泼尼松

Talanta79(2009)768–774Contents lists available at ScienceDirectTalantaj o u r n a l h o m e p a g e:w w w.e l s e v i e r.c o m/l o c a t e/t a l a n taSimultaneous voltammetric determination of prednisone and prednisolone in human bodyfluidsRajendra N.Goyal∗,Sunita BishnoiDepartment of Chemistry,Indian Institute of Technology Roorkee,Roorkee247667,Indiaa r t i c l e i n f oArticle history:Received1April2009Received in revised form29April2009 Accepted30April2009Available online9May2009Keywords:PrednisonePrednisoloneVoltammetryDopingBiologicalfluids a b s t r a c tA sensitive,rapid and reliable electrochemical method based on voltammetry at single wall carbon nan-otube(SWNT)modified edge plane pyrolytic graphite electrode(EPPGE)is proposed for the simultaneous determination of prednisolone and prednisone in human bodyfluids and pharmaceutical preparations. The electrochemical response of both the drugs was evaluated by osteryoung square wave voltammetry (OSWV)in phosphate buffer medium of pH7.2.The modified electrode exhibited good electrocat-alytic properties towards prednisone and prednisolone reduction with a peak potential of∼−1230and ∼−1332mV respectively.The concentration versus peak current plots were linear for both the analytes in the range0.01–100␮M and the detection limit(3 /slope)observed for prednisone and prednisolone were0.45×10−8,0.90×10−8M,respectively.The results of the quantitative estimation of prednisone and prednisolone in biologicalfluids were also compared with HPLC and the results were in good agreement.©2009Elsevier B.V.All rights reserved.1.IntroductionCorticosteroids affect biochemical events and cellular processes in tissues and organ of the body,hence,play a crucial role in human physiology[1].Synthetic corticosteroids also have important physi-ological activities,such as anti-inflammatory and anti-stress action and are regarded as the most effective treatment for topical dis-eases[2,3]and these drugs also have the potential positive effects on sports performance.Hence,synthetic corticoids are extensively abused by athletes in competitive games and their use has been forbidden by the International Olympic Committee and World Anti-Doping Agency[4–6].Prednisone(I)and prednisolone(II)are synthetic corticosteroids usually prescribed in the treatment of a wide variety of inflammatory diseases such as asthma,rheumatoid arthritis,various kidney diseases including nephritic syndrome, allergies and cluster headache[7–11].These are available in market in the form of tablets,capsules,injections,ointments and creams and the use of both the compounds is banned in sports under anti-doping rules[12–14].Simultaneous determination of prednisolone and prednisone has great significance to bioscience and clinical diagnosis since prednisone is a biologically inactive11-dehydro metabolite of prednisolone.In human system prednisone is con-verted to the bioactive moiety prednisolone,via reduction of the 11-oxo group by the liver enzyme,11-␤-hydroxydehydrogenase ∗Corresponding author.Tel.:+911332285794;fax:+911332273560.E-mail addresses:rngcyfcy@iitr.ernet.in,rngcyfcy@(R.N.Goyal).[15,16].In mammals including humans interconversion of pred-nisone to prednisolone was found after oral administration of either of them with somewhat favoured prednisolone[17–19].In view of the clinical importance and increased abuse of prednisolone and prednisone by athletes,it is considered desirable to analyze their concentrations in bodyfluids as well as in pharmaceutical formu-lations.In view of the importance of corticosteroids in human physi-ology many methods such as spectroscopy and chromatographic methods coupled with spectroscopy or diode array detector and chemiluminescence have been developed for the simultaneous or individual determination of prednisone and prednisolone in biolog-icalfluids[20–23].However,inspite of the fact that above methods are sensitive and widely used,they require special sample prepa-ration,time consuming extraction steps and long derivatization procedures.Polarography has also been attempted for determi-nation of prednisone and prednisolone,however,the two peaks were found to be seriously overlapping[24].In view of the increas-ing cases of doping by the athletes,simple,efficient,sensitive and reproducible analytical technique is needed to detect such cases at the site of games.Osteryoung square wave voltammetry has been used for the sensitive determination of large number of anabolic steroids[25,26].However,the determination in these studies is based on oxidation of steroids.Since blood and urine possess uric acid,dopamine,xanthine and ascorbic acid,etc.,a major problem is encountered due to interference of their oxidation signals with ana-lyte signal.Hence,attempts in the present method are focused on determination based on reduction of prednisone and prednisolone.0039-9140/$–see front matter©2009Elsevier B.V.All rights reserved. doi:10.1016/j.talanta.2009.04.067R.N.Goyal,S.Bishnoi /Talanta 79(2009)768–774769The present paper presents determination of prednisone and pred-nisolone in biological fluids using edge plane pyrolytic graphite electrode after modification with single wall carbonnanotube.2.Materials and methods 2.1.InstrumentationVoltammetric experiments were carried out using a comput-erized BAS (Bioanalytical Systems,West Lafayette,USA)CV-50W analyzer.A three-electrode single compartment cell system was employed,with a platinum wire as counter electrode and Ag/AgCl electrode (3M NaCl)as reference (Model MF-2052RB-5B).The edge plane pyrolytic graphite electrode (∼6mm 2)used as the working electrode was prepared in the laboratory by the reported method [27].The pH of the buffers was measured using a Century India Ltd.,digital pH meter (Model CP-901).All potentials reported are referred to Ag/AgCl electrode at an ambient temperature of 27±2◦C.High performance liquid chromatography (HPLC)experi-ments were performed on Agilent 1100series system with reverse phase column RP-18e (5␮M).The mobile phase used for HPLC experiments was a mixture of water:methanol (60:40)at a flow rate of 1.4ml min −1and absorbance of the eluent was monitored at 254nm.The JEOL JSM-7400F field emission scanning electron microscopy (FE-SEM)instrument was used to study the surface morphology of SWNT modified EPPGE.2.2.Chemicals and reagentsPrednisone and prednisolone were obtained from Sigma (St.Louis,MO,USA).Prednisolone containing tablets manufactured by different pharmaceutical companies were purchased from the local market.Single-wall carbon nanotube (SWNT)of purity >95%was purchased from Bucky,USA.Phosphate buffers of desired pH and ionic strength (0.5M)were prepared according to the method of Christian and Purdy [28]using analytical grade chemicals from Merck.The stock solutions of desired concentration of prednisone and prednisolone were prepared in methanol (A.R.).Other solvents and chemicals used were of analytical grade.2.3.ProcedureVoltammograms were recorded after mixing 2mL of the stock solution of appropriate compound with 2mL of phosphate buffer.The solution was deoxygenated by bubbling nitrogen for 8–10min before recording the voltammograms.The optimized operating parameters to record osteryoung square voltammograms (OSWVs)were:initial E :0.0mV and final E :−1600mV,square wave fre-quency (f ):15Hz,square wave amplitude (E SW ):25mV and potential step (E ):4mV.Control urine sample were received from healthy laboratory personnel and urine samples of patients undergoing treatment with prednisolone were obtained from the Institute Hospital.The samples were obtained after 4h of administration of prednisolonetablet (Nucort Forte 10mg).The human blood samples from three healthy volunteers were obtained from the Institute Hospital.The blood with EDTA as anticoagulant was ultra-centrifuged and the supernatant blood plasma obtained was taken for the analysis of prednisone and prednisolone.Urine and blood plasma samples were used for voltammetric analysis without any dilution since bio-logical samples did not show any reduction peak on scanning from 0.0to −1600mV.2.4.Fabrication of SWNT modified PGEThe bare edge plane pyrolytic graphite electrode was rubbed on an emery paper and washed with double distilled water and then touched softly onto tissue paper.The electrode was then ready for modification.A 0.05mg mL −1suspension of SWNT was prepared by dispersing 0.05mg SWNT in 1.0mL N,N-dimethylformamide (DMF)by ultrasonic agitation.Initially 10–100␮L of the solution was casted at the surface of EPPGE using a microsyringe and dried in a stream of hot air.It was found that the peak current for pred-nisone as well as prednisolone did not increase after 40␮L.Hence,for further studies 40␮L of this solution was coated onto the sur-face of the clean pyrolytic graphite electrode.The electrode was then kept in air for 8–10h and now the working electrode with a well-coated layer of SWNT on its surface was ready for experi-mental purpose.Initially the response of modified electrode was recorded in a solution containing 2mL phosphate buffer solution of pH 7.2and 2mL methanol,which was previously de-oxygenated with nitrogen for at least 10min.The absence of peak in voltammo-gram in the region 0.0to −1.6V versus Ag/AgCl and very low current values indicate that the modified electrode can be safely used for experimental purposes.A comparison of typical FE-SEM images of bare edge plane PGE with SWNT modified EPPGE is presented in Fig.1.3.Results and discussion3.1.Electrochemical behaviour of prednisone and prednisolone Fig.2presents a comparison of typical osteryoung square wave voltammograms of 10␮M of prednisolone and prednisone at the bare and modified edge plane pyrolytic graphite electrodes in phosphate buffer of pH 7.2.Well defined peaks for the reduc-tion of prednisone and prednisolone were observed at potentials ∼−1299and ∼−1412mV respectively at the bare electrode.The peak obtained for both the compounds is rather broad,suggesting slow electron transfer kinetics.However,with increase in concen-tration of prednisolone the second peak merged with background,hence,the attempts to use bare EPPGE for determination of the two steroids failed.At the SWNT modified electrode,well-defined reduction peaks were obtained,where,the reduction peak potential of prednisone and prednisolone shifted to less negative potentials,∼−1230and ∼−1332mV respectively and the peak current also increased.The above results suggested that the SWNT modified electrode promoted the electrochemical reduction of prednisone and prednisolone by considerably accelerating the rate of elec-tron transfer.One of the reasons for this catalysis is that metallic impurities in nanotubes act as a promoter by increasing the rate of electron transfer,thus,a negative shift in their reduction poten-tials is observed.The electrocatalytic activity of SWNT has been assigned by many workers to entrapped metals in the cavity [29,30].3.2.Effect of pH and square wave frequencyThe effect of pH on the reduction of both the drugs was studied in the pH range 2.4–10.0.The peak potential (E p )of both the drugs770R.N.Goyal,S.Bishnoi /Talanta 79(2009)768–774Fig.1.A comparison of typical FE-SEM image of (A)bare edge plane PGE and (B)SWNT modified edge plane pyrolytic graphiteelectrode.Fig.2.Observed osteryoung square wave voltammograms for the reduction of 10␮M of prednisolone and prednisone at bare EPPGE (—–),SWCNT modified EPPGE (—)and background PBS (pH 7.2)at SWNT modified EPPGE (......).Fig.3.(A)Observed dependence of peak potential (−E p )on pH for 10␮M prednisone ( )and 10␮M prednisolone ( )at SWNT modified EPPGE.(B)Plot of −E p versus logarithm of frequency (log f )of 10␮M prednisone ( )and 10␮M prednisolone ( )at SWNT modified EPPGE.(C)Calibration plot observed for prednisone ( )and prednisolone ( )at SWNT modified EPPGE at pH 7.2.was found to shift towards more negative potentials with increase in pH (Fig.3A).The variation of peak potential (E p )with pH was linear for both the analytes and the dependence of E p on pH obey the relations:−E p (pH 2.4−10.0)=[825.52+54.53pH]versus Ag/AgCl for prednisone−E p (pH 2.4−10.0)=[944.52+54.58pH]versus Ag/AgCl for prednisolonehaving correlation coefficients 0.9973and 0.9968respectively.The slope of the −E p versus pH curves was ∼55mV/pH for both the analytes,indicating that equal number of protons and electrons are involved in the electrode reaction.The influence of square wave frequency (f )on the peak current (i p )of both analytes was examined in the range of 5–75Hz.At fre-quency higher than 75Hz,the peaks merged with the background.The peak current (i p )of both the analytes was found to increase with increase in square wave frequency (f ).The dependence of peak cur-rent on square wave frequency was linear suggesting thereby that the electrode reaction for both the compounds is adsorption con-trolled [31,32].The variation of i p with f can be expressed by theR.N.Goyal,S.Bishnoi/Talanta79(2009)768–774771equations:i p(␮A)=0.2268f+0.4283for prednisonei p(␮A)=0.2215f+0.1444for prednisolonehaving correlation coefficients0.9958and0.9991respectively.The peak potential of prednisone and prednisolone were also found to shift towards more negative potentials with increase in square wave frequency and the plots of E p versus log f were linear in the frequency range5–75Hz(Fig.3B).The variation of E p with log f can be expressed by the equations:−E p(mV)=103.22log f+1107.4for prednisone−E p(mV)=98.775log f+1212.6for prednisolonewith correlation coefficients0.9988and0.9929respectively.Sucha behaviour indicates the nature of redox reaction as reversible[32].3.3.Individual determination of prednisone and prednisoloneThe square wave voltammograms were recorded for prednisone as well as prednisolone at different concentrations in the range 0.01–100␮M.It was found that the peak currents increased lin-early with increasing concentration(Fig.3C).The graphs between peak current(i p)and concentration from the data generated during OSWV studies were linear and the relation can be represented by the equations:i p(␮A)=0.3233C(␮M)+0.8415for prednisonei p(␮A)=0.1897C(␮M)+0.5815for prednisolonewith correlation coefficients0.9993and0.9945respectively,where i p is current in␮A and C is concentration in␮M.The observed sensi-tivities for prednisone and prednisolone are0.33and0.19␮A␮M−1 respectively,indicating that both analytes can be safely estimated in the given concentration range.The detection limit of pred-nisone and prednisolone was calculated by using the formula3 /b, where is standard deviation of the blank and b is slope of the calibration curve,and found to be0.45×10−8and0.90×10−8M respectively.3.4.Simultaneous determination of prednisone and prednisoloneThe main aim of the present study was to simultaneously investi-gate the electrochemical response of prednisone and prednisolone in human bodyfluids,as metabolism of prednisolone in human system proceeds through prednisone.The main problem encoun-tered with simultaneous determination in earlier reports was close reduction potentials due to which it was difficult to resolve the overlapped voltammograms[33].This problem was resolved in the present studies using SWNT modified edge plane pyrolytic graphite electrode.The simultaneous determination of prednisone and pred-nisolone at modified EPPGE was carried out byfixing the concentration of one compound and varying the concentration of other at pH7.2.Fig.4A shows square wave voltammograms for different concentration of prednisone keeping the concentration of prednisolone constant(10␮M).Thefigure clearly depicts that the voltammetric peak of prednisolone remains unaltered and the peak current remained practically constant.The peak current of prednisone increased with increase in its concentration.Similarly, Fig.4B shows square wave voltammograms obtained by varying the concentration of prednisolone keeping the concentration of pred-nisone constant(10␮M).The prednisolone signal increaseswith Fig.4.(A)Square wave voltammograms of mixture of prednisolone and prednisone at modified electrodes in0.1M phosphate buffer(pH7.2);(A)prednisone at a fixed concentration of prednisolone10␮M;[prednisone]:a=5,b=10,c=30,d=40, e=60␮M and(B)prednisolone at afixed concentration of prednisone;[prednisone] 10␮M;[prednisolone]:a=5,b=10,c=30,d=40,e=60␮M.increase in its concentration without affecting the prednisone sig-nal,which remains almost constant.Currents observed in both the cases for the varied component were essentially same as observed during the individual determination and obeyed the relation for the calibration plot.It was found that the reduction peak of nei-ther prednisone nor prednisolone interferes with each other in the studied concentration range and thus,the proposed method can be safely applied for their simultaneous determination.3.5.Interference studyIt was considered necessary to evaluate the influence of some electroactive interferents such as,uric acid,ascorbic acid,xanthine, albumin and hypoxanthine in the determination of prednisone and prednisolone,since these are common biological compounds present in noticeable amount in living systems.Study of their influ-ence on voltammetric response of prednisone and prednisolone was carried out by recording voltammograms for mixtures con-tainingfixed quantity(10␮M)of the analytes(prednisone or prednisolone)and varying concentration of each interferent in the range10–1000␮M.The addition of these interferents did not affect the peak current of prednisone or prednisolone and no new reduc-tion peaks were observed in the range0to−1.6V.This behaviour suggested that prednisone and prednisolone can be safely deter-mined in biologicalfluids.As determination is based on reduction of prednisone and prednisolone,it is understandable that common metabolites present in biologicalfluids do not interfere because none of them are reducible.772R.N.Goyal,S.Bishnoi /Talanta 79(2009)768–774Fig.5.Square wave voltammograms of control urine sample (a),urine sample after administration of prednisolone tablet (b),control urine spiked with 30␮M pred-nisolone (c)and control urine spiked with 30␮M prednisone (d)at SWNT modified EPPGE.3.6.Analytical applications3.6.1.Real sample analysisTo establish the utility of the developed method,prednisone and prednisolone was determined in human urine samples.In mammals including humans,interconversion of prednisolone to prednisone,has been reported,hence,residues of both steroids are found after oral administration of either of them [17–19].Square wave voltammograms of control urine sample from healthy volunteer (a),urine sample (sample 1)from patient undergoing treatment with prednisolone after 4h of administration of sin-gle dose of 10mg prednisolone tablet (Nucort Forte)(b),control urine sample spiked with 30␮M of prednisolone (c)and con-trol urine sample spiked with 30␮M of prednisone (d)at SWNT modified edge plane pyrolytic graphite electrode are depicted in Fig.5.As can be seen,two clear peaks at −1230and −1332mV are observed in voltammograms (c)and (d),which are due to reduction of prednisone and prednisolone respectively.Curve (a)presents voltammogram of control urine and does not exhibit peaks corre-sponding to prednisone and prednisolone,however,peaks at same potentials in curve (b)indicate that both these steroids are excreted in urine sample (sample 1)from the patient undergoing treatment with prednisolone.The concentrations of both the compounds in urine sample 1were determined using developed method and were found as 12.48and 7.12␮M for prednisolone and prednisone respectively.Three urine samples were analyzed and the results obtained are presented in Table 1.The concentrations of prednisone and prednisolone in human urine samples were also determined using HPLC and the results were compared with the ones observed by present method.For this purpose,various concentrations of prednisone and prednisolone standards were analyzed using HPLC and the calibration curves were obtained by plotting the peak area of the analytes peaks against concentrations.Typical HPLC chromatograms of prednisoneTable 1A comparison of prednisone and prednisolone concentration determined by SWNT modified EPPGE and HPLC in human urine after 4h of administration of 10mg of prednisolone tablet.SamplePrednisolone Prednisone Modified EPPGEHPLC Modified EPPGE HPLC 112.4812.007.127.20212.0612.207.447.80312.8012.607.606.60Fig.6.A comparison of typical HPLC chromatograms observed for (A)standard solu-tion of prednisone,(B)standard solution of prednisolone,(C)control urine sample,(D)urine sample obtained from patient undergoing treatment with prednisolone after 4h of administration of 10mg prednisolone tablet.and prednisolone are presented by curves A and B in Fig.6and exhibit peaks at retention time ∼7.990and ∼9.432min respectively.Urine samples were diluted 6times prior to analysis to minimize the complexity of matrix.Curve C in Fig.6presents chromatogram of control urine and does not exhibit peaks corresponding to pred-nisone and prednisolone.The urine samples obtained from patients undergoing treatment with prednisolone after 4h of administration of prednisolone tablet were then injected.Samples were then ana-lyzed using HPLC method and a typical chromatogram observed for sample 1is presented by curve D.Two well-defined peaks at reten-tion time ∼7.990and ∼9.432min were noticed corresponding to prednisone and prednisolone respectively.Other prominent peaks in chromatogram were observed at R t ∼1.090and ∼1.223min,most probably due to the presence of major urine metabolites like uric acid,xanthine,etc.,however,no attempts were made to identify them.Finally,the concentrations of prednisolone and prednisone in human urine samples were determined using calibration curves and found as 2.00and 1.2␮M respectively.As urine sample was diluted 6times before injection in HPLC,the concentration of pred-nisone and prednisolone in urine (sample 1)was determined as 12.0and 7.2␮M respectively.A comparison of prednisone and prednisolone determined by the developed method and HPLC is presented in Table 1and clearly indicates that the proposed method is in good agreement with those obtained by HPLC method.3.6.2.Analysis of prednisolone in pharmaceutical preparationsPrednisolone is normally prescribed for the treatment of vari-ous diseases,hence,the modified EPPGE was also used to analyze the prednisolone content in three common commercial medicinal samples,viz.Nucort Forte (Unimax Laboratories,Mfg.Lic.No.;32-B (H)),Omnacortil –10(Macleods Pharmaceuticals Ltd.,Mfg.Lic.No.;DD/313)and Wysolone*10,(Wyeth Ltd.,Mfg.Lic.No.;545).The tablets were grounded to powder,dissolved in methanol and then diluted so that the concentration of prednisolone falls in theR.N.Goyal,S.Bishnoi/Talanta79(2009)768–774773Table2Determination of prednisolone in commercial tablets using SWNT modified edgeplane pyrolytic graphite electrode.Sample Stated content(mg/tablet)Detected content(mg/tablet)Error(%)Nucort Forte109.83−1.7 Wysolone109.94−0.6 Omnacortil-10109.62−3.8working range.Following the proposed method the concentration of prednisolone in three commercial samples was determined using modified EPPGE.The results are summarized in Table2and clearly indicate that the prednisolone content determined by the proposed method is in good agreement with the claimed prednisolone con-tent in pharmaceutical preparations.It is also expected that the SWNT modified EPPGE has great potential for the determination of prednisone and prednisolone in pharmaceutical sample analysis.3.6.3.Recovery testRecovery experiments were also performed using standard addition method to evaluate the accuracy of the proposed method. The recovery tests of both drugs ranging from10to50␮M were carried out utilizing SWNT modified EPPGE.Three human plasma samples obtained from healthy volunteers were spiked with known amounts of standard prednisone and prednisolone subsequently followed by recording their voltammograms.In all the cases two separate well-defined peaks were observed with E p−1230and −1332mV corresponding to prednisone and prednisolone respec-tively.The concentration of the two compounds was calculated using calibration plots and the results observed are listed in Table3. The recoveries varied in the range from97.60%to101.50%in the case of prednisolone and from96.60%to103.28%in case of pred-nisone.The recoveries indicate that the accuracy of the proposed voltammetric method is good.3.7.Stability and reproducibility of modified EPPGEThe stability and reproducibility of the modified electrode was evaluated by monitoring the peak current responses daily at a con-stant prednisolone or prednisone concentration over a period of15 days.The experimental results indicated that the current responses showed a relative standard deviation of3.24%and4.12%for pred-nisone and prednisolone respectively.After15days,reduction peak potentials shifted towards more negative potentials and current values were also decreased.These results suggest that the modi-fied electrode can be safely used up to15days of its preparation, hence,possesses good stability.Table3Recovery results obtained for prednisone and prednisolone in human plasma sam-ples at the modified electrode.Prednisolone PrednisoneSpiked(␮M)Detected(␮M)Recovery(%)Detected(␮M)Recovery(%) Sample110.010.15101.509.8898.8030.029.8499.4730.24100.8050.050.60101.2051.64103.28Sample210.09.7697.609.6696.6030.030.34101.1330.96103.2050.049.9599.9051.08102.16 Sample310.010.00100.009.7697.6030.030.09100.3030.16100.5350.049.5099.0050.0100.00To examine the intraday reproducibility,repetitive measure-ments were carried out in a solution offixed concentration of either prednisolone or prednisone.The results of seven successive scans show a relative standard deviation of1.06%and1.64%respectively for prednisone and prednisolone.To ascertain the reproducibility of the results further,four electrodes with almost same area were modified with the same volume of SWNT solution in DMF and their response towards the reduction offixed concentration of pred-nisolone and prednisone was observed.The current obtained for the four independent electrodes showed a relative standard devi-ation of2.08%and1.84%respectively,confirming thereby that the results are reproducible.Thus,the modified electrode exhibits good reproducibility and stability for the determination of prednisone and prednisolone.4.ConclusionsThe results presented above clearly demonstrate that pred-nisone and prednisolone can be simultaneously determined using square wave voltammetric technique at SWNT modified edge plane pyrolytic graphite electrode.The most promising result of the proposed method is the use of pyrolytic graphite electrode.The modified electrode not only exhibited strong catalytic activity towards the simultaneous reduction of prednisone and pred-nisolone,but also increased the peak currents.Several studies have been devoted to investigate the reason of electrocatalytic activity of SWNT.It is now generally accepted that embedded metals present in SWNT and accessible tofluids are responsible for such catalysis. The impurity of metals in SWNT has been assigned to cause elec-trocatalysis[29].Hurt et al.[34]and Liu et al.[35]have reported that metal which is completely encapsulated in impermeable car-bon shells and not in contact withfluid does not play any role during bioavailability studies of nickel in SWNT.It is also reported that with purification of carbon nanotubes,the peak potential of dopamine shifts to more negative potential[30].Hence,such a metal is expected not to be electrochemically active.Thus,the “fluid accessible metal”is probably an important property of car-bon nanotubes(rather than total metal present)and is believed to be responsible for electrocatalytic activity of CNT.The method has been successfully applied for the determina-tion of prednisolone in commercial preparations.Both the steroids have also been determined in human blood and urine samples with adequate reproducibility and sensitivity.As both drugs are exten-sively abused by athletes for doping and both are interconvertable to each other,therefore,it is expected that the simultaneous deter-mination of these corticosteroids in bodyfluids would provide a simple and fast method for detecting the cases of doping at the site of competitive games.Thus,the proposed method is a fast, sensitive and reliable approach for simultaneous determination of both synthetic corticosteroids and a promising substitute to other reported methods owing to its simplicity,rapidity and lowfinancial input.The available sites for reduction in prednisolone are the keto groups at positions3and20and in prednisone the keto groups at positions3,11and20.Ketosteroid having a carbonyl group con-jugated with a double bond has been reported to undergo easier reduction in comparison to isolated keto groups[36].In prednisone reduction at carbonyl group,C-11has been reported to be hindered due to the presence of methyl group at position10[23,37].Thus,the probable active site for reduction in both ketosteroids is carbonyl groups at position3.Therefore,peaks at−1230and−1332mV are assigned to the reduction of carbonyl groups at position3to give corresponding hydroxyl groups in a2H+,2e−reaction.Their inter-conversion in liver indicates that carbonyl group at position11is reduced to CHOH group[17–19]in prednisone and hydroxyl group in prednisolone is oxidized to C O.。