Determination of osimertinib in human plasma, urine and cerebrospinal fluid.

Aim: Osimertinib (Tagrisso, AZD9291) has been approved for the treatment of patients with metastatic EGFRm T790M non-small-cell lung cancer. Results: Rapid and sensitive LC-MC/MS methods were developed for osimertinib and its metabolites, AZ13597550 and AZ13575104, in human plasma (low- and high-range), urine and cerebrospinal fluid. We discuss the challenges of these multi-analyte and multiple matrix assays. The methods have been successfully validated and used for the analysis of over 20,000 clinical samples, with successful incurred sample reproducibility. Conclusion: The assays have been shown to be selective, accurate and robust, providing high-throughput analysis during the clinical development of osimertinib.

Fast and simple screening for the simultaneous analysis of seven metabolites derived from five volatile organic compounds in human urine using on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry.

Recently, the International Agency for Research on cancer classified outdoor air pollution and particulate matter from outdoor air pollution as carcinogenic to humans (IARC Group 1), based on sufficient evidence of carcinogenicity in humans and experimental animals and strong mechanistic evidence. In particular, a wide variety of volatile organic compounds (VOCs) are volatized or released into the atmosphere and can become ubiquitous, as they originate from many different natural and anthropogenic sources, such as paints, pesticides, vehicle exhausts, cooking fumes, and tobacco smoke. Humans may be exposed to VOCs through inhalation, ingestion, or dermal contact, which may increase the risk of leukemia, birth defects, neurocognitive impairment, and cancer. Therefore, the focus of this study was the development of a simple, effective and rapid sample preparation method for the simultaneous determination of seven metabolites (6 mercaptic acids+t,t-muconic acid) derived from five VOCs (acrylamide, 1,3-butadiene, acrylonitrile, benzene, and xylene) in human urine by using automated on-line solid-phase extraction (SPE) coupled with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). An aliquot of each diluted urinary sample was directly injected into an autosampler through a trap column to reduce contamination, and then the retained target compounds were eluted by back-flush mode into an analytical column for separation. Negative electrospray ionization tandem mass spectrometry was utilized for quantification. The coefficients of correlation (r(2)) for the calibration curves were greater than 0.995. Reproducibility was assessed by the precision and accuracy of intra-day and inter-day precision, which showed results for coefficient of variation (CV) that were low 0.9 to 6.6% and 3.7 to 8.5%, respectively, and results for recovery that ranged from 90.8 to 108.9% and 92.1 to 107.7%, respectively. The limits of detection (LOD) and limits of quantification (LOQ) were determined to within 0.010 to 0.769 ng mL(-1) and 0.033 to 2.564 ng mL(-1) in this study. A stability study test included 3 freeze/thaw cycles during short-term storage at room temperature for 36 h and long-term storage at -20 °C for 1 month, and the CV (coefficient of variation) showed less than 8.4, 7.4 and 9.7%, respectively. To the best of our knowledge, this is the first study to provide simple, small injection volumes (40 µL) and a rapid LC-MS/MS method combined with an on-line SPE step for the simultaneous detection, identification, and quantification of seven metabolites derived from five VOCs in human urine for evaluation of the future risk of human exposure to volatile organic compounds.

Metabolism and pharmacokinetics of allitinib in cancer patients: the roles of cytochrome P450s and epoxide hydrolase in its biotransformation.

Allitinib, a novel irreversible selective inhibitor of the epidermal growth factor receptor (EGFR) 1 and human epidermal receptor 2 (ErbB2), is currently in clinical trials in China for the treatment of solid tumors. It is a structural analog of lapatinib but has an acrylamide side chain. Sixteen metabolites of allitinib were detected by ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The pharmacologically active α,ß-unsaturated carbonyl group was the major metabolic site. The metabolic pathways included O-dealkylation, amide hydrolysis, dihydrodiol formation, hydroxylation, and secondary phase 2 conjugation. The metabolite of amide hydrolysis (M6) and 27,28-dihydrodiol allitinib (M10) were the major pharmacologically active metabolites in the circulation. The steady-state exposures to M6 and M10 were 11% and 70% of that of allitinib, respectively. The biotransformation of allitinib was determined using microsomes and recombinant metabolic enzymes. In vitro phenotyping studies demonstrated that multiple cytochrome P450 (P450) isoforms, mainly CYP3A4/5 and CYP1A2, were involved in the metabolism of allitinib. Thiol conjugates (M14 and M16) and dihydrodiol metabolites (M5 and M10) were detected in humans, implying the formation of reactive intermediates. The formation of a glutathione conjugate of allitinib was independent of NADPH and P450 isoforms, but was catalyzed by glutathione-S-transferase. P450 enzymes and epoxide hydrolase were involved in M10 formation. Overall, our study showed that allitinib was metabolized by the O-dealkylation pathway similar to lapatinib, but that amide hydrolysis and the formation of dihydrodiol were the dominant metabolic pathways. The absorbed allitinib was extensively metabolized by multiple enzymes.

Biological monitoring for occupational acrylamide exposure from acrylamide production workers.

OBJECTIVE: We conducted a repeated-measurement study to (1) investigate the correlation between occupational exposure to airborne acrylamide (AA) and the time-dependent behavior of urinary AAMA, GAMA2, and GAMA3 and (2) calculate the estimated biological exposure index at the permissible exposure limit (PEL) level of 30 µg/m(3). METHODS: Forty-four workers were recruited--8 were AA-exposed and 36 were controls. Pre- and post-shift urine samples were collected from the exposed group in parallel with personal sampling for 8 consecutive days and only 1 day for the control group and analyzed using liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS). RESULTS: Post-shift urinary AAMA level was significantly associated with personal AA exposure (p < 0.001), indicating that urinary AAMA was a better AA exposure biomarker. The estimated urinary excretion of AAMA was 3.0 mg/g creatinine for nonsmoking workers exposed to the PEL of 30 µg/m(3). The median GAMA (the sum of GAMA2 and GAMA3)/AAMA ratio for exposed workers was 0.03 (range, 0.005-0.14), relatively lower than that of the nonoccupational group. CONCLUSIONS: Although sample size in this study was small, the repeated-measurement data provide useful reference for future studies related to biological monitoring of occupational exposure to AA.

Method for tracking nanogel particles in vivo and in vitro.

Hydrogels made of N-isopropylacrylamide (NIPA) can be synthesized in the form of highly monodispersed nanoparticles. After synthesis, NIPA hydrogel nanoparticles (nanogels) can be labeled by Alexa Fluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester through amine-terminated functional groups. This choice of dye is complementary to other biological labeling methods for in vivo studies. When the nanogel/dye nanoparticles are injected into rabbits, they can be imaged via tissue sectioning and confocal microscopy, while nanoparticle concentration can be determined by fluorescent microplate assays. Time-course persistence of nanoparticles in the circulatory system can be readily tracked by direct assay of plasma and urine samples using 485 nm excitation and 538 emission wavelengths to keep background fluorescence to nearly the same level as that found using an empty well. Depending upon how the nanoparticles are injected, circulatory system concentrations can reach high concentrations and diminish to low levels or gradually increase and gradually decrease over time. Injection in the femoral artery results in a rapid spike in circulating nanogel/dye concentration, while injection into the renal artery results in a more gradual increase.

Acrylamide exposure via the diet: influence of fasting on urinary mercapturic acid metabolite excretion in humans.

Acrylamide (AA) is carcinogenic in animals and classified by the International Agency for Research on Cancer as probably carcinogenic in humans. Regarding the AA contents of food the diet significantly contributes to the overall AA burden of the general population. However, it is unclear to which degree the diet, apart from smoking, contributes to the internal AA exposure. Therefore the influence of an AA-free diet on the excretion of urinary mercapturic acid metabolites derived from AA in three healthy volunteers fasting for 48 h was examined. Urinary AA mercapturic acid metabolites were considerably reduced after 48 h of fasting. The levels were even well below the median level in non-smokers. This confirms that the diet is the main source of environmental AA exposure in humans, apart from smoking. Other possible AA sources could be of minor quantitative importance only.

Mouse bone marrow micronucleus test results do not predict the germ cell mutagenicity of N-hydroxymethylacrylamide in the mouse dominant lethal assay.

N-Hydroxymethylacrylamide (NHMA), a mouse carcinogen inactive in the Salmonella assay and mouse micronucleus (MN) assay, was tested for reproductive effects in a mouse continuous breeding study. In that study, increased embryonic deaths were observed after 13 weeks exposure of parental animals to NHMA via drinking water (highest dose, 360 ppm); the results indicated the possible induction of chromosome damage in germ cells of treated males. An additional mouse MN test was conducted using a 31-day treatment period to better match the dosing regimen used in the breeding study; the results were negative. Additional studies were conducted to explore the germ cell activity of NHMA. A male mouse dominant lethal study was conducted using a single intraperitoneal injection of 150 mg/kg NHMA; the results were negative. A follow-up study was conducted using fractionated dosing, 50 mg/kg/day for 5 days; again, no increase in dominant lethal mutations was observed. NHMA (180-720 ppm) was then administered to male mice in drinking water for 13 weeks, during which three sets of matings occurred. Two weeks after mating, females were killed and the uterine contents were analyzed. Large, dose-related increases in dominant lethal mutations were observed with increasing length of exposure. The magnitude of the increases stabilized after 8 weeks of treatment. However, the frequency of micronucleated peripheral blood erythrocytes was not elevated in mice treated for 13 weeks with NHMA in drinking water. Thus, NHMA appears to be unique in inducing genetic damage in germ cells but not somatic cells of male mice.

Determination of acrylamide metabolite, mercapturic acid by high performance liquid chromatography.

A HPLC Assay was developed to identify and measure the metabolite of acrylamide, mercapturic acid, N-Acetyl-s-(propionamide)-cysteine (APC) in urine. O-phthalaldehyde (OPA) was utilized as a precolumn derivatizing agent in the assay. This derivative was isolated with a good selectivity by high performance liquid chromatography (HPLC) employing reversed phase ODS columns. The quantitation of the mercapturic acid derivative was reproducible and with a detection limit of 1 pmol. The average coefficient of variation for the runs carried out on the same day was approximately 4.6% at the range of 80-160 mumol.L-1 of APC, and the mean analytical recovery from urine samples was 94%. The metabolite of urine of workers exposed to acrylamide was identified as N-acetyl-s-(propionamide)-cysteine by HPLC chromatography and fluorescence scan and HPLC-Mass spectra. All results were identical with the authentic synthesized compound.

Characterization and quantitation of urinary metabolites of [1,2,3-13C]acrylamide in rats and mice using 13C nuclear magnetic resonance spectroscopy.

Acrylamide, widely used for the production of polymers and as a grouting agent, causes neurotoxic effects in humans and neurotoxic, genotoxic, reproductive, and carcinogenic effects in laboratory animals. In this study, 13C NMR spectroscopy was used to detect metabolites of acrylamide directly in the urine of rats and mice following administration of [1,2,3-13C]acrylamide (50 mg/kg po). Two-dimensional NMR experiments were used to correlate carbon signals for each metabolite in the urine samples and to determine the number of hydrogens attached to each carbon. Metabolite structures were identified from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison with standards. The metabolites assigned in rat and mouse urine are N-acetyl-S-(3-amino-3-oxopropyl)cysteine, N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine, N-acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)cysteine, glycidamide, and 2,3-dihydroxypropionamide. These metabolites arise from direct conjugation of acrylamide with glutathione or from oxidation to the epoxide, glycidamide, and further metabolism. Acrylamide was also detected in the urine. Quantitation was carried out by integrating the metabolite carbon signals with respect to that of dioxane added at a known concentration. The major metabolite for both the rat (70% of total metabolites excreted) and the mouse (40%) was formed from direct conjugation of acrylamide with glutathione. The remaining metabolites for the rat (30%) and mouse (60%) are derived from glycidamide. The species differences in extent of metabolism through glycidamide may have important consequences for the toxic and carcinogenic effects of acrylamide.