Amedeo De Nicolò (Presenter)
University of Turin
Bio: I work in the laboratory of clinical pharmacology and pharmacogenetics of the University of Turin, focusing on the therapeutic drug monitoring of several drugs, including antivirals, antihypertensives, immunosuppressors and endogenous compounds. I am currently a PhD student in Medicine and Experimental Therapy at the University of Turin.
Authorship: Amedeo De Nicolò (1), Debora Pensi (1), Michele Pinon (2), Clarissa Pisciotta (1), Pier Luigi Calvo (2), Antonello Nonnato (4), Renato Romagnoli (3), Francesco Tandoi (3) , Giovanni Di Perri (1) and Antonio D'Avolio (1).
(1) Unit of Infectious Diseases, University of Turin, Italy (2) Unit of Pediatric Gastroenterology and Hepatology, University of Turin, Italy (3) Liver Transpl. Center and Gen. Surgery 2U, University of Turin, Italy (4) Clin. Biochem. Unit, A.O.U. "Città della Salute e della Scienza", Turin, Italy.
| Short Abstract Although the use of tacrolimus and everolimus is currently guided through TDM in whole blood, this does not necessarily reflect concentrations in lymphocytes. In this work we describe a method for their dosage in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood with CPT vacutainers, washed twice and lysed. Drugs were extracted from cell lysates with on-line SPE platform and then separated and detected through reverse-phase UPLC-MS/MS. Validation parameters successfully fitted FDA and EMA guidelines and samples from pediatric patients resulted within calibration range. This method results eligible for use on real PBMC samples from pediatric patients. |
Long Abstract
Introduction: Nowadays the use of tacrolimus (TAC) and everolimus (EVE) to avoid graft rejection is widespread. Dose adjustments during treatment are currently guided through therapeutic drug monitoring (TDM) in whole blood. Since considerable inter-patient differences exist in blood composition (eg. hematocrit), whole blood concentrations do not necessarily reflect drug exposure in lymphocytes.
In this work an analytical method was developed for the simultaneous quantification of TAC and EVE in peripheral blood mononuclear cells (PBMCs), the actual target of treatment. Moreover, since up to now no formula is currently available for the evaluation of the correcting power of not-deuterated internal standards (IS) on analytes matrix effect, we proposed a formula for the calculation of the “IS-normalized” matrix effect.
Experimental: The isolation of PBMCs was performed through CPT vacutainers, centrifuged at 1600 g. Cell pellets were washed twice with NaCl 0.9% and counted with an automated cell coulter, determining cell number and mean cell volume (MCV). Cell extracts were lysed with water:methanol 30:70 and stored at -80°C.
For sample analysis, 100 ìL of calibrating solutions (containing specific TAC and EVE concentrations to spike standards and quality controls and only water:methanol for patients samples) and 40 ìL of internal standard solution (1000 ng/mL ascomycin in water:methanol 50:50) were added to cell extracts, then, after centrifugation at 21000 g, supernatants were collected in glass vials for the analysis. Drugs extraction was performed through an automated on-line SPE system (OSM®, Waters, Milan) with X-Bridge ® C8 10 ìm, 1 x 10 mm cartridges. The elution of cartridges was performed directly through the flow of mobile phases A and B (Water and methanol, respectively, both with ammonium acetate 2 mM + formic acid 0.1%) and peak separation was obtained through a gradient run, with a BEH C18 1.7 ìm, 50 x 2.1 mm column, at 45° for 6 minutes.
Method validation was conducted in accordance with FDA and EMA guidelines.
Besides the usual evaluation of matrix effect (post-extraction addition method), in this work we also evaluated the “IS-normalized” matrix effect, based on the formula nEM ={[(PAA-matrix/PAIS-matrix)/(PAA-neat/PAIS-neat)-1]*100}, where PA is peak area of the analyte (PAA-matrix or PAA-neat) and of the IS (PAIS-matrix or PAIS-neat). The RSD (or CV%) of this parameter was calculated considering the variability of [(PAA-matrix/PAIS-matrix)/(PAA-neat/PAIS-neat)*100].
Recovery of SPE was evaluated through the dedicated “advanced method development” function of the OSM, while the recovery of the pre-SPE steps was evaluated by comparing post-extraction spiked with pre-extraction spiked samples.
The method was tested on PBMC samples from 5 pediatric patients co-treated with TAC and EVE. Drugs amount have been converted in concentrations through the formula [Drug C] = Drug amount/(Cell number x MCV].
Results: Intra-day and inter-day precision and accuracies resulted all within the limits of acceptance indicated by FDA and EMA guidelines.
The lower limit of detection (LLOD) for TAC and EVE were 0.0195 ng and 0.0190 ng, respectively, while the lower limit of quantification (LLOQ) were 0.0975 ng and 0.039 ng, respectively.
Non interfering peaks were observed at the analytes retention times by testing 10 different blank PBMC lots.
Mean recoveries for TAC, EVE and IS were all above 91% and stable for all compounds (RSD <4.70%). Mean matrix effects for TAC, EVE and IS were -29.48% (RSD 3.35%), +17.45% (RSD 1.67%) and -31.71% (RSD 8.00%), respectively. Matrix effect was also evaluated at different cell concentrations (from 3x106 to 24x106 cell/mL). The determined mean RSD% of all QCs amounts was +13.6% for EVE and +5.9% for TAC. Considering only population lower than 12x106cell/mL, this value became 11.2% for EVE. Therefore, the validation was made with samples of 12x106 cell/mL and samples were diluted to this cells number.
The “IS-normalized” matrix effect resulted lower for TAC (mean 3.90%, RSD 9.50%) and high, but still highly reproducible, for EVE (mean 72.81%, RSD 8.39%).
All tested samples from patients resulted within the ranges of the calibration curve.
Interestingly, TAC and EVE concentrations within PBMCs resulted respectively 19.23 and 218.61 times higher than the hematic ones.
Results from tests on real samples evidenced a good correlation between intra-PBMC and whole blood concentrations for both TAC (r2=0.773, P= 0.05, not strictly tight) and EVE (r2=0.971, P= 0.002).
Discussion: In this work an analytical method was developed capable of successfully quantifying TAC and EVE concentrations in PBMC extracts, characterized by the use of automated on-line SPE to increase the injection volume (50 ìL), without increasing matrix effect. The adoption of on-line SPE allowed to quantify very low drugs concentrations, limiting the manual preparation steps to the spiking of standards and QCs and the addition of IS.
Furthermore, in this work we introduced a model for evaluating “IS-normalized” matrix effect: this parameter considers the effect of matrix effect in each sample for both IS and target analyte and represents the capability of the IS of successfully counterbalancing the variability in analytical performance due to matrix effect.
In analytical methods which use calibration curves prepared in matrix and not-deuterated IS, the mean value of “IS-normalized” matrix effect is successfully corrected by the matrix effect of the calibration curve, while the real amount of bias is due to its variability among different matrix lots (different samples and different blank PBMCs from healthy donors), expressed as the RSD%.
In our method “IS-normalized” matrix effect resulted very low and highly reproducible for TAC, confirming ascomycin as a very good IS for TAC, while the mean value resulted very high for EVE. However, its variability (RSD%) was very low (8.39%), indicating that the calibration curve prepared in matrix is capable of correcting the error due to matrix effect.
Conclusion: The presented method is capable of quantifying TAC and EVE in their active site. This could be useful as a “second-level” test in doubtful cases, in which the determination of whole blood concentrations seems to not reflect the clinical behavior of patients. The use of a relatively cheap IS and of an automated SPE platform allows obtaining precise results in a relatively fast and cheap way, making this method useful also in a clinical routine setting.
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