Carbon-Carbon double bond
group containing drugs
Alken~lgroup molecules play nn important mlc in tt~dicinal'cltcmis~~.
'l'hert
innumerable chemical reactions that invol\:e or p d u c c a camt,n-ch~n&juble h ~ n d
group as an intermediate or as an end p~xiucc.Since the discovery of the first rrcw
steroidal antiestrogen, several studies have h c ~ nconduct4 to daem~incthe tncehunisln
of action of this classes of compounds. However. despite cxpwssivc elli~hsin ritmy
laboratories, there exists no mechanism. which ndquatcly could explain Inxh tire
agonistic and antagonistic actions ofthc triplirnyl ethylene coriipurrds.
The other important derivatives wliicli arc well known 8s nnlihacteriul ligcnls nrr
highly effective phamiacruticals. Thcsc findings indicate that triphcnyl ctliylcnc
derivatives generally share a sitllilarity of action and that the dilkwnccs hctw~xn
flunarizine, olopatadine and clomifene arc nrcwly qu:mtitativc. l'hc devclopmmt ol'
sensitive and cflicient electroanalytical mc~htxlsIb the Jclcrtnitration ol' lhcsc dnrgs and
their metabolites in body lluids is essential for tcsli~igand eva1u:rtion c~fthese dmys.
Previously, fluorescenec and radio itiin~unoassiy tncthtrds
i~n:
~rl?l~liculthr the
determination of the carbon-carbon douhlc hond group corltaininl: drugs.
In addition to that various analytical methods, which utili~ctllin lilyc~or liquid
chromatography and spectrophotomctric or fluorescence dctcction in clrdcr to
dit'ferentiate the co~npoundand its n~ctabolites,hirve also hccn dcvclol,al. I:inirlly,
voltammetric analysis has boen studied for the clsctrc~licmicc~l
reduction of lhcsc l l ~ g Its
mercury electrode in literature.
The present work explalns broad spc~trum~rrplialylcthylcnc tlcrtvat~vcdrugs
which are clectrolyscd under optimum condrtions i~ndgotny to bc raluccd arc us fcdlown.
i Flunariz~ne
i Olopatdinc
i. C'lomifenc
Experimental
The description of the instrumentation and exp~.riarntaldcrtarls ,m prcstntal in
chapter 111.
Flunarizine and Olopatadine are ohtaincul h , ~ n I:ulk Indi:~ l.td.. Rangulorr:
Clomifene is obtained from Micro 1.ahs (Bangalom, I~idiil).I.iihl~ts\VCR p ~ ~ u mfmnr
d
local pharmacy. Britton-Robinson (R-R)hulkr of pH r~rrgc2.0-(r.0 wax ubtninni by
using acetic, phosphoric and boric acid of' 0.04 M tach and tho pl I of' Ihc solution was
adjusted using 0.2 M NaOH solution. All tlrc chonicds and so1valr.s uscxl wcw of
analytical reagent grade obtained from Mcrck (Mu~l~hai.
It~diii).
RESULTS AND DISCUSSIONS
1. FLUNARlZlNE
IUPAC Name : I-[bis (4-fl~1c~rophcuyl)mctliyll-4-[(2E)I-plrenylprop2-on-l -yl] pipcrwnc
Molecular fonnula: C'?n
H?t, b'?
N2
Molecular weight: 404.495dm01
Flunarizine is a drug classified as a calcium chinncl hltrkcr. 11 is a non-sclwtivc
calciuln entry blocker with caln~odulinbinding propctiics imrl Iiistaminc. t!1 hltrking
activity. Flunarizine inhibits the influx of extnccllular cillciurn chrc~ughn~yocanlialmtl
vascular me~nbranc p r c s by physically plugging Ihc ch:rnncl. 'I'hc dwrcaw in
intracellular calcium inhibits the contrirctilc prircsscs of' smm)th muxlc cells, causing
dilation of the coronary and systcrnic ;ir(cries, incruscd oxygen dclivcry lo the
myocardial tissue, decreastd total pcriphcrill rr:sistiulc~, c i e c r c ~ ~systcrnic
~l
hltxul
pressure, and d ~ r c a s dattcrload. It is cf'fcctivc in llrc prttphylaxis of~tliguns,tcclusivs
peripheral va.ular disease, vertigo, of central arid pcriphcral origin, and iiS an adjuvant in
the t h a ~ p yof epilepsy, It may help to rcducc Il~cscvcrity and dunlion of atiocks of
~aralysis.Flunarizine tablets were used k)r Prophylax~sof mlwtne. Pnyhylaxis of
vertigo and vestibular dlsorde~.pnjphylaxa ofprripheml ;uld ccn.hn>vasc.ulilrdin)ders.
High-~erfonnancethin layer chmn~atagraphicrnr.(hcd h a kwn d~velopodfor the
estimation of h w k i n e dihydnxhloride in cotnhin~rl d o w p form. The mcthcd
employed HPTLC alun~illulllplates prccoatd with ~ilic:!gcl (>OF1~ t[v statiollury phase
while the solvent system was toluene. The KF value uril.ohserv~dIO he 0.67M.02
flunarizine dihydrochloride'. Dcterniination of Co~~tcnt
of Mularizinc Hydmchloride
Capsules by HPLC method
using C ( 1 8) column. :icetonitrile-water (add 2.0y xtdiulil
laurylsulfate and 1 ml phosphoric acid to 320nil, water) (OX: 32) us the tnohile phase. Tbc
flow rate was 1.0 mL min- (-1); tho dct~xtionwa\,clcrigth w x i 251 nrn and cnlihrutinrr
curve was linear in the range of 10-90pg.ml.-(- I ) lix gcniposidc(r-0.W99). 'The average
recovery (n=7) was 99. 59% (RSD=0,7O?/o).1)ctcnninrrlion of llunnriritie in plusmn hy n
new high-performance liquid chromatography tncthod 3i1d to a bioaviiilshility study in
the rat was done by Aparicio et
Spc~trnphotomctricn i d h d ' has been c~tahlishdlilr
the detcrminaticln of astcmizole(l), terli.nadir~c(ll) i~nd Llutii~ri~i~tc
kydnrchloridc(ltl)
based on ternary complex fbmiation with ~wsinand Icitd(1l).f h c tiidh(u1 thws not involve
solvent extraction. The colour of the produced conlplcx is tneasur~ulat 547.5 ntn for (I)
and (Ill), while (11) is rneasurcxl at 540.7 nrn. A literittun: suwcy regarding quiintitativc
analysis of tlunarjzinc dihydrochloridc rcvcaletf that sttc~nplshave k n rndc lo dcv~iop
analytical methods for the estimation of thc drug ;tlonc i~nd111 cornhination with nrhcr
drugs by liquid chr(>matographic (1.C').
" I"
liuoro~nclr~" and sfxclropholorndric
methods. I'
Characterization of peaks
Flunarizine shows' a single wcll-dcfincd c;~thtwlicpcak in thc Ilriltm-Kohinwn
burner systelns studid over the pll r;ingc 2.0-12.0. 'l'ypical cyclic voilmtnogrnm is
shown in Fig.V.1. This p~.akmay he ;rssigncd lo thc faciic
carbon - carbon double hond group in flunarisine.
tufa
clcctrtm reduction of'
Cyclic voltammetric studirs
The plot of i, vs v"! is observed to bc llnw luld pll~singthmugh origin indicating
the difision controlled adsorption nature at the e l t ~ l n d surfucc,
c
This ohse~-vrliondong
with the expenmental constancy of iJv w~thscat1 mtc in cyclic \loltam~trctryof rhc
flunarizine has shown the electrode prima to hc frw S ~ I kinc~ic
U
conrpl~crliuns.
The value of Ep is found to incrc~sewith corrcarlration of the tlunurixinc to morc
negative potentials. This phenomenon has kvn obscnixl in thc nductic~nof mnny orgnnic
compounds md is the chawcteristic of thc irrcvcrsihlc pwcss. In cyclic voltan~nrctryno
peak is observcd on the reverse scan since the prttcys is imv~usihlc,h i incruse in the
perccntage of methanol in the voltamnletric tzst is SLYII to shin Ihc p a k potentials towards
more negative values with simultaneous cl~*.n.ascin dilTusion cunrnl, which lilay hr
explained as the increase in viscosily of the rncdiurn atld
ifuc lo the d~u.re3scin tllc
i l l ~
concentration of elcctroactivc species near the clc*.tndc surhcc.
Identification of the product
Elcctrochcmical rcducticm of' flunarizinc follows ii two clcctron coursc to (itm Ihc
corresponding saturated compound in tb cntirc pll ranging fiom 2.0-12.0
milliicoulomctric and contrc)llcd potential clcctrrrlysis rcsultn of llunilrizinc also confirm
the same. The nuinbcr of clcctrons involved in thc cl~vtrtxlcprircss l~ushmn dcterminctl
by millicoulolnetry is found to hc Iwr, in hltI1 mcdia. In lho controlled p~tcntial
elwtrolysis experiment, (he applicd potential is sct at -0.4 V
VS.
SC'E
iiI
pfi 4.0 d
electrolysis is allowed to proceed to compl~qionand the prtduct in identilied hy IR
spectral data. The disappear~nceof peak at iridicatcs Ihc forni;~tionof u the corrssp)nditiy
saturated product
Based on the results obtained from all the twh~liiques,the clemhwnicnl
reduction mechanism of flunarizine is p m p s d as ~ O I I Q ~ V S :
Adsorptive Stripping Voltanimctrlc studies
When the potential is scanned from -0.I V lo -0.4 V, lluni~rizinchas yielded o
single pcak at the working elwlnxle CIMDI; (Iig V.4). Consitlcring Ihc pcak cumnt in
order to establish the optimum conditions, the uxprrio~cnlalpilramclcrs are sludicd that
affect the adsorptive stripping volta~nnictricsignal. Stilndard addition ~ncthtxl hils hccn
employed for the determination of'llunarizinein phmttaceutical fonnulotions in hulk drug
and tablet dosage forms.
Effect of pH
The influence of pH on the AdSV rcsponsc has htm #tudltul at IIMI)E of ll~c
1.0 x 10.' M flunarillne with two supporting clwtrr)lytcs natnely Llrtom-Robinson hulrcr
and phosphate b u f k of pH In the range 0 0 lo 7 0 It can bc o k r v d from (Ry.V,7) that
the maximum peak current IS obtaned w ~ B-R
h bull'cr
tot. ptl
4.0. \$%en the pH has k n
increased from 2.0 to 12.0, the peak po~cntialsh a ~ hc ~ v nshlflcd towanis mare nqativc
values, indicating proton participation in the Auction prcxcss.
Effect of accumulation potential
The effect of the accumulation potential on thc AdSV signal hi~shru.11 studitxi with
an accumulation time of 60 sec. at HMDE for I .O x I 0 ' M tlun;~ririncsolulion (Fig.V.lO).
From the figure it is observed that the i,, valucs Ibr 11un;ui~iasis ~naairiiu~ii
at 0.0FA. I l r
maximum peak current at an accumulation potential
of
-0.23 V (or pH 4.0 is ohscrvd
because of an increased accumulation ntc. duc fo the more fuvori~hlcaligntncnt of the
molecules by the electric lield at the cltx~rcdcsolutio~iinterface.
Effect of accumulation time
The dependence of the pcak current r m t11c ;rccumulation tlnic :it a co~~ccntnrtion
level of 1.0 x 10.' M for llunarizine was showwl in (lig.V.11). Illis indicates increasing
pcak currents arc obtained up to 60 sw. For longer time ahotc (A) scw,the pci~kcurrent htrs
practically levelled ofl: For further analytical sfudics. ;UI :~ccu~rrulafion
li~nca f 6 0 .w.has
been chosen at HMDE.
lnterferences
The cftect of foreign lions and orgrmic coriip~undsw i ~ sdudid. It was Sound lhut
glucose, malt sugar, starch and dcxtrin have no influence cm lhc dctcnination
111'
flunarizjne. More than tenfolt1 ascorbic ac~dand phcnol i11tt~fcrtYI
with t h ~dctcrminnticm
:
of flundzint. Cationic and anionic surfactant.: havc ncglig~hlc i~lllucnce on Ihc
determination of flunarizinc.
Analysis
For the andytical determination of flunarizinc, A ~ S Vis found to br a suitable
technique due to its high sensitivity and r ~ ~ l u t i The
o ~ . hcst dcfinrd p a k for thc
analytical purposes is obtained at pH 4.0. Standard addition nlrth(rl is crnployrd for the
quantitative determination of llunarizine in phwnaceuricai Sonnulaticms.
Recommended analytical procedure
A stock solution 1.0 x 10'' M is preparcti by disscllutiolr of thc appnbpriale o~nount
of tlunarizine in methanol. Standard solutions :ur: prep;rrcd by dllution of this stk~k
solution with suitable amount of methanol, lrnl of the stantlid solution is trisli?rrrxl
into voltammetric ccll and made up with Y
1111 01'
111esupp~lingclc~tn)lyte and then
deoxygenated with Nz gas fhr I0 nlin. Allcr rwoniing Ihc voltan~rtiogriunssmall
increments of standard solution is addcd arid the volta~nmogramsure n~onlrxlnllcr each
addition under similar conditions.
Various experimcntal conditions arc cxpcricnc~viin onicr to rrbtoin thc most
striking AdSV signal for the detcnnination of llunirrizinc. 111 the prcscnt study the hcst
precision is obtained at pH 4.0 with ;I rest time of 2 SLX, pulsc aelplitudc of 50 niV und un
applicd potential of -0.25 V lbr flunarizinc. ('althration curva obtaind undw thee
conditions display a linear equation of currcnt intcnslty 'i,,' with ccinccntwtion 'c" for
irreversible scan. All experiments arc perfornlcd wttlt the optilnal AdSV puanrcten and
each point of the calibration curvcs is calculated iIIs thc avLragc value ol' Ihrcc succerr~ivc
measurements. Quantitative mcasurcnla~tsarc succcss1'ul in Ihc concmlralion Nngc of I .O
x
M (0 1.0 x 10 .'with the lower cicttytion lirnit ol' 1.72 x 10''. 'l'hc relolive standard
deviation and correlation ctw[]jci~ntsarc found lo hc 0.56, 0.999 mspwtivcly fbr I()
replicates.
The developed analytical proccrlurc has bc~tlapplrcd for the dctcnnination of
flunarizinein phllmaceutlcal formulattons Ior the analyvs of' l~ulliafl/lncin f(~rmulalbna
such
slbellum is cho~m..l'llc: quircd qudntlty or fbrmulation ctvrrcsp~ndinglo a
I.OXIO-~
M stock solution is accurately m r a s d a d trunsfenwl inlu'a SO ml volurnevic
flask and dissolved in methanol. A standard solution of 1x10.'M is prrFanhby dilution of
stock solution with required amount of methanol. Aner purging oxygen-frtt N1 gas into
are rccordd. Assay wsuits
this solution, voltamn~ogran~s
filr
Ilunarizine in fomlultitions
are given in (Table V.1). Recoveries of llunarizine (sihclium) in the m g e of 95.78 to
99.00 % which indicate that the accuracy ;md rrpnducibilily of pmpbsrxl adsoiplivc
stripping volt&metric method. The method is also qplicui for tubla dosage fcwms.
2. OLOPATADINE
IUPAC Name :((1 14-11-[3-(d1rncthylani1no)pn~pylick11~]-6,1
I.
d~hydrodtben/o[h,c]mp~n-2-y
1
Molecular formula: C~IHIINOI
Molcvular weight: 337.412 dmol
Olopatadine bolongs to the class ol' topic;~l ;~nt~;tllcryic
tlrugs uscrl fbr the
treatment of allergic conjunctivitis, allergic rhiuitis, chnmic uriticwia. cczana dcnnutitis.
psoriasis vulgaris and pruritius cutana)us."lt has herrl dcn~onslratcxlthiit olopatndine is
clinically superior to olhcr allcrgic drugs owing lo its dual aclivily a%~tihislamincund
ocular mast cell stabiliz~r.'~
'fhc cnicacy of the dnig is c~iilui~tcd
based on its ctTwtivc
diminishing action on the cxprcssion of ccll adhesion molwulc!; and in!lam~n:~litm
markers in conjuctival surfacc cells in patients with sursonal nllcrgic conjunctivitis." l'hc
eficacy and tolerability of olopatadinc and cron~olynsc~dium2%
compwed and it ia
i n f e d that olopaladinc had significant e f l i ~ on
t c ~ u l wsigns and syrnptoniv of allcryic
conjuctivitis than cromolyn 2% opthalimic solution. I" The incwascd ocular cr)mli)rt and
patient acceptance for olopatadine is mainly due to its rcstrictcd inlaaction with
membrane phospholipids thereby inducing less dgrcw of manhrrlne pctturhation and
release of intmcellular constituents. I' Rased on thc cr,njuctival allergcn challcngc mcdcl,
the efficacy, safety, c~ptimalconcentration and dosage of'olopatadine are cvaluatod in thc
lreatmenl of allergic conjunctivitis.
IK
The enzymes responsible for olopatadine nietnhlisnl
idmiifid and the cfftx~
of olo~atadineon human liver micrnsomai cytochmmt. p450 activities wm studid. "
The inhibitory action of Olopatadine on cytokinc genes in n1 b~ophilicleukmlia cells
investigated. 'O The physiological significanceof SIOO pmtcins cm hc ddcnnind h w d
on the selective interaction of these protci~iswith olopatudinc.
!' Oloputadine is also
l ~u.r.c~ilatuus
skin lesions in
found to improve the neuronal conditions of spinal c o ~ iuld
Pharmaceutical analysis is an in~ycbdant mpcct during drug
contact
production, drug developmcnt, phmacokinetics md pharmi~cologicalstudies. A number
of analytical methods have bccn developed fiv the detcnninution of Olipitndinc in
human plasma using High pertbmlance liquid chri1n1atogrsptiy-tnndcm tnnss
spectrometry. "-"
Recently olopatadinc has bcvn t1c.lcrniini.d in bulk drug fonn ant)
pharmaceutical formulation using Higli performance thin layer clirornu~ography"it'
UV Vis, spectrophotometry.27Voltlunmctric tcchniquc?i cwcupy a pn>rnincnt plucc in
drug analysis owing to its simple, rapid, cosl efrhtivc uitl highly sensitive w ~ dscllxlivc
procedures.
" The
aim of the present work is to cstahlish :I vciI!arn~nctricprtxcxlurc li)r
the determination of olopatadinc in bulk drug Ihnn and i n phann;~cculisalfi~nnulation.
The method developd is a simple, fast, less cxpensivc mctliod with high scnsilivity iind
selectivity.
Cyclic voltammetric study
Cyclic voltammogram was rccordcd by cycling ~ h cpolcnlial ol'llMDli fLorn 0.0
V to -0.4 V vs. AdAgCI at a scat rate 01' IIW) 1nV1s.A sinylc, well dctincd reduction
peak was obtained at -0.19 V for Olopa(atlinetluo to tltc 2c' rctluction of the c ~ ~ ~ y c l i c
carbon-carbon double bond. The ahscnce of the anodic peak on the rcversc scan indicirtcs
that the reaction is irrevasible. 'll~ccyclic voltammograms of thc Olopatadine of three
concentrations, (all.0 * 10" M, (b) 2.0 8 10" M and
(c) 3 . 0 * 10.'' M wcrc
regisled under similar contlitions (Figure V.2). Tho nunibcrs of clwtronq involvd in
the reduction reaction arc found to be two h a d on millicoulomdric studies.
Voltammetric determination of Olopatadinc at HMDE
By dissolving an appropriate amount of crloptlt;Nline in incthnncll. pr stock solution
of concentration 1.0 xlw3 M was prcpnrcd. A \\.orking s~luidurrlw a pmpnred f b m the
stock solution by dilution with douhlc distilld watcr. I .O ml of standard solution and 9.0
ml of BR buffer of pH 3.0 was placed in Uie clcx\roI)qic cell and purgd with nitmgln gas
for 15 minutes. The potential of thc clectrcde \vas negatively s c m n d from 0.0 lo -0.4 V
vs. AdAgCl. Olopatadine exhibited mrtvi~nun~
pH 3.0. The voltammograms of the blimk
M were recordcd under same
CUITCIII 91
-0.19 V in UK bulYer of
aid olopatadine of conccnlrution I x 10"
contliticms (Fig. V.5).
Based on the results obtatn~vi fn)ro dl ~ h ctcchn~qucs, 11ic dc~lmcliaaicul
reduction mechanism of olopa~adincis pn)poswl ,is Ibllows.
The pH of the medium plays an i~nportantmle nffrvtirlgthe mtc of nccumulntion
and the rate of electrode reduction reaction. I'hc intluencc of pH on tbc pok current ww
studied for 1 x 10' M of olopatadine hctwccn pli 2.0 to 10.0.7.11cpak
l current i n c w t s
with pH initially and reaches a maximum value at pH 3.0 und thon dwwrlsrts ns shown in
Fig. V.8. The increase in pH shiftrd the pc& p,tcntii~ls lo moh: ncgativc \+alucs
suggesting proton participation in the duction process.
Effect of accumulation potential
The effect of' accumulation potential on the pcuk signill was involipntcd at
HMDE for 1 x 10'" M olopatadinc. It was founrl that ip valuc wacllcs muinrum vnluc at
-0.8 V vs. AdAgCI (Fig.V.I I). Tllc maximum pcak current ;tt -0.8 V was duc to incrcw
in the rate of accumulation owing to the hvourahle orientation of molecules by the
applied electric field at the clwtrodc-solutitrninlcrljcc.
Effect of accumulation time
The variation of peak current with ircu~iinlation titnc w ~ sstudicd Ibr a
concentration of Ix 10"' M olopatadinc solurion. As lhc ~)rc-cot~cenlrfitiotl
limc increuscd,
the peak current incrcnses slowly and rcachcs a rnirxi~iiu~ll
vi~lucat
1(K) scc.
With furthcr
increase in accumulation time, no c l l i ~ on
t the peak signal was obscrvccl indicnting that
the electrode surface WiLs saturated with the analytc moleculcs. 'lhe plot bctwccn I, vs i,
is shown in Fig. V.14.
Effect of scan rate
The lnlluence of scan ratc on p k currcnt was ~nvc>t~gatcd
o v a the range of
20-200 mV/s. A plot of log ip vs. log v w;fi ohr~lncd;md the 11nct;rrquation wiis givcn
by log Ip (PA) -. 0.81 1 log v (mV/s) - 0.887 w~thR' 0.949. The value of thc s l o p was
found to be close to 1 indicating that the duction p m . a under study was ndsnrption
controlled at HMDE.
Recommended analytical procedure
Standard solutions arc prcparcd by dilution oSstcxk solution oScc,ncmimtion 1.0 x
10-3M with suitable amount of metllanol. 1 mL of thc stutlldud solution is t m s k n a d into
voltsunmetric cell and made up with 9 ml. ofthc suppofiing clwtrolyfc und then purging
with
N2
gas for 15 rnin to remove excess of' oxygen. Alter rcu-oniing thc valtnni~nirgrnms
small increments of standard soluti~nis addd and the voltan~~r~ogru~~is
nxonlcrl aflcr
each addition under similar conditions.
An aliquot (0.1 mg) of Olopine O.l% crphtl~drnisfi\nnulutioa wus taken in I000
rnL volumetric flask and a series of concentrations IO0, 200, 3(K), 400 and
S(l0 ngml.
was prepared. A recovery test was canid out on 111eabove conccnlmtions hmai on thc
proposed method. The average rccovcrics (% K) and tile rclotive standard deviation
(%RSD) for five rcplicarc dettminalions were givnl in I';~bleV.?.
A callbration plot was drawn hctwccn peak current and the concmtrution of
Olopatad~neover the range I .0 x 10 M to 4.0 x I0 ' M. It was ~bscrvcdthat the peak
current increases linearly u ~ t hthe conccntrittlon of Olopatadrnc. 'I'hc lrncnr rcgrwslon
equation was given by Ip (,LA) 0.221C' (pM) t 0 310 w~thR'
dctect~on(LOD = 3S,/1n) and lim~tsofqua~~l~t;~tron
(I.0Q
0 999 'Thc limrls ol'
10%Jm) wcrc crlculnlcd
u
q
1.92 ndml and 6.34 ndml respcctlvcly.
The developed proccxlure was applicd for thc tlctcrmination of hulk drug
Olopatadinr of concentration 1.0 x 10" M in thc ahs~nccof cxcipimts and ophthalmic
firmulation of cc>nc~!ntration1.0 x 10'" M in Ihc prewncc of'cxcipicnt?i.Five rcplicalc
determinations w ~ carried
~ e out in each
cast
u?ilnS lllc prs)pprr~d mdhcd. 7 % mwtn
~
recoveries (% R) and rclative standard ticviation (lKSII)
,i wcw given by 98.9 i 1.21 to
99.8
1.26 (without cxcipients) and OX.5 i 1.22 to 99.6 i 1.24 (with acipirmlr) with no
significant dimerace. ticnce the pmccxlurc could he urnidcrud as specific. The inter-day
and inba-day precision was establ~shedby dctrtnlirl~ngIIW bulk druy of cow.I .0xlO"M
on five successive days and five times on a s~ngIcday using fie pmtaol dcvcl*
The
mean recoveries (%R) and relattve standard de\~st~an
(OPRSD) vdurs art:99.8 (%R) and
1.8 (%RSD) for interday and 99.6(%R) and I.2(noRS[))for rntn-duy mwsurcmatts.
The results indicate the h~ghprecision af'thc proposal method.
lUPAC Name :2-(4-(2-chloro-1,2-iiiphcnylcthcnyl)phcnoxy)-N.N41ctl~yl-ethn1~~1ni1~e
Molecular formula: C26H28CIN0
Molecular weight: 406 g/mol
It acts as a seleclive estrogen rcvel)lor nitdulator (SEKM), similar to trirnoxifcn
and raloxifene. All three drugs are competitive inhihiturs of utrnyen binding to cstnlgcn
receptors and have mixed agonisl and anlagonisl itdivity dcpsndiny upon the t q c t
tissue. 2Y Clomifene is sold as a mixturc ol's~cru)iauner?;
(cis i111tltcins) zu-clomilonc nnd
en-clomifene, the first one being an estrogenic agonist untl the last onc srr cstrt)ycnic
antagonist. It is generally used as a stiniulant of ovarian f'unclion anti it is listd as a
doping substance on the World Anti-Doping Agency (WAIIA) pnjhihitcd list due to its
''
selective estrogen receptor modulator ci~~abilitics."' 'l'rcatntcnt ol' nsglrtive cKecls of'
anabolic androgenic steroid ahusc (c.g., gynecotnlstia) its wcll as a ncg;rtivc Swdhirck lo
testosterone lnctabolisln causing an indirect cnhanccrncnt of lhc ssnrln Icslr~slcronc
concentration is the main reason for the ban ol'tliis suhsta~~ce."
Excretion of several clomifenc mctnbr)liles and clotnifcnc ilsclf hus baa
dwribed afier in vivo md in vitro studics. Its mctahlism tidlows reactions
of'
N-
hydroxylation, N-deethylation and N-mcthoxylation."u Its relationship with plmrnatic
and urinary level variations of 17-hydroxpmgesterone, tcstoslcmnc and iuleiniziny
hormone has
described loo. '7.'K ~lomif'cnccilr~teha%k e n Sotmtl very ell'cctive in
the treatment of scrondary male hypogonadism in many caw.'' 'l'his has shown lo bc? a
much more attractive option than tcstostcn)ne rcplaccm~ntthwapy ('SRT) in many c a w
because of the reduced cost and convenience of taking a pill ns opibstd to tatodtronc
injections or gels.40 Unlike traditional TRT it alx) d(ws nclt shrink the Isles *id us a
result can enhance fertility. Traditional TRT can n'1rder a am sterile (ulthough with
careful monitoring and low-dose hCG ns an adjunct. this is both prcvmtt\blc and
reversible for most men).
Since clomifene citrate has not hcvn FIIA :~ppn)\"~Ik>r uw in males it is
prescribed oi'f-label. According to Craig Nidcrhcrgcr, haausu this drug is now generic,
no drug company would pursue FDA appm~iilb r use in lncrr now k u u s c of tilnitad
profit incentive, mostly due to the ~lativclysrrliill ~narkctptrtcntial. Howcvcr, thc single
isomer of clomifene, enclomif'tne under the hr;ind n;iorc ,41drcrn~I,is c u m ~ ~ t under
ly
phase 2 trials for use in
M~~hiinism
of actittr~of cloniilns has hilh estmgenic
and anti-estrogenic properties, but its precise n~~~iianisrn
oL' action has not k n
determined. Isotope dilution mass spatrornctry for ni~cnr- iuld truce-chilent
determination of c~ornifene~~,deteminati~t~~
hy liquid cl~n,mattrgraphy.nrass slxxtmnielry
of clomiphene isomers in the plasina of paticnts undergoing t ~ i i t ~ t i li)r
~ n thc
t induction of
ovulation,
44.40
Direct current ~liir~gl.ilphy
n11t1cyclic \ ' ~ l t n ~ ~ ~ nwen'
~ c t rusul
y to study
tarnoxiphene (TX), clorniphcnc (C'M) anti clilort~trianiscnc(('lil'). 'fhe dcpl'ndcnccs of
the limiting currents and half-wave potentials orr thc p11 of solulion, tcltlpcmture,
mercury head, ionic strength arid surhcc tcnsitm ofthc ullution wrrc studied.
""
Characterization of peaks
Cathodic pcak single, well-ilclinsd pcak
WAS
ohscrvctl for clomifcnc in tllc
supporting elcctrolytc Britton-Kobinsorr hu1li.r syslcms studid trvcr Ihs pi1 rmgc 2.012.0. This peak is attributed to the rcduclion of cahn-carbon Ooublc bond in two
electron process to its .corr~%pndinysaturated pnduct. According to thc cyclic
voltammograms we (&serv~xJthat the solut~onsc ~ grculcr
f
alkalinity, the reduction an: no1
well facilitated owing to the less availabrlity ol'prolons.
Cyclic voltammetric studies
When the potential was scanned fmni -0.25 to - 0.45 V. ulomifac shown wcll
defined peak at HMDE (Fig.V.3). Ci>osidering thc pllk in oder to cstuhlish the
an. evaluntrd i~ltersignal. No peaks
optimum conditions, the experimental piuu~i~etcrs
are observed in anodic scan indicating an irrevcrsihlc nrluction pnxllss. Suhscquent
scans exhibit a dramatic dec~aseof pcak current to a stahlc vduc xp-scnting the
response of the solution spwim. This is ohvious indication thu~clo~iiikt~c
llus an
adsorptive characteristic at the mercury clcctrodc. Such wcdic signal is nttrihutd to thc
cyclcisation reaction to fonn corresponding dcrivativt..
The reduction process in cloniikne is ditl'us~oncontn>lld adsorption pnrssr on
electrode surface was observcd by the lincllr plot ipvs, v".
Virlric of Ii,, is Ibund to
increase with concentration of the cl~)tuili.ncto nlorc ncgutivc pc)taitiuls. 'I'his
pheno~nenonhas been observcd in the reduction ol' mculy orgunic co~np~uads
and is thc
characteristic of the irrcversiblc procass. In cyclic volt;unmvtry no pcak
IS ohsrrvcd al Ihc
reverse scan since the prwess i s imvcrsihlc. i\n increase in thc pcrcaltagc trf mcthmol in
the voltammetric test is sccn to shin thc peak potentials towarcis morc ncgitivs values
with simultaneous docroase in ditYusion currcnt.
Adsorptive stripping voltammetric studies
Electrolysis of the elcctroactivc substance has kc11carricd out at -1.48 V vs.
SCE and the product fonnd allcr thc controllcxl potcnl~slelectrolysis is idrntilid and
confinnod ;~sthe saturated product of the corrusponding clcxlrcractive spocies. 'Iypical
voltarnmogralns arc shown in Fig.V.6. 'She dstcrm~nationof nunlher of' clutrnns (n)
involved during the eltttrodo process in clomifenc h ; ~hccn carried oul by
mi]Eicoulomctry.According to this tcchniquc, 'n' is li~und111 hcl two for clomifcnc in
acidic as we[[ ~5 neutral and basic ~iidia.(.'ontrolledplcntial clalrotysis is c111ploy~d
to get
r&ction pral~ct.It hi& kc11 catricd out in a nitulifiul ccll wilh a thrcc
clectrtde system. The pnduct ohtainud i \ slucltcrd by IK spcclroxopy. 'I'hc
disappearance of peak at 167s cm.' indicates that the douhlc h n d in
clomifene has
undergone reduction in the final product.
Electrode mechanism
Based on the experirncntal results ohtsinryf h\mall 111stwhniqucs anployd. u
possible el+roche~nicalreduction mechmisln h a kp11suwestd 011tk hwis of
protons and electrons involvcd in the reduction is as lir1lcnr.s:
:r
I
14
I*,
dnna,",ng .,q,
Effect of p l l
Thc influence of pH on the stripping voltatnn~ctricrcsponsc Ibr 1.0 x 10.' M
clornifene was cxarnined in Rritton Robinson h u f i of pl.1 2.0 to 6.0 nficr prcconcentration of thc clon~if'cneonto thc HMI)f; l i ~ r00 scc. A sinylc irrcvmihlc peak
was generated in solution of pH 3.0. 11 can bc cbhscncd from Fig. V.9, when thc pi? wiix
increased above 2.0, it shins to mow ncgiltivc potentials.
Effect of accumulation potentid
The dfect of acc~lnulationpotential on i\dSC' cumnt of the peak (11'clomifenr
has been investigated after prcconcmtration of the drug crnto thc ttMDE for 60 scc.
over the potential range 0.0 to -.05V. A niuclr illon. pnk dcvcloprd Tclr pcak curmil
was achieved at potential range -0.2 V (Fig V.12). .hcrtbrr. a pn.conccntmtro~i
potential of -0.2 V was chosen over the st of ~ h cstudy.
Effect of scan rate
When the scan rate has bccn varicd tiom 25 to 2 5 0 mVs I, the p k cumtit
increased linearly with the square rajt of scan mtc indicating dilrusitw crmtmllrxl
reduction reaction
. Other experimental pwimctcrs such us lempcnture trnd ioaic
strength were optimized. The stirring pcak currents were not modified wtrcn tlic
temperature varied between 25-50°C. The viiluc chosen was 30°C'. k a u s c it is mom
temperature.
Effect of accumulation time
The variation of peak current with accutnulutioti tinru w;ts studied fi~ra
concentration of 1x10'~M clomifcne solution. As the prc-concctitrutir,li limc incxilscd
the peak current increases slowly and rcrichk5 a rnuxioium value at XO sw. (lig. V.15).
With further increase in accumulatic~ntime, no cllcct on (he peak signal was ohscrvai
indicating that the clcctrocic surfiicewas saluratul with t l i ~analylc ~ O ~ W U ~ C S .
Recommended analytical procedure
A stock solution was prepared by dissc~iuliono f the ;~pprtrprialcaslounl of thc
elatroactive spccics in methane. 1.I) in[. (if stiindartl u~lul~on
is lranslirrcd inlo a
vol[ammetric cell and diluted with 9.0 niL of supporting clcctrolflr md dalxyycnnfd
with Nz gas for 10 min. Af\tx the voltamm~~gram
is
rccnrdcd, small incr~mcnts((1.2
mL) of standard solution arc added and lhcn uoilammrrgrums are m n r d d afkr cwh
addition under similar condition. In the pmcnt s~udy,the hest prrcision is chtaind a1 pH
3.0 with rest time of 2 sec., pulse amplitude or25 mV md u t ~qrplicd ptcntid -0.325V.
Under these conditions the cumnt was linear functicrll wit11 concartnttion ~ o S c l w ~ c t i v c
species in the range of I .O x 10" to I .O n 10.' M. Thc Io\vcr detc~tionlimit of 1.5 x 10" M
.
was obtained with cornlation cocffTciun1a11d wlati~cstu~rdilnldeviation 0.992 O/Q 0.17
%respectively.
The described pmc~durchas heen succc~sl'ullycmpl~~ycd
Ihr tlis detrrn~inntion
of clomifene in tablet forms. In 111r prcscnt study, phiznnoccu~iculIh~~nulnlion
of
namely clomifcne is e~nploycdIbr assay results. 'I-11c~vquircdqua~rtityof f'l~nnulution
is prepared corresponding to a I.()x 10' M Stock solutii~nis :tccuri~lcly111rrusud ttnd
transferred into a 50 mL volu~nctric Ilnsk con~aining 111cthiinol. I ~ n l .of lhc
formulation solution corresponding to n standard solution of concallrition 1 .O x 10''M
is diluted with 9 ml. of supporting clcctrolyte (p11 1.0) iind voltunmograms tlrc
recorded aRer purging with oxygen-free N2 giis Ibr ;I duration of 10 min. Assay wsults
for clomifene in formulations iuc given in Sahlc V.!. 'l'hc rcx.ovcrics of cl~bmifcncin
phannaccutical fomiulations ranging from 08.20nbto 9).I O Y C
:IIZohtrrinal.
Potential /V
Fig. V.2. Cyclic voltammograms olopatadinc. nt three different conccntrrEiun lcvel#
(a) 1.0~10-6M,(b) 2.0 x10l-6 M,
at pH 3.0
(c) 3.0 ~ 1 0 - 6M using ltMDE
1.2
-
1
0.8
0.6
0.4
0.2 '
0'
4.2
I
4.25
I
4.3
I
4.35
t
4.4
1
4.45
Potential / V
Fig. V.3. Cyclic voltammograms of clornifene us in^ l4MI)E at pi1 3.0
Potential (V)
Fig. V.4. Diffcrcntial pulse adsorptive stripping
voltamtnoyrarn
flunaridne concentration 1 xlO" M at I I M I ) E at pll3.0
of
Potenttal (V)
Fig. V.5. Differcntiul pulse adsorptive stripping voltammogra~nof
blank and h) Olopmtadinc
a)
ef concentration 1 xlfld M at
H M I W at p11 3.
.0.25
O
. 3
0 35
.0.4
.0.45
Potential /V
Fig. V.6. Dlfferentlal pulse adrorptivc stripping voltammagram ofclomifenc
conccntration 1 xlf14 M at HMDE at pH 3.0
4
\
-0.
Fig. V.7.
Effect o f p l l on the peak current ul llunarl~incof conc. 1.0xlO"hl 11 I1MI)E
Fig. V.H. EllPct of p H on Ihe peak current olOlopatadine of conr. 1 . 0 x l ~M
" at I1Ml)t
Fig.V.9. E k t o f plf on the peak current of ctomifcnc ofconc. 1.0w10* M
Fig. V.lO. Efftct of accuaulaUom potrmtial or prak curreat of nunarldnt at 11MI)E
Fig. V.13.
Effwt of ~crlin~tilu~iutr
t i ~ i w011 I)CI&
CUTTCII~
of OMIIW~~X~IIE
01
F i ~ ~ r1'.e15. Effect of ucca nlulatilrn liti~t.
en pcak ri~rrrs
t of r l o ~ i l i f v ~of~ c
-Sarnplu
-.,--
Amount
i Amaunt laken
fo u l ~ '
d
,
(ns/ml)
5 my
.
.
% RSI)
% Rucnv~y
[rldml)
I..~-~L..LH,~~II,-W..~+-IPIP-.-.---~
.rm-rrm,
Siklum
',
50
4781)
95,fH
0,9K
150
t 48.23
98.82
1.1
250
24735
08.8
0.9 1
350
34691
99.0
l,82
445.53
OK.88
1.2
450.
I
.
..
No.of detmirratimrs3 5
A_4C_.l....
.
-.-4,-
,
, ,
,
-
.
.
,
,
.
, ,
,
'
,
.
,
,
, ,,, , ,
,
, .
.;,.:
- . . . .: ,
,
,
,
,
: : . .
m
.
..
. ,
-
'.
,
,
.-
.
.
.. . . .. .. . .. .. .. ...., . .
,-
,
. , , ,- , , . :..
.. ,
,
.
'
"
,
.
'.
Tnblc V.2. \'oltammrtric asmy of Olopinc 2% ophthrlmic d u d o n
.lilUUIII WALll
(ngml)
-----
-
Oloplne
+
-
-
-
.-
ftund*
*YO
(ngn~l)
-- - .
-""
.-
Rwovcry
-
9 0 RSD
---*.
99 62
W 62
200
197 86
9N.W
1.22
300
286.73
VX V I
1.20
400
IYX.08
89 67
1.18
No of dctcnninrtions 5
Table V.3. Voltammctric array nf Clamid 10 mg tnhlrts
80
160
('lomid
I Orng
-
100
240
360
-
References
Palak P i e l and K a s h p p K. Bhnn, lSRN .bal!liwl Chemistry, Vulumc 20 (12)
(20 12).
ZHU Bi-jun. ZHAO Hi-mng. TIAN Hui.
WAN Kai-hua, Chin~vcf o u m l of
Pharmaceutical Analpis- 2005.01.
Wiwin Farina K;u?inasari. ticnny Chufilu~ty & iiuunwai Itrdnymlo, rhincse
Pharmaceutical Journal 2006. Vol. 41 Issuc (of)) : 4 3 - 4 1 5
X. Apiuicio, J. Gm. A. ('ampos. F.. Fc.n~andsr,1:. Cirlpi. Sountal ol'l'hsmruscuticnl
and Biomedical Analysis 6(?),1988, 167 173.
Kelani K, Bebawy 1-1. Ahicl-Fot~ah1. J I'hsrm Biotntd Anal. IOU9 I X(0):YXS-92.
M. W. LA),U. Silhcr, and S. Kicgdmli!\, Inun~tllof Chmmalographio Science,
2q3). 1982. 126--13 1.
E. C. Kwong and D. D. Shm. Journal c1S('hn1matogrqhy. 4 IJ(2). 1YH7.305 -37%
S. 1,aganicrc. E. Kwong, and I).
I). Shcn, Journal of' ('hnvnutogr:tphy, vol. 4X8(
2). 1989,407-416 .
E. C:. Kwong and D.I). Shm, Journal ol'('hron~u~o~ri~l,hy,
vol. 414, no. 2, pp. 365.
379, 1987.
S. Inganicrc. I.:. Kwong, iind TI. I). Slicn, .Ioum;~lrrf( ' I ~ n ~ r i i i r ~ r ~ g vol.
r s ~ ~488,
I ~ y no.
,
2, pp. 407--416, 19x9.
J. F. Pritchard, 1). W. Schncck. W. 3 . K ~ c I , i1nJ A. It. llaycs Jr., C'linicnl
Hitrhcrnislry, vtll. I I, no. 3 , pp. 121 125, 107K.
N. M. Sarlghavi and N. C i . Jivani, " I < s ~ t m a ~ or'
~ t apropr:inolol hytlrt~hloridc,"
Talmtil, VOI. 27. no. 7, pp. 59 1 --5'12, IVXO.
Ohmori K. Ilayashi K. Kaiw T. Ohshinla I;, Kohnyashi S. Yumwki
'r',
Mukouyama A. JapancscJournal of IJhannacology 200?; 8X:370-397.
Rascnwasscr LJ.
O'Hricn 'I"and Wcync J. I'urnnt Medical R~%san.hant1 Opinion.
2005: 2 I: 1377- 1387.
Avunduk AM, Tekelioylu Y, Turk A. Akytll N. (.'linical 'Thcrapcutics 2005; 27
:1 392- 1402.
KatelarisCH,Ciprandi G. Miswttm L. Turnsr FD, Rsstin [I. B d u x G.Clinical
T h ~ u t i c 2002;
s
24: 1561-1575.
Brockman HL. Momsen MM, Knudtslln JR, Millcr ST, GraBCi ard Yunni JM.
Ocular Immunology m d Intlam~aat~on.
Z(M3: I I: 147-268.
Abclson MR. .Annals of Allergy. Asthrna 6L Inrrnum~logy.IWS: 8 1: 2 1 1-218.
Kajita J, lnmo K. Fusc E. ICu\suhm 'T. a i d l i t h ~ y a s h H.
i
Drug Mctnb Diyxrsit.
2002; 30: 1 5 0 - I5 1 1.
Matsubam M, Masaki S, Ohnlori K, Kamilwa A, Ilusc!uwa K. I3iochcmid
P h m 8 ~ 0 l o g yZOO?; 292 : 1023 - 1030.
Okada M, 'l'okumitsu 11. Kuhota Y, Kohiiy;.?;hi K, t3iwhr1uicul and Bicrphysicul
Research Communicatic)ns ?OM:07: I 3 I5 13-76,
I-tamada R, Seikc M, Kaniijima 11. Ikctiii M. Ktrii~rnaEl. Ohtsu li. Ilurnpcm Journal
nl'Phannacology 20M; 547: 45.5 I .
Fujita K, Magan li, Kohayashi if.
Journal of Chrcjt~iatopri~phy
N IVVV;7.71 : 345
352.
Fujimaki K, 1 . c ~X -I), Kur~ia/iiwaT, Sato J, Sale K. Forensic Toxicology 2006;
24: 8 -10.
Wci L, llui %,.Dong-yaag L,, I'ci I!, Ji J. Jounial of ('hincsc Muss Spcctn~mctry
2006; 27 (4): 193-107.
Anand M, Ciandhi PUWIS,Nancy I>, (iandhi Snn~host~V,
Sahp;mdc 1';lrJmlmubh H.
International Jounial of('hcnlt~*.hKtwrrch ?010; 2(1): 1372-1175.
Suddhasattya [Icy, Vikram roddy Y, Swcllia 1%. S : n i d ~ qkurnar I), Muflliy I'N,
Sudhir kuniar sahtw~,Ilhiraj kutii;~r, Suhhasis f'atrt~ S iind Suhlrasis Moli~putt-i
International Journal ol' 1"iarniac y arid I'hiu~iiaccutical Scicnccs 20 10; 2(4): 2 12218.
(iupta VK, Jain R, Hadhapyari K. J:ldon N, Shilpi A. Voliarnrn~~ric
ra.hniquc!i for
the a s n y of phannaccu~icalsA rcvicw t\riirlyi~calRicchnnislry. 201 1 ; 408: 179196.
Shelly. W. Drapcr. M. W. Krislinim. V. Wong M. JaiTc, R . B. Ohsld. Ciyntxol.
Sum. 20OX. h3, 163.
Roslami-Htdjdjqncn, A. L m n d , At. S. Tucker. G.T. Lrdgcr, W. L. Phil.
D.Fcrtil.
Steril. 2W, XI, 1 187.
Coldstein. S. R. Siddhmti, S. Cinccia. A.V.: Ploulk. L.: I-luninn R q m l . (lpdatc
2000.6.3 12.
John. A. Linda D. Raja. W. Toxicol. Appl. Phannrteol. 1971,23.339.
Rossi S. culitor. Australian Mdicint?; H w d h w k Z(M. Adcluidc: Australian
Medicint3 H w d h w k ; 2(M.
M m k - E n g e l k c , U. Sigmund, Ci. O~litrn1:uin. (i. Ciayc-r. 1.I.Suhunxcr. W. Rtxctic
Adv. [hying Anal. ? 0 1 , 0 . 53.
Dc Luis,
D.A,: Allcr, K.; C'ur'llar, I.. 11.;I'crn~hil.('. Roti~m),li. Ann. Ma!. lnlcma
200 1, 18,480.
Rucnitz, P, hrrcndalc, K.(i~ujrgs. ( i . '!'hornpst)n, I'.
Mtwklcr,
('.
Nnnwati. N.
('ancer Rcs. 1987,47,3015.
Ruen~lz.1'. Baygley, J. Mtrklrr, r.B~tr.licm.I'tisnnncnl. 1983, 32, 2941.
Kuenita, P. Arrtndale. K.; Schmidt. W. I'hornpson. C'. Nitnavati. N.J. Mt4. Chon.
1080, 32. 192.
Rucnitz, P !)rug Metah 1)ispc)s. IOXI, 9,456.
l'uli~yK~lic-Okman,T.Kucuk. M. Allaner, S. I:crl~l. Slcrit. ?(K13, 80. 1330.
Miyake. A. Yoshirnoto, Y. tlinbtir. K.; W;~k~niolo.I{. 'f'crilki~we. N, Aaro, T.
'fanir~wa,0, Eur. 1. Ohstct. (;ynccol. Rc-pnnl. Fiitrl. 1987, 2 0 , 19.
('arlstriim. K. Fredricsson, 13. Int. J. Atidl.ol. IOHO. 3. 317.
loannidoukadis, SIella Wright, I'al J. Ncvly. H. I)cr~nol;Quinlnn, K~churtl(2(K)b).
Fcrtili[y and Sterility X6 (5): 1513.
Taylor. 1:r~rlerick; 1,evinc. 1.aurcncc (2010). Journal of' Scxwl Mnlicinr 7 (1 11 I):
269-.76.
Katninctsky, J L ~.fiemani.
;
Micah I. (2OW)). Expc71 Opinion on Invmliyetiond
Drugs I X (1 2): 1947.. 55.
Ilill. S.; Arutchclva, V.; Quinton. H. lI1tupi 12 (2) (2rMH3, IOV.
Klaus ( i . llcumann, Journal c)r('hrt>matography H, Volumc X4712).2(K)7, 296. 296.
48.
H.K. Crrue, C. GhuMt, A. Gregory. A R~~stmli-Hcdjcg:~w,
M.S. L ~ m d ,
Acrtdem~c l l n ~ t of Clinrud Pharn~ilcology, Llnlvwlty of Shcfidd, ROY]
Hallamsh~retiosp~tal.Shefield, UK.
49. Zblgn~ew Fijalek, Jwzy Chtxlku~sk~,Malgorzala Wnruwna Journal of
Elcctnlanalyt~calChanistry and lntcrfaclsl klwl~whern~stry,
!26(1 -?).I 987. 119.
136.
50.
Mw.anno M. Flacca I, de la 'l'crm S. BoWc F. I:u~'priin Journal of MLW
Sptvtmmotry 2008, I J(1): 17 1- 180.