A Simple and Sensitive Poly-1,5-Diaminonaphthalene Modified Sensor for the Determination of...

DOI: 10.1002/elan.201400688

A Simple and Sensitive Poly-1,5-DiaminonaphthaleneModified Sensor for the Determination ofSulfamethoxazole in Biological SamplesHimanshu Chasta[a] and Rajendra N. Goyal*[a]

1 Introduction

Sulfamethoxazole (SMZ) [N1-(5-methylisoxazol-3-yl) sul-fanilamide] (Scheme 1 a), a sulfonamide drug with anti-bacterial properties, is used for the treatment of humaninfections. Sulfonamide drugs are pervasively used in vet-erinary care and their use without a proper withdrawalperiod can cause accumulation of sulfonamides in eggs,milk, meat and honey as well as in fish [1–3]. Sulfona-mide drugs were used as a first chemotherapeutic agentand employed for the systematic prevention and cure of

bacterial infection in human beings. Activity of the sulfo-namide drugs has been associated with their competitionwith p-aminobenzoic acid (PABA) in the synthesis offolic acid for the growth of bacteria. Therefore, sulfadrugs act by inhibiting the bacterial growth rather thandirectly affecting the bacteria [4]. Recently, it is reportedthat modern classes of sulfonamides and related sulfonylderivatives are served as an effective inhibitor for grow-ing tumor cells, or for the medication of different types ofcancer [5–7]. SMZ has been extensively used for thetreatment of bacterial infections including urinary tract,pneumocystis carinii pneumonia, chronic bronchitis, me-ningococcal meningitis, acute otitis media, Whipple�s dis-ease and toxoplasmosis as well as in the treatment of op-portunistic infection in transplantation and for AIDS re-lated infection. Nevertheless, SMZ has also been reportedto exhibit different types of side effects, like hypersensi-tivity reaction, gastro-intestinal distribution (mainlynausea and vomiting) and various hematological disor-ders such as thrombocytopenia, sulfhemoglobinemia,megaloblastosis, eosinophilia and agranulocytosis [8–12].Hence, the determination of SMZ in various biologicalsamples and pharmaceutical formulations has been con-sidered of great significance for human health and qualitycontrol.

Literature survey reveals that various techniques havebeen used for the determination of SMZ, such as spectro-photometry [13], ratio spectra derivative spectrophotome-try [14], flow injection spectrophotometry [15], gas chro-matography-mass spectrometry [16], capillary electropho-resis [17], liquid chromatography [18], high performance

Abstract : Sulfamethoxazole (SMZ), an antibacterial sul-fonamide drug, has been selectively determined usingpoly-1,5-diaminonaphthalene (p-DAN) modified glassycarbon electrode (GCE). The modified sensor was char-acterized by field emission scanning electron microscopy(FE-SEM), electrochemical impedance spectroscopy(EIS) and cyclic voltammetry (CV). SMZ showed linear

response in the concentration range of 0.5–150 mM byusing square wave voltammetry (SWV) and the detectionlimit was found to be 0.05 nM with sensitivity of0.085 mAmM�1. The proposed sensor has been successfullyemployed to determine SMZ in the pharmaceutical tab-lets and human urine samples.

Keywords: Sulfamethoxazole · 1,5-Diaminonaphthalene · Glassy carbon electrode · Square wave voltammetry

Scheme 1. (a) Sulfamethoxazole (SMZ) [N1-(5-methylisoxazol-3-yl) sulfanilamide]; (b) 1,5-DAN.

[a] H. Chasta, R. N. GoyalDepartment of Chemistry, Indian Institute of TechnologyRoorkee, Roorkee -247667 (India)*e-mail: [email protected]

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liquid chromatography (HPLC) using an on-line clean-upcolumn [19], high performance thin layer chromatogra-phy [20], liquid Chromatography-mass spectrometry (LC-MS) [21] and visible and UV spectrophotometry [22].Most of these methods were time consuming, expensiveand required tedious extraction and separation steps. Re-cently, electrochemical methods have also attracted atten-tion towards the determination of biomolecules and drugsdue to their high sensitivity and selectivity, low cost andpossibility of analysis without requirement of sample pre-treatment. Hence, electrochemical methods have alsobeen explored in the recent years for the determinationof SMZ using [5, 10,15, 20-tetrakis (3-methoxy-4-hydroxyphenyl)porphyrinato] Cu(II) modified carbon pastesensor (TMHPP Cu(II)) [23], boron-doped diamond elec-trode [24,25], multiwall carbon nanotubes-Nafion modi-fied glassy carbon electrode (MWCNTs-Nafion/GCE)[26], CeO2-chitosan-nanocomposite modified glassycarbon electrode (nano CeO2/CHIT/GC electrode) [27],multiwalled carbon nanotubes modified with antimonynanoparticles (MWCNT-SbNPs nanocomposite) [28] etc.Most of these studies were carried out to determine SMZconcentration in food products, water and milk samplesand pharmaceutical tablets. In few studies analysis ofSMZ in biological samples has been carried out by spik-ing and recovery is reported due to which these studiesare of little significance. To the best of our knowledge, noeffort has been made for the determination of SMZ inhuman urine samples using voltammetry. As urine matrixis much more complicated than other systems, due to thepresence of several metabolites such as ascorbic acid, uricacid, dopamine etc., which interfere in the determination.Hence, analysis in urine is important from the view ofroutine clinical analysis. The aim of this study is to fabri-cate an electrochemical sensor for the simple and rapiddetermination of SMZ in human urine samples as well asin pharmaceutical formulations with high selectivity andsensitivity.

In last few years, application of conducting polymers inelectrochemical sensors and biosensors has increased dueto their reproducibility, electrochemical reversibility, lowcost and good stability. Conducting polymers like polyani-line, polypyrrole, polythiophenes, polyazulenes and poly-1,5-diaminonapthalene (p-DAN), have been used to pre-pare electrochemical sensors for the monitoring of com-pounds of biological interest. These polymeric materialshave played important role in improving the sensitivityand selectivity of the sensor, and to decrease fouling ef-fects [29–33]. p-DAN is found to have versatile applica-tions in the construction of chemically modified sensorsdue to its electroactive nature in both acidic and aqueoussolutions and also bears free NH2 groups as shown by FT-IR analysis [34]. The application of p-DAN conductingpolymer has been studied at different surfaces, such asplatinum and glassy carbon, pyrolytic graphite electrodewith gold nanoparticles and glassy carbon electrode withmultiwalled carbon nanotube in the selective monitoringof compounds [35,36]. p-DAN has been considered as

a promising candidate to form polymer�drug conjugatefor drug delivery purposes and it is successfully appliedfor the determination of various drugs as earlier reportedby our group [37–39]. Here, we used only p-DAN film tofabricate the electrode due the some of the advantages,such as easy synthesis, low cost in comparison to otherconducting polymers, good adhesive property and densestructure [40–42].

In the present paper, the electropolymerization of 1,5-DAN (Scheme 1b) in the acidic medium at glassy carbonelectrode (GCE) has been carried out. The modificationhas been confirmed by various techniques including fieldemission scanning electron microscopy (FE-SEM), elec-trochemical impedance spectroscopy (EIS) and cyclic vol-tammetry (CV). The p-DAN/GCE sensor has been usedfor the determination of SMZ in biological samples. Theexperimental parameters, which affect the response of themodified GCE were optimized in terms of pH, frequency,scan rate and applied potential.

2 Experimental

2.1 Chemicals and Reagents

Sulfamethoxazole, sulfuric acid, potassium chloride, po-tassium ferricyanide and 1,5-DAN were purchased fromSigma Aldrich, USA and used without further purifica-tion. The studies were accomplished in the pH range of2.4–11 using 1.0 M phosphate buffer solution [43]. Sulfa-methoxazole containing tablets were purchased from thelocal market of Roorkee. Human urine samples of patienttreated with SMZ were collected from the hospital ofIndian Institute of Techology, Roorkee after the permis-sion of ethical clearance committee. All other solventsand reagents used in the experimental work were of ana-lytical grade. Double distilled water was used throughoutthe experiments.

2.2 Apparatus

All the electrochemical work was performed by compu-terized bio-analytical system (BAS, West Lafayette,USA) CV-50 voltammetric analyzer. The electrochemicalcell setup was equipped with p-DAN/GCE as a workingelectrode, Ag/AgCl (Saturated 1 M KCl) as a referenceand platinum wire as a counter electrode respectively.

Field emission scanning electron microscopic (FE-SEM) images were recorded by using Zeiss ultra plus 55.Galvanostat VersaSTAT-3 (PAR, USA) was used for theelectrochemical impedance spectroscopic (EIS) studies.The pH of the phosphate buffer was measured usinga Thermo Fischer, scientific Singapore digital pH meter(Eutech pH 700).

2.3 Fabrication of p-DAN Film at the Surface of GCE

GCE was polished to mirror like surface with aluminaslurry and ZnO on polishing cloth, and thoroughly



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washed with double distilled water. The p-DAN/GCEwas prepared by electropolymerization of 1 mM 1,5-DANin 0.5 M H2SO4 using cyclic voltammetry, in which the po-tential range between �0.2 to +0.9 V s�1 (v/s Ag/AgCl)was applied at the surface of the GCE for 7 cycles (scanrate: 0.1 Vs�1) as reported in the literature [44]. Afterelectropolymerization, a potential of �100 mV was ap-plied for 100 s to facilitate the removal of adsorbed ana-lyte to get a clean modified surface and, then it wascleaned well with double distilled water. The surface mor-phology of the bare GCE and p-DAN/GCE was studiedby recording FE-SEM. A comparison of the FE-SEMimages observed for bare GCE and p-DAN/GCE is pre-sented in Figure 1 and clearly indicate that p-DAN hasbeen deposited at the surface of GCE.

2.4 Experimental Procedure

The stock solution of sulfamethoxazole (1 mM) was pre-pared by dissolving required amount in minimum amountof methanol (1 mL) and volume was made up to 25 mL ina volumetric flask. For voltammetric experiments, the de-sired volume of sulfamethoxazole was added to 2 mL ofphosphate buffer of pH 7.2 (1.0 M) and the total volumewas made to 4.0 mL with double distilled water. The opti-mum instrumental conditions for square wave voltamme-try were: initial (E): 0.200 V, final (E): 1.400 V, squarewave amplitude (Esw): 25 mV, square wave frequency (f):15 Hz, potential step (E): 4 mV. Optimized cyclic voltam-metric (CV) parameters used were: initial (E): 0.200 V,switching potential (E): 1.400 V final (E): 0.200 V, scanrate (v): 50 mV s�1 and full scale (�): 10 mA. CV studieswere performed after bubbling high-purity nitrogenthrough the solutions for 12–15 min. All the potentials re-

ported are versus Ag/AgCl (3 M NaCl) at an ambienttemperature of 25�2 8C. The surface of p-DAN/GCEwas cleaned after each run by using time based techniqueby applying a constant potential (�100 mV) for 100 s inbuffer solution of pH 7.2.

3 Results and Discussions

3.1 Electrochemical Characterization of p-DAN/GCEFilm

The electrochemical properties of bare and p-DANcoated GCE surfaces, were characterized by recordingimpedance spectra in 1 :1 solution of 5 mM K3[Fe(CN)6]and 0.1 M KCl solution in the frequency range of 0.1–100KHz. Figure 2 shows the impedance spectra of bare GCEand p-DAN/GCE. A Randle�s equivalent circuit was uti-lized for the impedance data as represented in inset ofFigure 2, where Rct is parallel combination of resistanceto charge transfer and Cdl is interfacial capacity. Thevalues for Rct were obtained by fitting the experimentaldata to the Randle�s equivalent circuit. The value of Rct

for p-DAN/GCE (850 W) was lower than that for thebare GCE (1100 W). These values confirmed the conduc-tive nature of the p-DAN film. These results also revealedthat the p-DAN film successfully adhered to the surfaceof electrode.

3.2 Cyclic Voltammetry

The cyclic voltammograms for 40 mM SMZ were recordedusing bare GCE and p-DAN/GCE in the phosphatebuffer solution (1 M) at pH 7.2 as represented in Figure 3.A well-defined oxidation peak at ~980 mV was observed

Fig. 1. Typical FE-SEM images of (A) p-DAN/GCE (B) bare GCE.



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for the oxidation of SMZ at bare GCE, which shifted toless positive potential by ~50 mV, with a marked en-hancement in peak current, at p-DAN/GCE. These re-sults clearly indicated that p-DAN film efficiently en-hanced the kinetics of the electrochemical oxidation ofSMZ. In the reverse scan, no peak was observed, whichclearly suggested that the oxidation of SMZ is irreversiblein nature at p-DAN/GCE. To ascertain the nature of theelectrode reaction, sweep rate studies were performed inthe range 10–300 mVs�1. The peak current of SMZ wasfound to increase with increasing sweep rates at p-DAN/GCE. The plots of ip/v

1/2 versus log v for SMZ was linearand clearly indicated that the electrode process of SMZ isadsorption controlled at p-DAN/GCE [45,46].

Electrochemical response of bare GCE and p-DAN/GCE was also examined by recording the cyclic voltam-mograms in the solution of 1 mM K3[Fe(CN)6] and 0.1 M

KCl to determine the effective surface area of the sen-sors. The enhancement in peak current and negative shiftin peak potential was found at p-DAN/GCE (Fig. 4) incomparison to bare GCE. The surface area was calculatedfrom the slopes of ip versus v1/2 plots using Randles-Sevcik equation and found as 0.039 and 0.124 cm2 forbare GCE and p-DAN/GCE, respectively. Thus, the effec-tive surface area of modified sensor was nearly threetimes larger than bare GCE.

3.3 Square Wave Voltammetry

Initially square wave voltammograms were recorded forthe 40 mM SMZ at bare GCE and p-DAN/GCE in phos-phate buffer solution of pH 7.2 under optimal SWV pa-rameters. A broad peak is obtained at the bare GCEhaving peak potential ~910 mV (curve a), whereas, at p-DAN/GCE the peak potential is shifted to ~850 mV(curve b), with significant enhancement in the peak cur-rent (Figure 5). The shift in peak potential to less positivepotential and enhancement in peak current indicate thatthe p-DAN film exhibits efficient electrocatalysis towardsoxidation of SMZ. Hence, p-DAN/GCE sensor has beenemployed for further detailed studies of SMZ determina-tion using SWV.

3.4 Effect of pH and Square Wave Frequency

The electrochemical behavior of SMZ at different squarewave frequencies and pH values was studied using p-DAN/GCE. The electrochemical oxidation of SMZ wasstudied in the phosphate buffer solutions ranging between2.4–11.0. The pH of the solution strongly influenced theoxidation peak potential (Ep) of SMZ and the Ep shiftedtowards less positive potential values with increase in pH

Fig. 2. Electrochemical impedance spectra at (A) p-DAN/GCE(B) bare GCE. The inset shows Randle�s equivalent circuit.

Fig. 3. Observed cyclic voltammogram for 40 mM SMZ at scanrate of 50 mV s�1 at p-DAN/GCE (—) and bare GCE (····) atpH 7.2.

Fig. 4. Comparative cyclic voltammograms of 1 mMK3[Fe(CN)6] in 0.1 M KCl using (a) bare GCE and (b) p-DAN/GCE at scan rate of 100 mVs�1.



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of solution. The linear relationship between Ep and pHcan be presented by the equation:

Ep ðmVÞ ¼ 1272�58:10 pH ð1Þ

having a correlation coefficient of 0.982. The slope valueof dEp/dpH of (58.10 mV/pH) indicates that the numberof protons and electrons involved in the oxidation isequal.

The influence of square wave frequency (f) on the oxi-dation peak current (ip) was examined at pH 7.2 using p-DAN/GCE. It was observed that the peak current for theelectrooxidation of 40 mM SMZ was increased linearlywith increase in square wave frequency in the range 5–200 Hz. It is suggested that adsorption of SMZ occurredat the surface of p-DAN/GCE, which also confirmed theinference obtained from cyclic voltammetry. The varia-tion of ip with f can be expressed by the equation:

ip ðmAÞ ¼ 0:190 f ðHzÞ þ 0:681 ð2Þ

having a correlation coefficient of 0.989.

3.5 Concentration Study

To analyze the effect of SMZ concentration on the oxida-tion peak current, square wave voltammograms were re-corded in the concentration range 0.05 mM to 150 mMusing p-DAN/GCE at pH 7.2 under optimized parametersof square wave voltammetry. Systematic increase in thepeak current was observed with increase in concentrationof SMZ as represented in Figure 6. It was observed thatwhen the concentration of SMZ increased the peak cur-rent linearly increased at p-DAN/GCE sensor. The ob-served linear calibration curve between peak current (ip)and concentration of SMZ at p-DAN/GCE sensor is illus-trated in Figure 7. Linear dependence of SMZ (after sub-

tracting background current) can be represented by theequation:

ip ðmAÞ ¼ 0:085 C ðmMÞ þ 1:050 ð3Þ

having correlation coefficient 0.995. The limit of detectionfor SMZ is calculated by using the relation 3s/b, wheres is the standard deviation of blank and b is slope of thecalibration curve and found to be 0.05 nM at p-DAN/GCE sensor. The limit of detection for SMZ at bareGCE is found to be 0.1 mM, Thus, it can be seen that elec-tropolymerization by p-DAN significantly lowered the de-tection limit as compared to the bare GCE. For any ana-lytical technique, it is necessary to validate the methodusing validation characteristics such as linearity, range, ac-

Fig. 5. Comparison of square wave voltammograms of 40 mMSMZ at pH 7.2 (i) curve a represents bare GCE, (ii) curve b rep-resents p-DAN/GCE and (iii) (- - -) shows (background) phos-phate buffer solution at p-DAN/GCE.

Fig. 6. Observed square wave voltammograms for (i) blankphosphate buffer solution (background) (····) and (ii) increasingconcentration of SMZ at (a) 0.5; (b) 5; (c) 10; (d) 20; (e) 40; (f)80 and (g) 150 mM using p-DAN/GCE in phosphate buffer solu-tion of pH 7.2.

Fig. 7. Calibration plot observed for SMZ using p-DAN/GCEat pH 7.2.



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curacy, precision, limit of detection and quantification.The typical validation parameters of present method areshown in Table 1.

In order to prove the accuracy and precision of the pur-posed method, analysis of varying concentration (low,medium and high concentrations) was done for threedays (n=3/day for within day and n=5/day for betweenday). The results of precision are expressed as relativestandard deviation and the accuracies are expressed asthe Bias %. The results of precision and accuracy param-eters are described in Table 2. The RSD obtained was inthe range of 0.18–3.03 % and the Bias % was in the rangeof 0.34–1.92% for varying concentrations, which indicategood accuracy and precision of the method [47–50].

3.6 Effect of Interferents

The effect of common interfering molecules, such as as-corbic acid, uric acid, xanthine present in urine and trime-thoprim (commonly given in combination with SMZ) hasbeen analyzed on the determination of SMZ. These mole-cules can alter the electrochemical sensitivity of p-DAN/GCE sensor for SMZ determination by influencing itspeak potential and peak current response. Interferencestudy was carried out by measuring the voltammetricpeak current response for fixed concentration of SMZ(5 mM) and varying the amount of interfering moleculesup to 100 fold excess at pH 7.2. In voltammograms, peakpotential of SMZ was obtained at +850 mV and other ad-ditional peaks were obtained at +100, +340, +720 and+1140 mV corresponding to the oxidation of ascorbicacid, uric acid, xanthine and trimethoprim respectively asshown in Figure 8. It was found that there is no change inthe peak current as well as in the peak potential for SMZoxidation up to 100 fold excess of the interfering mole-cules. These results clearly indicate the specificity of p-

DAN/GCE sensor towards the electrochemical oxidationof SMZ. Hence, p-DAN/GCE can be utilized for the de-termination of SMZ in biological samples and medicines.

3.7 Analytical Applicability

3.7.1 Determination of SMZ Content in PharmaceuticalFormulations

In order to assess the utility of p-DAN/GCE sensor inpharmaceutical industries, different commercially avail-able pharmaceutical samples containing SMZ, like Co-tri-moxazole tablet double strength (Cadila, industrialGrowth centre, Samba, State of J &K) and Septran(GlaxoSmithKline, D-5, M. I.D. C. Area, Paithan, Maha-rashtra) were acquired from the local market of Roorkee.The tablets were powdered and then dissolved in mini-mum amount of methanol and volume was made up to25 mL with double distilled water. The samples were sub-sequently diluted with phosphate buffer solution upto theworking range of SMZ. Applying the identical conditions,which were used in the concentration study, the concen-tration of SMZ in the various pharmaceutical sampleswas ascertained by recording square wave voltammo-grams at p-DAN/GCE sensor. The obtained results aresummarized in Table 3. It can be seen that the lablledvalues of SMZ on tablets were in good agreement withthe results obtained using p-DAN/GCE sensor and hencesuggested the good accuracy of the proposed sensor.

To check the validity of purposed method, results werecompared with reported HPLC method using student t-test and F-test [3]. At the 95% confidence level, calculat-ed t-value and F-value were less than the tabulatedvalues, indicating that there were no significant differen-ces between the data obtained using the two methods.The results are shown in Table 4. Hence, these resultsshow the excellent performance of the electroanalytical

Table 1. The validation characteristics for the determination ofSMZ using P-DAN/GCE sensor.

Validation Parameters Values

Concentration range (mM) 0.5–150Correlation coefficient (R2) 0.995Detection limit (nM) 0.05Limit of quantification (nM) 0.16Sensitivity (mA/mM) 0.085Standard error of slope (a, 0.05) �0.0054Standard error of intercept (a, 0.05) �0.3407

Table 2. Precision and accuracy data, within day and betweendays for the determination of different concentrations of SMZ atp-DAN/GCE sensor.

SMZ (mM) Within day (n=3) Between days (n=5)RSD (%) Bias (%) RSD (%) Bias (%)

0.5 3.03 �1 2.07 0.7250 1.53 �0.34 2.59 �1.92150 0.67 �0.47 0.18 �0.25

Fig. 8. Square wave voltammograms showing interferences of(A) ascorbic acid, (B) uric acid, (C) xanthine and (E) trimetho-prim at fixed concentration of SMZ (D) (5 mM).



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method using the p-DAN/GCE sensor when compared tothe HPLC method.

3.7.2 Determination of SMZ in Human Urine

In order to evaluate the applicability of the proposedsensor in human urine samples, determination of SMZwas carried out in urine of patients undergoing treatmentwith SMZ. The urine samples of patients were obtainedfrom the hospital of Indian Institute of Technology Roor-kee. The samples were collected after 6 h of oral adminis-tration of 800 mg Co-trimoxazole tablet. Prior to theanalysis, the samples were diluted three times with phos-phate buffer solution (pH 7.2) to minimize matrix com-plexity and square wave voltammograms were recorded.A well-defined anodic peak (Ep�850 mV vs. Ag/AgCl) atp-DAN/GCE sensor was obtained corresponding to theoxidation of SMZ. The other voltammetric peaks at~340 mV and ~1140 mV in the voltammogram are due tothe oxidation of uric acid and trimethoprim respectively,which do not affect the determination of SMZ in urinesamples. Ascorbic acid, which remains present in theurine samples, did not interfere in the determination ofSMZ, as it oxidized at ~100 V. The SMZ oxidation peakat ~850 mV is further confirmed by the spiking of urinesamples with the known amount of SMZ. It was observedthat the oxidation peak current of SMZ increased onspiking with SMZ, confirming thereby that it correspond-ed to the oxidation of SMZ, while peak currents due touric acid and trimethoprim remained unchanged. The re-sults of SMZ obtained in different urine samples aretabulated in Table 5. The SMZ concentration in urinesamples of patients was calculated by preparing standard

addition plot as demonstrated in Figure 9 and was foundto be 3.2 mM.

3.8 Stability and Reproducibility

To determine the reproducibility of p-DAN/GCE sensor,successive square wave voltammetric measurements of20 mM SMZ were recorded at pH 7.2 daily for 15 days.The modified electrode was used daily and stored in air.Marginal decrease of current sensitivity with a relativestandard deviation of about 3.52% was observed, whichrepresented the excellent stability of the p-DAN/GCE.The results of ten repetitive measurements during intra-day studies showed a relative standard deviation (RSD)

Table 3. Determination of SMZ in tablets using p-DAN/GCE.

Tablet Observed amount (mg/tablet) Observed amount [a] (mg/tablet) RSD (%) Bias (%)

Septran 400.0 395.10 0.92 �1.23Co-trimoxazole 800.0 780.54 1.51 �2.43

[a] The RSD for the determination was for n=3.

Table 4. Validation of data by comparing with reported HPLC method.

Labeled amount (mg) Found amount (mg) [a] t-test [b] F-test [c]Proposed method Published method

400 395.1�3.65 409�9 2.33 6.08

[a] Mean�SD, n=3. [b] Tabulated t-value at P (0.05) is 2.78. [c] Tabulated F-value at P (0.05) is 19.00.

Table 5. Recovery analysis of SMZ in urine sample of patient at p-DAN GCE.

Spiked (mM) Observed (mM) Actual [a] (mM) Recovery (%) RSD [b] (%) Bias (%)

0 3.20 3.20 – – –10 13.23 3.23 100.93 2.43 0.9320 23.05 3.05 95.31 3.46 �4.6940 43.15 3.15 98.43 1.54 �1.56

[a] The actual amount is observed – spiked amount. [b] RSD value for the determination was for n=3.

Fig. 9. The observed linear calibration curve between peak cur-rent and spiked SMZ concentration at p-DAN/GCE in urinesample.



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of 1.96 % in current response of SMZ, which confirmedthe excellent reproducibility of the developed sensor.Hence, the p-DAN/GCE exhibits efficient stability andreproducibility for the determination of SMZ.

4 Conclusions

In the present work, we described a method for the nano-molar concentration determination of SMZ in humanurine samples as well as in pharmaceutical preparationsemploying a p-DAN/GCE sensor. A well-defined peakfor the oxidation of SMZ appeared at ~850 mV at p-DAN coated GCE surface. Further, p-DAN film in-creased the electrocatalytic activity due to its high specificsurface area in comparison to the bare GCE surface. Theperformance of p-DAN/GCE sensor towards SMZ deter-mination is evaluated by a comparison of the detectionlimit and calibration range reported in the last few yearsas shown in Table 6. It can be concluded that the detec-tion limit at p-DAN/GCE is superior than papers report-ed in recent years. The developed sensor also demonstrat-ed the efficient ability to quantify SMZ concentration invarious pharmaceutical formulations with reliable accura-cy. The modified sensor has also been employed for thesuccessful analysis of SMZ in urine samples of patientsundergoing treatment with SMZ and good selectivity andsensitivity is observed.

Acknowledgement

One of the authors Himanshu Chasta (HC) is thankful tothe Council of Scientific and Industrial Research, NewDelhi for the award of Senior Research Fellowship.

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Received: November 20, 2014Accepted: December 15, 2014

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A Simple and Sensitive Poly-1,5-Diaminonaphthalene ModifiedSensor for the Determination ofSulfamethoxazole in BiologicalSamples



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