High-throughput assay of nitric oxide metabolites in human plasma without deproteinization by lab-on-a-chip electrophoresis using a zwitterionic additive

In order to develop a high-throughput assay for nitric oxide metabolites, nitrite (NO2-) and nitrate (NO3-), in biological fluids, we have investigated the simultaneous determination of them using an electrophoretic lab-on-a-chip (microchip capillary electrophoresis, MCE) technique. In this study, in order to establish an MCE assay process without deproteinization, the addition of a zwitterionic additive into the running buffer to reduce the adsorption of protein onto the surface of channel was investigated. Initially, some zwitterionic additives were investigated by making a comparison of relative standard deviations (RSDs) of the migration times for NO2(-) and NO3(-) on capillary electrophoresis. From the results of our comparison of the RSD values, 2% (w/w) N-cyclohexyl-2-aminoethanesulfonic acid (CHES) was selected. As a result of the application of the running buffer with CHES to the MCE process, the complete separation of NO2(-) and NO3(-) in human plasma without deproteinization was achieved within 1 min. Since the RSD values of the positions of the peaks were less than 2.3%, beneficial reduction effects on MCE were suggested. When we used an internal standard method in order to correct the injection volume, the RSDs of the peak heights and areas were less than 10%, and the correlation coefficients of spiked calibration curves ranging from 0 to 350 microM were 0.999 and 0.997 for NO2(-) and NO3(-), respectively. The limits of detection (S/N=3) were 53 microM for NO2(-) and 41 microM for NO3(-). Moreover, the correlation coefficients in excess of 0.99 between the MCE method and a conventional Griess method were achieved for both NO2(-) and NO3(-). Consequently, the possibility of establishing a high-throughput assay process was obtained by utilizing 2% (w/w) CHES to reduce protein adsorption.     

Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation

Nitrite and nitrate are main stable products of nitric oxide, a pivotal cellular signaling molecule, in biological fluids. Therefore, accurate measurement of the two ions is profoundly important. Nitrite is difficult to be determined for a larger number of interferences and unstable in the presence of oxygen. In this paper, a simple, cost-effective and accurate HPLC method for the determination of nitrite and nitrate was developed. On the basis of the reaction that nitrite is oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the determination of nitrite and nitrate was achieved by the following strategy: each sample was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and the second injection was to measure total nitrate including initial nitrate and the nitrate from the conversion of nitrite with the addition of acid potassium permanganate in the sample. The amount of nitrite can be calculated as difference between injections 2 and 1. The HPLC separation was performed on a reversed phase C₁₈ column for 15 min. The mobile phase consisted of methanol–water (2:98 by volume); the water in the mobile phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength was set at 210 nm. Additionally, we systemically investigated the effects of the concentration of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the amount of acidic potassium permanganate added to the sample on the separation efficacy. The results showed that the limits of detection (LOD) and the limit of quantitation (LOQ) were 0.075 and 0.25 μM for nitrate (containing the oxidized nitrite), respectively. The linear range was 1–800 μM. This developed approach was successfully applied to assay nitrite/nitrate levels in cell culture medium, cell lysate, rat plasma and urine.

     

Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation

Nitrite and nitrate are main stable products of nitric oxide, a pivotal cellular signaling molecule, in biological fluids. Therefore, accurate measurement of the two ions is profoundly important. Nitrite is difficult to be determined for a larger number of interferences and unstable in the presence of oxygen. In this paper, a simple, cost-effective and accurate HPLC method for the determination of nitrite and nitrate was developed. On the basis of the reaction that nitrite is oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the determination of nitrite and nitrate was achieved by the following strategy: each sample was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and the second injection was to measure total nitrate including initial nitrate and the nitrate from the conversion of nitrite with the addition of acid potassium permanganate in the sample. The amount of nitrite can be calculated as difference between injections 2 and 1. The HPLC separation was performed on a reversed phase C₁₈ column for 15 min. The mobile phase consisted of methanol–water (2:98 by volume); the water in the mobile phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength was set at 210 nm. Additionally, we systemically investigated the effects of the concentration of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the amount of acidic potassium permanganate added to the sample on the separation efficacy. The results showed that the limits of detection (LOD) and the limit of quantitation (LOQ) were 0.075 and 0.25 μM for nitrate (containing the oxidized nitrite), respectively. The linear range was 1–800 μM. This developed approach was successfully applied to assay nitrite/nitrate levels in cell culture medium, cell lysate, rat plasma and urine.     

Hadamard transform CE-UV detection for biological samples

A Hadamard transform-capillary electrophoresis-UV (HT-CE-UV) detection technique is described for the analysis of biological samples. Pseudorandom injections of sample and buffer according to a simplex matrix obtained from the corresponding Hadamard matrix is performed with conventional capillaries. Alternating injections are achieved with a novel capillary "T" connector created by drilling conventional capillary dimensions through a 1-cm diameter polycarbonate disc. This connector design coupled with a switching system allows for rapid, electrokinetic injections of solution into alternating sample and buffer capillary arms for UV detection. The standard mixtures of nitric oxide (NO) metabolites, nitrite and nitrate, dissolved in physiological saline solution are injected into the separation capillary according to an 83-element injection sequence to obtain a signal-to-noise ratio (S/N) enhancement of ca. 4.5 over a single injection. Nitrite, being the less concentrated metabolite in NO detection and thereby more difficult to detect, was calibrated with the HT-CE-UV method and a limit of detection (LOD) of 0.56 microM was obtained. Rat blood plasma was analyzed with this detection system and demonstrated to be comparable with NO metabolite concentrations of previously published results. This HT-CE-UV method is described where a unique reservoir tube design that contains 8-microL standard nitrite sample volumes is placed over the end of the capillary arm to explore low volume limits for biological samples.     

Nitrite and nitrate measurement in human urine by capillary electrophoresis.

Nitrite and nitrate have been widely used as markers for nitric oxide (NO) formation in vivo and represent the major NO oxidation products in biological fluids. In the present study, the use of capillary electrophoresis (CE) in the measurement of nitrite and nitrate in human urine is described. Urine samples were electrophoresed in an extended light path fused-silica capillary (104 cm; 75 microm ID) at an applied negative potential of 30 kV, and UV detection at 214 nm. Using electrokinetic sample injection (-6 kV x 20 s), we found that urine concentration, pH, sodium and chloride interfered with nitrite and nitrate detection. The detection of nitrite and nitrate was decreased when hydrodynamic sample injection was used (30 mbar x 60 s). However, basal levels of urinary nitrite (0.25 +/- 0.05 microM) and nitrate (591 +/- 115 microM) were detected and no interference by variations in urine concentration and pH was noted when hydrodynamic sample injection was used. Thus, hydrodynamic sample injection is convenient for the measurement of urinary nitrite and nitrate and avoids the effect of variations in urine matrices and pH on nitrite and nitrate detection.     

Multiplexed detection of nitrate and nitrite for capillary electrophoresis with an automated device for high injection efficiency

A simple automated nanoliter scale injection device which allows for reproducible 5 nL sample injections from samples with a volume of <1 microL is successfully used for conventional capillary electrophoresis (CE) and Hadamard transform (HT) CE detection. Two standard fused silica capillaries are assembled axially through the device to function as an injection and a separation capillary. Sample solution is supplied to the injection capillary using pressure controlled with a solenoid valve. Buffer solution flows gravimetrically by the junction of the injection and separation capillaries and is also gated with a solenoid valve. Plugs of sample are pushed into the space between the injection and separation capillaries for electrokinectic injection. To evaluate the performance of the injection device, several optimizations are performed including the influence of flow rates, the injected sample volume and the control of the buffer transverse flow on the overall sensitivity. The system was then applied to HT-CE-UV detection for the signal-to-noise ratio (S/N) improvement of the nitric oxide (NO) metabolites, nitrite and nitrate. In addition, signal averaging was performed to explore the possibility of greater sensitivity enhancements compared to single injections.     

Simultaneous determination of nitrite and nitrate in human plasma by on-capillary preconcentration with field-amplified sample stacking

A simple method for the determination of nitrite and nitrate in human plasma has been developed using CZE with minimal sample preparation. Field‐amplified sample stacking (FASS) was used to achieve submicromolar detection by dilution of the plasma sample with deionized water. In CZE, the separation of nitrite and nitrate was achieved within 10 min without adding EOF modifier. The optimal condition was achieved with 50 mM phosphate buffer at pH 9.3. The ninefold diluted plasma samples were injected hydrodynamically for 40 s into a 60 cm×75 μm id uncoated fused‐silica capillary. The separation voltage was 20 kV (negative potential) and UV detection was performed at 214 nm. The linearity curves for nitrite and nitrate were obtained by the standard addition method. The estimated LODs for nitrite and nitrate in ninefold diluted plasma sample were 0.05 and 0.07 μM, respectively. The LODs for nitrite and nitrate in original plasma samples were 0.45 and 0.63 μM. The intra‐ and inter‐day precisions for both analytes were <2.6% and the recovery ranged between 92.3 and 113.3%. It was found that nitrite was more stable than nitrate in the plasma after the sample preparation. This proposed method was applied to a number of human plasma samples and the measured nitrite and nitrate concentrations in human plasma were consistent with the literature ranges.     

The role of nitric oxide in cancer improved methods for measurement of nitrite and nitrate by high-performance ion chromatography

The short lifetime of nitric oxide (NO) in vivo impedes its quantitation directly; however, the determination of nitrite and nitrate ions as the end-products of NO oxidation has proven a more practical approach. High-performance ion chromatographic analysis of nitrite in biological fluids is hampered by the large amount of chloride ion (up to 100mmol/l) which results in insufficient peak resolution when utilizing conductimetric detection. Analysis of both anions in small sample volumes is also constrained by the need to minimise sample handling to avoid contamination by environmental nitrate. We report a means to remove Cl⁻ ions from small sample volumes using Ag⁺ resin which facilitates quantitation of either nitrite and nitrate anions in biological samples, using silica or polymer based ion-exchange resins with conductimetric or electrochemical and spectrophotometric detection. Including a reversed-phase guard column before the anion-exchange guard and analytical column also greatly extends column lifetime.     

Simultaneous spectrophotometric determination of nitrite and nitrate by flow injection analysis

The flow injection principle is used in the photometric determination of nitrite and nitrate with sulfanilamide and N-(1-naphthyl)ethylenediamine as reagents. An on-line copper-coated cadmium reductor reduces nitrate to nitrite. The detection limit is 0.05 μM for nitrite and 0.1 mM for nitrate at a total sample volume of 200 μM. Up to 30 samples can be analyzed per hour with a relative precision of ca. 1%.     

Spectrophotometric simultaneous determination of nitrite, nitrate and ammonium in soils by flow injection analysis

A new rapid flow injection procedure for the simultaneous determination of nitrate, nitrite and ammonium in single flow injection analysis system is proposed. The procedure combines on-line reduction of nitrate to nitrite and oxidation of ammonium to nitrite with spectrophotometric detection of nitrite by using the Griess-llosvay reaction. The formed azo dye was measured at 543 nm. The influence of reagent concentration and manifold parameters were studied. Nitrite, nitrate and ammonium can be determined within the range of 0.02–1.60 μg mL⁻¹, 0.02–1.60 μg mL⁻¹ and 0.05–1.40 μg mL⁻¹, respectively. R.S.D. values (n = 10) were 2.66; 1.41 and 3.58 for nitrate, nitrite and ammonium, respectively. This procedure allows the determination and speciation of inorganic nitrogen species in soils with a single injection in a simple way, and high sampling rate (18 h⁻¹). Detection limits of 0.013, 0.046 and 0.047 μg mL⁻¹were achieved for nitrate, nitrite and ammonium, respectively. In comparison with others methods, the proposed one is more simple, it uses as single chromogenic reagent less injection volume (250 mL in stead of 350 mL) and it has a higher sampling rate.     

Direct determination of nitrite traces in high ionic-strength samples by electrostatic ion chromatography using diluted acid solutions as eluent

An ion-chromatographic (IC) system with high selectivity for separation of nitrite is described. It is analogous to the EIC (electrostatic IC) previously reported and was established using 3-(N,N-dimethylstearylammonio)propanesulfonate (C23H49NO3S, a sulfobetaine type of zwitterionic surfactants) as the stationary phase and dilute aqueous HCl solutions as the mobile phase. Five inorganic anions, sulfate, chloride, bromide, nitrate, and nitrite were chosen as the model analytes and were analyzed using this EIC system. Sulfate was always eluted first, followed by chloride, bromide and nitrate. Nitrite, however, could be eluted either before or after nitrate, depending on the concentration of HCl in the eluent. An elution order nitrate< nitrite was always obtained simply by using >3 mmol L(-1) HCl as the eluent. For nitrite the detection limit was better than 2.1 x 10(-7) mol L(-1) (100 microL sample injection volume, S/N=3, UV at 210 nm). Bromide and nitrate could also be separated under these HPLC conditions. The detection limit for bromide was 7.2 x 10(-8) mol L(-1) and for nitrate 6.5 x 10(-8) mol L(-1). Both nitrite and nitrate in real seawater samples were successfully determined with direct sample injection using this EIC system.     

Evaluation of an ODS column modified with zwitterionic/nonionic mixed surfactants and its application to direct injection determination of inorganic anions.

An octadecylsilica (ODS) column modified with zwitterionic/nonionic mixed surfactants was evaluated for the direct injection determination of inorganic anions in biological fluids by ion chromatography. A zwitterionic surfactant (sulfobetaine-type) and a nonionic surfactant (polyoxyethylene-type) were used for a stationary-phase modification. When aqueous electrolyte solutions with concentrations of sub-mM to several mM were used as a mobile phase, the zwitterionic surfactant coated on the ODS surface exhibited unique separation selectivity for ionic species, while the nonionic surfactant coated on the ODS might have formed a hydrophilic network over the ODS surface and restricted matrix proteins from adsorbing on the stationary phase. Consequently, the mixed surfactant-modified column system allowed an efficient ion chromatographic separation of inorganic anions as well as a size-exclusive removal of column-fouling proteins. This separation system was applied to the direct injection determination of UV-absorbing anions in human saliva. The detection limits for nitrite, nitrate, iodide and thiocyanate were 3.1, 2.7, 4.5 and 25 microM, respectively, with UV detection at 210 nm (injection volume; 20 microl), and their relative standard deviations for 5 replicate measurements of saliva samples spiked with 100 microM each of those anions were 1.4, 0.9, 2.2 and 5.5%, respectively.     

A simple simultaneous flow injection method based on phosphomolybdenum chemistry for nitrate and nitrite determinations in water and fish samples

A direct spectrophotometric flow injection method for the simultaneous determination of nitrite and nitrate has been developed. The method is based on the oxidation of a phosphomolybdenum blue complex by the addition of nitrite and the decrease in absorbance of the blue complex is monitored at 820 nm. The injected sample is split into two segments. One of the streams was directly reacted with the above reagent and detected as nitrite. The other stream was passed through a copperised cadmium reductor column where reduction of nitrate to nitrite occurs, and the sample was then mixed with the reagent and passed through the cell of the spectrophotometer to be detected as nitrite plus nitrate. The conditions for the flow injection manifold parameters were optimised by experimental design and the concentration of nitrite and nitrate was determined in the linear range from 0.05 to 1.15 mug ml(-1) nitrite and 0.06 to 1.6 mug ml(-1) nitrate with a detection limit of 0.01 mug ml(-1) for nitrite and 0.025 mug ml(-1) for nitrate. The method is suitable for the simultaneous determination of nitrite and nitrate in fish and water samples with a sampling rate of 25+/-2 sample per hour.     

Large Volume Injection of Biological Samples Dissolved in a Non-Eluting Solvent: A Way to Increase Sensitivity and a Means of Automating Drug Determination Using Hplc

Abstract

The injected volume of a sample dissolved in the mobile phase of an HPLC system must be maintained as small as possible so as to minimize the loss in efficiency. Generally this requirement limits the sensitivity of HPLC methods devoted to trace quantity determinations of drugs in biological fluids. In order to avoid this limitation and to increase the effective sensitivity of HPLC methods for determination of drugs such as antrafenine, nifuroxazide and cipropride, the samples were dissolved in a non-eluting solvent and a large volume (> 100 μl) was injected on to the chromatographic column.

The above-mentioned compounds and their internal standards were dissolved in a series of eluting and non-eluting solvents and increasing volumes (5 to 1000 μl) were injected. Peaks corresponding to injections made in an eluting solvent showed retention times independent of the injection volume but their variances increased with the volume injected. In contrast, peaks corresponding to injections made in a non-eluting solvent, similar to the mobile phase, had a variance independent of the injection volume but their retention times increased linearly with the injection volume. The repeated injection of such non-eluting solvents had no influence on chromatographic behaviour. Peaks corresponding to compounds injected in a non-eluting solvent made with components different from those of the mobile phase had a variance independent of the injection volume but their retention times varied both with the injection volume and with the interval between injection.

The application of non-eluting solvents has been defined theoretically and it has been demonstrated that solutions composed of 25% of the mobile phase diluted with the least eluting of its components act as non-eluting solvents and can be injected in large volume without loss in efficiency. This feature could be used to inject all the samples volume or only part of it, manually or automatically, since any automatic injector can be used with large volumes.

Thus, using the relatively simple procedure of making injections with a non-eluting solvent it is possible to increase both sensitivity and the rate of sample analysis.     

A downsized flow set up based on multicommutation for the sequential photometric determination of iron(II)/iron(III) and nitrite/nitrate in surface water

In this work, a downsized flow set up designed based on multicommutation concept for photometric determination of iron(II)/iron(III) and nitrite/nitrate is surface water is described. The flow system network comprised a set of three-way solenoid valves, reaction coil and a double-channel flow cell, which were nested in order to obtain a compact and small-size instrument. To accomplish the downsizing requirement light source (LED) and radiation detection (phototransistor) were coupled to the flow cell. In order to demonstrated the effectiveness of the system, the photometer methods based on Griess reaction and 1-10-phenantroline for nitrite and iron(II) determination, respectively, were selected. Under computer control the set up provided facilities to handle four reagent solutions employing a single pumping channel, thus permitting also the determination of nitrate and iron(III) after its reduction to nitrite and to iron(II), respectively. The overall system performance was demonstrated working several days running standard solution, no significant variation of base line, linear response range and slop (less than 1%) were observed. The usefulness of the downsized system was ascertained by analyzing a set of surface water. Aiming to access the accuracy sample were also analyzed employing reference procedures and no significant difference at 95% confidence level were observed for the four analytes. Other profitable features such as analytical throughput of 40 determination per hour; relative standard deviation of 1%; linear response range between 50 and 300 μg l⁻¹ for nitrite and nitrate, 0.5-6.0 mg l⁻¹ iron(II) and iron(III); low reagent consumption 75 μg for nitrate/nitrite and 0.6 mg for iron(II)/iron(III) per determination; and 2.4 ml waste generation per determination were also achieved.     

Single-column ion chromatography of passive monitors for atmospheric pollution

Nitrite, nitrate and sulfate anions deposited on passive monitors were determined by using single-column ion chromatography. These monitors were exposed outdoors for various periods of time in an atmosphere with a very low degree of pollution, and they were used to evaluate the rate of deposition of some corrosive species on metallic materials. Two types of passive monitors were studied, namely sulfation and nitration plates. Comparison of the results obtained by using the classical turbidimetric method and single-column ion chromatography shows that turbidimetry is inappropriate for low sulfate concentrations (<2.5 mg/plate) deposited on sulfation plates. Thus, ion chromatography should be used to obtain a representative value of sulfate deposition because it allows the determination of sulfate in amounts as low as 200 μg/plate without further preconcentration. For nitration plates, ion chromatography permits simultaneous determination of nitrite, nitrate and sulfate from the same plate, whereas only nitrite can be determined by the classical colorimetric method. Both gluconate/borate and phthalate eluents were investigated and the results show that phthalate eluent is more appropriate for these plates. It was found that the pH of the sample has an important effect on the area of the sulfate peak. For both eluents, the area increases when the sample pH is lower than 6. This demonstrates the importance of an adequate sample treatment before injection in order to obtain reproducible and accurate values.     

Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS

The need for sensitive analytical technologies applicable to metabolic profiling of volume-restricted biological samples is high. Here, we demonstrate feasibility of capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (MS) with sheathless nano-electrospray interface for non-targeted profiling of ionogenic metabolites in body fluids of experimental animals. A representative mixture of the metabolites and body fluids of mice such as cerebrospinal fluid (CSF), urine and plasma were used as examples of low-volume biological samples for method evaluation. An injection volume of only 9?nL resulted in limits of detection between 0.7 and 12?nM for the metabolite mixture. The method allowed the detection of ～350 molecular features in mouse CSF (an injection volume of ca. 45?nL), while ～400 features were observed in mouse plasma and ～3,500 features in mouse urine (an injection volume of ca. 9?nL). The low-volume body fluid samples were analyzed directly after only 1:1 dilution with water, thereby fully retaining sample integrity, which is of crucial importance for non-targeted metabolic profiling. As little is known about the metabolic composition of mouse CSF, we identified a fraction of the molecular features in mouse CSF using accurate mass information, migration times, MS/MS data, and comparison with authentic standards. We conclude that sheathless CE-MS can be used for sensitive metabolic profiling of volume-restricted biological samples.     

Flow-injection determination of inorganic forms of nitrogen by gas diffusion and conductimetry

A flow-injection—conductimetric method was applied to the determination of ammonia, nitrate and nitrite at concentrations down to 5, 20 and 20 ng ml^?1, respectively. Ammonia was determined by merging the injected sample with an alkaline solution (NaOHEDTA) and passing the mixture through a diffusion cell. The ammonia released was collected by a flowing stream of deionized water that passed through a conductance flow cell. Nitrate and nitrite concentrations were determined after reduction to ammonia in alkaline medium using a column filled with metallic zinc. The ammonia produced was then measured as described above. About 60 samples per hour can be processed with a relative standard deviation of about 1%. Satisfactory agreement was observed between results for ammonia in samples of natural water and nitrate in tap and mineral water determined by the proposed method and by standard spectrophotometric procedures. Speciation can be achieved by adding sulphanilic acid to remove nitrite from the sample and determining the ammonia without the use of the column.     

High sensitivity analysis of nitrite and nitrate in biological samples by capillary zone electrophoresis with transient isotachophoretic sample stacking

Tissue level of nitrate and nitrite are established indicators of altered nitric oxide metabolism under various pathological conditions. Determination of these anions in biological samples, in the presence of high chloride concentration, using capillary zone electrophoresis suffers from poor detection sensitivity. Separation conditions providing excellent resolution and submicromolar detection sensitivity of nitrate and nitrite have been developed and validated. Simple sample preparation was applied that maintains nitrite stability in tissue extracts and at the same time allows transient isotachophoresis stacking of the analytes. Nitrate and nitrite concentrations in rat brain and liver tissue samples were determined in control and lipopolysaccharide treated animals.     

Simultaneous stepwise injection-photometric determination of nitrite and nitrate ions in aqueous media

A procedure for the stepwise injection-photometric determination of nitrite and nitrate ions has been developed. The procedure employs their subsequent determination by the reaction of colored azo compound formation after the reduction of nitrate ions into nitrite ions on a cadmium reducer. The analytical ranges for nitrite and nitrate ions are 2–15 and 5–50 mg/L (sample volume 2 mL, analysis time 14 min).