Research Article

Received: 23 September 2008, Revised: 27 February 2009, Accepted: 28 February 2009, Published online in Wiley Interscience: 18 June 2009

(www.interscience.wiley.com) DOI 10.1002/bio.1137

Copyright © 2009 John Wiley & Sons, Ltd. Luminescence 2009; 24: 448–452

448

John Wiley & Sons, Ltd.

Automated determination of asulam by enhanced chemiluminescence using luminol/peroxidase systemAutomated determination of asulam by enhanced chemiluminescence

Francisco García Sánchez*, Aurora Navas Díaz, Visitación Bracho, Alfonso

Aguilar and Manuel Algarra

ABSTRACT: A flow injection system with chemiluminescence detection for the determination of asulam, enhancer of the sys-tem luminol–H2O2–horseradish peroxidase, is proposed. The method shows a moderate selectivity against other pesticidesusually present in formulations of herbicides and in water. The procedure was applied to the determination of asulam in tapwater samples and a recovery study was carried out in order to validate the method. The obtained results show acceptablerecovery values (between 88.3 and 93.9%). The detection limit for asulam was 0.12 ng/mL. The precision of the methodexpressed as relative standard deviation was 1.55% (n ==== 8), at the 19 ng/mL level. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: chemiluminescence; asulam; luminol; enhancers, flow injection analysis; carbamate; pesticide

Introduction

Enhanced chemiluminescence (CL) describes the phenomenonof the light output increase in the chemiluminescent reactionproduced by a chemical system that satisfies the energeticrequirements to promote to the excited stated a chemical sub-stance that emits light when it is relaxed to the ground state,[1]

catalyzed by a compound that enhances the speed of a low stepin the overall luminescence.[2]

Several analytes have been studied as enhancers for thesystem luminol–peroxidase, mainly phenolic and amine deriva-tives,[3–6] 4-phenylylboronic acid,[7] 4-methoxyphenol, 4-hydroxy-biphenyl and 4-(1H-pyrrol-1-yl)-phenol,[8] and different polymers.[9]

Also the CL of the system enhanced by 4-iodophenol was appliedto hydrogen peroxide determination.[10,11] In several instancesthe analyte acts as inhibitor of the reference reaction, producinga decreasing in the speed of the CL reaction.[1] This situation canbe used to develop analytical methodologies to determine lowconcentrations of the inhibitor based on CL inhibition kinetic.Asulam can act as an enhancer or inhibitor depending on the con-centration range. Aromatic amines have been found to enhanceor inhibit luminol–peroxidase chemiluminescence,[12] dependingon the Hammet constants and redox potential of the compoundsor the time scale of recovery data.[2]

Flow injection with chemiluminescence detection (FI-CL) hasbeen extensively used for the determination of numerousanalytes in the environment at low concentrations.[12,13–16] Thistechnique includes high sensitivity and simple instrumentation.

Asulam (methyl 4-aminobenzene sulfonyl carbamate) is apesticide that acts by stopping the cell division and growth ofplant tissues, and can accumulate and remain in environmentwith a broad spectrum of biological activity.[17,18] Because of itshigh water solubility it is a potential water pollutant. The currentguideline value for asulam in drinking waters is 0.1 μg/L,[19] justi-fying the development of suitable analytical methods. Asulamhas been determined in several matrices by fluorescence[20] and

photo-CL[21] determination using flow injections systems, deriva-tization methods,[22] UV and electrochemical detection,[23] HPLC-fluorescence,[24] thin-layer chromatography[25] and electrophoreticdetection.[26] In a previous paper[27] a stopped flow chemilumi-nescence determination of asulam, based on its inhibitionaction over the system luminol–H2O2–HRP in the first instantsof the reaction, has been proposed. The aim of this work wasto develop a simple and fast procedure for determining asulamin freshwater, which involves an enhanced effect in the CL signalof the HRP reaction. No references have been found in the litera-ture search exploiting the enhancer character of asulam usingan FIA-CL system.

Materials and methods

Materials

Luminol (5-amino-2,3-dihydro-1, 4-phthalazinedione, 97%), horse-radish peroxidase (HRP), type VI-A (1100 U/mg) and Tris–HCl(99–99.5%) were purchased from Sigma (St Louis, MO, USA);hydrogen peroxide 6% (p/v) was purchased from Merck (Darm-stadt, Germany). Asulam (methyl 4-aminobenzenesulfanilylcarbamate (99.9%), from Dr S. Ehrenstorfer, Augsburg, Germany)was employed without further purification. Solutions were pre-pared in deionized water previously purified through a Millipore60 system (Bedford, MA, USA) and employed for all aqueoussolutions. Luminol stock solution (0.01 M) and HRP 0.1 g/L wereprepared in Tris–HCl (0.1 M) buffer solution (pH 8.5), and the

* Correspondence to: F. Garcia Sánchez, Department of Analytical Chemistry,Faculty of Sciences. University of Málaga. Campus de Teatinos s/n. 29071Málaga, Spain. E-mail: f_garcia@uma.es

Department of Analytical Chemistry, Faculty of Sciences. University ofMálaga. Campus de Teatinos s/n. 29071 Málaga, Spain

Automated determination of asulam by enhanced chemiluminescence

Luminescence 2009; 24: 448–452 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bio

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hydrogen peroxide (0.1 M) was prepared in borate buffer 0.05 M(pH 8.5).

The stability of the aqueous stock solutions of asulam wastested by preparing a solution containing 0.5 μg/mL, which waskept into a brown flask, protected from room lights and storedin a refrigerator at 4ºC. Every day its absorption spectrum wasrecorded and no changes were observed.

Apparatus and flow assembly

CL measurements were made with a luminescence spectrometer(Perkin-Elmer LS-50, Beaconsfield, UK), equipped with a quartzflow cell (5 cm of light path and 12 μL of volume) in the sampleholder and assembled to an FI system, which consisted of a coilof 20 cm of polyethylene tubing (0.5 mm i.d. × 1.5 mm e.d.) assem-bled to the liquid chromatograph.

A pump (L-6200, Merck-Hitachi liquid chromatograph, Darmstadt,Germany) was used to deliver the entire chemical: (A) luminol;(B) H2O2 and (C) peroxidase (HRP) at 1 mL/min of flow rate. Anautosampler AS-4000 and a D-6000 interface (Merck) were usedto inject sample and send data to an external computer. Threechannels were employed for sample solutions and were mixedin the tubing system (Fig. 1).

FIA-CL method

The flow injection consisted of a three-channel configurationwhere the standard solutions were incorporated into the stream,aided by means of the pump. The pump propelled the differentsolutions, (A) luminol, (B) H2O2 and (C) HRP, through the systemat a flow rate of 1 mL/min. When the injector was commutated,selected volumes of asulam were pushed by a carrier streamof the reagents. The CL produced in the flow cell was detectedby the fluorescence spectrometer at 425 nm emission wavelengthand the light source turned off. The apparatus was set in the

phosphorescence mode with 0.00 ms of delay time and 10 msof gate time. The slit width of the emission monochromator wasset at 20 nm and the photomultiplier voltage set to 700 V. Allmeasurements were carried out at 25 ± 0.1°C.

Sample assay

To avoid photodegradation, water samples were collected inbrown measuring flasks. These samples were held in the darkand refrigerated to 4°C until chemiluminescence analysis. Therecovery test and interference study were also performed in thesame procedure.

Results and discussion

Kinetic curve of the CL reaction

Previous experiments were carried out, employing the batchmethod, to study the kinetic behavior of the proposed method.In the batch mode, the experimental parameters were keptconstant; the typical response curve (intensity versus time) ofluminol (0.01 mol/L) was recorded with the kinetic characteristicof the CL reaction. The CL reaction was catalyzed by HRP (0.1 g/L)in the presence of H2O2 (0.1 mol/L) and a quick CL intensitypeak was obtained when asulam was injected into the flowsystem. After that the signal reached the baseline at 30 s. In thiscase the kinetic curve indicates that the CL method is rapid andsensitive enough and suitable for performing a determination ofasulam.

Optimization of the FIA-CL

Preliminary experiments showed that the CL reaction of luminolwas enhanced by asulam. Blank signals were obtained usingthe luminol–H2O2 system by itself and catalyzed by peroxidase

Figure 1. Schematic flow injection system for the determination of asulam.

F. Garcia Sánchez et al.

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according to the enhancement produced by asulam. The differ-ent ranges of concentration and composition were investigated,for the FIA system, to reach the optimal CL signal (Table 1).Values of 3.6 × 10−6 and 6.6 × 10−6 mol/L were chosen for luminoland H2O2, respectively, for further experiments. The concentra-tion of HRP was kept at 0.01 mg/mL in all experiments. Thus, byemploying H2O2 buffered with borate (pH 8.5) in the same chan-nel, the best reproducibility was obtained. The feed compositionof each channel was evaluated at different ranges, the best valuesbeing 10, 80 and 10% for luminol, H2O2 and HRP, respectively.The luminol percentage of 10% was selected because the inten-sity of the blank signal must be maintained at a level where theenhanced signal can be significantly different. H2O2 percentagewas selected at 80% because at 90% the signal decay was 10%,probably because the CL transient signal was very rapid and whenthe flow reached the photomultiplier the signal was decreasingin intensity. HRP composition was selected at 10% because higherpercentages decreased the signal intensity, probably due to theincreased speed in the CL reaction. In contrast, a 5% compositiongave a reduced signal because the speed of reaction was exces-sively low. In order to obtain satisfactory emission intensity, theflow rate of the reagent solution was explored. The CL emissionintensity was observed to increase constantly with the increase

of the flow rate, which may be due to the rapid rate of the CLreaction. However, at a high flow rate the CL emission becamemore turbulent and nonreproducible. Considering a compro-mise between emission and reproducibility, the flow-rate of1.0 mL was adopted for all experiments. A typical profile in theoptimum conditions in this FIA-CL profile of asulam with mixedCL reagents solutions is shown in Fig. 2.

Analytical characteristics of the CL-FIA method

Under the experimental conditions mentioned previously, thecalibration graph of area under CL peak vs asulam concentrationwas linear in the range 0.36–35 ng/mL, with a detection limit of0.12 ng/mL for asulam, based on a signal-to-noise ratio of 3. Theregression equation and correlation coefficient was:

A = 6 × 106 [asulam] + 2 × 106 and r = 0.990

where A is area under the peak and [asulam] is expressed in ng/mL.The calibration graph at concentrations higher than 35 ng/mLindicates loss of linearity at higher concentrations, which is typi-cal behavior of chemiluminescence reactions. According to theliterature, this behavior may be due to increased light quench-ing at high analyte concentrations or to leveling of the catalyticactivity of the peroxidase on the chemiluminescent at higherconcentrations.[28] The precision was calculated by analyzingsamples containing 19 ng/mL of asulam (n = 8) and it was 1.55%as relative standard deviation (RSD).

Influence of interference species

In order to assess the selectivity of the proposed method, theeffect of other pesticides that can be present in water at similarlevels was studied. All the interferences studied were herbicides.In addition, several synergetic formulations employed in thecontrol of dallisgrass, a perennial grass[29] and crabgrass[30] oractive against monocotyl and dicotyl weeds[31] use mixtures of

Table 1. Range of concentrations and compositions stud-ied to optimize FIA-CL system

Concentration Range of composition (%)

Chosen

Luminol 3.5 × 10−5 to 3.5 × 10−6 mol/L

10–60 10

H2O2 6 × 10−5 to 6 × 10−6 mol/L

30–90 80

HRP 0.01 g/L 5–40 10

Figure 2. Typical peak profile of the luminol–H2O2–HRP system enhanced by asulam. Flow rate, 1 mL/min, and high voltage, 700 V.

Automated determination of asulam by enhanced chemiluminescence

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atrazine and asulam. In Table 2 are ordered the results obtainedfor samples containing 19 ng/mL of asulam. The selectivity of theproposed method is acceptable, except in the case of atrazine,in which a severe interference is produced.

Application to tap water

The method was applied to determination of asulam in tap watersamples, collected at different locations in Málaga and Antequera,a village at north of Málaga (Spain). Samples of 5 mL, withoutfiltration, were spiked with asulam and 35 μL of each samplewas injected into the FIA system. Recovery results are shown inTable 3. The RSDs obtained demonstrate the method’s applica-bility for routine analysis. In Table 4 the proposed method iscompared with other previously published and the detectionlimits obtained represent a substantially improvement in thesensitivity.

Conclusions

The proposed flow injection procedure for the determination ofasulam in waters is simple, sensitive, quick and precise. Enhance-ment of the base reaction by means of asulam is a novel methodfor a procedure for the determination of asulam by an automatedmethod using standard instrumentation.

References

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Table 2. Interferences study at 19.75 ng/mL of asulamlevel

Interference Asulam:Interferente(w:w)

Recovery(%)

2,4,5-T 1:1 78.21:0.5 85.6

Diclorprop 1:1 88.6MCPA 1:1 88.7

1:1 69.2Atrazine 1:0.5 76.9

1:0.25 78.9Metamidophos 1:1 88.1Amitrole 1:1 88.9

Table 3. Recovery study of asulam in water samples

Malaga sample Antequera sample

Added (ng/mL) Recovery (%) RSD (%) Added (ng/mL) Recovery (%) RSD (%)

Asulam 0.28 (1.21) 94.5 0.89 0.28 (1.21) 88.3 0.630.69 (19.74) 91.5 0.14 0.69 (19.74) 93.6 0.401.39 (39.71) 91.6 0.35 1.39 (39.71) 93.9 0.23

Table 4. Comparison of the proposed method with published previously

Sample DL Recovery (%) RSD (%) Technique

Tap water 0.12 ng/mL 88.3–93.9 1.55 This methodTap water 0.35 ng/L 98.1 0.40 Stopped flow chemiluminescence inhibition[28]

Tap water 10 μg/mL 86 6.50 MECK-UV detection[29]

Tap water 10 μg/mL 88 5.80 MECK-electrochemical detection[24]

Tap water 0.04 ng 90–118 1.13 HPLC with derivatization[25]

Natural water 10.9 μg/mL 98 <5.3 CE-UV detection[27]

Natural water 0.9 μg/mL 99 <6.5 CE-electrochemical detection[27]

Water Samples 40 μg/mL — 4.1 Photochemiluminometric[22]

Water Samples 5 μg/mL — 1 FIA- fluorescence[21]

Soil 1 mg/mL — — TLC[26] Peaches 0.043 μg/mL 85–106 1.6 Synchronous fluorescence with derivatization[23]

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