Biomonitoring of occupational exposure to 5-FU by assaying α-fluoro-ß-alanine in urine with a highly sensitive UHPLC-MS/MS method.

5-Fluorouracil (5-FU) is one of the most widely used antineoplastic drugs handled by healthcare professionals (HCP). To monitor occupational exposure to 5-FU, a highly sensitive ESI-UHPLC-MS/MS method was developed for the assay of its main human metabolite, α-fluoro-ß-alanine (FBAL), in urine. After a derivatization step, solid phase extraction was used for the urine. Good linearity (r > 0.996), precision (CV < 14.76%), and accuracy (bias < 12.16%) were achieved. The lower limit of quantification (LOQ), 20 pg ml-1, is the lowest one published to date. Seven urine samples from 73 HCP exposed to 5FU were positive for FBAL, indicating 5FU contamination (9.6%). FBAL urine concentrations ranged from 25 to 301 pg ml-1. Such an efficient analytical tool combining high specificity with high sensitivity is essential for the reliable detection and routine biological monitoring of healthcare professionals occupationally exposed to this widely used antineoplastic drug. This method allows biomonitoring of occupational exposure to 5-fluorouracil in a routine manner, with the aim of assessing the effectiveness of collective and individual protective measures.

High throughput routine determination of 17 tyrosine kinase inhibitors by LC-MS/MS.

Several studies have shown that therapeutic drug monitoring of tyrosine kinase inhibitors (TKI) can improve their benefit in cancer. An analytical tool has been developed in order to quantify 17 tyrosine kinase inhibitors and 2 metabolites in human plasma (afatinib, axitinib, bosutinib, crizotinib, dabrafenib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, ponatinib, regorafenib, regorafenib M2, regorafenib M5, ruxolitinib, sorafenib, sunitinib, vandetanib). Drugs were arranged in four groups, according to their plasma concentration range: 0.1-200ng/ml, 1-200ng/ml, 4-800ng/ml and 25-5000ng/ml. Solid phase extraction was used and separation was performed with HPLC using a gradient system on a solid core particle C18 column (5×2.1mm, 1.6µm). Ions were detected with a triple quadrupole mass spectrometry system. This assay allows rapid determination of 19 TKI in less than 5min per run. This high throughput routine method will be useful to adjust doses of oral anticancer drugs in order to improve treatments efficacy.

Trough dabrafenib plasma concentrations can predict occurrence of adverse events requiring dose reduction in metastatic melanoma.

INTRODUCTION: Dabrafenib and trametinib bitherapy provides significant benefits in BRAFV600mut metastatic melanoma patients; however, adverse events (AE) occur, leading to dose reduction in 33% of patients. We aimed to investigate a relation between plasma dabrafenib and trametinib concentrations and occurrence of AE. METHODS: Plasma samples from metastatic BRAFV600mut melanoma patients treated with dabrafenib±trametinib were prospectively collected at trough concentration before any dose reduction. Dabrafenib and trametinib were measured by UPLC-MS/MS. Plasma threshold of concentration associated with dose reduction for AE was studied by ROC-curve analysis. RESULTS: Twenty-seven patients (13M/14F) were included. Dabrafenib trough plasma concentrations displayed high interindividual variability, ranging from 15.4 to 279.6ng/ml, mean±SD 58.7±61.1ng/ml. Trough trametinib plasma concentrations ranged from 4.1 to 23.8ng/ml, mean±SD 11.9±4.1ng/ml. Mean trough dabrafenib plasma concentration was higher in patients with AE requiring dose reduction (30%) than in other patients: 118.6ng/ml and 33.5ng/ml respectively (P<0.0001). Adverse events leading to dabrafenib dose reduction were all grade≥2. No differences in mean trametinib trough plasma concentrations were observed in patients requiring or not dose reduction. A dabrafenib trough plasma threshold of 48ng/ml can predict the occurrence of adverse events requiring dose reduction.

An UPLC-MS/MS method for the quantification of BRAF inhibitors (vemurafenib, dabrafenib) and MEK inhibitors (cobimetinib, trametinib, binimetinib) in human plasma. Application to treated melanoma patients.

Targeted therapies for cancers are fast-growing therapies. For instance kinase inhibitors such as BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) are increasingly used to treat malignant melanoma. The metabolic profile of these drugs can result in great interindividual variability, justifying therapeutic drug monitoring (TDM). We describe a rapid and specific method for quantification of 2 BRAFi (vemurafenib, dabrafenib) and 3 MEKi (cobimetinib, trametinib and binimetinib). Chromatography was performed on a Waters Acquity-UPLC system with CORTECS C18+ column, under a gradient of 10% acetic acid in water/acetonitrile. An Acquity-TQD® with electrospray ionization was used for detection. Samples were prepared by solid phase extraction (Oasis® MCX microElution) before injection in the system. Calibration curves ranges from 0.4 to 100µg/ml for vemurafenib, from 1 to 1000ng/ml for dabrafenib, from 0.5 to 500ng/ml for cobimetinib and binimetinib, and from 0.75 to 250ng/ml for trametinib. At all concentrations the bias was within ±15% of the nominal concentrations and precision was ≤15%. All results were within the acceptance criteria of the EMA guidelines on method validation. This rapid, sensitive and specific UPLC-MS/MS method can perform simultaneous quantification of targeted therapies used in malignant melanoma and is usable for routine TDM.

Highly sensitive LC-MS/MS methods for urinary biological monitoring of occupational exposure to cyclophosphamide, ifosfamide, and methotrexate antineoplastic drugs and routine application.

Highly sensitive ESI-LC-MS/MS methods were developed for urinary biological monitoring of occupational exposure to cyclophosphamide (CP), ifosfamide (IF), and methotrexate (MTX), which are hazardous antineoplastic drugs frequently handled by healthcare professionals. Extraction methods consisted of liquid/liquid extraction for simultaneous urinary CP and IF assays, and of solid phase extraction for the urinary MTX assay. A good linearity (r2>0.997), precision (CV<14.6%), and accuracy (bias<9.9%) were achieved for all compounds. The limit of detection (LOD) was 10pg/ml and the lower limit of quantification (LOQ) was 20pg/ml for all three drugs. Applying these methods in routine, more than 116 healthcare professionals occupationally exposed to antineoplastic drugs were monitored and 635 urines were analysed. Eleven healthcare professionals (9.5%) were found to be contaminated to at least one of the three antineoplastic drugs. Among analysed urines, 22 samples were found positives. The measured concentrations ranged from 20.1 to 1850pg/ml and, for six samples, concentrations were at CP trace level, between the LOD and LOQ values (10-20pg/ml). Such efficient analytical tools combining high specificity with high sensitivity are essential for reliable detection and routine biological monitoring of healthcare professionals occupationally exposed to these widely used antineoplastic drugs. These methods allow to monitor the healthcare professionals exposure to antineoplastic drugs in the aim to assess the effectiveness of collective and individual protective measures.

Relationship between imatinib trough concentration and outcomes in the treatment of advanced gastrointestinal stromal tumours in a real-life setting.

BACKGROUND: Imatinib has dramatically improved the prognosis of advanced gastrointestinal stromal tumours (GISTs). Clinical trial data showed that patients with trough imatinib plasma concentrations (Cmin) below 1100 ng/ml (quartile 1) had shorter time to progression, but no threshold has been defined. The main objective of this study was to investigate in advanced GIST whether a Cmin threshold value associated with a longer progression-free survival (PFS) could be specified. This would be the first step leading to therapeutic drug monitoring of imatinib in GIST. PATIENTS AND METHODS: Advanced GIST patients (n=96) treated with imatinib 400 mg/d (41 stomach, 34 small bowel, and 21 other primary site localisations) were prospectively included in this real-life setting study. Routine plasma level testing imatinib (Cmin) and clinical data of were recorded prospectively. RESULTS: Small bowel localisation was associated with an increased relative risk of progression of 3.09 versus stomach localisation (p=0.0255). Mean Cmin (±standard deviation) was 868 (±536) ng/ml with 75% inter-individual and 26% intra-patient variability. A Cmin threshold of 760 ng/ml defined by log-rank test was associated with longer PFS for the whole population (p=0.0256) and for both stomach (p=0.043) and small bowel (p=0.049) localisations when analysed separately. Multivariate Cox regression analysis found that Cmin above 760 ng/ml was associated with 65% reduction risk of progression (p=0.0271) in the whole population independently of the anatomical localisation. CONCLUSION: Concentration of imatinib significantly influences duration of tumour control treatment in GIST patients with a Cmin threshold of 760 ng/ml associated with prolonged PFS in real-life setting.

Therapeutic drug monitoring of imatinib in chronic myeloid leukemia: experience from 1216 patients at a centralized laboratory.

This study set out to examine in a large real-life cohort of patients with chronic myeloid leukemia (CML) the impact of imatinib threshold of 1000 ng/mL on molecular response, as suggested in a small subset of patients. Patient plasma samples were submitted from around France to a central facility, free of charge under the auspices of the European Treatment and Outcome Study (EUTOS) for CML. Submitting physicians were required to complete an 'imatinib monitoring request form', including details of why therapeutic drug monitoring (TDM) was requested, dose and duration of imatinib treatment, cytogenetic and molecular response, adverse events, and concurrent medications. Imatinib trough plasma concentration (C(min)) was measured at the central facility. Among 1985 eligible plasma samples analyzed, from 1216 CML patients, imatinib C(min) correlated positively with reported imatinib dose, but interpatient variability in C(min) was high (60%). A logistic regression analysis revealed that treatment duration and imatinib C(min) > 1000 ng/mL were significantly associated with major and complete molecular responses with odds ratios of 1.69 and 2.08, respectively. These data support in real-life setting that imatinib C(min) threshold of 1000 ng/mL is associated with major and complete molecular response and that TDM could play an important role in dose optimization.

Simultaneous determination of nine tyrosine kinase inhibitors by 96-well solid-phase extraction and ultra performance LC/MS-MS.

BACKGROUND: Tyrosine Kinase Inhibitors (TKIs) are a class of targeted drugs for the treatment of malignant pathologies. The metabolic profile of these drugs can result in great interindividual variability, thus therapeutic drug monitoring (TDM) is of importance. Here, a rapid and specific method for quantification of nine TKIs in plasma samples is described. METHODS: Chromatography was performed on a Waters Acquity-UPLC® system with BEH C18-50*2.1 mm column, under a gradient of ammonium formate-acetonitrile. An Acquity-TQD® with electrospray ionization was used for detection. Samples were prepared by solid phase extraction (Oasis® MCX µElution) and eluate was injected in the system. RESULTS: Calibration curves ranged from 10 to 5000 ng/mL for imatinib, its metabolite, nilotinib, lapatinib, erlotinib and sorafenib and from 0.1 to 200 ng/mL for dasatinib, axitinib, gefitinib and sunitinib. Peaks of each compound (retention time from 0.76 to 2.51 min) were adequately separated. The mean relative extraction recovery was in the range of 90.3-106.5% thanks to the use of stable isotopes as internal standard. There was no significant ion suppression observed at the respective TKI retention times. CONCLUSION: This rapid, sensitive and specific UPLC/MS-MS method is able to perform simultaneous quantification of nine TKIs in human plasma and usable for routine TDM.

Establishment of the medico-legal cause of death in two atypical cases of gas embolism in hospital: Importance of the scene.

Two cases of lethal gas embolism occurring in a hospital setting are presented. These did not differ with regards to the medical cause of death (MCOD), but did so with regard to the medico-legal cause of death (MLCOD). In the first case, the immediate recognition of a suspicious death and subsequent conservation of the scene led to a certain MLCOD (autolysis). In the second case, the death was initially treated as of natural cause. The subsequent disruption of the scene of death led to loss of evidence essential to establish the MLCOD, that was concluded to be undetermined. These cases illustrate the importance of medico-legal treatment of scenes of death to arrive at the MLCOD, and that the hospital setting is no exception especially as in view of potential medical liability.

[Therapeutic drug monitoring of tyrosine-kinase inhibitors in the treatment of chronic myelogenous leukaemia: interests and limits]. / Niveau de preuve du suivi thérapeutique pharmacologique des inhibiteurs de tyrosine-kinase dans le traitement des leucémies myéloïdes chroniques.

During the last decade, imatinib was current gold standard treatment of chronic myelogenous leukemia (CML), showing a great effectiveness. Thus, the Therapeutic Drug Monitoring (TDM), rarely applied in clinical oncology practice, did not appear necessary at the moment. However, the absence of response among patients and the metabolic profile of imatinib (involving the CYP3A4) suggested the existence of a great interindividual variability. During the last 4 years, studies about the pharmacokinetic/pharmacodynamic relationship have confirmed this variability and highlighted a relation between the trough concentrations of imatinib and the clinical response. An effectiveness threshold, close to 1000 ng/mL, seems to be correlated with better cytogenetic and molecular responses. Consequently, TDM could assist in investigation of the observance, the absence of response, the drug-drug interactions, but the proof of its utility requires complementary studies. In conclusion, the level of proof of imatinib TDM in LMC varies between levels "recommended" and "potentially useful".

[Not Available]. / Niveau de preuve du suivi thérapeutique pharmacologique des inhibiteurs de tyrosine-kinase dans le traitement des leucémies myéloïdes chroniques.

During the last decade, imatinib was current gold standard treatment of chronic myelogenous leukemia (CML), showing a great effectiveness. Thus, the Therapeutic Drug Monitoring (TDM), rarely applied in clinical oncology practice, did not appear necessary at the moment. However, the absence of response among patients and the metabolic profile of imatinib (involving the CYP3A4) suggested the existence of a great interindividual variability. During the last 4 years, studies about the pharmacokinetic/pharmacodynamic relationship have confirmed this variability and highlighted a relation between the trough concentrations of imatinib and the clinical response. An effectiveness threshold, close to 1000 ng/mL, seems to be correlated with better cytogenetic and molecular responses. Consequently, TDM could assist in investigation of the observance, the absence of response, the drug-drug interactions, but the proof of its utility requires complementary studies. In conclusion, the level of proof of imatinib TDM in LMC varies between levels "recommended" and "potentially useful".

Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia.

Using high-performance liquid chromatography-tandem mass spectrometry, we assessed trough imatinib plasma levels in 68 patients with chronic myeloid leukemia (CML) who responded or not to standard-dose imatinib, after at least 12 months' treatment. Mean trough imatinib plasma levels were significantly higher in the group with complete cytogenetic response (56 patients) than in the group without (12 patients; P = .03) and higher in the group with major molecular response (MMR) than in the group without (34 patients [1452 +/- 649 ng/mL] versus 34 patients [869 +/- 427 ng/mL]; P < .001). Regarding trough imatinib plasma levels and their discrimination potential for MMR, the area under receiver operating characteristic curve was 0.775, with best sensitivity (77%) and specificity (71%) at a plasma threshold of 1002 ng/mL. Therefore, monitoring of imatinib plasma levels could be very useful for the management of patients with CML or should at least be checked in the case of treatment failure or suboptimal response.

Fully automated analytical method for mycophenolic acid quantification in human plasma using on-line solid phase extraction and high performance liquid chromatography with diode array detection.

We have developed a new fully automated method for mycophenolic (MPA) acid quantification in plasma to optimize therapeutic drug monitoring of tranplant patients. This method involved solid-phase extraction on disposable extraction cartridges and reversed-phase high-performance liquid chromatography with diode array detection. Solid-phase extraction was performed automatically by an automated sample with extraction catridges system. After washing, MPA was eluted from the cartridge onto a Chromolith RP-18e column. MPA and the internal standard were detected at 306 nm. The retention time of MPA was 6.3 minutes. The developed method was linear from 0.2 to 20 microg/mL. The limit of quantification was 0.2 microg/mL. The method showed a good precision with intraday and interday variation coefficient less than 6%. The intraday accuracy ranged from 97.6% to 100.4% and the interday accuracy varied from 97.1% to 100.8%. The extraction efficiency was greater than 90%. This method is simple and shows a good specificity with respect to commonly co-prescripted drugs.

Quantification of imatinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry.

Imatinib, also known as Gleevec or Glivec, is a selective tyrosine kinase inhibitor currently used for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML) and for other malignant pathologies. We have developed a LC-MS-MS [corrected] method that could be used for imatinib therapeutic drug monitoring in plasma. After a liquid-liquid extraction, the imatinib and its deuterated internal standard were eluted on an XTerra RP18 column with a gradient of acetonitrile-ammonium formiate buffer 4 mmol/L, pH 3.2. Imatinib was detected by electrospray ionization mass spectrometry with multiple reaction-monitoring mode. The calibration curves were linear over the range 10-5000 ng/mL. The limit of quantification was set at 10 ng/mL. The bias was lower than 8%. Intra-day and inter-day precisions were lower than 8%. The extraction recovery was higher than 90%. This method is simple, adapted to routine application, and allows accurate therapeutic monitoring of imatinib. It can be used to evaluate patient adherence to daily oral therapy, drug-drug interactions, or pharmacokinetic/pharmacodynamic relationships.

Atypical antipsychotics: from potassium channels to torsade de pointes and sudden death.

Syncope and sudden death are features of schizophrenia that can be attributed to ischaemic heart disease, the use of antipsychotics (because of proarrhythmia or other reasons such as pharyngeal dyskinesia) or the psychiatric disease itself. Cases have been described with most antipsychotics and have led to the withdrawal, temporary suspension from the market or restricted use of antipsychotics, such as sultopride, droperidol, sertindole or thioridazine. Reviewing the available data shows that all antipsychotics tested affect the cardiac potassium channel, with the concentration that produces 50% inhibition (IC50) ranging from 1 nmol/L (haloperidol) to 6 micromol/L (olanzapine). Experimental in vitro or in vivo electrophysiological studies have shown a dose-dependent increase in the duration of the action potential with various degrees of indicators of serious arrhythmogenicity. However, this does not always translate clinically into an increased duration of the QT interval or increased risk of torsade de pointes or sudden death in clinical trials or pharmacoepidemiological studies. In turn, QT prolongation in clinical trials does not always translate to an increased risk of torsade de pointes or sudden death. The reasons for these apparent discrepancies are unclear and could be related to insufficiently powered field studies, low plasma and tissue drug concentrations with reference to in vitro data or drug effects on other receptors or ion channels that have a protective effect. Alternatively, risks that were not apparent from preclinical or clinical data could be related to the use of the drug in high-risk patients, metabolic interactions or other factors that would only be encountered in large postmarketing populations. The assessment of cardiovascular safety, both preclinical and during premarketing clinical trials, needs to be supported by appropriately powered pharmacoepidemiology studies.

Identification and quantitation by high-performance liquid chromatography of mancozeb following derivatization by 1,2-benzenedithiol.

Ethylenebisdithiocarbamate (EBDC) fungicides are the most important class of organic fungicides and exhibit a high degree of carcinogenicity, mutagenicity, and neurotoxicity. For that reason, the safe application of these fungicides in practice requires a convenient method for their determination, applicable to biological fluids. We describe a high-performance liquid chromatography (HPLC) assay. After elimination of the metal which defines the product (maneb, mancozeb, zineb.) with EDTA, the resulting EBDC is derivatized with 1,2-benzenedithiol to yield a cyclocondensation product, 1,3-benzodithiole-2-thione, which can then be quantitated by reversed-phase HPLC at 365 nm using a microBondapak C18 column. The mobile phase was methanol/H2O (70:30, v/v). The assay was linear from 0.25 to 100 microg/mL. Within- and between-day precision and accuracy for this assay were better than 9% and 6%, respectively. The lower limits of detection and quantitation were estimated to be 0.1 and 0.25 microg/mL, respectively. This simple new method has been applied to determine mancozeb concentration in rat urine samples from urinary excretion studies.

Pharmacokinetic interaction between high-dose methotrexate and oxacillin.

An 18-year-old man received two high-dose methotrexate cycles for the treatment of an osteosarcoma. Fifteen grams of methotrexate were infused over 6 hours. During the second cycle, co-administration of oxacillin (1g/8h) resulted in prolonged and marked elevation of methotrexate plasma concentrations. The patient experienced acute toxicity with renal failure, myelosuppression, mucitis, fever, and dermatologic abnormalities. After an initial improvement with folinic acid rescue and hemodialysis, the patient died. Oxacillin may thus inhibit the elimination of methotrexate.