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Stopped-flow Chemiluminescence Spectrometry to Improve
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7/18/2019 Stopped-flow Chemiluminescence Spectrometry to Improve
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Analyflca Chuntca Acta, 266 W92) 301-307
Elsewer Science Pubhshers B V Amsterdam
301
Stopped-flow chemiluminescence spectrometry to improve
the determination of penicillins based
on the luminol-iodine reaction
Sebastian Ventura, Manuel Sllva and Dolores PBrez-Bendlto
Depor tm en t f Ana l y t z ca i hemas t~ Facu l t y f Scmces Un wemty o f Chbba 14004 C&doba &nun)
(Received 22nd January 1992, rewsed manuscript recewed 19th March 1992)
Abstract
The hght generated m the reaction between lummol and &me was used for the m&rect stopped-flow
chemdummescence spectrometnc determmatlon of pemcdlins based m their mhlbltory effect on this reactlon The
reation was momtored v1a direct rate measurements on the formatron and decay steps of the chenulummescence
process m addrtron to peak-height measurements that were made for compmn The proposed method allows the
determmafion of pemcdhns m the range 1 X 10m6-1 2 X 10m4 M Hnth a relatwe standard donation of ca 2% and a
samphng frequency of 120 h-’ The method IS relatnrely free from the mterference of common exclplents
accompanymg pemcdhns m pharmaceutical samples Its figures of merrt (preclslon, rapldlty, sample consumption and
hablllty to automation) compare favourably with those of its batch counterpart, which entads a chenuluounescence
titration assay
xeywords Chenuhmunescence, Fluonmetry, Pemcdlms
Chemdummescence (CL) results from hght be-
mg enutted m a chenucal reaction Until recently,
chemdummescent reactions were regarded merely
as mterestmg phenomena and few attempts at
developmg analytxcal chenxal apphcatlons based
on them had been made However, the develop-
ment of extremely sensltlve and rehable mstru-
mentatlon has aroused much interest m such
reactlons lately [1,2] The wide vanety of such
apphcatlons currently avadable testify to the re-
markable sensltlvlty and selectlvlty of CL reac-
tions 111 everal areas of mterest In this context,
apphcatlons to drug analysis are stdl few m num-
ber but are growmg steaddy [3-51 Recent work
m this laboratory mvolved the search for CL
Correspo&ence to D PCrez-Bendlto, Department of Analytl-
Cal Chenustry, Faculty of Sciences, Umverslty of C&doba,
14004 C5rdoba (Spam)
reactlons to be nnplemented by stopped-flow
chermlummescence spectrometry (SFCLS) Hrlth
the aid of a modular stopped-flow system that
ensures rapld, reproducible nuxmg of sample and
reagents [6,7] and allows full mtenaty vs tnne
profiles to be obtamed, unlike contmuous and
flow-mJectlon systems These assets, together Hrlth
its low cost and great snnphc~ty, make the SFCLS
technique extremely attractwe In addition, thanks
to the kmetlc mformatlon offered by the whole
transient signal, the formatlon and decay rates of
CL can be related to the analyte concentration
vvlth a higher precision and selectlvlty than the
peak light mtenslty or the area under the hght
enusslon-tnne curve [6,7]
The antlbactenal drug pe~cdlm and various
closely related compounds are m wdespread use
nowadays
A
host of analyttcal procedures for the
determmatlon of pemcdlms 111 pharmaceutical
0003~2670/92/ 05 00 Q 1992 - Elsevler Science Pubhshers B V All r&s reserved
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3 2
S Ventura et al /Anal Chun Acta 266 1992) 301-307
formulations, fermentation broths and blologlcal
flmds have been reported, mcludmg tltrunetrlc
methods, based on the use of mdme [8], N-
bromosuccmmude [9], chloramme-T [ 101, sodmm
metaperlodate [ll] and potassium lodate [12] as
tltrants, spectrophotometnc methods, based on
vanous reactions such as that of umdazole Hrlth
mercury01) chloride [13], chrommm(VI) wth
metol [141, mnhydrm wth tm(II> chloride [15],
ammonium vanadate m sulphunc acid [16], hq-
md-liquid extraction methods using Azure B
[
171,
lodme or periodate 111 he presence of met01 and
sulphamlanude [18], enzymatic methods relymg
on reactions such as the hydrolysis of native pem-
clllrn to pemclllolc acid usmg lmmobrllzed or
dissolved pemcllhnase and direct detection meth-
ods based on measurements of reaction enthalpy
[193 or on momtormg pH changes due to penml-
101~aad formation [20,21] or mdrrect mdlmetrlc
detection of this acid m a flow mjectlon manifold
1221, and liquid chromatographlc methods, the
performance of which 1s llrmted by the use of
photometnc detectors [23,24] and must be boosted
by mcorporatmg a pre-column [WI or post-col-
umn 126,271 reaction or using an alternatwe de-
tection technique such as laser-based polarunetxy
[28] These procedures and others have been re-
viewed [29]
This paper reports on the use of the lodme-
lummol CL system for the indirect kmetlc deter-
mmatlon of pemcllhns Halogens are known to
react vvlth lummol m basic aqueous solutions to
yreld mtense CL Babko et al [30] were the first
to investigate the CL reaction between lodme
and lummol Later, Se& and Hercules [31] stud-
led its mechanism and potential apphcatlon to
the indirect titration of trace amounts of sub-
stances [32] that react with iodine [e g ,
arsemc(III), sulphur dloxlde and pemcrllm G]
Although the reaction between lodme and pem-
cdhn G takes over 2 h to complete, the reported
method results m a substantially shortened analy-
sis tune (ca 5 mm> In this work we used tins
reaction for the determmatlon of several pem-
cdlms by using the stopped-flow technique and a
commercially avadable spectrofluorlmeter as a CL
detection system Under the optnnum expermen-
tal condltlons, the reaction 1s very fast (the whole
transient signal can be acqmred m about 300 ms)
In addition, the stopped-flow technique nnproves
the accuracy and precision of the analytical mfor-
matlon obtamed with hrgh sample throughput m
only 2-3 s one can obtam an average of all
measurement parameters from four expernnents
The rehabdlty of the proposed method for the
determmatlon of pemc&n m pharmaceutical for-
mulations 1s discussed by evaluatmg the mterfer-
ences posed by various exclplents
EXPERIMENTAL
Reagents
All
chermcals and reagents were of analytlcal-
reagent or pharmaceutical grade Dlstdled water
was used throughout Aqueous solutions of pem-
cllhns (1
X
lo-’ M) were used to prepare work-
mg strength solutions by appropriate ddutlon
The lodme stock solution contamed 1 x 10V4 M
lodme m 1 X 10m3 M potassmm iodide and was
stored at 4°C A 2 X lob2 M stock solution of
lummol (Aldrich) was prepared by dlssolvmg
0 8858 g of the chermcal m 0 1 M carbonate
buffer (pH 10 80) m a 250~ml volumetnc flask
The 0 1 M carbonate buffer was prepared from
sodium carbonate and adjusted to pH 10 80 f 0 01
wth hydrochloric acid
Apparatus
The
instrumental set-up used consisted of a
stopped-flow module described elsewhere [33], a
Perkm-Elmer 650 10-5 spectrofluornneter, the
band width of the emlsslon monochromator of
which was set at 20 nm and the source shutter
was closed, and a data-acqmsltlon system based
on a laboratory computer (PC> equipped vvlth a
12-bit Metrabyte Dash-8 interface for data stor-
age and analysis The resulting data were pro-
cessed by three data evaluation methods usmg
software rotten by the authors pH measure-
ments were made with a Radiometer PHM62 pH
meter furnished with a combined glass-calomel
electrode
Procedure
The
sample and reagent solutions were rapidly
mixed m the stopped-flow cell by smmltaneously
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S Ventura et al /AnaL Chm Acta 266 1992) 301-307
303
mjectmg the contents of two dnve syrmges, one
of whch was filled v&h a solution prepared by
mtxlng m a M-ml vohunetnc flask the sample
solution contammg standard solution of each
pemcdhn (ca 1 X 10d6-1 2 X 10m4 M) and 10 ml
of lodme stock solution and made up to the mark
wth dlstdled water The other syrmge was loaded
Wlfh the lununol solution The temperature was
kept constant at 20 f 0 1°C throughout The hght
output of the reactlon takmg place m the cell was
detected by the spectrofluornneter at an emlsslon
wavelength of 425 nm The computer system
recorded the full signal vs tune profile and calcu-
lated the three measured parameters (formatlon
and decay rates and peak-height) and the concen-
tration of each pemcdhn
RESULTS AND DISCUSSION
Iodme has been extensively used for the mdr-
rect determmatlon of a large number of orgamc
compounds, mamly on account of its redox prop-
ertles The lodme uptake, which is proportional
to the analyte concentration, IS determined by
momtormg the decrease 111he absorbance of the
lodme-starch complex formed ChemAnnmes-
cence reactlons with rodme are also well known
[30-321, but have scarcely been exploited for ana-
lytical purposes This reactlon IS subject to mter-
ference by species that react with lodme and thus
a)
Fig 1 Effect of pemcdhn G on the CL lummol-lodme
system, (1) in the absence and (2) m the presence of the
antlblotlc [Pemcdhn G] = 2 X lo-’ M Other experunental
condmons as described m the text
exert an mhlbltory effect In this work we took
advantage of this phenomenon for the indirect
determination of pemdlms, a major class of an-
tlbactenal agents m wldespread use Under given
expernnental condltlons, this reaction can be
made pseudo-first order m the pemcdhn concen-
tration, so the enutted light mtenslty wdl be dl-
rectly proportional to the drug concentration
Figure 1 shows the tune courses of CL ob-
served by momtormg the lummol-lodme system
m the absence and presence of pemcdlm G (a
widely used member of the pemcllhn famdy) by
using the stopped-flow system on which the de-
b)
-10 -2; -10
Log [Lumlnol]
Fig 2 Dependence of the measured parameters on (a) the lummol concentration and (b) pH 0) Formation rate, (0) decay rate,
(a) peak-height For detads, see text
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304
S Ventura et aL Anal Chtm Acta 266 1992) 301-307
TABLE 1
Effect of potassmm l&de on the CL reactlon at a fmed
mdme concentration of 1 X lo-’ M
sensltlvlty to lodme, after which the effect of
various pemcdlms was assessed
[I&de] [I&de]/ Formation rate Decay rate Peak
(MI
[mdme] (V s-l)
w s-v
height
ratio
Q
25x10-5 25 2302 1352 0888
50x10-5 50 22 94 13 48 0862
10x10-4 10
22 83
13 42
0 835
80~10-~ 80 2268 13 39 0804
16x1O-3 160 22 14 13 13 0 785
24x1O-3 240
2198 13 11
0781
32~10-~ 320 2183 1298
0 750
40x10-3 40 2176 12 0 710
velopment of the proposed method rehes In the
expernnents below the spectrofluoruneter was set
at 425 mn and data were acqmred at a rate of 1
ms per pomt As shown, the CL mtenslty peaked
at about 60 ms and decreased to the background
level m about 300 ms
The mfluence of the hnnmol concentration on
the analytxal signal 1s shown m Fig 2a All the
measurement parameters used increased mth m-
creasmg hnmnol concentration up to 1 x lo-* M,
above which they remamed constant In order to
ensure maxunum sensitrvlty, a lumtnol concentra-
tion of 1 X lo-* M was chosen The effect of the
pH on the CL reaction 1s shown m Fig 2b The
three parameters used showed maxnna at pH
10 8, which was therefore used for subsequent
measurements Accordmg to the above results,
the lummol solution used to fill one of the drive
syrmges of the stopped-flow module was pre-
pared at a 2 X lo-* M concentration m 0 1 M
carbonate buffer (pH 10 80)
Study of txpenmental vanables
A senes of expernnents was conducted m or-
der to determme the selected analytical condo-
tlons for the CL determmation of pemdhns by
usmg the lummol-mdme reaction. The variables
stuQed were the hmunol, alkah (pH), mdlde and
lodme concentrations and the momtormg param-
eters were the formation and decay rates and the
peak-height Four separate measurements were
made under each condition or concentration
tested and averaged All concentrations stated
are mltlal concentrations m the reaction mixture
mediately after nuxmg The study of the reac-
tlon vatlables was carried out m the absence of
pemc~llm m order to achieve the best possible
Before the analytical features of the deternu-
nation of pemalhns were assessed, a maJor van-
able, namely the concentration of Iodme, was
studied In fact, only a lmear dependence on tlus
vanable would allow the mdlrect determmatlon
of pemcdhns based on then mhbltory effects on
thrs CL reaction One serious problem encoun-
tered m this context 1s the stability of lodme
solutions In order to mcrease the stablhty of the
aqueous solutions of mdme used they were pre-
pared m potassmm iodide Thus, for an nutlal
concentratton of lodme m the drnre syrmge of the
stopped-flow module of 1 X 10m5 M, the KI con-
centration was vmed between 2 5
x
10m5 and
4
x
10m3 M The formation and decay rates de-
creased shghtly with mcreasmg IU concentration
(ca 4-5 ), whereas the peak-height decreased
by ca 20 (see Table 1) A potassmm iodide
concentration of 1
X
10V4 M, which resulted m a
KI/I, ratio of 10, was chosen m order to achieve
TABLE 2
Analytxal features of the CL determmabon of mdme
Feature
Dynanuclmear range (M)
Sensltmty
Dete&on kt (M)
Prectslon R S D ) ( I (n = 11)
Method
Formation rate
106-10-S
26X106Vs-11mol-1
19x10-7
241
Decay rate
10-6-10-S
2 6 X lo6 V s-l 1 mol-’
2 1 x 10-7
294
Peak height
106-10-S
21X10sVImol-’
17 x 10-7
253
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S Ventura et al /Ad Ch Acta 266 1992) 301-307
305
the maxnnum possible sensltnnty This ratio was
be kept constant for all &me concentrations
assayed m further experunents Under these ex-
pernnental conditions, the three measured pa-
rameters varied linearly wth the mdme concen-
tration from 1 x 10m6 to 1 x lo-’ M
Table 2 hsts data relevant to the cahbratlon
graph The three approaches performanced smu-
larly 111 his respect. Lower concentrations of lo-
dme could be determmed, yet the analytical slg-
nal was lrreproductble, which 1s consistent urlth
the findmgs of Ham et al [34] m relation to the
stab&y of trace lodme solutions In fact, the
lummol-&me system has been used as a very
sensltnre approach to measurmg mdme concen-
trations vvlth a detection llrmt as low as 5 x lo-”
M [31] However, Ham et al showed the great
difficulty mvolved m handlmg mdme solutions at
concentrations below 1 x 10V6 M arrsmg from
mstablllty problems, even at tis concentration, tt
IS recommended that solutions be stored m tightly
capped, s&uuzed glass tubes, kept from light and
used wtthm 20 mm of preparation. We therefore
chose a 1 x 10m5 M lodme concentration m order
to avoid nreproduclble results and the need to
prepare a fresh &me solution before each senes
of measurements
naly t t cal eatures
After the CL reaction condltlons had been
established, the method was applied to the mdl-
rect determmatlon of pem&lms based on the
reaction between mdme and the pemdlms Be-
cause of the mstrumentatlon used to unplement
the stopped-flow techmque, It was first necessary
to determine m whuzh drwe syrmge the pemclllm
solution was to be placed There were two alter-
natwes m this respect either m the syrmge hold-
mg the humnol solution and or m the other,
which was filled wth the mdme solution In the
former mstance, the reaction between lodme and
pemcrllm must be faster than the CL reaction
whch developed to completion m ca 300 ms In
the latter, the elapsed tune could be used to
ensure that the two substances reacted quantlta-
tlvely The expenmental results revealed no m-
hlbltory effect from pemcdlm m the former m-
stance Also, the latter alternative was obviously
TABLE
3
Features of the deternmatmn of pemcdlms by the CL reactlon between lununol and mdme
Pemcdbn Measured
parameter
Pemullm G FormatIon rate
Decay rate
Peak he&t
Alllp1cdbll FormatEon rate
Decay rate
Peak he@
AXlIOIQJcdhn Formation rate
Decay rate
Peak he&t
Carbemalbn Formation rate
Decay rate
Peak height
Tlcarcdlul
Formation rate
Decay rate
Peak he&t
mear
range
am
2 x 10-6-3 x 10-S
2 x 10-6-3 x 10-s
2 x 10-6-3 x 10-s
4 0 x 10-6-15 x 10-4
3Ox1O-6-12x1O-4
40x10-6-90x10-~
1 x 10-6-2 x 10-S
1 x 10-6-2 x 10-s
1 x 10-6-2 x 10-s
1 x 10-6-2 2 x 10-S
1 x 10-6-2 0 x 10-s
1 x 10-6-2 1 x 10-s
2 x 10-6-3 2 x 10-5
2 x lo-+-28 x lO-5
2 x 10-6-3 0 x 10-s
Lmear regression
equation
Z( )=221+302x106C 0996
Z( )-583+292x106C 0994
Z( )=363+289x106C
0990
I( )-022+615xlO’C 0997
Z( )=172+903x10sC 0999
I( )= -133+104x106c 0999
I( )= -123+458x106C 0999
I( )
= -o75+468x106c
0999
Z( )=121+450x106C 0999
I( )= -069+408x106C 0998
1( )-141+451x106c
0998
Z( )= -002+435x106c
0998
Z( )=111+291x106C
0997
I( )= -1O6+334x106c 0999
z( b)=041+314x106c
0997
Corre
lation
Coeffi-
clent
(n = 7)
Detectton
bnut (M)
( o)
46x lo-’ 301
78x10-’ 3 67
86X 10-7
255
23 x 1O-6
134
25 x lo+ 227
24x lo+
116
30x10-7 208
49x10-7 641
55x10-7 205
34x10-7
118
5 1 x 10-7
201
57x 10-7
097
48x10-’
325
69x10-’ 3 78
80x10-’
3 05
(RSD)
(II = 11)
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Fig 3 Structures of pemcdlm analogues
time consummg We chose to place pemcllhn m
the drive syringe holding the lodme solution, so
the CL reaction took place umnedlately This
resulted m a high samplmg frequency, with no
appreclble detrnnent to the sensltlvlty
The CL intensity vs time curves recorded for
different amounts of pemcdlms were analysed by
usmg the three measurement approaches Five
pemclllms were assayed, VIZ pemcdlm G, ampl-
clllm, amoxycrllm, carbemclllm and tlcarcllhn, the
structures of which are shown m Fig 3 The
cahbratlon graphs for these pemcllhns were ob-
tamed by plottmg the percent mhlbltlon, I( ),
against then concentrations, which were calcu-
lated as
Z( ) = [(S, - S,)/S,] x 100
(1)
where S, and S, are the measured parameter m
the absence and presence of pemdhn, respec-
tively Table 3 lists data relevant to the cahbra-
tlons graphs for the pemclllms As can be seen,
the linear cahbratron ranges and slopes (analytl-
cal sensltlvrty) of the cahbratlon graphs are srml-
lar for the three measurement approaches, how-
ever, there are differences m each pemc~llm that
are directly related to the reactlvlty of each com-
pound on account of its structure Thus, the
highest sensltlvlty was achieved for amoxycllhn,
probably because of the presence of a hydroxyl
group m the benzene rmg of substltuent R
S Ventur a et aL Anal Chun Acta 266 1992) 301-307
Other sahent features of the determmatlon of
pemclllms investigated were as follows The de-
tection hnut was calculated as the mmunum pem-
cillm concentration resultmg in a statlstlcally slg-
mflcant I( ) accordmg to
cm_
ZDl_(I
/m
(2)
where Z&o/o) 1s the mmnnum value statlstlcally
obtained by replacmg S,-S, m Eqn 1 with three
times the standard deviation of the measured
parameter m the absence of pemc&n, and
m IS
the slope of the cahbratlon graph (analytical sen-
sitivlty) for each pemcllhn The values found
ranged from 3
X
10m7 to 2 3
X
lop6 M, depend-
ing on the pemclllm concerned The precision of
the proposed methods was determmed by usmg
eleven samples contammg 1 X 10m5 M pemclllm
The average relative standard devlatron was 2 3
The sampling rate drd not depend on the reaction
tune (300 ms), so it was calculated by takmg mto
account the time required to change the sample
solution m the drive syrmge of the stopped-flow
module, 1 e , ca 30 s A samphng rate of 120 h-’
was achieved
It was considered of mterest to compare the
figures of merit of the proposed method and
those of the CL tltratlon assay for pemcdlm G
only reported by Hardy et al [32] and based on
the same chemical system They used a 5
x
lo-’
M iodine solution, which may pose above-men-
tioned mstablhty problems, and succeeded m de-
termining pemclllrn G from 2 1
X
10e8 to 2 1
x
10e7 M [relative standard devlatlon (R S D ) 9 ]
in a 500~ml sample volume The reaction time
was about 6-7 mm Accordmg to the results m
Table 3, the method proposed here 1s less sensl-
tlve for the above-described reasons, yet 1s more
precise (average R S D 2 3 ) and faster (reac-
tion tnne ca 300 ms), uses samples more spar-
ingly (ca 5 ml 1s sufficient) and lends Itself more
readily to automation (use of the stopped-flow
technique for nuxmg sample and reagents) than
does the titration assay These assets endow the
proposed method with great potential for the
routme determmation of pemcdlms
In order to assess the potential analytical ap-
phcatlons of the proposed method, the effect of
some common exclplents used m pharmaceutical
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S Ventura et al /Anal Chm. Acta 266 1992) 301-307
TABLE 4
Recovery of 1x 10m5 M pemcfim G from solutions contam-
mg 100-fold concentrations of various exclplents
Exciplent
Recovery ( o)
Formation rate Decay rate Peak height
Glucose
103 0 93 5 1010
Sucrose
1001 1000 93 9
Lactose
977 990 93 4
Glycerol
95 4 97 6 1017
Talc
1019 964 101 1
Starch
913 919 925
preparations was studled by analysmg synthetic
sample solutions contammg 1 x lo-’ M pemcllhn
G and 1 X 10e3 M of each exciplent Any undls-
solved material was filtered before measurement
The recoveries obtamed are given m Table 4 No
mterference was observed from any of the exclpl-
ents tested except for the small perturbation from
starch (average recovery 919%), which 1s proba-
bly due to an interaction v&h lodme These re-
sults are of great mterest urlth regard to the
determmatlon of pemclllms m pharmaceutical
samples
The authors gratefully acknowledge financial
support from the DIGICyT
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