Untreated Urine Samples Research Articles

Solvent bar microextraction based on using deep eutectic solvent and multivariate optimization for simultaneous determination of antidepressant and antiepileptic drugs in biological fluids

AbstractMonitoring medications in biological fluids is an essential aspect of patient care, particularly in cases of altered mental status where accurate diagnosis, effective treatment, and even forensic examinations are crucial. In this study, a new approach combining hydrophobic-deep-eutectic and solvent-bar-microextraction (HDE-SBME) followed by a high-performance liquid chromatography-diode array detector (HPLC-DAD) was developed for the simultaneous determination of desipramine, clomipramine, as antidepressant and carbamazepine as antiepileptic agents in untreated human urine and plasma samples. The HDE solvents, synthesized using various ratios of menthol and fatty acids, were utilized in the SBME setups. Computational methods were employed to predict the structure and modes of interaction between HDE and the chosen analytes. Central composite design methodology (CCD) was used for multivariate optimization of the effects of different parameters influencing the extraction efficiency of the proposed method. Under optimized experimental conditions, the calibration graph of the spiked selected drugs in urine and plasma samples demonstrated excellent linearity (R2 ≥ 0.994), with limits of detection/quantification below 0.60/2.02 μg L−1. The extraction recoveries achieved were 88–97%, and the repeatability/reproducibility (RSD%, n = 5) was less than 6.12/7.57. The proposed method was successfully applied to determine selected drugs in patients' urine and plasma samples. The proposed method detects three drugs in patients' urine and plasma samples without using toxic volatile organic solvents. The proposed microextraction technique exhibited a confident sensitivity, feasible operation, and simplicity compared with other published methods. Thus, it can be considered a promising method for monitoring the therapeutic levels of specific antidepressant and antiepileptic drugs in urine and plasma samples.

Enzymeless electrochemical biosensor platform utilizing Cu2O-Au nanohybrids for point-of-care creatinine testing in complex biological fluids

Herein, we developed a novel, enzyme-free electrochemical biosensor that can directly quantify creatinine within complex biological fluids in a highly specific and sensitive manner. Our approach utilizes the synergistic effects of zwitterion-functionalized Cu2O-Au core satellite nanohybrids with sophisticated 3D granular structures, which were synthesized via a rapid room-temperature combinatory protocol. Through nanoscale zwitterion surface engineering, outstanding antifouling capabilities were obtained and interferents were effectively eliminated by generating a pseudo proton exchange membrane (PEM). This pseudo-PEM can potentially serve as an alternative to traditional methods of removing interference; thus, analytes can be directly detected in untreated biofluids. The optimized biosensor platform demonstrated a linear response to creatinine with exceptional reproducibility and an excellent limit of detection (LOD) in the following sensing ranges: (i) 10.0–200 µM (R2 =0.99, RSD=1.22 %, n = 10, LOD=0.72 µM) and (ii) 1.00–35.0 mM (R2 =0.99, RSD=1.32 %, n = 10, LOD=2.41 mM). These ranges are clinically relevant and cover the entire physiological range of creatinine in human serum and urine, respectively. Furthermore, we successfully detected creatinine directly in untreated human urine samples and observed a high correlation with clinically determined values (r = 0.98). Overall, our proposed biosensor platform offers significant improvements over existing methods and provides a reliable and efficient approach for practical point-of-care (POC) creatinine quantification.

Extending the range of metal ions SERS detection using hybrid plasmonic/ZIF-8 particles

Nanosensors based on surface-enhanced Raman spectroscopy (SERS) have emerged as a class of promising optical tools for the ultrasensitive quantification of metal ions of environmental and biological interest. A central bottleneck in this field is the availability of suitable surface receptors able to convert the selective binding with these vibrationless analytes into measurable SERS signals. In this work, we tackle this issue by employing a hybrid substrate comprising a highly SERS-active plasmonic core and a ZIF-8 metal-organic framework (MOF) shell. The ZIF-8 shell firmly captures aromatic receptors close to the plasmonic structure regardless of their intrinsic affinity for the metallic surface and without altering their ability to coordinate metal ions. Furthermore, it imparts molecular sieving abilities enabling the direct use of the SERS sensing platform in complex media such as biological fluids. This was demonstrated by using different classes of chromogenic reagents (bathocuproine, a 2,6':2′,2″-terpyridine derivative, and Arsenazo III) which were exploited for the SERS detection of both transition and alkaline earth metal ions (i.e., divalent copper, cobalt and calcium ions). Notably, we successfully applied this approach for the detection of Cu(II) in untreated urine samples for Wilson's disease diagnosis. Overall, we believe this class of multifunctional hybrid substrates will serve as a valuable material for expanding the applicability of SERS spectroscopy in real-life environmental and biomedical metal ions analysis.

Cadmium-free silica-encapsulated molecularly imprinted AuZnCeSeS quantum dots nanocomposite as an ultrasensitive fluorescence nanosensor for methamphetamine detection

One of the major challenges facing forensic drug analysis is the difficulty in detecting ultralow concentration of illicit drugs in biological matrices without the need for an extraction or a pre-treatment step. This work report on the development of a novel AuZnCeSeS quantum dots (QDs)-molecular imprinted polymer (MIP) nanocomposite fluorescent probe for methamphetamine (METH) recognition. Silica-coated AuZnCeSeS QDs were synthesized and characterized using spectrophotometric, spectroscopic and electron microscopy techniques. Via a free radical polymerization reaction, a thin layer of MIP shell with METH as the template was coated around the QDs surface leading to the formation of a QDs-MIP nanocomposite probe. The MIP coating passivated the QDs surface leading to radiative fluorescence enhancement of the bound QDs. Under optimum reaction conditions, METH was selectively and quantitatively detected via a fluorescence quenching reaction process. The unique selectivity of the nanoprobe for METH recognition showed clearly that METH was able to precisely re-bind to the MIP surface with size and shape reorganization. While the MIP shell functioned to provide the required selectivity, the AuZnCeSeS QDs functioned to fluorescently report the surface binding interaction. The use of a AuZnCeSeS QDs-non-imprinted polymer as probe to detect METH resulted in poor sensitivity and selectivity; hence, demonstrating the suitability of the AuZnCeSeS QDs-MIP nanoprobe to accurately detect METH. METH was detected within a wide concentration range from 0.05 to 50,000 nM with a detection limit of ∼0.02 nM (0.0036 ng/mL). The developed AuZnCeSeS QDs-MIP nanoprobe was efficiently used to detect METH in untreated urine sample with recovery efficiency from ∼100 to 110%.

Biomimetic multienzyme nanocomplex for efficient cascade reactions

In nature, living cells form multienzyme complex by spatializing enzymes in confined compartments to enable cascade reactions. As these reactions are highly efficient and crucial to cellular metabolism, it is important to understand their catalytic behaviors. Herein, we report a living cell-inspired design of nanoscale multienzyme complex (nanocomplex), in which different enzymes are densely packed with intact conformations as the cellular multienzyme complex does, allowing sufficient transportation of intermedia and creating efficient cascade reactions. Benefit from the similarity between the proposed nanocomplex and cellular multienzyme complex, the systematic study of the effect of enzyme ratios and quantity to the nanocomplex reaction efficiency can inspire the investigation of the basic rules of cellular catalytic behaviors. In addition, the nanocomplex can also contribute to boosting the detection sensitivity of multienzyme-based biosensing due to its unsurpassed efficiency. Using the nanocomplex fabricated with methoxy polyethylene glycol (mPEG), glucose oxidase (GOx) and horseradish peroxidase (HRP), glucose in untreated urine, plasma and saliva samples can be detected with a remarkable detection limit of 1.6 µM and a detection time of just 30 s, surpass the current glucose detection approaches. Remarkably, the proposed sensor can also perform glucose detection with preserved cascade activity under high temperature, acid environment, or after long-time storage. Overall, this work can potentially provide a general understanding of multienzyme catalytic behaviors and a promising platform of ultrasensitive biosensing for disease diagnosis and prognosis.

Profiling of Urine Carbonyl Metabolic Fingerprints in Bladder Cancer Based on Ambient Ionization Mass Spectrometry.

The diagnosis of bladder cancer (BC) is currently based on cystoscopy, which is invasive and expensive. Here, we describe a noninvasive profiling method for carbonyl metabolic fingerprints in BC, which is based on a desorption, separation, and ionization mass spectrometry (DSI-MS) platform with N,N-dimethylethylenediamine (DMED) as a differential labeling reagent. The DSI-MS platform avoids the interferences from intra- and/or intersamples. Additionally, the DMED derivatization increases detection sensitivity and distinguishes carboxyl, aldehyde, and ketone groups in untreated urine samples. Carbonyl metabolic fingerprints of urine from 41 BC patients and 41 controls were portrayed and 9 potential biomarkers were identified. The mechanisms of the regulations of these biomarkers have been tentatively discussed. A logistic regression (LR) machine learning algorithm was applied to discriminate BC from controls, and an accuracy of 85% was achieved. We believe that the method proposed here may pave the way toward the point-of-care diagnosis of BC in a patient-friendly manner.

Sensitive ‘two-steps’ competitive assay for gonadotropin-releasing hormone detection via SPR biosensing and polynorepinephrine-based molecularly imprinted polymer

The work reports an innovative bioassay for the detection of gonadorelin in urine, a gonadotropin-releasing hormone agonist widely used in fertility medicine and to treat hormonal dysfunctions. Gonadorelin is also a synthetic hormone listed by the World Anti-Doping Agency (WADA) and of interest in anti-doping controls. The main novelty relies on the development of a biocompatible, stable, and low-cost biomimetic receptor alternative to classic antibodies. Starting from norepinephrine monomer, a highly selective and sensitive molecularly imprinted polymer (MIP) was developed and optimized for optical real-time and label-free SPR biosensing. The selectivity has been addressed by testing a series of peptides, from high to low similarity, both in terms of molecular weight and primary sequence. Due to the very low molecular weight of gonadorelin (1182 Da), a ‘two-steps’ competitive assay was developed. Particular attention has been paid to the design of the competitor and its binding affinity constant towards the MIP, being a key step for the success of the competitive strategy. The SPR assay was first optimized in standard conditions and finally applied to untreated urine samples, achieving the sensitivity required by WADA guidelines. The MIP, tested in parallel with a monoclonal antibody, gave comparable results in terms of affinity constants and selectivity towards possible interfering analytes. However, the biomimetic receptor appears clearly superior in terms of sensitivity and reproducibility. This, together with its preparation simplicity, the extremely low-cost of the monomer and its reusability for hundreds of measurements, make polynorepinephrine-based MIPs powerful rivals to immune-based approaches in the near future for similar applications.

Interpretable machine learning model to detect chemically adulterated urine samples analyzed by high resolution mass spectrometry.

Urine sample manipulation including substitution, dilution, and chemical adulteration is a continuing challenge for workplace drug testing, abstinence control, and doping control laboratories. The simultaneous detection of sample manipulation and prohibited drugs within one single analytical measurement would be highly advantageous. Machine learning algorithms are able to learn from existing datasets and predict outcomes of new data, which are unknown to the model. Authentic human urine samples were treated with pyridinium chlorochromate, potassium nitrite, hydrogen peroxide, iodine, sodium hypochlorite, and water as control. In total, 702 samples, measured with liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, were used. After retention time alignment within Progenesis QI, an artificial neural network was trained with 500 samples, each featuring 33,448 values. The feature importance was analyzed with the local interpretable model-agnostic explanations approach. Following 10-fold cross-validation, the mean sensitivity, specificity, positive predictive value, and negative predictive value was 88.9, 92.0, 91.9, and 89.2%, respectively. A diverse test set (n=202) containing treated and untreated urine samples could be correctly classified with an accuracy of 95.4%. In addition, 14 important features and four potential biomarkers were extracted. With interpretable retention time aligned liquid chromatography high-resolution mass spectrometry data, a reliable machine learning model could be established that rapidly uncovers chemical urine manipulation. The incorporation of our model into routine clinical or forensic analysis allows simultaneous LC-MS analysis and sample integrity testing in one run, thus revolutionizing this field of drug testing.

Multidimensional Liquid Chromatography Employing a Graphene Oxide Capillary Column as the First Dimension: Determination of Antidepressant and Antiepileptic Drugs in Urine

Human mental disorders can be currently classified as one of the most relevant health topics. Including in this are depression and anxiety, which can affect us at any stage of life, causing economic and social problems. The treatments involve cognitive psychotherapy, and mainly the oral intake of pharmaceutical antidepressants. Therefore, the development of analytical methods for monitoring the levels of these drugs in biological fluids is critical. Considering the current demand for sensitive and automated analytical methods, the coupling between liquid chromatography and mass spectrometry, combined with suitable sample preparation, becomes a useful way to improve the analytical results even more. Herein we present an automated multidimensional method based on high-performance liquid chromatography-tandem mass spectrometry using a lab-made, graphene-based capillary extraction column connected to a C8 analytical column to determined five pharmaceutical drugs in urine. A method enhancement was performed by considering the chromatographic separation and the variables of the loading phase, loading time, loading flow, and injection volume. Under optimized conditions, the study reports good linearity with R2 > 0.98, and limits of detection in the range of 0.5–20 µg L−1. Afterward, the method was applied to the direct analysis of ten untreated urine samples, reporting traces of citalopram in one of them. The results suggest that the proposed approach could be a promising alternative that provides direct and fully automated analysis of pharmaceutical drugs in complex biological matrices.

Rapid time-resolved luminescence based screening of bacteria in urine with luminescence modulating biosensing phages

Urinary tract infections (UTIs) are a common problem worldwide. The most prevalent causative pathogen of UTI is Escherichia coli, focus of this study. The current golden standard for detecting UTI is bacterial culture, creating a major workload for hospital laboratories - cost-effective and rapid mass screening of patient samples is needed. Here we present an alternative approach to screen patient samples with a single-step assay utilising time-resolved luminescence and luminescence modulating biosensing phages. Filamentous phage M13 was biopanned for binding luminescence quenching metal (copper) and further E. coli. The screening assay luminescence modulation was further enhanced by selecting right chemical environment for the functioning phage clones. Semi-specific interaction between phage, target bacteria and metal was detected by modulation in the signal of a weakly chelating, easily quenchable lanthanide complex. In the presence of the target pathogen, the phages collected quenching metal from solution to the bacterial surface changing the quenching effect on the lanthanide label and thus modulating the signal. Our method was compared with the bacterial culture data obtained from 70 patient samples. The developed proof-of-principle screening assay showed sensitivity and a specificity at the 90% mark when compared to culture method although some samples had high turbidity and even blood. The detection limit of E. coli was in the range of 1000–10 000 colony forming units/mL. Untreated urine sample was screened and time-resolved luminescence signal result was achieved within 10 min in a single incubation step.

Development of a multi‐functional concurrent assay using weak cation‐exchange solid‐phase extraction (WCX‐SPE) and reconstitution with a diluted sample aliquot for anti‐doping analysis

In addition to the development of adequate screening methods for multiple compounds, the World Anti-Doping Agency (WADA) requires anti-doping laboratories to analyze prohibited substances and their metabolites from various classes. This task presents a difficult challenge for all agencies and interests involved in the field of doping control. A screening method is reported in which hybrid sample preparation was performed using a combination of weak cation-exchange solid-phase extraction (WCX-SPE) and the 'Dilute and Shoot' strategy in order to take advantage of both the methodologies. Target substances were extracted using a WCX cartridge and reconstituted with a diluted sample aliquot that included 20% of an untreated urine sample. The target substances were further analyzed by high-performance liquid chromatography/triple quadrupole mass spectrometry (LC/MS). The SPE procedure was optimized using a cartridge-washing step, elution conditions, and elution volume. The cartridge-washing step, which was performed using 10% methanol, improved the overall recovery of target substances. Since the recovery was observed to vary according to the pH of the eluting solution, we applied an elution step using both an acid and a basic organic solvent to achieve complementary recovery. Reconstitution of the diluted aliquot sample was performed to recover the polar substances. The method was validated and applied to real samples in accordance with the external quality assessment scheme of WADA and to the previously reported samples that had provided positive test results. This novel method using hybrid sample preparation and LC/MS could be useful to screen multiple classes of the 264 targeted substances in anti-doping analysis.

Rapid Detection of Copper in Biological Systems Using Click Chemistry.

A fast (1 min), straightforward but efficient, click chemistry-based system that enables the rapid detection of free copper (Cu) ions in either biological fluids or living cells without tedious pretreatment is provided. Cu can quickly induce the conjugation between graphene oxide (GO) and a fluorescent dye via click reaction. On the basis of the high specificity of bioorthogonal reaction and the effective quenching ability of GO, the assay studied in this paper can respond to Cu ions in less than 1 min with excellent selectivity and sensitivity, which is the fastest sensor for Cu as far as it is known. In addition, the application of this system is verified by performing assays in living cells and untreated urine samples from patients suffering from Wilson's Disease. Such a Cu detection system shows great promises in both fundamental research and routine clinical diagnostics.

Competitive amperometric immunosensor for determination of p53 protein in urine with carbon nanotubes/gold nanoparticles screen-printed electrodes: A potential rapid and noninvasive screening tool for early diagnosis of urinary tract carcinoma

Since p53 protein has become recognized biomarker for both diagnostic and therapeutic purposes in oncological diseases with particular relevance for bladder cancer, it is highly desirable to search for a novel sensing tool for detecting the patient's p53 level at the early stage. Here we report the first study on the development and validation of a novel disposable competitive amperometric immunosensor for determination of p53 protein at subnanomolar levels, based on p53 immobilization on gold nanoparticles/carbon nanotubes modified screen-printed carbon electrodes. The assay protocol requires the use of single anti-p53 mouse monoclonal antibody (DO-7 clone), able to recognize both wild-type and mutant p53. The developed immunosensor as well as the protocol of the electrochemical immunoassay were optimized by means of an experimental design procedure to assess the suitability of the device to be validated and applied for the determination of p53 in untreated and undiluted urine samples. It was found that the developed competitive immunodevice was able to achieve wide linear range detection of wild-type p53 from 20 pM to 10 nM with a low detection limit of 14 pM in synthetic urine samples, suggesting the sensor's capability of working in a complex sample matrix. The excellent performance results also in terms of selectivity, trueness and precision, coupled with the advantages of an easy preparation and low-cost assay in contrast to other methods which require very complex, time-consuming and costly nanostructured architectures, makes the developed competitive immunosensor an analytically robust diagnostic tool, valuable for implementation of screening and follow-up programs in patients with urologic malignancies.

A new metabolomics-based strategy for identification of endogenous markers of urine adulteration attempts exemplified for potassium nitrite.

Urine adulteration to circumvent positive drug testing represents a problem for toxicological laboratories. While creatinine is a suitable marker for dilution, detection of chemicals is often performed by dipstick tests associated with high rates of false positives. Several methods would be necessary to check for all possible adulterants. Untargeted mass spectrometry (MS) methods used in metabolomics should theoretically allow detecting concentration changes of any endogenous urinary metabolite or presence of new biomarkers produced by chemical adulteration. As a proof of concept study, urine samples from 10 volunteers were treated with KNO2 and analyzed by high-resolution MS. For statistical data evaluation, XCMSplus and MetaboAnalyst were used. Compound identification was performed by database searches using an in-house database, Chemspider, METLIN, HMDB, and NIST. Principle component analysis revealed clear separation between treated and untreated urine samples. In detail, 307 features showed significant concentration changes with fold changes greater than 2 (79 decreased; 228 increased). Mainly amino acids (e.g., histidine, methylhistidine, di- and trimethyllysine) and purines (uric acid) were detected in lower amounts. 5-HO-isourate was found to be formed as a new compound from uric acid and, e.g., imidazole lactate concentrations increased due to the breakdown of histidine. This metabolomics-based strategy allowed for a broad identification range of markers of urinary adulteration. More studies will be needed to investigate routine applicability of identified potential markers exploring urinary conditions of their formation and stability. Selected markers might then be integrated into routine MS screening procedures allowing for detection of adulteration within routine MS analysis. Graphical Abstract ᅟ.

PD65-05 URINE EXPRESSION OF TIMP1, SERPINB1, AND SEMENOGELIN 2 MAY DIFFERENTIATE MEN WITH LOW-RISK OR NO EVIDENCE OF PROSTATE CANCER FROM MEN WITH HIGH-RISK OR METASTATIC DISEASE

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 2017PD65-05 URINE EXPRESSION OF TIMP1, SERPINB1, AND SEMENOGELIN 2 MAY DIFFERENTIATE MEN WITH LOW-RISK OR NO EVIDENCE OF PROSTATE CANCER FROM MEN WITH HIGH-RISK OR METASTATIC DISEASE Sarah Prophet, Adam Feldman, Mary Fergus, and Bruce Zetter Sarah ProphetSarah Prophet More articles by this author , Adam FeldmanAdam Feldman More articles by this author , Mary FergusMary Fergus More articles by this author , and Bruce ZetterBruce Zetter More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2955AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES While serum PSA and other available diagnostic biomarkers can provide valuable guidance for assessing the risk of prostate cancer (PCa), limitations in accuracy persist. Novel biomarkers with improved performance characteristics are needed. Using mass spectrometry-based proteomics, we have identified three urinary proteins with significantly different expression patterns across PCa stages. This study evaluates the expression of these potential biomarkers in urine. METHODS Urinary protein concentrations of three proteins, TIMP1, serpinB1, and semenogelin 2, were assessed via Western blot and ELISA for 160 total urine samples. Each patient group (control, Gleason 6 PCa, Gleason ≥8 PCa, and metastatic PCa) had 40 samples. Urine protein was isolated using Amicon Ultra-15 Centrifugal Filter Units for Western blotting. ELISAs were performed using untreated raw urine samples. Immunohistochemistry (IHC) was performed on prostate tissue sections for all three proteins of interest. RESULTS TIMP1 levels were statistically higher in control and Gleason 6 PCa urine samples than for Gleason ≥8 and metastatic disease (2.19 ± 1.7 vs. 1.23 ± 1.13 ng/mL; p = 0.002). Expression of serpinB1 was significantly higher in men with Gleason 6 PCa than those with high-grade Gleason ≥8 PCa (0.71 ± 0.49 vs. 0.23 ± 0.33 ng/mL; p = 0.003). Metastatic PCa had significantly higher semenogelin2 concentrations in urine than healthy men (155.68 ± 74.3 vs. 81.55 ± 55.6 pg/mL; p = 0.02), and expression levels seem to rise with disease progression. IHC staining of tissue sections corroborated these findings. CONCLUSIONS Our results indicate differences in urinary concentrations of TIMP1, serpinB1, and semenogelin 2 across PCa stages. These novel biomarkers allowed distinction between men without prostate cancer or low-risk disease and those with high-risk or metastatic disease. These proteins represent potentially valuable non-invasive prostate cancer biomarkers and warrant further investigation. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1268 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Sarah Prophet More articles by this author Adam Feldman More articles by this author Mary Fergus More articles by this author Bruce Zetter More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Molecularly imprinted probe for solid-phase extraction of hippuric and 4-methylhippuric acids directly from human urine samples followed by MEKC analysis.

Hippuric acid (HA) and 4-methylhippuric acid (4-MHA) are metabolites as well as biological indicators for toluene and xylenes, respectively, and their determination in urine samples is very important, in order to monitor the occupational exposition to these solvents, ensuring a safe working environment. Thus, this paper describes the synthesis and characterization of a probe impregnated with molecularly imprinted polymers (MIPs) for the solid-phase extraction of HA and 4-MHA directly from untreated urine samples followed by micellar electrokinetic chromatography (MEKC) analyses. The MIP probe selectivity was compared to the non-imprinted polymer probe. The MEKC separations were carried out in 50 mmol/L sodium tetraborate pH 10.0/0.5 mmol/L cetyltrimethylammonium bromide aqueous solution, with a constant voltage of -15 kV. The system variables were optimized to provide ideal conditions for the extraction and desorption of the analytes, as well as for the MEKC analyses. The method was linear from 0.5 to 5.0 g/L for both analytes, with correlation coefficients > 0.994. Precisions and accuracies, expressed as relative standard deviation and relative error, were < 20.0 and within -15.4 to 16.6%, respectively, in accordance with the United States Food and Drug Administration recommendation. The MIP probe has proven to be simple, cheap, resistant, and synthetically reproducible, being successfully used to analyze both HA and 4-MHA from real samples.

Estimation of the presence of unmetabolized dialkyl phthalates in untreated human urine by an on-line miniaturized reliable method

At present, human exposure to dialkyl phthalates is assessed through urinary measurement of their metabolites due mainly to contamination in their analysis by their ubiquitous presence. An on-line miniaturized method and the processing of the untreated urine samples have been the key factors for minimizing contamination and achieving unbiased results. Di(2-ethylhexyl) (DEHP), diethyl (DEP), dibutyl (DBP) and mono-ethylhexyl (MEHP) phthalates in urine samples have been included in the study; MEHP as metabolite of the main dialkyl phthalate such as DEHP. On-line in-tube solid-phase microextraction (IT-SPME)-capillary liquid chromatography (CapLC) with diode array detection (DAD) is employed. The detection limits (LODs) achieved in urine were between 0.5 and 1.5μg/L. Eighteen urines were processed. DBP and DEHP were found in nine and five samples, respectively and DEP in three of them. MEHP was only detected in one of the eighteen samples analyzed.

New procedure for rapid identification of microorganisms causing urinary tract infection from urine samples by mass spectrometry (MALDI-TOF)

IntroductionMatrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry is widely established as a technique in clinical microbiology laboratories for the identification of microorganisms. Using this technique, it is also possible to obtain the identification of microorganisms from untreated urine samples. MethodsIn this study, a differential centrifugation protocol and a criterion for validation of the results in order to achieve microbial identification from untreated urine samples are proposed. Additionally, the sensitivity of the analytical procedure in monobacterial urine samples has been evaluated. ResultsA 90% sensitivity (confidence interval of 81.96%–94.84%) was obtained in urine samples with bacterial counts of ≥1×105CFU/ml, and it was possible to improve the percentages of direct identifications from urine samples with bacterial counts of <1×105CFU/ml. ConclusionIt is concluded that the MALDI-TOF system is both fast and reliable in the identification of individual microorganisms from untreated urine samples with counts of ≥1×105CFU/ml.

Detection and quantification of 8‐hydroxy‐2′‐deoxyguanosine in Alzheimer's transgenic mouse urine using capillary electrophoresis

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the major forms of oxidative DNA damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8-OHdG by using CE-LIF detection. The method involved the use of specific antibody to detect the DNA lesion (8-OHdG) and consecutive fluorescence labeling. Next, urinary 8-OHdG fluorescently labeled along with other constituents were resolved by capillary electrophoretic system and the lesion of interest was detected using a fluorescence detector. The limit of detection was 0.18 fmol, which proved sufficient sensitivity for detection and quantification of 8-OHdG in untreated urine samples. The relative standard deviation was found to be 11.32% for migration time and 5.52% for peak area. To demonstrate the utility of this method, the urinary concentration of 8-OHdG in an Alzheimer's transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages, such as high separation efficiency, good selectivity, low limit of detection, simplicity and low cost of analysis.

Boronate affinity solid-phase extraction based on functionalized magnesia–zirconia composite for enrichment of nucleosides in human urine

A novel boronic acid functionalized magnesia–zirconia (ZM) based material was facile synthesized by Lewis acid–base chemisorption. The elemental analysis, inductively coupled plasma atomic emission spectrometer (ICP-AES) and Fourier transform infrared spectrometer (FT-IR) have proven the successful attachment of 3-aminophenylboronic acid derivative onto ZM surface. In the present work, the affinity of the resulting material towards cis-diols binding was evaluated by using it as the sorbent for solid-phase extraction of 4 nucleosides (namely cytidine, uridine, guanine and adenosine). The results show that such boronic acid modified magnesia–zirconia composite microparticles were excellent for capturing nucleosides in the system with a wide range of pH and high salt concentration, which could be effectively released using TFA–ACN–H2O (5 : 50 : 45, v/v/v) solution as elution. The 4 nucleosides spiked to untreated urine sample (i.e., no diluting or pH adjustment) were prominently enriched with recoveries in the range of 86–109% and LOD values in the range of 0.003–0.010 μg mL−1. The constructed calibration curves range from 0.05 to 2.0 μg mL−1 showing good linearity correlation coefficient (R ≥ 0.9961) and precision (RSD < 9%). The proposed material would allow for an alternative extraction strategy for identification and quantitative analysis of cis-diols structure compounds in biological samples.