Population Normalization with Ammonium in Wastewater-Based Epidemiology: Application to Illicit Drug Monitoring

Fluctuations in ammonium (NH4+), measured as NH4-N loads using an ion-selective electrode installed at the inlet of a sewage treatment plant, showed a distinctive pattern which was associated to weekly (i.e., commuters) and seasonal (i.e., holidays) fluctuations of the population. Moreover, population size estimates based on NH4-N loads were lower compared to census data. Diurnal profiles of benzoylecgonine (BE) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) were shown to be strongly correlated to NH4-N. Characteristic patterns, which reflect the prolonged nocturnal activity of people during the weekend, could be observed for BE, cocaine, and a major metabolite of MDMA (i.e., 4-hydroxy-3-methoxymethamphetamine). Additional 24 h composite samples were collected between February and September 2013. Per-capita loads (i.e., grams per day per 1000 inhabitants) were computed using census data and NH4-N measurements. Normalization with NH4-N did not modify the overall pattern, suggesting that the magnitude of fluctuations in the size of the population is negligible compared to those of illicit drug loads. Results show that fluctuations in the size of the population over longer periods of time or during major events can be monitored using NH4-N loads: either using raw NH4-N loads or population size estimates based on NH4-N loads, if information about site-specific NH4-N population equivalents is available.

Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

Exploration of the contribution of isotope ratio mass spectrometry to the investigation of explosives: A study of black powders and ammonium nitrate fertilisers

The increasing number of bomb attacks involving improvised explosive devices, as well as the nature of the explosives, give rise to concern among safety and law enforcement agencies. The substances used in explosive charges are often everyday products diverted from their primary licit applications. Thus, reducing or limiting their accessibility for prevention purposes is difficult. Ammonium nitrate, employed in agriculture as a fertiliser, is used worldwide in small and large homemade bombs. Black powder, dedicated to hunting and shooting sports, is used illegally as a filling in pipe bombs causing extensive damage. If the main developments of instrumental techniques in explosive analysis have been constantly pushing the limits of detection, their actual contribution to the investigation of explosives in terms of source discrimination is limited. Forensic science has seen the emergence of a new technology, isotope ratio mass spectrometry (IRMS), that shows promising results. Its very first application in forensic science dates back to 1979. Liu et al. analysed cannabis plants coming from different countries [Liu et al. 1979]. This preliminary study highlighted its potential to discriminate specimens coming from different sources. Thirty years later, the keen interest in this new technology has given rise to a flourishing number of publications in forensic science. The countless applications of IRMS to a wide range of materials and substances attest to its success and suggest that the technique is ready to be used in forensic science. However, many studies are characterised by a lack of methodology and fundamental data. They have been undertaken in a top-down approach, applying this technique in an exploratory manner on a restricted sampling. This manner of procedure often does not allow the researcher to answer a number of questions, such as: do the specimens come from the same source, what do we mean by source or what is the inherent variability of a substance? The production of positive results has prevailed at the expense of forensic fundamentals. This research focused on the evaluation of the contribution of the information provided by isotopic analysis to the investigation of explosives. More specifically, this evaluation was based on a sampling of black powders and ammonium nitrate fertilisers coming from known sources. Not only has the methodology developed in this work enabled us to highlight crucial elements inherent to the methods themselves, but also to evaluate both the longitudinal and transversal variabilities of the information. First, the study of the variability of the profile over time was undertaken. Secondly, the variability of black powders and ammonium nitrate fertilisers within the same source and between different sources was evaluated. The contribution of this information to the investigation of explosives was then evaluated and discussed. --------------------------------------------------------------------------------------------------- Le nombre croissant d'attentats à la bombe impliquant des engins explosifs artisanaux, ainsi que la nature des charges explosives, constituent une préoccupation majeure pour les autorités d'application de la loi et les organismes de sécurité. Les substances utilisées dans les charges explosives sont souvent des produits du quotidien, détournés de leurs applications licites. Par conséquent, réduire ou limiter l'accessibilité de ces produits dans un but de prévention est difficile. Le nitrate d'ammonium, employé dans l'agriculture comme engrais, est utilisé dans des petits et grands engins explosifs artisanaux. La poudre noire, initialement dédiée à la chasse et au tir sportif, est fréquemment utilisée comme charge explosive dans les pipe bombs, qui causent des dommages importants. Si les développements des techniques d'analyse des explosifs n'ont cessé de repousser les limites de détection, leur contribution réelle à l'investigation des explosifs est limitée en termes de discrimination de sources. Une nouvelle technologie qui donne des résultats prometteurs a fait son apparition en science forensique: la spectrométrie de masse à rapport isotopique (IRMS). Sa première application en science forensique remonte à 1979. Liu et al. ont analysé des plants de cannabis provenant de différents pays [Liu et al. 1979]. Cette étude préliminaire, basée sur quelques analyses, a mis en évidence le potentiel de l'IRMS à discriminer des spécimens provenant de sources différentes. Trente ans plus tard, l'intérêt marqué pour cette nouvelle technologie en science forensique se traduit par un nombre florissant de publications. Les innombrables applications de l'IRMS à une large gamme de matériaux et de substances attestent de son succès et suggèrent que la technique est prête à être utilisée en science forensique. Cependant, de nombreuses études sont caractérisées par un manque de méthodologie et de données fondamentales. Elles ont été menées sans définir les hypothèses de recherche et en appliquant cette technique de façon exploratoire sur un échantillonnage restreint. Cette manière de procéder ne permet souvent pas au chercheur de répondre à un certain nombre de questions, tels que: est-ce que deux spécimens proviennent de la même source, qu'entend-on par source ou encore quelle est l'intravariabilité d'une substance? La production de résultats positifs a prévalu au détriment des fondamentaux de science forensique. Cette recherche s'est attachée à évaluer la contribution réelle de l'information isotopique dans les investigations en matière d'explosifs. Plus particulièrement, cette évaluation s'est basée sur un échantillonnage constitué de poudres noires et d'engrais à base de nitrate d'ammonium provenant de sources connues. La méthodologie développée dans ce travail a permis non seulement de mettre en évidence des éléments cruciaux relatifs à la méthode d'analyse elle-même, mais également d'évaluer la variabilité de l'information isotopique d'un point de vue longitudinal et transversal. Dans un premier temps, l'évolution du profil en fonction du temps a été étudiée. Dans un second temps, la variabilité du profil des poudres noires et des engrais à base de nitrate d'ammonium au sein d'une même source et entre différentes sources a été évaluée. La contribution de cette information dans le cadre des investigations d'explosifs a ensuite été discutée et évaluée.

Brain damage in methylmalonic aciduria: 2-methylcitrate induces cerebral ammonium accumulation and apoptosis in 3D organotypic brain cell cultures.

BACKGROUND: Methylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage. METHODS: To explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose-response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3. RESULTS: Among the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose-response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA. CONCLUSION: Surprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium accumulation is secondary to cell suffering and/or cell death. Local accumulation of ammonium in CNS, that may remain undetected in plasma and urine, may therefore play a key role in the neuropathogenesis of methylmalonic aciduria both during acute decompensations and in chronic phases. If confirmed in vivo, this finding might shift the current paradigm and result in novel therapeutic strategies.

Ammonium-induced impairment of axonal growth is prevented through glial creatine.

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.

Ammonium alters creatine transport and synthesis in a 3D culture of developing brain cells, resulting in secondary cerebral creatine deficiency.

Hyperammonemic disorders in pediatric patients lead to poorly understood irreversible effects on the developing brain that may be life-threatening. We showed previously that some of these NH4+-induced irreversible effects might be due to impairment of axonal growth that can be protected under ammonium exposure by creatine co-treatment. The aim of the present work was thus to analyse how the genes of arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), allowing creatine synthesis, as well as of the creatine transporter SLC6A8, allowing creatine uptake into cells, are regulated in rat brain cells under NH4+ exposure. Reaggregated brain cell three-dimensional cultures exposed to NH4Cl were used as an experimental model of hyperammonemia in the developing central nervous system (CNS). We show here that NH4+ exposure differentially alters AGAT, GAMT and SLC6A8 regulation, in terms of both gene expression and protein activity, in a cell type-specific manner. In particular, we demonstrate that NH4+ exposure decreases both creatine and its synthesis intermediate, guanidinoacetate, in brain cells, probably through the inhibition of AGAT enzymatic activity. Our work also suggests that oligodendrocytes are major actors in the brain in terms of creatine synthesis, trafficking and uptake, which might be affected by hyperammonemia. Finally, we show that NH4+ exposure induces SLC6A8 in astrocytes. This suggests that hyperammonemia increases blood-brain barrier permeability for creatine. This is normally limited due to the absence of SLC6A8 from the astrocyte feet lining microcapillary endothelial cells, and thus creatine supplementation may protect the developing CNS of hyperammonemic patients.

Low concentrations of 3-hydroxy glutarate leads to ammonium accumulation and non-apoptotic cell death in developing brain cells

We previously showed in a 3D rat brain cell in vitro model for glutaric aciduria type-I that repeated application of 1mM 3-hydroxy-glutarate (3-OHGA) caused ammonium accumulation, morphologic alterations and induction of non-apoptotic cell death in developing brain cells. Here, we performed a dose-response study with lower concentrations of 3- OHGA.We exposed our cultures to 0.1, 0.33 and 1mM 3-OHGA every 12h over three days at two developmental stages (DIV5-8 and DIV11-14). Ammonium accumulation was observed at both stages starting from 0.1mM 3-OHGA, in parallel with a glutamine decrease. Morphological changes started at 0.33mM with loss of MBP expression and loss of astrocytic processes. Neurons were not substantially affected. At DIV8, release of LDH in the medium and cellular TUNEL staining increased from 0.1mM and 0.33mM 3-OHGA exposure, respectively. No increase in activated caspase-3 was observed. We confirmed ammonium accumulation and non-apoptotic cell death of brain cells in our in vitro model at lower 3-OHGA concentrations thus strongly suggesting that the observed effects are likely to take place in the brain of affected patients. The concomitant glutamine decrease suggests a defect in the astrocyte ammonium buffering system. Ammonium accumulation might be the cause of non-apoptotic cell death.

Contribution of isotope ratio mass spectrometry to the investigation of improvised explosives: isotopic study of black powders ans ammonium nitrates

The establishment of legislative rules about explosives in the eighties has reduced the illicit use of military and civilian explosives. However, bomb-makers have rapidly taken advantage of substances easily accessible and intended for licit uses to produce their own explosives. This change in strategy has given rise to an increase of improvised explosive charges, which is moreover assisted by the ease of implementation of the recipes, widely available through open sources. While the nature of the explosive charges has evolved, instrumental methods currently used in routine, although more sensitive than before, have a limited power of discrimination and allow mostly the determination of the chemical nature of the substance. Isotope ratio mass spectrometry (IRMS) has been applied to a wide range of forensic materials. Conclusions drawn from the majority of the studies stress its high power of discrimination. Preliminary studies conducted so far on the isotopic analysis of intact explosives (pre-blast) have shown that samples with the same chemical composition and coming from different sources could be differentiated. The measurement of stable isotope ratios appears therefore as a new and remarkable analytical tool for the discrimination or the identification of a substance with a definite source. However, much research is still needed to assess the validity of the results in order to use them either in an operational prospect or in court. Through the isotopic study of black powders and ammonium nitrates, this research aims at evaluating the contribution of isotope ratio mass spectrometry to the investigation of explosives, both from a pre-blast and from a post-blast approach. More specifically, the goal of the research is to provide additional elements necessary to a valid interpretation of the results, when used in explosives investigation. This work includes a fundamental study on the variability of the isotopic profile of black powder and ammonium nitrate in both space and time. On one hand, the inter-variability between manufacturers and, particularly, the intra-variability within a manufacturer has been studied. On the other hand, the stability of the isotopic profile over time has been evaluated through the aging of these substances exposed to different environmental conditions. The second part of this project considers the applicability of this high-precision technology to traces and residues of explosives, taking account of the characteristics specific to the field, including their sampling, a probable isotopic fractionation during the explosion, and the interferences with the matrix of the site.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Reversible congestive heart failure associated with primary carnitin deficiency: report of a case and review of the literature.

A 5-year-old previously healthy boy was admitted for abdominal pain and vomiting. Physical examination showed tachypnoe (32/min), hepatomegaly and painful palpation of the upper right abdominal quadrant. Laboratory tests were normal except for elevated ammonium (202mcmol/l). Chest X-ray was performed, showing cardiomegaly and interstitial edema. Transthoracic echocardiography revealed dilated left cavities and LV hypertrophy together with a diffuse hypokinesia and LVEF of 30-40%. Diuretics and ACE-inhibitors were introduced. At that time, the differential diagnosis for the DCM included myocarditis, congenital or genetic, metabolic or autoimmune disease. The next day, the boy underwent cardiac magnetic resonance (CMR) examination, showing a severe dilatation of the LV with an end-diastolic diameter of 50mm and a volume of 150ml. LVEF was 20% with diffuse LV hypokinesia (Fig. 1). No late enhancement was present after Gadolinium injection, ruling out myocarditis. Further laboratory metabolic analysis indicated severely decreased total and free carnitin levels and low renal carnitin reabsorption, corroborating the diagnosis of primary carnitin deficiency (PCD). Carnitin substitution was initiated. The clinical condition rapidly improved. No symptoms of heart failure were present anymore. A follow-up CMR performed 9 months later confirmed the recovery. LV end-diastolic volume decreased from 150ml to 66ml, LVEF increased from 20% to 55% (Fig. 2). Late enhancement was absent after Gadolinum injection (Fig. 3).Carnitin is required for the transport of fatty acids from the cytosol into mitochondria during lipid breakdown. 75% of carnitin is obtained from food, 25% is endogenously synthesized. PCD is an autosomal recessive disorder resulting from impairment of a transporter activity, caused by mutation of the SLC22A5 gene. Incidence is about 1 in 40'000 newborns. Diagnosis is usually made at age 1 to 7. Three forms of PCD are described. In the form associated with cardiomyopathy, the disease is progressive and patient die from heart failure if not treated. Substitution of L-Carnitin leads to a dramatic improvement of disease course.This case underlines the crucial role of etiologic diagnostics in this reversible form of DCM. Early diagnostics and therapy are critical for the prognosis of the patient. This is furthermore an example of a role played by CMR in the diagnostic work-up of heart failure and its follow-up under therapy.

Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity.

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 microM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 microM chloroquine.

Orthogonal organic and aqueous-based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous-based buffer for tissue section preparation before matrix-assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water-acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14-day mouse fetus whole-body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on-tissue MALDI analysis compared with solely conventional organic rinsing.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.