Development and validation of a combined enzymatic-digestion/mass spectrometry assay for Tacrolimus quantitation in cardiac biopsies.
J Chromatogr B Analyt Technol Biomed Life Sci
; 1152: 122215, 2020 Sep 01.
Article
em En
| MEDLINE
| ID: mdl-32615534
ABSTRACT
Recent studies report strategies for analysing immunosuppressive drugs in brain, liver and renal tissue, mostly in animals we developed and validated a two steps combined enzymatic digestion/mass spectrometry assay to quantify Tacrolimus (TAC) in heart biopsies. Our aims were to avoid sample loss and sample contamination during the laboratory preparation, and to limit matrix effects in the electrospray ionization source (ESI) of the mass spectrometer. Enzymatic tissue digestion followed by a liquid-liquid drug extraction in the same vial of reaction allowed us to reach both our aims. The assay was assessed for selectivity, matrix effect, linearity, Lower Limit of Quantification (LLOQ) and Detection (LOD), accuracy and precision, according to the "Guideline on Bioanalytical Method Validation (EMA). A stable isotopically labelled (SIL) analogue (13CD2-TAC) was used as internal standard. The chromatographic separation of the analyte took 6 min. The observed linear range of quantification was 0.0162-0.520 ng in terms of TAC added to the biopsies (by 50 µL of the corresponding working solutions). The limit of detection and the lower limit of quantification (LLOQ) were 0.008 and 0.0162 ng, respectively. Both the mobile phases contained ammonium acetate and formic acid that promote the formation of ammoniated precursor ions that can be easily fragmented ([M + NH4]+, TAC m/z 821.3; 13CD2-TAC m/z 824.3). The calibration curves were generated by plotting analyte-to-internal standard peak area ratios versus TAC amount (ng) added to the biopsies, and using a weighted (1/x) linear regression. Curves were not forced to pass through the origin. Swine hearts were employed as blank matrix for all the analytical method validation procedures but, after approval by the ethics committee (by "Fondazione IRCCS Policlinico San Matteo" Protocol 20190032933), TAC was also quantified in endomyocardial biopsies from informed and consenting heart transplant patients. The study was funded by Fondazione IRCCS Policlinico San Matteo (RC08017617), as a part of the clinical studies on the maintenance of immunosuppressive therapy in cardiac transplant patients. Tacrolimus concentrations in patients biopsies were expressed as ratio between the detected amount of TAC (ng) in the tissue and the weight of the tissue itself (mg).
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Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
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Biópsia
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Tacrolimo
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Imunossupressores
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Miocárdio
Tipo de estudo:
Guideline
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Prognostic_studies
Aspecto:
Ethics
Limite:
Animals
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Humans
Idioma:
En
Revista:
J Chromatogr B Analyt Technol Biomed Life Sci
Ano de publicação:
2020
Tipo de documento:
Article