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Analytica Chimica Acta 762 (2013) 14– 24

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

jou rn al hom epa ge: www.elsev ier .com/ locate /aca

eview

review on determination of steroids in biological samples exploitinganobio-electroanalytical methods

aurabh K. Yadava, Pranjal Chandrab, Rajendra N. Goyala,b,∗, Yoon-Bo Shimb,∗∗

Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, IndiaDepartment of Chemistry and Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735, South Korea

i g h l i g h t s

Review article on steroids determi-nation from 2009 to present.Nanomaterial, molecular imprintingpolymer modified and immunosen-sors are discussed.Detection limit is similar to chro-matographic techniques for manysteroids.Future prospects using nanocon-ducting polymer modified sensorand microchips suggested.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 29 September 2012eceived in revised form9 November 2012ccepted 22 November 2012vailable online 12 December 2012

a b s t r a c t

The applications of nanomaterial modified sensors, molecularly imprinting polymer based, aptamerbased, and immunosensors have been described in the determination of steroids using electroanalyticaltechniques. After a brief description of the steroids and assays in biological fluids, the principles of elec-trochemical detection with the advantages and the limitations of the various sensors are presented. Thenanomaterial modified sensors catalyze the oxidation/reduction of steroids and are suitable for sensingthem in environmental samples and biological fluids. The determination of steroids based on their reduc-

eywords:teroidsopingarbon nanotubes

tion has been found more useful in comparison to oxidation as the common metabolites present in thebiological fluids do not undergo reduction in the usual potential window and hence, do not interfere inthe determination. The sensors based on immunosensors and aptamers were found more sensitive andselective for steroid determination. Conducting polymer modified bio-sensors and microchip devices are

mmunosensorslectrochemical sensors

suggested as possible future prospects for the ultra sensitive and simultaneous determination of steroidsand their metabolites in various samples.

© 2012 Elsevier B.V. All rights reserved.

ontents

1. Electrochemical sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162. Biosensors (antibody, aptamers and enzyme based) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213. Comparison of electroanalytical with chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

4. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Department of Chemistry and Institute of BioPhysio Sensorel.: +82 51 510 2244; fax: +82 51 514 2430.∗∗ Corresponding author at: Department of Chemistry, Indian Institute of Technology Ro

E-mail addresses: rngcyfcy@iitr.ernet.in, rngcyfcy@gmail.com (R.N. Goyal), ybshim@pu

003-2670/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aca.2012.11.037

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Technology, Pusan National University, Busan 609-735, South Korea.

orkee, Roorkee 247667, India. Tel.: +91 1332 285794; fax: +91 1332 273560.san.ac.kr (Y.-B. Shim).

Chimica Acta 762 (2013) 14– 24 15

Yoon-Bo Shim received his Ph.D. in Department ofChemistry at Pusan National University, South Korea,in 1985. He is working as a professor at Departmentof Chemistry and a director at Institute of BioPhysioSensor Technology (IBST), Pusan National University.His current research interests are the developmentof bio-(protein, DNA, enzyme, etc.)/chemical-sensors,electroanalytical method of trace biological, organicspecies with modified electrodes, electron transferof organic compounds and proteins on the biomem-branes, and characterization of conducting polymersand their applications.

Rajendra N. Goyal did his Ph.D. at Roorkee University,Roorkee, India in 1975. He was Postdoctoral Fellow atthe University of Oklahoma, Norman, USA, from 1979to 1982 and again from 1989 to 1991. He was visit-ing Professor at Kyoto University, Japan in 2006. He isworking as Professor at Department of Chemistry, IITRoorkee, India since 1996. His current research inter-ests are molecular electrochemistry, developmentof sensors and biosensors for biologically importantmolecules and doping agents.

memssffiFimOtnaimdhdrohthss(raouctvhwe

S.K. Yadav et al. / Analytica

Saurabh K. Yadav received his M.Sc. from BanarasHindu University in the year 2011. He is working asJunior Research Fellow of CSIR, New Delhi for Ph.D. ondetermination of biomolecules at nanomaterial andconducting polymer modified electrodes.

Pranjal Chandra received his M.Sc. in Microbiol-ogy in 2005 and M.Tech in Biotechnology from Indiain 2008. Currently he is a Ph.D. student at Institute ofBioPhysio Sensor Technology (IBST), Department ofChemistry, Pusan National University, South Korea.His research interest includes medical/clinical diag-nosis for cancer cells, DNA, RNA, and bio-markersusing nano-biosensors and microfluidic devices.

Steroids are organic compounds found in animals, plants andicroorganisms. Some common steroids found in animals include

stradiol, progesterone, epitestosterone and testosterone (sex hor-ones). A large number of therapeutic drugs also possess steroidal

keleton in their respective chemical structure such as dexametha-one and betamethasone. The basic skeleton of a steroid possessesour fused rings out of which three are cyclohexane (A–C) andourth (D) is a cyclopentane ring. The basic skeleton and number-ng system of different carbon atoms in steroids are presented inig. 1. In most of the steroids methyl groups are present at pos-tions C10 and C13 and an alkyl chain (or substituted alkyl chain)

ay also be present at C17. In many steroids functional groups likeH, CHO, CO or COOH may also be attached to the ring or present in

he alkyl chain. The structure of some common endogenous (thataturally occur in human system) and exogenous steroids (thatre administered from outside) is presented in Fig 1. The admin-stration of exogenous steroids produces positive effects including

uscle growth, appetite stimulation, increased red blood cell pro-uction and bone density [1–3]. In the medical therapy, steroidsave been used in treating variety of diseases including topicaliseases, inflammation, anemia, neoplasia including breast cancer,ebuilding of muscles after debilitating disease and in the treatmentf osteoporosis in postmenopausal women [4]. Although, steroidsave been proved beneficial for the treatment of various diseases,hey also cause serious environmental toxicity and exhibit healthazards [5]. With regard to the risk assessment for the naturalteroidal hormones such as; testosterone, progesterone and theirynthetic analogs; such as zeranol, TbOH, and melengestrol acetateMGA) have been found to cause neurobiological, developmental,eproductive and immunological effects, as well as genotoxicitynd carcinogenicity. The continuous exposure and widespread usef corticosteroids are also alarming. Workers involved in the man-facturing process are certainly exposed to corticosteroids. Theumulative exposure to such an environmental pollutant may leado long-term consequences on the health of the susceptible indi-iduals and cause ocular medical disorder such as cataracts, ocular

ypertension, and open-angle glaucoma [6]. In another case, aidely used steroidal hormone, “testosterone”, is known to cause

mbryotoxicity in daphnia magna [7].

Apart from these issues, the major concern of the misuse ofsteroids is in competitive games. The abuse of steroids at suprather-apeutic doses is a problem not only in the world of sports, but alsoamong non-athletes to improve physical appearance and to becomemore bold and courageous [8,9]. Hence, the use of a large num-ber of steroids is banned in competitive sports by the InternationalOlympic Committee (IOC) and World Anti-Doping Agency (WADA)as they are extensively used by the bodybuilders and athletes forthe purpose of enhancing athletic performance [10,11]. The Inter-national Olympic Committee has set a limit for several steroids inurine as the upper limit, beyond which an athlete is suspected ofdoping [12]. Several competitors have been found to be doped withsteroids during competitive games. Thus, use of steroids in com-petitive sports has been considered against the ethics of sports andalso possesses a health risk to the person, hence, IOC and WADAhave prepared a list of banned substances in human sports, whichis reviewed each year [13]. In the view of such an environmen-tal and clinical importance of steroids, several attempts have beenmade over the years for the detection of steroids using diverseanalytical techniques. Generally, the biological samples containingthese steroids are analyzed using gas chromatography coupled withmass spectrometry (GC–MS) or liquid chromatography coupledwith mass spectrometry (LC–MS) or similar hyphenated techniques[14]. The determination by these techniques is time consuming,requires derivatization, sample pre-treatment, hydrolysis, etc., andthe procedure requires specialized skills for analyses. The turn ofthe last few years has seen interest in the development of elec-trochemical techniques for the analyses of steroids as most of thesteroids are electroactive (oxidizable or reducible). After the dis-covery of nanomaterials, antibody, aptamer, etc., more selectiveand sensitive detection of steroids has been achieved. These mate-rials have been extensively used for the nano-biosensor fabricationcoupled with the electrochemical detection in the recent years.Generally, electrochemical techniques are simple, easy to oper-ate, less expensive, portable and the detection can be achieved inless time and, hence, it is expected that they can be used as the

first tool to detect the steroids in various environmental, clinicalsamples and the cases of doping at the site of competitive games.The general strategy based on steroids determination involves

16 S.K. Yadav et al. / Analytica Chimica Acta 762 (2013) 14– 24

d che

vTbai

Frai

Fig. 1. The numbering pattern in steroids an

arious types of transducer and read out methods as shown in Fig. 2.his review focuses on the determination of steroids using nano-

iosensor (C60, carbon nanotube, nanogold particles, antibody andptamers) for the sensitive and selective detection published dur-ng the last three years. The advantages and limitations of these

ig. 2. General steroid detection strategy based on various type of transducers andeadout methods. (A) AuNPs, (B) CNTs, (C) antibody, (D) receptor, and (E) thiolatedptamer modified surface. (More details of sensor design and detection can be seenn corresponding references.)

mical structures of some common steroids.

methods are discussed and a brief view on future prospects is alsomentioned.

1. Electrochemical sensors

A typical electrochemical sensor consists of a sensing electrode(or commonly called as working electrode), a counter electrodeseparated by a thin layer of electrolyte and a reference electrode(usually saturated calomel electrode or Ag/AgCl electrode). Thechange in current is monitored with change in concentration ofanalyte by using suitable voltammetric technique. A variety of bareand modified electrochemical sensors based on carbon paste, glassycarbon, pyrolytic graphite, molecularly imprinting polymers (MIP),C60, carbon nanotubes, nanocomposites, etc. have been developedfor the determination of steroids. The typical details of commonlyused nanomaterials used in nanosensor fabrication with their scan-ning electron microscopic images (SEM) are presented in Fig. 3.An electrochemical sensor for 17�-estradiol based on the molec-ular imprinting polymer membranes was constructed by Yuanet al. [15]. 6-Mercaptonicotinic acid (MNA) and 17�-estradiol (E2)were first assembled on the surface of platinum nanoparticles –modified glassy carbon electrode by the formation of Pt S bondsand hydrogen-bonding interactions, and subsequently the polymermembranes were formed by electropolymerization as shown inFig. 4. Finally, a novel MIP sensor was obtained after the removalof 17�-estradiol. Under optimal conditions, the sensor exhibited alarge adsorption capacity and high selectivity. The analytical per-formance of the sensor is compiled in Table 1. The sensor had been

successfully used to analyze 17�-estradiol in water samples. Thereported strategy was novel, inexpensive and easy to operate andsuggested the use of molecularly imprinted sensor as an effec-tive tool for monitoring of environmental endocrine disruptors.

S.K. Yadav et al. / Analytica Chimica Acta 762 (2013) 14– 24 17

F ne C6

P

HlstTd

3afhrsabpo

(

ig. 3. Some commonly used nanomaterials for nanosensors fabrication (A) fullereGE and (C) SWCNT and SEM after its deposition at PGE surface.

owever, no attempt was made to determine 17�-estradiol in bio-ogical samples. In general, the major short comings with MIP basedensors are long extraction time, degradation of sensing layer dueo the change in external environment and reproducibility [16].hus, not many MIP based detection has been documented for theetermination of steroids.

9-Fluoro-11�,17,21-trihydroxy-16�-methylpregna-1,4-diene-,20-dione, commonly known as dexamethasone, is a syntheticdrenocortical steroid. It is considered as a doping agent as it isrequently abused by the athletes in sports such as cycling andorse racing to improve the performance. To prevent its misuseelated to euphoria and pain suppression, the use of dexametha-one is controlled and restricted by some sport federations. Hence,

ttempts have been made to determine dexamethasone in theiological fluids. Goyal et al. [17] used fullerene C60-modified edgelane pyrolytic graphite electrode (EPPGE) for the determinationf dexamethasone using square wave voltammetry (SWV). A

Fig. 4. The formation mechanism of MReprinted from Ref. [15] with the permission of Elsevier).

0 and SEM after its deposition at glassy carbon surface, (B) edge and basal plane of

well-defined reduction peak was observed in the pH range2.4–10.9. At pH 7.2, the peak current of dexamethasone at mod-ified electrode was found to increase ∼4 times in comparison tobare EPPGE with shift of peak potential to less positive potentialsby ∼50 mV, indicating the importance of C60 in its sensitive detec-tion. The modified electrode was applied for the determination ofdexamethasone in human blood plasma samples of the patientsundergoing treatment with dexamethasone and a validation withHPLC indicated a good agreement between the two methods. Inanother study, Oliveira et al. [18] used square wave adsorptivestripping voltammetry at hanging mercury drop electrode for thedetermination of dexamethasone and two reduction peaks wereobserved. The first peak was observed at −0.26 V and second peak

at −0.84 V and corresponded to the reduction of carbonyl groupat C20 and C3 positions respectively. The second peak was moresignificant at pH 2 and peak current decreased with increase inpH and it was utilized for the determination of dexamethasone.

IS using electropolymerization.

18 S.K. Yadav et al. / Analytica Chimica Acta 762 (2013) 14– 24

Table 1A comparison of analytical performance of electrochemical sensors for some commonly used steroids for doping.

Steroid Electrode Dynamic range (M) Detection limit (M) Real samples Technique Refs.

17�-EstradiolMIP/PtNP/GCE 3.0 × 10−8–5 × 10−5 1.6 × 10−8 No DPV [15]Ni/GCE 5 × 10−6–1 × 10−4 0.1 × 10−6 No CV [48]

Dexamethasone

C60/EPPGE 5 × 10−8–1 × 10−4 5 × 10−8 Blood SWV [17]HMDE 4.9 × 10−8–6.1 × 10−7 2.5 × 10−9 No SWV [18]MWCNT/PE 0.15 × 10−6–10−4 0.09 × 10−6 Urine SWV [20]SWCNT/CTAB/EPPGE 1 × 10−9–1 × 10−4 9.1 × 10−10 Urine SWV [21]

HydrocortisonePyrolytic graphite (edge plane) 1.0 × 10−7–2 × 10−6 88 × 10−9 Serum SWV [22]Pyrolytic graphite (basal plane) 5.0 × 10−7–10 × 10−5 49 × 10−8 Serum SWV [22]

Prednisolone �-Cyclodextrin/CPE 5.6 × 10−7–2 × 10−5 4.8 × 10−7 Urine DPV [25]

BetamethasoneSWCNT/EPPGE 1.0 × 10−9–40 × 10−9 0.5 × 10−9 Blood SWV [27]SWCNT/CTAB/EPPGE 0.5 × 10−9–100 × 10−9 0.25 × 10−9 Urine SWV [28]

TestosteroneSWCNT/EPPGE 5.0 × 10−9–1 × 10−6 2.8 × 10−9 Urine SWV [30]3D-disk-ring micro Electrode 3.5 × 10−11–3.5 × 10−8 4.3 × 10−11 No Chronoamperometry [31]

Epitestosterone SWCNT/EPPGE 5.0 × 10−9–1 × 10−6 4.1 × 10−9 Urine SWV [30]

PrednisoneSWCNT/EPPGE 1.0 × 10−8–1 × 10−4 0.45 × 10− Urine SWV [40]C60/EPPGE 5.0 × 10−8–5 × 10−5 4.8 × 10−8 Urine, blood SWV [41]

Prednisolone

SWCNT/EPPGE 1.0 × 10−8–1 × 10−4 0.90 × 10−8 Urine SWV [40]C60/Au 1.0 × 10−6–1 × 10−4 26 × 10−9 Urine, blood DPV [42]NanoAu/ITO 1.0 × 10−6–1 × 10−4 90 × 10−9 Urine, blood DPV [42]MIP-MWCNT paste 8.0 × 10−8–16 × 10−7 5 × 10−8 Blood DPV [43]

Nandrolone C60/EPPGE 1.0 × 10−11–5 × 10−8 1.5 × 10−11 No SWV [46]

TriamcinoloneGCE 0.5 × 10−6–1.27 × 10−4 2.54 × 10−7 Urine DPSV [48]GCE 3.8 × 10−8–1.27 × 10−4 2.54 × 10−8 Urine SWSV [48]SWCNT/EPPGE 0.1 × 10−9–25 × 10−9 8.9 × 10−10 Urine SWV [49]

Methylprednisolone SWCNT/EPPGE 5.0 × 10−9–5 × 10−7 4.5 × 10−9 Urine SWV [50]

FlumethasoneC60/EPPGE [Peak 1] 0.5 × 10−6–1 × 10−4 13.8 × 10−8 Blood SWV [51]C60/EPPGE [Peak 2] 0.5 × 10−6–1 × 10−4 27.5 × 10−8 Blood SWV [51]

Estrone Ni/GCE 5 × 10−6–1 × 10−4 0.1 × 10−6 No CV [52]

17-Ethynyl-estradiol Ni/GCE 5 × 10−6–1 × 10−4 0.12 × 10−6 No CV [52]

Estriol Ni/GCE 5 × 10−6–1 × 10−4 0.1 × 10−6 No CV [52]

SWCNT/EPPPGE, single wall carbon nanotube modified edge plane pyrolytic graphite electrode; SWCNT/CTAB/EPPPGE, single wall carbon nanotube and cetyltrimethy-lammonium bromide modified edge plane pyrolytic graphite electrode; MWCNT/PE, multi wall carbon nanotube modified pencil electrode; HMDE, hanging mercury drope try; DM nickle

TiTrmifRomvnomwasoeoisaope

lectrode; SWSV, square wave stripping voltammetry; SWV, square wave voltammeIP-MWCNT, molecularly imprinted polymer-multiwall carbon nanotube; Ni/GCE,

he determination of dexamethasone was carried out at pH 2n pharmaceutical tablets and good correlation was observed.he study was interesting; however, it was not applied in theeal sample analysis for dexamethasone detection. Furthermore,ercury is toxic to environment and its use has been banned

n several countries [19]. Thus, this method cannot be appliedor point of care determination of dexamethasone in real sense.ezaei et al. [20] used the electrochemical oxidation behaviorf dexamethasone at a multiwalled carbon nanotube (MWCNT)odified pencil electrode for its determination. The square wave

oltammetry and electrochemical impedance spectroscopy tech-iques were used. The oxidation of dexamethasone occurred inne oxidation peak (Ep +0.80 V at pH 5.0) on the surface of theodified electrode and the strong adsorption of dexamethasoneas noticed. The effect of several interfering species was evaluated

nd the recovery of dexamethasone in blood plasma and urineamples was reported. The information on analytical performancef the sensor is summarized in Table 1. The method is simple, how-ver, no information about the requirement of cleaning the surfacef modified electrode after each run (due to adsorbed material)s provided. In addition, the interference due to various chemicalpecies present in real biological matrix (such as dopamine, uric

cid, ascorbic acid) is expected much more in the studies basedn oxidation as most of these species are oxidizable in the normalotential range of 0 to +1.0 V. The effect of surfactants on thelectrochemical determination of dexamethasone at an EPPGE

PV, differential pulse voltammetry; DPSV, differential pulse stripping voltammetry; modified glassy carbon electrode; CV, cyclic voltammtery.

modified with single-walled carbon nanotubes (SWCNTs) has alsobeen investigated [21]. The unique electrocatalytic properties ofSWCNT along with the synergistic adsorption of cationic surfac-tant, cetyltrimethylammonium bromide (CTAB) on SWCNT leadto sensitive voltammetric response of dexamethasone with thereduction peak at ca. −1.195 V at pH 7.2. The limit of detectionand sensitivity of dexamethasone are summarized in Table 1.The SWCNT coated EPPGE had good stability and reproducibility.A comparison of the observed results of proposed method withHPLC clearly indicates that the results of both the methods are ingood agreement as shown in Table 2. The product obtained afterthe reduction of dexamethasone has also been characterized byusing 1H NMR and carbonyl group at position 3 of dexamethasonehas been found to reduce. The analytical utility of the developedmethod was evaluated by applying it for sensing dexamethasonein human urine after 3 h of oral administration and also in variouspharmaceutical preparations. The chemical species present inbiological samples did not interfere as they were nonreducible.The method is instant, simple and accurate and can be easilyapplied for detecting dexamethasone in various samples.

A comparison of basal- and edge-planes of pyrolytic graphitehas been reported [22] toward the determination of hydrocorti-

sone, a corticosteroid used for chronic lung disease in neonates[23,24]. The reduction of hydrocortisone at edge plane exposedsurface occurred at less negative potentials in comparison to basalplane exposed surface. In SWV, the shift in Ep to less potentials was

S.K. Yadav et al. / Analytica Chim

Table 2A comparison of observed concentration of dexamethasone in human urine after3 h of dexamethasone administration at SWCNT modified EPPGE in the presence of1 �M CTAB and by using HPLC.

Spiked (�M) Found (�M) by SWV Actual concentration (�M) by

Modified EPPGE HPLC

Sample 10.0 3.138 3.138 3.1405.0 8.142 3.142 3.143

10.0 13.024 3.024 3.020Sample 2

0.0 3.254 3.254 3.2575.0 8.283 3.283 3.284

10.0 13.240 3.240 3.243Sample 3

0.0 3.175 3.175 3.1725.0 8.135 3.135 3.131

10.0 13.280 3.280 3.281

RR

1baaTseisvitmatttma

svfitmfiiioop

iaflihrieomSfiao

eprinted from Ref. [28] with the permission of Elsevier.SD value for determination was less than 2.8% for n = 3.

10 mV and current was ∼4 times larger at EPPGE as compared toasal plane. The peculiar catalytic activity of an EPPGE has beenssigned to the crystal orientation on its surface. The high percent-ge of the edge orientation results in the high catalytic activity.he edge orientation on the surface of an EPPGE serves as an activeite for the reduction of hydrocortisone, where the graphite lay-rs are perpendicular to the disk surface and are separated with annterlayer spacing of ∼3.35 A. Surface defects occur in the form ofteps exposing the edges of the graphite layers. A comparison of thealues of hydrocortisone excreted in urine after 2 h of oral admin-stration obtained by electrochemical and HPLC clearly indicatedhat the results obtained by the two methods were in good agree-

ent. The calculated values of F-test and paired t-test were 1.63nd 1.26, respectively, which were less than their tabulated valueshereby indicated that there was no significant difference betweenhe precision of both the methods at 10% level. As the determina-ion was based on the reduction of hydrocortisone, the common

etabolites present in urine such as dopamine, uric acid, ascorbiccid did not interfere in the determination.

The determination of three steroids, prednisolone, dexametha-one, and hydrocortisone was carried out using differential pulseoltammetry (DPV) at a carbon paste and �-cyclodextrin modi-ed carbon paste electrode [25]. All the three steroids reduced inhe range −1.1 to −1.2 V and the peak current at �-cyclodextrin

odified electrode was ∼2.5 to 3 times larger than the unmodi-ed electrode. The recovery of the steroids has been carried out

n spiked urine and blood serum samples and recovery of 96–97%s shown. The method has an advantage that no prior treatmentf real samples is needed; however, simultaneous determinationf these steroids is not possible due to the overlapping reductionotentials

Betamethasone, a synthetic corticosteroid, is included by WADAn the doping list of pharmacological forbidden substances. Thethlete is considered as doped when its concentration in bodyuids exceeds a specific threshold [26]. As testing of the prohib-

ted substances in athletes at the site of competitive games is aighly challenging task, several approaches based on chromatog-aphy were suggested for the determination of betamethasone andts derivatives, alone or in combination with other active ingredi-nts in biological matrices and pharmaceutical products. In viewf such interest, Goyal et al. [27] proposed an electrochemicalethod based on SWV for the determination of betamethasone at

WCNT modified EPPGE. The effective surface area of the modi-ed electrode was found to increase by 2 times after modifications compared to the bare EPPGE. The reduction of betamethasoneccurred in a well-defined reduction peak and detection limit at

ica Acta 762 (2013) 14– 24 19

modified electrode was ∼25 times lower as compared to the bareEPPGE (Table 1). The voltammetric response to betamethasone didnot change due to matrix complexity of blood serum and results ofanalysis of betamethasone by HPLC and electrochemical methodswere in agreement. Further, to increase the sensitivity of detectionof betamethasone, a cationic surfactant (CTAB) was additionallydoped with SWCNT by Goyal et al. [28]. By this method, betametha-sone was directly detected in the urine samples of pregnant womenundergoing treatment with betamethasone. The SWCNT–CTABnanocomposite film further enhances the electrochemical responseof betamethasone as compared to SWCNT modified EPPGE. Theincrease in peak current and shift of peak potential to less neg-ative potentials can be explained by the fact that when SWCNTmaterial was dispersed in the solution of cationic surfactant CTABrather than dimethyl formamide, positively charged CTAB adsorbedonto the electrode surface through hydrophobic interaction withstrong adsorptive properties to form a compact monolayer on theelectrode surface with high density of positive charges. As a conse-quence, electron rich betamethasone easily reaches at the surfaceof the electrode. The interference due to common metabolitespresent in the urine matrix is not observed. The detection limitobserved is shown in Table 1. Thus, SWCNT–CTAB nanocompositefilm was found to be better than simple SWCNT modification forthe determination of betamethasone and detection can be made ata concentration much lower than the threshold value mentionedby WADA.

Testosterone, chemically known as 17�-hydroxyandrost-4-en-3-one, is the principal endogenous androgenic–anabolic steroid inhumans. Testosterone abuse is widespread among sportsmen will-ing to increase strength, aggressiveness, and recovery; making itthe most frequently reported substance in steroid misuse. The uri-nary testosterone/epitestosterone (T/E) ratio in healthy persons is1 and the ratio >6 is considered as an indication of exogenous useof testosterone enhancing compounds and is taken as the hall-mark of the drug abuse [29]. The (T/E) ratio was studied by Goyalet al. [30] through the simultaneous determination of (T) and (E) inhuman urine samples using SWCNT/EPPGE. The peak potential forthe reduction of the two compounds in SWV showed a peak poten-tial separation of 0.186 V at pH 7.2 and low detection limit wasachieved (Table 1). The practical utility of the developed methodwas demonstrated by analyzing (T) and (E) in urine samples ofthe normal persons and the patients undergoing treatment with(T) and exogenous administration of (T) was found to cause anincrease in T/E ratio as shown in Table 3. The validation of the devel-oped method was also carried out by using HPLC and an excellentagreement between the two methods was observed. The inter-ference due to related steroids on the voltammetric response oftestosterone and epitestosterone was also examined. Betametha-sone, methylprednisolone and triamcinolone exhibited reductionpeaks at −1.102, −1.285 and −1.232 V confirming thereby that thisvoltammetric sensor is specific for the reduction of testosteroneand epitestosterone at −1.152 and −1.338 V respectively. There-fore, both the steroids can be safely determined in the biologicalfluids using the proposed method. In another study, Laczka et al.[31] used three-dimensional disk-ring microelectrode array devicefor the detection of testosterone. Each device had a microelectrodearray that consisted of a large number of individual microdisks,which were used as the substrate for immunofunctionalizationand assay performance. One micrometer above it, a second micro-electrode array, consisting of microrings was used as the workingelectrode for the electrochemical monitoring using chronomaper-ometry. The method involved a complicated electrode preparation

and enzyme tagging, however, a low detection limit for testos-terone was obtained (Table 1).

Prednisone and prednisolone are synthetic corticosteroidsusually prescribed in the treatment of a wide variety of

20 S.K. Yadav et al. / Analytica Chimica Acta 762 (2013) 14– 24

Table 3A comparison of observed concentration of testosterone and epitestosterone at SWNT-modified EPPGE and by using HPLC after 8 h of testosterone administration.

Sample Testosterone concentration (nM) as determined by Epitestosterone concentration (nM) as determined by T/E

Voltammetry HPLC Voltammetry HPLC Voltammetry HPLC

Normal male urine (control)1 120 122 90 86 1.33 1.422 150 146 110 113 1.36 1.293 190 192 180 184 1.05 1.04Patient urine1 660 652 115 117 5.74 5.572 820 829 140 145 5.86 5.723 790 798 135 139 5.85 5.74

RT

ioo[admv1[noetotSmiaCatEtastciM[tatad(lnTigtp0Taite

eprinted from Ref. [30] with the permission of Elsevier.he RSD value for determination was less than 3.1% for n = 3.

nflammatory diseases. These are available in market in the formf tablets, capsules, injections, ointments, and creams and the usef both the steroids is banned in sports under antidoping rules32–34]. Individual or simultaneous determination of prednisolonend prednisone has great significance in bioscience and clinicaliagnosis since prednisone is a biologically inactive 11-dehydroetabolite of prednisolone. In human system prednisone is con-

erted to the bioactive moiety prednisolone, via reduction of the1-oxo group by the liver enzyme, 11-�-hydroxydehydrogenase35,36]. In mammals, including humans interconversion of pred-isone to prednisolone was found after oral administration of eitherf them with somewhat favored prednisolone [37–39]. Hence, sev-ral attempts were made during the last few years to determinehese steroids using electrochemical methods [40–43]. The effectf fullerene C60 modification on various substrates was studiedoward reduction potential of prednisone [41] at pH 7.2 using SWV.mall reduction peaks at −1.420 and −1.375 V were observed atodified BPPGE and gold respectively. In the case of C60 mod-

fied indium tin oxide (ITO), the reduction peak was observedt −1.353 V which shifted to −1.311 V at C60 modified GCE. At60 modified EPPGE, a marked enhancement in the peak currentlong with the shift of the reduction peak toward lower poten-ial (−1.252 V) was clearly observed. Hence, it was deduced thatPPGE was a better substrate for modification by fullerene C60 forhe determination of prednisone. The C60/EPPGE was satisfactorilypplied for the determination of prednisone in various biologicalamples (Table 1). A comparison of the results with HPLC indicatedhat the sensor was sensitive and the results were comparable. Aatalytic application of MIP prepared by thermal radical copolymer-zation using methacrylic acid, prednisolone template mixed with

WCNT and graphite powder was employed by Rezaei and Zare43] to determine prednisolone in biological samples using elec-rochemical oxidation. The peak potential of the oxidation peakt pH 4.0 was observed at ∼0.3 V in DPV. To achieve better sensi-ivity, MWCNT was also used in the preparation of MIP. The MIPs a recognition element improved the selectivity of the sensor foretermination of prednisolone and a low detection limit is observedTable 1). However, the preparation of MIP is tedious and simi-ar detection limit for prednisolone has been achieved by usinganosensors, which are more convenient and quick to prepare.he electrochemical determination of prednisolone has also beennvestigated at fullerene-C60-modified gold (C60/Au) electrode andold nanoparticles modified indium tin oxide (nanoAu/ITO) elec-rode in phosphate buffer of pH 7.2 [42]. During oxidation ofrednisolone, an anodic peak with peak potential (Ep) at 0.570 and.400 V appeared at nanoAu/ITO and C60/Au electrode, respectively.he experimental results revealed that gold nanoparticles as well

s fullerene (C60) promote the rate of prednisolone oxidation byncreasing the peak current (ip) and oxidizing at lower peak poten-ials as compared to the respective bare electrodes due to theirlectrocatalytic effect. A comparison of the electrocatalytic activity

of nanoAu/ITO with C60/Au indicated that the C60/Au electrode wasabout ten times more sensitive in comparison to nanoAu/ITO elec-trode (Table 1). The developed method was successfully applied forthe quantification of prednisolone in blood serum samples. How-ever, the presence of uric acid and dopamine seriously interferes inthe analysis of urine samples even after 16 times dilution of urine.The simplicity, sensitivity, and short analysis time of the developedprocedure make it useful for fast analysis to detect prednisolone invarious samples. To overcome the interference of common speciespresent in blood and urine an attempt was made to determineprednisone and prednisolone based on their electro-reduction [40].The determination was carried out at SWCNT/EPPGE using SWVat pH 7.2. The modified electrode exhibited good electrocatalyticproperties toward prednisone and prednisolone reduction with apeak potential of −1.230 and −1.332 V respectively. The electro-catalytic activity of SWCNT modified EPPGE cannot be accountedfor only by embedded metals [44] or the semi-infinite diffusionmodel alone. The thin layer diffusion as reported earlier at SWCNTmodified glassy carbon electrode may also contribute to the elec-trocatalytic property [45]. As both steroids are extensively abusedby the athletes for doping and both are interconvertable, therefore,the simultaneous determination of these corticosteroids in bodyfluids is expected to provide a simple and fast method for detectingthe cases of doping.

Nandrolone, an anabolic androgenic steroid, is banned by theIOC and WADA as it is extensively used by the athletes for thepurpose of doping. The determination of nandrolone at a varietyof surfaces modified with C60 has been reported [46]. The inves-tigation clearly indicated that EPPGE serves as the best substratefor fullerene modification in comparison to the other conven-tional surfaces like ITO, gold, GCE and BPPGE. The use of EPPGEis thus found advantageous for the electrochemical determinationof nandrolone because of its highly reactive edge plane sites whichallow low overpotentials, high sensitivity, low detection limit, andimproved signal to noise characteristics [47]. Another, vital infor-mation reported in the studies [46] is the role of embedded metallicimpurities of fullerene. It has been clearly demonstrated that withthe successive removal of metallic impurities from fullerene, thepeak potential shifts to more positive values and there is a markeddecrease in the peak current of nandrolone as shown in Table 4.Thus, the catalytic activity of fullerene toward the oxidation ofnandrolone is assigned to the embedded metallic impurities thatare accessible to the fluids. About 98% pure fullerene is found suit-able for the surface modification and further purification employingcomplicated procedure is not required. The possibility of themethod for the determination of cases of doping in urine and bloodsamples is also suggested.

Triamcinolone chemically known as 9�-fluoro-11�,16�,17�,21-tetrahydroxy-l,4-pregnadiene-3,20-dione isa synthetic steroid of the glucocorticoid family which is amimic of natural hydrocortisone. Vedhi et al. [48] determined

S.K. Yadav et al. / Analytica Chimica Acta 762 (2013) 14– 24 21

Table 4Effect of embedded metallic contents of fullerene C60 on the peak potential and peak current of nandrolone in phosphate buffer of pH 7.2.

Sample Metallic content in (%) Nandrolone

Fe Cu Co Ni Ep (mV) ip (�A)

Untreated fullerenea 0.416 0.191 0.007 0.392 456 11.7Purified fullerene 0.162 0.164 0.001 0.263 492 7.25

ts1attlGmcw(ot(satwtcc

motAoftpatposna

1dTnotTbosms

tpts

Super-purified fullerene 0.099 0.071

a 98% pure fullerene.

riamcinolone at a glassy carbon electrode (GCE) using anodictripping voltammetry. The determination was carried out at pH3 in 50% methanolic solution at a potential of ∼−1.6 V, which wasn abnormally high potential for glassy carbon electrode. Similarly,he determination at physiological pH is preferred in comparisono pH 13. Due to the problem of over potential and low detectionimit for triamcinolone detection at bare glassy carbon electrode,oyal et al. developed a new, selective, and highly improvedethod for the detection of triamcinolone [49]. In the latter

ase nanosensors composed of SWCNT/EPPGE and C60/EPPGEere fabricated. The detection limit was drastically improved

∼200 times), which clearly indicated the successful applicationf nanosensors in triamcinilone detection. The interference dueo several related steroids was also examined and prednisoloneEp −1.332 V), methylprednisolone (Ep −1.285 V) and betametha-one (Ep −1.124 V) did not interfere in the determination. Thepplication of the developed protocol for the determination ofriamcinolone in human urine samples of patients being treatedith triamcinolone was also demonstrated and a comparison of

he observed results with HPLC indicated that the results wereomparable. The method is simple and can be applied to detect theases of doping.

SWCNT/EPPGE has also been used for the determination ofethylprednisolone in biological fluids [50]. The blood serum

btained after 10 h of oral administration of methylprednisoloneablet from the patients was examined without dilution using SWV.

well-defined peak at −1.285 V was observed at pH 7.2. The devel-ped protocol is simple, rapid, specific, reproducible, and sensitiveor the determination of methylprednisolone. Flumethasone, a cor-icosteroid, was also determined at C60/EPPGE and two reductioneaks were observed at pH 7.2 [51]. The detection was carried outt both the peaks and the detection limit at the first peak was lowerhan second peak (Table 1). The recovery in urine and blood sam-les is observed in the range 97–103% and the method is superiorver chromatographic methods as no pretreatment of biologicalamples prior to analysis is needed. Hence, it is an attractive alter-ative to the chromatographic methods with good reproducibilitynd recovery along with a low detection limit.

The individual determination of four steroids viz., estrone,7�-estradiol, 17�-ethynylestradiol, and estriol based on their oxi-ation was carried out by Muna et al. [52] at nickel modified GCE.he modification was carried out by depositing 20 �L of 10 mMickel nitrate solution at the surface of GCE. The voltammogramsf all the four steroids were overlapping due to similar peak poten-ials and hence their simultaneous determination was not possible.he Ni–GCE electrode was found to be resistant to electrode foulingy phenoxy radicals and intermediates generated during oxidationf steroids, however, the detection limits observed for all the fourteroids was relatively high (Table 1). No attempt was, however,ade by the authors to determine these steroids in the biological

amples.The methods described above are based on the direct electron

ransfer process between sensor and steroid molecules. The majorroblem expected in such methods is the overlapping of peaks inhe case of simultaneous determination and the stability of sen-ors for reproducible results. The sensor surface fouling due to

0.001 0.086 554 5.43

adsorption is another difficulty often encountered. Due to theseshort comings, several biosensors based approaches have also beenused for more stable and sensitive detection of steroids and aredescribed in next section.

2. Biosensors (antibody, aptamers and enzyme based)

The classical direct electrochemical reduction or oxidation ofvarious steroids was well explored for their detection in variouscomplex matrices. However, most of these techniques suffer limi-tations due to the less sensitivity, selectivity, and electrode fouling.To overcome these problems in the last few years several detectionmethods based on immunosensor, peptide sensor, aptamer sensor,enzyme based sensor have been developed for the selective andsensitive detection of various steroids. Additionally, multienzymefunctionalized mesoporous silica nanoparticles, graphene, etc.based sensors have been also reported for highly sensitive steroidsdetermination. Immunosensors are ligand-based biosensors andthe fundamental basis of determination of all immunosensors is thespecificity of the molecular recognition of antigens by antibodiesto form a stable complex. The detection principle of immunosen-sors can be electrochemical, optical, or microgravimetric [53].Recently, an electrochemical immunosensor for the rapid and sen-sitive detection of estradiol has been reported by Ojeda et al. [54].In this study a disposable screen printed electrode was modifiedwith p-aminobenzoic acid followed by covalent binding of strep-tavidin and immobilization of biotinylated anti-estradiol for thedetermination of estradiol using competitive immunoassay. Thesignal was obtained through peroxidase-labeled estradiol by mea-suring the amperometric response at −0.2 V using hydroquinoneas redox mediator. The details of the immunosensor fabricationused are shown in Fig. 5. The detection limit was impressive;however, this method lacks to differentiate structurally relatedcompounds and other hormones. Moreover, the electrode sur-face was nonconducting in nature which can drastically affectthe sensitivity of the steroid determination. A comparison of theanalytical performance of various immunosensors is presentedin Table 5. In another study, a dual electrochemical immunosen-sor for the multiplexed determination of adrenocorticotropin andcortisol has been developed [55]. The screen-printed carbon elec-trode was modified with aminophenylboronic acid, on which thecorresponding adrenocorticotropin and cortisol antibodies wereimmobilized. Competitive immunoassays involved biotinylatedadrenocorticotropin and alkaline phosphatase labeled streptavidin,or alkaline phosphatase labeled cortisol. Further, differential pulsevoltammetry upon 1-naphtyl phosphate addition was employedto monitor the affinity reactions. This method also suffers disad-vantage due to the nonconducting electrode preparation and therequirement of multistep preparation. Additionally screen printedelectrodes used in these studies [54,55] are not user friendly,possess short electrode life time and poor inter electrode repro-ducibility and hence, limits the applications. Thus, in a very recent

study a nanoconducting immunosensor surface was developedand applied for the electrochemical determination of progesteroneusing enzymatic reaction [56]. A significantly lower detection limitof 0.08 ng mL−1 compared to previously reported methods was

22 S.K. Yadav et al. / Analytica Chimica Acta 762 (2013) 14– 24

i-estr(

ouelacabi(Tcpw

TA

mAa

Fig. 5. A protocol used for the preparation of antReprinted from Ref. [54] with the permission of Elsevier).

bserved, indicating thereby that the determination of steroidssing a nanoconducting surface coupled with enzymatic or non-nzymatic catalytic reaction may allow their detection at lowevels. The male steroidal hormone “testosterone” is tremendouslyttempted by various research groups using chromatography andhromatography based immunoassays [57,58]. However, scarcettempts have been made to use electrochemical techniquesased immunoassay. In one study, a disposable electrochemical

mmunosensor based on protein A-functionalized magnetic beadsMBs) was developed for the determination of testosterone [59].he anti-testosterone was immobilized onto MBs and a direct

ompetitive immunoassay involving testosterone labeled witheroxidase (HRP) was performed. Determination of testosteroneas carried out by amperometry at −0.2 V upon H2O2 additions

able 5 comparison of analytical performance of some steroids determined by various electroc

Steroid Electrode Potential(mV)

Linear range

Estradiol carbon electrode Screen printed carbonelectrode

−200 0.0036–0.92

Adrenocorticotropin Aminophenylboronicacid/screen printed

+250 5 × 10−5–0.1

Cortisol Same +250 0.27 nM–1.3Progesterone mAbP4-Au NP-Cys-gold

disk– 0.25–22.3 nM

Testosterone Screen printedcarbon/protein AFunct. magnetic beads

−200 0.017 nM–0.

Norethisterone Ab1/TH-GS/GCE – 3.3 × 10−11–17�-Estradiol Estrogen receptor

Modified gold electrode1 × 10−9–1 ×

AbP4-AuNP-Cys-gold disk, anti-progesterone monoclonal antibody on a modified gold

b1/TH-GS/GCE, primary antibody (Ab1) immobilized onto thionine (TH) and graphene shll are immunosensor.

adiol-Biotin-Strept-ABA-g-SPCE immunosensor.

using hydroquinone as the redox mediator. The usefulness of thedeveloped immunosensor was demonstrated by analyzing humanserum spiked with 1 and 10 ng mL−1 testosterone. Authorsexpected the interference of the structurally related compoundsin the determination of testosterone. Thus, it would be challeng-ing to make more accurate diagnostics tools which differentiatebetween the structurally similar steroids. In another study, testos-terone was determined using an electrochemical immunosensorusing gold nanoparticle–carbon nanotubes composites. The abilityof gold nanoparticles for antibody immobilization with the synergicelectrocatalytic effect was demonstrated by coupling gold nanopar-

ticles and carbon nanotubes for the sensitive determination oftestosterone. Nor ethisterone, an anabolic steroid, has been deter-mined by Wei et al. using an electrochemical immunosensor based

hemical methods using immunosensors.

Detection limit Real samples Technique used Refs.

nM 0.0028 nM Spiked urine Amperometry [54]

ng mL−1 0.04 pg mL−1 Serum Amperometry [55]

8 �M 0.1 nM Serum Amperometry [55] 0.25 nM Spiked bovine

serumSWV [56]

17 �M 0.006 nM Spiked serum Amperometry [59]

3.3 × 10−8 M 1.2 × 10−11 M – Amperometry [60] 10−7 M 1 × 10−13 M Spiked urine SWV [62]

disk with gold nanoparticles lodged on a self assembled monolayer of cysteamine;eet (GS) modified glassy carbon electrode. Except Ref. [62] which is a peptide sensor

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n graphene sheets and multienzyme functionalized mesoporusilica nanoparticles [60]. A sandwich protocol was used and a lowetection limit was observed (Table 5). Although, immunosensoromposed of monoclonal antibodies can selectively detect variousteroids, it possess major disadvantage such as; denaturation ofntibodies due to external factors, reusability, shelf life of the sen-or, and most importantly its antibodies require animal models,ence expensive. Thus, it is extremely desirable to develop diag-ostic methods for the detection of various steroids using otherelective bimolecular ligands.

In this context, a very few aptamer based sensor for steroidetection are reported. Aptamers are small oligonucleic acids thatpecifically bind to the target molecule. The advantages of aptamersre that they can be regenerated, highly stable to external fac-ors, and does not require animal models for generation [61]. Kimt al. [62] selected DNA aptamers that specifically bind to 17�-stradiol through the SELEX (Systematic Evolution of Ligands byxponential enrichment) process from a random ssDNA. In thistudy, the DNA aptamer was immobilized on the gold electrodehrough the avidin–biotin interaction. The cyclic voltammetry (CV)nd square wave voltammetry (SWV) values were measured tovaluate the chemical binding to aptamer. When 17�-estradiolnteracted with the DNA aptamer, the current decreased due to thenterference of bound 17�-estradiol with the electron flow pro-uced by a redox reaction between ferrocyanide and ferricyanide.he aptamer sensor in this case was more sensitive than previoustudies designed for chemical sensing. Thus, it would be advanta-eous to use aptamers for the determination of various steroids inuture.

Most of the methods reported so far required enzyme tagging,ue to which an additional step is needed. Furthermore, enzymeagged secondary antibodies can easily denature at unfavorableemperature or pH. Thus, some more advancement is needed interoid determination to avoid enzyme tagging.

In a new perspective, a label free impedance method is alsottempted for the determination of estrogen using the estrogeneceptor biosensor [63]. The electrochemical impedance spec-roscopy is a label free technique and has been widely applied forhe biomolecules detection in recent years [64]. The impedance

ethod relied on the detection of change in electrode surface resis-ance in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1, v/v) as

redox indicator. The developed biosensor gives a linear responser2 = 0.992) for 17�-estradiol with a significantly lower detectionimit (Table 5) as compared to those obtained with other detection

ethods. This is quite promising approach and can be easily appliedor other steroids detection, however, the detection can be furtherignificantly improved using a conducting polymer–nanomaterialsomposites [65,66] in place of the simple gold plate electrode.

. Comparison of electroanalytical with chromatographicethods

In general chromatographic methods coupled with mass spec-rometry are widely used in laboratories for the determinationf steroids and their metabolites. To achieve higher sensitivitynd low detection limit, gas or liquid chromatographic tech-iques are usually coupled with mass spectrometry or tandemass spectrometry [67–69]. Gas chromatography coupled with

riple-quadrupole mass spectrometry or two dimensional gas chro-atography combustion-isotope ratio mass spectrometry has also

een used for the determination of steroids [70,71]. No doubt

hese techniques are capable of simultaneously determining num-er of steroids and their metabolites, however, these techniques arexpensive and analysis requires derivatization for making the com-ounds volatile in the case of gas chromatography, whereas, solid

ica Acta 762 (2013) 14– 24 23

phase extraction, hydrolysis and liquid–liquid extraction are usu-ally needed for HPLC. Thus, analysis by chromatographic methodsis laborious, time consuming and requires special skills for analy-sis. Also the analysis is carried out in well established laboratory faraway from the site of games. Electroanalytical methods on the otherhand are simple, easy to operate, and can be applied for the point ofcare onsite determination of steroids due to its miniaturization. Acomparison of chromatographic techniques with electroanalyticaltechniques has been reported in the literature for the determina-tion of variety of compounds and similar detection limits have beenreported [72]. For number of steroids such comparisons have alsobeen made (Tables 2 and 3) and it is found that the detection limitin both the cases is comparable. This suggests that for the individ-ual determination of steroids the results of electroanalytical andchromatographic techniques are similar. The major drawback inthe case of chromatographic techniques is the requirement of largesample volume due to pre-treatment requirement, whereas, in thecase of electroanalytical techniques coupled with microchip needstremendously small volume sample [73]. In addition the advan-tage of electroanalytical methods is that biological samples canbe analyzed without any prior treatment in most cases. Also thecommon metabolites present in biological fluids such as uric acid,dopamine, ascorbic acid do not interfere in the determination basedon electro-reduction as they do not undergo reduction. However,for the simultaneous determination of steroids and their metabo-lites in a mixture, electroanalytical techniques do not appear to bemuch useful due to overlapping reduction or oxidation potentials ofsteroids. Thus, both the techniques have their advantages and lim-itations and the choice for the selection of the technique dependsupon the availability of time for analysis and whether determi-nation of individual steroids or screening of steroids content isrequired.

4. Conclusions and future prospects

Electrochemical nano-biosensors have found a unique place inthe determination of steroids in recent times due to their simplicity,sensitivity, portability, and ease of operation. Unlike chromato-graphic techniques, where derivatization and other pre-treatmentsof biological samples are required before analysis, electrochemicalmethods are used without any derivatization or pre-treatment. Dueto the abuse of steroids in doping during competitive games, it hasbecome necessary to develop simple methods to determine themin biological samples at the site of competitive games. This reviewsummarizes various electrochemical nanosensors (C60, CNT, etc.)and biosensors (aptamers and antibodies, enzymes) based methodsthat have been used for the determination of steroids commonlyused for doping in competitive games during last few years. Aftera brief description of the respective steroid, the important resultsare summarized and the analytical performance of various sensorsis compiled. The advantages and limitations of the methods havealso been highlighted. It has been suggested that a further decreasein the detection limit of steroids is expected by the use of con-ducting polymer modified electrodes or nanomaterial-conductingpolymer composite sensors [74–77]. Microfluidic device coupledwith electrochemical detection (MF-EC) has also attracted consid-erable attention during last few years for the determination ofbiomolecules [78,79]. The details of fabrication of such systemshave been reported in the literature [79]. As microfluidic systemrequires small volume (∼10 �L) and offers very high sensitivity,inherent miniaturization of the detection/separation system, low

cost, portability, compatibility with mass fabrication, and onsiteanalysis, it is expected that the technique will be highly useful inthe simultaneous determination of steroids and their metabolites.Also MF-EC technique has an advantage of achieving ultra sensitive

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etection in short time [78,79]. This device has not been explored soar for the determination of steroids in biological systems. It maye beneficial to explore MF-EC to simultaneously detect severalteroids and their metabolites in human urine samples at the sitef competitive games as a first tool for the test of doping.

cknowledgements

One of the authors is (RNG) thankful to the Council of Scien-ific and Industrial Research, New Delhi and Department of Sciencend Technology, New Delhi for funding projects related to thenalysis of doping agents. Thanks are also due to the Korean Fed-ration of Science and Technology, South Korea for awarding Brainool Fellowship to RNG and IIT Roorkee, Roorkee (India) for sanc-ioning leave of absence. The work is also supported by Nationalesearch Foundation grant funded by MEST, South Korea (Granto. 20100029128).

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