A water stable Eu(III)-organic framework as a recyclable multi-responsive luminescent sensor for efficient detection of p-aminophenol in simulated urine, and MnVII and CrVI anions in aqueous solutions.

A novel 3D Eu(iii) metal-organic framework (Eu-MOF-1) formulated as [Eu(L)(H2O)(DMA)] (L = 2-(2-nitro-4-carboxylphenyl)terephthalic acid) has been successfully synthesized under solvothermal conditions and characterized by structural analyses. Eu-MOF-1 displays a new 3D framework containing EuIII ions, ligand L, and coordinated DMA molecules and water molecules. The fluorescence investigations indicate that Eu-MOF-1 emits bright red luminescence, and shows relatively high water stability and outstanding chemical stability under a relatively wide range of pH conditions. It is noteworthy that Eu-MOF-1 can quantitatively detect p-aminophenol (PAP) which is a metabolite of phenylamine in human urine. More significantly, Eu-MOF-1 is the first reported multi-responsive luminescent sensor for detecting the biomarker PAP, and MnVII and CrVI anions with high selectivity, sensitivity, recyclability and relatively low detection limits in aqueous solutions. Furthermore, the possible sensing mechanisms of Eu-MOF-1 for selective sensing have also been explored in detail. Eu-MOF-1 could be an ideal candidate as a multi-responsive luminescent sensor in biological and environmental areas.

Human metabolism and urinary excretion kinetics of the UV filter Uvinul A plus® after a single oral or dermal dosage.

Hexyl 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoate, better known under its trading name Uvinul A plus® is a UV filter mainly used in sunscreens, but also present in other cosmetic products with a maximum concentration of 10% (w/w) according to the EU directive. In this study we investigated the human metabolism after a single oral and a single dermal dose of Uvinul A plus®, respectively. Samples collected within 72 h of administration were analyzed with a newly developed UHPLC-MS/MS method. Results of the study revealed three major urinary metabolites, namely 2-(4-amino-2-hydroxybenzoyl)benzoic acid (AHB), 2-(4-(ethylamino)-2-hydroxybenzoyl)benzoic acid (EHB) and 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid (DHB), representing 52% of the administered oral dose. The three major metabolites are further converted into four minor metabolites with an additional hydroxyl group in the aniline moiety. Toxicokinetic parameters (amount excreted, tmax, elimination constant and half-life t1/2) and conversion factors were determined for the three major metabolites. The conversion factors were used to estimate the mean daily exposure to Uvinul A plus® in spot urine samples from 58 volunteers not intentionally exposed to Uvinul A plus® derived from a pilot study. The three major metabolites were quantifiable in 26% of the samples. In 35% of the samples, at least one major metabolite could be quantified. The daily systemic exposure to Uvinul A plus® was estimated to approximately 8.1-9.3 µg/d by applying the combined conversion factor for all three major metabolites. In conclusion, a very low systemic exposure to DHHB was observed with regard to the no observed adverse effect level (NOAEL) as an established threshold for chronic uptake.

A validated UPLC-MS/MS method for the determination of urinary metabolites of Uvinul® A plus.

Uvinul® A plus (DHHB) is a synthetic benzophenone derivative mainly used in sunscreens, and also in other skin care products. The compound is authorized by the EU as UV filter and a maximum concentration of 10% in consumer products is permitted. Despite its high production volume and usage in consumer products,to date, no information about the systemic exposure to Uvinul® A plus in humans is available. Therefore, we developed a human biomonitoring method which allows the simultaneous determination of three major metabolites of Uvinul® A plus in human urine samples. Furthermore, three minor metabolites of Uvinul® A plus were identified by ion trap experiments. Urine samples were enzymatically hydrolyzed, extracted via liquid-liquid extraction with ethyl acetate, and analyzed by means of UPLC-MS/MS. The final method was validated according to FDA guidelines and applied to 58 urine samples retrieved from the general German population. The three major and specific metabolites of Uvinul® A plus were found in about 36% of the samples, proving the suitability of the method for future human biomonitoring studies.

Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control.

Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.

Male urinary paracetamol and semen quality.

The endocrine-disrupting properties of paracetamol have been previously demonstrated in rodent studies of abnormal sperm morphology and diminished testosterone production, in addition to epidemiologic studies of diminished couple fecundity. In this study, we examined the relationship between paracetamol and its metabolite p-aminophenol quantified in a single spot urine and semen quality among 501 male partners of couples planning for pregnancy. Men provided a urine specimen and two fresh semen samples collected approximately one month apart and underwent 24-h analysis for 35 semen quality parameters. Paracetamol and p-aminophenol were quantified in urine by ultra-high-performance liquid chromatography coupled with an electrospray triple quadrupole mass spectrometry. The relationship between natural-log-transformed urinary paracetamol and p-aminophenol rescaled by their standard deviation and 21 Box-Cox-transformed, 14 non-transformed semen parameters was assessed using linear mixed-effects models. The median concentrations (IQR) of urinary paracetamol and p-aminophenol were 15.5 ng/mL (5.44, 73.5) and 978 ng/mL (500, 1596), respectively. Following adjustment for creatinine and age, a 1-standard deviation increase in log-transformed urinary paracetamol was associated with a reduction in beat cross-frequency and an increase in DNA fragmentation [ß (95% CI): -0.59 Hz (-1.16, -0.03) and 0.05% (0.01, 0.09), respectively]. These findings were corroborated in models of categorical chemical concentrations; higher concentrations of paracetamol remained associated with reduced beat cross-frequency and increased DNA fragmentation. A 1-standard deviation increase in log-transformed urinary p-aminophenol was associated with a reduction in sperm head area [ß (95% CI): -0.1 µm2 (-0.18, -0.02) and width -0.02 µm (-0.04, -0.01)]. However, only the association with sperm head area remained statistically significant in models of p-aminophenol quartiles. Our findings suggest that adult male urinary paracetamol is associated with sperm motility and DNA fragmentation, while the metabolite, p-aminophenol, is predominantly associated with sperm head morphometry.

Visual and colorimetric detection of p-aminophenol in environmental water and human urine samples based on anisotropic growth of Ag nanoshells on Au nanorods.

A simple, sensitive, selective and high-resolution colorimetric method has been developed for the detection of p-aminophenol in environmental water and human urine samples. In the presence of p-aminophenol, silver ions are reduced to silver atoms and subsequently Ag nanoshells anisotropically grow on the surface of Au nanorods to generate orange slice-like Au@Ag core-shell nanocrystals, thereby resulting in the blue-shift of longitudinal surface plasmon resonance band of Au nanorods accompanying a sharp-contrast multicolor change. Using Au@Ag core-shell nanocrystals as the transducer, sub-micromolar p-aminophenol can be detected by the colorimetric method and 10 µmol L(-1) p-aminophenol can be visual readout by the naked eyes. Furthermore, a simple, cheap, portable test kit is constructed for the visual assay of urinary p-aminophenol without complicated sample pretreatment and sophisticated instruments. The proposed colorimetric method has the potential for the rapid and on-site analyses of p-aminophenol in environmental water and human urine samples.

Application of ultrasound-assisted emulsification microextraction for simultaneous determination of aminophenol isomers in human urine, hair dye, and water samples using high-performance liquid chromatography.

Aminophenol isomers (2-, 3-, and 4-aminophenols) are typically classified as industrial pollutants with genotoxic and mutagenic effects due to their easy penetration through the skin and membranes of human, animals, and plants. In the present study, a simple and efficient ultrasound-assisted emulsification microextraction procedure coupled with high-performance liquid chromatography with ultraviolet detector was developed for preconcentration and determination of these compounds in human fluid and environmental water samples. Effective parameters (such as type and volume of extraction solvent, pH and ionic strength of sample, and ultrasonication and centrifuging time) were investigated and optimized. Under optimum conditions (including sample volume: 5 mL; extraction solvent: chloroform, 80 µL; pH: 6.5; without salt addition; ultrasonication: 3.5 min; and centrifuging time: 3 min, 5000 rpm min(-1)), the enrichment factors and limits of detection were ranged from 42 to 51 and 0.028 to 0.112 µg mL(-1), respectively. Once optimized, analytical performance of the method was studied in terms of linearity (0.085-157 µg mL(-1), r (2) > 0.998), accuracy (recovery = 88.6- 101.7%), and precision (repeatability: intraday precision < 3.98%, and interday precision < 5.12%). Finally, applicability of the method was evaluated by the extraction and determination of these compounds in human urine, hair dye, and real water samples.

N-Acetyl-4-aminophenol (paracetamol), N-acetyl-2-aminophenol and acetanilide in urine samples from the general population, individuals exposed to aniline and paracetamol users.

Epidemiological studies suggest associations between the use of N-acetyl-4-aminophenol (paracetamol) during pregnancy and increased risks of reproductive disorders in the male offspring. Previously we have reported a ubiquitous urinary excretion of N-acetyl-4-aminophenol in the general population. Possible sources are (1) direct intake of paracetamol through medication, (2) paracetamol residues in the food chain and (3) environmental exposure to aniline or related substances that are metabolized into N-acetyl-4-aminophenol. In order to elucidate the origins of the excretion of N-acetyl-4-aminophenol in urine and to contribute to the understanding of paracetamol and aniline metabolism in humans we developed a rapid, turbulent-flow HPLC-MS/MS method with isotope dilution for the simultaneous quantification of N-acetyl-4-aminophenol and two other aniline related metabolites, N-acetyl-2-aminophenol and acetanilide. We applied this method to three sets of urine samples: (1) individuals with no known exposure to aniline and also no recent paracetamol medication; (2) individuals after occupational exposure to aniline but no paracetamol medication and (3) paracetamol users. We confirmed the omnipresent excretion of N-acetyl-4-aminophenol. Additionally we revealed an omnipresent excretion of N-acetyl-2-aminophenol. In contrast, acetanilide was only found after occupational exposure to aniline, not in the general population or after paracetamol use. The results lead to four preliminary conclusions: (1) other sources than aniline seem to be responsible for the major part of urinary N-acetyl-4-aminophenol in the general population; (2) acetanilide is a metabolite of aniline in man and a valuable biomarker for aniline in occupational settings; (3) aniline baseline levels in the general population measured after chemical hydrolysis do not seem to originate from acetanilide and hence not from a direct exposure to aniline itself and (4) N-acetyl-2-aminophenol does not seem to be related to aniline nor to N-acetyl-4-aminophenol in man.

UPLC-QTOF/MS metabolic profiling unveils urinary changes in humans after a whole grain rye versus refined wheat bread intervention.

SCOPE: Non-targeted urine metabolite profiling has not been previously exploited in the field of whole grain (WG) products. WG products, particularly rye, are important elements in a healthy Nordic diet. The aim of this study was to identify novel urinary biomarkers of WG rye bread (RB) intake in a randomised crossover study with RB versus refined wheat bread (WB). METHODS AND RESULTS: UPLC-QTOF/MS metabolite profiling was applied to urine from a 2 × 4 wk crossover intervention with RB versus WB in 20 subjects. Sixteen metabolites were revealed as major contributing biomarkers. The most discriminative metabolite after the cereal intervention was identified as 3-(3,5-dihydroxyphenyl)-1-propanoic acid sulphate, which was excreted to a higher extent after the RB versus WB intervention. Other alkylresorcinol metabolites were identified, as well as enterolactone glucuronide, azelaic acid, 2-aminophenol sulphate and its benzoxazinoid precursor 2,4-dihydroxy-1,4-benzoxazin-3-one. Our study also suggests that nitrogen-containing metabolites are other major markers. However, other methodologies will be needed to elucidate their final structure. CONCLUSION: The present non-targeted metabolite profiling proved to be a useful approach to identify major urine metabolites discriminating RB intake from that of white wheat bread. Once validated these markers could help evaluate compliance to healthy Nordic diets.

SARM-S4 and metabolites detection in sports drug testing: a case report.

Recently, pharmaceutical industry developed a new class of therapeutics called Selective Androgen Receptor Modulator (SARM) to substitute the synthetic anabolic drugs used in medical treatments. Since the beginning of the anti-doping testing in sports in the 1970s, steroids have been the most frequently detected drugs mainly used for their anabolic properties. The major advantage of SARMs is the reduced androgenic activities which are the main source of side effects following anabolic agents' administration. In 2010, the Swiss laboratory for doping analyses reported the first case of SARMs abuse during in-competition testing. The analytical steps leading to this finding are described in this paper. Screening and confirmation results were obtained based on liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. Additional information regarding the SARM S-4 metabolism was investigated by ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS).

Simultaneous determination of N-acetyl-p-aminophenol and p-aminophenol with poly(3,4-ethylenedioxythiophene) modified glassy carbon electrode.

A sensitive and selective method was developed for the determination of N-acetyl-p-aminophenol (APAP) and p-aminophenol (PAP) using poly(3,4-ethylenedioxythiophene) (PEDOT)-modified glassy carbon electrode (GCE). Cyclic voltammetry and differential pulse voltammetry were used to investigate the electrochemical reaction of APAP and PAP at the modified electrode. Both APAP and PAP showed quasireversible redox reactions with formal potentials of 367 mV and 101 mV (vs. Ag/AgCl), respectively, in phosphate buffer solution of pH 7.0. The significant peak potential difference (266 mV) between APAP and PAP enabled the simultaneous determination both species based on differential pulse voltammetry. The voltammetric responses gave linear ranges of 1.0×10(-6)-1.0×10(-4) mol L(-1) and 4.0×10(-6)-3.2×10(-4) mol L(-1), with detection limits of 4.0×10(-7) mol L(-1) and 1.2×10(-6) mol L(-1) for APAP and PAP, respectively. The method was successfully applied for the determination of APAP and PAP in pharmaceutical formulations and biological samples.

Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator andarine (S-4) for routine doping control purposes.

Selective androgen receptor modulators (SARMs) are potent anabolic agents with tissue-selective properties. Due to their potential misuse in elite sport, the World Anti-Doping Agency (WADA) has prohibited SARMs since 2008, and although no representative drug candidate has yet received full clinical approval, recent findings of SARMs illegally sold via the internet have further supported the need to efficiently test for these compounds in doping controls. In the present communication, the mass spectrometric characterization of urinary metabolites of the SARM Andarine (also referred to as S-4) compared with earlier in vitro and animal studies is reported. Liquid chromatography interfaced to high-resolution/high-accuracy (tandem) mass spectrometry was used to identify phase I and II metabolites, confirming the predicted target analytes for sports drug testing purposes including the glucuronic acid conjugates of the active drug, its monohydroxylated and/or deacetylated product, the hydrolysis product resulting from the removal of the compound's B-ring, as well as the sulfate of the monohydroxylated and the deacetylated phase I metabolite. The obtained data will support future efforts to effectively screen for and confirm the misuse of the non-approved drug candidate Andarine.

Automated spectrophotometric assay for urine p-aminophenol by an oxidative coupling reaction.

Urine p-aminophenol (PAP) concentration serves as a biological marker for occupational exposures to aniline. We report the development of a rapid, simple spectrophotometric method for quantification of urine PAP concentration using a chemical autoanalyzer (Olympus Reply). The method involves oxidative coupling of PAP with an aromatic compound, xylenol, that contains an electron-donating group, based on an electrophilic aromatic substitution reaction catalyzed by sodium periodate. A calibration curve is constructed in the same matrix, urine, as the unknown samples to be analyzed. In this way, potential matrix interferences are largely avoided. The linearity range of the method is 20 to 400 mg/L. Time-course studies show that the color formation by reaction of PAP with xylenol is rapid and essentially complete within 5 min. Within-run and day-to-day reproducibility data at medium (50 mg/L) and high (200 mg/L) concentrations yield CV's <5.0%. Several prescription drugs and drugs of abuse, as well as related compounds, gave negative tests for interference in the procedure. Clinical applications of the method are illustrated by data for (a) PAP concentrations in 255 urine samples from workers at a rubber plant, and (b) PAP elimination in serial urine samples from 5 volunteers after an oral dose (500 mg) of acetaminophen. In summary, the new method has the advantages of automation, operational simplicity, and suitability for monitoring workers for exposures to aniline.

Quantitative colorimetric determination of urinary p-aminophenol with an automated analyzer.

We developed an automated colorimetric method for the quantitative determination of p-aminophenol with a Cobas Mira analyzer. The procedure can be used for the biological monitoring of human exposure to aniline. An absorbed aniline dose is extensively oxidized to p-aminophenol, which is excreted in urine mainly as glucurono- and sulfo- conjugates. After enzymatic hydrolysis, we reacted the free compound with resorcinol in the presence of manganese ions to form an indophenol dye, which is measured at 550 nm. Excellent accuracy (102.8%, 103.9%, and 96.8% at 2.5, 50, and 90 mg/L, respectively) and precision (7.7%, 2.1%, and 0.8% CV for within-run and 11.1%, 4.7%, and 4.6% for total reproducibility at 2.5, 50, and 90 mg/L, respectively) were achieved over a linear concentration range of 2.0 to 100 mg/L. The detection limit was 0.9 mg/L and no significant interference (except for o-aminophenol) was found for several investigated drugs and related compounds. The proposed method was used for a stability study and to analyze several samples from an occupational health screen.

High resolution NMR spectroscopic studies on the metabolism and futile deacetylation of 4-hydroxyacetanilide (paracetamol) in the rat.

Paracetamol (4-hydroxyacetanilide, acetaminophen) was synthesized with the acetyl group labelled with C2H3 (paracetamol-C2H3), and dosed to rats i.p. at 25 mg/kg (N = 5) and 40 mg/kg (N = 3) body weight. Paracetamol, with a 13CH3 in the acetyl group (paracetamol-13CH3) was also synthesized and dosed to rats i.p. at 40 mg/kg (N = 3). The metabolism and excretion of the 2H-labelled compound was followed in the rat using 600 MHz 1H and 92.1 MHz 2H NMR spectroscopy of urine collected 0-8, 8-24, 24-32 and 32-48 hr post-dosing. The metabolism of paracetamol-13CH3 was also monitored using 600 MHz 1H NMR spectroscopy of urine collected 0-8, 8-24 and 24-48 hr post-dosing. For paracetamol-C2H3 the total recovery of the sulphate, glucuronide and N-acetyl cysteinyl metabolites via the urine accounted for 61.2 +/- 14.1% of the 25 mg/kg dose and 61.4 +/- 8.8% of the 40 mg/kg dose. For paracetamol-13CH3 the recovery was 102.7 +/- 3.7% indicating that the low % urinary recovery with the C2H3-labelled drug is the result of isotope effects on the disposition of paracetamol. In the case of the paracetamol-C2H3, quantitative 1H NMR analysis of urine showed that 13.3 +/- 0.5 and 10.0 +/- 1.2 mole % (25 and 40 mg/kg, respectively) of the urinary paracetamol sulphate recovered following dosing of the deuterium labelled drug had the C2H3 acetyl groups replaced by C1H3 acetyl groups from endogenous sources. In the case of the paracetamol-13CH3 8.9 +/- 0.7 mole % of the sulphate conjugate had also been transacetylated to paracetamol-12CH3. There was no significant difference between the level of futile deacetylation observed for the deuterated and 13C-labelled drug. Overall these data indicate a high level of deacetylation followed by reacetylation (i.e. futile deacetylation) prior to excretion of paracetamol via the nephrotoxic intermediate 4-aminophenol. The level of deacetylation is much higher than has previously been thought which may cast new light on the role of 4-aminophenol in the development of paracetamol induced nephrotoxicity.

A spectrophotometric assay for the detection of 2-aminophenols in biological samples.

A sensitive and efficient spectrophotometric assay is described for the determination of 2-aminophenols in biological samples. Using ferric ions as a metal catalyst, 2-aminophenol dimerizes in an acidic aqueous environment to 2-hydroxyisophenoxazin-3-one, an intensively colored dye. The newly developed assay is suitable for the detection of 2-aminophenols in the micromolar range. The paper demonstrates that this chemical assay is also applicable for the determination of 2-aminophenols substituted with a halogen at the ortho, meta, or para position of the aromatic ring with respect to the amino moiety, lambda max, and the molar extinction coefficient varying with the substituent pattern. Results obtained for fluorinated 2-aminophenol detection in biological samples by either this new method or 19F NMR are similar. This observation corroborates that the newly developed assay is suitable for detection of 2-aminophenols in biological samples.

Para-aminophenol and structurally related compounds as intermediates in lipofuscin formation and in renal and other tissue toxicities.

P-aminophenol is considered a minor nephrotoxic metabolite of phenacetin and acetaminophen (paracetamol) in man. Our experiments show that p-aminophenol readily undergoes oxidative polymerization during incubation in human blood or plasma, to form melanin, as a component of soluble lipofuscin. Haemolysis accompanies this process in whole blood. Unmetabolized phenacetin and acetaminophen do not form soluble lipofuscins. Long-term excessive use of phenacetin or acetaminophen has been associated with chronic renal disease, haemolytic anaemia, and increased solid lipofuscin deposition in tissues. Excessive use of phenacetin has also been associated with cancer of renal pelvis and bladder. It appears to us that p-aminophenol and other o- and p-aminophenol metabolites of these drugs are intermediates not only in the etiology of chronic renal disease, but in the other developments as well. P-aminophenol and other ex(end)ogenous aminohydroxyphenyl, aminopolyhydroxyphenyl, polyhydroxyphenyl and polyaminophenyl compounds with these groups in ortho and para positions (such as 3-hydroxyanthranilic acid, 6-aminodopamine, dopamine, p-phenylenediamine, etc.) can undergo autoxidations and metal-catalyzed and enzymatic oxidations in man to produce toxic (semi)quinones(imines), (semi)quinonediimines and reactive oxygen species. After depletion of antioxidants these very reactive (semi)quinones(imines) and (semi)quinonediimine intermediates, many of which are precursors of plasma soluble lipofuscins and melanoproteins, react with essential proteins, DNA, other macromolecules and can cause or contribute to renal and other tissue toxicity, haemolytic anaemia, neoplasia, and granular lipofuscin formation. The reactive oxygen species can also deplete antioxidants, damage essential proteins, DNA, and other macromolecules, and thereby injure cells and extracellular matrix.