GffiF[M#2

GffiF[M#2
]MFflIIUMEWIEW

2.1 SULPHAMETTIOXAZOLE.
2.1.1 Spcctroscopic determin!tion of tulphonchtonalc.
Nasinja er al. (l) dcveloped n i6l)id, simple and sensirive spectrophoronctric
netiod for lhe deteminalion of lome sulf. dtuss The nethod is based on the
ro.bation oforesc yellowcoloEd @ ptoduct by llE diuzolization ofstrlfonmidcs,
v'2., dapsone, sulfalhiazotc, sulfidiozi'rc, $'lfacetamid€. sulphrmethox'zole
sulfane@ine, sulfaguoidin. &d srilfldihidina followed bv a couplin8 reacl'on
wilh 3-dinopnenol in aqucous Dcdiu. AbsorDanoc of dE resullins otuBc vellow
prcduct *s Ecasued at 460 M sd 0lc cololr ws slablc for 6 dals at 27 'C Bccr s
law ws obeyed in thc conanF.lion Erg. of o.o5_8 0 FrlnL !t the wavelenBh of
hdinu absorption. Thc hcthod ws s!@6tfully cnptqtd for lle.letctMlIDn
or sulfondid.s itr varioE phmseuiiol Pepu.tio$ dd comon excipicnts 6ed
s rdditiEs in phamaccuticlb do not inr.rf.rc in th. prcPdcd ncthod
Lop.z-M.nin.2 cl .1., (2) d*lop.d ! simdtdcos mcthod for thc
dct€frinatlon of binary nixtuEs in Ph@reutical md v€tc.in ry prodncls of
rinethopdn dd sllphdneftor@l. of sulfmclhoxtTvtid&ine bv the biwiate
calihntion speclrophotonctdc n thod. I'lE r.sllts obtaincd w@ conpeed with
ttose fron derivanrc sp.choPhoton ty Th. slatistical eQhation of the method
bias showed that the proposcd Prcccdurc k conpelbh with comonlv usd nst_
deriwtir spectiopbotonctry.
cens et al, (3) sinulie.ously ddemined sulphmethordole 'id
tihethoprim in tablets witi robust Egrcssion bv spectrophdbmeut' The robusl
regssion equations sre dedvcd mathcDaticdlv lo rcsolv. the overlapPng speclr'a
For $e dct€mination of sulPhucthoxuolc and trimethoPrin e tPpropriat€
mount of sulphmclhoxuolc tlblat ws dissolred in dd dilutd with absolule
ethmol, rhe lbsorbmc. of thc aoludon ws m.asured frcn 265 to 300 m a15_m
inlcdils. The @ntcnc of sulPhdcthor@lc &d trindnopnn w€E calculttcd wnh
the dedved equalio$. Bcc/s law was obcvcd uplo 20 dd 32 !8/nl-
suhnmethor@le sd trimcthop.iF.
56
rsPectivelv' for

Liu d al , (4) iivsligarcd tlG sidukaneous spectioplotometiic deiemnrotion
of two componenls in compolnd sulphdmcthoxazolc by flexible tolcronce sinpl.x
merbod. Powdcrcd samplc conhining sulphamcthox@lc .nd hinethoprin Ms
dissolved i. anlydrous cthanol Thc aoluiion w$ mixed wilh 0.lM llcl and heated
at 5o"C on . vatcr baih for 10 min. Ancr cooliig, thc aolutio. Ms dillted with 0. I M
HCI &d liltcred. Fihmtc I nL wd dihtcd to 50 nL wilh 0. I M HCl. The ablorbmc€
w6 nes@d Aom zlo to 290 M at 2-N i .nals vr a r.ascd blanl R€sulls
show.d ccovcncs ed RSD, Esp.ctircly, of 998% and 0.38% ror
sulrhdcthoMt. ed IOO-25% 4d t.l l% for trihcthoprin Th. m.lhod ws also
applicd to thc dctcminrtid oI sulphmclhoxlal. &d tim.thoPrnn in itrFctions.
ctuccs-Bleo .t al., (5) inEnisltld thc dclcmidrios of rh. diibactcrial
drug sutpbmctho@lc in ph.mr*lticd pEpdtions contlining trm.l[oprim bv
sD.otolluorin iy .ii.r dcriEti2lrio. w r flmcscminc Wh.n both darvtes rc
pBdt a sclcctive dclihliztior with lborcacanin of sulPlmcthox@le 9w &
intens. nuorc$.nt dcivltiw with no intcrf.rcne fton thc olh.r @npoud TlE
Frction wd opdniad ro obr.h lhc b;st 6.lyric.l PcrfoMcc Th. dctftilon limit
md lhe low.r linil of qtdtilalion of sulphmcthoeolc in tt. rcqcdon ncdiun {s
t.l ngnL. TtE m.thod e$ !ppl'.d lo tI[* phmac.uliql prepmnoni conra'nmg
bo$ conponnds, wilhout dny s.pndtion 3t p! aquncd.
Ribone et al., (6) .v.lu.l.d th. analvsb of sulPhnethox@le dd
fiindl'oDri'n in bin.y ixturc, uld t-n.y Itixtu*t of sulplotelhox@lc
rnnc$opifr and phneoptridin. hydto.hloride bv spectrophoiometrv in
conjucdon with muldvnnetc p.nid haslaqldcs calibration In lhe dalvsis or
tnblels, a porlion of lincly grolnd niteri6l ws sonicakd, the solution was iiliered
md the filFAte was dilubd to IOO mL ed ile absotption speotrun was neasued t'
the rdgc 200-600 m. R.covc.ica df stbres in slhinedc nixtures w'r€ 933-
105.8% for birary nixtur.s ed 100'1062% for iemary nixtures, whilst for
phrmAceutical pEpltntions, thc rccovdies were 93-lll.8 and 86 4-99 9"/c
resplctiEly, foi binary sd tciary nixtuics

Fcm!.dd .r !1., (7) irBtis$.d a now inj.ction $Nd for simulhncoG
d.tcminllion or sulphmcrh@tc od Einctbopin bv uing Scphtd.x sP C_25
for co.li.uoc on-linc lcp.niiotr Dd olid phs. UV F nlduction. A now'duouglt
s.isor bscd @ itrtcgntioD oflpc.to otomctri. d.t ction od thc dill4nt kincti*
of sdyt.3 rdcnlion on ! 3olid 3uppo.t w popo3d for rh. sinuxd@lt
dd.min ion of sulphM.oloruol. dd F,h.thoPdn. Tlt ssor tsponds lin'alv
in rhc m.$unn8 tugc of 5G250 dd lG70 $g/nl fo. sulPhm.thoxMlc ond
tim.thopdn, 6P.ctivcly. Thc mh.dEltgcs oflh. s.isor 4 sinPlicitv' nPiditv
dd low rcrgcnts @nsunption.
Br!y.ij$! .t .1., (8) &Elop.d ! spdtoPhotomctric relhod for lh'
.lctminrtun of sulphlncthox@lc ,nd EiFdltopdn in bintry nixtuEs od in
tatlcB- Il|c .qucou solutior of th. individu.l dru85 sd th'ir bintry nirnes *ft
s.d.d on zlro ordd utl o. fist ddi*tiE .t thc swlcnglh bct*ld 200' 300 m
ud .t ln. DH of 4.5 Thc 2.!@tdq lpccf. of lhc comPouG {* conPlcl'lv
ovdr!.ppins. Ho*s. tn. f6t{aivili* ss ofi.Ed bcnd t'Pdtiotr sd hhe
timctnoDrin ws .lct mincd fron lhc $6or!se at 2376 @ wiltl n'glisibl'
@nEibution from sulDhmcthox@l. Likcwis 3ulphmc0!or@l' wa dctcmincd
al a wrcld$h of 259 m wncn lmctupnm w'e cding do ThG lin'&
c.libdtion gdpht w.a obtti..d fo|l_25 F8/mL of sulPham'lhdtzol'
N|8rnj. ct tl., (9) Md iEilbdib.nzvl 6 t tro€l couplins tsd for th' 6Pid'
3.l.diE a.d sinPlc tp.cEoPlotom.lrc d.cmin.tion 6l sulfonmid' dcrivtlits
Th. mcthod i! b.sed on tlE fomrlio! of violct colorcd e prcdud bv O'
.liriatio. of sulfonmidcs, viz 3ulftthi@lc, sulftdiainc' 3ulfac'tmid'
$nohmclhoMl., sulfsrroilc, lulf.gididie sd 5ulhdinidin' followcd bv !
couplins dction sith imjtodibdii{ in .lcohot m.diM Aborbtncc of ihc
rciuking viot t @ p.odEt * lBund it 5?G580 m sd rh' colou B stdl'
for 24 h !t 2? "C. B.ct lrw *!s ob.vcd i! dt. @lc.ntadon r'ngc of 0 05'6 0
!g/n[ ,t lnc svelcnSlh of nrximun ttBotption Thc mcfiod *!t succ'ssfullv
cmploy.d for th. &tcmidtion of 3ulforni&s in utio$ phlmeuticd
58

prcparations a.d comon axciPicnt lsd 6s addiliv's in phamacclticals do nol
interfere i. thc propdcd ncthod
Lopez Etoz €t al. (lo) dcwlopcd ' flow'inj'crion-fluoimefiic ft'lhod for
sulphame$oxaalc in phamaccuiicat pr'ptraions a'd biological fluids vd'os
sples weie dnsotv.d in cihanol ed ftltced Th' filtare sas iniected nto a str€ad
of0.olM ,-phlluldirldctvdc in 0 ?M HrPOr lnd nerged with a strcam of 0 olM ts
iercaptoc$.nol i. o ?M H:?Oa Thc tcsult'ng soluti@ flow'd throlgh a reactor corl
demostaltcd at 4O"C ud into a now ccll for lluorinctric det'ctio' 5t 412 m
(cxcitatiotr al 302 m) Thc svstcn Oow nt' ws I ml-/nin' The calihFtion graPrl
vN hta. for OOI'25 Fg/mL of 3llphamethox'ele *idr a detcction linil of ?
L@ .l al, (ll) dcrcoin d sllphmcthddole in tablets bv tu€llfenhded
sp.cEonuorimcEv. Shpl. tsblels *s disslEd sil diht€d with
anhydrcus cthanol. Ponions of rha sip'm tnr solution ffi€ dilurcd with Hlo to
giE a lo pg/nl sotudon of sulPhdncthoitalc Ponio of lc'rale buff'r o' PH 4
dd etylFnclhylMoniM bronidc w'r stdcd dd 'tihr'd with tl'O to 25 hL
Th.ouoresccnc€ int.$itv w6 ncasu'd at 346 m (€xciralion at 261 m) The
c'libBtion snPh w6 lincd Aon so'8oo nyn[ of sulphm'thox@le wfih '
detection li;it of 16 ng/n! Thc soluion vas stable for 30 nin Co-fomulated
brmehoprin did not int rfcr.
Fe.s et !1, (12) dcv.lopcd spcctoPltotom'ric iettod for 1he ealvsh of
coopoud sulpbmlthoxeol' tabl't! bv ! charS''1r&sfd re&tion Powdercd
iablets, conbining sulPhan.thoxazole' wd' dissolved in dil HCI and the solut'on
w6 filtded dd diht d wiih H2O A Porhon was tcated with of clhsolic 0 15M Pbenrcqninone.
0 sM HCl, 'th'nol 'nd H:O followcd bv aod heaiins at 6ooc for I b
md the absorboce was ncEurcd Nt 500 iin R'oov'ics *€t' >99'lo ftct Ms no
59
dl., (13) inwstiSat d colodmetric deteoination of
pham!*uticd dosagc fomt Powdetd hblet o' sNpenrion

.quivalent to IOO n8 of sllphde1llor@le w6 dissolEd and dilnted wifi ncthaol
A ponion of this soludon wd futthd dilured with nclhdnol and trc'Ld with
dethsnolic O.l% 4-di;cftyluinochidsaldelryde solntion dd sethanolic ophorphoiic
tcid sotuiion Aicr 15 ni,, thc mian[€ w6 dilded wirh n'thdol dd
lb. .bso'bdce 6f th. Gstlting $luiion was n'$ur'd 't 545 M vs a rcrgcnt blanl
Thc cali[dtion $aph ;s r€ctilincar for O 4 to 4 8 pg /mL of sulphaB'thox@le'
Zhaig et al, (14) intstiSatcd the simultan'ous dclemin'don of two
conponcnB of sutptrlmcino@h comPosite in 1,4-dioxa'e bv dctivtlive litrer
rav.lcnsft sPecEopholon.trv. Sdpl. B dissolv'd in dd dihtcd tilh l'4-
dioxanc , f tercd, 2 5 nL of th. filtatc Ms dilut'd wit! l'4-dioxua dd I nL
porlion of the solution is fuflh.r diluled wilh ),4'dioMn' Cobo'ntrotio's of
trim.rhoprin dd sulpho6.tho@1. in solutio! e dct'din€d bv ddivative tni'c_
w.wt.nsrh speclroPholoNllv eith tiicsume al229' 24? 5 dd 25?5 M for
I,4-dioxde dd 23?,3, 259 3 &d 289,2 m for ttim'rhoprin Awdgc r"ov'nca of
1,4-dioxane antl ttimcthoprin v.r. 99? dd 1OO% resp€otirclv' ed ther
cor.sponding @.ff of wiaiion wd 061 dd 039% Calibabon srlphs Mc
rcctilince for np to 20 4/nL of trnethoPiin 6d uP ro 30 rym! of
zhou et al., (15) develop€d ! oPid nethod for the d€remination or tuee
componenc in lrim.tlDPrin ed 3llphmcthoezolc conpound itblcts bv partial-
hishquar.s Uv sp.cttophoton.Fv. APpropriai' mounts of sulphmcliiox''lc '
sulphadieinc lnd trincthoprin srrndrd solution wcrc nixed @d dilutd wi$ 0 lM
NsOH. The abso$$ce oflhe sotutidifron 246 306 m is ncasured at intedah of2
nN vs o.lM Nroll. Thc oplimlm dine'sionaliiies of sllphnmclhxdorc
sllphadiuine and tdmclhoprin trcat d by panial l'6l sq@s ar' 4' I dd 8
respccdely, ed coresPonding mcan recoverics al tlE 95% conndencc levcl *crc
t9.5 + o.s7q 100.8 + O.?'/. ed 99.5 * o.?"/o Thc prcPos'd ncllod has a shon
opc.ating rinc, 8i*s morc accutc sd tliablc Gsults dd is cspccialtv aPplicablc
60

Zhotr €t at.. (16) sinultatr.o$tv d.temin d sulptm'thoxuolc dd
EinErhoPrin by sPeckopholonety. A smPle niitut ol sulPlructltox@le and
uirncd$uirn w.s .lhsolvcd in llro followcd !v tlrc nddnior ol0 tM llcl' Thc
atsort'nncc ulues of rhc solutiot *rc neasutd al 240 and 260 nn and
sulphanchox@lc tnd rrinc6@.in ec.c dercmin.d Bina qlibrltiot sdpxs
B*is law w6 obcy.d for up lo 12 ng/L of sutphd.dox@le and fiin€lhopnm'
The rccolen.s werc 99.7-lOO.1% wilh RSD of2.3-2 6% The n.thod was enploved
in ihc ua\sn of cohpoud sinomir t blctr ud inj.ction sluto's
Muoz ct al., (t4 d*lop.d t Oldinetric mdnod for thc dct@hation or
sllphMrclhoxablc in nhnmreutical prcpnations in con6in.hon with trim'lhoprin
by inchsion in Scyclodexlrivea. Po{deted tablcls or capsulcs conhini!8
sulpbmlhox@le ws dlllclcd eith ctbnol, tilcad' wNhed with lhc sc
solvdt md dilukd lo 5o bL. A gonion of th. ,olurion ss dihtcd widl
cthanol/Hro (l:I) followed by frdncr ditution witb .cdatc bufer oIpH 4 and0 lMF
-cyclodcxtrin (LCD) id 8M u@. Thc bixturt ws diluted witt HrO sd ethmol to
gi* a lf/o fin.l cfisolic conGnt Fluorcsencc neduci.nts of ihc
sulphmclhox@le - !.CD cmpld (l:l) w@ m.d..t 342 m (.xcitation ar 261
m) The calibdlion g6ph ws lincar ftom o ln Islml ot sulphmethoszolc
Dct.ction @d qudlilation limits wft 0.023 4d 0.0?6 pgln! of sulphmethoxdole'
csp.ctiEly. Fot phm.@ltical Prcplntions conhinhg sulph@thoMle 6d
irimethoprin there Ms oo intcrfftncc fion tdncthopnn up to a 2:l of
sulphaB.dbxeoh to trin€lhoPnm.
Endoh et al, (18) snrdi.d (hc u.tulne$ of .nztme munoa$av d s
Malttical mdnod for sulphm.thox@lc rcsiduc d?l.minariotr in 6inal tissnes
Tissue or plasna *m ?-fold dilnt€d vith ptosPhilc buf€r ol PH , 2, homogeniz'd
and ccifiifuged, ArtilulPhmetho@lc elibodv was imobili&d o' a nidoit'
dd th. ctuonogcnic rcadt soluti;n 2,2-einobisl{thvlb'hzdri@lincsulfo'ic
acid 1vd added. An* ! prcdefn d rimc, Na! $lulion ws add'd ro nop fi€ rcacion
6l

.nd nbsorb.ncc of thc platc ws n B!rcd al 415 m. Th€ cdibdnon Stph Ms
Pd& Z. G. cl al., (19) inresti8ted rh. dct minalion of sulphmcrhonzole
aid fiinetltoprin i! sdphd.tho@I. corposire by synchrcnous nuotimetry. Ai
appropnatc amout of dried md powdRd t bh smpl. Ms naccratcd wilh €thanol
for 12 li .fier nllratio4 thc fdhdc w6 dihted Nith no.c.draml to 25 nL.
synchronous-reming fluorin.ty w @icd out or thc solulioi wih cmi$io! &d
€xcit4tion bandpa$cs s€1 at 2.5 mt the sign.l du. to sulplamethoxeole ws
n.as@d at 270 nn, }ifiilc drc lriocllop.in signal *s m€asucd ar 234 m. The
lnr0r mngcs fo. nrlphamcthoxazolc and trincthoprim weie 0 l?-4 nnd 012-3 l
C/nl. epcctiwfy. RcoErid w 95.5-105.7o/r.
lssa nrd Anitr, (20) dev.loped spcctrophoro,netf,c Dethod for nicro-
dctcminrlion of solphdcthdal. and Eim.thoprin urif,a aliain and
quinaliaft The mclhod is bas.d otr chargc-tansfcr cobplcx fomalion of thc drug
vith rliz n o. quinalidin in !&alir. m€dirm sd ncasNha ihc al'sorbec. of tnc
complex at 510 or 555 nm. Bccls lrw w.s obcyed for 10-130 pgliL of
sulphmeihox@lc ed 10-l9O rtglnl ofitincth@rim. Thc ncthod is sinpler th0n
thc U.S.P. ncthod ud equally !ccuat.. RSD w.E 0.46-0.65%for 30-160q/ml of
sulphdcdDx@lc dd 0.58-0.66% for 2G 190 rVhL of ftihcthoprin.
Abdcl-Hay .nd El-W.lily.. (21) sinutr&.otrsly sa\zed ! rcma.y mixtuE of
phamac.uticals conhhing Eibcihopd4 sulphdcdDx@lc ed phcn@pyndine
hydochorid. using third"d.drliiE ud z@-ord.r photodiod.-my
spccirophoronety. The uplinldes of tle thlid-deri€tive spectr^
^t 242 276 ffi
^nd
w.re ne6@d for lh. d.t.@in tioD of t r.thopdn, sulphm.do@olc,
rspectivcly, dd the cbsorbecc at 450 m lor the of plu@pyddinc hydrcchlorid..
Cal'bnlion glphr wce lin.ar for 420, IG50 md 2.J-12.5 rglml of irFcihopriq
sulpnmcihoxeolc edph@eopFidilehydroc onde, respectively.
f.nm&z ct rl., (22) dceloped r Aow-ntroud scdor bascd on iilesrarion
of spechophoiobetic det*tion dd rhe diifehr tindns of retentiot/elution ot
62

anall.s on a solid suPpon for fic simulldcos dct€mimtion of sulphm.thoxtzoh
nnd tinclhoprin The inlrinsic absoddcc of bolh conponnds
dn€crly on the slid pne at 269 m. Using two tltcmat€ sotutro.s, O lmM HCI and
.c.tdc bufcr pH 5.0, rlE $sor rcspord. lincely in thc n6uing 6ngc of 50'250
aid lO-tO rtg/ml with detcction liniG of9.5 ed 0.6l]e/ml- for sulph.m.lhox.zole
and rincthoprim, respectivcly.
Fan d al.. (2, inv€stigatcd . npid and s.nsirive flow injcclion
sp.ctrophotometric nidhod lor lb. detemination of sulfadiizinc and
sulphnnElloxazole The helhod b bscd on thc ditzotization oI sulfonamid. witb
sodiun nitrile, dd a couplins rcacnon ofth. di@o conpound wilh o_naPhthvllhinc
Th. oprimuh etpcrinenl.l conditio @ obtaired by usi.g th. cont ou.d and
wci8htcd Enttoid sinpld ne0Dd, Tn Incs mges fot th. d.t mjnltion of
sulfadi&i.c dd sulphamcthMlc {@ 0.2-20|rglnl- dd 0 l'20 rryhL, lnd th.n
d.tccrion linits w@ 0 06lr8/ml- md 0.05 pgltrq rcstecliEly Th. ncrhod ws uscd
to dcrcmine sulfadi@ine dd sulphmcthoxmle in thmaccuticds .nd unnc
HBsoun!., (24) d.vclopcd simultntr ous spectrophotometiic hcthod fo!
d.t.mirarion of sulphm.thoMlc ad tin.thoprin in drugs CNsh.d tabl.ts w.rc
dissolv€d in mcheol ud thc soltlion was dituicd to loo n! Stnp dosagc foru
wcE diluled with ncth6ol. Ptpdd smpl.s *d. da\6ed bv sPecropholohctry'
with AbsoQlon mesuencnt! ion 2OG35O M at 2 m irteMls Th. spc.lrd dda
was subFded to cldsical lca$-squaas (CLs) usins lne conputcr PtosEn
Altcmadvcly thc fist-d€ri€tivc sP..6! wcrc obtained @d !$d for Eg'cssio'
analysis usi.8 lhe ONGIN comPutd progim Using the tust-deriv'tiv' nclhod
3llplumethox-otc {d hincthopdm *crc dckmined ol 283 and 240 nm,
rcspcc(ilcly. The calibr.tion Erapls v.ic linen. f.om 4-20 p8lnl for botl! dnrgs 'nd
$c mca Ecoveri$ of sulphudhox@lc and trinedroPnm wet' 10864% And
lol.47%, rd cctivcly UsinB CLS, rlrc m.an rccoveiies for bo$ dNgs wcrc l00p/o
wilh RSOof0.85%.
63

Hlsain cl al., (25) deElop.d ondrdw specEoptolomctic in.tlrd for
sulplamEln.x.z.lc in lhe prcs€f,e of trinctlbpnm in tablcis nsing Coic(lv)
Ponions oa stand&d sulphanehoxuoh solulion in lM sllphuric ncid w.s reacled
wnh exc.ss of o 5% C€(IV) ed dilulcd wiih H:O. The soluliot wns.quilibra!.d ror
2 nin wilh lO nl, CHCh and lh. org.nic lry{ Ms Emoved, dricd orcr a.hvdrcus
Na,SOr and irs a|)3orbm* ws m.Du.d at 460 m vs a rca8cnt blsk B.cds law
wc obeyed fron 0.075-0 35 he/nl of sulphd.Ihox@l€ Th. m.thod wd apPlied
to thc ealysis of sulptMehor@lc i! powdcs ed tablets also contlining TMP.
Resulls agrced well wit thore obtaif,cd by thc Brilish PhamacoDociA ncthod
Dai .t al., (26) invstigated statistical simulttion spectrophoton€iry and 'ts
applic ion to the asay for indiuduat nedlcm€nLs jn trimcilDpdn dd
sulphd.$ox@lc cmpoud tabl.ts Grcud ablers wd€ sonicll.d ti$ 0 lM Hcl
dd dildcd with o.rM NaOH to 50 nli 4 hL ofthe trlrat ls tutth.r dilutcd vidt
o.lM N'OH lo 50 nL for scmitg of absorption wilh meducm.nt of ab$Ibdcc
6fucs 217,239,24 t.243,252,254,156,25A,2a4.2A6 ^d 288 M' The contenls
of the thrcc conponeqts of sllPhMclhoxdole, sulphadieine and tiincthopnn in
the conpouid drug w{e calculaled by rh. statisdcal simulation sp.cttophoionetlc
Ren et al., (2?) inrestiga$d th. dalvsis of sdPhm.thowole bv Pl'S-NlR
iliftrs. rcflcct !c. spfttophotomcty Powd.td mptc w3 dirccdy Pack€d
into th'
sdplc hold.! of h inlegration bdl fot scsding betw€cn ll00 sd 2500 M The
resulring drl. were @alysed by padial least-sq@res in ordcr lo delemn'
sulphamethox@le, InGrlerence Aon h.$esim st€date and stnrcl was effectivelv
orercon., The RSD of pr.diction of sllphd.thoxeole in refcr'nce ud sample
fraGrirk w(c O. t l% fud 0.567', rcapcctiv.lv. Re.overi.s wcrc 98 9' ! 00 4%
K.ryosh. dd Ann.di, (2S) tihulidcouslv det min'd tulphecthoxdolc
od lriiclhop.im in co-trinox@l. iibl.ls bv li6t-denut* mR spccEoneav
Gronnd tablcis conlaining sulphd.rho@le sd himethoprin wcrc cxEact'd with
ac.tone/chlotofom (3r?) wilh sonicllion for 5 nb The extiact wss iltcled 'nd dre

liltr.rc wls injccrcd into r ni6o flov-.lU lit!:d wilh Z.S. wi'do*s and ! p.lhlcn8rh
of 0.5 m. FiBt-ddintirc FmR sp.cr.! wr. rcco.dcd ftoh 1650 to I l0O cm r.
Simllrln.our rLr.mintiid of sulDhndnox.El. sd Eih.rhoprin w .Ifat.d .t
I 166 cm' a.d | 138 cn', cpdtiEly, Ti. c.libdtion grtphs wcG lin.u up !o 12
$d l0 ng/nL for sulphm.dio,@lc ed timcthopii, r.sp*tiv.ly ud thc
dcrccr'on lnnrs wcc 9.a dd 1.0 rynL r6petiEly. Tnc asulc wcrc in good
lEcmcrt with tfiosc ohrncd by ttc bnlilh Ph'lrcpei. ofiici.l n.thod. for th.
Su ct !1., (29) dct tuitud lrlpbmdnoxltl. in @mpoud
sulphdcrhox@lc t blcts by fBl ddintiE r|tio sp!.EomcEy. Po$d.rcd hbl.G
tuplc cbnt inilg sqlphrncborlzdc liincthoprin. c'crc di$olrcd ed dilut d widl
O.lM NrOH. Thc .b6orbsc of thc $l!tio! fls n.socd tt 2J0 dd 252 M,
Gsp.ctiEly. Drrs wcc obt hed sd rhc fi6t ddiqtiw drio sp.ciM of
sulph&ndhorszolc eG u.d for th. !D!l}si5 by lpplying th. P.ak_zlro n lhod .l
25t m. Thc cllibntio! griph s lin.at non l-15 Fglnl of $tlptMcthqazolc
Th. mcs @vcry of ll5 fdlllof .dded salphmdDxtzolc sa 100t6.
Mih.d.re .r al., (30) inrlslisitcd r r.v fiBr{.dwtirc phot@hdidlly-
induccd lluorcsccnc. dahod for thc d.lcmilalion of sulphm.lhoxMlc in nilk A
50:50 (v/v) nEtlbnol:wlcr mcdium, Uv idditliotr tnc oa2 nin, 8d pll 5 7 wcrc
lix.d, Sr.nd.rd cdditiotr ncthod * ed with lh. d.riEtis !p4lr. of thc incrct*
spcctri .s andfiol si8mt. For sulptuctttor@I. dcl.min.tion in doss, dicct
inccs. of fluoesq@ sigr.l n.s@ M usd. In both c4t thc ncthod ws
suihbl. for suloh.mct[o@lc @n4nt tiotr' bctwen 0,5 dd 2.5 PPm
2.1.2 El.ctroplorcticd.tcmlutionof rElphltnclhoretc
Tdhiro cl lli (31) d*lopd oPilluy clc.EoPhorctic ncrhod for lhc
sinolreou det@i!.ti@ of $&d.tbox@l. dd Eimc|lioprin in Dlsni.
Sulfmcthox@lc ud ttin.thoDriD crrtlcGd &oF hd.n plttn. rvith .lhvl &.t t.
*cr stlyed .t 20 kV ed 25 'C $in8 l5 m phosphdc buficl GH
6.2) $ dtc
cldFol},l.. ThG d.t ction w by uv.l 220 M. Tl. limit of quntilication wa
65

10.00 pslnL for sulfametho@lc &d 2.00 */niL fd i.rm.rhoprim. TIG r.mv.ry
w6 >99% for bolh @npo!n&. Ttn nerhod enabl.d lhc d€tection or
sulIaneihoxAzolc lnd hin.thoprim in plasha of patients ,ft* orll ingcstion of their
Edas Ncvrdo ct al., (32) d*lop.d a c.pitl&y anc .l.ctroPhorsis n.ftod
to sprst sulfadi@inc, sdphmethoxe|., irin€fiopim, bronlExine and suaacol
by uinB r tusd{ilica .apillary- Ttc *prntion wd dicd out at 30 kv dd 25
dcerc.r C in a I 5 nM phosphatc buffd ldj6tcd to pH 6 2 as cl.croltrc under th*c
coDditions, thc run iimc ws 6 mh ed the linih ofqudtification w.rc about I mg/L
for cvery componcnt. The method t€s aPplicd to phmecutical prcPardions dd
rhe esulrs provid.d recorc.i.s clo$ to l00el..
Li et al , (33) dchined fie dis@iatior conttants of trincthoprin dd
sdphmcfiox@le md their @ncdiitioB in etdnor@lc r.bhrs bv capilary
a.e cl.ctroPhorcsis.Trinethoprim ed sulPhanehox@lc wcrc s.pmtelv or in
combimrio\ dctcmimd by CZE, on s u.coatcd fus.d{ilica caPillary op'raled 'l
e applied voltsge of 20 kV, with 30niM acdate buffd of pH 5 55 as ruming bufer
and d€tection 254 m. Thc cclibtation grlPhs for trimcthoprim md
sulphafrcthoxazol€ wcG linw &on 21.6'472 6d20.2404 mdL rcsp€ctitlv' wilh
coft€spondine dcl.ction limits ofo.48 u.t O O28 ng/I. rcspcctiElv RccoEi6 w4
99.6-10l.5% Thc dissociation conttols of trinethopnm a.d sulphdethox@lc
wer€ 6.6 dd 5,9, .espectivcly. Thc li.thod w.s also applicd io th. dctcminadon of
Trinethoplii nnd sulphmethoxMlcinco'trinoxuole tabl.ls
2.1.3 Tifinetric nethods for tulphanethoxazol. determin.tion.
lsopoulos., (34) inv.sngat.d th. @ltcis of vdo$ rc'v slirhtlv wah
sohbl. dru8,s in Nccud DcdiM ed d.l.mincd sulPhddho@le in prc fom
by poFnriomcFic dd visua! tihltion sulPtuetho@tc ws dissolEd in aquss
o.o5M ccryllrimclhylamooiu brcnidc aurfact nt 6d dihcd to 200 mL Mln dE
sme solvcnr A podion of the solution conlainine 10_30 ot 150'500 ng r"s ntraled
pokniionrclricrlly wiitr O.O5M N.OH,.nhs nanuallv using th.66i and second
66

d€rilalivcs oflhe titralio. cutu or altomliicolly at pH 7.51 or 842 for the rwo
sulphmellbxszole conce.tralions, resPectively. For vnnal titrntions, ten drops of
phenol rd o! phcnolphthalein wcr. 6ddcd to th€ sulphmethox@lc aolution beforc
tilrltion Th. dDg was detemincd i! thc mg. 0.051-0.5mM Th. rcsult3 agr€ed
wi$ thosc obt.ircd by the ofiicial n.thod,
Abdut Kual Ner ct !1., (35) dcrclopcd lndircct porc imetric tiEation
medrod for lhc dctemi@ilon of sulPhmcthoxeole in tbc pEs.trcc of ttimethoprin
ih co-tiiraale kblets nsinS copp.t bascd mercurv film .lcotiodc Ground t'bl€ls
were stidcd wilh O.5M NIOH for 30 dn Aftd filiratiod, the filbate Ms adjsted io
pll 8.2 wnh O.lM Naoll/o.lM llNOr lhcn nixed witt 0 2M t ictl'nNlmine/nitmt€
brfl.r of pH 8.2 and o.lM ASNOr' UM.actcd AgNOr wd titraled against 0 rM
NIISCN 6ina . Cu-!6cd H8-filn .lccrodc Undd optinum co'ditions rne
metl$d enobl.d itcrmination of l-lo ng tulphde$orMlc wnh FlsD (t = 7)
L32% and rccov.ry of 99.88%. No inr?rfcmcc * cas.d by crcipidts Prcsnt in
dosagc foms R.sults obtained for sutphanehox@lc in co_trin@l' tablcts
conpscd w.ll wih those oftu BP mcthod
Lidg €t al., (36) inwstiSared qumtitative d't'dinalion or
sulphamdhox&olc by seodd{ditn* dif{€rtial pnls. poldoSraphv' Slod&d
sutph8cthox@L solution, s di$olrcd in 50 mL IO% fiCl bcfoG addi4 50 nL
HrBO,/KCI bur.r ofpH 9 dd H'O lo 100 n! Sdond{"istirc dillcrcniia! puls'
potdosnphy was Perlomed with ncsutencnt or the peak at - 1 45 v (vr As/AscD
fte cllibtatioo Enph fot sutphan.thdatulc was lin€d non 008-08n \4 wtth
d.kction lioil of 8.6nM Excipicnts did not interfeE' Ihe m'thod wAs applied to
sulphm.lhox@le tablets, wilh rcco\rry of loo% @d R'sD of 0 5%'
Zh.n8., (3?) detemincd thc @nt nt of sulPhm'thoxdot' in conpodd
sinonin labl.rs by intcmtl idicalot n.lhod. Grcund tabl'tr cqui€t'nt lo 05 g
sutphlmclhox@le wcG dis$ltd in 25 n! *m HCI (l:2) $d 25 hL H'?o An€r
cootin& rhc sohtion ws EPidly tilit d with o lM NaNO, Bcforc th' cnd-point 3
dops of o.15% methyl€ne btue dd 4 drcps ot 01% o@se vellow wcie added @d
61

h. titralion *d continu.d until a grc.n.nd-point ws ob3eftd Tte Esultt wct.
comped with thosc obraircd by thc d.ad+top nc6od 6d speclrophotonetly.
Liu d al., (3 8) delemjncd sulphm.thowole h conpoud sinonin tableb by
tirrinetry. Finety Brbund r.blcb cquihl.nt to sifrild, eqnalso.3 s of
sulphanethoxMle wde dissolrcd in 20 mL of DMF. After the addilion of l0 mL of
Hlo and 5 drol)s of phenolphth.lcin, rh. ninure wd titratcd with o.iM NaoH dd
E conc.. of sulphtu Ahoxaelc wos cnlcllated, R.ooveries vere qndnlatirc cnd
resuhs agreed with those obtained by both dual wavelenClh sp€cFopholomelric ud
Abdul Kdal et al., (39) d.vcloD.d . sidplc ed mpid indit.t por.nlionclnc
tination of sulphm.rho@le ir thc prc*lcc of Eimethoprim contailed in cotrin@l.
lableG. The methoil is bs.d on th. fomation of a @mpl€x ol
sdphdcthox,aL wiln a tnoM .163 of silw ions dd thc titatio! of lmer.d
silEr ion potentiomehically wing s in?xp.nsivc lab-m.de @pper bdcd hdcury
film €lstrode (CBMFE). Thc titrlion conditio.s wcE opdni4d for fic
debminado. of |0-10.0mgof iolphMcthox@I. in puE &d dosaee foms Thc
pr€cision md acc@cy of thc mcthod w.5 4sc$cd by rh. aPplicalion of lrck of fit
test sd othd siatistical B.tho&. Oitioll n.e rcotrry dd Elative sianddd
dcviatiom obtained weE 99.88% sd 1.32% (n-7) respcctirely. No interrd.rc. was
cEused by other ercipieni. prcsnl in phannaccutical dos!8e foms. The applicaion
olihis mcthod for sulphaftelhoxoale 0s5ny in lhc nasenc€ of trihethopiim in lflblets
ws di.hted by the conpdsor of tsulk obtaicd by the proposd nethod with
that of rie Brirish Phmrcopoch (BP) n.ihod.
zhd el al., (40) Itrvcstigdcd oscillopoldog.aphic ph@entictl @lvsis md
ddemiied SMZ Gulphu.thouol.) md TMP (ttinethopdi) in sinonin
conposne t blers. Tablets w.rc pllFi*d dd colv.rtcd into a sludon for dnect
detcnnindrior ol knbdhoprnn nr a suPponins .lstroly|e of I M aceric a€i todiun
acetak by oscilognphic volhMcEy with mc6@ncnt of lhc peak potcntial at ' l 5
V The calibration gEph *6 lin.u ftoE I ?.3pM to 0 346n vtl. For ddeminttion of
68

sulpnamctnoxazole, 5 mL lhc lcst sohiion w.s diluled wnh H,O lo 20 nL and 20 nL
6M HCI and 0.5 g (Br wcrc added prior to oscilloBraphic chro.opoiennoner'c
tirotion with KBrOr slandnrd solulion ns tifiaot uniit incnions .pped.d (€nd-point
There w.s no co-inteiference beNecn th. two drugs,.nd excipi€nb did not nrteder€
eilher. Resul$ werc comparabl. with tho$ publthcd in the Chi.cse Phamacopoeia
2,1.4 Ch.onrlogrlphic d€terminotion of 3ulph.nethoxrzol€:
Akay fld ozl(m., (41) d.slop.d ! sidplc, sdsitire, prKise a.d npid
rcvds.d-pnas hish p.rfomecc liquid clrcmalographic ncthod with ullraviolet
detection for rhe sinult n.ous e.lyr'! of rimcthoPrin ud slbnmethox@le md
applied to rh. dctemination in @mcEiat pham.ccunc.l pftp@tions. Thesc
mmpomds @ welt scPmtcd or t Bon&Pal C(18) Evesc ph6e colum u'ns a
nobih ph4c @nsisrcd of . mixturc of mcrbdol:wdcr (60:40; v/v) adjNted ro pH 3
wilh lf/. orfiophosphoric &id .t r llow hl. of 1., ml-/nin. Thc proposed n.lnod
w lin r in lh. mgc IO,G5O.O $/n! for sdpndetho@l. The linit of
dctection ws 0.45 ed l.2l Fg/nL for !'ncthoPlin dd sulpbdethox@le,
Van Eeckho €t al., (42) dcrcloP.d a scnsitiv!, ltsh perfomace liquid
chronatoeraphylmdcm nss spccrmcttic hclhod with on'linc dtraction dd
smplc clem up for thc scrc.nii8 ed confimation of Gsidues of snlfonanides in
kidney. The sulfonamides d. cxt$ctcd ton honogedizcd kidnev sith nerh&ol
Afier centrifugllion of lhe cxhact, m diquol of thc ext act was di@llv itrject d on
ite LCA'ls/Ms systen with furthr dtraction and clem up of fie smple onnine
Detection of the ealtl.s ws .chi.vcd ty positive elccFospnv ioniztion (ESl)
Iotlo{ed by nullipla rcaclion monito.ing The ncthod wd validated for
sulfadinethoxine, snlfqni.ox in., sllfdcthazinc, sulfme@ine. sul&thiuole
sulphmethox@le, sllfadidin. dd $lflPtridin. Calibotion curcs resulthg fron
spiled bld* kidn€y smd$ ot th. lO-200 Fglkg lcE! showed g@d linear
comlation The linitt of dctcctioo (LOD' ruscd Aom 5 ro 135 Fglrs Th'
recoyeries tuged fron 78 to 82%. Th. mcthod is sunabb for roulioc malvsis
69

Hltlcr d al., (43) d.rclopcd ! fst sd @n .r.ctiE mclhod to .xtnct od
qumtily rcsidues of vct rin.ry .ntinicrobial .g.nt! i. anin.l nduc by liquid-
liqlid dtr ction .nd liquid clsmliognphy-nass spcctromcty. Th. codPounds
inE risat.d includ. six sulfonmidcs" on m.tlbolilc, ud lnncthopdm. Tlc ndhod
was O.domcd wiihoul 3mpl. cld up, tulatiE sldddd dditlioB ot lhc
r.cov.rica wfi. la the 5% widri! t!. smc d.y rd l.s the 20% bct*r.n da's.
TIE linit of querilioiion w bclow O,l mg&a liquid nduc slurry for .ll
conpouds 6d @lib6tion cufts ottliftd fton *b.ct! of tPik d smplct w.rc
lin d up to.l4l of 5 ng/k8liqlid '@e, .ept for lrincthop.im.
Klin.s sd Moky, (aa) datdLd ctmlbgnphic .onditiolt to .nlul..
modd nixtue of fod sulfoMid6 Gdphmdhox@Iq sulfmoxolc, sulfisoi@lc
dd eulhm.thoxydidinc) n phrh! using ! tffi.d phs. HPLC Cl8 coll|m dd t
nobit phsc composcd of bcth{ol &d phosphlic bufiq (60:40; v/v) .t pH 5.0.
Thc dcrccrion Ms nrd. using ar .lecEochcmic.l dct clotr tith thc Pototi.l sct at
l.! V. tn cas. of srlfondid* d@bcinttion of dM! with nctheol was nor
.d@tagcos rhs .xtrction with oErnic aolcnts. Thc rccov!ry of &alvt d
rtrlaon dcs N$ n' ltc hnsc oa 95-9?6, ll[ dctcclion liniG *cru l0 nysl ol
xu ct rl., (45) inrcstistcd HPI,C &tcmination of ditslution of compolnd
sulph!'n.thox@lc od trinclhop.im lsblctt. For th. HPt! n.$od, lhc Dio6il
Ctr column wos usc4 sd th. nobiliphe qs n.thanol-brrcr tolution (a0:60),
th. now r . wae | .O nrl-/mi|!' dctcction sEl.ngth {s d 240 M, inicction rclunc
w$ lo pl; hydMhlodc !ci4 dktil.d ${t!r dd pH 5-8 phosphst. blft r. Tnc linctr
rcepon* tulcd fron loc5oo rryol- of sulPh@clhor@lc. Thc n.tlod it sihpl.
Bc6s Nchdo .t -d., (46) tinulblcorilv d.l.nin.d
sulphan.thornyrid@inc, sulphmcfior@1., 3ulPhdimchoriic, iin.ihoPrim 6d
bomhcxitrc by HPLC otr ! K$n6il Ctr @ludr slidicnr chtio! with ciFdt buffcr
of pH 3 (mobil. phe 7{yh.thuol (tiobilc Ph$c 4 tnd UV d.lcction 3t 255 nm
?0

Th. olibntion gnphs EE lincd fot up to 16 tru/L .id d.rccriof linns src i. th.
Ms. 0.05-0.34 mg/1. RSD w4 {.5% for 4 mg/L of .ach sulfonamidc. Th.
m.thod wrs applicd to comdill ttrcm.ry pcpurionr,
Fcns ct .1., (4, ihutlNaly deGdircd thrc. compo..nr5 or
trincthoprim-sulf.diehc-lulplMcrb@lc tsblcts by thin{ycr c.lmm.togFphic
rctNins. Powdccd t!bl.t! cod.inhg sdph.diein., sulphm.tlor@lc md
nin.thoffim scr. dissohd 6d dilutcd wirh cihdol- thc solltio. wa filt r!d. A
ponion of lhc lilrdc w tpplicd lo iilic! gcl CFrs lnd tlE platca *cG d*lopcd
eiilt ctlryl lccolrcHclr (2:l) $r@t d with tLO. Ttc phld w@ s.am.d !t 2?5
nm for r!lph.di&in., 265 m for sulphmtho'@lc ed 285 M fo. rimclhoFin.
An .xt nd st |..d n.thod slr os.d for qurntir.lion. Th. elibntion gnplB Ec
lin.a. fm 0.044.65 Fg of sulphdctho'@lc. Th. rc@wiB wc 98.4-100.r%.
VE. .r .1.. (48) isl.r.d, idmrili.d od dcr.nincd sulphh.rhoxblc .nd is
k'oM n.r.boliG3 h hMu pl.s ed uirc by hish-pcrfoBa4 liqrid
chron.rogopty.Fd lll the ]\/-hydro:y nctrbolic, dicthylainc v.r .ddcd ro
inpow .itlytc solubility. Udlc sMpl.r w.rc c. ritug.d ed thc aupc6 art
src dilur.d by addirion of phosph.rc Duller of pH 5. PI'lm wB d.prctcitrizd *ith
lccto.ilrilc. HPLC w3 wilh . 5 t&n Sphcd$rb ODS colunn *itlt . P.lliculd-p.ckcd
sutd @lum dd t lind-8ndic nobil. pha..
Ahy dd Ozlrn (49) d*lop.d . siEplc, s.BidE, pFi* ud 6pid
rcvcBcd-ph8c HPLC mctnod with Uv &tFlion fo. th. linulta.oB dctcmimdon
of Ein rhoprim sd rulphh.rho@1. rd .ppli.d ro ..mcrchl phmreutiol
pr.p.ndons. Thcac.onDoud5 e wclt rcp.nted on ! Bddap.k Ctr Mcd-phac
collm by usire ! hobiL ph.sc co ininS of .q!68 60010 E.th|nol djBtcd to pH
3 with l0e/6 onhophosphoric &id rt ! flow 6tc of 1.8 hvmit- Thc n.0Dd wd
lincd in thc nng. 2.G10.0 qlrnl, for Eincllopdm and 10.0-50.0 r8lnl fo.
sulpldnc$oxrzolc. Tlrc LOD wcrc 0.45 and l.2l ltS/ml foi trimcthoprin and
sdphdcthox@lq rcsp@tircly- Th. trEthod w rpplicd to ihc 8sy of comcrcitl

Thc c.lib.lrion g.lphs v.a lin.u for up ro 16 ns/L rnd ddcction linits w.c in lhc
ong. 0.05-0.34 mg/L. RSD wcE

tablet a.d oral suspension dosagc foms cont.ini.g
D.teminalio. of sulphd.thoxMolc 6nd rih.lhoprih in Injcction by HPLC
wN nrvcatisatcd by Chi nnd Yu, (50). S!'Iplc wns dilutcd wiln mobile pl'asc Tl'e
solutioh were separat€d .nd det min.d by HPLC o. a HlTersil ODS2 colum ,
opdated ar 35'c, with KH:Poy'ncthsol (75:25) s nobil. phsc dd dct.ction al
230 m. 'l lE .alibdtion Sraphs for sulphm.lhox@lc a.d tri'n.thopnn weE linear
fron O.O5-0.85 md O.0l-0.1? F8, rcsp*tiv.ly. R.covclcs wn€ 99 5-99 r"/' with
RSD of L2- l .36Y..
Kcbria*ad.h .t al., (5 l) dd.lop.d a higlt-Pcrf@ce liquid
cho@toSrnpiic n.dlod for tlE sinultt!@u Ealysis of slbnddhox@le tnd
tinetlbprim in humrtr plish.. Pl.snr smPlB fton adult subjccts ontainins
sulph@crnox@1. dd sllPhd.thox@lq wr. vond-mix.d wirh Ph€nol 3olution
(intend standdd) md pml|oric eia rcl[ti@, Ttc hjxtu. va dlrituged ed a
pmrion of supcmai.nt B 4.l'5.d or ! Novqal Ctr &altdcal colllm wilh
acetonitrild2onM dialmonim hrdrcgcn phosphatc bufer of pH 6 (13:8?) a
nobib pnase and dcLction !l 235 ni. CalibFtio. 8Epfis wcE li!.d uP to 60 dd uP
ro 2 Fglr[ in plasm!, resp.ctiv.ly, for sulPhdc$otszolc dd iriDethopin (/ =
09993 md 0.993t, rcspcctitly). Thc dctcction limits of sulPhamelhoxuole md
trihefiopdn in plasna wae lO ed 5 ng/ml, rcsp.ciiv.ly. Th. ncthd M appli.d
to a phamacokinedc sNdy of sulphm.thoxazolc dd nimethoprin in plstu of
Roefln d dl., (52) detrlopcd i Evdsed-Phsc high'perfommc€ liquid
chromarography method fot in. dct dnin.lion of colrinoxuole i. childrcn realed
for naldia Hcpdi'iad pls,sa wcs vort xinixed for with lM tichloroac€tic acid
and centrifuged dt 35OO g for lO min A poniotr oflhe suPem.t.nt w$ nix.d Nith
nobile ph6e, modilied with lM NaOH. A ponion of thc resulting solnhon tas
injecbd into a Nova-Pa* Cl8 colud, cquiPPcdwih a Rcsolve'tM c13 prcolum
12

Md opcnrcd with thc nobilc ph$. Al I nunin ald da.cdon .t 230 m. Thc
olibdtion clre ws lind | -300 rtglhl fd lllphrmcthorolc.
Fes .r !1., (53) tinult rcouly id. ificd 3olphu.Oox@lc, lulph.didin.
sd t imclhoplin h t!b1.t5. S.hplc.ontlidng sulphdcth@1.. obtdncd fi6n
powd4d LbLt5. w3 di$olrcd n elonc. Exoin$ion of lhc cluonAtogan ws
@dc. UV .1254 M. sotphD.lho@lc, tulph.di&inc sd Eih.thoprim 3pols v.c
d ,Ilr nl@s of 0.82, 0.7 and 0.56, FspedEM tm. .s thct 3t ftLnls
Rcncw sd Htuns, (54) dcvdopcd ! rebut $d $Biti€ ncthod fd thc
dctcction of fluoroquinolonc!" sulfomidcs dd irinctlopdn. Th. .nr.ccd
$tibioriG w4 e.lyrrd lsing liqlid chroruiogEPhy .lccrrosPdv ms
spcctom.ry. scl.ctcd ion mo.itodn& Qurntificltid of$tibiotica w$ a$sd
both by intdsl dud..d ud st'ndrd .ddition ndhods- A€68c e@uiB for .
6nE. of Mitcwltd m.td6 *G ?7 ro lzyh tq , I F8/I- spi*ing condh.lion
Thc mcthod dctcction linits of $tibiotica in d.ionizd wttcr, fm.l 6d i.cond.rv
cmud dngcd from i lo ? ns[, iotn 20 to 50 ndl, and fren 30 to 90 n8&,
w0ng ct .1., (5t) inEstigrlcd thc binding of tulPhmcthox@|. to h!m.n
*rum albumin by ! @mbiEd_ tchniquc of nicredi.lFit wnh liqlid
chrem ogmphy. Hunan scm dbMir in PhosPhlt. bu{t r of pH 7 4 *a mircd
wirh sulphmcfiox@lc sd fic $l'tion w incuba|d st 37oC A nicroditlsis
probc Mr plac.d in th. 6irtu., *it[ photPh . blffcr of pH 7 4 a thc p..tusion
slutio.. Ailr t2 ninut s thc di.$s!t w.! @lLctcd for 30 nin !t . p..flsion Fr.
of I Fvmin. A podion of thc di.ly$L wu .Iily*d on ! SPh.rilo.b C 18 @l!m *ith
aqucoB 40el' Dcthanol condininS &clitc blffd of pH 5 9 s Fobil. Pta. md
dctcctio. .l 270 M. Unboud sulphd.thox@1. conccniratioN and binding
foctioG v.rc d.t mhcd. Th. nca! rc@vcry of lh. dd! w3 41 8%wi&. RSD or
2.3Yo.
Zuo ..d UchikM., (56) linulbnco$ly d.t min.d sulphldiu,nc,
sulphad.thox@t. dd trih.thopin i! multidrug t!bl.t! bv RPLC sllphadi@n.'

sulphafretho*zol€ dnd himethopin solution w.rc s.p.nted and deiemined by
HPLC on . YwG-CjsH? colurn opcrnt d .t 4o!c with phosphale bufl€r oftH 5.9
/m.lhrnol (4rl) 6s hohil. phdr. ud d.t.ction .r 240 m. The oatibration eltplls for
sulphadiuine, snlphm.rhox@lc sd trin.thoprin werc lin.ar lrcm 24-120, 24J20
and 9.6-43 ng/L, respccliEly, Awrgc r.cotrrics wcc 99.1-1004%, with RSD or
0. I8-0.85%. Excipiedts did nor intcrf.r..
Yang €i al., (lO d.rclodad HPLC mcthod fo. lhe detedi.ation of
sulphmeihoxeol., trin thopdh rnd pcdnilo.c e.tate in iableB. Sople Ms
dissolv.d od dilured b | 00 mL wirh Ecthdol md liltc.cd. A ponion of th. filEatc
B r€at€d wnh ph€naccin (intcmd 3htud) in m.thdol dd dilut€d with nore
nrctl,lml. Po.rions of rnc solulior wcrc m0lysd fo. sulph.ncthor@lc ,
rrimcthop.in , md pEdnisotrc leliitc by HPLC on a ]'wG-CtrHv colllm with
aqueous 70plo frethanol adjudcd ro pH 6.6 with gleill acctic .cid 6 nobile phse
dd d.rdion ar 240 m. C.lib6tion gEphs vcc lincar fron 20-30, 4_16 and 0-5-3 5
'1elml, espetivcly, for rulphmcihorelc, lrih.thoPrin , 6d pEdnisone lc.lat€
Moorc dd Brcu*cr., (58) inv.stigricd hich-p*fommce liquidcbrematoghpnic
cvalmtion of rh! Jf"c of mut t*ur."nr of timcthoPnn &d
sulpbmethox@lc stability in scM. Sttnded scM sdplcs wcrc spiked with
sulph@ethuire (inr.md st !ddd). Onc lhird of th. sdPhs w.re beated, @c third
werc kept ar 37"C md onc third wd. 5lorcd at -20'C for 2 months. Portions of
reated and lntrealcd saDplcs q.rc nixcd wiih ciEatc blflct of pH 5.5 dd the
minm qas applied lo . pr.condidonid Ctr Balctbond SPE cartridge Trimcdrcprn
and sulphmethoxuolc w.r. .lui.d *iih ncthdol, Thc clual. ws €vapo6t.d io
dl}aess uder a sheam ofN, at 45'C Md tlc rcsiduc w6 dissolvcd in mobile phae
Portions wcE ualys.d by HPLC on a Bondapal Crs colum with r% acet,c
acidacetonitile (4119) as nobile phasc sd delecdon at 240.n No sratisdcallv
sisnificdi diffdences wdc foud b;sc.n th. untteated and ttaied souPs for
T.inethoprim dd sllphmclhoxuol.,
14

2.1.5 Inmuno.$ay t*hriqu. for ulphsm.thorarolc determinrlion.
Yang el .l (59) dcv.lolJ.d relatircly sinpt. coupled solid_phse daaction
tadioimunosssy method tor scrcdi.g anivo! nonilo.ing tetracycline and
sulfoiobid. compounds in walei. Thc rcsulls indicate thnt TC, oxtreEarychne
(oTc), ohlortetracycline (CTC) of tts invcstis.ied 5 Tcs, a.d sulranethdinc
(sMT). sulfanethoxdole (SMX), sulfadincihoxd. (SDM) and sulfathiMole (STz)
of the inwsligated 6 SAs in wder rnatrix crGs-rcact to a simild degEe within each
family in RIA.nd SPSRIA. SPVRIA mAlysis of lh.sc srnl,les wG compated wilh
sPMiquid chrooatoer.lhy (Lclm.ss spcctomcty (Ms) qlmtification of 5 Tc
md 6 SA conpounds. R.suhs of ln. study ildic.t that SPE/RIA cm he d .f.ctivc
rechnique for no.ibnng etibiodd conpo!.ds in varcB susPcctcd ro b.
cont ninated with rh4e cmp!un&. Thc @uplcd s.lhod provids a sufljcicndv low
dd.crion limil (0.05nicroc/L) to scrcft l&gc a@Ple s.ls .r dviromdlallv
Kalnytova et al., (60) dcsiE+qi ! dss s64Ptiblc itmdos$or for FIA ol
s fuethox@lc rcsiducs in liquid ptudu.is. Tle imuosedor is bocd on
piercelectric transducer. Haptcn-poEi! conjugaic (SMX-Di@-BSA) imobilizd
on the p€ljni.@ly sildi4d al.ct od. surf@. oI piraelecFic qutrlz crystal wis
uscd s lh€ lioEc.ptor costi!8. OPtimialior of $c FlA conditions pcmfted to
develop a sinple and exprc$ procaCulc for oncatep detection of sulfmettoxele
in a smple ud fudher rcgcn r6iiotr of thc biorc@ptor lav* 'fhe measdi.g lmges
re I lo 50 ne/ml ed the detcction limit is 0,15 n8/ml. 'rhe det€ctio. rcsuhs wcre
conpded with the IIPLC data Th. tduoiag* of the new Procedure ae ils
simplicily ud opid provision ofthc elrsis r.sulk.
15

2.2 FUROSf,MIDE:
2.2,! SDcctrothorom.tric dctominorion of luroscnidc
F.na.o cl al., (61) simult.neously dci.mined furcsenidc and amiloridc
hydeNo.idc ia phmaeurical fonuhto$, throuSh speropholohctric panial
lest srtu.rcs (rls-l) n€tlod- Po$dcEd t l,l.rs w.E.xhct d with ordrrnol ud
wce dilu&d to 50 nL eith H,O. A ponioi (l nL) B tat(cn and flnhcr dilut d to
25 nL siin a@eons 50elo meth&ol. Thc absorbee of the diluled soluiion was
scanncd from 250-150 m dd the data vcrc Fssfered to a PC for chemoncbc
evaluaiion using the Mailab 5 3 p!o8r4, Thc resdis for synthetic nixtur.s of 8-13
nCL of furcsemide md l-i.6 n8& of miloride byrliochlorid. eAv. r.lalive
sldd&d deviarior (RSD) of 0.1-1.3ry. ed r.covcn.s of 99.59 ,nd 101.047q
esp.cdwly. For thr€e comrcial fodul.tiotrs, th€ Bdi! w.re in good 4t .n.nl
sirh rlDsc obt.ined by a standard EPLC nclhod.
Ag.tonovieKubin ct d., (62) spccEophoton Eic'ly det mincd furosmid.
and its paUadim GD dmplex. T.tilch wcE exEacted with Mt r dd thc solurion
Nas adjustcd io pH 9. A potlion of thc soluiion Ms h€at€d with 50 nM PdCl2 itr
HCl, 2 M KCl, Britton-Robinson buffd soluiion GH l0) md HrO. Afiei inixins lhe
absorltion was mcasued at 526 M Bccas law w.s obeyed from 0 25 to 3 5 nM
frus.mid.. Th.lower limit of *nsirivity ras 8.41 Fg /ml Recoverv of furoscnrd.
from hbl.$ dd @poulca Mr 97,2% sd 98,57q rcspectiwlt lnd thc cocn or
mriatio. w.s 2.5%.
Lin cr al., (63) €stablish.d E 3p.caophotonetric nethod to nc$u.c thc fc.
fscion or hisl y-boud drugs in 3crun for a @se of non4t 'oid.l dri
innimnalory dtues (NSAlDs) &d for ftucmide The nethod dep.nds or d!
pri!rciplc thai drus rmsir fron dialysti. lo setu6 is p.oportional io sctum binding
reqnncs neitherisotopic dru8 prep.Btiotu nor spccifio drug 6says, is iMependenl of
.xtr.ction efliciency from thc diil$.le and requires no mesuEmcnB from lh€
16

scr!'b co,npatuent. The 'nclhod w$ valid for ! folFfold ch.nse in scrutu:didlys.ie
Incs Tonl et al., (64) simulrd@Gly d.t mh d miloride &d fuo$nidc in
phmac.uric.l fomdatios by f6t digrhl ddiEiiE sp.cFophotohctry. Thc ddgs
wcr. ext&t€d fron foduhtions uing lIC!, diso|v€d in cthdol a solv.nt.
Subs.qu.ntly the smplcs werc .mhat d dircctly by fiEt disital dcriEtirc
spcctophotometiy, The sinultmcos dclcmh.tion of turcsenide sd amiloridc
wcre cMied out at 241.4 ed 343.6 m, rcap.ciiwly. h both cases, thc zcro ctossng
nppro.ch wos used. Wh.n both compoods e. pr*.nt log.thd h i sampl., it B
possible to qu&tiry one in thc pns.oe of thc orh€r, without mutual in|rfcrcncc.
Thc dctcdnirahotr dgc w foud to h. of6.9 x lor lo 16 x 10 r dd 6,8 x lo3 to 8
x lor mol/L, for eilo.idc dd irosmidc, ..sp.ctirly. A c@d ld.l of
rcpcabbihy GSD) of 0.9 ed 0.6% ws ob*Ncd for amilo.ide tnd furosdidc.
rspeii!.Iy. Thc m€fiod ws lpplicd 10 lhe d.tcdimtion of th.se dru85 E
plBmaceutical fomulatioc tilholt iltcrf..cnces ftom @mon cxcicpints
Scviudo'Cabea et .1., (55) d.veloped & exlractiE sP.ctropboromcF c
mcllbd for flrosen de del.mination with sodiun 1,2-naphthoqninone'4{llplonde
in ph@a*ulicsl fomultlions. Smplc containing 200 ng& turosenidc wAs h.at.d
nt ?O"C for 30 ninut€s at pH 7.5 af,d t.7 nM !,2-naphthoquinone_4{ulfonotc. After
.oolin! to room tcnpdanrc, tne miiturc w.s shalen wiih 5 n! isomvl alcohol ror
2 min tollosed by entrifug.rion for 5 mi.. Thc absotbdcc offie orga.ic phrsc wd
'ncsurcd fron 3oo-?00 m. For qtibnlion 8xapt., fie ealvrical sign.l ws
ncdurcd at 498 m. The ncrhod s lppli.d to irj6tion solutions md t blcc. Thc
inlcd.rcncc fron laclose s e .xcipicnt d be elininacd by using pH 4 5 bv th.
lsc of aeet&te bufler. 11€ detcction linit wa L2 nrr- for flro*midc
ziv&ovic .t al., (66) €stiblishcd n sPecaophotonetric m.rhod for the
detemination of flrosmidc s its iron(llt) conpl* in ph@aceutical prcpdiiion!
Powd.rcd tablet was exFacted with .$eol, th. extracl wd cenrifoged dd thc
supcmatalt soltrtiotr Ms dilutcd wilh .lhsol. A 5 til ponion ws njs.d wiih I M

F€Cl3 a.d lM KCI ddrhe pH Adjust dto 5.7. The abso$occ wd neasured aft*5
nin at s L3 m. B.eis law was ob.ycd from I to 8 mM.
Mouslafa et .l , (67) dd.mincd fuo$hide speckophotonchically rt 225,
273, md 326 m. Bccis law w.s obcycd fmm 2 5 to 25 rt8 /dL of furoscnide in
ethmol. Recovery was tlom 89.99 to 100,79'lo, By this method, flrosenide c.i be
identified sd dciemincd in rsw hat rials sd phm.ceudcal prcp@tions.The ntio
ofthc n!&e absoddces cd be u$d in $scssiig dibilrty
GdsMl, et al., (68) conp*rtirly cklulc two diflcretrr spectolhotonett
ftethods loi simulaancous .stiDation ofspircnolacton. ed fGdidc tron conbircd
dosaae foms. ctuhcd t!bl.1s qft disolEd in squ.ons mcthanol ad malysed by
muhiwa*lengd sp4rioplDlom.try &om 200-300 m. Fru.nide dd spiionolactonc
rEre detcmincd Bing thch ahsorbdc. ndim rt 223 and 242 nr4 csp€cn@ry.
Altenatively, fict{cdvanE spccli'ii,hoton.ty 6 cdicd out Tle c ting
calibntion graphs weE lincd up ro 20 rrynl for fdsmide dd 30 q/nl- for
spircool&ronc Th. snhiMv.lcnllh r6ults w.rc bend rhan nsl-derivativ€ resulh
The 6ct-d.rintiv€ m.thod r.qun d a tcording sp.cLophotomet€i but ws sinpler
dd shorie.lhe thc multiwlv.lcngth n.thod.
c'rciq ct .1, (69) in*sligrtd flow injection spectropnobneFic
detmination of fru.nidc or sulph.lhiuoh in phmacelticah. Powdered lableh
equivaleni to 40 mg frus.'tridc oi tO ng sulphathieole *ere sonicated for5 ninnt€s
wilh lM NaOH, then diluted to 100 ri! Nith H,O. hFclion solutions were dissolvcd
in H,O. Poriions (?2 FL) wde inj.ct d iito. carier stlcah orH,O (12 nl/n'r)
which nqged wit\ . sEcu (1.2 triririn) of 3nM PdCl, in Britton-Robinson bulfo
ofpH 5 and pased through. rcador (3 d x 0.5 m id ) '.aintained at 55"C before
th. absodmc. ws D..sue

t0blcB or cdpsule w€re dissolwd in mcth..ol od diluted to 100 mL witl nctlaol,
0.lN NaOH or 0.lN HCL Thc absorbec. olthc aotulion m n arured at 255. 288
or 258 m. r.sp.ctively (for total .onr.nr of spionolactone dd funid.) !f,d at
310, 360 or 145 rul! rcspccrivcly (for frus.nide 6ntcn0. B*r's h* w$ oucycd
with usc of rll rhee slmi! RSD w@ 1.4-2.r% dd recow.ies w.n 98.6-99.9vo.
Thc 6ults codelated well with thoe obt incd by d HPLC ncthod.
Ed@, ud Tipnis., (?D ils3tisrtcd @other sinple spccnophotomctnc
h.thod for fta€nide dd amiloid! hydrochloride fron combin.d ptamacc ical
dosiga. Cround iableis contdidng 40 n€ of fruscnidc wr€ nlEasonically dissolved
ilr 50 Nl- of nrclnmol Tlrc solution wds dilutd to t00 nL with 'nctlitrol, O lM
N'OH or 0 I M HCl, and filiercd. Th. lbso.bancq of th€ filtrate qs n.lsurd at 260,
243 ud 260 m! respcctircl, foi thc dcr.minadon of thc totll contcnt of
auroscmid. .nd amilorid€ hldrochloridc tnd .r 33n, 337 dd 395 nn for amiloridc
hydrochloddc Th. nrosmid. conc.nE rion w6 calolat€d fren $e dilldnas in
thc coGspo ing tbsorbeces. B4ls l.w wd obey€d for sll thEc E.thods
R.coveri.s wcrc >98%. Iltcr ed inta-.tly RSD nry€d from I 6-2 ?% $d l 0-
2.9dl., rcspectivcly. Rcsdls agEed wdl with lhos obhined by s HPLC mclhod
Zivmovic €t a!., (72) invBligated Extracnon specrophobn.trlc
dct.mination of frusemide s its iron (lll) conplcr. Frus€mide wd r.octed with
cxc.ss F.Cl3 in the pr€smcc ofNHaSCN (pH 6.5) ed Hcl The ed conPlq was
cxtracicd with chlorofom sd lbc tlsbanct of the slution wd n.asur.d al 463
m. Bccis lN wd obertd &om L5 to l3.5rnM ofAuseni.le
s;try er d., (73) d.Elopcd s dh.ctiv. sp€cEophoton€nic mdhod for
ftosnide usng n€thyldc violct. Powdctd tablc$ equiFlcnt to 40 ns of
b.Elhiuide or A6mide, wdc cxt.ctcd with chlorofm 6d dilutcd widr vaicr'
Thc rcsidue was dissolved in ac.ton. Nnd dE soludotr vas lilt red ftc filhaic was
cvapor.lcd to drynss md fie residuc wss dissolved ed diluted! as apPropriatc' with
HrO Injeclion soluiion equival.nt 40 ng of fi&senide was diluted with H:O to a
conc.ntBtion of 4OO ps /frL. Ponions of solotion weE treared with phosPhat. bufi.r

pH ? ed I mL of rqneolr 0.05% m.rhylcr. violct. Th€ nixtue B dilltcd 1o 20
ml- with I{,O and exF@r.d with CHqJ Aner 5 min, lhe absorb&c. of lhc cxtract
rvas neasued at 540 m. B..t s law M ob.ycd fron I to l0 pg /nL offurcenide
Rccovcries werc qudtitative ed rcsults llrccd wcll wiih lhose oflh€ U.S P method.
No interfere.ce *as obseNed.
ksopoulos., (?4) invsirgatcd a Spectrophotonefic nefiod for micro
quanlities of tusemide using iso&d hctdro-polyanions of nolybdenum(Vl) B
oxidiznrg asc.ts. S.mplc solulioh w$ mir.d witl molybdophospho.ic acid solulion
od sulphuic acid, dilur.d wilh H,O ro 22 nl, dd heaied at 98' fo! 25 nin. Alt t
cooling fie solution Ms dihtcd ro 25 nL with H,O, nix€d md set sidc for 15 min
in the d&L Th. !bsrb6c. wa mcEed .r 690 o. ?00 m. B..r's law ws obcyed
for 5 to 200 rg /tril of Ac.did.. Thc n tho.ls w.c of cquiulent aelncy and
pRisior\ md rcsults did not direr slgninddy from dosc of lfic U.S.P. XMII
Rao et al , (?5) d4lopcd coloincE c ncthod for &rBenide il puc .ltug sd
fomulatioG. FlNnidc dbsolEd in nlthr,ol ed diluicd b 15 nL with mctb&ol
dd trcared *iih 5 nL of mctheolic 2 8% sulphlric acid al 60' to ?0" for 30
ninutes. And cooling dd dilfion to 50 trd. th. soluiion is fiIi.rc4 I nL ofiiltratc
was mixcd wilh 5 nL of H2O d.d th. solution wG cooled to 15' befoe being treiled
with 2 mL of2M sulpbuic acid ud I nL of2% NaNO, solution. Aiet 3 mih, I mL
of 4% .monim sulphdatc solution wN .ddcd ed swded. An€r 15 min, lh.
absoibMce is ne$ured at 523 M. Be.ls law b obcy.d lor 2 lo t0 rA /nL The
m.thod is applied to d€rcdinc tlosmidc i. tlblets and inj.ction solution md
rcsulls agree wel with cxp.ctcd vrlEs.
2.2.2 ChronrtogHpnic dct€rninttion olf!t6.mid.:
C@d et al, (76) dalop.d flow injcctio. md I{PLC m.rhods for
deimination ofnnos.mid. usi.g puls.d mpcrcmclric dcl4tlon,l nicroelecEod.s.
Dxpernnental conditions tuch s pH 6d phosPlalc [ufier cone.lrtlion vcrc
30

opdnircd lsins squ.rc *ov. Klriftnctry (SWV), A s'il.ble aiprcm.t c
dcr.ction of furoFmidc ws rchi.v.d lsing ! PAD prcgrm consisdng of a two+t p
potendal rvrv€fonh with ahcm.dng eodic and crlhodic pol&iation. A lin€ar
cal'bdtron srsth wa oh$n.d lor fuoscmid. in th. 5 O x lO'.1 o x 104 mol/L
conentration ransc. HPLC-PAD at carbon librc nico.lccrodB wd urd for the
det min.rior of f@s.nid. in $. prcsn@ of srcnl thiour&il .lrues &d
oxyr.rracy.linc (OTC). Tfu nobil. phs. sclcct.d ws a 25:75 acctoniEil€:
NaHrPO. nirtur.. A lin & olib ion goph w oblsiGd for fimsoidc in rh. t lo
100 FM tu9., with . limii of dd..tion of 0.55 ttM, Th. uscfrIncss of this n.thod
for ln. d.tcminltion of f@s.mid. in F l spl.r w €mluatcd by p..foming rh.
&llysis of conundciNl nil( s$npt.t 3pik d wilh turcldide. A mcd rcco*ry or
95+^5olo fnoscmidc wa obtlimd.
C.rdr-Broch .t al., (7?) dcsqih.d t simplifcd Mg|l-PcrfomaE liqlid
clFn.log6phic proccdue fo! dclmirldon of nF*6idc with UV dcr.dion, t
Cl8, rcv!.s.d-pha$ colun! Nnd nic.lhr nobil. phes ofsodim dodccyl sulPhatc
(SDS) ed prcpeol !l pH 3 buffcEd witlt ph$ph.tc syst m- Thc nixtr of
f@s.midc dd il5 d.g6d.tid produc6 which s fomd lpon light.xposition a
rc$lEd witn s nobilc phnc of0.04 M SDS-2% PrcPdol.
3l
Scpaadon of tuGddc
fom its common inpuiti.s dd th. hydolyiic product, 4-chlorc-5-
sulph@oyla.theillic acid, wrs .lso po$ibl.. Thc ulidity of thc proccdurc wa
chcclcd by.natyling 2? phmaccuti.ds comcrci.lis.d in s.vcnl couines Thc
labct claih tcrc.nlaecs md cocflici.nts of vliation w.c in thc 95-102% ed 0 05'
1.3"/o rang6, r.lpcctircly, TIE rcaolB sho*d that th. proc.dd is suitable for
outine ealvsis ofthc diurctic.
cl-saharly., (78) dcvclopcd simllid.ous hiSh-pc.foftancc liquid
chrorutoStaphic ssay of n&snid. sd proproolol HCL ad .PPlicd !t 'n a
phamacolindic study,Ttu dru$ w.E.lulcd tltougb. nuolcosil C(13) coluo. with
a nobile phas. coiposcd of O.o2 M polssiun dihy.lrc8.n phosphate and

acctoiitrile (80:20. v/, .4josied to pH 4.5 and tc cmu.nt fron lhc coludn was
n$tritor.d al215 rn. The proccdure ws also applicd lor th€ assay offurosemid. nr
dw nateriab, pham&euiical fomnl*iotu &d in rabbits' plsh4 using propmolol
hydbcl orid. s d intcmd stddrd. Thc linca coaco!?tion rdgc or th. ssry
wss O l-2OO .nd 5-2OO l4l nL for turosmide ud propranolol tydiochlodde,
rcspcctiv.ly. Thc itrter dd inEa{ay a$ay pEcision dd accur.cy show.d
rproducibility ed eood linc.nty.
Reuwijk et al., (?9) iivsng.tcd n hi8t-pcrfomance tiqnid chronalosrapltic
metnod using ltuoresccnc. detection for lh€ simulioeous dctcmiutioo of
tlrosnidc and flnitondc. rlE cluonabgrlPhic tyttcn wa bdd on rc@c.d-phac
ion-p.ir chromatoSraplry with sodiM dodecylsulphak 6 ion'Diiring ageoi Thc
sme countq ion was $cd fot lhc ion-pdr liqlid-liquid extraction io clhvl tc€tatc.
Thc nininum dclectabl€ @n@nt ation.houls to 0 3 ng ofi@s.nidc ed 003 .g
of.nilondc pcr mL ofplasm'. The;;plicabiliry of th. nethod was demonsrated bv
ihe onfllysis of plasma samplcs ttlcn fron volule.6 recciving bo$ ddgs
AbdclHmid, (so) inBriStt d ! 6pi( spftific {d *nsitiE hish-
Ntfonsancc tiqlid chonato$aphy-ras spechomcttic (LC-MS) assav for lhe
dekmrndlion ol turosehide in hds plNma usin8 d(lof'nac as tu inlemil
shndard (tS). Borh conpounds w€E;;tuckd Aom humd plsna widn cthvl ac'tat
ai DH I and v.re chbnltograPh€d $i!g Shin-?act OLC'CN cdum 6d t mobile
ph.se conshiing of acctonilrilc dd 20 mM Monium.c€hte bufler soluiion pH ?'
4:l (v/v) at a flow ralc I nL/nh. Furossid. and IS w€rc ddccted bv na$
sp.ctronct r opcmted in thc n.gatiw singl. iotr monitoring mod' using AICI6 an
ioniation proc*s .r n/z 329.2 6i 294.r, r.spcctiElv Thc ssiv lin'ditv of
fuosemide was connmcd orer the rtngc 50_2,000 !g/tr[ Dclcciiotr limit for
n&*nid. ir pl6na I lO ne/ml-. Thc dcElopcd LC-MS prcc'du' was lppli'd
for the detcmination of fi. phmacokhetic ps.iclc6 of nlosehidc afier m onl
adminisEation of t4bl.t fomulatim (40 mg) to two healihv nalc volunte'G The
calcuhted p!@.tcs wd. in 8@d .gicmdl wifi thc rcPoned v,l!'s'

clrda.Bfoch ei at., (81) invcsdgated a seNitiv., selectivc ud efiicient
nicellar liquid chionaroataphic CMLC) proeduc for thc dctcminaiion or
fu@snidc in urin€ sdplcs by dicct inj.ctiotr sd Uv d.tection The prccedurc
nAlcs lsc of a C,s revers.d-pha* coluN od . nic.Uu nobilc phasc oI0 05M
sodiM sulfAlc 6% v/v prop$ol dd phosPhale bufid at PH 3 to r.solve inosemidc
Aon i|l phorocbemi€l dcgndation Podu.is.
8l
TIE limil ofquandficalion @ 0 15 |r
8/mL a.d th. RSD of th. int€cday 6say v6 0 8'0 04% il the G82 pgln]- teec
comonly admidstded d$8s abo did not interf€t The propos.d MLc procedur'
pcmits th. r.Pid md rcProduciblc n.lswmmt of low letlr of flroseside in a
Nava-Ocupo er al., (s2) devcloped h simptined ncrhod to quantiry
furosenidc in nrine by hiEh-p..fodtncc liquid chronatograPhv md traviol'l
detslion. Ulnc smptcs, Aom patictrts 8iM a 20 ng lablet of furcsdi& wcrc
c€ntifugcd at 4OOO g for 5 nh, ed ! 100 !L portion of supcmatdt was a"lvs'd
direclly by HPLC on a J Fn Cl8 Sphcrisorb colum wilhmedwolH:O co'iainlng
3% acetic lcid (2:3) s nobil. pl6e Dd &tccliotr at 280 m Thc dlibFlor Sraph
rv6 lincar from 0.750-250 pytrll- of fuosdnidc with a d€kction linit of 0 200 p
g/ml-. Th. .ccu6cy of thc d.imilnion ws in rhc rangc 92-lO3% with sianddd
dcvialions of+0 l-4.2 r€/r.L.
Midro et .1., (83) studied thc Phololvsis ed hvdtulvsis in aqu@$ solul'oi
of furose,nide . Furoseridc wN pui6.d by IIPLC lnd afid dcgr'ddtion the troducts
weE analys.d by HPLC on a ODS q.li@ with 20% tcetonitril' in 0 05"/o HiPOa
adjustcd to pn 3 4 mobiL pts. (1.5 nl-/bin) ud photodrode@v deEcton
Fmsmide and its degradation Products also *dc cmmi'ed bv negttive_iotr MS'
Photode8radntion was shom to inslw displacnent of cblorine bv r hvdtoxvl
grcup wh.mt hy&olysis i! qu.ors 3olutiotr invol€d acvl group nigation wi$in
the sllc!rcnidc mol€culc.
Abou.Auda et al, (S4) dcrcmincd frtosnidc i! plasma and uine usiig
Hieb-pc'fomsce liquid cluoMiognPhlc nc6od sd uscd n in bioav'ilabilitv

sndies. Fnrosefride was .xlreted ftum plasfr5 or urinc with di€lhyl clher a.d lhe
Esidue dissolv.d in fie HPLC nobilc ph!*. A 20 !L snDlc vd dalysed on a Cl3
or ! Esolvc sphencal Cl8 ollm for pl4ma dd urb. @ly*s, EsP.cti\tty, wirh
0.01M KH,POa bulIer ofpH 3 dnobilcphae ed nuodnetric deteclio. at389nn
Calibotion graphs wd. lin.r for 0,5-50 pglhL of tuos.mid€ il Pl$ma
virh aqu!trriliurion hnjt of5 ng/ml-.
84
md uri.c
Milk .r al., (85) Qurntifi€d turosdide ton sctus and tissuca usins hish'
perfomarcc liquid chrcmatognphy. ScNn rissu€ extrict were spik.d with 5 Fl p_
nitrophenol (intdal dand&d) rd .cidilicd wi6 5 !L of 2M H]PO. Th€ nrtur.
rB €xractcd with .6yl acela& ud iic o.Srnic phd.I evapor €d lo dtt4css
under ! str.e ofNr. Thc rsid@ M3 dis$lEd in 30 BL nobih phasc a'd s l5 l1l
portion was e.\sed by HPLC on . r.6sed-phlsc ODS colum wirh 0 olM
KH,PO. of pt'l 5.5 @ntriling 3ry; ;.tuol d nobil. ph& ed d.rcclion lt 235
nn Thc .alibralion Snph was lin.d !D to 500 ps/nl f!rcserid. wnh . d'l'ction
Iinit of

cxtaction. Sctun cont ining known adounls of futosenide , was filtercd and
ilrjccted o o iltr IIPLC coluinn using llt aulosepler md a six-pon switchihg volve.
Scpandon *.s cff.ctcd on . dalytical ODS colun. wirh grtdimt €lution wnh
0.02M pnosphsre bur€r/.4ronitriL for 4.6 nin with *run prct in dd €ndogctuus
srbsiances wished to wnnc. Th€ dalttc furoknidc wf iniroduced to lhe analylicil
column op.r .d at 4o'C md chicd Mln 0.02M phosphat butrd of pH
7&etoniEilc (13:?) @ntAinilg rsdM lctta-n-butyl.moriM b'onide s nobilc
ph6es rnd dctcciion at 271 nm. Thc c.libndon gr.ph w.s lin.ar frcm 5-1000nynL
walrlE .t al., (88) Conped two dEaction n.thods for detefrin.tion or
proprsolol dntl ndosmid. in nMs PI@a by nitcd modc chromtog pltv
(ls;jni.oal sianddd) was
Pl.sba contriDns propsotol, ntumid€ md Pindolol
mix.d wirh 2M aeuc &id 4d nrcmidc ws dFactcd into I nL diclhvl
.lhcr/hce. (13:?). Thc rcF.iDing dqEou Ph4. was lr.ar€d wii! 50 FL lM NaOH
rnd propanotol and IS w.rc exrJactcd into r nL dicthvl €lher' The pooled cxfiacc
w.G mpootcd to drtnc$ udd N: rnd cconsn$t d h 100 |rL loodMsodium
.cchte of pH 4/acetoniril. (6?:33); nobil. phse A portion {at udv*d bv HPLC
on n revedcd-phse cdlioo cxchMgc colm. witb a nobile pl$. flow ratc of I
numin and derecdon at 230 m All.mttivcly. onli.. €xloction wa pc'fomcd bv
injcction of @ple od ro a Cl8 Ht?6il collm Rcdveries wft 90?5% ed
gOos% fdr turoscmi{c rnd proPmolol, rcsPcclivclv' trsins Iiqui{l-liloll cxtnclion
76.32o/r a d 9o.soela .csP.cti*ly, using collm extraciion The calibt'non
^dd
gnphs wrc lincd for 2l-3OO ne/D! firotchide ed 50'400 ry/nl poPmolol'
Baroso et al.. (?9) d.lefrin.d pi!.ianide flnd flrorenidc in phtmaoclticrls
md hus.n uine by hid-p€rfomMc. Iiquid ct&natogaphy w,th mperomeh'c
dctcctio. Udn. ws .cidificd wilh 2 r.L rM (Hr?O{ of PH 3 25 dd 'nEcl.d wilh
4 mL ethyl rcetate. Thc orgaric pne ws evaPoot d udd N: al 40"c ad the
residue was dissolved in 2 s! mobilc phasc Po*dcr.d tablels wcre exfiactcd with
m.theol. Po.tiotrs of rh. r.lultinc soludons w.r€ dslvs.d on a colum of Bond'Ptr

Cr . Calibration graphs wd. lin.s for 15-500 nglnl and 0.5-10 Fs/nL of
wosemide or pirctdid.. Re@v..ica of 6 lglnr @*njdced 2 !B/nL pidanid.
ftun urine aveFgcd 92.?./r &d 94.4% apdtively.
Bdoso ci .1., (90) simllrd.ouly d.icmined th. nirmtcrcn. ad fur6.mid.
in ph@aceuiic.l fodulalions sd winc by HPLC-EC, Th. an.ltt6 wde extract.d
with metndol and ulrsonicalcd. Thc mixture ws nl€rcd, dilurcd with nerhuol
.nd porrions of th. dilur. filt atc *.ic'ftnld dilutcd witn hobit€ phe. urinc ws
adj$t€d to pH l0 ud lnut@nc md frloscnide w@ citractcd Mth crhyl tcctatc.
Tle deanic layd us cmov.4 Mpollted to dirie$ undd N2 &d rhe Esidu.
ledissolved in 2 mL niobile pha!., Portio$ o{ the mobile phas. dilded sohiions
wer da\sed by HPLC on a Bmdapak Cu col(m wifi aqucoa 30plo acetonihilc,
.djust d ro ' pH of 5.5 6 molilc i!is. snd dperemctic dct ction at +1.3 v "
Ag/Agcl. calibhlion grapl$ wee liics uF to 5 ed l0 n8/l- rcspcctiElv, of
ti.nteme and f!rcsemide, with dctcctio! limits ofo.l sd 15 nc/nl. EsPectirclv
Recoleris of th. analtls Aon urinc teated with I qglhl- of himlerene Md 3 p
g/mL of fuosenidc were qMtilativc.
Shaitl, (91) d.veloped sd illidat€d a liquid{hromatosophic nethod for
the dereminadon of tuosmide, in boviic mitk Milk M dcftncd dd dcprotcinaicd
dd &aly*d by LC on a Spherisorb ODScol@ equipPcd with a suard coluhn of
similar mnt€rial, oD.rated !t 35'C with .c.ioni tril e/phospnat buffo adjusted to DH 3
with s5% HrPor (3:7) s oobile phasc dd fluorescencc d.tection at 410 nn
(excir.tion at 2?2 m) Aveogc rcc;*4 of fircsnid. sPikcd at 5 lo dd20 Ppb
w@ |08,9l dd 857a r6pstiEly, Thc co@spoDding RSD wrc 14 8 dd 6% Th'
method M €lidat d using nilk ohtaincd fron a @w dos.d iniEEnoulv with 500
vrce d ol., (92) invesrisated lft detemination of furoscnide wirn its rcvl
sluc@nid. i. hMe pl$ma sd uinc by neds of dn€ct gradicnr hisn_
perfomance liquid-chrctubgraphic daltsi3 wfth tuorcsc.nc..lctcctiod Ponions
of the splc solution w€rc @lts.d on ! .olllm Cp Sph.risoft ODS, equjppcd
36

wilh n BMrd colunn packed wirh pclli.ule r.vesed-phasc,whl fluorincric
detection at 405 M (excitarior at 345 m) The det€crion linirs for furosemide and
its acyl glucuonid€ were 5 ng/br ed l0 ng/nr, Esp.criv.ly; corcsponding
.v.rag. Rcov.ri.s wqe 91.5yr u4 a5,52yr. Thc nerhod Mt usd lo study rh.
phmokincdcs offr@scmidc od $. cffcds ofprob.n cid
Macuu. ct al., (93) I!rcstis.td lutomared high-p.rfom.ncc liquid-
chrondtogr.phic ncthod for dcteftitunon of flro*ftide dog plasni. CalibEtiotr
'n
sdphs for furosenide wd€ rcctilince &om 0. l- l0 rslmr.
Millq et al., (94) Inrestigated rcwrs.d-phse ion-pair liquid-cbofratosraphic
melhod for dekmining rh€ impuiiti.s of fuosmide . Sdple wd dissolved in
mobil. phas. ud the solution w4 applicd orto a colm of octylsiM siliq dd
cn'tioi wns ndjuslcd lo pII T md dclcclcd.t 254 nn Thc mcthod w8 conP.rcd
{irh rhc proposcd mcthod ofthc Ed. P., ed r U.s.P. n€thod.
Sidlu and Chd6 (e5) invcsngikd sihple nic'osdlc high-pcrromdce
Iiquid-chromdogfrphic m€thod for d.tminatio. offurosemide in n.onar.l Pl6na
Plarna was protcin prccipitat d *ith eetodihile, cenirifuged md lbc suPcmaiant
w.s evsporat.d uoder &r at 55'. Thc r.sidue wa dissolled ih mobilc Phasc ed a
ponioo of th. sohtion 6 daltsed by HPLC. A colum Bond.Fi( Cl8 was used,
wirh . nobil. phase of lcctoniril. - 0,08M Gphosphoric acid (pH 2) md
flud$c.nc. d.tetion ar 4lO @ (GxcitAtion al 270 m): naproxm was used d
intcml srdddd. CdibFtio. t6ptr! wcrc rcctilincd fq 0.1 to 25 F€/nL of
Fdthiog et a1,, (96) inre3ti8ltld .xrcmahtanddd hisl-p$fomeoc liquid-
chronatoghphic n€thod for q@tirari\r dci.nu@lior of filosenid. in plasma bv
using solid-phas exbaction dd o'ihe clution. IlE ctlihFtion s.tph
a7
was rccrilined
for 25 to l0OO !s /n! of fie$nidci dt. linit or ddecdon was 1.8 n8 /ol. For
dctcminadon of 250 ng /o! of l, thc cocn of vdiaion wN 5. l%. R.coEncs wcr€
80 o 85%. Both inrdal od dtcmat strldard pmc.dlBs Nrc .vdrhtcd; the
*t.mrl st ndatd mchod gavc supdior rcsulrs.

R.euwijk .t .1.. (94 inEsiig.tcd sindkn@6 dercmination of furoscmide
ud milond. in plosmr nsine high.p.rfomma liqnid chrcnaioeEphy with
fluorcscencc det crio. Plasma ws .xtacrcd with cthyl acelale. A po!'tion of the
solurion *as subjccted to HPLC on . stsss colum ofNucleosil 100 Cl8 equipped
with n sinils gudd colum. ft. nobilcphasc flow mt. ws 0.6 nunin wr&
fluorcscncc dctFlion at 413 m (dcil.iion !t 360 m). Thc olib6tion smphs v@
rectilinc& for -

Frusenide wa ellted with ncthmol contli.ing phosphoric acid, fld a portion w.s
tnalysed by FPLC on a column ofPRP-I .id gurrd colunn oI Lichrosorb RP-18.
'llE nobile plase was 3% HrPorac€bnihilc (6?:33), md nuorim€lri! d€leclion was
4l0 nn (excitation at 268 rm). Thc cnliur.lion gr.ph was rcclilih€ar foi l0 to 500
ng/nL oaFnsenide. The dct.ction limilwns sinilsr, equalsl ng /nL. R€coveies of
100 md 50 ng of frusoide wcr. 96 | to 103.3%and 94,0io 104 37q rcsp.clively.
Rusel er al., (l0l) inrstiSst.d solid-phfe cxtracdon of furcsenidc ftom
plana md ud.c md s$s.qucndy andy*d by high-perfomd@ liqdd
chrcmatosnphy. Frus.mjd. sd d.sn.rhylnaproxcn (int.mal standrd) rerc
cxt'actcd fm ptsma dd uirc on a Cl8 qa.nidge by ehtion wiin of m€lhmol. A
0onio. ofd'c elu.tc was nnnltscd on a Lichosorb RP-l3.olumn opemled.t 40o
wirh phosphat bufc. (!H 3.0) - mcthrnol (13:12) a nobile phe dd fluorimeErc
detection at 400 m (excitltion .t 275 M} Calibatio. sEphs
39
wcr€ rectilincd for
O.O I to O.I ed o.1 to 2.5 rtg /mL of fino*hidc i! plama and for 0.5 to 25 p8 /nL in
uinc, dd lh. dcr.dion lidit wN5 !0 tg /mL in bofi natriccr. Rw€n€s w@
quantitattu, dd the intr.-dsay @cn of wiation wcrc 1.8 to 3-2% fo. platu and
l.l ro 2.v/, fot urine. DtuCs coMonty adninistecd wilh fr[os€mide did not
sinen et al, (102) simultd.ously dalyed 6&senide dd bunerdide in
ho6e plasm. using high-p.rfomM. Iiquid chronatosnphy Plasma ws acidined
witlr Hcl md e{rAcred with cHrclr, cdryl acciatc or light petroleuD The exract
was evaporalcd lo dry.ass under N ed th. t sidrc ws dissolved in nobil. phase for
HPLC on a colmn ofC8 nat risl with .cctonitrile - 0.01M HrPo{ (?:13) a nob'le
phase ed fluorescence dctcction st 409 nn (cxcilation at 235 m) Cdibialion
eopts
werc r&tilinear fiom l0 to 500.!9 /nL.
Sood er al, (lo3) inftsdgltcd a us.ful miccsc'lc he$od for nonnoring
f@smidc by ligh-p.rfoms@ liqlid chsm.toeraphyTne nobil€ phase Siws
sh$p p.alG wilh shon ctcnrion tin s, aod us. onc*tcp ncthod for sanple
pnc6sing beforc irj*lion.

Krnatr ct sl., (104) iln4srig.i.d hign-p.rfomec. liquid-cht!fulognphic
ncrl'od for thc ddcmin.tion of furosmid. Bins an clccnochcnical ddcctor.
Furcscnid. wa dctcmi!.d wirh B. of r .dum of ODS micdal, m.rhmol -
0.0aM KH2PO4 of pH 2.0 (trl) B hobil. phs..nd dcr.crion .r +t.30 v vs. Ag -
AgCl. ,tllc {Ibdtion g.ph w{ @lili! Aom I to l0 F8 /r.l of f@!cmid. $d
thc fihn of d.tetion wls 0.5 ry *hich k . I c'fold imporcncnt @mprrcd with Uv
Sood .t.1., (105) in%tiglrcd r 6pid nic.o 3c!1. m.thod for d.Lmin.tion of
nroscnidc ii pl.$d rd uinc. P.oncrhdol N tddcd to plasm d uinc $ intcnal
st rdad b€forc dctcminiion of Ausnidc by HPLC on a colutu of Bondapal<
ctuip|Ed wilh o guud olu'nn of Dondlprt Pl'cnyl, will !q. 44% acdonilrilc
cont idng 0.4 g/L ofN! dodeyl $dphd. ed 6 mUL of lc.tic acid (pH 2.5) a
mobilc phe dd dct ction by n6ui!8 flumiq.nc. wifi .xch.tion .t 225 M.
Tn. catiborion gnph w rcctilin u up to !t lcat 20 Fg /nll- and rc@rcry w.t
PinLnon d rl., (106) irBtirttcd 3cp|raion of fitroscnidc , phcnylbuh4o.
tr oxyphcnbuttrzonc in tlsma by dircct injcction on to intcnll surtac. '+Bcdpha.
coltm$ *irh rysrcn.ric optinizldon of sclccrivity.Phsh. w* Mlr5cd
dirccdy d coluhnr of GFf'S5-80, with dd.ction .t 241 m. Opliouh nobilc
pnsc co.diions Er. o.lM phosphid - THF (9:D ofpH ?.5 and 0.6 mumin. Oood
rcslurion of mixruca co.tsiring .ll thE. drugr ws obt.incd. Th.* or$nic
nodili.rr THF, prepr!-2d sd rc.tditilc, wn cslual.d !t Elying pH nlucs.
Lovc! ct rl., (104 ilBligltcd io inpt@ thc HPLC n.thod for
.Ltcmin.tion of fuos.nidc i' dtim sd uinc. Acidic plama sMplct w.c
dhrccd wirh mcdryldc ciloridc @ntthing rn inhal rt .dard, 6-
d.sm.thylnlprex.n. Tn. .xF&!! vcE cwponrcd !o d.yr.s3 ud th. Bidu* wae
dissolEd in n.thaol. Um. !mpl.! wrc indbttcd wirh tsglucurenidac
Mbiot tchp. fo. 15 nin ud thc prct.is wcr. prccipitltcd *ith lcctoniFilc
co .inils fuoscni&- An r 6Ein€iEs. i ponion ofdE aoPoar.rt solution wtr
90

irjcctcd inlo ih. chredatognph for ddcmin.rion of ftuchidc. A colunn pekcd
with Zolbd ODS s.s urcd with fluorin.Eic d.t.ction 389 m (.xcn.rion n 235
n'n). Thc mobil. plE. for pl.snr wa pnosphrtc GH 3.5) - mcthaol (ll:7) whilc
rhrl for unn w .ccric &id (!H 3.5) - re$ool (3:2); botl mobil. phs6 wF
lsd rt 3 mumin. Th. rcspotric w Grilircd for plMr fren o.0l ro 15 rrynL of
f@midc ud for uin ton 0.5 0o 200 rryml- Thc tMEry of turoscmidc frem
phsna w$ 69.E% !t rhc 2.0 pginl- lcrcl and 98.6% non urinc 0r 6. 5.0 rtg/nl
Gucmouch. ct rl., (108) d.cmincd of nros@idc in nr pl$n. ainS HPLC
&d liqlid scintill.tion. Filtcd ot pblm smplcs wce rlyscd by HPLC on .
@lutu ot Bondrpal CI8 cquipp.d wth i gurd olum of Cl8 Co6il. ftc
optimizd mobilc ph.s; wls lcctrtc bufld (9H 5.3) .o linirs 22% of rcronirilc
and dctdtion ms by lb$rtdcc .t 254 ad 280 nm. Th. dcierio! linit of frucnid.
vs O.0l ng/L. Th. @ff. of winion w 5 to 6%.
Br@ .t al., (109) coDplEd .xF.crion sd pFipitation n.thods for lhc
HPLC dctcminltion of fdos.hidc in plsn. ed udtrc. Th. acidifi.d sdplcs of
pbsna or urinc cxtlacicd 6d an.lyrcd by HPLC on. coluru plcl.d with ODS witn
, nobil. phrsc of m.ibeol - H,O - .elic ocid (40:57:3) for cthd .xFlcrion or
!4ronitrilc - phosph{. (7:13) fo. r@iolitilc prccipidtion. Fluoasenc. dct*tion
wrs !r 460 to 600 nn (.&ir.tion !t 330 to 400 nn) TIE *nsniviti.s of thc ncthods
for pllsn! w.c 0.26 Fs /trrl (pptn.) dd 0.05 rtg /nL (cxrnction); for uinc thc
scisitiviry wds 0.5 Fs /nL (.xr6ction). Fo*nid. Ms stablc in frca. plsmn .
uin. for $ lc.rr I l3 ud 204 dryi s!.ctiEly.
Cu.morclE cr d,, (llo) ilv6ti8licd optinal condnions for aoalysir of
tubs.nidc in pl.sm. by HPLC fd inprovn.nt upon rhc prcision of l0 to 15%
achicvcd prcvioudy in HPt,c udyli! of thc nulanidc. A syscnaric srldy Ms
madc of th. cf.cts of6. pH, buf.r concn. 3d lc.toniril. contcnt of 0!. nobil.
phs* on Etcrrior psmd6. A 30 cn rMltti.lt cdum rnd. 2 cm gwd colrm,
borh coniaining Cl8 silica, w.rc u.d, wiin & Uv. d.tcctor ed ph.nob.6nonc
9l

sodiun as intemnl siandard. Th. d.r.crion limit ws l0 ng/nL in pldfra. Analyses
of skndard soluyion showcd cocft of vdi.tion of:s%. Us. of the nerhod in a
plannacokinetic study of luman subj.ds is exeiplilied
Uchino €t al, (llt) quanitatidy dct mined furosemid. in plasm4 plasma
water, ui.e ad asiies fluid by hid-pcrfomilce liqlid chromatosraphy. Thc
sanple ws mrxed wirh piel&idc 6 iDi.mrl simd.d md exhacted in the prescncc
of phosphale soluiion (!H L5) wifi mc$ylene clloride. The orsanio ph6e was
traied wilh lnM NaOH, th. .q. phls *as nixcd with the phosphaie solution and
€rfacied with CIt CI: od dic finrl cxFaci was 6alys.d by HPLC on ! st inless-
slecl oolunu of l{itachi Oel 30ll at 30o wi$ 65% of.$dol in 0.02M HCIOr -
NaClOr {pH 2.0) a nobile phe ed fluorimct ic dccction a14l0 m (dcilation at
263 M} The dctcctioi linn w$ 20 ng /oL and th. calibntiotr gaph w6 @tilincd
frem 20 to 2000 ng /nL.
snedden et al., (tl2) Ilv*dsacd r s.trritirc n l[od for fuseoid. h
plana dd uinc by high-p€rfoddcc liquid ctuon.togruphy.
Keranms et al., (l 13) lnv6ri8sr.d Spccinen nedling and hiBh-perfomdcc
liquid-chrodatographic detcmination of nrosmidc. Fuosmide ws d.temin€d in
vaidyr er al., (ll4) d*l;pid u HPLC n rhod for the sinultarcous
detemimrion of fruscndde ed spircnohctonc h phmrceu[cal p4&ations. Thc
dngs Ne.e dissolved in n.tldoland the mixtuE was injeclcd orlo a Hr?{sil BDS
Cl8 colum. The nobile pha. w.s acctoniEil./0.01M tehabutyl Nonium
hydro8eD sulfate (l : l) nd dci.ciion wd .t 238 m. Ilte calibntion Braph vd lrnetr
frofr l34oo ng/nl for frlrscdidc ad iz-looo I8/mI- for spnonolacton€.
Arseku ct al., (l 15) bvcstig cd sinllt ncos dcimination of diloride
hydelionde dd Ausnid. b phadeutid prcpurtiotrs by HPTLC. Povdctd
tablets €quiElcnr ro 25 mg mito.id. hydrochlo.idc nd 200 ng &lemide werc
ullrasonicated wiih l0 n! H:O for l0 hin, 20 trO nethdol wd addcd rolowed by
ulrrenicarion for a furrh.r l0 min, Th. slm. ws nade up to 50 mL ed tnc
92

mixture was filtered A podioo of thc tltratc ws $catcd with i.tcmal sddard
(rydrochloiothi$zide) and nad. up to 100 mL with methmol. A portion of the
solurion w.s appliedto A silica gelHPTLC plst The plate was developed with elhyl
acetntcrnclnrrcUl3% Nllr (75:lO:8) lid dcnsiloneuic scanning at 27s rub.
Qnontnatior was bsed on peit drca. rhc mcthod was applied to two comercial
phamaceuical preparali ons wilh r.cow ri.s >99.37"/0dnd RSD

elecnochemical dcrcctio. .l 0.90 V. The c.librdtion grDhs wcre recliline.r fron
0.25to5nB/nLof frusehid.ins.Mdd0.5to l0ng/nLollrusenideinuiine.
Critond. ed J.y.rL,, ( I 19) dcicmir.d Irus.nide and spironolacionc ton a
combined phamaceltical dosagc by HPLC. Powdccd t.blcts were dissolvcd in
nobile phas. to l0O n! ed th. rcaullitrS sotutiod wd lill.rcd. A 5 nL portion of
rh. filr.lle ws dil ed to 100 nL wifi th. nobilc phsc ud dalysed by HPLC on d
colum of Excalihu Sphcrisorb with 0.02M NaH2PO4 - nethmol (3:2) a nobile
phas. md dctection !t 242 M. Calibotion g6phr wd. r.clilin.d for 50 to 700 Fg
/n[ of frt$midc o. spnonollctonc. nE n.thod Ms sinpl. a.d hpid.
Ampurc cl .1., (120) inBtigrtcd Hid-p..fom&.c liquid

in aq. 30% flcelonilJils (pH 3,5) !s hobile phBe od nuorescencc d€tsclion at 415
nm Gxcnation at 333 nm). Th. cllibration Sr.ph is recilineA.for25 ns/mL to l0lls
/mL ol fdseride in sem lnd 1,25 io 25 rt8 /nL in urinc, lhc linits of detectio.
being 2.J ed 5.3 ng /mL, respecrivcly,
Rao ud Ra8llurcd,, (124)invcalgstcd 8ig!-prc$urc liquid-cluomatoSraphic
estination of Ausnide in wioN .lo6ag. foms. Tiblcls wcr€ dissolv€d in, and
injeclion solution v.s dilut.d wi$ nobil.phsc, nerhanolic 0.0'M NaacetaleofpH
7.8. Fos.nide is thcn dct mincd, wirh cltlorFopuid. as intcnal sranded on ! p
Bondap.l NH2 .olm, wi[r dc|clion at 254 nm. Thc .alibFrion gdph wB
Eca h€e up to ?l Fg ed rhc d.t otiotr linir wa 0.5 rg. This mclhod dlos
idcnlifistion ol lhc prcsnatirs in E fomulations, and dcr.cdon ofdc inpu.ily,
4drlotu-5-sulphmoyl&th.uilic eid-
2.2.3 Tit.inctric netiodr for furc$mid. dd.rminltion.
Nikolic rnd vcldvic., (125) inv6ti8.r.d a couloEcrric titration mcthod for
the dclemination of blnctrnid. sd f@scnidc. Buct nid. ed Aucmide wc
dcrcdin.d by couloncEic tiE rio4 with .lcctiogcrcFtcd Cl in ! supFrrrng
dKrrolyre of 0.5 M HrSO. - 0.2M NaCl dd D.thyl otugc a indicator. Th. m.rhod
is &c€uale od reproducibl. dd cu b. appli.d to phmaccutcars.
Shrnma cr .1., (126) Dadopld ! nN tilrimciric nethod for n'cro
dcrem,nar,on or f rus.tude D phmetuuc6l pcpdlrioN
2.2.4 ElNt.ophor.ii. m.tho& for fur6.mid. dctcrnin.tlon:
Quaglia et !1., (127) inEsrigd.d th. dcimidtiotr of thc biidins of ! &-
blockd drus, frusemidc |nd c.toiixo.c to scM ptotcins by caPillaty zone
elecFophoresh. A tused4ilic. capilary filled wiLh s solution of lCI, frusenide or
celtnaone in 70mM phosphatc/lM NlOg buffcr ofptl7.4 w$ subj.cted to czE at
l0 kv dd 35'C wjth hjcclion of bufln,.{ duing 2.5 s D td.t, md Uv-visible
detection. Calibntion grlphs bAsed on tI. d.. of the neSaiirc p.al wet lincd for I -
lO, l-tOO md 8-40 rt8/nl- lcl , fius.mid. or ce{ttiuone resPcctively. The nelhod
95

vrs us.d in a modified HumD.l-Drcycr proccdur. 6d results for frusmide or
cefiiaoie are conpared with thosc ofoth{ mcthods.
Cadavska and Thomm, (128) dcvclopcd a rapid analytical nethod lor
furos€mide in hunnn uinc by c.pillary .lectropbolcsis wiih ls.r indnced
nuorc$ence and €lcchospny ioniarion-ion tnp ms3 sp.ciloncFic dctcction. Data
produced by injcction or pbin or dilutcd pdi€nt urin.s werc confmed w rhose
obtained v,o dalysb of uin!ry solid-ph.sc .xn&t' Tl s is in contrst to 3olutc
delslion viz UV absorbmcc for which th. .xE ction of turos.midc is Equir€d.
CZE-LIF lnd CE-MS with injation of pl.in or dilut d ui.. r.pr*.nt simplc, npid
and ath&tivc urinary sc@ning {d confim ion lssrys for ftucnidc i. prlic.r
2.2.5 Ntrd@r mrgnciic r6onrnc. 3pdtrucolic .s.r! for forc3.mida
Hm! ed Lru-cm .i al,, (129) inwstig.t d slability indicating prcton
nuclelr nagn lic resons* sp.ctroscopic mcfiod fo. fu.oscmidc in tablets dd
injetiotu. crodd tlblcts w.c di$okd in n.theol 6d erFitug€d prio, to
cnpontiotr ro drynss; itrjstion mpoul.s wd. fr.*-dricd. Thc inrch'l standd4
buhol, vG added and lh. solution ws udys.d on t 90 MH2 vdd EM 390
specirom.t€r sl 35oC *ilh . s*.p tim. of 5 hins and seccp widln of 2 ppm A scI
of letr ry hctic fomulatiotu w.r. salyscd Siving rcoowics of 996% of
fNsemide ed 98.9% of 4{hlorctuul{moyluthtuilic &id, its dcshdation
product. Elev.o com€rcial iablck dd irjcct0bhs *crc dalyscd.
2.2.6 Elechochemic.l det.rmlnsfi on of frrosmide.
Besona Bdoso et al., (l30) iovcstis.tcd.n cl.ctrochenical deled.inadon of
the pnetmide and furoscmidc in phamaceutical fomuhiions and urine Powdded
phmacenticals wcre exraded {ith n.thanol A.d filtcr.d. The filttale was diluted
for ualysis. U ne was acidilicd with KHTPOT solution !i pH 4.25 and vortex.d with
ethyl .cetak for 20 min 'rle orgriic phsc wis washed wilh o,lM phosPhiie al pH
?.5 .nd de. e*porAted ro drlicss at 4o'C undd Nr. The rcsiduc was dissolved in
nEtlmnol for rn.lysis. For e.cb nnrlylc, lhc volt.mm.lric p.nk lllal occuned.! 03_
96

|. I v * usd for quandrarivc puqFs6. Diflcrcntial pulse volianmety was crricd
outataslep polential of 4'nV and . pul$ rnpliludc of 50 nV. Calibniion Braphs fol
pcak crftnr dd conccntorion wcE liner uD to 69 lM pir€lanide and 5s rM
rutosefride.Th€ det€clion limic wcc 0 ll8 pM pnciamide lnd 0 I5pM frusenidc
The recovenes of4.7 mg of.ach Maltl. from spik d urine we.€ 99 2%.
2.2,? lnnunoNs.yforfurdchidcd.icrninrtion:
Stanker et al., (l3l) devclop€d 0 monoolonrl antibody-b.sed inmuno.ssay lo
detect turcseiide in cow's hil*, Ttc ELISA lor detcdng fdosenide used ihe anti-
fmsemide mtibody, FURO-?3. Miorotirr. pl4res w€re coded vith a fuos.mide
BSA coniugnte. And bbc*in8 6d w.5hitrg th. pldes, milk dd andbody solulion
w.re add.d to each wcll @d incubatcd fo. I h ar 37"C. The platcs scre wsh.d again
od 100 rL pcrexide conjlg.r.d goat ati-noBc otibody *s addcd. Aftc. a
fnrthd ircubation p..iod of t t rt 37'C, K-bte subsFatc v6 add.d md th. platcs
wdc rcad i, a microtit c phtc rcldcr. Thc dd.clion liijt for tusscmid. in nilk *$
sinsh €r ar, al32).vdut.d hiSlr-pcrfo.muc. liqnid chromaiosraphy
(HPLC). €nzlre-linlcd immunosorbcnt asay (ELISA) and panicle-concentr.tion
fluore$ence imunoa$.y (PCFIA) mcthods fot the scrcenin& quantitation and
phamacoldnetic study of fruscmidc in ho$cs. Thc ELISA md patticle conccnranon
Ituorscence imunoa$ay (PCFIA) wtrc usd $snti.lly 6 previously rePonedi
The ELISA and PCFIA methods wd. sinph and quict but they did noi g've
rcproducible rcaults for frusenid. in plama oi unnc.
2.2.3 Chemilumin€scence detcrminntion of furG.mid€:
Pa6 .r al, (133) inwrrig.r.d chcniluninescence now injcctioi
detcdiMtio. of futuknidc b&d on ! rlod.nin. 6G $nsiliz€d cerilm(I9 r.ihod.
A sanpl. w6 i...ned inlo a dgcnt slrcln fodcd onlinc by mergi.g lmM Cc(lV)
in 0.5M H,SO. dd l0 pM rhodlnin.6G. Th. chenilunincscenc. s'cnal qt
d.lKled using a photomultiplicr tubc. Thc calibnlion gdph ws linear fot l-Jo rM
97

wilh n dcl@tion |nnir ofo.22 rM. Thc.*ovcriq ol lO ns furcs.midc fre'n 3pitcd
phm@utic.l prcp.ntions w.G 98- | 0?/d
Jankows&i ct al., ( 134) invesrigltcd Oc &€mi.rrion md phm@ki.di6 of
a r@.niddhilori& dds @hbin.ti6- PI&E cdtlining thmp.utic lMk of
turcshidc 4d miloridc w$ lddcd to nitr@p.m d p,.!4in (int rml slodlrd! for
6r!!64idc ad milorid., r6petiEv). Fuomi& w dccctcd |l 280 nm;
uildidc by ooorimcric ddcction 14la m (qcilrtion 362nn). C.libntion
Snph! wE lir.u ftoh 3G3000 lg/nl mll 0.5-30 nglml for turoscni.lc ad
hiloid., EpetiEly. Th. rcl||od E rypli.d to r pi'm@tincdc inqtislti@
of rh. two cmpound. .dhiniri4d s . dnt cdbinrtid.
98

2.3 NIMESULIDE.
2.1.r S p€ct.oph oton.tric d.t rmin.llon of rinsulide
Naeaaja er al., (135) us.d nov.l coupling rcagcnts for the scnsniv.
speclrophotonetic detemination of mcaulidc in eifier pft fom or in ils
phmaceutical pEparations The ncthods baFd on the diuonsaiion of rcduccd
nimesnlide, followed by cither cbrfphg wiih alcoholic iminodibe.zyl in acid
nedium ro cn. ' d.ep blue colou.dproducr (lmbdanh;Iooo mr orcoupline
siih 3-uinophmol in acidic ncdiM io p.oduce o ordrge rcd @lou€d prodlci
(lmbda nd. of 470 m)- Bolh tlc m.thods ae hiel y Eprcdlcibh 8d hwc
becn appli€d to a sidc wicty of phma.culical prepmdons dd thc rcsults
comp&. favombly with rhc .c!oi.d ncthod. Comor tripients us.d d
addiriv.s in phmaEuric.l pEp*rtions do .ot intcrfce in 0E prcposed nethorls.
cho*dary .1 al, (136) i.Estigatcd ! n.w sp.crrophobnetic method for
thc detedination of ninesulidc, ?owdcrcd tabhts di$olvcd in nclhdol wcr.
mix€d vith Z. dcl dd conc.nEctd HCl, l€ft to stand for 30 mi., filtercd 6d tb.
volufre hade up ro lodmLwirho liil HCl, A I mL portion Ms nired wilh I mL
5 N HCI and I nL NaNO, Gn ro stmd for 5 nin, mned with I nL 0.5%
arMonium sulfamaie and l.fi lo sldd for 2 nin, N-(l-naphthyl)e1hyl.n€didnine
dihydrochlondc was added, th. tub. Ms hlatld at 100"C for 30 nin in a w.l.r
barh, cooled and tl€ volme nrd. up to l0 nL with H:O The purDle colour was
n4ued at 550 m. B.cis l.w wl;bbcycd Aom o4o pgltr[ dd ln. colour was
stoblc for >r2 h (€= 95 570). Th. RSD dd rc@v.d.s rcr€ in th. reg.s 1.5-2
dd 93- l0lolq rcspsliEly.
Altiloz adDsd.,(137) inEitiS&:d lhc ilcLmiMtion ofnimcsulitlc in
phmaeutical dosag. fom by *@nd otdd d.dntivc UV sprclropholomctry
The sdmt rh€ d.sEe of d.liEti;rr rary.s of waEt.ns$ md n_valnc wcr.
ctoscn in ord€r to oplinia lhc corditions. Tle concentialion ofninesulidc in ns
solntions in ethaiol and chlorofom wcrc det€mined between the eavd€ng$

an8.s of 200 ad 500 m ed in th.lincdity nnses of2.0"90.0 |lg/nl in.lhahol
lid 20-50.0 l€/ml in chlorofom by using the values oblaincd from thc second
d..ivative UV specrrum of thc suhr.rcc. Thc dweloped nc$od was acclrate,
chmdrm .t '1:, (138) delopcd ieo sinple dd a@unrc ulit'viol.t
spcctrophoton Eic merhods wi0t bcttcr dci{tl@ @gc for catinrlio. of
nimcsrlidc in pu€ fom od in solid do$8c foor\ using aceto irilc N th. solv.nt
syncnr. Tnc Inrc.nlyi.ngcoanim*ulidc i. $oll tlt nedo& w.s foud to bc I0-
50 Fglhl at a lmbda ux of 300 nm. The deiection linit as p.r th. .tur
propagation tneory wa found to be 0.46 Fg/nL dd 1.04 rc/ml, lcapcctively, in
50% v/v ond looplq v/v acclonil.ilc. Thc dcvclopcd mcllods yerc cmploycd wilh
hiSh d.gre. of Frcision ed accMcl for lh. Btinatid of total drug cof,Lni in
thft. colM@ial ta6lcr fomulalioa of nimulide
Mrinalinj, ct d., (139) riEu|nn ocly .siimt d nin*ulid. ed
chloEoxazone in 6bl€t doeg. fom thro!8l sp.crrophoton€lri. mcthod.Tlte
n.hod is ba*d on the s of sEullll.ous dqunioc to cahulatc nimcaulidc dd
chloEox.zone frcn absorbances of roluriom of samplc in O.lM NaOH 'n.aslrcd
at 244 tnd 395 m. B.eds law Ms obcFd for 2-10 ^d 25125 p9lnL of
nimcsulidc dd cNoturM.!, r.sp.ctircly. Thc method Ms applicd to iablcls
contrininB 100 ud 50 ns of nimBulidc &d cNoEox@tr€, respccdtly, giving
rsults which agE wiIn cbin d Elu.
Naralgmd .t al., 040) inBtiSrGd r sinplc @lorin rric n.lhod for the
dcl.mbation of nin€sulidc ion tu phmdeutical p..pdaiion T$lcG
containios nimcsulide wcc dissohtd in acctonitdle.Tb€ filhat obtnined was
dilutcd to 100 n[ md l0 n! of thc solution was tufther dilui.d to 100 m!. A
samph ws nixed with aceloniFilc and 2N NaOH ud finally dilutcd to l0 mL.
Thc .bso.bdc. wa ncawd !t 399 m. Thc calibratio! Frpb
50 Pg/nL. Th. r€cove.y w 100.09/r
100
was line.t fron 5'

tilshnri ct nl, (l4l) invcsrig cd flmrinctric dcrcrminarion ofnin sulidc
with,v-(l-n.phlhyl)ethyldediuh.. PowdcEd tabl.b conraining ninesulidc
wft dissolv.d in mclhanol md trc.tcd wilh IM HCI and Z. dust Aftcr st nding
ro. 45 nin, drc mixture Ms nltr.d thronBh cotion wool .nd thc r€sidne w.s
washed lith n.thanol TIE filttatc w6 nelhalized with lM NaOH.nd diluted
widr ILO io a known conc€nFaliotr. Ponions olthc rcduccd nihcsulide solution
werc diazoliz.d with o lM HCI rn.l N.NO, ftr 5 min. Amn@ium sulfam.tc ws
add.d followcd by I DL rv-(l-naphthyl).tiyl€ftdidin dft.r 5 niA md dilur.d
wilh wl.r lo l0 nL. FluoEscdcc m..su€d at 427 nn Gxcnation d 309 nn)
Beels law w6s obeyed fiom 0,55-2,75 tteilnl,. The RSD was 0.92%. Recov.rics
mgcd fron 99.5+0.?5% to 100.4+0.?%. Comon dcipieits did nor interfeE.
L $ini 6d Rcd.ly., 042) ilEstie.tcd fou sieplc ud 36sinrc visiblc
sp.cbphotoncFic n.tho& for rhc a.y of nincsulidc cirhcr in pw fom or in
pham@utical fomulltions. Mcrho& A md B de b!s.d on the ond.iiw
coupling betw..n the redlced produd ofnimc$nidc (RNMD) sndp-/V,^.-din€rhyl
phmylenediaminc dihydbchlolidc (DMPD) in prcscnce of chlomine-T (CAT)
or 3-mcthyl-2-bcMihi@lino!. hydr@rc hy&octdond. (MBTH) in pBencc of
fcnic cllorid. (F. lU) to fom coloed produ€ts wnh r mr ar 540 m ud 600
nn. r€sp.cliv.ly. Method C wa bdcd otr thc di@tiation of RNIV D Mth €xccss
.it ous acid and cstimting th. coftum.d ENO: wilh cr.syl fast violei acct t.
(CFVA). M.thod D vgs bsed on i!! fomatior of the colourcd chuso-rdsf{
complcx, wlrcn RNMD is trcaGd virh nerol (r-nctltyl miiothenol sulphaGr
PMAP) in F.s.ne ofpota$iM drclrom.t . All wirblcs wcr. oprimted dd dc
reacroi scqunce wd pEscnt.d. The @rcmlrdion
eproduciblc wi$in a Glative dddud deviation of l.oplo R.cowdes &e 986-
t40.2v,.
Chowdary ct 41., (la3) inrsnSlrd specEophoton.tnc d.t mimrion of
ninesulid. using 3-neIhyl-2-be@thi@liion. hydMnc hydrochloride reag.nr
l0t

Ni'ncsulide vas dissolvcd in nclluot and rrcatcd with Zn dust and conc.nEaled
HCl. Aficr sondins for I h, rh. mixturc was tilt red and th€ residue sas washed
widr metbdol, The filE.t w.5 rdjlstcd ro 100 nL with HlO then tuth€r diluted
to ! concentration of 100 F&h!. Podions of tte reduc€d nnnesulide solurion wer€
nixed wilh 1.5 nL 3-methyl-2-bcMorhiuolinone hydrazone hydrocdoide and
left for 2 nin, th.n 2 mL fcnic chloddc lolurion was !dd€d. The mi{nre ws lefi
for I0 nin, dihted to lO mL wiih H:O, hd it! .bo.bdc. mesnred at 600 m G
= 12 270). Beeis ldw Mr obcy.d Aom 5.20 rVnI or ninesnlid. Th. n.rhod
was applied to tableti ed sspensioc; r.sults compdcd favo@bly with a UV
metltod. Re@verics nnged Aom 99-l0l%. Comon excipicnLs did not interfde.
23.2 Cnromrto€rrphi.n.thod!forth..r.ly!i!ofnimdulid€
Parn'd. md Sola, (144) ilvcst Sat d a simplc, npi4 r@.oducibL ed
shbility indidIing hiSh p.fom$ce lhidlyd chrcrutosEphic n€ihod fo. ln.
dalysis of nim6ulidc both thc bllk dn8 sd &on phm&cu!@l fomtrlations.
Thc mobile phac s.lectcd @nsist' of Cyclohcxdc-Ehylacctate [60:40, v/v]
Delsiton.Lic ualysis of nincsulidc was ceicd out at 295 m. Tlte @libt?tion
cuNo ofninesulidc in medrdotw$ lincd in thc tdSc of 100-900 ng. Tbc linils
of detection and qudtiiation wcr. 50 dd 100 ne, rcspcctiwly, The ftcovery of
.imesnlide Ms abonl 99.5%.
Khaks. .nd UdlpA, (145) dcvdoP.d n rapid md sensilive high_
pcrfomece liquid chromdtoSrsphc mcthod fo. detlmination of nimesulide in
hune plasnfl. The chromatogr.phic syst m u*s I revcsed-phas€ Cl8 colum
wilh Uv-vis delection at 230 m Mbbilc-phse consistcd ofphosphatc buff€r (pH
5.5lnelhdol-ac.toniFilc (50120:30, v/v) !t a flow{ate of 1.4 mvmin The
calibralio. ctr* was linc& in th. coo..fiation rangc of0.O5-5 FE/nL, dd fte
lower linit ofdetcction w 30 ne/![.
Mil.jlovic €t al., (146) dav.loPcd, &d validatcd a thin_lavd
chbmtognphic (ILC) ncthod for Auuri$dve dalysis of ninesulide in hufrai
ptsmr. ed rpplicd to a phmeoldn.lic study of hc.lthy volmlecc
102

Chromaloerrphy was pofomcd oo silica g.l 60 Fn4 Ttr plates, developed w 1
tolu€ne-aceionc, md.valu cd dcnsiromchically ai 1,= 310 m in rco€ctance-
absorbance modc. Tle RF was 0.5025, Thc mellrd presenled is chamcbnrd by
high sensiliviry (0.1 n8/L) Th. ncihod was used to delemine ihc
phmcokinetio Fofih of nin.sutidc in hcrlthy volnntees aftd otal
KoFrikoE ei al, (147) studi.d Photoch.rucal st bility of nimesulide dd
obsryed the pr$dcc of dcgradation p@duc6 h th. cxpos.d smple, 2-phenoxy-
4-nihodiline *as ddccted by HPLC $dxis dd sllfo c acid Ms detected by
TLC analFis.
Rljm dd ?atil., (148) ilE3litltcd s GWs.d pha+HPLC for
sinuhaneous eslimariotr of tiaidirc ed nimcaulid. in r'blct{ a chr6nsorb RP
13 cotm rvas uc4 wirh o.lM .moniM a@tat/mclhmovacctoni$ile (3:5:2)
at pH 6 as mobile phase with dct ction ai 210 nn. Thc calibation ghph sas litr4r
fion 2-20 FglnL for tianidinc 6d lO-100 FElhL for nincstrlid.. Codcsponding
lin't3 of d.lNtion w.r. 0.04 &d 0.09 ppm
sane dd Gadsrl., 049) .sdnatcd simultmeously p&'cetamor,
cl ozoxazone .nd ni,nesutide Uy fiPTLC ssmpl.s wcre dissolved in nedlanol
dd the solution was nixcd with diclofcnac sodiun (inimal stmddd) S@ples
w€re sponed od precoatcd silha g.l 60 F& Plahs dd ddeloped wilh
CHcly'netheovtoluene (6 + I + 1.5) as mobile phasc dd d€nsitonenic del.crion
al 2?O m. Calibrgtion g.lpht wcr. lincd ir lhc rdeca 0.0607'0 1322 nVmL for
Syed .t al, (150) d.velopcd simplc ed npid HPLC, GC, and TLC
proceddes for d€tection dd dclmi tion of nimcsulide Usc ofthese techniqnes
hs enabLd sp&.tion of implriii* sd lhc prccusor in th€ bulk naterial dd i.
fomrlations. lsocBtic Gre.d-ph&i. HPLC w.s perfom.d on Shinpact Cr.
@tl|m with mctbmolMtd-ac.tic tcid (67:32:1, v/vr), as mobilc phae md Uv
dete.tid 230 m. Cdibdtion curws sr. li..d o*r lh€ co.cdraoon mge
t03

the nethod w6 suco.ssfully uscd to ealys. conucrcial solid dosaee fom
Iabell.d to contain 100 ng ofnih.sulidc and 50 ng ofcamyloline.
Zmpkd er al, (l5a) sinulrmeously deremined ninesulide and
chlouoxhzone i. phmdccutic.l dosage foms by reveae-phase hisn-
perfomance liquid chrom.tognphy. Ponions of a nixed ninesnlide and
chloEoxdone stsdrd solution in acctoniirilc wcr. alal'sed by HPLC o. a
Inensil colum wifi Hro/ac.tonirile (2r3, v/r, .djusted to pH 3.5 with,-
phosphonc acid, 6 nobile ph.s. md Uv dctcctio. .l 240 m. calibmtio. gnphs
wer€ li!€e for dmesnlid. dd chloEoeonc, pith co@spording detection limits
Rm dd P.ril., (155) sinuh.n.olsly cstim.ti.d nimedidc dd
p@etanol in rlbleb by F6c phsc HPLC, siry a Lichiotph@ RPl8e
colllm wiln H:O/ecto trilc (2:3) s thc nobil. phe 6d d.t ctiotr !t 210 m
Rctention timcs Erc upto 4 hin. Lin@ catibFtion gdphs wcE obtain d for 2-20
lg/triL dncsulide dd 10n00 lglnl pa$cd.mol.
camsco; (156) dcvclopcd ! simpl. nclhod fo. dcicminatio. or nim€sulide
in nt blood smplcs !y high-pcrfomlncc liqlid ch$n.tograPhy.The nethod
includes a si.gh .xtractio. of.cidii.d btood with diethyl cth.r. Blood ertracls
w€E urlys€d on a Ews.d-Phs. coltfu, .lut d with a milturc of
hetheovphosphale buffd (pH 5) and dcl.cled by absorboce !t 290 @
Reiention limes for the inimal sieded and nimcsulide werc 4.2 @d 8.2 nin,
respechEly. The nethod ws lin.e in tic mgc 0.2-5 pglml, and the deteclion
linil oflhe m€fiod v.s 40 ng/nl,. This ncthod b 3lilable fo! dctemination ol
nimesulide after oBl administntiotr lnd could bc ucd for Dh@cohitetic snrdies
of rhc drug in rh. rtt wing sdrllvolMc amples.
Nav.lsmd et al., (15?) inEstiglGd . SsschronatosraPhic method for the
dctmination of nin.sutid. fton iis phdmac.ulical p.cpanlion. SmPles wrc
dalys.d on a mctat cdlm co.tcd qith 10p,6 SE 30 oi Chrcnosorb wAw &d
I05

opcraled at 260oC wilh N, as caric. 8as
r06
(30 mumin) atrd FID Dctcctor and
inicctor rverc sl al 275oC. Thc inrmnl si..dard wd lriclosan nin.srlide vas
Iine& over the roge 2.5-20 rngln!, wilh. midnu dctectabl. conerirarion of
0.5 nymL Recovcry wN I00.67%, Excipicnts did not ini..f€E.
Ar3ekd a.d Sawmr, (lJ8) dct mir.d nimcsulide in phmaceutical dos.8e
foms by HPTLC. Powderod iabl.is, contai.ing nimesulide pere extacted wnl
nrcdr ol dilured wirh me[mol, Rcslliing solurion wos applied as 6 mn bands lo
silicr gel 60 F!! plates. Thc ptets *{c dcvclopcd {or 6 cn with
nethmovtoluene (lr4), d4 rn{ drying, wcrc scmed d@itonceically with a
Cdae scen{ at 324 M, rccordirg thc pcar &eas. Ihe calihdation gntt wd
lin d fJon 20-200 nglfl ofnimcaulid., with ! dctcction linit of0.26 n8/FL. Thc
rwragc rmovcrics of lG30 &d l-3 dg, Fsp@tiEty.
Jawomwicz cr .1., O59) dcv.lop.d & inpord hiel-pcdomcc liquid
chln'toshphic c.y ror nib4dil9. Tr.ltcd pl8M Portio6 ree &alys.d by
HPt C otr a HlTe6il BDS Ctr colutnn with 80nrM cillle ,nd phosPhatc buf.r of
pH 3/nethmol (8:17) s mobilc phs. &d detcction ai240 m. The calibFliof,
glaph w$ lined from 0.5-10 Ps/n[ ofnincsuli& in pldna. oer rhe calibnrion
ruge, t[e rmov.ri.s ofnimc$ulid. v.rc in ihc rege 91.5-96.2%.
Navalsud et al., (160) d.v.lop.d a figh perfomdc. thin layd
chronatognplic nethod for lie dc|cmination of nimosulide Aon phmaceutical
p.cpdations. Tabl.B conr.ining nin.sulid., w.re dissolved in nethmol nd
nixed wilh inteoal stddard, th. volm. w&s nad. up to r00 nL vith ncrhmol,
lO !L portioN !rer€ spon d onro ncthdol-wsh.d Mdc'( pFcoated silica 8.1
6oF,e platcs which wac d*top.d wih cNoofotD/tolumc (9: l) d nobnc phse
MIh detection at 300 m. Thc calibotion gnph wa lincd from l-2-8 Pg
cdini et al. (l6i) d.cmin.d th. nain urinary n€labolila ofniDctulide in
l@ thsugh hiSh pcrfom&cc lhi! laycr chrcnalo8raphic nctnod md
chmclerizd by Mass-spcc$onct r. Uri!. M ccntriirged, lhe supemrht {as

ad.iuscd io pH 7.0 ad applied ro a Exr.clut SPE colum. Nincsulid. ud is
ncrabolites w.r. clutcd wirt | 0 vol@s CH:CI: containing 2 5% popm-2-ol, rh.
coDbincd cxtracts vcrc @n@ntEr.d ro d.yncss and thc rcsiduc M dissolwd in I
nL &etonitrile. Urine ws a.ljust d to pH 4.5 with sodium aceiate buf.r lnd
tr.ated with tsgluclroniddstaryhulfatasc fot 36 h at l7'C. Unne Ms adjusred to
pH ?.0 md extnct.d as for fEe hdabolitcs. All netabolites were ohractenad by
TLC on on (iesels€l C!254 *ith by EIMS dcicction. ?oriions of ACN solutions
weE dalysd lor ninesulide dd it! n.taboliles by HPLC on a Supelcosil LC-18
DB cdlm with a Supelgad LC.IS 8!rd colum with endicnt chtion with
AcN/phosphate bdf.r solurion ofpH 3 &d d.r.odon .t 230 M. Thc calibration
gdph wa lin€& from 25-5000 ne/D! of nimcsulide ud its n€tabolitcs in uinc
vith a ddcction lidl of I0 n8/ml.
Alrftz d '1., (162) ddcmilcd nih6 idc in r.blct' by hjgn p.rrom44
tiquid chroMtographic mcthod with .l.cttoch.ni@l detectid. HPLC Ms
perfomed on a Po6il C-18 mlom.quippcd wift a Cl8 prc@lum *iih
phosphai. bulIer of pH 3/m.1hsol (3:2) 6 nobilc ph8e md el€otrochcmidl
derectio. in the puls€ dode .1 1200 nV. Thc d.t€ction md qudtitsiion limils
$crcIt'M.nJ25!V rcsDE('vdv. $lGr d($o(l'cnicil dele.ion wis used,
od 2.2 rM md 5.8 pM, .€sp.ctively, wh.n diode-mly detection was used. Thc
trEthod w.s suirablc foJ dctcmrining nnNsulidc in t.blcls.
Oiachcni dd Tcnconi,, (163) dcl.mined ncsulide and
hydroxlarinesulide in human plsna by high-perfomdce li+id €tuohalogophy
CrlibFdon g6phs wae li..d froh 5G5OOO dd 25-2500 n€lnl for nimsulidc
ed hydroryninsdid., cspc.tivcly. It. RSD Me l-19.2% (, = 5) dd
rcoEn.s w.re > l0@/o
Sutd .t at., (164) srudi.d r uniquc d.labolne oI lim.sulidc in !nn.
lhtoust Gs chronatosr.phic, FIIR |'d NMR Dclhods Urin. ws nixcd with
l0?

amoniu'n sulfitc burcr of pH 9.5 ud ancr .ddins 3 s XAD-2 Esi. thc solution
w nired for 15 nin. Aftr filtation lhc rcsin M Bhed widi l5 nL H,O .nd
clltcd with CH2CI/ethyl a..t.G (2:D, Thc dr.ct ws disolsd in 200 rl
n.lhmol md appli.d ro . Tt,c platc. An r d.v.loping with CH,Cl,/.thyl ler.tc
(9il), J00 El of lolr 4ueous almoniM hydrond. dd 7 mL eiher wer. .dd.d.
The mixtuc w6 rcnex€d, thc eth.r l.y.r was filt Ed md concentrated und.r N:.
The residue Ms dalysed for 4-mirG2-phenoxy-nethd6ulfonmilide by (i) CC-
MS o. a colutr coaled with DB XLB-14 opdated wirh tenpcratuE progftming
from 70-3004C at 20 oc/minj (/t FIIR on a P.*in-Elner 16 PC spcclrohct r
3398,3330,3069,2818, 1612. 1584 150?, 148?, 1325, 1213, ll53 cmrbddsr
dd (,i4 |NMR on a Brukd AMSoo srccEomcter. An unlroM rubslme ws
d.tectcdwtich%id. in.dsamct3bolitc of nim.rdid..
NoDioli .t al., (165) d*loFd. High-pcrfomd@ liquid{hrchalognphic
ncthod for ihe detemiulioo of lihdulidc ud ils inpuili€s.IlE smplc wa
rnrlysed on a colmn of Bond.p.l Cl8 with methdol : phosphal. bntrd ofpH
7.0 (l l:9) as nobile phac 6d dctcction at 230 nm. The Espo.se ws r.ctilincd
fro'n 0.04 to 0.32 pg /r!L of nin*ulidc md tlr linirs of d.tcmination for lour
synlhclic precurso8 ond two ifrpuriiies wcre froh 0. I to 0.5%.
csroldi €i il., (166) sinultan.ously del@ined ninesulidc .nd
hydroxrdnesulid€ in hMd pt.sma 6d uirc by high-perfomscc liquid
cl@alog.aphy. Plsoa or uii. wa mncd *ith nelh&olic tolbulamid. (inrchal
nd.lrd) dd acidificd to pH I vi$ conc. HCI- T[. ni,{tutc wd qF.ctld ridr
bdzcne dd enhiflgc4 thc orgrnic llyd *a Mponted ro dryncss, dd thc
r$iduc, containing ncsulid. ed its ncrgboln. htdrcxyninesdidq wd distoltd
in thc mobile Dhs.. Tt. HPLC wa cMi.d out on a lo+n Cl3 colu6 with r
mobilc phas€ ofsobM phGph.tc bufl.. (!1l5.0) - n.tnuol (l:l) dd d.i.ciion at
230 m. Calibmtion c.aphr wcr. tle{iln.u. Recov.ri.s of nin6llide non pl.smi
108

nd urinc wcr. 93 and 98vq Esplctiv.ly.Thc dct ction limii for both nihcsulidc
and hydoxynin.sulidc in bolh plami ud linc w6s 50 .g/ nL.
2,3,3 El.ctrochcmic.ldctomlnrtionolnihsulid.t
Cllad.o ct !1, (167) bvcadgarcd a oow lmpcromctric ddcmi.ation or
ph.maccuticds with onti!. .l.crod. surhc. an wt.Thc .cf.tcd syttcd vas
madc up of t*o plEll.l flow ch@ds thlt sh&cd thc rcltmmciric dctccto' or
tubul.r coffauotio'\ whos. novcn nt ii th. nuifold follow.d th. con4pt of
mulri-sit location ofdcrcclor. Fo. nin*ulidc, ! scri* ofrccovdy citdimcnc
w@ oroecdcd with and a mqn uluc of lol.l% *ith ! R.S.D. of 0.7/o ws
Alv.rcz ct trI., (163) dcrcrdnrcd nincaulid. by dilTeE.rial Pulsc
polrrcgraphic in pharmacculic.ls, Tt,bt€t conhining l0o mg nimcsulidc, ws
scD.ndcd in al@hol, 3onic.rcd .nd diluicd to 50 mL. A 2.5 nL poni@ of thc
sohtion wa ta&.n 6d dihtcd to 50 mL with .thanolic 40mM B.inon-Robi$on
bllTd of pH 7. Thc r.sultinS $lution ws i.ssf.ftd io a pollrosnphic c.ll
.rtrippcd rvnh n d.oppina-HA{lccood., i SCE rcfftna clFtrod. and 3 Pt viE
cout i .lccdod., Polegnm ** rc@dcd Aom -0.3 ro -0.6 v uting lhc
dir.ren&l pulsc tfthniq@ virh a puls. hcisht of-50 nv dd a pul* i.c al of
40 nr. Th. d.r.crion linit w$ found to h.2 5 rM, Th..rciti.nts $@lly pt s.nr
i. ihe phmaqcuric.l pr.perdio$ did tut int rfcrc.
Fule.to ct al.. ( 169) dBiSr!.d .4.rid.nts lo optinis. ud v.lidate
$c adsorprivc atrippinE vollamh.tric dcimi..tion of nincsulid.. A Plackctt-
Bumd matrix dd . Dochl.d d.sign vd. utcd for ihc scG.nins n.p 8nd
rcspons. suf.c. itudy. Gspcctircly. A full fadorial dcaisn wss .hploltd for
inremediarc p.ecieiotr dctemiNrion. Applying thc optimised condnions, the
linee mng. wns 3,32 " lo-t to 3.85 " l(l-'M an

M.lte$ et.l; (170) d.EloDcd . Epid n.thod fd q@dncadon ofnindulidr
ii 6bbit aqucou lunor. Thc Drtys* we p.rfomcd by hid-pcfoma.G
liquid ctrcnatogdphy using r C(lE) rcversed-phrs. mlum with Uv dcrfttion at
300 m. The nobil. phasc co$i!t.d of @ronitrilc-water otrtrining l%
hietlrylanine adjusted ro pH 3.2 wirh .rrhophosphoric acid. The etention tin.
*as 4 5 nin. A simplc pt.-tr.am.nt with acctonitrile was used to deprckinie
lqueo6 hu6or samplcs, Thc lidit of qumtiiation ras 50 sg/n|-. Th€ r.cov.rt
Ms over 90yr TIE rcl.tionshilJ bctw.cn tcal 6es and concenir.tion was lincar
owr rh. mse bctwer 0.05 dd 2.5 /mL. 'IXe 6say prcvided Sood
Eprcd@ibility ed &c@cy sd presd to bc suitabl. fq phalr]r@kinctic
lt0

2.4 LACTULOSE
2.{.I SperrophoroD.Ficd.tdnin ion of htulole:
Aiiic ct aL, (liD dcrclopcd s ncw cnrynoric spmFoneFic 6say for rhc
d.t.mination of lactuldc in milt sahplcs. Thc h€thod Ms ldcd on tbc trF ofF-
galaclosidse which hy&obs6 lactulos Siving fructose ad gal.ctose, a.d ftu tosc
dchydrogenase which .cac6 vith auctosc in pr.sence of a lcF@oliun salt, giving a
cololed compound wnicb can be dclcci.d lsing sDectropholomet€r al 570 nn Thc
alsay showod a letulose detcction limit in nilk of about l0 ng/L, .liner nnge of
sinilr 300 mg,/L ed. r.lativ. slmdad dcviatjon of5%. This proceduc wB found
3uit.bl. for d'e qutit tion of llctulos in milt alter ote h.at rrsE enr preccas.
Nagendi!.ed F&. (l?2) inpmd the coldimeiric nethod for lhc
.stimation of lactulo* il lrctosc - ls.rulor. mixrur.s. The nethod for rhc dtin.tion
of tlcfirlosc ws modifi€d by .ltc!i!g th. enc.nEation of alconol GcrcaFd) ed
rcctic acid (ds@cd) add.d to th. c&ti6 mixnrc. This Esult.d in a two-fold
incas. ir secitivity dd also feilitltcd u incr.ase in smple wlumq Laclos. did
nor inlcrf.rc in tne estimrion of up to 300 [g of lactulose. Valu$ of lactulote
6iinsled in lactos€ - kch os. nixtuts using lhis method. conped well wi$ ihosc
obhin€d by TLC involving a prior sepdation of l.ctolose.
Noldtrop €t al., (173) i.vcstig|ied autonated enzynilic 'ss.ys for th.
dctcmimtion of intestinal pmc.bili9 prob.s in u.inc 0&ush lactulosc &d laciosc.
Sample, trierheolminc - MgSO{ bullcr lolltion dd Egala€tosidac aolution wcrc
incubat d in a Cob*-Bio sdplc crp !t 3t' for 1.5 ro 2 hos. A funhcr 50 rL of
smplc w6 nixcd with ?5 FL of lhc buft{ slulion ed ts.d to dcr.min. frc.
gl!.or€ and ftuctose. Tfie nain Erg.nr as.mir of the ealyser conlai.cd a nirturc
of h.xotiMse to facilitatc phosphoryl.tion of th. monosacchdidcs and glucosc-6-
phosph.re d.hydrog.nas. 10 pronotc ihc fom.rion of sluconat. 6-phosp[at ond
NADPH, vhich ws detemined sp.ctrophotonetrically at 340 m. Tlc difircnc. in
rcsporse bcleen the awo smplc ponions repesentcd lile samplc laclosc

conc.nn ion Glucos.-6-phosphar. isoncrasc ws then added to both nixtuB from
tlc st.n E B.nt cnpj thc r.sulting difl@ncc in response b.tw.cn lh. srnple ponions
r.pEsnt d th. smple lactulosc @rcotntion. Tlc c.libntior gnph ws dlilinear
for both loctose dd l&!]losc @nc.nErtion up ro sinild, equab 2 g/L, dd th. limit
of dcrcmination ws 25 ng/L. Th. rtu Ecov.nes of lactos. dd l&tulosc weE
102.5 hd t,47a rcsp@livcly. Anbinos, grlactos., D@itol, ntrnos., rh.motc,
sorbosa .nd sucrcse did not inl.rfcE in cilh.r Nsay when ptcscnt at 29 2mM, and
intcrfcr.nce trcm naltosc, tureosc and xylos. was 0.5%. Ii the laclosc a35ay, high€r
intcrfcrence was obsened &on rchrlosc (5.570) dd tuctose (3.27'), bui fiat f.on
lactutos (5 8dlt niell reflcct rhe Drcs.n@ of lactose in the conncrcial lactulose
Lilov., (l?4) itrEstigarcd th. quudt tivc detcnninarion of lacolosc.
Lactulorc solllion rvee mircd wi|l| 50 mL or I M oxalic lcid tolllio. (pH 0.64) ond
0.5 nL of 25nM 2-thiobrlituic .cid iolrtion of pH sinile, .q@k 2.5. Thc
mixnr. v$ heat€d on a boiling w.rd bath for I hour, th€n €oolcd, a.d th.
absorbnnc. w6 measurcd at 430 m, L5ctos., Sltrcose ed galacto& (up to 3 tincs
tle arhon.l of lactulose) did nor int rftrc, nor did snall amounh ofgluco.ic acid,
TIF detectioh linit wa 0,4 lnol /nL md co.fl of vdiatio. wee

chdgEi in circular dichroiBh only l&tulos. 8i.vc s sctire specia wiln tunax 416-5
m. The Esults aE lttribut d ro letulos bridging thc two boronic acid groups ro
fom an inhanolecular l:l conpl.x, Th.0uor.sc.nc. itrteGily of the fllocsc..i
dibotu ic rcid was incre.sed in th. p!ca.nc. of l&tulose due lo the ngrdilication
efeor A sinilar increae in th. absorption bands was also obseMd allowine
lact'rlosc to be se.sed by sp.cFoscopic methods.
2.4.2 f,nryn.tic de&rninstion ol l.ctuld.l
L@ma et al., 077) irvcstiSstcd co?ratic rcrctions fo. thc dekmination of
sugd ir food sdpl6 6ing thc difd. ial pH technjqle. All ATP couplcd
r.acrioro, whd perfodcd rr rcut l or modcBtcly altalin€ ptl. produe d
&idincatioi of thc rcaclion nirhr. Thc d.tdtion of sndl pH chua* - 0. I mpH ir
a @nst nt bufiering €ap&ity slution n.l.s n po$ible to qnmdry, orcr a widc
c@cot rtion @gq vdiou ddyr.s {nh v..y hi8h pEcision a a@ucy.
Glucose, AEb*, glycc6l ed glucotiic acid on bc salyr.d in l.s thu I ni. wilh
. sinslc st€p reaction. Incrc.s.d snsilivily in somc sppli@tio6 cd h. obtlin€d by
cycliry E&tions, 4A a ldmse Factio! follor.d by a phosphar.se Baction, in a onc
step analysis, as rcquired for l.ctulos. assay in milla a usetul indicator of h.at
r.ahn nt dam8.. A s.nsitivity w.ll bclow 0,ln]i4 in th. onsinal dilk s@ple has
Amine d nl., (178) dcv.lopcd a llpid nedod for 0E deremination of
lactulose i. nilt usinS Seliwanofs rcmtion.ftc nethod was bas€d on Seliwanoffs
Faction to distinguish betwcen UHT rnd stdiliscd nilk. L&nnos€ was deimined
dircctly in milk with no trcatnlnt. Arlysb of raw milk dd itr cdbohydrat€s
showcd tnat thc onty intdfcrne wi, dE rclatirly hiel concentration of l.ctosc.
which imposes tfte lowd linit of dct ction. Snall veiations of lactnlose durins he{t
t@tndt could bc obseded whs rh. $so ancc *as nedu€d as.inst pE-trdlcd
milk (blstrk) This nclhod rhow.d a lild rugc betw€cn 17-170 ng /dL dd a
det€ction linir lo*d rhd ftar of rhc olficial HPLC mcthod This norcl prccedur.
Ill

wd co(p.rcd with . cotlercillly lEibblc cn?Drlic n.thod ad thc rcsults
obtai.cd coel.tcd wcu.
Sckinc .id HAll., (179) @nsEtscd.biocnsrfortrctulos. inmilkbs.don
u oz'E. rc.ctron i! which lrctulos. w$ hydrolysd to D-auctosc ed D-gd.closc
by tsgalactosida* which had bc.n imohiliad on ! didysis ncobM. toccd'.r
with liuclosc d.tydrca.N., B.d to oldiz th. Iiudo* ro 5-kcto-D-&ucto* whilc
its.lf bcinc rcdued. Ttc rcdued fom wa oEdi4d by lctdlhitrulnld-
Gllcyuoquirodincllrc, whioh wn pt lcd on ! c.rbon p.st ring clccFode.
Cuncnt ws nca!.Ed .t 200 nV B. AtAtCl. T|t. d.tcciion limit *s I rtM dd dtc
calibErior g6ph w liftt up to 30 |lM. RccoEry ws 103.6 and 104.8p The
cldEod. showcd . sclcctivily for l&nio!. lo00 ine hidc. lhD for l.ctos..
R.s!lr5 cofrh.d *.ll (. = 0.979) witl lhc by dzynic photonctry.
Mrys .r al., (lEo) inlcltis;J.uoonld dctcmh.tion of hctuldc ir dlk
BinS I Gnzync cd6r ud tld lulysis with iit gntcd di.lyris. T1. mctbod wa
bed on thc Gnzyhic hrdelylb of lrcnioi. to ftucto$ lnd 8d.do$ follo*.d by
thc s@dtion of ftuctosc by di.lysi! ud ilt d.tetion by &ucto$ d.hydo$ne
(FDH) cltatysd ondaiion wilh fcfricrrd& $ .l.cEon .@ptot. Th. m.fiod B
.pplicd lo fi. oslysi! of milt( 3dpla uring r srdd.td-sddition c.libdtid
proc.du.. Llctutos. @ndr.tions of up to l2,28hM wr. m.annd dd ihcs
.Bult3 w.rc qonfim.d by . photoh.hic ncthod.
Mscotri ct al,, (l8l) inEstig4.d hc.t-t rt d mil( diff.Mlitlion by a
s..sitis !&lutos sr.y. Thi &$iriE .nzynstic 6ry s3 spplicd to 90 sMpl.t
of wious dilk catesorics, ruch !s, pstMizcd mil} A.sh 6d hisrH.nPmturc,
UHT nilk, and in-cont.ind st.iili4d milk. Rcaulk lhowcd that n srs po$iblc to
dbtitrsubh, by hctulosc .ontcn! no! oily in-.onl.ird sr.rilizcd mi|r (?44 ngl),
indiact UHT (341 ns/L) &d di@r UHT (165 ms&), but .bo UET dilk prcduc.d
by nild r.chnologics s@h B mjft ldlcd by hn|3ion (107 m8/L), higlt-icmpcdnn€
pasl.uriz.d milk (58 ng/L), and low t np.nturc Pstcuird milk (3 5 ng,{,)
l14

Fci.r.ed Codsch., (182) pcdodld intd hbootory studics on p'cchion
che.tnstics of lralytic.l n.dbd! fd lhc da.didtion of lactulos in nilt.Tn.
Intcnarion.l Staidnrd mcrhod (lSO) q3 lsd in lt hbohtorid to study ih.
prccision of thc GcmD Omcial mcdtod (enzrmic n ihod), at cquind by Ccman
L!w. For UHT miu( rmplcs (lo0 !) @nrrinina 28 mg oflactulosc, thc Epot,bilily
wB I ng wilh r $edard d.viltro! of0.35 mg sd. cpredlcibiliiy of3 nU with ,
shdrrd d.vi.rion of Lo6 nsi for sGfiliz.d milk rdpl.s (l0o s) contliniq 105 ng
of lactulosc, rhc corcspordi.S slca wF 3, | ,06, 8 and 2.83 me.
2.4.J Chrom.roer.Dhicmctho&forltctuldcd.l.rmi..tion.
Sa'z .t rl., (183) invalsll.d tl. GB CkonatognPhic bchlviour of
dLscclEidc rincthyllilyl oincs. awnty fivc disrcch.rid.'s wa .onrcncd to
rinclhylsilyl oxim.r ad itrj@ted ;!'two catill.ry @lunN of diftcrcnt Fluilics
(mcthyl 6d phcnylncthyl iili@ne). Illcn Konts rctdtior indiccs r@ dctmin.d
!t thc l.m9.Blucr, Mulhun.G d.tr eElylis w u.d in od.r ro oh.h rcl.lions
b.twen rd.nrion .nd 3om. lEshlrl f6ner. Ph.nylm.thyl tilicon. ha b.t!.r
cnDn{o8r+hic chMct ristics for th. 6ay.d cohPoud., Th. iwo @lmN cu
providc us.tul chFnErogdphic irT;iDltion wh.n . Ery cmplcx dkechrid.
mixbrc nas io bc dnrly4d,
Rds ct rl., (184) dct miftd lhc lactulo* and mmitol in unne by hiSh
rcsolution gs chtonatogrtphy. Uiin. seplc I sPilcd with itusnol md
3rcches (inl.mrl rt rd.'d5 tor lrtio$ ed mMitol, rcsPcctEly) dd 3hrLn
witn Amh.dilc XAD-2 rdir lor 30";i!. PodioB of suDcmt !l wc mocd 4d
cuporlt d lo drync!! udcr t 3tcM of Nr. Thc rcsiduc ers db$lsd h DMSO and
t!.l.d wi$ NrOH sd CttJ fo. l0 nir w.ld w tdd.d ed lhc mixturc m3
dblcGd with Ctlclt. Thc oAlnio Pbsc w dticd ow noleculd sicE rid
Mpoocd lo drync$ uEd4 r slr.u ofNr- Thc Bid@ w3 dis$lv.d iI CHrCl, ud
a ponion ws aMlyscd by GC on ; &luu @.t d wirh Ov-l? ed opcdtcd with
t np.r!tur. progrmiry Aom l2O'C to l4o'C tl3'C/nin lrd lhci to 240'C at
lo'C7min $ilh H, cai,i.r gls sd FlD. Thc cdibntion 8r.Ph wls lincar frcm 100-
|l5

6400 riSll- for lactulose and naDitol rnd th. ddetio. linits wre 7.5 dd 4.4 p&
(rpeclively, of lactulose dd mannitol.
Frhadi et !1., (185) dcvelopcd a capillary colum GC nelhod for thc
me.surem€nt of unn.ry su(alose Md thr.c o$er suear probes includi.g, sucrose,
lactulose sd mamitol for usc in ,. vivo studies of intestina! p.meability. Th.y
cohpaied tne capillary method with a packrd colum GC netnod and inresigated i
possible role for sufaldsc as ! probc for th. nesur.ment of vhole Cutpemeability.
Somple pretMtion w.s npid ond limplc Tnc abow four suaas w€re delcclcd
picciscly, without interference. Th. netabolnft of snSds w6s cvrlu.led by
incubating the intestinal baciqia wilh d iscosnold nixture of mmilol, l&tulose
md sucnlose at 37"C for I t h. Thc us. of thc CCGC n€fiod inp.o\td lhe dci.cl'on
of slc€lose d 6npd.d to ?CGC, It dso incrcded ilE smitiviry of dctcction by
200-2000 fold. Tte nry method for dE bcs@ndt of unrary sucnlosc pcmirs th.
simuhd@6 q@tiration of slcres., nMitol sd lachlo*, dd t npid, sinplc,
scNnivc, accurate ddrep.oduciblc.
Nas et al., (186) inv*iigacd elbaG or l&tulose prep@tion us.g eas
ctuomto8rplry. Dri.d smpl. w$ dtuoMd in ptridin , reacled wil! exc.ss
h.x@cthyldisileme ud Fin thylchlorosilse rcagents at 3O-35oC for 2 h, thcn
malysed on an OVnT colum opd.ied wiii ienpemtuE progr@hg fron 100'C
to 285oC ar looc/min, N2 !s cariiii gd3 a.d FID. The method w$ applied to
dioerenl sdnplcs includinS dDsc from siomee slability studres. Rccownes wcrc
95.5-98.8 md 109.?-l13.8%for lactulosc ed laclose Espectively.
lt6
Maninez ei al., 087) dcrclopcd a Fpid ssliquid chrcnatognphic nelhod
for rhc ddemination bf lactulosc ald mmilol in uine dd appli€d in sodics of
intesinat pedeability. Uri.c sdplc vs tpiked wiir m.thyl c-D_slumPynnosidc
d irtcnal srsndadr for nr@ilol .!d lactulosc, rcspe€titly. Thc spiled solutioo
rv.s cvaporarcd ro dryncss undcr N2 5O'C ond dncd ovcr P?Or. Tnc dried Esiduc
w6 ireared wirh plndin€r'{O-bis(ridc$ytsilyl) acehide/ chlorotrinerhvhilan.
(2:2:l). Ponions of thc mixtur. *.rc ealys.d by CC on a tused{ilica colunn colted

wiln SPB-I and opeoted wirh tenp$ntufe prosromins froh 250'C to 300"C at
l5'C/ini wilh N, as c.nier g.s and IlD. Th€ calibFtion sdph wus linenr aro'n l0-
800 08 of lactulose &d mitol. Aw.lgc Ecoveries of n@itol and lactolose owr
ft calibration rmg€ lu€ 97.2 ed 10.5.9'lo, rcspdtirlt Tnc nc(hod w.s applied
to thc atudy of lilmid nacftncc dcraio. 6rios, d ! n.N of 4tiharing inr.$inal
pcd..bility, in infants f€d hbd nilk.
C.lli .t al., (138) dcv€lop.d d rdpid ga+tuoMrosr.phic 4$y of lactulose
.nd mdnnitol fo. estimating inrcatinal pcmeabiliry. Urin. sampl. wss nixcd with
Duolit MB resin md a ponion of supcmatant was dried under N2 at 60'C The
rcsidlc was incubated with lydrcxymirc chlorhydGtc in pyridin. at 60'C for 20
min ed o. coolira r ponion tincthylchlorosild./bis (Eim.thylsilyl)
trifltorolccthide % added. Thc dixtuc w6 oalrsed for lactulosc ed mmilol
by GC on a colllm @.Ld witl! HPI with t nFBture progl,ming fron 150-
270oC at 8oc/min wilh N2 s cdid g$ ud FID. 'ftc crlibntior gBph wai lined
from 0.1-to I pg of llcfirlos dd mMitol. Recovcri* wcre 91,5 a.d 92.5% for
lActulos. and mmitol, resp.ctiwly, Th. ncthod w6 applied to d.tcmin. inc sugd
Dumas €t al., (189) uEstislr.d gas chrcmatoslaphic application to lbe
lactulosc-mmitol intestidl pc@.lbility t.st. Uine I dcsaltcd with Ambdit
MB8 rsi.. filhtc Ms nixcd with myoinositol (int hal stadard for mamitol) dd
tunios. (int m.l stmd'rd for l&tulo*) The sohoon %.wpont.d in wuo: the
residuc Bas nix€d with prriditrc ed silyl.ting rea8.rt, Tn-Sil TBT and the nixnle
w.5 hcnted ,r 60"C for 30 nin. Portions of the r.action mixture {crc 6nilys.d on a
column coated wit! DBl, op.Bt d with t mp. progrming fron 250-300'oC at
8o'C/min, silh N2 as cdi.r 8as md FID. Calibhtio. Sraphs wctc lincar for 0.5-3
g/L of hmitol od for 25"300 ig/L of lactulose, pith &tectio. lidils of 5 and 3
nB/L cspecliwly. hra- hd int.!-ssry RSD wcr€ 2.7 6nd 8.9% for nNilol, a.d
6.8 md 9j% for llctulos.. Th. nc6od was suitabl€ fo! moniloring thc ralio of rhe
rwo suges n' i.resrinal l'.mabaliry r6ls.
\t7

Hu and Mao., (190) iMstigrred dcremination of lactulosc by gas
chrcnotosnphy. smpl. @ nix.d *ith N-Eincthybilylinid@le solution in
pyridine, lnd thc mixtE w hcar.d |r 70" ro ?5" for 30 nin. Afrcr cooling to @m
t mp, lhc aolution wa nixcd witl' irrdal standad soturion of t Eacosnf,. in
6cetone. Th. solution was injtrted o! to s cdm oI5% of OV-17 on Chronosorb
w'Hp. operded .t 205', with N 6 caicr els dd FID. Thc coefi. ofwiation for lh.
da.mimtior oflactulosc w 2'l.
vovk et al., O9D iLElopcd d oalrarcd ndnod for d.tmin ion of
kctulose 6d nannitol in uift on thc lmc eiN HPTLC plar., Th. method.nlbtcs
densitomelric qunntificaiion of lactulGc by use of fluor*c.nce node, and m.nnitol
by use of absorytioo mod€ aner d.tcction with AeNOr Gagctri
MoEtro ct d' (192) inBriglr.d rhe efrat of high prc$E oD isohfiiariotr
tnd deSmdation of lacto* in .lkali!. n dis. Thc conbif,c.t .ft cts of r.mpcraturc
ord hic!-prcasur. trearnents on thc ismc.iario!-dcshdsrion of laciosc (1070) ii
bnsic media wero slodied. Tle fomdion of isomeric dis0cciarides (lactulosc atrd
cpilactose) aid g.h€rore dccrcdcd by thc application of hish piesure in .qucols
sodiun hyd.oxidc 4 mM (pH 10.2) dd 8 trlM (!H 10.6), dd c!fton e buttu bE
10.0). In addilion, no subst ntial c.lor d.v.lopmmi wr obsen d h tha 3odim
hydroxide sysicns for high ud snnospheric pressur., whcreas the application of
lrigh pEssnre lcd to a noticelble dlcraae. of color d.v.lopncni h the cdbonale-
c.ocreso.r al., (193) @uplcd eUoliose dd lacrllosc rith mmiid a.d
detcmined usirg ion*rchdgc chrD(ography with puls.d mp€roneEic d.tcction,
for inv$tisation of intestinal pemcNbility. Slsds w€le noovered in lrinc colhcted
for 5 h. Analysis qs cdied out by lsing eiotrrxchege cbsmatoguphy with
pulsed mp@nctric der.cdon. B8.litrG s.pebor ofth..bov. cdbohydrarcs was
.chiered rvithio 13 nin by NirA t Crrbop& PA-I00 cdum ud lincd grldient
.lution. The c.libntion c!ft for caoh 8ugfi b liner ro 4OnM. Thc linit of suge
dclecliod is 0,0lmM. Recorcry ofsuSE| preb6 is bctwccn 98.2 ad I00%,
3

Zhou and Ji., (194) d.vclop.d ! HPLC ncthod aor $r!y of lactulose onl
solutions. L.cnrlose s@ple Ms dilutcd wnh nobile pha$. Portions of rhe solurion
were analys.d for lachnose by HPLC on a Phenoncn.x Nucl.osil amino-bonded
silic. gcl colum with acetonihih/lomM NaH,POa buffe., .djuslcd to pH 6 with
Hrpor (81:19) s mobile p!6e 6d difi.ulirl RI detcctior.'Ih. calibation gdph
for l&tulosc wa lincar nom 50-400 rg. The rrcdg. E@v.ry w6 looelo wirh an
RSD of | %. TIE solurion w shblc for 24 h. Therc w6 no intc.fcrcicc.
Sosa and SeM.. (195) dccmhcd cartohydr es in food samplcs by qpilla.y
€lectropho..sis with irdirect UV d.tcclion. CE sep@tions wcrc c.nied oul on tused-
silica c.pillarieswilh the capillary conditioned with 20 nM 2,6-pyridin.diceboxylic
acid wiih 0.05 mM oetyltimelhylaMoni@ hy&onde b r.v.6c fic dnection of thc
elcctro-os'notic 0ow S@pls wcr injcctd with pe$uc fo. 6 s and thc volLgc *t
at -25 kV md nrdirccl UV nonn;ng lsins a diorte my d.ietor al 350 nm
G.feEnc. Mv.lmgth wa 2?5 M). Undr optinised @ndirion! $. mcthod could
separat. 23 caibohydnies. Thc d.t clion limits for Auctosc, gtucose md sudosc
wet in n\e dg. l2- 16 me,/L vith a lincd mee of 50' 10000 mg/L.
Msilio et al., (196) dcwlopcd sinnlian€ous HPLC netlod for the
delemination witl tisht scattcring &ierion of hcblose dd mmitol in shldics of
'ni4iioal pcm.dility in paediatriG. Smpl4 of E@ wce purili.d by SPE on a
Mdid.d crr cariridgc rhd t.r.d wiih Ambdlir. IRA-400 Gsin dd dilur.d
bcfore a..lysis. Ceuobiose wa .d&d a i .mal standard. HPLC ras perfom.d at
85"C on a l0 !m Alltech ?00 CH C&bohyd8te Anatysis colum, Thc mobil. phase
Ms H1O and delection ws with an cwpontive light scalt.riig dctccior. Calibotion
saphs (not lined) weE.ostructcd for 3.125100 ng/L dd 12.5400 ng/L or
l.ctulosc and nannibl, rcsDcctiwly. Dct ctiotr Iinits wcE 0.82 .nd 0.65 mg/L for
hctulosc dd mitol, espsriEly. R.@qies Etr8€d f6m 97-101% fo. both
suga6. lnt rfceoce frcm gleos. tuclos. dd galacto* war not obscded
8ao et .l., ( 197) i.v€srieaicd dircct malysis of namitol, lActulose and slucose
in urinc sAmples by hid-perfomdc. anion-dch4ge chromaiogFphy wilh pulse
9

am!.rcncFic d.tcction and applicd to clinical cEluataon ofint€sii..l pcmabiliry it
hunan imunod€fici.ncy vitu inf.cdotr. Hunrn uin. ws nix.d with H,O
containing mclibiosc (intemal st@ddd) md a poniotr was filtered throuSh a cellulos
a*iatc menbrue. Thc filliate ws ualysed by HPtl on a 8.5lh CarboPac MA-l
aoio.-cxchanse colunn with 6$cbr.d suud colum, 480mM NIOH s nobilc
ph$. and pulsed mpercneEi. dcrRtion. TIE calibrariob grapns w.r. lin..r up to
lO n ol nnd derecrion limits werc d, 12,47 ond 52 pno! of 'nmnitol, glucose,
l.ciosc and lflctulosc, respccrively.
R@yalke6.i d, (198) dcrclop.d sinplc dd snsitivc nultisugd-probc eut
pcmctbility test by hig[-p6fom&c. liquid chrom ogrsply wnh nuoEsceno.
l.b.llihs. Urine s.npl€ frcn subjccts was mix.d witb of fllorcn-9-yln.thyl
chlorofomate hydrdirc solutio! i!;toniril. &d hcatcd for 16 h at 65.C ed pH
4.6, Aftd c@ling, 5 FL of supem.r4t M ualy*d on a Sphdisorb ODS mlum
with isobutyl .lcotol/p(opan-2-ovH,O (3J:4,t) I mobib pnae and fluorin.kic
d€t*lion at 315 m Gxcir.tion at 270 m). TE rcapoN. wN lincar in rh. rmge I'
1250 pnol ed thc dctcction linit3 w.rc 0.0J pnol for l6cdos. 6d 0 0l pmol for
rhamosc, xylos. &d 3-O-ndiyLD'glu6$, corcstondirg to utu.ry
co.ccnlr{ions of 0 | lrM ud 0.02 pM, rcsp€cdEly.
Flcmins ct al, (199) dcvctoped & HPLC method for simulhneous
det.minatio. of lactulos. md nmitol in serun .ftcr oral disesrion of t€sr sugs.
Snm ets Eeatcd with intetu l st ldard (Felibiosc) and ie-cold 5-sulfosalicylic
acid Ite mpl. was ccnrifirgcd dd rhc supmatat ms d6alt.d on ! mixed ion-
cxchange rcsin and recenirifu8cd. A ponion of thc aupemtei was oalysed o! a
Cdbopdck PAr dion-*cldge colue.quip!.d with a PAI gudd colum wilh
Nlopzinc acel,tc s clu.nl ed olbed el€cEochcmiql detection wi(h d Au
wotki.g .l€cLodc &d e AglA8Cl rcf*ncc cLctro&, Perr lleiglts wee ued for
qunnlittrtion Thc hiio of conccfiaiions of lactulde md maMiiol in serun
dscrininared well bciveen patidis wirh a nom.l biopsy ud ihose wirh villdus
120

dlrophy. Thc method protdcs an alr.h iE 10 Bing urinc which may b. ls.tuI fot
snmllcnildLen rnd lhc on.lysis tinr h reduoed fronr i h to 90 mnr
Miki lltrllcr ct nl , (200) devclop.d. hpid and simultaneous qu0ntific.tion of
rhrnno!, nmnd md la.niosc in uinc by HPtl for sdnaring ini.siimt
p.m.lbiriry in tedbhic phcde.Urin ws wrr.x-nix.d wirt D!oh. MB 5l 13
ior-.rch.nee resin for 0nd th. oixtur. ws cenhifugcd, Thc supematsr was fiherd
tud a portion Nas tualys.d by HPLC on s Ksmasil coium wirh aqu.ous ?0%
accloniEile s nobilc ph* dd RI dciccrion- C.libnrion graph w@ lin.d up ro
20mM for lactulosc (LAC), nmjlol (MAl0 ud Lrhamos (RflA) dd rhc
coftsPoding dclcciion limits w.c 0.05,0.05 and 0lnM, respccliEly, Avcrage
lccovcries w.re 99.3, loo.2 md I 05: t%, rcspfttirrr for 2nM or addcd LAC, MAN
and RHA to uinc. Th. ndnod B u*d for th€ 6a\ris of LAC, MAN sd RHA ii
thc lnnc oI infdls as . n es of ss.$ing inresrinat p.mcabiliiy in childM.
Maninez a.d Oldo., (201) d.Gctcd snall molnrs of letulosc in dairy
produots by thinlaycr clronatography in tieared ud unreaied Milk.
Dendene ci al., (202) studicd rlic scp@tion oftaciose, lactulose and gslactose
Uy liquid cluonatogmpny uing adonic ion#hdgc r.sin @lums. S.pmrions
wcrc pcrfome.l on r colomn pack d with Do{cx ACSOW in K', Cr" or Na' fom;
adjustcd lo a consla.l t.mpdatu.. with H,O as clucni The eluare conccnbarions
wcc nonitored with rbe u$ of o online Eaacroh.td det€ctor. Thc factors
cfr.crn,s lhc chbnrnlosmphic acoa;io. w€rc cwtuatcd md inorl'oratcd imo a
nathcmtical nodcl. Tnc nathd.riql mod.l s.rirt ciorily sinutred rh. s.p&3tion.
Emph6is is placed on the scaling up of th. nethod for indoshial use.
Soeoscn eL il , (20t devclopld ! novel HPLC mctlrcd for rlE snnullaneous
quantncation of monosaccbdidcs.tu-d die@hdid.s $ed in 16Ls of inrsturl
runcdoD md pem.ability. Spikcd uhi ed uinc ccorcrcd aner o6t ldminisFation
of sugas conlaining hmose, la.tutosc, x/los€ dd lo-m.rhyl8lucos€ werc dilured
50.250 fold ed portions wdc inject.d onto e @ion{xchsgc colum of Carbop&
PAl00 wiih elution with NaOE sd puled mpdom.tric dctc.tion wiih Au dd
t2l

As/AsCl cl.lkodes is wortins and r.fercnce €lcct odes. rcspcctively. Catibrotion
Sdphs verc line& from 20-100 mg& fo. all four sugN with a detecrion linit ot O.l
rl.m'ns er al , (2Oa) analrsed hulupl. su8! probej 'n unn. ed ptan; b)
high-perlomancd anion-cxch&ge chronatography widr putscd elecnochchicat
dct€ction ud lpplied in t[e ss.$ndt ot inr.stinal plmabitity in hlntu
imuod.lici.ncy vids inf@rion. Dillh:d uin jmplc M d.saltcd on Anbdn
1R-120 Pl.smd Ms nixcd wirh n.libiose solurion (intchal nanddd), the mixturc
ws dcproieinared with 5-sulfosalicylic ..i4 ccntrifug.d dd thc supemrrnt soturion
B nix.d wilh ion-dchmgc ftsin |!d agah dEifiBed. Tfu supdaianr soloti@
was d.lyscd by eioF.xchdrg€ HPLC o. a Cebopac PA-l oollm. Pube{t
cl4lrochemical detediotr M ar -0,01 V. C.lib.riion gBpl$ wcE rcctili.cd up to
15, 300 md 40 me/L of mirol, 3-Gbedytglu.olc ad l&r!tcc, Esp.cristy.
Recoveries were 89-105%.
willens el al., (205) n*ureJ'uilary suges by HPLC itr itc cstinaiion of
iniestinal pcm.ability. U.in w6 subj.cr.d ro enFitugarion dd rh€ suphata.r
solution wis dilut€d $/ith the mobit. phse. Analysis was by HPLC ar 85.C on a
colum oI Sls{Pal I wirh ! nobilc ph6e flow-Btc of 0.5 nu'nin md Gfrlctive
indd dct.crion. Tle calibnrion gmphr weE @ritin.d from 12-50 nC/L of tactuto*
and ndnnol aid the respc.tir Ecovlh.s w€rc 99 &d I oo%.
Kyn.ston et al.. (206) sirDulhcously q6titied n@i!ol, 3.O-h€lhylglucose
and lactulorc in uin. by HP[-c with pd*d elcciocheniat dcrcction for ur. jn
studies of inlesrinal pcm.sbility. Urift smptc *d diluicd ud nix.d wilh
nelibios (intemar stdded). TIE sohtion w.s d$salcd with washed hydroscn-ion
.:chdsc $ii\ nix.d thoronshly liid cenhifrlged. A ponior of the sup.mar.nt re
atu\^ed by HPLC on a col@ of C&bopsc PA-l &ionrxchdg. opemre

o-nethylslucose ad 40 ng/m|- of hctul6c. Thc d.te.tion linn *a 0.4 mg/mL
lactulos Tne oveFll cocff. ol veiltio! wcr. L8 to 5 6% R.coveries w.re 89
99.5v".
vd der voorde. €t al., (207) lFd an alrotu!.d rcboiic systcn for thc
extFciion and onlinc high-pdfomuc. liquid-chronatoSraphic ddysis of lactulosc
in pham@utical fomulations A.utohatic systcm dcrelop€d consisb ofr Zymatc
robot sysrem inteerated wirh two HPLC ststcms md h Gilson 232 etosupler with
iwo Gilson 401 dilutds ed two Rhcodync injcction pons. A peEonol conputcr witt
dedicoted softwN is ue.l to coltrol lh. suros@pld md to comuicatc with lhc
rcbot and rhe inte8rntors fo. ilc calcltlrion of thc contmt of the smplcs .trd
cenmtion of the rcport Thc n hod w6 ulidatcd by the detmimtion of th.
Itculose contcnt in a laclulosc syrup. Conpfison *ith esulis obt8in.d by Danual
analysis showed ro significdt difi.cne.
olano et al., (208) inEniS.r.d rh. dd.mimtion of frce cdbohydmt s sd
Anadori 6mponn& fofr.d at th. .aly st.g4 of notr€n4dic bromin8 in
milk.Thc fomalion ol frcc cebohy.lrarcs (lict!los., epilactose dd Cal&tosc) and u
N-.cetyl,FN{l-deoxy-Dl&tulosl-yl}L-lysine w6 nonnorcd by Gc Md HPLC,
respectiwly, i! milk nodel sysl.ms oonl.inhg l.crose, Nr-acctyl-lysine dd salts.
The model syslems wer€ cithcr hcddd at 120' oi fteeze&i€d od stored al45' ed
0,44 aw, GC was c.dcd out on o column of2% OV-17 with N 6 cd,er s.s and
t.mp. prc8@ed tom 200q to 2700 .1 15' per nin. Lacdose was fom.d .l
hiehd lereh tho -N-aetyt,eN{l{coxy-Dllctulos-lyl)-L-lysitc duing hcatins,
whercd the oppositc ws lruc duing lloFgc oflyophifisltes.
ve Ri.l dd Olidd. (209)'irv6tig.d selcctivity @nhol in thc aion-
cxch'nge c!rctutognphic d.cdn.tion of e@hdides in dairy prcduc6 using
pnls.d up.roneEic d.tectio.. ln th? s.p&atio of fiuctose D*al&tor, glu@e,
l.ctose lacnnosc, mrltosc dd mallFligoncrs wift pulscd @pqomcFrc d.kctioi
at +0. I V, +0.6 V md {.8 V for, mobilcphasc sndi.nts wdc uFd h a li.cd tlre.'
stagc E{sinon. Dionex C&boPac ?Al analtticd dd gwd colws wcr€ lscd
t23

Ch.n8c of ..tion from N.+ ro Li+, K+ or l.tr.butylmonium had linh .ficct
R.coverics of s{cchrides in hilk w.rc 98,2 to 10t.8% with rcpc.iability of0.? lo
5.6v".
t24
vclhaa .t al., (210) invcstig.d high-pcrfoMc. liquid cnrcubgnphy or
clction nixturcs from th. ondldon dd dcs6dation of l.closc. Su8&3 wcE
detemined widbur inrcrf.rcncc &om th. oxid.tion or dcgr.dation products. HPLC
on an RP-18 @lrmn wd ucd to ddmi& l&tulo*. A colum ol BA-X8 in Cl-or
SO42- fom Ms $cd to d.tcminc oxidrlion d d.gndrion Prcducts, rclP*titlx
*nhour inGrf.dc. frm tl! sus.rsi !di.l UV (212 m) ed r.fr&lirc ind.x
Kunhad, 8., ct al (2tl) dct mdncd 1..tu16. ozy@c![y *ith e of the
Biomct 1000 3y3tcn. Milt wls dilutc4 lh. elunon wa ctcd.d to GmoE f.1dd
protcin &d r poniotr of rh. filtr.c w ircub.t d with tsgd.cr6id.* solurion for 3
h 45". O{id.rion mixturc, H,O, ud qi.l$. slurid wc.c dd.d dd aflcr 3 h tt
,o' HrO s add.d. Thc crlibntion c@t *!s rcctilind up to 150 mg of l.ctulso.
p.r 100 g of sdplc ud thc @lL of wi.tion of 0.83%; r.corcti.3 trgcd &on
nminc ct.l., (212) d*loired. npid 6d sindtamu nahod fo.
&tcmination of lactul$. .nd n|nniol h uinc, by HPLC with Puls.d mpcrorcFic
det.clion, for usc in studi.s of inlsdld p.mc.bility. kctulos. ud nMitol werc
dctcftii.d in dcsdtcd uinc by HPLC on a .ollM of Dio.cx ltPlC'AS6 wilh
o. l5M NIOH s molril. phe dd p'rlsd dp.rcm.Fic d.tcction. calib6tion grophs
wft Ectilincf for o.l to 3000 n8/l df l.ctulo$ od hadlor'
V!@her et rl.. (213) comptrcd lhc lwo dctection modcs, rcfr&tivc ird.x md
ultra-viol.t, for thc dctcminltion of laculosc by high'pc.fomecc liquid
chronaioerafiy. Sianda.d solution of tactulosc w.E dn.lyscd bv HPLC on ! colunn
pack.d with Hr?asil APS 2 ed otdsl.d !t 38'isocEtically A rcttctiE ind.x
d.t.cto' m.l Uv dctcctor ( lto M) i ic u!.d simuttancously Th. Uv dctcctor sav.

cucr peak dele.tion ind Ms lastcr ro sct up dr& ih. efr&tiv. index detectoi which
[M rncmal slability poblems.
t25
Flick cr !1., @la) inEstigltcd thiniryer ckonatographic detcmination of
lrinary cxcr€tion of l&tulosq simplifi.d ud appli.d lo cyttic fibrosis paticnG,Tlt€
smtle was appli.d ro . layer of Silica eel 60 for TLC witn prcpe-2"o1 - q. t%
Nd:&o' solution (3:l) as Eobilc phas.. And the solvent tont had noved 65 Fm
th. plaie wd {tricd ed fte hcnlos.m loc.t d by sPnying tn. pl.le with dilul.
.th&olic rubnlic !cid.
Mdtincz-Csto, L; et al. (215) rwiq€d ihe chrcmatographic t chniques
lvail.ble for thc scpar.tion dd dctcminltion of laclulosc in nixtuEs with oihet
*bohy&otes. Pls& chrcmtognphy including p.pd, TLC sd paP.F
clcctrophor.tic t chriqu.s @ survlttd, Thc choi@ of $c *pmtion s,sl.n od
method of d.tection lo be Ul.d 'n colunn LC e o!0in d. Tie ae of&rivatiz3tion
t.chniqu€s in GC discused.
OIuo d al, (216) invcstiSlt.d thc ealysis of fre cadohyrlratcs in milk
using nicrc-pack.d collllm- Milk M traled with ph.nyl+slucosidc (intcDal
dddud), dilur.d wirh D.rhuol, ed filt cd. An diqlot is €u!o@ted, dd the
c&bohydrares wcr. d.ristizld with lrinethylsilylimiddolc. ttrtos., Saltcto*,
l.ctulos and epilacio$ *rc det dincd by CC on a st.inles$st€el colunn paclcd
with 2% of OV-17 on non{ileizcd Volrsphcr A-2. D.tccion @ dooc by FlD, fld
lhc &ftp. w6 progrm€d Aon 200' Ocld for 2 nin) to 270' at 15' pcr min. For
lactulo*, the arcogc aoeft of wi.tion oq I n@th w$ 5.8% and rccoEri.s werc
betveen 63.4 dd l0 | .6% fd 0. I ro 2.0 ng /nL.
Dchnnnry ind Holldd( ct al,, (21?) developcd liqlid-chronabSrdphic
nethod for .sti'lriing urinary sugd lo study intestinal Pemeabiliiy. Thc sanPle
*d nixed with N'OH eolution ed mtitug.d Th. rup.mtant solutior *as mixed
wnh anbdlit MB-3 ion{xchdg. ir_sin ed lh. supcmabt solunon w.s rakcn for
aMlysis. Th€ HPLC wd pc.fom.d oo ! colud of CHO-620 cation-exchanSc t.si.
wirh H?O .s mobile phsc dd rctsctiv.-ind€x detection. Lnctulose, rhmnose md

namnol w€r ftsolv.d md could b. dctdrun.d. Rcctilin.u crlibhtion gtrphs v€rc
obhnr.d foi nt to 5 g/l ofcach slen
vo Ricl ed Oliend er sl, (218) developed lrish-perfomancc liquid
chromatogmphy of suears on ! nix.d c ioFexchMgc rcsin colum. Mono', d; dd
ti{.cchuidcs w.rc scplatcd rec$tully on a @lum of HPX-65A Add4tdysd
hydrolysis oI suga6 ar elevaied HPLC rcnp. could bc supprc$.d by partially
conv.flitrg the r€sin into the Pb2+ {om. Thc sel.ctivity could be irtu.nced by
v.ryinS the Ag+ - Pbz+ rltio. Thc @obirarions mdtotriosc - sucrcse ed l&tulosc -
glucosc rcqnned addnional *ldtion on d @odilicd HPX{54 colum. Thc
mclhod was applicd cspccinuy to d.iry-prcduct foo&, including infant fomulae.
Beach md Menzies., (219) bwstis.ted the dctefrination of laci'rlose and
soy! oligosa@hdides in infmt fomub. kctdosc dd soya oligosechlrides w@
dcl.mincd by chrem.b8mphy on Wlatnd No 3 papcr for 24 h. wilh popanol -
acctic &id ' H2o ( 13 r3:5) as nobil. ph6.. fte infmt fomulae edF.d wre roud
to conrdr

separalcd by liquid chromatography tl 69 'C on a colum p5clcd with Ani.cx A-29
dion

L
2
3.
5.
128
ABa6j. P, Yafiirqie HS, R.ju CR vaetha RA, Nls€ndn P, Hellnih.
Kunnr MS. Ami.oplenot as a nov€l coupling agcnt for llc
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53(12), t295,1300.
OFz-Mtrrlincz, Lr LoDezdc- lD., P. L; d€,Leon-Rodri8ucz, l-.i Ycpcz-
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ud Biom.dicd tualrlis,200a 30 (l), 7?-8r_
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Liu, J, H.; Zlou M. D,i Wen, L,i Dcte, K. J. 189. Simutid.ous
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3.
IO.
t2
ll.
t4.
t5.
co.linuous on-lin. 3cpamtion lnd solid phrse UV r.nsducior. , Pham
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'n
Nr8:tr.rlo P, Suilhn KR, Vasmd. RA, YorhiEjan HS. lminodibcnzyl $ I
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t29

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t30

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ll.
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132

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47
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t55