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



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



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



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



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)



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



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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stopped-flow chemiluminescence spectrometry to improve

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