A supramolecular fluorescence sensor array for the differentiation of multiple anions and prediction of iodine in artificial urine using machine learning.

The simultaneous discrimination and detection of multiple anions in an aqueous solution has been a major challenge due to their structural similarity and low charge radii. In this study, we have constructed a supramolecular fluorescence sensor array based on three host-guest complexes to distinguish five anions (F-, Cl-, Br-, I-, and ClO-) in an aqueous solution using anionic-induced fluorescence quenching combined with linear discriminant analysis. Due to the different affinities of the three host-guest complexes for each anion the anion quenching efficiency for each host-guest complex was likewise different, and the five anions were well recognized. The fluorescence sensor array not only distinguished anions at different concentrations (0.5, 10, and 50 µM) with 100% accuracy but also showed good linearity within a certain concentration range. The limit of detection (LOD) was < 0.5 µM. Our interference study showed that the developed sensor array had good anti-interference ability. The practicability of the developed sensor array was also verified by the identification and differentiation of toothpaste brands with different fluoride content and the prediction of the iodine concentration in urine combined with machine learning.

Relative contributions of urine sulfate, titratable urine anion, and GI anion to net acid load and effects of age.

Models of acid-base balance include acid production from (1) oxidation of sulfur atoms on amino acids and (2) metabolically produced organic acid anions. Acid load is balanced by alkali from metabolism of GI anions; thus, net acid production is equivalent to the sum of urine sulfate and organic anion (measured by titration in urine), minus GI anion. However, the relative contributions of these three sources of acid production in people eating free choice diets, and presumably in acid-base balance, have not been well studied. We collected 26 urines from 18 normal subjects (10 male) and 43 urine samples from 34 stone formers (17 male) and measured sulfate, organic anion, and components of GI anion and acid excretion in each; values were expressed as mEq/mmol creatinine. Mean values of the urine components, except creatinine and pH, did not differ between the sexes or groups. Urine organic acid and acid production varied directly with age (p ≤ 0.03). In a general linear model of acid excretion, the coefficients for sulfate, organic anion, and GI anion were 0.34 ± 0.09, 0.49 ± 0.12, and -0.51 ± 0.06, respectively, p ≤ 0.005, and the model accounted for 54% of the variance. A model for urine ammonia gave similar results. Urine organic anion is a significant contributor to total acid production and may be responsible for an increase in acid production with age.

Simultaneous Determination of Inorganic Anions and Cations in Water and Biological Samples by Capillary Electrophoresis with a Capacitive Coupled Contactless Conductivity Detector Using Capillary Filling Method.

An analytical method for concurrent analysis of inorganic anions and cations has been developed using a capillary electrophoresis (CE)-capacitively coupled contactless conductivity detector (C4D) system. Although hydrodynamic and electrokinetic injection techniques have been widely used in CE, we employed a capillary filling method (CFM) for the analysis of inorganic ions. The procedure is relatively simple and has the advantage that CMF does not require pressure control and vial exchange. Three anions (chloride, sulfate, nitrate) and five cations (ammonium, potassium, sodium, magnesium, calcium) were successfully separated and detected at ppm levels within 80 s using a 9 mM histidine/15 mM malic acid (pH 3.6) containing 50 mM N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate as background electrolyte. Applying this analytical condition, the electroosmotic flow is negligible and anions and cations were migrated concurrently to different polarities according to their electrophoretic mobility. Obtained raw data showed stepwise increases in detected conductivity due to the migration of sample components, which expresses as peak profiles by differentiation of electropherograms. The RSD values of the peak area and migration times for the anions and cations were satisfactory and were less than 5.15 and 2.04%, respectively. The developed method was applied for the analysis of inorganic anions and cations in commercial mineral waters, tap water, urine, and exhaled breath condensate. These results indicate that the CE-C4D system with CFM is suitable for the rapid analysis of inorganic anions and cations in various samples.

Carrot-derived carbon dots modified with polyethyleneimine and nile blue for ratiometric two-photon fluorescence turn-on sensing of sulfide anion in biological fluids.

In this article, a facile and green synthesis of carbon dots (CDs) was developed by using natural carrot as new carbon source. After direct hydrothermal carbonization for 5h at 180°C, CDs were prepared facilely. Then, CDs were conjugated with polyethyleneimine (PEI) and Nile Blue (NB) chloride to produce CDs/PEI/NB nanocomposites under electrostatic interactions. Upon excitation at 800nm, two-photon fluorescence (TPF) of the nanocomposites was observed, with TPF peaks of CDs at 415nm and NB at 675nm. The addition of Cu2+ could lead to TPF quenching of CDs via inner filter effect, but hardly any impacted on TPF of NB. Afterward, the added S2- combined with Cu2+ to form stable species that caused the separation of Cu2+ from CDs surface and the TPF recovery of CDs, with negligible effects on TPF of NB. Herein, a new CDs-based ratiometric TPF turn-on probe of S2- was developed and showed a good linear relationship (R2 =0.9933) between ratiometric TPF intensity (I415/I675) and S2- concentration (0.1-8.0µM), with a low detection limit of 0.06µM. This probe was highly selective and sensitive toward S2- over potential interferences in real biological fluids, with high detection recoveries.

Comparison of capillary electrophoresis-mass spectrometry and hydrophilic interaction chromatography-mass spectrometry for anionic metabolic profiling of urine.

In order to assess the utility of a recently developed capillary electrophoresis-mass spectrometry (CE-MS) method for the study of anionic metabolites in urine, a comparison was made with hydrophilic interaction chromatography-MS (HILIC-MS) using negative electrospray ionization. After optimization of the HILIC conditions, a gradient employing 10mM ammonium acetate (pH 6.8) in acetonitrile-water (5 min 90% acetonitrile followed by 90%-50% acetonitrile in 10 min) was selected, providing baseline separation of five representative anionic test metabolites. Relative standard deviations (RSDs) for HILIC retention times and peak areas were below 0.2% and 7.7%, respectively, and detection limits were in the range 0.04-2.21 µM. Metabolites in rat urine could also be analysed in a reproducible way with retention time and peak area RSDs below 0.6% and 13.6%, respectively. The CE-MS and HILIC-MS methods were compared in terms of reproducibility, sensitivity, selectivity and coverage of the anionic urinary metabolome. In general, peak area RSDs were similar whereas HILIC-MS yielded better retention-time repeatability and up to 80 times lower detection limits (expressed in injected concentration) for test metabolites as compared to CE-MS. Rat urine analysis by HILIC-MS provided detection of 1360 molecular features compared to 347 molecular features revealed with CE-MS. Of these, a number of 144 molecular features were found with both HILIC-MS and CE-MS, which showed on average 10 times higher peak areas in HILIC-MS. The HILIC retention and CE migration times of the common features were clearly not correlated. The HILIC and CE behavior of the test metabolites and 16 putatively identified common features were evaluated involving their physicochemical properties, indicating a markedly different separation selectivity, and thus significant degree of orthogonality of HILIC and CE.

Hydrophilic interaction chromatography-mass spectrometry for anionic metabolic profiling of urine from antibiotic-treated rats.

Hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) was used for anionic metabolic profiling of urine from antibiotic-treated rats to study microbial-host co-metabolism. Rats were treated with the antibiotics penicillin G and streptomycin sulfate for four or eight days and compared to a control group. Urine samples were collected at day zero, four and eight, and analyzed by HILIC-MS. Multivariate data analysis was applied to the urinary metabolic profiles to identify biochemical variation between the treatment groups. Principal component analysis found a clear distinction between those animals receiving antibiotics and the control animals, with twenty-nine discriminatory compounds of which twenty were down-regulated and nine up-regulated upon treatment. In the treatment group receiving antibiotics for four days, a recovery effect was observed for seven compounds after cessation of antibiotic administration. Thirteen discriminatory compounds could be putatively identified based on their accurate mass, including aconitic acid, benzenediol sulfate, ferulic acid sulfate, hippuric acid, indoxyl sulfate, penicillin G, phenol and vanillin 4-sulfate. The rat urine samples had previously been analyzed by capillary electrophoresis (CE) with MS detection and proton nuclear magnetic resonance ((1)H NMR) spectroscopy. Using CE-MS and (1)H NMR spectroscopy seventeen and twenty-five discriminatory compounds were found, respectively. Both hippuric acid and indoxyl sulfate were detected across all three platforms. Additionally, eight compounds were observed with both HILIC-MS and CE-MS. Overall, HILIC-MS appears to be highly complementary to CE-MS and (1)H NMR spectroscopy, identifying additional compounds that discriminate the urine samples from antibiotic-treated and control rats.

A portable lab-on-a-chip instrument based on MCE with dual top-bottom capacitive coupled contactless conductivity detector in replaceable cell cartridge.

A new design for a compact portable lab-on-a-chip instrument based on MCE and dual capacitively coupled contactless conductivity detection (dC(4) D) is described. The instrument is battery powered with total dimension of 14 × 25 × 8 cm(3) (w × l × h), and weighs 1.2 kg. The device consists of a front electrophoresis compartment which has the chip holder and the chip, the associated high-voltage electrodes for electrophoresis injection and separation and the detector. The detection cell is integrated into the device housing with an exchangeable plug-and-play cartridge format. The design of the dC(4) D cell has been optimized for maximum performance. The cartridge includes the top-bottom excitation and pick up electrodes incorporated into the cell and connected to push-pull self-latching pins that are insulated with plastic. The metal frame of the cartridge is grounded completely to eliminate electronic interferences. The cartridge is designed to clamp a thin fluidic chip at the detection point. The cartridges are replaceable whereby different cartridges have different detection electrode configurations to employ according to the sensitivity or resolution needed in the specific analytical application. The second compartment consists of all the electronics, data acquisition card, high-voltage modules of up to ±5 kV both polarity, and batteries for 10 h of operation. The improved detector performance is illustrated by the electrophoresis analysis of six cations (NH4 (+) , K(+) , Ca(2+) , Na(+) , Mg(2+) , Li(+) ) with a detection limit of approximately 5 µM and the analysis of the anions (Br(-) , Cl(-) , NO2 (-) , NO3 (-) , SO4 (2-) , F(-) ) with a detection limit of about 3 µM. Analytical capabilities of the instrument for food and medical applications were evaluated by simultaneous detection of organic and inorganic acids in fruit juice and inorganic cations and anions in rabbit blood samples and human urine samples are also demonstrated.

A kinetic method for expeditious detection of pyrophosphate anions at nanomolar concentrations based on a nucleic acid fluorescent sensor.

A kinetic method has been developed for highly sensitive and selective detection of pyrophosphate anions (PPi) by using a fluorophore labelled single-stranded-DNA-Al(III) complex system. The facile and spectrum-tunable fluorescent sensor enables rapid detection of PPi in urine and cell lysate solutions without the need for complex pre-cleanup procedures.

Analysis of renal stones by capillary isotachophoresis.

An analytical method for the determination of the composition of renal stones by capillary isotachophoresis with conductometric detection was developed. Using different leading/terminating electrolyte systems, the qualitative and quantitative analysis of organic compounds (urate, xanthate, oxalate) and inorganic ions (phosphate, Ca(2+), Mg(2+), Na(+), NH(4)(+)) species commonly present in mixed renal stones in three separate steps can be carried out with limits of detection about 10 µmol/L. The developed method was validated by the analysis of real samples and can be used for urinary calculi classification. In addition, it was verified that this method can also be employed for the determination of the above mentioned analytes in some other samples (bones, teeth) concerning apatite biominerals (fluoro-, carbonate-, chloro-apatite).

Critical parameters for the analysis of anionic oligosaccharides by desorption electrospray ionization mass spectrometry.

Sulfated oligosaccharides derived from glycosaminoglycans (GAGs) are fragile compounds, highly polar and anionic. We report here on the rare but successful application of desorption electrospray ionization (DESI) - LTQ-Orbitrap mass spectrometry (MS) to the high-resolution analysis of anionic and sulfated oligosaccharides derived from the GAGs hyaluronic acid and heparin. For that purpose, key parameters affecting DESI performance, comprising the geometric parameters of the DESI source, the probed surface and the spraying conditions, applied spray voltage, flow rates and solvent composition were investigated. Under suitable conditions, the DESI technique allows the preservation of the structural integrity of such fragile compounds. DESI enabled the sensitive detection of anionic hyaluronic acid and heparin oligosaccharides with a limit of detection (LOD) down to 5 fmol (≈10 pg) for the hyaluronic acid decasaccharide. Detection of hyaluronic acid oligosaccharides in urine sample was also successfully achieved with LOD values inferior to the ng range. Multistage tandem mass spectrometry (MS(n) ) through the combination of the DESI source with a hybrid linear ion trap-orbitrap mass spectrometer allowed the discrimination of isomeric sulfated oligosaccharides and the sequence determination of a hyaluronic acid decasaccharide. These results open promising ways in glycomic and glycobiology fields where structure-activity relationships of bioactive carbohydrates are currently questioned.

Effect of urine pH changed by dietary intervention on uric acid clearance mechanism of pH-dependent excretion of urinary uric acid.

BACKGROUND: The finding reported in a previous paper - alkalization of urine facilitates uric acid excretion - is contradictory to what one might expect to occur: because food materials for the alkalization of urine contain fewer purine bodies than those for acidification, less uric acid in alkaline urine should have been excreted than in acid urine. To make clear what component of uric acid excretion mechanisms is responsible for this unexpected finding, we simultaneously collected data for the concentration of both creatinine and uric acid in serum as well as in urine, in order to calculate both uric acid and creatinine clearances. METHODS: Within the framework of the Japanese government's health promotion program, we made recipes which consisted of protein-rich and less vegetable-fruit food materials for H + -load (acidic diet) and others composed of less protein and more vegetable-fruit rich food materials (alkaline diet). This is a crossover study within some limitations. Healthy female students, who had no medical problems at the regular physical examination provided by the university, were enrolled in this consecutive 5-day study for each test. From whole-day collected urine, total volume, pH, organic acid, creatinine, uric acid, titratable acid and all cations (Na+,K+,Ca2+,Mg2+,NH4+) and anions (Cl-,SO42-,PO4-) necessary for the estimation of acid-base balance were measured. In the early morning before breakfast of the 1st, 3rd and 5th experimental day, we sampled 5 mL of blood to estimate the creatinine and uric acid concentration in serum. RESULTS AND DISCUSSION: Urine pH reached a steady state 3 days after switching from ordinary daily diets to specified regimens. The amount of acid generated ([SO42-] + organic acid - gut alkali)was linearly related with the excretion of acid (titratable acid + [NH4+] - [HCO3-]), indicating that H + in urine is generated by the metabolic degradation of food materials. Uric acid and excreted urine pH retained a linear relationship, as reported previously. Among the five factors which are associated with calculating clearances for both uric acid and creatinine, we identified a conspicuous difference between acidic and alkaline diets in the uric acid concentration in serum as well as in urine; uric acid in the serum was higher in the acidic group than in the alkaline group, while uric acid in the urine in the acidic group was lower than that in the alkaline group. These changes of uric acid in acidic urine and in serum were reflected in the reduction of its clearance. From these observations, it is considered that uric acid may be reabsorbed more actively in acidic urine than in alkaline urine. CONCLUSION: We conclude that alkalization of urine by eating nutritionally well-designed alkaline -prone food is effective for removing uric acid from the body.

Sensitivity enhancement in capillary electrophoresis-mass spectrometry of anionic metabolites using a triethylamine-containing background electrolyte and sheath liquid.

Analyte responses in CE-ESI-MS using negative ionization are frequently relatively low, thereby limiting sensitivity in metabolomics applications. In order to enhance the ionization efficiency of anionic metabolites, BGEs and sheath liquids (SLs) of various compositions were evaluated. Pressure-induced infusion and CE-MS experiments showed that addition of triethylamine (TEA) to the BGE and SL enhanced analyte intensities. A BGE consisting of 25 mM TEA (pH 11.7) and an SL of water-methanol (1:1, v/v) containing 5 mM TEA was selected, providing separation and detection of ten representative test metabolites with good reproducibility (migration time RSDs<1%) and linearity (R(2) >0.99). This BGE yielded lower limits of detection (0.7-9.1 µM) for most test compounds when compared with common CE-MS methods using a BGE and SL containing ammonium acetate (NH(4) Ac) (25 and 5 mM, respectively). CE-MS of human urine revealed an average amount of 231 molecular features in negative ionization mode when TEA was used in the BGE and SL, whereas 115 and 102 molecular features were found with an NH(4) Ac-containing BGE and SL, employing a bare fused-silica (BFS) and Polybrene-dextran sulfate-Polybrene (PB-DS-PB)-coated capillary, respectively. With the CE-MS method using TEA, about 170 molecular features were observed that were not detected with the NH(4) Ac-based CE-MS methods. For more than 82% of the molecular features that were detected with the TEA as well as the NH(4) Ac-containg BGEs (i.e. common features), the peak intensities were higher using TEA with gain factors up to 7. Overall, the results demonstrate that BGEs and SLs containing TEA are quite favorable for the analysis of anionic metabolites in CE-MS.

Microfluidic chip-capillary electrophoresis for two orders extension of adjustable upper working range for profiling of inorganic and organic anions in urine.

To meet the need for onsite monitoring of urine anions, a microfluidic chip-capillary electrophoresis device was designed, fabricated and tested to extend the upper CE working range for an enhancement up to 500 fold (100 fold for sample dilution and 5 folds for CE injection) in order to analyze highly variable anionic metabolites in urine samples. Capillaries were embedded between two PMMA plates with laser-fabricated microchannel patterns to produce the microfluidic chip-capillary electrophoresis to perform standard/sample dilution and CE injection with adjustable dilution ratios. A circular ferrofluid valve was incorporated on-chip to perform cleanup and conditioning, mixing and dilution, injection and CE separation. Under optimized conditions, a complete assay for four samples can be achieved within an hour for 15 anions commonly found in urines. Satisfactory working ranges (0.005-500 mM) and low detection limits (0.5-6.5 µM based on S/N =2) are obtained with satisfactory repeatability (RSD, n=5) 0.52-0.87% and 4.1-6.5% for migration time and peak area, respectively. The working ranges with two orders adjustable upper extension are adequate to cover all analytes concentrations commonly found in human urine samples. The device fabricated shows sufficiently large experimentally verifiable enhancement factor to meet the application requirements. Its reliability was established by more than 94% recoveries of spiked standards and agreeable results from parallel method comparison with conventional ion chromatography method. The extension of the upper CE working range enables flexible onsite dilution on demand, a quick turn-around of results, and a low-cost device suitable for bedside monitoring of patients under critical conditions for metabolic disorders.

Evaluation of CE methods for global metabolic profiling of urine.

In this study, the usefulness of noncovalently coated capillaries with layers of charged polymers is investigated to obtain global electrophoretic profiles of urinary metabolites covering a broad range of different compound classes in a highly repeatable way. Capillaries were coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS), or with a triple layer of PB, dextran sulfate (DS) and PB. The bilayer and triple layer coatings were evaluated at acidic (pH 2.0) and alkaline (pH 9.0) separation conditions, thereby providing separation conditions for basic and acidic compounds. A representative metabolite mixture and spiked urine samples were used for the evaluation of the four CE methods. Migration time repeatability (RSD<2%) and plate numbers (N, 100,000-400,000) were similar for the test compounds in all CE methods, except for some multivalent ions that may exhibit adsorption to oppositely charged coatings. The analysis of cationic compounds with the PB-DS-PB CE method at low pH (i.e. after the EOF time) provided a larger separation window and number of separated peaks in urine compared to the analysis with the PB-PVS CE method at low pH (i.e. before the EOF time). Approximately, 600 molecular features were detected in rat urine by the PB-DS-PB CE-MS method whereas about 300 features were found with the PB-PVS CE-MS method. This difference can be attributed to reduced comigration of compounds with the PB-DS-PB CE-MS method and a related decrease of ion suppression. With regard to the analysis of anionic compounds by CE-MS, in general analyte responses were significantly lower than that for cationic compounds, most probably due to less efficient ionization and to ion suppression effects caused by the background electrolyte. Hence, further optimization is required for the sensitive CE-MS analysis of anionic compounds in body fluids. It is concluded that the selection of a CE method for profiling of cationic metabolites in urine depends on the purpose of the study. For high-throughput analyses, the PB-PVS CE-MS method is favored whereas the PB-DS-PB CE-MS method provides a more information-rich metabolic profile, but at the cost of prolonged analysis time.

Electroosmotic flow-balanced isotachophoretic stacking with continuous electrokinetic injection for the concentration of anions in high conductivity samples.

An EOF counter-balanced ITP boundary has been used to stack anions from high conductivity samples during continuous electrokinetic injection of the sample. In a polystyrenesulfonate/poly(diallyldimethylammonium chloride) polyelectrolyte coated capillary, the time at which the ITP boundary exited the capillary could be prolonged by balancing the movement of the boundary with the EOF. Using a bis-tris-propane electrolyte, the ITP boundary was removed from the capillary within 7min, while when using triethanolamine the ITP boundary was still at 30% of the capillary after 2h of injection. Using these systems, the sensitivity of a mixture of simple organic acids in 100mM Cl(-) was improved by 700-800-fold using bis-tris-propane with a whole-capillary injection of the sample and 5min of electrokinetic injection at +28kV, and 1100-1300-fold using triethanolamine and 60min of electrokinetic injection under the same conditions. The potential of the method to be applicable to high conductivity samples was demonstrated by stacking a whole capillary filled with urine spiked with naphthalenedisulfonic acid, with limits of detection 450 times lower than those achievable with a normal hydrodynamic injection.

[A new technique for urinary oxalate ion determination].

The authors propose a technique for the determination of urinary oxalate ions by high performance liquid chromatography, which may be used for clinical and scientific purposes.

Use of dietary cation anion difference for control of urolithiasis risk factors in goats.

OBJECTIVE: To determine correlations between dietary cation anion difference (DCAD) and urine pH, urine specific gravity, and blood pH in goats. ANIMALS: 24 crossbred goat wethers. PROCEDURES: Goats were randomly assigned to 1 of 4 DCAD groups (-150, -75, 0, or +75 mEq/kg of feed) and fed pelleted feed and ground hay for 7 days. The diet was then supplemented with ammonium chloride to achieve the assigned DCAD of each group for 7 days. Urine was obtained for pH and specific gravity measurements at hours -3 to -1, 1 to 3, 5 to 7, 9 to 11, and 13 to 15 relative to the morning feeding. Blood pH was determined on alternate days of the study period. RESULTS: Goats in the -150 and -75 mEq/kg groups had a urine pH of 6.0 to 6.5 two days after initiation of administration of ammonium chloride, and urine pH decreased to < 6.0 by day 7. Goats in the 0 mEq/kg group had a urine pH from 6.0 to 6.5 on day 5, whereas urine pH in goats in the +75 mEq/kg group remained > 6.5 throughout the trial. Urine specific gravity differed only between the -150 mEq/kg and the -75 mEq/kg groups. Blood pH in the -150 mEq/kg group was significantly lower than that in the other groups. CONCLUSIONS AND CLINICAL RELEVANCE: Goats in the 0 mEq/kg DCAD group had a urine pH of 6.0 to 6.5 five days after intitiation of feeding the diet, and that pH was maintained through day 7, without significant reduction in blood pH. This may serve as a target for diet formulation for the prevention of urolithiasis.