Towards clinical adherence monitoring of oral endocrine breast cancer therapies by LC-HRMS-method development, validation, comparison of four sample matrices, and proof of concept.

Oral endocrine therapies (OET) for breast cancer treatment need to be taken over a long period of time and are associated with considerable side effects. Therefore, adherence to OET is an important issue and of high clinical significance for breast cancer patients' caregivers. We hypothesized that a new bioanalytical strategy based on liquid chromatography and high-resolution mass spectrometry might be suitable for unbiased adherence monitoring (AM) of OET. Four different biomatrices (plasma, urine, finger prick blood by volumetric absorptive microsampling (VAMS), oral fluid (OF)) were evaluated regarding their suitability for AM of the OET abemaciclib, anastrozole, exemestane, letrozole, palbociclib, ribociclib, tamoxifen, and endoxifen. An analytical method was developed and validated according to international recommendations. The analytical procedures were successfully validated in all sample matrices for most analytes, even meeting requirements for therapeutic drug monitoring. Chromatographic separation of analytes was achieved in less than 10 min and limits of quantification ranged from 1 to 1000 ng/mL. The analysis of 25 matching patient samples showed that AM of OET is possible using all four matrices with the exception of, e.g., letrozole and exemestane in OF. We were able to show that unbiased bioanalytical AM of OET was possible using different biomatrices with distinct restrictions. Sample collection of VAMS was difficult in most cases due to circulatory restraints and peripheral neuropathy in fingers and OF sampling was hampered by dry mouth syndrome in some cases. Although parent compounds could be detected in most of the urine samples, metabolites should be included when analyzing urine or OF. Plasma is currently the most suitable matrix due to available reference concentrations.

Single-Cell Screening of Tamoxifen Abundance and Effect Using Mass Spectrometry and Raman-Spectroscopy.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.

Determination of tamoxifen and its metabolites using micelle to solvent stacking in nonaqueous capillary electrophoresis.

Micelle to solvent stacking was implemented for the recently established NACE-C(4) D method to determine tamoxifen and its metabolites in standard samples and human plasma of breast cancer patients. For stacking, the standard samples and extract after liquid-liquid extraction (LLE) were prepared in methanol and the resulting sample solution was pressure injected after a micellar plug of SDS. Factors that affected the stacking such as SDS concentration, micelle, and sample plug length were examined. The sensitivity enhancement factor (peak height from stacking/peak height from typical injection of sample in BGE) was 15-22. The method detection limits with LLE were in the range of 5-10 ng/mL, which was lower than the established method (where the LLE extract was also prepared in methanol) with reported method detection limits of 25-40 ng/mL. The intraday and interday repeatability were in the range of 1.0-3.4% and 3.8-6.5%, respectively.

Smart coumarin-tagged imprinted polymers for the rapid detection of tamoxifen.

A signalling molecularly imprinted polymer was synthesised for easy detection of tamoxifen and its metabolites. 6-Vinylcoumarin-4-carboxylic acid (VCC) was synthesised from 4-bromophenol to give a fluorescent monomer, designed to switch off upon binding of tamoxifen. Clomiphene, a chlorinated analogue, was used as the template for the imprinting, and its ability to quench the coumarin fluorescence when used in a 1:1 ratio was demonstrated. Tamoxifen and 4-hydroxytamoxifen were also shown to quench coumarin fluorescence. Imprinted and non-imprinted polymers were synthesised using VCC, methacrylic acid as a backbone monomer and ethylene glycol dimethacrylate as cross-linker, and were ground and sieved to particle sizes ranging between 45 and 25 µm. Rebinding experiments demonstrate that the imprinted polymer shows very strong affinity for both clomiphene and tamoxifen, while the non-imprinted polymer shows negligible rebinding. The fluorescence of the imprinted polymer is quenched by clomiphene, tamoxifen and 4-hydroxytamoxifen. The switch off in fluorescence of the imprinted polymer under these conditions could also be detected under a UV lamp with the naked eye, making this matrix suitable for applications when coupled with a sample preparation system.

Is the step-wise tiered approach for ERA of pharmaceuticals useful for the assessment of cancer therapeutic drugs present in marine environment?

Methotrexate (MTX) and tamoxifen (TMX) cancer therapeutic drugs have been detected within the aquatic environment. Nevertheless, MTX and TMX research is essentially bio-medically orientated, with few studies addressing the question of its toxicity in fresh water organisms, and none to its' effect in the marine environment. To the authors' knowledge, Environmental Risk Assessments (ERA) for pharmaceuticals has mainly been designed for freshwater and terrestrial environments (European Medicines Agency-EMEA guideline, 2006). Therefore, the purpose of this research was (1) to assess effect of MTX and TMX in marine organism using the EMEA guideline, (2) to develop an ERA methodology for marine environment, and (3) to evaluate the suitability of including a biomarker approach in Phase III. To reach these aims, a risk assessment of MTX and TMX was performed following EMEA guideline, including a 2-tier approach during Phase III, applying lysosomal membrane stability (LMS) as a screening biomarker in tier-1 and a battery of biochemical biomarkers in tier-2. Results from Phase II indicated that MTX was not toxic for bacteria, microalgae and sea urchin at the concentrations tested, thus no further assessment was required, while TMX indicated a possible risk. Therefore, Phase III was performed for only TMX. Ruditapes philippinarum were exposed during 14 days to TMX (0.1, 1, 10, 50 µg L(-1)). At the end of the experiment, clams exposed to environmental concentration indicated significant changes in LMS compared to the control (p<0.01); thus a second tier was applied. A significant induction of biomarkers (activity of Ethoxyresorufin O-deethylase [EROD], glutathione S-transferase [GST], glutathione peroxidase [GPX], and lipid peroxidation [LPO] levels) was observed in digestive gland tissues of clams compared with control (p<0.01). Finally, this study indicated that MTX was not toxic at an environmental concentration, whilst TMX was potentially toxic for marine biota. This study has shown the necessity to create specific guidelines in order to evaluate effects of pharmaceuticals in marine environment which includes sensitive endpoints. The inadequacy of current EMEA guideline to predict chemotherapy agents toxicity in Phase II was displayed whilst the usefulness of other tests were demonstrated. The 2-tier approach, applied in Phase III, appears to be suitable for an ERA of cancer therapeutic drugs in the marine environment.

A human telomeric G-quadruplex-based electronic nanoswitch for the detection of anticancer drugs.

An electronic nanoswitch is described based on the conformational change of the DNA sequence in the presence of stabilizing ligands. The new electrochemical biosensor was prepared by modifying a screen-printed graphite electrode (SPE) with functionalized SiO2 nanoparticles [(SiO2-N-propylpiperazine-N-(2-mercaptopropane-1-one) (SiO2@NPPNSH)] and Au nanoparticles (AuNPs). These nanoparticles are able to immobilize thiolated G-quadruplex DNA structures (SH-G4DNA). The SH groups on the SiO2@NPPNSH nanoparticles provide a good platform for stabilizing AuNPs on the surface of the electrode. This is due to the fact that AuNPs are able to bind to the organic SH groups on the SiO2@NPPNSH. The SH-G4DNA binds to the modified electrode by a AuNPs-S bond. The structure of SiO2@NPPNSH was characterized by scanning electron microscopy (SEM), thermo-gravimetric analysis (TGA) and infrared (IR) spectroscopy. The morphology of the modified electrode was characterized by SEM. The interaction between G4DNA and the anticancer drug, Tamoxifen (Tam), was studied in Tris-HCl buffer and [Fe(CN)6](3-) using cyclic (CV) and square wave voltammetry (SWV). The G-quadruplex formation and the interaction mechanism were identified by circular dichroism (CD) measurements. The CV current was seen to decrease with increasing concentration of Tam due to interaction between G4DNA and Tam. This biosensor is a simple and useful tool for selecting G-quadruplex-binding ligands.

Bioconcentration of (15)N-tamoxifen at environmental concentration in liver, gonad and muscle of Danio rerio.

Pharmaceutical compounds (PCs) are ubiquitous in aquatic ecosystems. In addition to the direct ecotoxicological risk presented by certain PCs, others can accumulate inside organisms and along trophic webs, subsequently contaminating whole ecosystems. We studied the bioconcentration of a bioaccumulative PC already found several times in the environment: tamoxifen. To this end, we exposed Danio rerio for 21d to (15)N-tamoxifen concentrations ranging from 0.1 to 10µg/L and used an analytic method based on stable isotopes to evaluate the tamoxifen content in these organisms. The evolution of the (15)N/(14)N ratio was thus measured in liver, muscle and gonads of exposed fish compared to control fish. We succeeded in quantifying (15)N-tamoxifen bioconcentrations at all the exposure concentrations tested. The highest bioconcentration factors of tamoxifen measured were 14,920 in muscle, 73,800 in liver and 85,600 in gonads of fish after 21d exposure at a nominal concentration of 10µg/L. However, these bioconcentration factors have to be considered as maximal values (BCFMAX). Indeed, despite its proven stability, tamoxifen can be potentially partially degraded during experiments. We now need to refine these results by using a direct analytic method (i.e. LC-MS/MS).

The first electrochemical MIP sensor for tamoxifen.

We present an electrochemical MIP sensor for tamoxifen (TAM)-a nonsteroidal anti-estrogen-which is based on the electropolymerisation of an O-phenylenediamine‒resorcinol mixture directly on the electrode surface in the presence of the template molecule. Up to now only "bulk" MIPs for TAM have been described in literature, which are applied for separation in chromatography columns. Electro-polymerisation of the monomers in the presence of TAM generated a film which completely suppressed the reduction of ferricyanide. Removal of the template gave a markedly increased ferricyanide signal, which was again suppressed after rebinding as expected for filling of the cavities by target binding. The decrease of the ferricyanide peak of the MIP electrode depended linearly on the TAM concentration between 1 and 100 nM. The TAM-imprinted electrode showed a 2.3 times higher recognition of the template molecule itself as compared to its metabolite 4-hydroxytamoxifen and no cross-reactivity with the anticancer drug doxorubucin was found. Measurements at +1.1 V caused a fouling of the electrode surface, whilst pretreatment of TAM with peroxide in presence of HRP generated an oxidation product which was reducible at 0 mV, thus circumventing the polymer formation and electrochemical interferences.

Development of a CD-MEKC method for investigating the metabolism of tamoxifen by flavin-containing monooxygenases and the inhibitory effects of methimazole, nicotine and DMXAA.

A selective and low-cost CD-MEKC method under acidic conditions was developed for investigating the N-oxygenation of tamoxifen (TAM) by flavin-containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N-oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl ß-CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis-Menten constant (K(m)) of 164.1 µM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V(max)) was 3.61 µmol/min/µmol FMO1; 50% inhibitory concentration and inhibition constant (K(i)) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti-tumour agent, were investigated.

An HPLC-DAD method for the simultaneous quantification of opicapone (BIA 9-1067) and its active metabolite in human plasma.

Opicapone (BIA 9-1067) is a novel catechol-O-methyltransferase inhibitor presently under clinical development as an adjuvant in the pharmacotherapy of Parkinson's disease. This report describes the development and validation of a bioanalytical assay for the simultaneous quantification of opicapone and its active metabolite (BIA 9-1079) in human plasma. The method herein reported is based on high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) and the sample preparation consists of a plasma protein precipitation step followed by liquid-liquid extraction. Chromatographic separation of the analytes (opicapone and BIA 9-1079) and the internal standard (tamoxifen) was achieved in less than 10 min on a reversed-phase C18 column at 25 °C by applying a gradient elution program using a mobile phase composed of 0.05 M monosodium phosphate solution adjusted to pH 2.45 (A) and acetonitrile (B) pumped at 0.8 mL min(-1). Opicapone and the internal standard were monitored at 271 nm while BIA 9-1079 was assessed at 257 nm. Calibration curves of both analytes were linear (r(2) ≥ 0.997) in the concentration range of 25-3000 ng mL(-1) and their limits of quantification were established to be 25 ng mL(-1). The overall precision did not exceed 13.2% and the accuracy was within ±11.1%. Several drugs potentially co-administered with opicapone were tested and they did not interfere at the retention times of the analytes (opicapone and BIA 9-1079) and internal standard. The method was then successfully applied for quantifying opicapone and its active metabolite (BIA 9-1079) in plasma samples obtained from a healthy subject enrolled in a clinical trial.

Development of a UPLC-MS/MS method for the determination of ten anticancer drugs in hospital and urban wastewaters, and its application for the screening of human metabolites assisted by information-dependent acquisition tool (IDA) in sewage samples.

In the present work, the development, optimization, and validation (including a whole stability study) of a fast, reliable, and comprehensive method for the analysis of ten anticancer drugs in hospital and urban wastewater is described. Extraction of these pharmaceutical compounds was performed using automated off-line solid-phase extraction followed by their determination by ultra-performance liquid chromatography coupled to a triple quadrupole-linear ion trap mass spectrometer. Target compounds include nine cytotoxic agents: cyclophosphamide, ifosfamide, docetaxel, paclitaxel, etoposide, vincristine, tamoxifen, methotrexate, and azathioprine; and the cytotoxic quinolone, ciprofloxacin. Method detection limits (MDL) ranged from 0.8 to 24 ng/L. Levels found of cytostatic agents in the hospital and wastewater influents did not differ significantly, and therefore, hospitals cannot be considered as the primary source of this type of contaminants. All the target compounds were detected in at least one of the influent samples analyzed: Ciprofloxacin, cyclophosphamide, tamoxifen, and azathioprine were found in most of them and achieving maximum levels of 14.725, 0.201, 0.133, and 0.188 µg/L, respectively. The rest of target cancer drugs were less frequently detected and at values ranging between MDL and 0.406 µg/L. Furthermore, a feasible, useful, and advantageous approach based on information acquisition tool (information-dependent acquisition) was used for the screening of human metabolites in hospital effluents, where the hydroxy tamoxifen, endoxifen, and carboxyphosphamide were detected.

Estrogen-related receptor gamma disruption of source water and drinking water treatment processes extracts.

Environmental chemicals in drinking water can impact human health through nuclear receptors. Additionally, estrogen-related receptors (ERRs) are vulnerable to endocrine-disrupting effects. To date, however, ERR disruption of drinking water potency has not been reported. We used ERRgamma two-hybrid yeast assay to screen ERRgamma disrupting activities in a drinking water treatment plant (DWTP) located in north China and in source water from a reservoir, focusing on agonistic, antagonistic, and inverse agonistic activity to 4-hydroxytamoxifen (4-OHT). Water treatment processes in the DWTP consisted of pre-chlorination, coagulation, coal and sand filtration, activated carbon filtration, and secondary chlorination processes. Samples were extracted by solid phase extraction. Results showed that ERRgamma antagonistic activities were found in all sample extracts, but agonistic and inverse agonistic activity to 4-OHT was not found. When calibrated with the toxic equivalent of 4-OHT, antagonistic effluent effects ranged from 3.4 to 33.1 microg/L. In the treatment processes, secondary chlorination was effective in removing ERRgamma antagonists, but the coagulation process led to significantly increased ERRgamma antagonistic activity. The drinking water treatment processes removed 73.5% of ERRgamma antagonists. To our knowledge, the occurrence of ERRgamma disruption activities on source and drinking water in vitro had not been reported previously. It is vital, therefore, to increase our understanding of ERRy disrupting activities in drinking water.

Volumetric Absorptive Microsampling as a New Biosampling Tool for Monitoring of Tamoxifen, Endoxifen, 4-OH Tamoxifen and N-Desmethyltamoxifen in Breast Cancer Patients.

INTRODUCTION: In this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method and LC-MS.MS assay was then developed and validated for the simultaneous analysis of tamoxifen and its three active metabolites. METHODS: VAMS extraction was performed in methanol by sonication-assisted extraction method for 25 min after 2 hof VAMS drying. Separation was carried out using Acquity UPLC BEH C18 column (2.1 x 100 mm; 1.7 µm), with a flow rate of 0.2 mL/min, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 min. The multiple reaction monitoring (MRM) values were set at m/z 358.31>58.27 for N-desmethyltamoxifen, m/z 372.33>72.28 for tamoxifen, m/z 388.22>72.28 for 4-hydroxytamoxifen, m/z 374.25>58.25 for endoxifen, and m/z 260.26>116.12 for propranolol. RESULTS AND DISCUSSION: The lower limit of quantification value (LLOQ) was 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 1.50 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Accuracy (%bias) and precision (%CV) were within 20% for LLOQ and 15% for other concentrations. There were no interference responses >20% of the LLOQ and 5% of the internal standard. The level of ion suppression in all analytes was less than 7%. The preparation system developed in this study successfully extracted more than 90% of analytes from the matrix with precision below 15%. Carryover was shown to be below 6% in all analytes. Stability of analytes in VAMS was demonstrated for up to 30 days, under room temperature storage in a sealed plastic bag with desiccant. This method was successfully applied to analyze tamoxifen and the metabolites level in 30 ER+ breast cancer patients.

Tamoxifen metabolite isomer separation and quantification by liquid chromatography-tandem mass spectrometry.

Tamoxifen (Tam), the antiestrogen used to treat estrogen receptor-positive breast cancer is a pro-drug that is converted to its major active metabolites, endoxifen and 4-hydroxy-tamoxifen (4-OH-Tam) by various biotransformation enzymes of which cytochrome P450-2D6 (CYP2D6) is key. The usual Tam dose is 20 mg daily; however, the plasma active metabolite concentrations vary due to common genetic variants encoding the biotransformation enzymes and environmental factors (e.g., concomitant drugs) that inhibit these enzymes. Effective treatment depends on adequate Tam conversion to its active isomers. To monitor metabolite plasma levels, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantitate Tam, N-desmethyl-tamoxifen (ND-Tam), and tamoxifen-N-oxide (Tam-N-oxide), and the E, Z, and Z' isomers of endoxifen and 4-OH-Tam. Known standards were used to identify each metabolite/isomer. Quantitation of these metabolites in plasma was linear from 0.6 to 2000 nM. Intra- and inter-assay reproducibilities were 0.2-8.4% and 0.6-6.3%, respectively. Accuracy determined by spike experiments with known standards was 86-103%. Endoxifen, 4-OH-Tam, and their isomers were stable in fresh frozen plasma for ≥6 months. This method provides the first sensitive, specific, accurate, and reproducible quantitation of Tam and its metabolite isomers for monitoring Tam-treated breast cancer patients.

Mutagenicity of tamoxifen DNA adducts in human endometrial cells and in silico prediction of p53 mutation hotspots.

Tamoxifen elevates the risk of endometrial tumours in women and alpha-(N(2)-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with alpha-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N(2)-tamoxifen and dG-N(2)-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both alpha-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for alpha-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only alpha-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC-->TA transversions. Application of the LwPy53 algorithm to the alpha-acetoxytamoxifen spectrum predicted strong GC-->TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N(2)-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.

Occurrence of anticancer drugs in the aquatic environment: a systematic review.

Water contamination with pharmaceutical products is a well-studied problem. Numerous studies have demonstrated the presence of anticancer drugs in different water resources that failed to be eliminated by conventional wastewater treatment plants. The purpose of this report was to conduct a systematic review of anticancer drugs in the aquatic environment. The methodology adopted was carried out in compliance with the PRISMA guidelines. From the 75 studies that met the specific requirements for inclusion, data extracted showed that the most common anticancer drugs studied are cyclophosphamide, tamoxifen, ifosfamide and methotrexate with concentrations measured ranging between 0.01 and 86,200 ng/L. There was significant variation in the methodologies employed due to lack of available guidelines to address sampling techniques, seasonal variability and analytical strategy. The most routinely used technique for quantitative determination was found to be solid-phase extraction followed by LC-MS analysis. The lowest reported recovery percentage was 11%, and the highest limit of detection was 1700 ng/L. This indicated the inadequacy of some methods to analyse anticancer drugs and the failure to obtain reliable results. The significant heterogeneity within methodologies made it difficult to compare results and draw conclusions, nevertheless, this study aids in the extrapolation of proposed recommendations to guide future studies and reviews. Graphical abstract.

Quantification of drugs encapsulated in liposomes by 1H NMR.

Liposomes are one of the most important and extensively studied drug delivery system due to their ability to encapsulate different kinds of drugs. Exploiting the advantages of 1H Nuclear Magnetic Resonance (NMR) spectrometry, we established a rapid and easy method for quantification of drugs encapsulated in liposomes. An internal standard, pyridine, was used for quantitative determination of drug concentration. Two different drugs were involved in this work, one hydrophilic, methotrexate disodium salt, and another hydrophobic, tamoxifen. The specificity and selectivity of the suggested method were evaluated by the absence of overlapping of at least one signal of each drug with pyridine in the NMR spectrum. The accuracy and precision of the method were assessed by adding a known amount of each drug to unloaded liposomes. Results obtained by quantitative NMR (qNMR) were validated and confirmed by comparing with two other traditional techniques, Ultraviolet-Visible (UV-vis) spectrophotometry and High-Performance Liquid Chromatography (HPLC). It was found that the results were consistent with the ones obtained from our proposed qNMR method. Considering all the experiments conducted in this study, we deliberate that qNMR can be a suitable tool for the determination of drugs encapsulated in liposomes.

Development and validation of an UPLC-MS/MS method for the therapeutic drug monitoring of oral anti-hormonal drugs in oncology.

A liquid chromatography-mass spectrometry assay was developed and validated for simultaneous quantification of anti-hormonal compounds abiraterone, anastrozole, bicalutamide, Δ(4)-abiraterone (D4A), N-desmethyl enzalutamide, enzalutamide, Z-endoxifen, exemestane and letrozole for the purpose of therapeutic drug monitoring (TDM). Plasma samples were prepared with protein precipitation. Analyses were performed with a triple quadrupole mass spectrometer operating in the positive and negative ion-mode. The validated assay ranges from 2 to 200 ng/mL for abiraterone, 0.2-20 ng/mL for D4A, 10-200 ng/mL for anastrozole and letrozole, 1-20 ng/mL for Z-endoxifen, 1.88-37.5 ng/mL for exemestane and 1500-30,000 ng/mL for enzalutamide, N-desmethyl enzalutamide and bicalutamide. Due to low sensitivity for exemestane, the final extract of exemestane patient samples should be concentrated prior to injection and a larger sample volume should be prepared for exemestane patient samples and QC samples to obtain adequate sensitivity. Furthermore, we observed a batch-dependent stability for abiraterone in plasma at room temperature and therefore samples should be shipped on ice. This newly validated method has been successfully applied for routine TDM of anti-hormonal drugs in cancer patients.