a espectrometria de massa em dois estágios para quantificação de tacrolimo em matrizes de Linfócitos T (CD4+) e b do sangue periférico
• Beneficiário: Nayara Duarte de Oliveira
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 15/11389-1 - Monitoramento das concentrações intracelulares e dos efeitos farmacológicos do tacrolimo em linfócitos t (cd4+) e b do sangue periférico de receptores de transplante renal
• Beneficiário: Nelson Zocoler Galante
• Modalidade de apoio: Auxílio à Pesquisa - Regular