Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 DI 10.1186/s40543-015-0080-3 RESEARC ARTICLE pen Access Electrochemicl behvior of nticncer drug 5-fluorourcil t crbon pste electrode nd its nlyticl ppliction Shikndr D. Bukkitgr nd grj P Shetti * Abstrct Bckground: A set of pyrimidine nucleobse present in ll living systems s component of nucleic cid constitutes urcil together with thymine nd cytosine. A diverse physiologicl ctivity is exhibited by mny -substituted urcil derivtives. In oncology, 5-U is widely used s n importnt nticncer drug. Methods: Electrochemicl behvior ws studied using cyclic voltmmetric method, nd the nlyticl ppliction ws studied using differentil pulse voltmmetric method. Solution p hs been mesured by p meter. Results: The process on the surfce of electrode ws found to be irreversible nd diffusion controlled. The chrge trnsfer coefficient, heterogeneous rte constnt, nd the number of electron trnsferred were clculted. Possible rection mechnism tking plce on the surfce of electrode ws proposed. Clibrtion plot constructed using differentil pulse voltmmetric technique ws used for quntittive nlysis in phrmceuticl nd humn urine smple. Limit of detection (LD) nd limit of quntifiction (LQ) were clcultedtobe12.25nd40.8nm,respectively. Conclusions: Inthepresentwork,wedescribedtheelectrochemiclbehvior of nticncer drug nd its determintion in humn urine nd phrmceuticl smples. The method shows the development of sensor for selective nd sensitive determintion of 5-U. Keywords: Electro-oxidtion, Crbon pste electrode, 5-fluorourcil, Voltmmetry, Phrmceuticl smples Bckground Ribonucleic cid consists of pyrimidine bse clled urcil which forms bse pir with denine. or biosynthesis of nucleic cid in tumors, urcil is preferentilly used (Rutmn R.J. et l. 1954). A drstic chnge in the biologicl properties of urcil resulted from the substitution of hydrogen tom t fifth position by hlogen tom (Voet D. nd Voet J.G. 1995). Amongst the vriety of urcil derivtives reported s ntitumor nd ntivirl gent, 5-U hs cquired position of prticulr importnce. or the tretment of solid tumor of the brest nd rectum, 5-U hs been used extensively s n ntineoplstic gent (eidlberg C. nd Ansfield.J. 1963). ne of the mjor mechnisms responsible for ntitumor ctivity of 5-U is by inhibition of thymidylte synthesis (rtmnn K.U. nd eidelberger C. 1961). Detiled studies hve pointed to 5-U interference * Correspondence: npshetti@kleit.c.in Deprtment of Chemistry, K.L.E Institute of Technology, Gokul ubli-580030, Krntk, Indi with DA nd protein synthesis, becuse of conversion to the corresponding ribose nucleoside nd substitution into RA, s n eqully importnt mechnism of toxicity (Myers C.E. 1981). The studies on oxidtion reduction behvior of compounds of biologicl significnce is of considerble vlue, s they provide deep insight into the biologicl relevnt redox rections of these compounds. Although the ctul biologicl redox rections my be of more complexity due to enzymtic interctions, much more informtion cn be derived from the study of these compounds in queous solution of known p. Electrochemicl methods hve proved to be sensitive for the determintion of orgnic molecules, including drugs nd relted molecules in phrmceuticl dosge forms nd biologicl fluids nd their oxidizble property (Pdmini V. 2010; egde R. et l. 2008). Crbon electrodes, especilly pste electrodes, re widely used in the electrochemicl investigtions becuse of their low bckground current, wide potentil windows, chemicl 2016 Bukkitgr nd Shetti. pen Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License (http://cretivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde.
Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 Pge 2 of 9 inertness, low cost, nd suitbility for detection of vrious orgnic nd biologicl compounds (Genxi L. nd Peng M. 2013). Mny dvntges such s very low bckground current, low cost, lrge potentil window, simple surfce renewl process, nd esiness of minituriztion of crbon pste electrode (CPE) re widely pplicble in both electrochemicl studies nd electronlysis. In ddition, esy fbriction of the electrode cn be chieved by incorporting different substnces during pste preprtion which results in the so-clled modified electrode with desired composition nd predetermined properties (Khoobi A. et l. 2013; Mokhtri A. et l. 2012; Díz C. et l. 2013; Gholivnd M.B. nd Mohmmdi-Behzd L. 2014; Mzloum- Ardkni M. et l. 2010; Roof J.B et l. 2007; Dönmez S. et l. 2014). According to the literture, no works re reported on voltmmetric method for the determintion of 5-U t CPE. The focus behind this work is to develop suitble experimentl condition to investigte the electrochemicl behvior of 5-U nd its determintion in phrmceuticl smple nd humn urine smple. As compred to the other reported works, the sticking feture for this is, no prior extrction step is needed in urine nlysis. In the present work, the electro orgnic rections t the surfce of electrodes nd results of one more such study re presented. ere, we preferred to use bre CPE becuse of the strong dsorption of nitrogen tom on such mteril (Scheme 1). Methods Apprtus nd chemicls Electrochemicl nlyzer (CI Compny, D630, USA) ws used to study the electrochemicl deeds of the drug under investigtion t n mbient temperture of 25 ± 0.1 C. A three-electrode system consisting of crbon pste electrode s working electrode, pltinum wire s counter electrode, nd Ag/AgCl (3 M KCl) s reference electrode were used in 10-ml single comprtment. In ll the mesurements, bckground subtrction ws mde. The phosphte buffer solutions rnging 3.0 11.2 p (I = 0.2) were prepred ccording to literture (Christin G.D. nd Purdy W.C. 1962), nd p of the solutions ws mesured by p meter (Elico Ltd., LI120, Indi). 5-U (Sigm-Aldrich, USA) ws used to prepre 1.0 mm stock solution in double distilled wter (6.5 10 6 Ω). Double distilled wter nd nlyticl grde chemicls nd regents without further purifiction were used throughout the experiments. Preprtion of electrode The CPE ws prepred by mixing 1.0 g of grphite powder nd 0.5 ml of prffin oil in smll gte mortr, nd this mixture ws then homogenized. A portion of the resulting pste ws pcked firmly into cvity of polytetrfluoroethylene tube (PTE). The surfce of the electrode ws smoothed ginst weighing pper nd rinsed with wter. The pste ws crefully removed prior to pressing new portion in to the electrode fter every mesurement. The resulting electrode ws noted s CPE. Prior to use, the CPE ws ctivted in phosphte buffer solution of p 7 by cyclic voltmmetric sweeps between 0.4 nd 1.4 V with scn rte 50 mvs 1 - (Mlode S.J. et l. 2013). Rndles-Sevcik formul cn be used to clculte the electro-ctive re of the electrode using cyclic voltmmetric technique nd K 3 e (C) 6 1.0 mm s probe t different scn rtes in 0.1 M KCl s supporting electrolyte. At T = 298 K nd for reversible process, the eqution is s follows (Mlode S.J. et l. 2012): Ip ¼ 2:69 10 5 n 3=2 A 0 D 1=2 R υ 1=2 C 0 ð1þ Scheme 1 Chemicl structure of 5-U In Eq. (1), for 1.0 mm K 3 e (C) 6 nd 0.1 M KCl s supporting electrolyte, Ip refers to the nodic pek current, n is the number of electron trnsferred during the electrode rection equl to 1. A 0 is the surfce re of the electrode, D R is the diffusion coefficient equl to 7.6 10 6 cm 2 s 1, υ is the scn rte, nd C 0 is the concentrtion of K 3 e (C) 6. rom the slope of the plot of Ip vs. υ 1/2, the re of the electrode surfce ws clculted to be 36 ± 014 cm 2.
Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 Pge 3 of 9 Smple preprtion To crry out the phrmceuticl nlysis, 5-U tblets were grounded using mortr nd frction corresponding to stock solution of 1 mm ws weighed nd completed to the volume with double distilled wter in 100-ml clibrted flsk. After soniction for 10 min, to ffect complete dissolution suitble liquots of the cler superntnt, liquid ws tken nd diluted with buffer solution of p 7. The oxidtion pek current of 5-U ws mesured using differentil pulse voltmmetric technique. Stndrd ddition method ws to study the ccurcy of the projected method nd the interference from excipients used in dose forms. Results nd discussion Electrochemicl behvior of 5-U In order to understnd the electrochemicl behvior of 5-U, cyclic voltmmetric technique ws used. Between the rnge 3.0 11.2 p, one well-defined irreversible oxidtion pek ws observed. In ig. 1, voltmmetric behvior of 5-U in phosphte buffer solution (p = 7, I = 0.2) is represented, curve () corresponds to buffer solution nd (b) nodic pek corresponding to 5-U oxidtion. There ws no pek observed on the reverse scn, therefore electrode process is supposed to be irreversible. Since successive cyclic voltmmogrm showed decrese in the pek current due to dsorption of 5-U or its oxidtion product, the oxidtion pek corresponding to the first sweep ws only recorded. Influence of ccumultion time Pek current cn be gretly ffected by the dsorption of nlyte on the surfce of the electrode. It is importnt to fix the ccumultion time to improve the sensitivity of the electrode towrds the nlyte. pen circuit ccumultion with cyclic voltmmetric technique ws use to study the effect of ccumultion time on pek current (ig 2). The effect of ccumultion time ws studied in the rnge of 0 150 s. ne hundred twenty seconds ws employed s optiml ccumultion time in further experiments since mximum pek current ws observed t 120 s. Effect of supporting electrolyte Electrochemicl behvior of the nlyte under investigtion strongly depends on the p of the solution. By optimizing the p conditions, shrper response ccompnied with higher sensitivity cn be obtined. ence, phosphte buffer solution over the p rnge 3.0 11.2 ws used to study the electrochemicl behvior of 5-U (ig 3). It ws observed tht the pek potentil shifted to less positive vlues with increse in the p of the buffer solution (ig. 3(A)). The liner reltionship between Ep nd p cn be expressed s follows: Ep ðv Þ ¼ 0:0595 p ðv Þ þ 1:5652 : R 2 ¼ 0:9952 rom the plot of Ip vs. p (ig. 3(B)), it is cler tht the best result with respect to sensitivity ccompnied -24.0-21.0-18.0 b -15.0-12.0-9.0-6.0-3.0 3.0 0.8 0.9 1.0 1.1 1.2 1.3 Potentil / V (Vs. Ag/AgCl, 3M KCl) ig. 1 Cyclic voltmmogrm behvior of 5-U, phosphte buffer (p = 7, I =0.2M),() blnk crbon pste electrode, nd (b) crbon pste electrode 1.0 10 4 M; 5-U scn rte: 50 mvs 1 ; ccumultion time: 120 s (t open circuit)
Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 Pge 4 of 9 Ip/µA 7.2 7.0 6.8 6.6 6.4 6.2 6.0 0 50 100 150 200 t/s ig. 2 Vrition of the cyclic voltmmetric nodic pek current with ccumultion time with shrper response ws obtined with p = 7.0, hence, it ws selected for further work (egde R.. et l. 2009). The pek current depends on the deprotontion nd protontion form of the electro-ctive species in electrochemicl cell. At p 7, protonted nd deprotonted form of 5-U domintes. And grdul chnge of specition of 5-U occurs in the p rnge 7 to 9. urther, the mgnitude of current is directly proportionl to the rte of the electrochemicl rection. ence, it is pprent to conclude tht the oxidtion of 5-U is very high t p 7 (Ion P. et l. 2005). Influence of scn rte Reltionship between pek current nd scn rte gives constructive informtion bout electrochemicl mechnism. At different scn rtes, the electrochemicl behvior of 5-U ws studied by using cyclic voltmmetric technique (ig. 4). The dependence of the pek intensity Ip (μa) upon the scn rte υ (Vs 1 ) (ig. 4(A)) ws crried out to ssess whether the -12.0-1 -8.0-6.0-4.0 Ip/µA Ep/V 1.3 1.1 0.9 0.7 2 4 6 8 10 12 1 p 8.0 6.0 4.0 2 4 6 8 10 12 p h i A B g f e d c b -2.0 2.0 i 0.8 0.9 1.0 1.1 1.2 1.3 Potentil / V (Vs. Ag/AgCl, 3M KCl) ig. 3 Influence of p on the shpe of nodic pek. p () 3.0, (b) 4.2, (c) 5.0, (d) 6.0, (e) 7.0, (f) 8.0, (g) 9.0, (h) 1, nd (i) 11.2. (A) Vrition of pek potentil with p for 1.0 10 4 M 5-U (B) Vrition of pek current with p for 1.0 10 4 M 5-U 1.4
Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 Pge 5 of 9-21.0-18.0-15.0 2.0 1.5 1.0 0.5 A 0 0.1 0.2 0.3 0.4 / Vs -1 1.2 1.2 C 1.2 1.1 1.1 1.1-2.5-1.5-0.5 0.5 log /Vs -1 Ep (V) s -12.0-9.0-6.0 log Ip(µA) 0.5 B -0.5-1.0-2.5-2 -1.5-1 -0.5 0 log /Vs -1 s -3.0 3.0 0.8 0.9 1.0 1.1 1.2 1.3 Potentil / V (Vs. Ag/AgCl, 3M KCl) ig. 4 Cyclic voltmmogrms of 1.0 10 4 M 5-U in 0.2 M buffer solution t p 7.0 t scn rtes of () blnk, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5, (g) 6, (h) 7, (i) 8, (j) 0.10, (k) 0.12, (l) 0.14, (m) 0.16, (n) 0.18, (o) 0.20, (p) 0.24, (q) 0.28, (r) 0.32, nd (s) 0.36Vs 1.(A) Dependence of pek current on the scn rte (y = 3.9616x +0.2793:R 2 =0.9884).(B) Dependence of logrithm of pek current on logrithm of scn rte (y =0.571x + 0.4405: R 2 =0.9939).(C) Reltionship between pek potentil nd logrithm of scn rte (y =457x +1.1984:R 2 =0.9877) process on crbon pste electrode ws under diffusion or dsorption-controlled. A liner reltionship which is of typicl diffusion controlled process ws observed for the influence of squre root of scn rte on pek potentil, nd the eqution cn be expressed s follows: Ip ðμaþ ¼ 28433 υ 1=2 mvs 1 0:1465 : R 2 ¼ 0:9877 Stright line with slope of 0.57 (ig. 4(B)) closer to the theoreticl vlue of 0.5 for purely diffusion controlled process ws obtined for the plot, logrithm of nodic pek current vs. logrithm of scn rte (Mlode S.J (b) et l. 2012), corresponding to the following eqution: log Ip ðμaþ ¼ 571:7 log υ mvs 1 þ 0:4405 : R 2 ¼ 0:9939 And lso, the pek potentil shifted to more positive vlues on incresing the scn rte, which confirms the irreversibility of the oxidtion process (Brown E.R. nd Lrge R.. 1964), nd liner reltionship between pek potentil nd logrithm of scn rte (ig. 4(C)) cn be expressed by the following eqution: EpðV Þ ¼ 45:7 log υ mvs 1 þ 1:1984 : R 2 ¼ 0:9877 or n irreversible electrode process, ccording to Lviron (Lviron E. 1979), Ep is defined by the following eqution; Ep ¼ E 0 þ 2:303RT αn log RTk0 þ αn 2:303RT αn logυ Where α is the trnsfer coefficient, k 0 is the stndrd heterogeneous rte constnt of the rection, n is the number of electron trnsferred, υ is the scn rte, nd E 0 is redox potentil. rom the slope of E p versus log υ, vlueofαn cn be clculted. Tking T =298 K, R =8.314 JK 1 mol -1,nd =96,480 C mol -1,thevlueofαn ws clculted to be
Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 Pge 6 of 9 1.2. According to Brd nd ulkner, (Allen J. B. nd Lrry R.. 2004) α cn be clculted s α ¼ 47:7 E p E p=2 mv Where E p/2 is the potentil where the current is t hlf the pek vlue. rom the bove eqution, vlue of α ws to be 0.56. The number of electrons trnsferred in electrode oxidtion ws clculted to be 2.3 2. ence, 5-U my be ssumed to undergo two protons nd two electron trnsfer in the electrode rection. If the vlue of E 0 is known, the vlue of k 0 cn be determined from the intercept of the bove plot. rom the intercept of Ep versus υ curve by extrpolting to the verticl xis, t υ =0,the vlueofe 0 cn be clculted from Eq. 2 (Shetti.P. et l. 2012). rom the intercept of Ep versus log υ which ws found to be 1.199, E 0 nd k 0 were clculted to be 1.13 nd 1.7 10 3 s 1, respectively. Rection mechnism Electrochemicl oxidtion of 5-U proceeds with the removl of two electrons nd two protons. In the first step, removl of π-electrons from the ring to give species (1) tkes plce. As the species (1) is highly unstble, it is redily ttcked by ions to give species (2). urther removl of one proton nd one electron gives the finl product. The oxidtion of C=C depends on the electron density vilble t π-bond. The introduction of electron withdrwing group t the fifth position my exert pull on the electrons of π-bond due to inductive effect nd therefore the electron density my decrese. owever, the mesomeric effect of hlogen tom plys n importnt role, nd electron density increses t π-bond nd hence oxidtion becomes esy. Scheme 2 Concentrtion vrition Since differentil pulse voltmmetry technique gve shrper nd well-defined peks even t low concentrtion of 5-U, it ws preferred to develop voltmmetric technique for determintion of 5-U. The phosphte buffer solution of p = 7.0 ws selected s the supporting electrolyte for the quntittive determintion of 5-U. Differentil pulse voltmmogrms obtined with incresing mounts of 5-U s shown in ig. 5. In the rnge of 1 10 7 4 10 5 M liner clibrtion curves were obtined for 5-U. The liner eqution ws -e - 2 - + 5- U (1) (2) - + -e - 6-oxo 5- u Scheme 2 Possible electrode rection mechnism of 5-U
Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 Pge 7 of 9-4.0-3.5-3.0 5.0 4.0 3.0 2.0 1.0 t -2.5-2.0-1.5 t 0 1 2 Concentrtion -1.0-0.5 1.0 1.1 1.2 Potentil / V (Vs. Ag/AgCl) 1.3 1.4 ig. 5 Differentil pulse voltmogrmms of 5-U t crbon pste electrode t different concentrtions () blnk, (b) 3,(c) 5,(d) 10, (e) 15, (f) 20, (g) 25, (h) 30, (i) 35, (j) 40, (k) 50, (l) 60, (m) 70, (n) 80, (o) 90, (p) 100, (q) 120, (r) 150, (s) 170, nd (t) 200 μm. (A) Plot of current vs. concentrtion of 5-U Ip= μa ¼ 1:5301 c þ 0:7042 : R 2 ¼ 0:9974 The dsorption of 5-U or its oxidtion product on the electrode surfce devites the linerity for more concentrted solution. ive different determintions were used to develop the sttisticl dt relted to clibrtion curve (Tble 1). Limit of detection (LD) nd limit of quntifiction (LQ) were clculted to be 12.25 nd 40.8 nm, using following eqution (Shetti.P. et l. 2009) Tble 1 Chrcteristics of 5-fluorourcil clibrtion plot using differentil pulse voltmmetry t crbon pste electrode Linerity rnge (M) 1 10 7 to 4 10 5 M Slope of the clibrtion plot (μa M 1 ) 1.530 Intercept (μa) 0.704 Correltion coefficient (r) 0.997 RSD of slope (%) 0.88 RSD of intercept (%) 0.87 umber of dt points 19 LD (M) 12.25 nm LQ (M) 40.8 nm Repetbility (RSD %) 1.6 Reproducibility (RSD %) 2.7 LD ¼ 3S = m ; LQ ¼ 10S = m S is the stndrd devition of the pek currents nd m is the slope of clibrtion curve. The detection limits reported t different methods for 5-U re tbulted in the Tble 2. This method ws better s compred to other reported methods (u X. et l 2013; Strddi S. R. nd ndibewoor S. T. 2011; Bde I. et l. 2002; rs K.A et l. 2009; Tinrong Z. et l. 2011). Repetbility of the electrode renewed every time ws studied for every Tble 2 Comprison of detection limits of 5-fluorourcil by different reported methods Method LD (nm) Reference Glssy crbon electrode modified with bromothymol blue nd multi-wlled crbon nno-tube 267 u X. et l 2013 Glssy crbon electrode medited by surfctnt cetyltrimethyl mmonium bromide 20.1 Strddi S. R. nd ndibewoor S. T 2011 Spectrometry 30.69 Bde I. et l. 2002 PLC 70.89 rs K.A. et l. 2009 Ionic liquid modified crbon pste 13 Tinrong Z. et l. 2011 electrode Crbon pste electrode 12.25 Present work
Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 Pge 8 of 9 Tble 3 Anlysis of 5-fluorourcil in tblets by differentil pulse voltmmetry nd recovery studies Lbeled clim 100 Amount found (mg) 96.72 RSD (%) 3.7 t test of significnt 0.43 test of significnt 0.96 Bis (%) 2.3 Added (mg) 1 ound (mg) 0.97 Recovered (%) 97 RSD (%) 1.6 Bis (%) 3.0 Averge of five determintions severl hours within dy, nd percentge RSD hs been clculted to be 1.6 %. As to the reproducibility between dys, it ws similr to tht of within dy repetbility if, the temperture ws kept lmost unchnged. The reproducibility RSD hs been clculted to be 2.7 %. Tblet nlysis nd recovery test Commercilly vilble tblets were used for the nlysis of 5-U recovery test ws crried using stndrd ddition method. Grounding the tblets to powder nd dissolving it in distilled wter, the concentrtion of 5-U tblet ws prepred in such wy tht they fll in the rnge of clibrtion plot. The proposed procedure ws pplied to the nlysis of 5-U in commercilly vilble tblets. Stndrd ddition method ws used, nd the recovery studies were performed. Identicl conditions were employed for the nlysis of the tblet s used for plotting clibrtion plot. Differentil pulse voltmmetry technique ws used, nd results obtined were hving good greement with the content mrked in the lbel. In different smple, the recoveries lie between 93.9 nd 99.3 % with RSD of 2.1 % (Tble 3). Tble 4 Influence of potentil interferents on the voltmmetric response of 1.0 10 4 M 5-fluorourcil Interferent Concentrtion Signl chnge (%) xlic cid 0.1 0.87 Citric cid 0.1 2.66 Lctose 0.1 1.70 Sucrose 0.1 0.85 Dextrose 0.1 1.75 Glucose 0.1 2.47 Gum cci 0.1 3.41 Strch 0.1 0.95 Tble 5 Appliction of differentil pulse for the determintion of 5-fluorourcil in spiked humn urine Urine spiked (10 5 M) Detected (10 5 M) Recovery (%) RSD (%) Urine smple 1 0.1 98.5 2.9 Urine smple 2 0.2 92.8 3.1 Urine smple 3 0.5 99.5 2.9 Urine smple 4 0.8 99.9 2.9 Urine smple 5 1.0 96.6 3.0 Averge of five determintions Effect of interferents To evlute the effect of interferents, 0.1 mm 5-U ws used. The Tble 4 shows tht 100-fold of citric cid, gum cci, oxlic cid, sucrose, nd ure did not interfere with the voltmmetric signl of 5-U. The tolernce limit ws less ±5 %. The tolernce limit is defined s the mximum concentrtion of the interfering substnce tht cused error less thn ±5 % for determintion of 5-U. Urine nlysis nd recovery test or the determintion of 5-U in humn urine smple differentil pulse, voltmmetric technique ws used. Drugfree humn urine smples were obtined from helthy volunteers who gve their informed consent, filtered through filter pper, nd stored frozen until the ssy ws crried out. The study ws pproved by the Institutionl Review Committee of K. L. E. Institute of Technology (KLEIT/ IRC/2015-16/01). By spiking the drug-free urine smple with known mount of drug, the recovery study ws crried out. or the determintion of spiked 5-U in urine smple, clibrtion grph ws used. ive urine smples were used for the detection, nd obtined results re tbulted in Tble 5. The recovery determintion ws in the rnge from 92.8 to 99.9 % with RSD of 2.97 %. Conclusions In the present work, oxidtion of 5-U in phosphte buffer solution (p = 7) ws successfully crried out. The electrode process of 5-U is diffusion-controlled nd irreversible. Suitble electrode rection mechnism ws proposed. A differentil pulse voltmmetric technique ws developed for the determintion of 5-U in phrmceuticl dose nd humn urine smples. As compred to other methods, the proposed method offered n improvement in simplicity nd ccurcy. Competing interests The uthors declre tht they hve no competing interests Authors contributions PS nd SDB designed the experiment nd drfted the mnuscript. Experimentl prt nd clcultions were crried out by SDB. PS is the corresponding uthor. All the uthors hve red nd pproved the finl mnuscript.
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