Background
Anticancer drugs are notoriously characterized by a low therapeutic index, the introduction of therapeutic drug monitoring (TDM) in oncologic clinical practice could therefore be ...fundamental to improve treatment efficacy. In this context, an attractive technique to overcome the conventional venous sampling limits and simplify TDM application is represented by dried blood spot (DBS). Despite the significant progress made in bioanalysis exploiting DBS, there is still the need to tackle some challenges that limit the application of this technology: one of the main issues is the comparison of drug concentrations obtained from DBS with those obtained from reference matrix (e.g., plasma). In fact, the use of DBS assays to estimate plasma concentrations is highly dependent on the chemical-physical characteristics of the measured analyte, in particular on how these properties determine the drug partition in whole blood.
Methods
In the present review, we introduce a critical investigation of the DBS-to-plasma concentration conversion methods proposed in the last ten years and applied to quantitative bioanalysis of anticancer drugs in DBS matrix. To prove the concordance between DBS and plasma concentration, the results of statistical tests applied and the presence or absence of trends or biases were also considered.
A novel LC-MS/MS method was developed for the quantification of the new cyclin dependent kinase inhibitors (CDKIs) palbociclib and ribociclib and the aromatase inhibitor letrozole used in combinatory ...regimen. The proposed method is appropriate to be applied in clinical practice due to the simple and fast sample preparation based on protein precipitation, the low amount of patient sample necessary for the analysis (10 μL) and the total run time of 6.5 min. It was fully validated according to FDA and EMA guidelines on bioanalytical method validation. The linearity was assessed (R2 within 0.9992-0.9983) over the concentration ranges of 0.3-250 ng/mL for palbociclib, 10-10000 ng/mL for ribociclib and 0.5-500 ng/mL for letrozole that properly cover the therapeutic plasma concentrations. A specific strategy was implemented to reduce the carryover phenomenon, formerly known for these CDKIs. This method was applied to quantify the Cmin of palbociclib, ribociclib and letrozole in plasma samples from patients enrolled in a clinical study. The same set of study samples was analysed twice in separate runs to assess the reproducibility of the method by means of the incurred samples reanalysis. The results corroborated the reliability of the analyte concentrations obtained with the bioanalytical method, already proved by the validation process. The percentage differences were always within ±10% for all the analytes and the R2 of the correlation graph between the two quantifications was equal to 0.9994.
Lenvatinib (LENVA) is an oral antineoplastic drug used for the treatment of hepatocellular carcinoma and thyroid carcinoma. LENVA therapeutic drug monitoring (TDM) should be mandatory for a precision ...medicine to optimize the drug dosage. To this end, the development of a sensitive and robust quantification method to be applied in the clinical setting is essential. The aim of this work was to develop and validate a sensitive, rapid, and cost-effective LC-MS/MS method for the quantification of LENVA in human plasma. On this premise, sample preparation was based on a protein precipitation and the chromatographic separation was achieved on a Synergi Fusion RP C18 column in 4 min. The method was completely and successfully validated according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines, with good linearity in the range of 0.50-2000 ng/mL (R≥0.9968). Coefficient of variation (CV) for intra- and inter-day precision was ≤11.3% and accuracy ranged from 96.3 to 109.0%, internal standard normalized matrix effect CV% was ≤2.8% and recovery was ≥95.6%. Successful results were obtained for sensitivity (signal to noise (S/N) ratio >21) and selectivity, dilution integrity (CV% ≤ 4.0% and accuracy 99.9-102%), and analyte stability under various handling and storage conditions both in matrix and solvents. This method was applied to quantify LENVA in patient's plasma samples and covered the concentration range achievable in patients. In conclusion, a sensitive and robust quantification method was developed and validated to be applied in the clinical setting.
The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), ...strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50-7500 ng/mL for imatinib and 10-1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 μL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 μm, 2x50 mm) while the trapping column was a POROS R1/20 (20 μm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients' finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.
Gold nanoparticles (AuNPs) covered with mixtures of immiscible ligands present potentially anisotropic surfaces that can modulate their interactions at complex nano–bio interfaces. Mixed, ...self‐assembled, monolayer (SAM)‐protected AuNPs, prepared with incompatible hydrocarbon and fluorocarbon amphiphilic ligands, are used here to probe the molecular basis of surface phase separation and disclose the role of fluorinated ligands on the interaction with lipid model membranes and cells, by integrating in silico and experimental approaches. These results indicate that the presence of fluorinated amphiphilic ligands enhances the membrane binding ability and cellular uptake of gold nanoparticles with respect to those coated only with hydrogenated amphiphilic ligands. For mixed monolayers, computational results suggest that ligand phase separation occurs on the gold surface, and the resulting anisotropy affects the number of contacts and adhesion energies with a membrane bilayer. This reflects in a diverse membrane interaction for NPs with different surface morphologies, as determined by surface plasmon resonance, as well as differential effects on cells, as observed by flow cytometry and confocal microscopy. Overall, limited changes in monolayer features can significantly affect NP surface interfacial properties, which, in turn, affect the interaction of SAM‐AuNPs with cellular membranes and subsequent effects on cells.
The structure that matters. Patterning of gold nanoparticles due to self‐assembling monolayers of amphiphilic immiscible ligands determines their behavior at the nano–bio interface.
Fluorescent, imprinted nanosized polymers for the detection of irinotecan have been synthesised using a napthalimide polymerisable derivative (2-allyl-6-2-(aminoethyl)-amino napthalimide) as ...functional monomer. The imprinted polymers contain ethylene glycol dimethacrylate (EGDMA) as a cross-linker and were prepared by high dilution radical polymerisation in dimethylsulphoxide (DMSO). The material was able to rebind irinotecan up to 18 nmol/mg with good specificity. Fluorescence emission at 525 nm (excitation at 448 nm) was quenched by increasing concentrations of irinotecan via a static mechanism and also in analytically useful environments as mixtures of human plasma and organic solvents. This allowed the direct detection of irinotecan (in the 10–30 μM range) in human plasma treated with acetonitrile; the limit of detection (LOD) was 9.4 nM, with within-run variability of 10% and day-to-day variability of 13%.
•Dried Blood Spot (DBS) can overcome the limits of traditional sampling for TDM.•A DBS-based LC-MS method for palbociclib, ribociclib, and letrozole was developed.•The method was applied to analyze ...patient DBS samples.•DBS-to-plasma conversion was performed using different strategies.
Therapeutic drug monitoring (TDM) is strongly suggested to define the proper drug dosage to overcome inter- and intra-patient variability in drug exposure, which is typically observed with oral anticancer agents, such as palbociclib (PALBO), ribociclib (RIBO) and letrozole (LETRO), all approved for the treatment of HR+, HER2- locally advanced or metastatic breast cancer (BC). Optimal TDM implementation requires a blood sampling organization that can be hampered by limited availability of health and laboratory personnel. Dried Blood Spot (DBS) sampling is proposed to overcome such limitations. The aim of this work was the development of a new LC-MS/MS method to analyze DBS samples containing PALBO, RIBO, and LETRO. Analytes extraction from DBS was performed by adding a methanolic solution containing the corresponding internal standards. LC-MS/MS analysis was performed using a LC Nexera (Shimadzu) system coupled with an API 4000 QTrap (SCIEX) mass spectrometer. The chromatographic separation was performed on a Luna Omega Polar C18 column (Phenomenex). The method was applied to 38 clinical samples collected by finger prick. The influence of hematocrit and spot size, sample homogeneity, stability, and correlation between finger prick and venous DBS measurement were assessed. The analytical validation was performed according to EMA and FDA guidelines.
The analytical range of the method was 1 to 250 ng/mL for PALBO, 40 to 10000 ng/mL for RIBO, and 2 to 500 ng/mL for LETRO, where linearity was assessed, obtaining mean coefficients of determination (R2) of 0.9979 for PALBO, 0.9980 for RIBO, and 0.9987 for LETRO). The LC-MS/MS method runtime was 6.6 min. Incurred sample reanalysis demonstrated reproducibility, as the percentage difference between the two quantifications was lower than 20% for 100% of PALBO, 81.8% of RIBO, and 90.9% of LETRO paired samples. Intra- and inter-day precision (CV (%)) was lower than 11.4% and intra- and inter-day accuracy was between 90.0 and 106.5%. DBS sample stability at room temperature was confirmed for 2.5 months. A positive correlation was observed between DBS and plasma concentrations for the 3 drugs, Lin’s concordance correlation coefficients obtained by DBS normalization applying a selected strategy were 0.958 for PALBO, 0.957 for RIBO, and 0.963 for LETRO. In conclusion, a fast, easy, and reproducible DBS LC-MS/MS method for the simultaneous quantification of palbociclib; ribociclib and letrozole was developed to be used in clinical practice.
•LC–MS/MS method simultaneously quantifies sorafenib, regorafenib and metabolites.•FDA-EMA guided validation demonstrated the robustness and reliability of the method.•This method is applicable to ...the clinic: short run time and easy sample preparation.•Therapeutic monitoring of sorafenib and regorafenib was conducted on 10 HCC patients.
A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous quantification of sorafenib (SORA), its N-oxide active metabolite and of regorafenib (REGO) and its two active metabolites regorafenib N-oxide and N-desmethyl regorafenib N-oxide in hepatocellular carcinoma patients’ plasma.
A proper analytes’ separation was obtained with Synergi Fusion RP column (4 μm, 80 Å, 50 × 2.0 mm) using a gradient elution of 10 mM ammonium acetate with 0.1% formic acid (mobile phase A) and methanol:isopropanol (90:10, v/v, mobile phase B) containing 0.1% formic acid. The analysis was then performed by electrospray ionization in negative mode coupled with a triple quadrupole mass spectrometry, API 4000QT, monitoring two transitions for each analyte, one for the quantification and the other for confirmation. The method could be easily applied to the clinical practice thanks to the short run (7 min), the low amount of patient plasma necessary for the analysis (5 μL) and the fast sample processing based on protein precipitation. The method was therefore fully validated according to FDA and EMA guidelines. The linearity was assessed (R2≥0.998) over the concentration ranges of 50−8000 ng/mL for SORA and REGO, and 30−4000 ng/mL for their metabolites, that appropriately cover the therapeutic plasma concentrations. The presented method also showed adequate results in terms of intra- and inter-day accuracy and precision (CV ≤ 7.2% and accuracy between 89.4% and 108.8%), recovery (≥85.5%), sensitivity, analytes stability under various conditions and the absence of the matrix effect.
Once the validation was successfully completed, the method was applied to perform the Cmin quantification of SORA, REGO and their metabolites in 54 plasma samples collected from patients enrolled in a clinical study ongoing at the National Cancer Institute of Aviano.
•Development of a new LC–MS/MS method to quantify sunitinib and N-desethyl sunitinib.•2-Method useful for therapeutic drug monitoring and applicable to clinical routine.•Validation according to ...EMA-FDA guidelines on bioanalytical method validation.•Quantitatively reconversion of E-isomers without the necessity of light protection.•Application of the method to a pharmacokinetic study in patients.
Sunitinib is approved for advanced renal cell cancer, imatinib-resistant or -intolerant gastrointestinal stromal tumors and pancreatic neuroendocrine cancers. It is prescribed at a fixed dose but its plasma exposure shows large inter-individual variations. Taking into account the narrow therapeutic window and the positive exposure-efficacy relationship, there is a robust rationale for its therapeutic drug monitoring. In fact, a target plasma concentration of sunitinib plus its active metabolite, N-desethyl sunitinib, ≥50 ng/mL was suggested. In order to quantify sunitinib and N-desethyl sunitinib in patients’ plasma, we developed and validated a new LC–MS/MS method applicable to clinical routine. In solution, sunitinib and N-desethyl sunitinib undergo to photo-isomerization and many published methods overcome this problem by conducting the entire procedures of samples collection and handling under strictly light-protection. Our method is based on a simple and fast procedure that quantitatively reconverts the E-isomer of both analytes, obtained during sample draw and processing without light-protection, into their Z-forms. Moreover, our method uses a small plasma volume (30 μL) and the analytes are extracted by a rapid protein precipitation. It was validated according to EMA-FDA guidelines. The calibration curves resulted linear (R2 always >0.993) over the concentration ranges (0.1–500 ng/mL for sunitinib, 0.1–250 ng/mL for N-desethyl sunitinib) with a good precision (within 7.7 % for sunitinib and 10.8% for N- desethyl sunitinib) and accuracy (range 95.8–102.9% for sunitinib and 92.3–106.2% for N-desethyl sunitinib). This method was applied to a pharmacokinetic study in one patient treated with sunitinib. Moreover, as incurred samples reanalysis is an established part of the bioanalytical process to support clinical studies, its assessment was performed early in order to assure that any reproducibility issues was detected as soon as possible. The percentage difference between the two runs resulted within ±20% in all the re-analysed samples for both sunitinib and N- desethyl sunitinib.
Lenvatinib (LENVA) is an oral antineoplastic drug used for the treatment of hepatocellular carcinoma and thyroid carcinoma. LENVA therapeutic drug monitoring (TDM) should be mandatory for a precision ...medicine to optimize the drug dosage. To this end, the development of a sensitive and robust quantification method to be applied in the clinical setting is essential. The aim of this work was to develop and validate a sensitive, rapid, and cost-effective LC-MS/MS method for the quantification of LENVA in human plasma. On this premise, sample preparation was based on a protein precipitation and the chromatographic separation was achieved on a Synergi Fusion RP C18 column in 4 min. The method was completely and successfully validated according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines, with good linearity in the range of 0.50–2000 ng/mL (R≥0.9968). Coefficient of variation (CV) for intra- and inter-day precision was ≤11.3% and accuracy ranged from 96.3 to 109.0%, internal standard normalized matrix effect CV% was ≤2.8% and recovery was ≥95.6%. Successful results were obtained for sensitivity (signal to noise (S/N) ratio >21) and selectivity, dilution integrity (CV% ≤ 4.0% and accuracy 99.9–102%), and analyte stability under various handling and storage conditions both in matrix and solvents. This method was applied to quantify LENVA in patient’s plasma samples and covered the concentration range achievable in patients. In conclusion, a sensitive and robust quantification method was developed and validated to be applied in the clinical setting.
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