Simultaneous Detection of Clenbuterol and Higenamine in Urine Samples Using Interference-Free SERS Tags Combined with Magnetic Separation.

Sports doping remains a significant challenge in competitive sports. Given that urine analysis is the standard for detecting doping, developing rapid, sensitive, accurate, and high-throughput methods for stimulant detection in urine is crucial. Surface-enhanced Raman scattering (SERS) tag-based immunoassays have emerged as powerful analytical tools known for their high sensitivity and specificity, holding particular promise for stimulant detection in urine samples. However, both the Raman signals of typical SERS tags and sample matrices are within the Raman fingerprint region (<1800 cm-1), which could lead to spectrum overlap, potentially reducing detection accuracy and sensitivity. By recognizing this, we designed a competitive immunoassay that integrates two types of zero-background SERS tags and magnetic separation. These innovative SERS tags exhibit distinctive Raman peaks within the Raman-silent region (1800-2800 cm-1), effectively mitigating potential spectrum overlap with background sample signals. Moreover, magnetic separation not only enhances operational simplicity but also improves the system's anti-interference capability. Using clenbuterol (CL) and higenamine (HM) as model targets, the SERS-based competitive immunoassay demonstrated sensitive detection of individual CL or HM standards, with limits of detection (LODs) of 0.87 and 0.71 pg/mL, respectively. In multiplex mode, CL and HM can be simultaneously detected with LODs of 1.0 and 0.81 pg/mL, respectively. Furthermore, the recovery rates in urine samples ranged from 83 to 116% (relative standard deviation, RSD ≤ 6.4%) for CL and from 82 to 103% (RSD ≤ 5.1%) for HM, further confirming the reliability of the SERS-based immunoassay for practical applications.

Nano-scale multi-spectroscopic analysis of the anti-fibromyalgia drug pregabalin based on dihydropyridine ring formation with sustainability and whiteness evaluation.

This research addresses the challenge of analyzing pregabalin, a primary amine in a zwitterionic structure, which is difficult to evaluate due to its lack of chromatophore. The study introduces a derivatization assessment using Hantzsch's multicomponent organic reaction to enhance the detectability of pregabalin by forming a highly fluorescent dihydropyridine derivative. This process involves the condensation of pregabalin with acetylacetone and formaldehyde, yielding a yellowish-green compound measurable both colorimetrically at 338 nm and fluorimetrically at an emission wavelength of 486 nm (λexcitation = 408 nm). The reaction conditions were thoroughly optimized to obtain the highest possible sensitivity, reduce reagent and time consumption, and use safe solvents. The developed method displayed high sensitivity and linearity in the concentration ranges of 4.0 - 20.0 µg/mL in colorimetric assay and reached a nano-scale analysis level of 40 - 2000 ng/mL with a detection limit down to 10 ng/mL when adopting the fluorimetric measurement. Both assessments were rigorously evaluated for their performance, adhering to the International Conference on Harmonization (ICH) standards. The accuracy of these methods was confirmed through the recovery rates of real samples, showing 98.9 ± 0.2 % for colorimetric and 98.2 ± 0.7 % for fluorimetric assessment. The excellent sensitivity of the suggested spectrofluorometric approach led to its use in the measurement of PRG in spiked human urine samples, resulting in particularly good recoveries ranging from 95.3 to 102.8 %. Meanwhile, the Need, Quality, Sustainability (NQS) index looks into how necessary the method is, execution quality (evaluated using the RGB 12 algorithm), and how it fits with the Sustainable Development Goals (SDGs), underlining the benefits of employing natural reagents. The developed approach showed superiority in sensitivity and sustainability compared to previous analytical approaches for pregabalin.

Development of a CZE-MS/MS method with dynamic pH junction sample pretreatment for analysis of kratom psychoactive alkaloids in urine.

BACKGROUND: Kratom is a herbal substance belonging to the group of new psychoactive substances. It contains psychoactive indole alkaloids mitragynine and 7-hydroxymitragynine. At low doses, they act as psychostimulants and at higher doses they mediate an opioid-like effect. The increasing misuse of kratom requires the development of analytical methods that will accurately and reliably identify and quantify its psychoactive alkaloids in biological samples. Therefore, the development of effective, precise, and reliable green analytical methods that are easy to implement in practice is of great importance. On-line combination of capillary zone electrophoresis with tandem mass spectrometry (CZE-MS/MS) seems to be a promising solution. RESULTS: We present a novel green approach based on capillary zone electrophoresis - tandem mass spectrometry (CZE-MS/MS) method with on-line dynamic pH junction sample pretreatment to identify and determine mitragynine and 7-hydroxymitragynine in urine samples. The separation was performed in a background electrolyte composed of 100 mM formic acid (pH 2.39). The dynamic pH junction was ensured by injection of a short plug of 12.5 % NH4OH before the sample. Under optimal conditions, the developed method was validated and parameters such as linearity (r2 > 0.99), precision (2.2-8.7 %), accuracy (89.2-102.5 %) or stability of the sample (86.6-114.7 %) met the defined FDA guideline criteria (%RSD and %RE values where within ±15 %). Introduction of a simple in-capillary preconcentration strategy based on dynamic pH junction enabled significant improvement in analytical signal intensity and also the applicability of the method. Applying the presented approach, high sensitivity was achieved as indicated by limit of detection values, which were 0.5 ng mL-1 and 2 ng mL-1 for mitragynine and 7-hydroxymitragynine, respectively. Greenness of the proposed approach was confirmed by the AGREE metrics (score 0.63). The application potential of the developed method was successfully verified using blinded urine model samples. SIGNIFICANCE: For the first time a fully validated CZE-MS/MS method for kratom alkaloids determination was introduced. The presented novel method is a cheaper and more ecological alternative to conventionally used chromatographic techniques what was clearly confirmed by its greenness evaluation and comparison with previously published liquid chromatography (LC) approaches. In-capillary sample pretreatment (dynamic pH junction) has been demonstrated to be an effective and fast tool in bioanalysis, minimizing the number of pretreatment steps and the manipulation with the sample. Moreover, LOD values comparable to those obtained by LC methods were recorded. High potential for the implementation of this approach into the toxicology environment in the near future is expected.

Changes in urinary metabolomic profile show the effectiveness of a nutritional intervention in children 6-12 years old: The ALINFA study.

Diet plays an essential role in health and disease. Therefore, its determination is an important component of many investigations. The aim of the study was to evaluate the effect of a nutritional intervention on the urinary metabolome in children aged 6-12 years. Also, it was intended to identify biomarkers of diet quality and dietary intake. A 2-month, randomized, controlled, parallel trial was conducted in Spanish children. The analyses focused on the ALINFA group, which followed a full-fixed meal plan including healthy products, ready-to-eat meals, and healthy recipes. Diet quality was assessed by the KIDMED index and dietary intake by a food frequency questionnaire. Untargeted metabolomic analysis on urine samples was carried out, and multivariate analyses were performed for pattern recognition and characteristic metabolite identification. PLS-DA and Volcano plot analyses were performed to identify the discriminating metabolites of this group. 12 putative metabolites were found to be the most relevant to this intervention. Most of them were products derived from protein and amino acid metabolism (N-Ribosylhistidine, indolacrylic acid, and peptides) and lipid metabolism (3-oxo-2-pentylcyclopentane-1-hexanoic acid methyl, Suberoyl-L-carnitine, and 7-Dehydrodichapetalin E). All these metabolites decreased after the intervention, which was mainly associated with a decrease in the consumption of fatty meat and total fat, especially saturated fat. In turn, N-Ribosylhistidine and Suberoyl-L-carnitine were negatively associated with diet quality, as well as able to predict the change in KIDMED index. In conclusion, the changes observed in urinary metabolome demonstrate the effectiveness of the ALINFA nutritional intervention.

First voltammetric analysis of two possible anticancer drug candidates using an unmodified glassy carbon electrode.

Dimethyl 2-[2-(1-phenyl-4,5-dihydro-1H-imidazol-2-yl)hydrazinylidene]butanedioate (DIHB) and 8-(3-chlorophenyl)-2,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3,4-dione (HDIT) are promising candidates for anticancer agents, the first analytical procedures of which are presented in this paper. The commercially available unmodified glassy carbon electrode (GCE) was used as a sensor for the individual and simultaneous differential pulse voltammetric (DPV) determination of these possible anticancer drugs. The findings concerning the electrochemical behaviour indicated that DIHB and HDIT display at GCE, as a sensor, the oxidation peaks at 1.18 and 0.98 V, respectively (vs. Ag/AgCl, 3.0 mol L-1 KCl) in the 0.125 mol L-1 acetate buffer of pH = 4.5, which were employed for their quantification. Various experimental parameters were carefully investigated, to achieve high sensitivity in voltammetric measurements. Finally, under the optimised conditions (t of 60 s, ΔEA of 75 mV, ν of 225 mV s-1, and tm of 2 ms), the proposed DPV procedure with the GCE demonstrated broad linear sensing ranges (1-200 nmol L-1-DIHB and 5-200 nmol L-1-HDIT), boasting the detection limits of 0.18 nmol L-1 for DIHB and 1.1 nmol L-1 for HDIT. Moreover, the developed procedure was distinguished by good selectivity, repeatability of DIHB and HDIT signals and sensor reproducibility. The practical application of this method was demonstrated by analysing the urine reference material without any prior treatment. The results showed that this environmentally friendly approach, with a modification-free sensor, is suitable for the sensitive, selective and rapid quantification of DIHB and HDIT.

A newborn Screening Programme for Inborn errors of metabolism in Galicia: 22 years of evaluation and follow-up.

BACKGROUND: There is a notable lack of harmonisation in newborn screening (NBS) programmes worldwide. The Galician programme for early detection of inborn errors of metabolism (IEM) was one of the first NBS programmes in Europe to incorporate mass spectrometry (July 2000). This programme currently screens for 26 IEMs in dried blood and urine samples collected 24-72 h after birth. RESULTS: In its 22-year history, this programme has analysed samples from 440,723 neonates and identified 326 cases of IEM with a prevalence of 1:1351. The most prevalent IEMs were hyperphenylalaninaemia (n = 118), followed by medium chain acyl-CoA dehydrogenase deficiency (MCADD, n = 26), galactosaemia (n = 20), and cystinurias (n = 43). Sixty-one false positives and 18 conditions related to maternal pathologies were detected. Urine samples have been identified as a useful secondary sample to reduce the rate of false positives and identify new defects. There were 5 false negatives. The overall positive value was 84.23%. The fatality rate over a median of 12.1 years of follow-up was 2.76%. The intelligence quotient of patients was normal in 95.7% of cases, and school performance was largely optimal, with pedagogic special needs assistance required in < 10% of cases. Clinical onset of disease preceded diagnosis in 4% of cases. The age at which first NBS report is performed was reduced by 4 days since 2021. CONCLUSIONS: This study highlights the benefits of collecting urine samples, reduce NBS reporting time and expanding the number of IEMs included in NBS programmes.

Designing a Simple Electrochemical Genosensor for the Detection of Urinary PCA3, a Prostate Cancer Biomarker.

This study investigates the feasibility of a simple electrochemical detection of Prostate Cancer Antigen 3 (PCA3) fragments extracted from patients' urine, using a thiolated single-strand DNA probe immobilized on a gold surface without using a redox probe. To enhance the PCA3 recognition process, we conducted a comparative analysis of the hybridization location using two thiolated DNA probes: Probe 1 targets the first 40 bases, while Probe 2 targets the fragment from bases 47 to 86. Hybridization with PCA3 followed, using square wave voltammetry. The limit of detection of the designed genosenors were of the order of (2.2 ng/mL), and (1.6 ng/mL) for Probes 1 and 2, respectively, and the subsequent sensitivities were of the order of (0.09 ± 0.01) µA-1 · µg-1 · mL and (0.10 ± 0.01) µA-1 · µg-1 · mL. Specificity tests were then conducted with the sensor functionalized with Probe 2, as it presents better analytical performances. The electrochemical results indicate that the designed sensor can clearly discriminate a complementary target from a non-complementary one. A further modeling of the calibration curves with the Power Law/Hill model indicates that the dissociation constant increases by one order of magnitude, confirming the ability of the designed sensor to perfectly discriminate complementary targets from non-complementary ones.

No change in 24-h sodium intake estimated from spot urine in Norwegian adults from 2006 to 2019: the population-based Trøndelag Health Study (HUNT).

OBJECTIVE: Monitoring time trends in salt consumption is important for evaluating the impact of salt reduction initiatives on public health outcomes. There has so far not been available data to indicate if salt consumption in Norway has changed during the previous decade. We aimed to assess whether average 24-h salt intake estimated from spot urine samples in the adult population of mid-Norway changed from 2006-2008 to 2017-2019 and to describe variations by sex, age and educational level. DESIGN: Repeated cross-sectional studies. SETTING: The population-based Trøndelag Health Study (HUNT). PARTICIPANTS: In each of two consecutive waves (HUNT3: 2006-2008 and HUNT4: 2017-2019), spot urine samples were collected from 500 men and women aged 25-64 years, in addition to 250 men and women aged 70-79 years in HUNT4. Based on spot urine concentrations of Na, K and creatinine and age, sex and BMI, we estimated 24-h Na intake using the International Cooperative Study on Salt and Blood Pressure (INTERSALT) equation for the Northern European region. RESULTS: Mean (95 % CI) estimated 24-h salt intakes in men were 11·1 (95 % CI 10·8, 11·3) g in HUNT3 and 10·9 (95 % CI 10·6, 11·1) g in HUNT4, P = 0·25. Corresponding values in women were 7·7 (95 % CI 7·5, 7·9) g and 7·7 (95 % CI 7·5, 7·9) g, P = 0·88. Mean estimated salt intake in HUNT4 decreased with increasing age in women, but not in men, and it did not differ significantly across educational level in either sex. CONCLUSIONS: Estimated 24-h salt intake in adult men and women in mid-Norway did not change from 2006-2008 to 2017-2019.

Zirconium-based hydrophobic-MOFs as innovative electrode modifiers for flibanserin determination: Exploring the electrooxidation mechanism using a comprehensive spectroelectrochemical study.

Three different types of Zr-based MOFs derived from benzene dicarboxylic acid (BDC) and naphthalene dicarboxylic acid as organic linkers (ZrBDC, 2,6-ZrNDC, and 1,4-ZrNDC) were synthesized. They were characterized using X-ray diffraction analysis (XRD), X-ray photoelectron spectroscopy (XPS), Fourier-transform IR spectroscopy (FT-IR), and Transmission electron microscopy (TEM). Their hydrophilic/hydrophobic nature was investigated via contact angle measurements; ZrBDC MOF was hydrophilic and the other two (ZrNDC) MOFs were hydrophobic. The three MOFs were combined with MWCNTs as electrode modifiers for the determination of a hydrophobic analyte, flibanserin (FLB), as a proof-of-concept analyte. Under the optimized experimental conditions, a significant enhancement in the oxidation peak current of FLB was observed when utilizing 2,6-ZrNDC and 1,4-ZrNDC, being the highest when using 1,4-ZrNDC. Furthermore, a thorough investigation of the complex oxidation pathway of FLB was performed by carrying out simultaneous spectroelectrochemical measurements. Based on the obtained results, it was verified that the piperazine moiety of FLB is the primary site for electrochemical oxidation. The fabricated sensor based on 1,4-ZrNDC/MW/CPE showed an oxidation peak of FLB at 0.8 V vs Ag/AgCl. Moreover, it showed excellent linearity for the determination of FLB in the range 0.05 to 0.80 µmol L-1 with a correlation coefficient (r) = 0.9973 and limit of detection of 3.0 nmol L-1. The applicability of the developed approach was demonstrated by determination of FLB in pharmaceutical tablets and human urine samples with acceptable repeatability (% RSD values were below 1.9% and 2.1%, respectively) and reasonable recovery values (ranged between 97 and 103% for pharmaceutical tablets and between 96 and 102% for human urine samples). The outcomes of the suggested methodology can be utilized for the determination of other hydrophobic compounds of pharmaceutical or biological interest with the aim of achieving low detection limits of these compounds in various matrices.

Urinary biomonitoring of fuel ether oxygenates using a needle trap device packed with a novel molecularly imprinted polymer surface modified Zeolite Y.

This study introduces an innovative needle trap device (NTD) featuring a molecularly imprinted polymer (MIP) surface-modified Zeolite Y. The developed NTD was integrated with gas chromatography-flame ionization detector (GC-FID) and employed for analysis of fuel ether oxygenates (methyl tert­butyl ether, MTBE, ethyl tert­butyl ether, ETBE, and tert­butyl formate, TBF) in urine samples. To optimize the key experimental variables including extraction temperature, extraction time, salt concentration, and stirring speed, a central composite design-response surface methodology (CCD-RSM) was employed. The optimal values for extraction in the study were found to be 51.2 °C extraction temperature, 46.2 min extraction time, 27 % salt concentration, and 620 rpm stirring speed. Under the optimized conditions, the calibration curves demonstrated excellent linearity within the range of 0.1-100 µg L-1, with correlation coefficients (R2) exceeding 0.99. The limits of detection (LODs) for MTBE, ETBE, and TBF were obtained 0.06, 0.08, and 0.09 µg L-1, respectively. Moreover, the limits of quantification (LOQs) for MTBE, ETBE, and TBF were obtained 0.18, 0.24, and 0.27 µg L-1, respectively. The enrichment factor was also found to be in the range of 98-129.The NTD-GC-FID procedure demonstrated a high extraction efficiency, making it a promising tool for urinary biomonitoring of fuel ether oxygenates with improved sensitivity and selectivity compared to current methods.

Simultaneous detection of mycotoxins and pesticides in human urine samples: A 24-h diet intervention study comparing conventional and organic diets in Spain.

Pesticides and mycotoxins, prominent chemical hazards in the food chain, are commonly found in plant-based foods, contributing to their pervasive presence in the human body, as evidenced by biomonitoring programs. Despite this, there is limited knowledge about their co-occurrence patterns. While intervention studies have demonstrated that organic diets can significantly reduce pesticide levels, their impact on mycotoxin exposure has been overlooked. To address this gap, this study pursued two objectives: first, to characterize the simultaneous presence of mycotoxins and pesticides in human urine samples by means of the control of the biomarkers of exposure, and second, to investigate the influence of consuming organic foods on these co-exposure patterns. A pilot study involving 20 healthy volunteers was conducted, with participants consuming either exclusively organic or conventional foods during a 24-h diet intervention in autumn 2021 and spring 2022 to account for seasonal variability. Participants provided detailed 24-h dietary records, and their first-morning urine samples were collected, minimally treated and analysed using LC-Q-ToF-MS by means of a multitargeted method in order to detect the presence of these residues. Results indicated that among the 52 screened compounds, four mycotoxins and seven pesticides were detected in over 25% of the samples. Deoxynivalenol (DON) and the non-specific pesticide metabolite diethylphosphate (DEP) exhibited the highest frequency rates (100%) and concentration levels. Correlations were observed between urine levels of mycotoxins (DON, ochratoxin alpha [OTα], and enniatin B [ENNB]) and organophosphate pesticide metabolites DEP and 2-diethylamino-6-methyl-4-pyrimidinol (DEAMPY). The pilot intervention study suggested a reduction in ENNB and OTα levels and an increase in ß-zearalenol levels in urine after a short-term replacement with organic food. However, caution is advised due to the study's small sample size and short duration, emphasizing the need for further research to enhance understanding of the human chemical exposome and refine chemical risk assessment.

Sensitive, reliable and simultaneous determination of Fingolimod and Citalopram drug molecules used in multiple sclerosis treatment based on magnetic solid phase extraction and HPLC-PDA.

An analytical methodology has been developed for trace amounts of Fingolimod (FIN) and Citalopram (CIT) drug molecules based on magnetic solid phase extraction (MSPE) and high performance liquid chromatographic determination with photodiode array detector (HPLC-DAD). Fingolimod is used in treatment of Multiple sclerosis (MS) disease and sometimes antidepressant drugs such as citalopram accompany to treatment. Both simultaneous analysis of these molecules and application of MSPE with a new adsorbent has been performed for first times. Fe3O4@L-Tyrosine magnetic particles has been synthetized and characterized as a new magnetic adsorbent. Experimental variables of MPSE were examined and optimized step by step such as pH, adsorption and desorption conditions, time effect, etc. Analytical parameters of the proposed method were studied and determined under optimized conditions according to international guidelines. HPLC analysis of FIN and CIT molecules was performed by isocratic elution of a mixture of 50 % Acetonitrile, 40 % pH:3 phosphate buffer and 10 % methanol with flow rate 1.0 mL min-1. The chosen wavelengths in PDA was determined as 238 nm for FIN and 213 nm for CIT. The limits of detection (LOD) for proposed method were 6.32 ng mL-1 for FIN and 6.85 ng mL-1 for CIT molecules. RSD % values were lower than 5.5 % in analysis of model solutions including 250 and 500 ng mL-1 of target molecules. Recovery values by means of synthetic urine and saliva samples were in the range of 95.7-105.4 % for both molecules.

Selective Extraction of Diazepam and Its Metabolites from Urine Samples by a Molecularly Imprinted Solid-Phase Extraction (MISPE) Method.

In this research, a molecularly imprinted polymer (MIP) was synthesized by precipitation polymerization using oxazepam (OZ) as a template molecule and was subsequently applied as a selective sorbent for the extraction of diazepam (DZP) and its metabolites in urine samples using an SPE cartridge. OZ, temazepam (TZ), nordiazepam (NZ) and DZP were analyzed in the final extracts by high-performance liquid chromatography with diode array detection (HPLC-DAD). The SPE extraction steps were optimized, and the evaluation of an imprinting factor was carried out. The selectivity of the method for OZ versus structurally related benzodiazepines (BZDs), such as bromazepam (BRZ), tetrazepam (TTZ) and halazepam (HZ), was investigated. Under the optimum conditions, the proposed methodology provided good linearity in the range of 10-1500 ng/mL, with limit of detection values between 13.5 and 21.1 ng/mL and recovery levels for DZP and its metabolites from 89.0 to 93.9% (RSD ≤ 8%) at a concentration level of 1000 ng/mL. The proposed method exhibited good selectivity, precision and accuracy and was applied to the analysis of urine samples from a real case of DZP intake.

Preparation of branched polyethyleneimine-assisted boronic acid-functionalized magnetic MXene for the enrichment of catecholamines in urine samples.

Herein, a magnetic borate-functionalized MXene composite with multiple boronic affinity sites was fabricated by embedding Fe3 O4 nanoparticles with 4-formylphenylboronic acid functionalized Ti3 C2 Tx nanosheets and served as sorbent for the simultaneous extraction of catecholamines (CAs) in urine samples. The morphology and structure of the magnetic materials were investigated using scanning microscopy, vibrating sample magnetometer, X-ray photoelectron spectrometer, and X-ray diffraction. The introduction of polyethyleneimine can amplify the bonded boronic acid groups, thereby effectively improving the adsorption capacities for CAs based on the multiple interactions of boronic affinity, hydrogen bonding, and metal coordination. The adsorption performance was investigated using the kinetics and isotherms models, and the main parameters that influence the extraction efficiency were optimized. Under the most favorable magnetic solid-phase extraction condition, a sensitive method for the analysis of CAs in urine samples was developed by combining magnetic solid-phase extraction conditions with high-performance liquid chromatography detection. The findings illustrated that the proposed approach possessed a wide linearity range of 0.05-250 ng/mL with an acceptable correlation coefficient (R2  ≥ 0.9984) and detection limits of 0.010-0.015 ng/mL for the target CAs. The research not only provides a notable composite with multiple boronic affinity sites but also offers an effective and feasible measure for the detection of CAs in biological samples.

The pharmacokinetics of 38% silver diamine fluoride in healthy children with dental caries: a quasi-experimental study.

PURPOSE: Silver diamine fluoride (SDF) is a medication used for the arrestment of dental caries. This study aims to determine the pharmacokinetics (PK) of silver and fluoride following SDF application in healthy children with dental caries. METHODS: A total of 15 subjects, aged 4 to 10 years, with at least one cavity on a primary tooth, were recruited for this study. Urine samples were collected at baseline, first 24 h (F1) and second 24 h (F2) after SDF treatment for analysis of silver and fluoride content. Hair samples were also collected at baseline and at 7, 14, 30, 60, 75, and 90 days after SDF treatment to analyze silver content. RESULTS: Participants with under or over-collection of urine, or failure to provide urine collection were excluded for fluoride analysis. As a result, eight subjects' urine samples were eligible for fluoride analysis. Significant correlations were observed between baseline urinary fluoride levels and F1/F2 levels. Pairwise comparisons from Friedman's test showed significant differences between baseline and F1 fluoride levels. For silver analysis, 15 subjects were studied. F1 urinary silver levels were higher than baseline and F2 levels. Subsequent to SDF treatment, hair silver levels displayed fluctuations around the baseline. None of the participants reported adverse effects, and all caries teeth ceased progression within 30 days. CONCLUSIONS: The urinary fluoride levels after SDF treatment, although higher, were not clinically significant. Urinary and hair silver levels were negligible. Therefore, SDF appears safe to be used among children.

The diagnostic accuracy of the GeneXpert ESBL-ampC prototype assay for rapid PCR-based detection of extended-spectrum beta-lactamase genes directly from urine.

IMPORTANCE: Early identification of complicated urinary tract infections caused by ESBL-producing Enterobacterales has the potential to limit the use of carbapenems to those patients without alternative antibiotic options and avoid the empirical use of carbapenems in patients without ESBL-producing bacteria. The purpose for such a test will differ by setting and ESBL prevalence rates. Countries with low ESBL rates and cephalosporins as empiric treatment (e.g., The Netherlands) will need a rule-in test to decide to use carbapenems, while countries with high ESBL rates and empiric carbapenem treatment will need a rule-out test for ESBLs to de-escalate therapy early. Anyway, such as a test would-at least theoretically-improve patient care and reduce selective pressure for the emergence of carbapenem resistance.

Synthesis and application of quaternary amine-functionalized core-shell-shell magnetic polymers for determination of metabolites of benzene, toluene and xylene in human urine samples and study of exposure assessment.

As production processes have evolved, airborne concentrations of benzene, toluene and xylene in many workplaces are already well below the occupational exposure limits. However, studies have shown that low levels of exposure to benzene, toluene and xylene can still cause health effects in people exposed occupationally. However, there is no literature on health risk assessment of internal exposure. In view of this, an analytical method based on quaternary amine-functionalized core-shell-shell magnetic polymers (QA-CSS-MPs) was developed for the determination of seven metabolites in urine by MSPE-UPLC-DAD-HRMS. Furthermore, an improved QuEChERS method for the extraction of seven metabolites from human urine samples was introduced for the first time and satisfactory extraction rates were achieved. In addition, QA-CSS-MPs microspheres with core-shell-shell structure were designed and synthesized, and the morphology, composition and magnetic properties of the materials were fully characterized to verify the rationality of the synthetic route. Subsequently, QA-CSS-MPs microspheres were used as magnetic solid-phase extraction (MSPE) adsorbents for the purification of urine extracts, and UPLC-DAD-HRMS was used for the detection of seven metabolites. As a result, this method allows the accurate determination of seven metabolites in urine samples over an ultra-wide concentration range (0.001-100 mg/L). Under optimal experimental conditions, i.e., 2% hydrochloric acid in urine for the hydrolysis and 20 mg of QA-CSS-MPs for 5 min purification, the spiked recoveries of the seven target metabolites ranged from 81.5% to 117.7% with RSDs of 1.0%-9.4%. The limits of detection (LODs, S/N≥3) for the established method were in the range of 0.2-0.3 µg/L. The developed method was applied to 254 human urine samples for the determination of seven metabolites. The results showed that the concentration distributions of three xylene metabolites in urine, 2-MHA, 3-MHA, 4-MHA and total MHA, showed statistically significant differences for occupational exposure (p<0.001). In addition, the results of the internal exposure assessment showed that there is a high potential health risk associated with occupational exposure processes.

Prenatal exposure to triclosan assessed in multiple urine samples and placental DNA methylation.

A previous study reported positive associations of maternal urinary concentrations of triclosan, a synthetic phenol with widespread exposure in the general population, with placental DNA methylation of male fetuses. Given the high number of comparisons performed in -omic research, further studies were needed to validate and extend on these findings. Using a cohort of male and female fetuses with repeated maternal urine samples to assess exposure, we studied the associations between triclosan and placental DNA methylation. We assessed triclosan concentrations in two pools of 21 urine samples collected among 395 women from the SEPAGES cohort. We used Infinium Methylation EPIC arrays to measure DNA methylation in placental biopsies collected at delivery. We performed a candidate study restricted to a set of candidate CpGs (n = 500) identified in a previous work as well as an exploratory epigenome-wide association study to investigate the associations between triclosan and differentially methylated probes and regions. Analyses were conducted on the whole population and stratified by child's sex. Mediation analysis was performed to test whether heterogeneity of placental tissue may mediate the observed associations. In the candidate approach, we confirmed 18 triclosan-associated genes when both sexes were considered. After stratification for child's sex, triclosan was associated with 72 genes in females and three in males. Most of the associations were positive and several CpGs mapped to imprinted genes: FBRSL1, KCNQ1, RHOBTB3, and SMOC1. A mediation effect by placental tissue heterogeneity was identified for most of the observed associations. In the exploratory analysis, we identified a few isolated associations in the sex-stratified analysis. In line with a previous study on male placentas, our approach revealed several positive associations between triclosan exposure and placental DNA methylation. Several identified loci mapped to imprinted genes.

Comparison of Human Papillomavirus Genotype Detection in Paired Urine and Self-Collected Cervical Swabs: A Pilot Study.

OBJECTIVES: With the objective of establishing a simple, cost-effective, and effective screening tool for the screening of Human Papilloma Virus infection, the study was undertaken. MATERIAL METHODS: This pilot study was conducted on 20 urine samples of women whose cervical swabs were tested positive while screening for Human papilloma virus in asymptomatic women. RESULTS: HPV genotypes were detected in 94% (16/17) patients in urine samples by real-time PCR while a 100% detection rate (15/15) was observed in the cervical swab samples. The results of the urine and cervical swab samples, tested by the TRUPCR ®HPV high-risk genotyping kit, are shown in Table 2. HPV genotype 68 was found in 82.3% urinary samples and 100% of self-collected vaginal swab samples. Out of 16 positive urine samples, 2 were positive for HPV genotype 16 and 5 were positive for HPV genotype 18, and in cervical swab testing out of 15 positive samples, 3 were positive for HPV genotype 16, and 5 were positive for HPV genotype 18. Diagnostic accuracy of urine was found to be 98.8% (95% CI 79.43% - 100.00%). CONCLUSION: This pilot study aims to assess the accuracy of urine samples in the screening of HPV infection among asymptomatic women and establish the distribution of prevalent HPV genotypes. This may further contribute to standardizing the urine and cervical swab testing methods for cervical cancer screening strategies.

Simplex Droplet Digital PCR Assays for the Detection of TERT Promoter Mutations in Urine Samples for the Non-invasive Diagnosis of Urothelial Cancer.

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter region are highly frequent in urothelial cancer (UC), and their detection in urine (cell-free DNA from the urine supernatant or DNA from exfoliated cells in the urine pellet) has demonstrated promising evidence as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumour-derived mutations in urine requires highly sensitive methods, capable of measuring low-allelic fraction mutations. We developed sensitive droplet digital PCR (ddPCR) assays for detecting urinary TERT promoter mutations (uTERTpm), targeting the two most common mutations (C228T and C250T), as well as the rare A161C, C228A, and CC242-243TT mutations. Here, we described the step-by-step protocol uTERTpm mutation screening using simplex ddPCR assays and give some recommendations for isolation of DNA from urine samples. We also provide limits of detection for the two most frequent mutations and discuss advantages of the method for clinical implementation of the assays for the detection and monitoring of UC.