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Photodegradation and Flow-Injection Determination of Dithiocarbamate Fungicides in Natural Water with Chemiluminescence Detection
Amir WASEEM, Mohammad YAQOOB, and Abdul NABI
Department of Chemistry, University of Balochistan, Quetta, Pakistan

A simple and rapid flow-injection method is reported for the determination of dithiocarbamate fungicides (maneb, nabam and thiram) based on chemiluminescence detection. The method involves the photodegradation of dithiocarbamate fungicides via UV light in an alkaline medium. Photoproducts are then reacted with luminol in the absence of an oxidant. Linear calibration graphs were obtained in the range 0.01 4.0 mg L1 for maneb and nabam and 0.05 1.0 mg L1 for thiram with relative standard deviations (n = 4) in the range 1.0 2.6%. The detection limits (S/N = 3) of maneb, nabam and thiram were 10, 8.0 and 5.0 ng mL1, respectively, with a sample throughput of 100 h1. The method was successfully applied to determine these dithiocarbamate fungicides in spiked natural water samples. (Received March 10, 2008; Accepted June 10, 2008; Published March 10, 2009)

Introduction
Dithiocarbamates (DTCs) are a group of organosulfur compounds that have been extensively used as pesticides in agriculture for more than five decades, with some products already being introduced in the 1930s. Today, the yearly consumption (of DTCs) is between 25000 and 35000 metric tones.1 Most of the DTCs are applied as fungicides, and some are classified by the World Health Organization (WHO) as being hazardous.2 Maneb (manganese ethylenebis(dithiocarbamate)) is used exclusively as a broad-spectrum contact fungicide. The principal diseases controlled by maneb are early and late blight of potato and tomato, downy mildew and anthracnose on a number of vegetables, and the so-called rot diseases of fruits, such as apricots, peaches and grapes. It is also used for the seed treatment of small grains, such as wheat.3 Nabam (disodium ethylenebis(dithiocarbamate)) is a wellknown dithiocarbamate fungicide widely used against a variety of diseases on fruits, vegetables, field crops and turf. Nabam is combined with metallic sulfates for a variety of protection.4 Thiram (tetramethylthiuram disulfide) is a dimethyl dithiocarbamate compound used as a fungicide to prevent crop damage in the field and to protect harvested crops from deterioration in storage or transport. Thiram is used as a seed protectant and to protect fruit, vegetable, ornamental and turf crops from a variety of fungal diseases. Thiram is available as dust, flowable, wettable powder, water-dispersible granules, water-suspension formulations and in mixtures with other fungicides.5 In the body, carbon disulfide is formed from the breakdown of thiram and which contributes to the toxicity of thiram to the liver.6 The chemical structures of maneb, nabam and thiram are given in Fig. 1. To whom correspondence should be addressed. E-mail: yaqoob2001@hotmail.com

Various analytical methods have been reported for the determination of maneb, nabam and thiram, such as spectrophotometry,710 gas chromatography,11,12 gas chromatographymass spectrometry,13 high-performance liquid chromatography,14,15 polarography and voltammetry,16 capillary electrophoresis,17 Fourier-transform infrared spectroscopy,18 fluorometry19 and flame atomic absorption spectroscopy20 in water, foodstuff and commercial samples. Kubo et al.21 have reported the FI-CL method for the determination of dithiocarbamate fungicides (ziram, moncozeb and propineb), based on luminol-CL in the presence of catalyst in an alkaline solution with limits of detection (S/N = 3) of 2.0, 0.1 and 0.5 ppb, respectively. Girotti et al.22 recently reported a chemiluminescent enzyme-linked immunosorbent assay

Fig. 1

Chemical structures of dithiocarbamate fungicides.

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Table 1 Comparison among several methods for the determination of thiram, nabam and maneb Detection technique Analyte Thiram Thiram Thiram Maneb Maneb Nabam Thiram Nabam Maneb Thiram Maneb Nabam Thiram Matrix Synthetic mixture, commercial and forti ed samples Soil River water Commercial formulations and crops Tomatoes Tap water Water, wheat, barley and oat Water, food, commercial drug and wheat grain Honeybee Natural waters Calibration range 20 400 mg 10 mL1 0.5 25 mg L1 Up to 20 mg mL1 0.2 3.0 mg mL1 0.1 5 mg L1 20 600 ng mL1 5 600 ng mL1 0.25 2.56 mg mL1 0.5 5 mg mL1 9 15000 ng mL1 0.01 4.0 mg L1 0.01 4.0 mg L1 0.01 1.0 mg L1 Limit of detection 0.3 mg mL1 0.33 mg L1 0.161 mg mL1 0.0033 mg cm2 0.45 mg kg1 10 ng mL1 1 ng mL1 NG 2 mg mL1 9 ng mL1 10 ng mL1 8.0 ng mL1 5.0 ng mL1 R2 value 0.9754 NG NG NG 0.9993 0.942 0.996 0.999 NG NG 0.9987 0.9998 0.9987 Sample rate/h NG NG NG NG NG NG 80 NG NG 100 Ref. 7 8 9 10 14 15 19 20 22 This work

Spectrophotometry Spectrophotometry Spectrophotometry Spectrophotometry LC-DAD-UV HPLC UV FI- uorometry FAAS CL-ELISA FI-CL

NG, not given; FI- uorometry, ow injection uorometry; FAAS, ame atomic absorption spectrometry; LC-DAD-UV, liquid chromatographydiode array-ultraviolet detector; CL-ELISA, chemiluminescent enzyme-linked immunosorbent assay; CL, chemiluminescence.

(CL-ELISA) using luminol and horseradish peroxidase as labeling enzyme and polyclonal antibodies for the detection and quantification of thiram in honeybee extracts. Comprehensive reviews on thiram pesticide in environmental and pharmaceutical samples have also been reported.23,24 These methods are sensitive and accurate; however, many of these involve expensive methods, and toxic solvents; they are slow and require the development of extremely complex gradients for separation. Table 1 gives a comparison among several methods for the determination of thiram, nabam and maneb in terms of the sample matrix and the sensitivity. This study reports on a FI-CL manifold for the determination of maneb, nabam and thiram with limits of detection of 10, 8.0 and 5.0 ng mL1, respectively, and a sample throughput of 100 h1. The light from a low-pressure UV lamp was used for the deriving of the pesticide in NaOH, and then the photoproducts were reacted with luminol to generate CL. The manifold parameters were optimized for the analysis of spiked water samples.

of the compound in UHP water. Stock solutions (0.01 mol L1) of cations (Ca2+, Mg2+, Fe2+, Fe3+, Cu2+, Zn2+, Mn2+) were prepared in 0.01 mol L1 HCl solution. NH4+ in UHP water and anions (Cl, PO43, CO32, HCO3, S2, SO42, NO3, and NO2), prepared in UHP water and various working solutions from these stock solutions were prepared in UHP water for interference studies. Instrumentation and procedures The flow injection chemiluminescence manifold used for this work was previously reported.25 A peristaltic pump (Ismatec Reglo, 4 channels, Switzerland) was used to deliver the sample carrier and reagent solutions. All manifold tubing used was tygon (1.02 mm i.d., Ismatec, Switzerland). A rotary injection valve (Rheodyne 5020, Anachem, Luton, UK) was used to introduce dithiocarbamate standards into a UHP water stream, and merged with a stream of NaOH. This stream was allowed to pass through a photo reactor made up of PTEF tubing (200 cm in length) rolled over a thin glass plate with an exposed area (4.5 5.5 cm), and placed in front of a compact UV lamp (4 watts, Model UVGL-25, UVP, Upland, USA) at a distance of 1.0 cm, which produces a short wavelength of 254 nm. The photo reactor was covered with aluminum foil to minimize any stray light. The resulting photoproduct was then merged at a T-piece with the luminol reagent and allowed to travel 1.5 cm before passing through a glass spiral flow cell (2.0 mm i.d., 2.5 cm diam.) positioned in front of an end window photomultiplier tube (PMT, 9798B, Electron Tubes Ltd., Ruislip, UK). The PMT, glass coil and T-piece were enclosed in a light-tight housing. The PMT was kept at 800 V via a 2 kV power supply (Burle, PF1053, USA). The detector output was recorded using a chart recorder (Kipp & Zonen BD 40, Holland).

Experimental
Reagents and solutions All reagents used were of analytical grade; they were supplied by Merck (Darmstadt, Germany), and solutions were prepared in ultra-high-purity (UHP) deionized water (Elga, Purelab Option, UK). Nabam and thiram (Dr. Ehrenstorfer GmbH, Germany) stock solutions (200 mg L1) were prepared in ethanol and stored at 4C in a brown bottle in the dark. A maneb (Dr. Ehrenstorfer GmbH, Germany) stock solution (200 mg L1) was prepared in 0.01 M EDTANa2 and stored at 4C in a brown bottle in the dark. Subsequent standard solutions were prepared daily by serial dilution of the stock solutions with UHP water. A luminol stock solution (1.0 102 mol L1) was prepared by dissolving 0.177 g of luminol (5-amino-2,3-dihydro-1,4phthalazinedione, Sigma, St. Louis, MO) in a dilute sodium hydroxide solution, followed by sonication for 10 min. A working luminol solution was prepared by diluting the required volume in borate buffer. A sodium hydroxide solution was prepared freshly before use by dissolving the required amount

Results and Discussion

Photodegradation and luminol CL Studies of dithiocarbamates in aqueous solution have shown that they undergo UV photolysis and photo-oxidation. A variety of products were identified, including sulfur, sulfur dioxide, carbon disulfide, amines, hydrazines, thioamides and

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medium.3032 The organic compounds with oxidation potential of less than 0.5 V (vs. SCE) were capable of undergoing singleelectron transfer to 1O2 to generate O2 preferentially in aqueous solvents.33 The possibility for the direct conversion of 1O2 to OH by biological reductants, like glutathion and NADPH34,35 and sulfide/thiol-containing compounds, can reduce dissolved oxygen to O2 in alkaline solution has also been suggested.36 Kubo et al.21 reported the generation of hydroxyl radicals using electron-spin resonance studies of dithiocarbamate fungicides, namely ziram, moncozeb and propineb in 0.4 M NaOH. To check the involvement of ROS, the testing solution, luminol solution and sodium hydroxide solution were purged with nitrogen or oxygen for 5 min. When the dissolved oxygen was removed from the solutions by the purging nitrogen, the CL intensity decreased by about 20%. In contrast, when solutions purged with oxygen were used, the CL intensity increased markedly. The results indicated that dissolved oxygen is necessary for the luminol chemiluminescence reaction with dithiocarbamates. In order to examine if the reactive oxygen species participated in the CL reaction, the scavengers of the reactive oxygen species, such as ascorbic acid and mannitol, were added into the reaction system, respectively. The CL intensity was greatly decreased in the presence of these scavengers of radicals. These results showed that there is reactive oxygen species involved in the CL reaction. Upon the application of UV light, a strong CL signal was observed, possibly due to the generation of an excess of superoxide radicals, which is required for completion of the luminol CL reaction as the hydroxyl radical initially formed.21 They would have reacted with luminol to initiate light-emitting pathways.37,38 When the sample merges with luminol, the effect of this enhanced superoxide concentration is to increase the concentration of a hydroperoxide intermediate, and thus enhancing the light emitting pathways where it had already been initiated by hydroxyl radicals. The CL spectra of luminol in an alkaline medium with and without photocatalyzed dithiocarbamate fungicides were examined using the optimized FI-CL manifold. A very weak CL signal was observed when the sample was introduced with the UV lamp when in the OFF position. However, the signal markedly increased when the lamp was turned ON with the wavelength maximum at 425 nm. It is well known that the peak at 425 nm is the typical CL spectra of luminol, which suggests that the possible emission species is excited, 3-aminophthalate.39,40 Optimization of the FI manifold The FI-CL manifold previously reported25 was used for the determination of maneb, nabam and thiram. Various oxidants e.g., H2O2, KMnO4, Na2S2O8 and KIO4, over the range of 5.0 105 5.0 103 mol L1 were tested, and all of these generated CL with poor reproducibility and a high/noisy background. However, in the absence of these oxidants, a steady baseline with reproducible CL signals was achieved. Therefore, no oxidizing agent was used in the FI-CL manifold. For the photodegradation medium, NaOH (0.1 mol L1) and various buffers (CO32, PO43 and Na2B4O7, 0.1 mol L1, pH 11) were tested. Both CO32 and PO43 buffers generated CL signals with high background, while Na2B2O4 buffer generated a weak CL signal, as compared to NaOH. Therefore, a NaOH solution was used as the photodegradation medium. In order to establish the optimal conditions for the lowest possible detection limit of nabam, maneb and thiram in water samples, the effect of various parameters was investigated. These were the NaOH, borate buffer pH and luminol concentrations, the sample volume and the flow rate of the sample carrier and reagents streams. All of

Fig. 2 UV spectra of (a) maneb, (b) nabam and (c) thiram in NaOH, (1) without and (2) with UV irradiation.

thiourease.25,26 The major metabolites of thiram include carbon disulfide, dimethylamine, tetramethyl thiourea, tetramethyl hydrazine and sulfur.25 To assess the effect of UV irradiation on maneb, nabam and thiram (5 mg L1), UV-Vis spectra in an alkaline medium were obtained with the lamp ON and OFF. UV/Vis scanning was performed in the range of 200 400 nm using a UV/Vis spectrophotometer (Shimadzu, 1701, Japan). Figures 2(a) 2(c) shows UV-Vis spectral differences of fungicides with and without irradiation. Maneb and nabam have shown two absorption bands with lmax at 255 and 285 nm. In the case of maneb, a third peak was also observed at 222 nm, presumably due to the EDTA-Mn complex. Thiram has shown two absorption bands with lmax at 255 and 283 nm (Fig. 2(c)) before UV irradiation, which are not clear, as reported previously,27 while the third unknown absorption band at 365 nm was observed after UV irradiation. These absorption bands of maneb, nabam and thiram were still present after irradiation; however the absorption intensity was much lower, which shows the conversion of these fungicides into photoproducts. The spectra of these fungicides were the same for both water and NaOH. A variety of transient reactive oxygen species (ROS), including singlet oxygen (1O2), superoxide anion/radical (O2), hydroxyl radical (OH) and hydrogen peroxide (H2O2), are produced in surface water illuminated by sunlight.28,29 These ROS are highly redox-active due to the presence of an unpaired electron and/or their redox potential, and thus play an important role in the degradation of organic pollutants in natural environments. It was reported that superoxide anions/radicals (O2) are generated upon the UV irradiation of organic compounds in the presence of molecular oxygen in an aqueous

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Table 2 Effect of parameters on the determination of maneb, nabam and thiram (n = 4) Parameter Flow rate/mL min1 Injection volume/mL PMT voltage/V Range studied 0.5 2.5 30 180 750 950 Optimal value 2.0 120 850

The maximum CL intensity was observed at 5.0 105 mol L1 of the luminol concentration, and a further increase resulted in non-reproducible CL signals with high background. Therefore, a luminol concentration of 5.0 105 mol L1 was selected for subsequent studies. The CL response varied with the age of the luminol solution, and therefore, it was always prepared 24 h before use. The effect of the flow rate and the sample volume on the CL response was investigated in terms of the sensitivity, reagent consumption and sample throughput. The flow rates for each of the three channels were simultaneously investigated over the range 0.5 2.5 mL min1, and a flow rate of 2.0 mL min1 for all three channels gave the maximum CL response with a steady baseline and reproducible peak height. A sample injection volume of 120 mL gave the highest CL signal and was used for the economy of sample consumption. The effect of the PMT voltage over the range 750 950 V was optimized for the maximum CL signal-to-noise ratio CL response increased linearly with the PMT voltage, but 850 V was used for all subsequent studies, because it gave the best signal-to-noise ratio (Table 2).
Fig. 3 Variation of the CL intensity of pesticides (0.5 mg mL1, 60 mL). (a) NaOH concentration for a photodegradation medium: luminol, 5.0 105 mol L1 in borate buffer. (b) Borate buffer (0.1 mol L1) pH with optimal NaOH concentrations. (c) Luminol concentrations in borate buffer (0.1 mol L1 pH 11).

the studies were carried out with the 0.5 mg L1 (60 mL) standard solution and a PMT voltage of 800 V. The influence of the NaOH concentration on the determination of nabam, maneb and thiram was studied as a photodegradation medium over the range 0 1.0 mol L1. Figure 3 shows that the maximum CL intensity was observed at 0.1 mol L1 NaOH for nabam and 0.5 mol L1 for maneb and thiram. Therefore, these solutions of NaOH were subsequently used as photodegradation medium. The efficiency of luminol chemiluminescence depends on the reaction pH. In the proposed FI-CL system, the effect of the borate buffer pH (0.1 mol L1) for luminol was investigated over the range of 9.5 12, and the maximum CL intensity was observed at pH 11; the pH of the waste with this buffer was found to be slightly greater than 12. However, the rapid mixing (6 mL min1 total flow rate) in the flow cell may have a pH of between 11 to 12, which may be thought to be the optimum pH for this reaction. A NaOH solution of different concentrations was also investigated to check the response of luminol (5.0 105 mol L1) for CL enhancement. The maximum CL intensity was observed at a concentration of 0.1 mol L1 NaOH, but this CL intensity was lower (20%), as compared to borate buffer (0.1 mol L1, pH 11). Therefore, borate buffer (0.1 mol L1, pH 11) was selected and used for subsequent studies. The effect of the luminol concentration in borate buffer (0.1 mol L1, pH 11) on the determination of dithiocarbamate fungicides was studied over the range 0.5 20 105 mol L1.

Analytical figures of merit Under the optimum conditions, linear calibration graphs of CL intensity versus the concentration of maneb, nabam and thiram were obtained. The figures of merit corresponding at three dithiocarbamate fungicides are summarized in Table 3. The sample throughput was about 100 samples h1. The method was satisfactorily applied to determine these dithiocarbamate fungicides spiked in water samples. Interferences The interference of foreign species present in water at environmentally relevant concentrations was investigated by analyzing solutions containing 0.1 mg mL1 maneb, nabam and thiram. The tolerable foreign species were taken as a relative error not greater than 5%. No interference could be found when 2500-fold SO42, HCO3, CO32 and Cl, 1000-fold Ca2+, 100-fold humic acid, Mg2+, Zn2+, NH4+ and NO3 were added to the 0.1 mg mL1 maneb, nabam and thiram, respectively. Positive interferences were observed by 10-fold PO43, NO2 and S2 and 1.0-fold Fe3+ and Fe2+. Suppressive interferences were observed by 1.0-fold Cu2+ and Mn2+. However, the concentrations of PO43, NO2 and S2 in unpolluted surface and ground waters were generally 0.1, <0.02 and 0.1 mg L1 respectively. Cation interferents were removed off-line by passing through a micro-column containing an iminodiacetate chelating resin (Chelax 100, 50 100 mesh, sodium form, Sigma). Alternatively, an EDTANa2 solution of concentration 5.0 103 mol L1 can also be used to mask cation interferences. Application to natural waters The applicability of the proposed method based on photodegradation and the luminol CL reaction was checked by analyzing natural water samples from different areas. Lakewater (from Hanna valley, Quetta), tap-water (from university)

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Table 3 Analytical characteristics of maneb, nabam and thiram Figures of merit Linear range/mg mL Correlation coef cienta (r2) Calibration equationb RSD range, % (n = 4) Limit of detection/ng mL1 (S/N = 3)
1

399

Maneb 0.01 4.0 0.9987 I = 274.15c + 1.92 1.4 1.8 10

Nabam 0.01 4.0 0.9998 I = 294.56c + 0.39 1.2 2.6 8.0

Thiram 0.01 1.0 0.9987 I = 318.48c + 3.32 1.0 2.2 5.0

a. The correlation coef cient was calculated using 10 different concentration of each analyte. b. I, Intensity (mV); c, concentration (mg mL1).

Table 4 Results of recovery tests of maneb, nabam and thiram in natural water samples (n = 6) Sample Lake water Tape water Irrigation water Spiked/ mg mL1 0.1 0.5 0.1 0.5 0.1 0.5 Recovery, % Maneb 102 3 101 2 100 2 103 4 101 1.5 100 4 Nabam 99 1.5 102 2 99 3 98 2 102 3 101 2.5 Thiram 101 1.8 98 2.5 98 2.5 100 3 100 1 99 3

2. 3. 4. 5.

and irrigation-water samples were collected into acid-washed (10%, v/v HCl) polypropylene bottles. After collection, the samples were filtered through a cellulose membrane filter (cellulose acetate; pore size, 0.45 mm, 47 mm diam.; Whatman, Maidstone, UK) to remove the suspended solids, if any, kept refrigerated in the dark at 4C. All of the water samples were spiked with 0.1 and 0.5 mg mL1 of maneb, nabam and thiram within the linear range of the methods. The obtained data are given in Table 4. The recoveries found were in the range of 98 2.0 to 103 4.0%.

6. 7. 8. 9. 10. 11. 12. 13.

Conclusions
Phototransformation studies were found to be clean, cheap and reproducible analytical tools. The analytical strategy for pesticide determination based on photoreaction and chemiluminescence was successfully applied to the determination of dithiocarbamate fungicides in spiked water samples. The technique is simple and rapid (100 h1 sample throughput), and the recoveries were within an acceptable range for pesticide residue analysis. Appropriate dilutions are required when the method is applied to real samples as the maximum residue limits for dithiocarbamates (expressed as CS2), which are allowed by the European Union to be 2 7 mg kg1.5 The method has limits of detection of 10, 8.0 and 5.0 ng mL1 for maneb, nabam and thiram, respectively. The method is better in terms of sensitivity and sample throughput than the methods reported previously.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Acknowledgements
The authors are grateful to Higher Education Commission, Pakistan for financial support in the form of research project No. 639.

24. 25. 26. 27.

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