Diluted Urine Samples Research Articles

Dispersive liquid-liquid microextraction using a low transition temperature mixture and liquid chromatography-mass spectrometry analysis of pesticides in urine samples

Biomonitoring is a potent tool to control the health risk of people occupationally and non-occupationally exposed. The latest trend in bioanalytical chemistry is to develop quick, cheap, easy, safe and reliable green analytical procedures to analyse a large number of chemicals in easily accessible biomatrices such as urine. In this paper, a new dispersive liquid–liquid microextraction (DLLME) procedure, conceived to treat urine samples and based on the use of a low transition temperature mixture (LTTM), was developed and validated to analyse twenty pesticides commonly used in farm practises. The LTTM was composed of choline chloride and sesamol in molar ratio 1:3 (ChCl:Ses 1:3); its characterization via differential scanning calorimetry identified it as an LTTM and not as a deep eutectic solvent due to the occurrence of a glass transition at -71 °C. The prepared mixture was used as the extraction solvent in the DLLME procedure, while ethyl acetate as the dispersing solvent. The salting out effect (50 mg mL−1 of NaCl in a diluted urine sample) improved the separation phase and the analyte transfer to the extractant. Due to the high ionic strength and despite the density of ChCl:Ses 1:3 (1.25 g mL−1), the LTTM layer floated on the top of the sample solution after centrifugation. All extracts were analysed by high-performance liquid chromatography coupled to mass spectrometry. After optimization and validation of the whole method, lower limits of quantitation were in the range of 0.02 – 0.76 µg L−1. Extraction recoveries spanned from 50 to 101 % depending on the spike level and analytes. Precision and accuracy ranges were 3-18% and 5-20%, respectively. The extraction procedure was also compared with other methods, showing to be advantageous for rapidity, simplicity, efficiency, and low cost. Finally, urine samples from ten volunteers were effectively analysed using the developed method.

Paper-Based Competitive Immunochromatography Coupled with an Enzyme-Modified Electrode to Enable the Wireless Monitoring and Electrochemical Sensing of Cotinine in Urine

This paper proposes a combined strategy of using paper-based competitive immunochromatography and a near field communication (NFC) tag for wireless cotinine determination. The glucose oxidase labeled cotinine antibody specifically binds free cotinine in a sample, whereas the unoccupied antibody attached to BSA-cotinine at the test line on a lateral flow strip. The glucose oxidase on the strip and an assistant pad in the presence of glucose generated H2O2 and imposed the Ag oxidation on the modified electrode. This enabled monitoring of immunoreaction by either electrochemical measurement or wireless detection. Wireless sensing was realized for cotinine in the range of 100–1000 ng/mL (R2 = 0.96) in PBS medium. Undiluted urine samples from non-smokers exhibited an Ag-oxidation rate three times higher than the smoker’s urine samples. For 1:8 diluted urine samples (smokers), the proposed paper-based competitive immunochromatography coupled with an enzyme-modified electrode differentiated positive and negative samples and exhibited cotinine discrimination at levels higher than 12 ng/mL. This novel sensing platform can potentially be combined with a smartphone as a reader unit.

Accuracy evaluation of the C. elegans cancer test (N-NOSE) using a new combined method

Early cancer detection is critical for effective treatment. N-NOSE (Nematode-NOSE) is a simple, inexpensive, and highly sensitive cancer screening method based on the chemotaxis of the nematode Caenorhabditis elegans, which shows evasive action from the urine of healthy individuals while being attracted to the urine of cancer patients. Initially, N-NOSE relied on chemotaxis indexes obtained with 10-fold dilutions of urine samples. However, cancer tissue size and concentrations of cancer odors differ among cancer patients. In this study, we examined the accuracy improvement of N-NOSE method by using two types of dilutions, 10-fold and 100-fold. We have conducted N-NOSE tests with urine samples from 32 cancer patients (esophageal, gastric, colorectal, gallbladder, cholangiocarcinoma, breast, malignant lymphoma, and acute myeloid leukemia) along with 143 healthy subjects. Our data showed a significant difference in the N-NOSE at 10-fold dilution between the two groups (p < 0.0001), with an area under the ROC curve (AUC) of 0.9188 based on receiver operating characteristic (ROC) analysis. N-NOSE index at 100-fold dilutions was also significantly different between the two groups (p < 0.0001), with an AUC of 0.9032 based on ROC analysis.In this clinical study, we further improve N-NOSE with a combined method of two dilutions (10-fold and 100-fold) of urine samples, which results in a markedly improvement in cancer detection sensitivity of 87.5%. N-NOSE sensitivity improvement was significantly high even for early-stage cancer detection, which is in stark contrast with the sensitivity of detection using blood tumor markers (CEA, CA19-9 and CA15-3). These results strongly suggest that the N-NOSE test by this new combined method strikes a good balance between sensitivity and specificity.

Technical note: Evaluation of a colorimetric point-of-care test for measuring urine ammonium concentration in periparturient dairy cattle

Urine ammonium concentration ([NH4+]) provides a clinically useful indicator of the magnitude of nutritionally induced systemic acidification in dairy cattle when urine pH < 6.1. The objective of this study was to evaluate the analytical performance of a low-cost point-of-care colorimetric test in measuring urine [NH4+] in dairy cattle consuming an acidogenic ration. A method comparison study was performed using 154 urine samples from 43 periparturient Holstein-Friesian cows. We compared urine [NH4+] measured by an indophenol blue colorimetric test (MColortest, Merck KGaA, Billerica, MA; test method) with levels measured by formaldehyde titration (reference method). Diagnostic performance was evaluated using Pearson correlation coefficient (r), Passing-Bablok regression, Bland-Altman plot, and binary logistic regression. Urine [NH4+] measured by the colorimetric test was strongly correlated (r = 0.98) with urine [NH4+] measured by formaldehyde titration. Method comparison studies indicated that the colorimetric test provided acceptable test performance when urine [NH4+] < 80 mmol/L. Logistic regression analysis indicated that the area under the receiver operating characteristic curve for the colorimetric ammonium test was high at 0.985 when used to identify formaldehyde titration [NH4+] > 10 mmol/L, equivalent to urine pH <6.1. At the optimal cut point ([NH4+] > 11 mmol/L) for the colorimetric test, sensitivity = 0.94, specificity = 0.97, positive likelihood ratio = 27.6, and κ = 0.89, indicating excellent test performance. We conclude that the indophenol blue colorimetric test provided an accurate, low-cost, and practical on-farm test for measuring urine [NH4+] in diluted urine samples from dairy cattle consuming an acidogenic ration.

Dilute-and-Shoot HPLC-UV Method for Determination of Urinary Creatinine as a Normalization Tool in Mycotoxin Biomonitoring in Pigs.

A simple, rapid, and accurate HPLC-UV method was developed for the determination of creatinine in pig urine. Usually, it is determined in urine in biomonitoring of xenobiotics to correct for variations in dilutions of urine samples. The colorimetric method (based on Jaffe reaction), which was mainly used for this purpose in mycotoxin biomonitoring, is not a reliable approach for pig urine. Therefore, a novel and accurate HPLC method for creatinine determination was developed. The sample preparation was based on the dilute and shoot approach. An HPLC separation was performed with a porous graphitic carbon column with an aqueous mobile phase to achieve satisfactory retention time for creatinine. The method has been successfully validated, applied for the determination of creatinine in pig urine, and compared with other methods commonly used for that purpose—a colorimetric method based on Jaffe reaction and commercial ELISA test. The developed HPLC method shows the highest precision and accuracy for pig urine samples. Finally, the method was applied as a normalization tool in LC-MS/MS mycotoxin biomarkers analysis. The standardization to a constant creatinine level (0.5 mg/mL) enables similar matrix effects for eleven mycotoxin biomarkers for pig urine samples with different creatinine levels.

Getting Specific About Urine Specific Gravity

Research Article| February 01 2020 Getting Specific About Urine Specific Gravity AAP Grand Rounds (2020) 43 (2): 18. https://doi.org/10.1542/gr.43-2-18 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Getting Specific About Urine Specific Gravity. AAP Grand Rounds February 2020; 43 (2): 18. https://doi.org/10.1542/gr.43-2-18 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search toolbar search search input Search input auto suggest filter your search All PublicationsAll JournalsAAP Grand RoundsPediatricsHospital PediatricsPediatrics In ReviewNeoReviewsAAP NewsAll AAP Sites Search Advanced Search Topics: urinalysis, urine specific gravity, urinary tract infections, nitrites Source: Shaikh N, Shope MF, Kurs-Lasky M. Urine specific gravity and the accuracy of urinalysis. Pediatrics. 2019; 144(5): e20190467; doi: https://doi.org/0.1542/peds.2019-0467Google Scholar Investigators from the University of Pittsburgh conducted a retrospective analysis to determine whether urine specific gravity (SG) affects the accuracy of other components of the urinalysis. Children <24 months of age with a visit to the study ED between 2007 and 2013 were eligible if they underwent bladder catheterization and had an automated urinalysis and urine culture performed within 3 hours of each other. Children with genitourinary anomalies were excluded. Investigators evaluated the accuracy of urinalysis components used for diagnosis of UTI by comparing the positive likelihood ratios (LRs) in dilute versus concentrated urine samples. Dilute urine samples were defined as those with an SG <1.015, and concentrated samples were defined as those with an SG ≥1.015. UTI was defined as ≥100,000 colony-forming units (CFUs) per milliliter of a uropathogen from specimens obtained via clean catch or ≥50,000 CFU/mL from specimens obtained via catheterization. Urinalysis components evaluated included leukocyte esterase (LE; a positive test defined as ≥1), nitrite, white blood cell count (WBC) per high-power field (hpf; a positive test defined as ≥5), WBC/mm3 (a positive test defined as ≥10), bacteria/hpf (a positive test defined as ≥ few), and bacteria on Gram-stain (a positive test defined as ≥ any). Investigators also used predictive models to calculate how many children would be overtreated or missed with and without SG being used in the interpretation of other urinalysis components. There were 10,078 children included in analysis. Of these, 39.4% (n=3,966) had concentrated urine, and, overall, 6.1% had a UTI (n=617). The positive LRs for LE and bacteria/hpf (ie, their ability to rule in UTI) were similar in dilute and concentrated urine. However, WBC/hpf and WBC/mm3 had lower LRs in concentrated (vs dilute) urine, and nitrite and bacteria on Gram stain had higher LRs. Despite this finding, there was no change in predictive models for the number of children overtreated or missed with or without inclusion of SG in combination with urinalysis components. The investigators conclude that SG influences the accuracy of some components of the urinalysis but that inclusion of SG in decision-making would result in little change to clinical practice. Dr Bechtel has disclosed no financial relationship relevant to this commentary. This commentary does not contain a discussion of an unapproved/investigative use of a commercial product/device. UTIs are common bacterial infections in the pediatric population.1 An uncontaminated sample of urine for urinalysis and culture is generally required before starting any presumptive antimicrobial treatment. The urinalysis serves as the necessary screening test to determine the need to start empiric antibiotics, especially if pyuria and bacteriuria are present.2 Despite the widespread use of urinalyses by clinicians to make the presumptive diagnosis of UTI, urine SG has not previously been incorporated into their interpretation. For example, the urine SG has not been considered... You do not currently have access to this content.

A Dilute-and-Shoot LC–MS/MS Method for Urinary Glyphosate and AMPA

A method for the simultaneous determination of glyphosate and aminomethylphosphonic acid (AMPA) in urine was developed using a “dilute-and-shoot” sample preparation. A 1:1 dilution of urine sample in 15 mmol L−1 heptafluorobutyric acid (HFBA) was prepared prior to being injected into the LC–MS/MS system. The chromatographic separation was achieved by a Gemini C6-Phenyl analytical column, with the gradient elution of 15 mmol L−1 HFBA in water and acetonitrile. The limits of detection and quantitation of both compounds were 2.5 ng mL−1 and 5 ng mL−1, respectively. The sample preparation time was achieved in 2 min using only 0.1 mL of urine sample. The validated method was successfully applied to glyphosate and AMPA determination in farmer urine samples.

Urine Specific Gravity and the Accuracy of Urinalysis.

A recent study in young infants found that different cutoffs maximized the accuracy of the urine white blood cell count in dilute versus concentrated urine samples. We aimed to confirm this finding and to determine its impact on clinical care. We conducted a retrospective analysis of data gathered on consecutive children <24 months of age with visits to the emergency department during a 5-year period. We evaluated the accuracy of screening tests for urinary tract infection (UTI) in dilute and concentrated urine samples. We also calculated the number of children who would have been treated differently in a hypothetical cohort of 1000 children presenting with fever had urine specific gravity (SG) been taken into consideration. We included 10 078 children. The ability to rule in UTI (as measured by the positive likelihood ratio [LR]) was similar in dilute and concentrated urine for the leukocyte esterase test (11.76 vs 10.71, respectively). The positive LR for urine white blood cell count per high-powered field was higher in dilute urine (9.83 vs 6.12). In contrast, the positive LR for the nitrite test was lower in dilute urine (20.54 vs 47.44). Despite these differences, we found little change in the number of children treated with antibiotics in predictive models that took urine SG into consideration. Although we found that urine SG influences the accuracy of some components of the urinalysis, its inclusion in the decision-making process had negligible effect on the clinical care of children with UTI.

SAT-144 AN ULTRASENSITIVE SURFACE ENHANCED RAMAN SCATTERING (SERS)-BASED ASSAY FOR KIDNEY INJURY MOLECULE-1 (KIM-1)

Kidney injury molecule-1 (KIM-1) is a biomarker of acute and chronic kidney injury. However, dilution of urine samples to minimize the impact of pH and solute on results can push the concentration levels to below the lower limits detection of conventional ELISAs. Additionally, small samples from infants, children or from non-human renal injury models such as mice limits analyte detection. Here we develop an ultrasensitive (sub-pg/ml) assay utilizing surface enhanced Raman scattering (SERS) to measure KIM-1. Capture and detection antibodies of a commercially available KIM-1 ELISA kit (R&D Systems) were utilized. [email protected] nanocuboids ([email protected]) with 16 nm shell thickness, assembled on silicon wafers, were derivatized with cysteamine followed by glutaraldehyde to covalently attach the capture antibody. Separately, [email protected] were derivatized with the Raman reporter 4-aminothiophenol (pATP), glutaraldehyde followed by the detection antibody. After analyte capture on the silicon wafer, washing, incubating with the derivatized detection antibody and washing, the silicon wafers were subject to SERS. Among several prominent Raman bands, the intensity band at 1080 cm-1 of pATP was proportional to the concentration of human recombinant KIM-1 spiked into artificial urine. The urine of control individuals and patients with kidney disease was diluted prior to assay and the results compared to that using the conventional ELISA. The Raman intensity band at 1080 cm-1 of pATP was proportional to the KIM-1 concentration from to 400 pg/ml. As little as 1fg/ml above background could be reliably detected. The KIM-1 concentration, determined in patient urine using both the conventional ELISA and the SERS assay, was found to be synonymous with a correlation coefficient of 0.93 ( p<0.002). A similar strategy was used to develop a sub-pg/ml assay for the pro-inflammatory/myokine molecule IL-6. Using the power of SERS, capture and detection antibodies of a conventional ELISA kit were modified to measure sub-pg/ml amounts of KIM-1. This sensitivity allows dilution of samples to eliminate the influence of pH and/or solute concentration that would otherwise affect conventional ELISA results. Also the sensitivity of the assay allows the use of small samples of urine or plasma from infants, small children or from small animals such as mice with experimental renal diseases. It would be possible to detect preclinical changes in KIM-1 prior to disease manifestation. This assay strategy can be adapted to multiplexing to measure two or more analytes simultaneously in one sample by utilizing different Raman reporter molecules on different detection antibodies, an appropriate mix of capture antibodies on the silicon wafer, and monitoring non-overlapping Raman bands specific for each reporter.

Bract as a novel extraction phase in thin-film SPME combined with 96-well plate system for the high-throughput determination of estrogens in human urine by liquid chromatography coupled to fluorescence detection

In this study, an environmentally friendly and high-throughput method was developed for the determination of estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2) and estriol (E3) in human urine by liquid chromatography-fluorescence detector (HPLC-FLD). A biosorbent (bract) was proposed as extraction phase for Thin-Film SPME combined with 96-well system. The characterization of the biosorbent was performed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The optimizations were carried out through univariate and multivariate approaches with optimal conditions comprised of urine samples diluted 40-fold, liquid desorption performed in methanol and addition of 20% (w/v) of NaCl in the sample. Considering an extraction/desorption cycle using the 96-well plate system, the sample preparation time was 1.7 min per sample, which contributes to the high-throughput of the method proposed. The analytical parameters of merit were determined and satisfactory results were achieved, including limits of detection ranging from 0.3 μg L−1 for estradiol to 3 μg L−1 for estrone, while limits of quantification varied from 1 μg L−1 for estradiol to 10 μg L−1 for estrone. The correlation coefficients ranged from 0.9947 for estrone to 0.9999 for estriol. The accuracy and intra-assay and intermediate precisions (RSD) were evaluated through extractions in diluted urine samples (40-fold) spiked with each analyte (1, 200 and 400 μg L−1 for E3; 0.1, 200 and 400 μg L−1 for E2; 0.5, 200 and 400 μg L−1 for EE2 and 10, 200 and 400 μg L−1 for E1). The relative recoveries (n = 3) ranged from 71 to 105%, intra-assay precision (n = 3) varied from 1 to 17% and intermediate precision (n = 9) ranged from 2 to 19%. The method developed can be successfully used for the quantification of estrogens in human urine samples.

Characterization of urease derived from Citrullus lanatus (watermelon) seeds to estimate total Kjeldahl nitrogen in human urine

ABSTRACTA urease extract prepared by decanting liquid from a suspension of finely ground Citrullus lanatus (watermelon) seeds was characterized and applied to dilute urine samples to demonstrate a low-cost field method to estimate total Kjeldahl nitrogen (TKN) concentrations in human urine. The extract exhibited a Michaelis-Menten constant, Km, of 3.00 mM urea and a specific activity of up to 12.2 U/mg protein at an optimum pH of 8.1. A statistical F-test on 54 samples demonstrated that TKN can be estimated as the total ammonium-nitrogen recovered upon addition of urease in dilute fresh and stale urine samples. The total ammonium-nitrogen in urine samples determined after treatment with watermelon seed urease was consistent with that determined using traditional acid digestion techniques. The extract retained 85% of its initial capacity after three months of refrigeration. The effectiveness of this method to assay nitrogen in unbuffered urine samples will be useful in nitrogen analyses in nutrient recovery and urine or slurry storage contexts. Accordingly, this study is useful in understanding the kinetics of a plant-derived urease acting in dilute urine.

Hyperhydration-Induced Decrease in Urinary Luteinizing Hormone Concentrations of Male Athletes in Doping Control Analysis.

Low urinary luteinizing hormone (LH) values have been discussed as a marker to detect steroid abuse. However, suppressed LH concentrations related to highly diluted urine samples could be a misleading indication of anabolic steroid abuse. One aim of the present study was to examine the effect of hyperhydration on the interpretation of LH findings during doping control analysis and to investigate different possibilities to correct volume-related changes in urinary LH concentrations. Seven healthy, physically active, nonsmoking White males were examined for a 72-hr period, using water and a commercial sports drink as hyperhydration agents (20ml/kg body weight). Urine samples were collected and analyzed according to the World Anti-Doping Agency's technical documents. Baseline urinary LH concentrations, expressed as the mean ± SD for each individual, were within the acceptable physiological range (7.11 ± 5.42IU/L). A comparison of the measured LH values for both hyperhydration phases (Phase A: 4.24 ± 5.60IU/L and Phase B: 4.74 ± 4.72IU/L) with the baseline ("normal") values showed significant differences (Phase A: p < .001 and Phase B: p < .001), suggesting the clear effect of urine dilution due to hyperhydration. However, an adjustment of urinary LH concentrations by specific gravity based on a reference value of 1.020 seems to adequately correct the hyperhydration-induced decrease on the LH levels.

Detection of antimuscarinic agents tolterodine and fesoterodine and their metabolite 5-hydroxymethyl tolterodine by ion transfer voltammetry at a polarized room-temperature ionic liquid membrane

Ion transfer voltammetry at a polarized hydrophobic room temperature ionic liquid (RTIL) membrane was used for evaluation of the diffusion coefficients and the standard Gibbs energies of ion transfer of the protonated antimuscarinic agents tolterodine (TOL), fesoterodine (FES), and their common metabolite 5-hydroxymethyl tolterodine (5-HMT), as well as for their determination in the aqueous samples and urine. An analysis of the pH effect provided the parameters characterizing their lipophilicity both in their ionic and neutral forms confirming a remarkably low lipophilicity of 5-HMT. The application of the ion transfer voltammetry for a monitoring of the enzymatic hydrolysis of FES to 5-HMT was demonstrated. For determination of protonated TOL, FES, and 5-HMT in the aqueous samples, linear calibration dependences were plotted in the range 2.0–12.5 μmol L−1 with the mean limit of detection (LOD) 0.43 μmol L−1. Analogous linear calibration dependences were constructed for 10times diluted urine spiked with 2.0–12.5 μmol L−1 TOL, FES, and 5-HMT with the mean LOD 0.65 μmol L−1. Unfortunately, determination of 5-HMT in diluted urine samples was interfered by an unknown ionic urine component, which somewhat increased its particular LOD.

Development and validation of a hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry method for rapid simultaneous determination of 19 free amino acids in rat plasma and urine.

Determination of amino acids in biofluids is a challenging task because of difficulties deriving from their high polarity and matrix interference. A simple, reliable and high-throughput hydrophilic interaction UHPLC-MS/MS method was developed and validated for the rapid simultaneous determination of 19 free amino acids in rat plasma and urine samples in this paper. Hydrophilic method with a Waters Acquity UPLC BEH Amide column (100 × 2.1 mm,1.7 μm) was used with a gradient mobile phase system of acetonitrile and water both containing 0.2% formic acid. The analysis was performed on a positive electrospray ionization mass spectrometer via multiple reaction monitoring. Samples of 10 μL plasma and 50 μL urine were spiked with three deuterated internal standards, pretreated with 250 μL acetonitrile for one-step protein precipitation and a final dilution of urine samples. Good linearities (r > 0.99) were obtained for all of the analytes with the lower limit of quantification from 0.1 to 1.2 μg/mL. The relative standard deviation of the intra-day and inter-day precisions were within 15.0% and the accuracy ranged from -12.8 to 12.7%. The hydrophilic interaction UHPLC-MS/MS method was rapid, accurate and high-throughput and exhibited better chromatography behaviors than the regular RPLC methods. It was further successfully applied to detect 19 free amino acids in biological matrix.

Synthesis of molecular imprinted polymer nanoparticles followed by application of response surface methodology for optimization of metribuzin extraction from urine samples

A molecular imprinted polymer was prepared with precipitation polymerization technique and applied as a sorbent for selective extraction and enrichment of metribuzin herbicide prior to high performance liquid chromatography. Optimization of critical variables affecting the efficiency of molecularly imprinted solid-phase extraction (MISPE), such as sorbent mass, sample pH and flow rate of sample, volume, concentration, and flow rate of elution solvent was done by employing central composite design (CCD) of the response surface methodology. Two separate models were developed for the adsorption and recycling steps. The analysis of variance (ANOVA) demonstrated that, experimental data were excellently fitted to the proposed response models. The optimum operating conditions were: a sorbent mass of 25 mg, sample pH 6.19, sample flow rate of 2.15 mL/min, and a 5 mL portion of methanol/acetic acid with 92.7:7.3 (v/v) ratio and flow rate of 2.1 mL/min for the extraction process. Under the optimized conditions, the linear range was obtained from 20 to 120 µg/L (R2 = 0.999) and the lowest detectable concentration (LOD) and the lowest quantitative concentration (LOQ) were calculated as 5.75 and 19.86 µg/L, respectively. Finally, the designed MISPE method was successfully applied to determine trace amount of metribuzin in real samples. The diluted urine samples were spiked with metribuzin at 4 levels and extracted with recoveries ranging from 93.82 to 97.84% and the relative standard deviation (RSD) less than 4.8%.

High proportion of false negative urinary tract infections among dilute urine samples

High proportion of false negative urinary tract infections among dilute urine samples

A Graphene Oxide‐DNA Electrochemical Sensor Based on Glassy Carbon Electrode for Sensitive Determination of Methotrexate

AbstractMethotrexate (MTX) was used as an anti‐cancer drug, but its excessive use can cause serious side effects, it was necessary to monitor MTX in vivo. In this report, DNA was immobilized on a glassy carbon electrode (GCE) modified with graphene oxide (GO) to develop an electrochemical sensor for sensitive determination of MTX for the first time. The adsorptive voltammetric behaviors of MTX on DNA sensor were investigated using differential pulse voltammetry (DPV). The peak current response of guanine in DNA was used as a determination signal of MTX in acetate buffer solution pH 4.6. Voltammetric investigations revealed that the proposed method could determine MTX in the concentration range from 5.5×10−8 to 2.2×10−6 mol L−1 with a lower detection limit of 7.6×10−9 mol L−1 (S/N=3). The method was applied to detect MTX in human blood serum and diluted urine samples with excellent recoveries of 97.4–102.5 %. Compared with the previous studies, the DNA/GO/GCE electrode constructed by us based on the change rate of guanine current (R%) in DNA, proportionally reflecting the MTX concentration, is simple and sensitive.

Method for Screening and Confirming Meldonium in Human Urine by High- Resolution Mass Spectrometry and Identification of Endogenous Interferences for Anti-Doping Testing

Meldonium is a drug for treating ischemia by expanding the arteries but it can also enhance the performance of sports players. The World Anti-Doping Agency (WADA) has included it in the list of prohibited substances since 2016. Meldonium is one of the challenging substances for anti-doping testing because it is difficult to recover by general liquid-liquid or solid phase extraction due to its permanent charge and high polarity. Therefore, high-performance liquid chromatography (HPLC) is cur-rently used by injecting a diluted urine sample (known as the “dilute-and-shoot” strategy). There is no loss of target compounds in the extraction/cleanup procedure but its high matrix effect could interfere in their separation or detection from the endogenous urinary compounds. We report a single method using high-resolution mass spectrometry that can be used for both screening and confirmation, which follows the “dilute-and-shoot” strategy. In this method, the endogenous compounds` interfering peaks in the mass spectrum are separated at a high resolution of FWHM 140,000, and the results are suitable for substance detection follow-ing the WADA guidelines. The interferences in the obtained mass spectrum of the urine matrix are identified as acetylcholine, lysine, and glutamine by further analysis and database searching. Validation of the method is performed in routine anti-doping testing, and the limit of detection is 50 ng/mL. This method uses simple sample preparation and a general reverse phase HPLC column, and it can be easily applied to other substances.

Synthesis and Analysis of Glucuronic Acid-Conjugated Metabolites of 4-Bromo-2,5-Dimethoxyphenethylamine.

In the study reported here, two glucuronic acid-conjugated metabolites of 4-bromo-2,5-dimethoxyphenethylamine (2C-B)-a ring-substituted psychoactive phenethylamine-were chemically synthesized for the first time and a method for analyzing them in urine was developed. β-D-Glucuronide of 4-bromo-2,5-dimethoxyphenylethylalcohol was successfully synthesized using methyl 2,3,4-tri-Ο-acetyl-1-O-(trichloroacetimidoyl)-α-D-glucuronate as a glucuronyl donor and boron trifluoride diethylether complex as a Lewis acid catalyst. β-D-Glucuronide of 4-bromo-2,5-dimethoxyphenylacetic acid was synthesized by condensing 4-bromo-2,5-dimethoxyphenylacetic acid and benzyl D-glucuronate followed by benzyl group deprotection based on catalytic hydrogenation. Two glucuronic acid-conjugated metabolites of 2C-B in urine were qualitatively and semiquantitatively evaluated via direct liquid chromatography/mass spectrometry (LC/MS) analysis of a diluted urine sample. The simple method proposed is expected to be useful for studying the metabolic fate of 2C-B.

Liquid chromatography-tandem mass spectrometry assay for the quantification of niraparib and its metabolite M1 in human plasma and urine.

Niraparib (MK-4827) is a novel poly(ADP-Ribose) polymerase (PARP) inhibitor currently investigated in phase III clinical trials to treat cancers. The development of a new drug includes the characterisation of absorption, metabolism and excretion (AME) of the compound. AME studies are a requirement of regulatory agencies and for this purpose bioanalytical assays are essential. This article describes the development and validation of a bioanalytical assay for niraparib and its carboxylic acid metabolite M1 in human plasma and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample pre-treatment involved protein precipitation for plasma and dilution of urine samples using acetonitrile-methanol (50:50, v/v). Final extracts were injected onto a SunFire C18 column and gradient elution using 20mM ammonium acetate (mobile phase A) and formic acid:acetonitrile:methanol (0.1:50:50, v/v/v) (mobile phase B) was applied. Detection was performed on an API5500 tandem mass spectrometer operating in the positive electrospray ionisation mode applying multiple reaction monitoring (MRM). The assay was successfully validated in accordance with the Food and Drug Administration and latest European Medicines Agency guidelines on bioanalytical method validation and can therefore be applied in pharmacological clinical studies.