Simultaneous determination of ascorbic acid, dopamine, uric acid and xanthine using a nanostructured polymer film modified electrode

This paper describes the simultaneous determination of ascorbic acid (AA), dopamine (DA), uric acid (UA) and xanthine (XN) using an ultrathin electropolymerized film of 2-amino-1,3,4-thiadiazole (p-ATD) modified glassy carbon (GC) electrode in 0.20 M phosphate buffer solution (pH 5.0). Bare GC electrode failed to resolve the voltammetric signals of AA, DA, UA and XN in a mixture. On the other hand, the p-ATD modified electrode separated the voltammetric signals of AA, DA, UA and XN with potential differences of 110, 152 and 392 mV between AA-DA, DA-UA and UA-XN, respectively and also enhanced their oxidation peak currents. The modified electrode could sense 5 μM DA and 10 μM each UA and XN even in the presence of 200 μM AA. The oxidation currents were increased from 30 to 300 μM for AA, 5 to 50 μM for DA and 10 to 100 μM for each UA and XN, and the lowest detection limit was found to be 2.01, 0.33, 0.19 and 0.59 μM for AA, DA, UA and XN, respectively (S/N = 3). The practical application of the present modified electrode was demonstrated by the determination of AA, UA and XN in human urine samples.     

Simultaneous determination of epinephrine, uric acid and xanthine in the presence of ascorbic acid using an ultrathin polymer film of 5-amino-1,3,4-thiadiazole-2-thiol modified electrode

This paper describes the simultaneous determination of epinephrine (EP), uric acid (UA) and xanthine (XN) in the presence of ascorbic acid (AA) using electropolymerized ultrathin film of 5-amino-1,3,4-thiadiazole-2-thiol (p-ATT) modified glassy carbon (GC) electrode in 0.2 M phosphate buffer solution (pH 5). Although bare GC electrode resolves the voltammetric signals of AA and XN, it fails to resolve the voltammetric signals of EP and UA in a mixture. However, the p-ATT modified electrode not only separates the voltammetric signals of AA, EP, UA and XN with potential difference of 150, 120 and 400 mV between AA-EP, EP-UA and UA-XN, respectively but also shows higher oxidation current for these molecules. The p-ATT modified electrode exhibits excellent selectivity towards the oxidation of EP, UA and XN in the presence of 40-fold higher concentration of AA. Further, the p-ATT modified electrode was also used for the selective determination of EP in the presence of 40-fold higher concentrations of AA, UA and XN. Using amperometric method, we achieved the lowest detection of 40 nM EP and 60 nM each UA and XN. The amperometric current response was increased linearly with increasing EP concentration in the range of 4.0 × 10⁻⁸ to 4.0 × 10⁻⁵ M and the detection limit was found to be 27 × 10⁻¹¹ M (S/N = 3). The practical application of the present modified electrode was demonstrated by determining the concentration of EP in epinephrine tartrate injection and XN in human urine samples.     

Electropolymerized film of functionalized thiadiazole on glassy carbon electrode for the simultaneous determination of ascorbic acid, dopamine and uric acid

The present study reports the simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA) in 0.20 M phosphate buffer solution (pH 5.0) using electropolymerized ultrathin film of 5-amino-2-mercapto-1,3,4-thiadiazole (AMT) on glassy carbon (GC) electrode. The bare GC electrode does not separate the voltammetric signals of AA, DA and UA. However, electropolymerized AMT (p-AMT) modified GC electrode not only resolved the voltammetric signals of AA, DA and UA but also dramatically enhanced their oxidation peak currents when compared to bare GC electrode. The enhanced oxidation currents for AA, DA and UA at p-AMT modified electrode are due to the electrostatic interactions between them and the polymer film. Using amperometric method, we achieved the lowest detection of 75 nM AA, 40 nM DA and 60 nM UA at p-AMT modified electrode. The amperometric current was linearly increased from 200 nM to 0.80 mM for each AA, DA and UA and the lowest detection limit was found to be 0.92, 0.07 and 0.57 nM, respectively (S/N = 3). The practical application of the modified electrode was demonstrated by the determination of DA in dopamine hydrochloride injection.     

Facile synthesis of graphene hybrid tube-like structure for simultaneous detection of ascorbic acid,dopamine, uric acid and tryptophan

In the present work, a tube-like structure of graphene hybrid as modifier to fabricate electrode for simultaneous detection of ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp) was reported. The hybrid was synthesized by a simple method based on graphene sheets (GS) and 3,4,9,10-perylenetetracarboxylic acid (PTCA) via π–π stacking interaction under ultrasonic condition. The combination of GS and PTCA could effectively improve the dispersion of GS, owing to PTCA with the carboxylic-functionalized interface. Comparing with pure GS or PTCA modified electrode, GS–PTCA displayed high catalytic activity and selectivity toward the oxidation of AA, DA, UA, and Trp. Moreover, cyclic voltammetry, different pulse voltammetry and scanning electron microscopy were employed to characterize the sensors. The experiment results showed that the linear response range for simultaneous detection of AA, DA, UA, and Trp were 20–420 μM, 0.40–374 μM, 4–544 μM and 0.40–138 μM, respectively, and the detection limits were 5.60 μM, 0.13 μM, 0.92 μM and 0.06 μM (S/N = 3). Importantly, the proposed method offers promise for simple, rapid, selective and cost-effective analysis of small biomolecules.     

Simultaneous determination of ascorbic acid,dopamine and uric acid based on tryptophan functionalized graphene

A new type of tryptophan-functionalized graphene nanocomposite (Trp-GR) was synthesized by utilizing a facile ultrasonic method via π–π conjugate action between graphene (GR) and tryptophan (Trp) molecule. The material as prepared had well dispersivity in water and better conductivity than pure GR. The surface morphology of Trp-GR was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. The electrochemical behaviors of ascorbic acid (AA), dopamine (DA), and uric acid (UA) were investigated by cyclic voltammetry (CV) on the surface of Trp-GR. The separation of the oxidation peak potentials for AA–DA, DA–UA and UA–AA was about 182 mV, 125 mV and 307 mV, which allowed simultaneously determining AA, DA, and UA. Differential pulse voltammetery (DPV) was used for the determination of AA, DA, and UA in their mixture. Under optimum conditions, the linear response ranges for the determination of AA, DA, and UA were 0.2–12.9 mM, 0.5–110 μM, and 10–1000 μM, with the detection limits (S/N = 3) of 10.09 μM, 0.29 μM and 1.24 μM, respectively. Furthermore, the modified electrode was investigated for real sample analysis.     

Simultaneous determination of ascorbic acid,dopamine, uric acid and tryptophan on gold nanoparticles/overoxidized-polyimidazole composite modified glassy carbon electrode

A novel electrode was developed through electrodepositing gold nanoparticles (GNPs) on overoxidized-polyimidazole (PImox) film modified glassy carbon electrode (GCE). The combination of GNPs and the PImox film endowed the GNPs/PImox/GCE with good biological compatibility, high selectivity and sensitivity and excellent electrochemical catalytic activities towards ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp). In the fourfold co-existence system, the peak separations between AA–DA, DA–UA and UA–Trp were large up to 186, 165 and 285 mV, respectively. The calibration curves for AA, DA and UA were obtained in the range of 210.0–1010.0 μM, 5.0–268.0 μM and 6.0–486.0 μM with detection limits (S/N = 3) of 2.0 μM, 0.08 μM and 0.5 μM, respectively. Two linear calibrations for Trp were obtained over ranges of 3.0–34.0 μM and 84.0–464.0 μM with detection limit (S/N = 3) of 0.7 μM. In addition, the modified electrode was applied to detect AA, DA, UA and Trp in samples using standard addition method with satisfactory results.     

Modified platinum electrode with phytic acid and single-walled carbon nanotube: Application to the selective determination of dopamine in the presence of ascorbic and uric acids

A platinum (Pt) electrode modified by single-walled carbon nanotubes (SWNTs) and phytic acid (PA) was investigated by voltammetric methods in buffer solution. The PA-SWNTs/Pt-modified electrode demonstrated substantial enhancements in electrochemical sensitivity and selectivity towards dopamine (DA) in the presence of L-ascorbic acid (AA) and uric acid (UA). The PA-SWNTs films promoted the electron transfer reaction of DA, while the PA film, acting as a negatively charged linker, combined with the positively charged DA to induced DA accumulation in the film at pH under 7.4. However, the PA film restrained the electrochemical response of the negatively charged AA due to the electrostatic repulsion. The anodic peak potentials of DA, AA and UA could be separated by electrochemical techniques, and the interferences from AA and UA were effectively eliminated in the DA determination. Linear calibration plots were obtained in the DA concentration range of 0.2-10 μM and the detection limit of the DA oxidation current was determined to be 0.08 μM at a signal-to-noise ratio of 3. The results indicated that the modified electrode can be used to determine DA without interference from AA and UA, while ensuring good sensitivity, selectivity, and reproducibility.     

Poly(3,4-ethylenedioxythiophene-co-(5-amino-2-naphthalenesulfonic acid)) (PEDOT-PANS) film modified glassy carbon electrode for selective detection of dopamine in the presence of ascorbic acid and uric acid

Poly(3,4-ethylenedioxythiophene-co-(5-amino-2-naphthalenesulfonic acid)) (PEDOT-PANS) film modified glassy carbon electrode was prepared by electrochemical polymerization technique. The properties of modified electrode was studied. It was found that the electrochemical properties of modified electrode was very much dependent on the experimental conditions, such as monomer oxidation potential and pH. The modified electrode surface was characterized by scanning electron microscopy (SEM). The PEDOT-PANS film modified electrode shows electrocatalytic activity toward oxidation of dopamine (DA) in acetate buffer solution (pH 5.0) and results in a marked enhancement of the current response. The linear sweep voltammetric (LSV) peak heights are linear with DA concentration from 2 × 10⁻⁶ to 1 × 10⁻⁵ M. The detection limit is 5 × 10⁻⁷ M. More over, the interferences of ascorbic acid (AA) and uric acid (UA) were effectively diminished. This work provides a simple and easy approach for selective determination of dopamine in the presence of ascorbic acid and uric acid.     

Simultaneous electrochemical sensing of ascorbic acid,dopamine and uric acid at anodized nanocrystalline graphite-like pyrolytic carbon film electrode

Nanocrystalline graphite-like pyrolytic carbon film (PCF) electrode fabricated by a non-catalytic chemical vapor deposition (CVD) process was used for the simultaneous electrochemical sensing of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The electrode was studied with respect to changes in electrocatalytic activity caused by a simple and fast electrochemical pretreatment. The anodized electrode exhibited excellent performance compared to many chemically modified electrodes in terms of detection limit, linear dynamic range, and sensitivity. Differential pulse voltammetry (DPV) was used for the simultaneous determination of ternary mixtures of DA, AA, and UA. Under optimum conditions, the detection limits were 2.9 μM for AA, 0.04 μM for DA, and 0.03 μM for UA with sensitivities of 0.078, 5.345, and 6.192 A M⁻¹, respectively. The peak separation was 219 mV between AA and DA and 150 mV between DA and UA. No electrode fouling was observed and good reproducibility was obtained in all the experiments. The sensor was successfully applied for the assay of DA in an injectable drug and UA in human urine by using standard addition method.     

One step fabrication of nanoelectrode ensembles formed via amphiphilic block copolymers self-assembly and selective voltammetric detection of uric acid in the presence of high ascorbic acid content

A novel one-step approach to glassy carbon nanoelectrode ensembles (NEEs) with the pores of 20-120 nm in radii has been developed using an amphiphilic block copolymer [polystyrene-block-poly (acrylic acid)] self-assembly. This procedure is simple and fast, and requires only conventional, inexpensive electrochemical instrumentation. Electrochemical methods were used to characterize the NEEs prepared using this new procedure. The NEEs drastically suppressed the response of ascorbic acid (AA) and resolved the overlapping voltammetric response of uric acid (UA) and AA into two well-defined peaks with a large anodic peak difference (ΔE_pa) of about 310 mV. The peak current obtained from differential pulse voltammetry (DPV) was linearly dependent on the UA concentration in the range of 0.25-50 μM at neutral pH (PBS, pH 6.86) with a correlation coefficient of 0.999, and the detection limit was 0.04 μM (S/N = 3). The NEEs has also been demonstrated to be applicable in the detection of UA in serum and urine samples with excellent sensitivity and selectivity. The NEEs will hopefully be of good application for further sensor development.     

Preparation and characterization of PtAu hybrid film modified electrodes and their use in simultaneous determination of dopamine, ascorbic acid and uric acid

A novel biosensor was fabricated by electrochemical deposition of platinum and gold nanoparticles (nanoAu) with l-Cysteine on glassy carbon electrode. It was found that the nanoAu particle size distribution range was (50-80 nm), and the platinum particle size range was (200-300 nm). The hybrid film could be produced on gold and transparent indium tin oxide electrodes for different kind of studies such as electrochemical quartz crystal microbalance (EQCM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD) and electrochemical studies. The PtAu hybrid film was applied to the electro catalytic oxidation of dopamine (DA), ascorbic acid (AA) and uric acid (UA) at pH 4.0 using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The modified electrode was quite effective not only to detect DA, AA and UA individually but also in simultaneous determination of these species in a mixture. The overlapping anodic peaks of DA, AA and UA were resolved into three well-defined voltammetric peaks in CV and DPV. The catalytic peak currents obtained from CV and DPV increased linearly with concentration. The relative standard deviation (% R.S.D., n = 10) for AA, DA and UA were less than 2.0% and DA, AA and UA can be determined in the ranges of 0.103-1.65, 0.024-0.384 and 0.021-0.336 mM, respectively. In addition, the modified electrode also shows good sensitivity, and stability. Satisfactory results were achieved for the determination of DA, AA and UA in dopamine injection solution, vitamin C tablets and human urine samples.     

Cu nanoparticles incorporated polypyrrole modified GCE for sensitive simultaneous determination of dopamine and uric acid

Cu nanoparticles have been electrochemically incorporated polypyrrole film that was used for modification of the glassy carbon electrode surface. The performance of the electrode has been characterized by cyclic voltammetry and atomic force microscopy. The electrode has shown high electrocatalytic activity towards the oxidation of dopamine (DA) and uric acid (UA) simultaneously in a phosphate buffer solution (pH 7.00). The electrocatalytic oxidation currents of UA and DA were found linearly related to concentration over the range 1 × 10⁻⁹ to 1 × 10⁻⁵ M for UA and 1 × 10⁻⁹ to 1 × 10⁻⁷ M for DA using DPVs method. The detection limits were determined as 8 × 10⁻¹⁰ M (s/n = 3) for UA and 8.5 × 10⁻¹⁰ M (s/n = 3) for DA at a signal-to-noise ratio of 3.     

Electrodeposition of gold nanoclusters on overoxidized polypyrrole film modified glassy carbon electrode and its application for the simultaneous determination of epinephrine and uric acid under coexistence of ascorbic acid

A novel biosensor was fabricated by electrochemical deposition of gold nanoclusters on ultrathin overoxidized polypyrrole (PPyox) film, formed a nano-Au/PPyox composite on glassy carbon electrode (nano-Au/PPyox/GCE). The properties of the nanocomposite have been characterized by field emission scanning electron microscope (FE-SEM), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD) and electrochemical investigations. The nano-Au/PPyox/GCE had strongly catalytic activity toward the oxidation of epinephrine (EP), uric acid (UA) and ascorbic acid (AA), and resolved the overlapping voltammetric response of EP, UA and AA into three well-defined peaks with a large anodic peak difference. The catalytic peak currents obtained from differential pulse voltammetry increased linearly with increasing EP and UA concentrations in the range of 3.0 × 10⁻⁷ to 2.1 × 10⁻⁵ M and 5.0 × 10⁻⁸ to 2.8 × 10⁻⁵ M with a detection limit of 3.0 × 10⁻⁸ and 1.2 × 10⁻⁸ M (s/n = 3), respectively. The results showed that the modified electrode can selectively determine EP and UA in the coexistence of a large amount of AA. In addition, the sensor exhibited excellent sensitivity, selectivity and stability. The nano-Au/PPyox/GCE has been applied to determination of EP in epinephrine hydrochloride injection and UA in urine samples with satisfactory results.     

Simultaneous detection of ascorbate and uric acid using a selectively catalytic surface

The direct and selective detection of ascorbate at conventional carbon or metal electrodes is difficult due to its large overpotential and fouling by oxidation products. Electrode modification by electrochemical reduction of diazonium salts of different aryl derivatives is useful for catalytic, analytical and biotechnological applications. A monolayer of o-aminophenol (o-AP) was grafted on a glassy carbon electrode (GCE) via the electrochemical reduction of its in situ prepared diazonium salts in aqueous solution. The o-aminophenol confined surface was characterized by cyclic voltammetry. The grafted film demonstrated an excellent electrocatalytic activity towards the oxidation of ascorbate in phosphate buffer of pH 7.0 shifting the overpotential from +462 to +263 mV versus Ag/AgCl. Cyclic voltammetry and d.c. amperometric measurements were carried out for the quantitative determination of ascorbate and uric acid. The catalytic oxidation peak current was linearly dependent on the ascorbate concentration and a linear calibration curve was obtained using d.c. amperometry in the range of 2-20 μM of ascorbate with a correlation coefficient 0.9998, and limit of detection 0.3 μM. The effect of H₂O₂ on the electrocatalytic oxidation of ascorbate at o-aminophenol modified GC electrode has been studied, the half-life time and rate constant was estimated as 270 s, and 2.57 × 10⁻³ s⁻¹, respectively. The catalytically selective electrode was applied to the simultaneous detection of ascorbate and uric acid, and used for their determination in real urine samples. This o-AP/GCE showed high stability with time, and was used as a simple and precise amperometric sensor for the selective determination of ascorbate.     

Simultaneous electrochemical determination of norepinephrine,ascorbic acid and uric acid using a graphene modified glassy carbon electrode

This paper describes the simultaneous determination of ascorbic acid (AA), norepinephrine (NE) and uric acid (UA) using a graphene modified glassy carbon electrode (GME) in pH 4.0 phosphate buffer solution. The electrochemical behaviors of AA, NE and UA at a bare glassy carbon electrode (GCE) and the GME were studied by cyclic voltammetry. Bare GCE failed to resolve the voltammetric signals of AA, NE and UA in a mixture, whereas the GME not only resolved their voltammetric signals, but also exhibited excellent electrocatalytic activity towards their electrochemical oxidation. The oxidation peak currents of AA, NE and UA were linearly proportional to their concentrations over the range of 1.0.0–1000.0, 0.6–45.0 and 1.0–100.0 μM, respectively, and their detection limits were 1.2, 0.10 and 0.60 μM, respectively, The modified electrode is of excellent sensitivity and selectivity, and has been satisfactorily used for the simultaneous determination of AA, NE and UA in their ternary mixture.     

Voltammetric Determination of Uric Acid at a Fullerene‐C60‐Modified Glassy Carbon Electrode

Glassy carbon electrode modified by microcrystals of fullerene‐C₆₀ mediates the voltammetric determination of uric acid (UA) in the presence of ascorbic acid (AA). Interference of AA was overcome owing to the ability of pretreated fullerene‐C₆₀‐modified glassy carbon electrode. Based on its strong catalytic function towards the oxidation of UA and AA, the overlapping voltammetric response of uric acid and ascorbic acid is resolved into two well‐defined voltammetric peaks with lowered oxidation potential and enhanced oxidation currents under conditions of both linear sweep voltammetry (LSV) and Osteryoung square‐wave voltammetry (OSWV). At pH 7.2, a linear calibration graph is obtained for UA in linear sweep voltammetry over the range from 0.5 μM to 700 μM with a correlation coefficient of 0.9904 and a sensitivity of 0.0215 μA μM^?1 . The detection limit (3σ) is 0.2 μM for standard solution. AA in less than four fold excess does not interfere. The sensitivity and detection limit in OSWV were found as 0.0255 μA μM^?1 and 0.12 μM, for standard solution respectively. The presence of physiologically common interferents (i.e. adenine, hypoxanthine and xanthine) negligibly affects the response of UA. The fullerene‐C₆₀‐modified electrode exhibited a stable, selective and sensitive response to uric acid in the presence of interferents.     

Selective Detection of Uric Acid in the Presence of Ascorbic Acid and Dopamine Using Polymerized Luminol Film Modified Glassy Carbon Electrode

Electrochemically polymerized luminol film on a glassy carbon electrode (GCE) surface has been used as a sensor for selective detection of uric acid (UA) in the presence of ascorbic acid (AA) and dopamine (DA). Cyclic voltammetry was used to evaluate the electrochemical properties of the poly(luminol) film modified electrode. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) have been used for surface characterizations. The bare GCE failed to distinguish the oxidation peaks of AA, DA and UA in phosphate buffer solution (pH 7.0), while the poly(luminol) modified electrode could separate them efficiently. In differential pulse voltammetric (DPV) measurements, the modified GCE could separate AA and DA signals from UA, allowing the selective determination of UA. Using DPV, the linear range (3.0×10^?5 to 1.0×10^?3 M) and the detection limit (2.0×10^?6 M) were estimated for measurement of UA in physiological condition. The applicability of the prepared electrode was demonstrated by measuring UA in human urine samples.     

Poly(isonicotinic acid) modified glassy carbon electrode for electrochemical detection of norepinephrine

A glassy carbon electrode (GCE) was modified with electropolymerized films of isonicotinic acid in pH 5.6 phosphate buffer solution (PBS) by cyclic voltammetry (CV). The modified electrode showed an excellent electrocatalytical effect on the oxidation of norepinephrine (NE). In PBS of pH 7.4, the oxidation current increased linearly with two concentration intervals of NE, one is 4.0×10⁻⁷ to 1.0×10⁻⁵ M, the other is 1.0×10⁻⁵ to 2.0×10⁻⁴ M. The detection limit (S/N=3) obtained by DPV was 6.0×10⁻⁹ M. Then the modified electrode was used to determine NE in an excess of ascorbic acid (AA) by difference pulse voltammetry. The peak potentials recorded in a PBS of pH 7.4 were −68 and +111 mV versus SCE for AA and NE, respectively. The high selectivity and sensitivity for NE was found to be due to the very distinct attracting interaction between NE cations and the negtively charged poly(isonicotinic acid) film in pH 7.4 PBS. The proposed method exhibited good recovery and reproducibility.     

Electropolymerization of Thin Film Conducting Polymer and Its Application for Simultaneous Determination of Ascorbic Acid,Dopamine and Uric Acid

In this paper electropolymerization of a thin film of para‐phenylenediamine (PPD) is studied at glassy carbon electrode (GCE) in sulfuric acid media by cyclic voltammetry. The results showed that this polymer was conducting and had a reproducible redox couple in the potential region from 0.0 to 0.4 V in phosphate buffer solution. This modified GCE (p‐PPD‐GCE) was applied for simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA) using differential pulse voltammetry (DPV). The p‐PPD‐GCE in 0.1 M phosphate buffer solution (pH 5.0) separated the DPV signals of AA, DA and UA with sufficient potential differences between AA–DA and DA–UA and also enhanced their oxidation peak currents. The oxidation currents were increased from 2.0 to 2000.0 µM for AA, 10.0 to 1250.0 µM for DA and 50.0 to 1600.0 µM for UA. The detection limits were evaluated as 0.4, 1.0 and 2.5 µM for AA, DA and UA, respectively (S/N=3).     

Voltammetric determination of salicylic acid in pharmaceuticals formulations of acetylsalicylic acid

The electrochemical oxidation of salicylic acid (SA) has been studied on a glassy carbon electrode using cyclic voltammetry and differential pulse voltammetric (DPV) method. SA gives a single irreversible oxidation wave over the wide pH range studied. The irreversibility of the electrode process was verified by different criteria. The mechanism of oxidation is discussed. Using differential pulse voltammetry, SA yielded a well-defined voltammetric response in Britton-Robinson buffer solution, pH 2.37 at 1.088 V (versus Ag/AgCl). The method was linear over the SA concentration range: 1-60 μg ml⁻¹. The method was successfully applied for the analysis of SA as a hydrolysis product, in solid pharmaceutical formulations containing acetylsalicylic acid (ASA).