Anal. Chem. 1086, 58,1725-1729
1725
front viewing, this configuration is not as good as a true back-viewed arrangement. The emission passes through a shorter dye path length, and the distance depends on the excitation beam width and its centering on the QC cell face. Thus, it is especially important to select a blocking filter that cuts off any of the dye emission that is self-absorbed. Another difficulty with the triangular cell is that the transmittance of the excitation into the cell is quite sensitive to the polarization of the excitation beam. To avoid problems from this source, a polarizer must be placed in front of the QC or the system must be calibrated for transmission effects under all excitation conditions. This latter point is necessary because the polarization of the excitation beam is both wavelength and slit width dependent for most monochromators. Finally, we stress that the errors reported here arise from severe overlap between the emission and absorption. Dyes free of self-absorption should be free of this error source. Metal complex QC’s such as tris(2,2’-bipyridine)ruthenium(II) have negligible absorption-emission overlap and extremely flat spectral response (9). They should, thus, be given further attention as QC materials.
lations, and E. R. Carraway and G. Reitz for their help in running absorption and emission spectra. We thank K. Schoene for his work on an earlier analog version of the comparator. Registry No. RhB, 81-88-9.
ACKNOWLEDGMENT We gratefully acknowledge Nelson Ayala for his invaluable technical assistance with the spectrofluorometer and computer system, Seth Snyder for his consultations on data manipu-
RECEIVED for review October 8,1985. Accepted February 3, 1986. We gratefully acknowledge support of the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the NSF (Grant 82-06279).
LITERATURE CITED (1) Parker, C. A. Photoluminescence of Solutions ; Elsevier: New York, 1968. (2) Calvert, J. G.; PmS, J. N., Jr. Rwfochemisby; Wiley: New York, 1966. (3) Bowen, E. J. Proc. R . SOC.London, Ser A 1936, 154, 349. (4) Melhulsh, W. H. J . Res. Natl. Bur. Stand., Sect. A 1972, 76, 547. (5) Cehelnik, E. D.; Cundall, R. 8.; Lockwood, J. R.; Palmer, T. F. Chem. Phys. Left. 1974, 27, 586. (6)Taylor, D. G.;Demas, J. N. Anal. Chem. 1979, 5 1 , 712. (7) Taylor, D. 0.;Demas, J. N. Anal. Chem. 1979, 51, 717. (8) Mandal, K.; Pearson, T. D. L.; Demas, J. N. Anal. Chem. 1980, 52,
2184. (9) Mandal, K.; Pearson, T. D. L.; Demas, J. N. Inorg. Chem. 1981, 20, 786. (10) Kopf, U.; Helnze, J. Anal. Chem. 1964, 56, 1931. (11) Demas, J. N.; Pearson, T. D. L.; Cetron, E. J. Anal. Chem. 1985, 57, 51. (12)Demas. J. N. Optical Radlation Measurements; Mielenz, K., Ed.; Academic Press: New York, 1982;Vol. 3. (13) Ayala, N.; Demas, J. N.; DeGraff. B. A. Anal. Chem., in press.
Fluorometric Reaction Rate Method for Determination of Hydrogen Peroxide at the Nanomolar Level Jose Peinado and Fermh Toribio
Department of Biochemistry, Faculty of Veterinary, University of Cdrdoba, CBrdoba, Spain Dolores Pgrez-Bendito*
Department of Analytical Chemistry, Faculty of Sciences, University of CBrdoba, CBrdoba, Spain
A klnetic nonenzymatk method Is proposed for detenninath of hydrogen peroxide at the nanomolar level and applied to the analysis of several types of coffee,tea, and mlik samples. The method Is based on the Mn( 1I)tatalyzed oxidation of 2-hydroxynaphthaldehyde thlosemlcarbazone (HNTS) by H,O,. The reactlon is monitored spectrofiuorometrlcaily by measuring the lnltlal rate at the excitation and emission wavelength of the oxidation product. The kinetic study involved obtaining the rate law and evaiuatlng and optlmlring the chemical variables. Salient features of the method are (a) a llnear caHbratbn graph Is obtained from 50 to 2000 nM; (b) no enzymes are required; (c) it tolerates moderate amounts of various metal Ions.
Hydrogen peroxide is often determined at the micromolar level after reaction with a chromogenic hydrogen donor and a coupling agent in the presence of peroxidase ( 1 , 2) or by decomposition with peroxidase and oxidation of an indicator compound ( 3 , 4 ) . However, other procedures for the determination of hydrogen peroxide are based on its decomposition, promoted by a transition metal, with concomitant oxidation of a “marker substrate” to form a product that yields the analytical signal. 0003-2700/86/0358-1725$01.50/0
Thus, the most sensitive methods for determination of hydrogen peroxide involve chemiluminescence (by means of the luminol/Cu(II)-catalyzed oxidation reaction) (5),photometry (6-8)) and fluorometry (9-13), involving the use of peroxidase as catalyst of the indicator reaction. This peroxide has also been determined photometrically by means of Mo(VI)-catalyzedI--H202 system, but this method is subject to the limitations imposed by any species reacting with iodide (7).In all cases, their determination ranges are between 0.1 and 20 pM. There are also some methods for determination of hydrogen peroxide by the FIA technique (14-1 7) in similar or wider concentration ranges than those mentioned above and using peroxidase as well. Despite the fact that hydrogen peroxide is one of the compounds most frequently used in indicator redox reactions in nonenzymatic catalytic kinetic analysis, its kinetic determination has not yet been carried out. Hydrogen peroxide has been determined by FIA on the basis of Mn(I1)-catalyzed oxidation of hydroxynaphthol (A = 645 nm) at concentration ranging from 3 pM to 0.15 mM (18). The proposed method is quite selective and very sensitive, since it permits the determination of hydrogen peroxide at the nanogram-per-milliliter level. This method is based on the Mn(I1)-catalyzedoxidation of 2-hydroxynaphthaldehyde thiosemicarbazone (HNTS) by hydrogen peroxide. This re@ 1986 American Chemical Society
1726
ANALYTICAL CHEMISTRY, VOL.
58, NO. 8, JULY 1986
dox-catalytic system was formerly used for the kinetic-fluorometric determination of manganese(I1) (19). The method is applied to the determination of HzOzin various coffee, tea, and milk samples. There has been a recent report on the mutagenic activity of coffee in S. typhimurium TA 100 (20). Mutagens present in coffee are originated in the coffee toasting process (21). Its mutagenic activity can be supressed by using catalase or peroxidase, but not superoxide dismutase. Therefore, hydrogen peroxide may play a certain role in coffee mutagenesis. Freshly prepared instant coffee (15 mg/L) contains 260 pM hydrogen peroxide. However, this amount of hydrogen peroxide could account for less than 10% of the mutagenicity of coffee (20). Hydrogen peroxide is continuously produced in coffee solutions. In this paper, we have determined the hydrogen peroxide content in different types of coffee samples (green, normal, toasted, instant, and decaffeinated) by using the nonenzymatic redox indicator reaction proposed herein. The H202content in tea after heating is quite hq& however, its analysis has not yet been carried out by any method. In contrast, milk is one of the few foods reportedly analyzed for HzOz,that is why we have chosen it.
EXPERIMENTAL SECTION Reagents and Equipment. All solvents and reagents used were of analytical grade. A standard manganese solution (1.0 g L-') was prepared by dissolving 1.0 g of pure manganese in the minium volume of hydrochloric acid (1+ 1)possible and diluting to exactly 1:l with hydrochloric acid (1+ 9). The stock solution was diluted as required immediatelyprior to use. A 0.01% (w/v) HNTS solution in ethanol was also prepared. This solution was stable for at least 1week. The HNTS solution was prepared by a modification of the Sah and Daniel method (23). Aqueous hydrogen peroxide (0.5 mM) was standardized by acid permanganimetr y. Fluorescence was measured on an Aminco-Bowman spectrofluorometer furnished with a device that permits direct recording of fluorescence/time graphs at fixed excitation and emission wavelengths. The instrumental parameters used were: sensitivity x 10; excitation and emission slits, 16.5 nm and photomultiplier slit 11.0 nm. Under these conditions, 0.1 pg L-' quinine sulfate yielded a fluorescence signal of 16% full-scale deflection. Procedures. Kinetic Fluorometric Determination of Hydrogen Peroxide. To a 10-ml calibrated flask add, in this order, 1.5mL of HNTS solution in ethanol,variable volumes of hydrogen peroxide solution, 5 mL of ethanol, and 1+ 5 ammonia solution. Add redistilled water to the mark and add 2.5 of manganese(II). Mix and transfer a portion to a 10-mm quartz fluorometer cell maintained at 30 f 0.1 "C. Since the oxidation reaction is very fast, ethanol and water are both preheated at 30 "C. Wait 15 s before starting to record the fluorescence intensity A(,, = 390 nm, , A, = 450 nm) as a function of time. Monitor the reaction in the absence of hydrogen peroxide under s i m i i conditions. The reaction rate is calculated from the difference between the slopes of the two fluorescence/time plots. Determination of Hydrogen Peroxide in Coffee, Tea, and Milk. The coffee samples were prepared in a filter coffee pot from 25 g of powdered coffee and then liophilized. The solutions of liophilized coffee were prepared in redistilled water, with no sample pretreatment or masking. Each tea solution was prepared from one commercial tea bag in 100 mL of hot redistilled water that was boiled for 2 min and cooled. The solutions were assayed directly and the amount of Hz02 present was calculated from the difference between the reaction rate obtained in the presence and absence of catalase (100 U/mL). The milk samples (pasteurized, uperized, and sterilized) were prepared as follows: To 1mL of milk, 25 pL of glacial acetic acid and 2.5 or 5 ppm of HzOz were added; the solution was mixed and centrifuged, and the supernatant was assayed. RESULTS AND DISCUSSION Catalytic Effect of Mangangese(I1) on the Oxidation Reaction. The fluorometric characteristics of HNTS in an
I
25
6.1 X M M [NH,] < 11.7 X [NH,] > 11.7 X lo-' M 5.25 x lo-" M < [H+] < 1.26 x lo-" M 2.46 X lo-, M < [NH4+]< 9.58 X lo-, M [Mn2*] < 4.53 X lo4 M 4.53 x lo4 M < [Mn2+]< 7.57 X lo6 M IMn2+1> 7.57 x lo4 M
1 -2 1
-112 -1 1 2 -1
initial rate fixed time
linear range, pmol 0.05-2.0 0.07-2.0
% re1 std dev"
1.20 4.27
Table IV. Tolerance for Foreign Ions in the Determination of 34 ng mL-' H202by the Initial Rate Method
F-, S202BrOC Sr(II), Mo(VI), Ca(II), SzO:-, acetato, oxalato Mg(II), Na(I), K(I), C1-, Br-, I-, citrato Hg(II), Cd(II), SO4-, tartrato Se(IV), Zn(II), Sn(IV), Sn(II), Be(I1) Fe(III), EDTA, SCNCu(II), Fe(II), Pb(II), Ni(I1) Co(II), Ag(I), P2052-
[HNTS] < 5.6 X M M [HNTS] > 5.6 X [NH,] < 6.7 X M 6.7 X lo-' M < [NH,] < 11.9 X lo-' M [NH,] > 11.9 X M 5.25 x 10-l1 M < [H+] < 1.26 x lo-" 2.46 X lo-, M < [",+I < 9.58 X M 2.73 X lo4 M < [Mn2+]< 10.92 X lo4 M
312
-1 1
0 -1 -1 1 1
Table V. Hydrogen Peroxide (pM) Content in Commercial Coffee" and Tea
aObtained by 11 determinations on 1 pmol L-l (34 ng mL-') of H202.
ion added
partial order
-3
Table 111. Linear Calibration Ranges and Relative Standard Deviations for the Kinetic-Fluorometric Determination of HzOz method
without H202 variable and concentration range
amt tolerated [ion1I [HZ021 500 300 200 100 75 50 20 15 10 7.5
was obtained for solutions containing different amounts of hydrogen peroxide, under the optimum conditions stated in the Experimental Section. To these curves, two well-established methods for construction of calibration graphs were applied. Table I11 shows the concentration range over which the calibration graphs are linear and the relative standard deviations for the determination of hydrogen peroxide by the initial-rate and fixed-time methods. Only the initial portion of the reaction was utilized (1-3 min) in the initial-rate method. For the fixed-time method, the fluorescence was measured at 3-min intervals. The initial-rate method was selected on account of its higher precision. The variable-time method, owing to its narrower determination range, provided poorer results. Interferences. For the determination of 34 ng mL-' of hydrogen peroxide (1nmol mL-') by the initial-rate method, the ionic species assayed can be tolerated up to the levels listed in Table IV. Pyrophosphate ion interferes through the formation of a complex with manganese(II1). Metals such as copper, cadmium, iron, cobalt, and nickel, which interfere in the classical luminiscence method of luminol for determination of H20z,are tolerated in moderate amounts of this method. Determination of Hydrogen Peroxide i n Coffee, Tea, and Milk. References about the determination of Hz02in coffee are scarce. The hydrogen peroxide content of different types of commercial coffee (bean, instant, and green coffee) was assayed by the procedure described in the Experimental Section. The results found are shown in Table V. The hydrogen peroxide content obtained by the proposed method
sample
kinetic method
contrasting method (oximeter)
coffee normal toasted Marcilla Saimaza sugar toasted Marcilla instant coffees Nescaf6 Nescaf6 (decaffeinated) Nescaf6 (sugar toasted) Mokanor (sugar toasted)
245 258
238 263
265
226
257 301 249 286
267 304 242 263
160 171
140 152
Tea Lipton Hornimans "All coffee solutions were 20 mg/mL.
and the contrast one are coincident. Nagao et al. (22) reported that 15 mg/mL of instant coffee contained 260 pM hydrogen peroxide. With catalase (200 U/mL), the Hz02 concentration of an instant coffee solution (20 mg/mL) is decreased to 50% ( t = 5 min) and becomes undetectable after 30 min. The H2OZcontent returned to its initial value upon heating at 90 "C. When aliquots of 25 r L are taken, a linear relationship has been found between the hydrogen peroxide generated and the concentration of coffee, for solutions in the range 3-20 mg/mL. For solutions of concentrations above 20 mg/mL, the volume of the aliquote must be reduced, since the caffeic acid present gives an orange hue in alkaline medium, which interferes with the fluorometric measurement. As can be seen from Table V decaffeinated coffee is highly concentrated in hydrogen peroxide and we have tried to exploit this fact. Weber and Schwedt (24)reported the presence of certain metals such as Fe, Cu, and Zn in coffee, generally linked to flavonoids. Iron(II1) acts as catalyst of HNTS oxidation (although its catalytic effect is not as powerful as that of manganese) (19),and according to Michelson et al. (25), iron is involved in the generation of activated species of oxygen. Decaffeinated coffee is obtained by extraction with water (26). Therefore, the hydrogen peroxide concentration calculated for decaffeinated coffee can be increased by iron present. In fact, the use of EDTA results in a gradual decrease in the hydrogen peroxide concentration down to 39% of its initial value (t = 30 min). We have checked by atomic absorption spectrometry (AAS) that decaffeinated coffee contains about 50% more iron than normal coffee (probably due to the large volumes of water required for its elaboration). With catalase, hydrogen peroxide is eliminated from decaffeinated coffee; thus, the positive effect of iron observed is
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
Table VI. Hydrogen Peroxide Content in Milk milk pasteurized +2.5 ppm +5.0 ppm uperized +2.5 ppm +5.0 ppm sterilized +2.5 ppm +5.0 ppm
found, ppm
recovery, %
2.42 4.80
96.8 96.0
2.46 4.87
98.4 97.4
2.41 4.93
96.4 98.6
probably due to its participation in the generation of hydrogen peroxide. This is in agreement with the finding that superoxide dismutase (250 U/mL) increases in 15% the hydrogen peroxide concentration determined in a solution of 20 mg/mL decaffeinated coffee. To our knowledge, no determinative procedure for hydrogen peroxide in tea had been described so far. We have observed the generation of very small amounts of Hz02in the process of preparation of the tea solution. When the solution was boiled for 2 min, the HzOz content increased. Table V also shows the results obtained in the determination of H202 by the kinetic method and the contrast method (oximeter). The amount of hydrogen peroxide was calculated from the difference between the values obtained in the absence and presence of catalase, since the matrix exerts an additional effect, enhancing the response. Table VI shows the results obtained in the determination of HzOz added to milk, as well as the calculated recovery.
CONCLUSIONS The kinetic method for determination of H202proposed herein is one of the most sensitive described so far. Moreover, it affords speed, selectivity, and precision levels similar to those provided by enzymatic methods, but with the added advantage of requiring no enzyme. The kinetic determination of HzOz as a substrate in nonenzymatic catalyzed indicator reactions had not been approached to date, probably because this substance is used at high concentrations, which makes its determination scarcely interesting from an analytical point of view. The high sensitivity achieved by the use of this method can he accounted for on the grounds of the reaction mechanism itself. Experiments under way-the results of which will be the subject matter of a forthcoming paper-have demonstrated that, hydrogen peroxide acts as an activator of man-
1720
ganese(II), which takes part in the regeneration by interaction with intermediate manganese(II1) formed, thus increasing the reaction rate. This finding is consistent with a similar behavior observed for ethanol and ascorbic acid. The kinetic method for determination of ascorbic acid (by atmospheric oxygen) with HNTS (27) reported by our team is as sensitive (identical slope of the calibrated graph) as the one proposed in this paper.
ACKNOWLEDGMENT The authors thank Carmen Pueyo for providing coffee samples and for his helpful discussion and Concha Benitez for technical assistance in AAS. Registry No. HzOz,7722-84-1; Mn, 7439-96-5; HNTS,741040-4.
LITERATURE CITED (1) Richmond, W. A.; Poon, L. S.; Chan, S. G. Clin. Chem. (Winston-&/em, N.C.) 1974, 20. 470. (2) Kubota, S.; Okada, M.; Imahori, K.; Osawa, N. Igako no Ayuml 1983, 724, 22. (3) Hugget, A.; Nizon, D. Biochem. J . 1957, 66. (4) Salomon, L.; Johnson, J. Anal. Chem. 1959, 3 7 , 453. (5) Kok. G.; Holier, T.; Lbpez, M.; Nachtrieb, H.; Yuan, M. Environ. Sci. Techno/. 1978, 72, 1072. (6) Shiga, M.; Saito, M.; Kina, K. Anal. Chim. Acta 1983, 753, 191. (7) Frew. J. E.; Jones, P.; Scholes, G. Anal. Chim. Acta 1983, 755, 139. (8) Tamaoku, K.; Murao, Y.; Aklura, K. Anal. Chim. Act8 1982, 736, 121. (9) Guilbauit, 0.; Kramer, D.; Hackley, E. Anal. Chem. 1987, 3 9 , 271. (10) Guilbault, G.; Brignac, P.; Zknmer. M. Anal. Chem. 1988, 40, 1256. (11) Kelly, G.: Christian, G. Anal. Chem. 1981, 5 3 , 250. (12) Lichtenberg, L. A.; Weiiner, J. Anal. Biochem. 1988, 2 6 , 313. (13) Taitsu, K.; Okura, Y. Anal. Biochem. 1980, 709, 109. (14) Guiberg, E.; Attyat, A.; Christian, G. J . Autom. Chem. 1980, 2 , 189. (15) Rule, G.; Seitr, W. Clin. Chem. (Winston-Salem, N . C . ) 1979, 2 5 , 1635. (16) Madsen, B. C.; Kromis, M. S. Anal. Chem. 1984, 5 6 , 2849. (17) Lundback. H.; Johansson, G.; Hoist, 0. Anal. Chim. Acta 1983, 755, 47. (18) Yamane, T. Bunseki Kagaku 1984, 33, E203. (19) PBrez-Bendko, D.; Peinado, J.; Toriblo, F. Ana/yst(London)1984, 709, 1297. (20) Nagao, M.; Takashaki, Y.; Yamanaka, H.; Sugimura, T. Mutat. Res. 1979, 68, 101. (21) Kosugi, A.; Nagao, M.; Suwa, Y.; Wakabayashi, T.; Sugimura, T. Mufat.Res. 1983, 776, 179. (22) Nagao, M.; Suwa, Y.; Yoshizumi, H.; Sugimura, T. Banbury Rep. 1984. No. 77. (23) Sah, P. T.; Daniels, T. C. Recl. Trav. Chim. Pay-Bas 1950, 6 9 , 145. (24) Weber, 0.;Schwedt, G. Fresenius' 2.Anal. Chem. 1983, 376, 594. (25) Superoxide and Superoxide Dismufases; Micheison, A. M., McCord, J. M.. Fridovich, I., Eds.; Academic Press: New York, 1977. (26) Katz, S. N. SenburyRep. 1984, No. 77. (27) Peinado. J., Ph.D. Thesis, Cbrdoba University, Cbrdoba, Spain, 1965.
RECEIVED for review December 23, 1985. Accepted April 1, 1986.
