Anal. Chem. 1907, 5 9 , 769-773
might be attributed to the weaker coordinating ability of oxygen than the quinolyl nitrogen atom which has excellent coordinating ability (23,24). The low Li' selectivity of 14 and 15 should greatly be attributed to the increasing freedom of C-C bond rotation and larger size of pseudocavity compared with trimethylene derivatives. Thus, the most important factors for the ligand are cavity size, donor atom type and number, chain conformation, and substituents on the carrier skeleton. On the membrane surface the carriers substitute one water molecule after the other around the lithium ion with ether oxygens and aromatic nitrogens. Then the ligands in the complexed form have a much more lipophilic exterior than the uncomplexed form. The complex can now diffuse across the membrane. The release action of Li+ a t the opposite boundary on the membrane is likely to be the reverse of what has been described above. In fact, in our preliminary report on the cation transport by these carriers through liquid membranes, the same tendencies of the Li+ selectivity have been observed (17).That is, in comparison with the results of the ion transport through liquid membranes by these carriers, i t should be noted that the electrochemical ion selectivity in the PVC membrane electrodes based on these carriers changed almost in parallel to the selectivity of ion transport. As Winkler has suggested (25), the optimal ligand for lithium ion would be one which substitutes for water molecules in a stepwise fashion and is itself flexible enough to change conformation sequentially to a more compact structure.
LITERATURE CITED (1) Canessa, M.; Adragna, N.; Solomon, H. S.; Connolly, T. M.; Tosteson, D. C. NewEngiandJ. Med. 1980, 302, 772. (2) Schou, M. J. J . Psychlab. Res. 1968, 6 , 67.
769
(3) Tosteson, D. C. Scl. Am. 1981, 244, 164. (4) Gadzekpo, V. P. Y.; Hungerford, J. M.; Kadry, A. M.; Ibrahim, Y. A.; Xie, R. Y.; Cristlan, G. D. Anal. Chem. 1986, 58, 1948. (5) . . GMai, M.; Fledler, U.; Pretsch, E.; Simon, W. Anal. Lett. 1975, 8 ,
85s:
(6) Zhukov, A. F.; Erne, D.; Ammann, D.; Guggi, M.; Pretsch, E.; Simon, W. Anal. Chim. Acta 1981, 131, 117. (7) Metzger, E.; Ammann, D.; Asper, R.; Simon, W. Anal. Chem. 1986, (8) (9) (10) (11) (12) (13) (14)
(15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)
_58._ .132. _-
Olsher, U. J . Am. Chem. S O ~1982, . 704, 4006. Aalmo, K. M.; Krane, J. Acta Chem. Scand., Ser. A 1982, A36, 227. Kimura, K.; Kitazawa. S.; Shono, T. Chem. Lett. 1984, 639. Kitazawa, S.; Kimura, K.; Yano, H.; Shono, T. J . Am. Chem. SOC. 1984, 106, 6978. Kitazawa, S.; Kimura, K.; Yano, H.; Shono, T. Analyst (London) 1985, 770, 295. Xie, R. Y.; Cristian, G. D. Anal. Chem. 1988, 58, 1606. Ammann, D.; Morf, W. E.; Anker, P.; Meier, P. C.; Pretsch, E.; Simon, W. Ion-Sel. Electrode Rev. 1983, 5 , 3. Ion-Selective Electrodes in Analytical Chemistry; Freiser, H., Ed.; Plenum: New York and London, 1976. Kimura, K.; Ishikawa, A,; Tamura, H.; Shono, T. J . Chem. SOC.,Perkln Trans. 2 1984, 447, and references cited therein. Ryba, 0.;KnizBkovl, E.; Petrlnek, J. Collect. Czech. Chem. Commun. 1873, 36, 497. Hiratani, K.; Taguchi, K.; Sugihara, H.; Okada, T. Chem. Lett. 1986, 197. Srinivasan, K.; Rechnitz, G. A. Anal. Chem. 1969, 4 7 , 1203. Morf, W. E.; Simon, W. Ion-Selective ElectrWres in Analytical Chemistry; Freiser, H., Ed.; Plenum: New York and London, 1976; Chapter 3; pp 211-286. Hiratani, K.; Okada, T.; Sugihara, H. Bull. Chem. SOC.Jpn. 1988, 59, 2015. Hiratani, K.; Taguchi, K.; Sugihara, H.; Iio, K. Bull. Chem. SOC.Jpn. 1884, 57, 1976. Hiratani, K. Bull. Chem. SOC.Jpn. 1985, 58, 420. Vogtle, F.; Weber, E. Angew. Chem., Int. Ed. Engl. 1979, 78, 753. Tummler, B.; Maass, G.; Vgtle, F.; Sieger, H.; Heimann, U.; Weber, E. J . Am. Chem. SOC. 1979, 101, 2588. Winkler, R. Neurosci. Res. Program Bull. 1976, 74, 139.
RECEIVED for review August 6, 1986. Accepted November 6, 1986. This work was supported in part by grants from the Science and Technology Agency.
Determination of Histamine by Derivative Synchronous Fluorescence Spectrometry Carmen Gutibrrez, Soledad Rubio, Agustina Gbmez-Hens, and Miguel ValcPrcel* Department of Analytical Chemistry, Faculty of Sciences, University of Cdrdoba, Cdrdoba, Spain
First- and second-derlvative synchronous fluorescence spectrometry have been used to develop straightforward, fast methods for determination of histamine based on its reaction with o-phthaldiaidehyde. Calibration graphs are linear In the ranges 0.2-2000 ng/mL for the first-derivative method and 0.1-2000 ng/mL for the second-derivative method, with detection limits of 0.16 and 0.06 ng/mL, respectively. The second-derlvative synchronous method has been satisfactorily applied to the dlrect determlnatlon of thls amlne In wlne and canned tuna samples with no prior separation step.
Synchronous fluorescence spectrometry ( I ) is a simple modification of the conventional fluorescence technique affording higher selectivity thanks to the narrowing of spectral bands and the simplification of spectra (2). These features make this technique suitable for use in the resolution of
mixtures involving spectral overlap problems. The maximum fluorescence intensity obtained with synchronous scanning appears when the wavelength increment (AA) corresponds to the difference between the wavelengths of excitation and emission maxima and coincides with the maximum signal obtained in the emission spectrum of conventional scan. Thus, synchronous fluorometry does not improve analytical signals obtained by conventional fluorometry. The derivative fluorescence technique ( 3 ) can be used to enhance minor spectral features and reduce the effect of many spectral interferences. However, the signal-to-noiseratio (SNR) generally worsens with increasing derivative orders (4), although in some cases the SNR of the derivatives exceeds that of the normal spectrum ( 5 ) . The combination of synchronous and derivative techniques (6) results in a host of advantages including increased selectivity and rapidity and decreased cost. In addition, the SNR values obtained by differentiation of the synchronous spec-
0003-2700/87/0359-0769$01.50/0 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
trum are greater than those obtained by differentiation of the conventional spectrum. This is so because the amplitude of the derivative signal is inversely proportional to the bandwidth of the original spectrum (5, 7) and, as stated above, a salient feature of synchronous fluorometry is the narrowing of the spectral bandwidth in relation to conventional fluorometry. When adequate instrumental conditions are used, this increase in amplitude results in higher SNR values for the derivative synchronous spectrum than those obtained in the zero-order spectrum recorded by conventional fluorometry. Therefore, the use of the synchronous scan reduces the degradation of the SNR in the derivative spectrum, one of the most significant disadvantages of derivative spectrometry. The applicability of derivative synchronous fluorescence spectrometry has been shown in the determination of mixtures (B-II), some of which could only be satisfactorily resolved by time-consuming or expensive techniques. This technique is applied in this paper to the determination of histamine with the purpose of improving the detection limit and selectivity of the methods described for this amine, as well as to avoid the separation step entailed in its determination in food samples. Histamine occurs in food primarily as the result of the microbial decarboxylation of histidine. Consequently, this amine is a normal constituent of fermented foodstuff such as wine and foods with a relatively high histidine concentration which have been exposed to microbial degradation, such as tuna fish. Histamine has been pointed out as the causative agent in a number of food poisoning episodes, but no safety level for histamine in foods has been established, and potential hazards to consumers are difficult to predict because of individual susceptibilities to food containing the amine. However, the control of histamine content in foods, especially in fish products potentially rich in free histidine, should be recommended. For surveillance purposes, a reliable and fast method for histamine is indispensable. The method currently accepted for analysis of histamine (12) involves several extraction steps followed by column chromatography owing to the lack of specificity of the colorimetric diazonium coupling assay. This method is tedious and time-consuming and, in addition, is unreliable when applied to the analysis of food samples containing less than 5 mg of histamine per 100 g (13). At present, fluorometric methods based on the condensation reaction of histamine with o-phthaldialdehyde (14-17) are used routinely in the analysis of histamine in foods and are reportedly relatively specific. However, interferences of a different nature have often required including separation steps. The method described in this paper is based on the o-phthaldialdehyde reaction. The method uses second-derivative synchronous fluorescence spectrometry and, in addition to its simplicity, rapidity, and low cost, improves the detection limit and selectivity of Shore's method (141, reportedly the best for histamine detection ( 1 7 ) . This has allowed its direct application to the analysis of histamine in wine and canned tuna samples. EXPERIMENTAL SECTION Apparatus. Fluorescence measurements were performed on a Perkin-Elmer fluorescence spectrophotometer, Model MPF-43A, fitted with 1-cm cells and a xenon-arc source. The temperature of the spectrofluorometer cell compartment was kept at 22 "C with a circulating water bath. A spectral band-pass of 5 nm was set for the excitation and emission monochromators. For synchronous fluorescence measurements, both monochromators were locked together and scanned simultaneously at a rate of 8 nm/s. Derivative spectra were obtained by electronic differentiation of the signal from a Perkin-Elmer derivative accessory, Model H 200-0507. Six differential time constants, selected by the mode switch, were available. The differentiation constant increases as the mode decreases. The mode selector was set in position 6 for
all measurements. The spectrofluorometer response (time constant) was set at 0.3 s. We used a series of fluorescence polymer samples daily to adjust the spectrofluorometer to compensate for changes in the source intensity. No correction of the instrumental response was made. Reagents. A 1 g/L (free base) stock solution of histamine dihydrochloride (Aldrich) was prepared in doubly distilled water and stored at 0-4 "C. Standard solutions of lower concentrations were prepared daily by diluting the appropriate volume. A 0.1 % (w/v) o-phthaldialdehyde (OPT) solution was prepared by dissolving 25 mg of reagent (Merck) in 5 mL of ethanol and was made up to the mark with doubly distilled water. All chemicals used were Analytical Reagent Grade. Procedure. Sample volumes containing 1 ng/mL (secondderivative method) or 2 ng/mL (first-derivative method) and 20 pg/mL of histamine, 6 mL of 0.1 M sodium hydroxide, and 0.3 mL of 0.1% OPT solution were mixed in a 10-mL standard flask. After 6 min, 0.4 mL of 1M sulfuric acid was added and the volume was made up to the mark with doubly distilled water. The firstor second-derivative spectrum, obtained by synchronous fluorescence, was recorded by scanning both monochromators together with a 80-nm constant difference between them. The excitation monochromator was scanned from 300 to 460 nm and the emission monochromator from 380 to 540 nm. The instrumental parameters were as above. First- and second-derivative measurements were carried out peak to peak (18), Le., by measuring the difference between the derivative signal at two wavelengths corresponding to an adjacent maximum and minimum, given as relative fluorescence intensity and expressed as AI. First-derivativemeasurements were made at 432-470 nm and second-derivative ones at 46C-494 nm. The fluorescence intensities of these derivative signals were directly related to the histamine concentration from the previously plotted calibration graphs. Determination of Histamine in White Wine. This determination only required the appropriate dilution of the sample to obtain a concentration level of histamine in the lower region of the calibration graph, in order to avoid the possible interference from the sample matrix. No previous treatment of the sample was necesary. The diluted samples were treated as described under Procedure. Determination of Histamine in Canned Tuna. After the oil was removed, 10 g of well-mixed tuna sample was homogenized with 50 mL of methanol and transferred to a 100-mL standard flask which was placed in a water bath at 60 "C where it was kept for 15 min. After the mixture was cooled, the volume was made up to the mark with methanol, the contents were transferred to a capped polypropylene centrifuge tube, and the tube was centrifuged at 2000 rpm for 5 min. A portion of the extract was diluted 1 : l O with methanol and an adequate volume of this solution was treated as described under Procedure so that its histamine concentration lay in the lower region of the calibration graph. RESULTS AND DISCUSSION The conventional emission (Aex 360 nm), synchronous, and first- and second-derivative synchronous (AA 80 nm) fluorescence spectra of the reaction product of histamine with OPT are shown in Figure 1. The synchronous spectrum shows a narrower spectral bandwidth than that of the conventional emission spectrum, which could result in improved selectivity of the reaction of OPT with histamine, but the maximum fluorescence intensity obtained from both spectra is the same so that no sensitivity improvement is gained with the synchronous scan. However, when the synchronous signal is derived, both the analytical signal and the SNR are greater than those obtained by the former method. Therefore, the association of derivative and synchronous techniques results in increased selectivity and decreased detection limits. Effect of Variables. The optimization of variables was carried out so that the maximum analytical signal was obtained for the first- and second-derivative of the synchronous spectra. There is a variety of instrumental variables affecting derivative synchronous spectra, which endows this technique
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
2o
771
I
1
LOO
LBO
560 h h r n l
Flgure 1. Conventionalemission (Aax 360 nm) (-e - -), synchronous (AA 80 nm) (-.-.-), and first- (---) and second (-) derivative synchronous fluorescence spectra of the histamine-OPT condensation product. [histamine] = 10 ng/mL.
L20
L60
500
540
A(nrn1
-
Figure 3. Effect of the wavelength scanning speed on the first- (- -) and second (-) derivative synchronous fluorescence spectra of the histamine-OPT condensation product: (A) 2 nm/s; (6)4 nmls; (C) 8 nm/s.
L20 L60 500 5LO
Flgwe 2. Effect of AA on the first- (---) and second- (-) derivative synchronous fluorescence spectra of the histamine-OPT condensation product: (A) 60 nm; (6)80 nm (C) 100 nm; (D) 120 nm.
with great versatility. They have a decisive effect on the intensity, number, and distribution of maxima and minima in these spectra. Figure 2 shows the effect of the constant wavelength interval between both monochromators (Ax) on the first- and second-derivative synchronous spectra. An increase in this variable results in a bathochromic shift of the maxima and minima. These spectra change in shape from AA = 100 nm owing to the modification of the synchronous spectrum. The best analytical signal ( A I ) is obtained for AA = 80 nm, which corresponds to the difference between the maximum excitation and emission wavelengths. Another important instrumental variable is the wavelength scanning speed as it determines the U/dT value obtained from the differentiation acessory. As shown in Figure 3, the AI value
improves as the scanning speed increases, this effect being more noticeable for second-derivative synchronous spectra. For a given time interval, a greater variation of the fluorescence intensity is differentiated and, therefore, a better analytical signal is obtained. This may pose some problems in the resolution of mixtures of analytes since a high scanning speed causes a simplification of the spectrum and, possibly, spectral overlap. However, when the purpose is the determination of an individual species, a high scanning speed is preferable, provided that the analytical signal is proportional to the analyte concentration. The differentiation constant has an effect similar to that of the scanning speed on these spectra: the spectral resolution decreases and the AI value is maximum as this variable increases. The shape of the derivative synchronous spectra does not change appreciably with the spectrofluorometer time constants (0.3,1.5, and 3.0), but the analytical signal obtained decreases as this time constant increases. The chemical variables do not affect the shape and distribution of bands of the derivative synchronous spectra, but they do affect the AZ values. As is well-known (14,15), the condensation reaction between histamine and OPT develops optimally in a basic medium, where histamine occurs as a free base. This reaction takes some time to develop; once elapsed, the determination is carried out in an acidic medium, where the fluorescence intensity of the condensation product is higher. All these variables have been tested in the derivative synchronous mode and the optimum values found are very similar to those described in the conventional method: initial pH 12.2-12.5, reaction time = 6-9 min, and final pH 1.5-6.0. The OPT concentration does not affect A I above 1.5 X M. The fluorescence intensity is independent of the order of reagent addition. The optimum interval of temperature is 15-27 O C . Above the upper limit the analytical signal AI
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
Table I. Signal-to-Noise Ratios firstb
zero'
scanning speedc(8 nm/s) scanning speedC(4 nm/s) scanning speedC(2 nm/s)
secondb 10 ng/mL 1 ng/mL
10 ng/mL
1 ng/mL
10 ng/mL
1 ng/mL
80.0
45.0 44.2
99.8
78.3
44.1
46.5
43.9 27.1 14.4
115.0
80.5
13.1
71.2 14.5 8.5
43.9
115.0 63.1 19.0
71.2 10.0 3.0
81.0
mode 6d mode 5d mode 4d
99.8 78.5
46.1
18.8 14.0
63.7
Conventional or synchronous spectrum. bDerivativesynchronous spectrum. Mode 6. Scanning speed (8 nm/s). starts to decrease, especially for the second-derivative spectrum. Hakanson and Ronnberg (16) have described a modification of the original methods for histamine (15, 19) that uses a temperature of -20 "C and achieves a detection limit of 1 ng/mL. However, it is always troublesome to work a t such a low temperature in routine analyses. Signal to Noise Ratio. As indicated above, the use of derivative spectrometry may result in reduced SNR values. However, it is generally desirable to have the largest possible SNR in order to obtain the most precise results and the lowest detection limit possible (20). A study has been carried out that compares the SNR values obtained when the conventional emission spectrum (zero-derivative order) and first- and second-derivative synchronous spectra are recorded (Table I). Five superimposed spectra of sample and blank were recorded in each case to obtain the noise values, and the variation in the amplitude of the maxima was measured. The measurements of the derivative spectra were carried out peak to peak, as described in the procedure. The SNR values were calculated according to St. John et al. (21). As Table I shows, the SNR decreases as the analyte concentration, the scanning speed, or/and the differentiation constant (mode) decrease, consistent with the Green and O'Haver study ( 3 ) . Because the derivative signal is obtained with respect to time rather than wavelength, all the variables including the time factor markedly affect the SNR. Thus, an increase in the scanning speed results in a more rapidly changing fluorescent signal in the derivative circuit, with a significant improvement in the SNR. Table I shows that all the SNR values obtained when firstor second-derivative scan is used are lower than those obtained for the zero-order spectrum, except when the scanning speed and the differentiation constant maxima are used. These conditions were chosen as optimum in our method. This increase in the SNR of the derivative spectra in relation to the conventional emission spectrum, which is not frequent in derivative spectrometry can be attributed to the bandwidth narrowing of the synchronous spectra in relation to the conventional one. Thus, for two coincident bands of equal intensity, the derivative amplitude of the sharper band is greater than that of the broader band (7). This has been verified by the authors by calculating the SNR values of the first and second derivatives of the conventional emission spectrum recorded under the same optimum conditions. Thus,the SNR value obtained for the second derivative of the conventional spectrum of a sample containing 10 ng/mL of histamine was 56.2, whereas for the second derivative synchronous spectrum of the same sample, as shown in Table I, was 115.0. This improvement in the SNR is the origin of the low detection limit afforded by this methodology. Features of the Analytical Method. Linear calibration graphs are obtained by plotting different histamine concentrations against the values obtained for the signal AIat4,, from the first-derivative synchronous spectra and for the signal AZm4% from the second-derivative synchronous spectra. The linear concentration ranges are 0.2-2000 ng/mL for the first
Table 11. Effect of Various Species on the Determination of 1 ng/rnL of Histamine tolerate ratio of species to histamine 1000:1
1001 50:l 40:l 101
species added putrescine, cadaverine, spermine, tyramine, NAD, 1-methylhistidine,3-methylhistidine, 1-methylhistamine,1-methyl-4-imidazolaceticacid, Fe(III), Mg(II), Ca(II), EDTA norepinephrine, tryptamine epinephrine, Cu(I1) histidine spermidine
Table 111. Determination of Histamine in Wine
sample 1
source
sherry
histamine, rg/mL added found4 0.0
2.0 4.0 2
sherry montilla
5
6
a
mglaga penedks
102.5
8.5
13.4 18.0
98.0 95.0
12.5 17.3 22.5
94.0 100.0
0.0 10.0
badajoz
105.0
12.1
5.0 5.0 4
8.0 10.1
0.0 10.0
3
% recovery
0.0 10.0 20.0
16.0 26.4
35.0
104.0 95.0
0.0 2.5 5.0
2.5
0.0 1.0 2.0
1.3 2.3
100.0
3.3
100.0
5.0
100.0
7.3
96.0
Mean of three determinations.
derivative and 0.1-2000 ng/mL for the second derivative. Pearson's correlation coefficients ( 7 ) for the calibration graphs of first and second-derivative scan are 0.998 and 0.999, respectively. The detection limits, as defined by IUPAC (22), are 0.16 and 0.06 ng/mL for the first- and second-derivative mode, respectively. Under the same experimental conditions, but with conventional fluorometry instead of synchronous derivative fluorometry, the detection limit obtained with our instrument was 0.4 ng/mL. To test precision, three series of samples (n = 11)covering the concentration range of interest (0.8, 10.0, and 300 ng/mL) for each method were assayed. The percent RSD values obtained were f6.40, 11.51, and 12.51, respectively, for the first-derivative method, and 12.96, f0.76, and f2.75, respectively, for the second-derivative one.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
Table IV. Determination of Histamine in Canned Tuna
sample 1
2
histamine, mg/ lOOg added found" 0.0 5.0 10.0
0.0 5.0 10.0
3
4
5
6
0.0 5.0 10.0
% recovery
4.1 9.0
14.2 20.0 25.0 30.5 6.3 11.2
16.5
98.0 101.0 100.0 105.0 98.0 102.0
0.0 5.0
14.4
10.0
19.8
108.0 108.0
0.0 5.0 10.0
6.5 11.3 17.4
96.0 109.0
0.0
3.5
9.0
5.0
8.8
106.0
10.0
13.4
99.0
Mean of three determinations. To study the selectivity of the determination of histamine, several potentially interfering amines and related compounds frequently accompanying histamine in foods and several inorganic ions were added to samples containing 1 ng/mL of histamine. The results obtained are summarized in Table 11. None of the species tested interfered at the same level as histamine and, in some cases (e.g., spermidine), the tolerated concentration was higher than in the conventional method (14). This increase in selectivity has allowed this method to be applied to two different types of food samples without having to resort to the previous separation step which is usually necessary in this type of determination. Wine Samples. The determination of histamine in wine is usually carried out by the Shore method (14), in which histamine is removed from the matrix prior to its reaction with OPT by extraction with 1-butanol (two or three extractions are necessary to obtain an adequate recovery), washed with sodium hydroxide, and back extracted with a hydrochloric acid solution. This separation step is rather time-consuming for the routine assay of histamine. The second-derivative synchronous fluorescence method for histamine has been applied to the direct determination of this amine in six samples of Spanish white wine. The only treatment needed was dilution to a level where the histamine concentration lay in the lower region of the calibration graph. This dilution avoided the possible interference of the matrix. The concentration level of histamine in each wine and the
773
analytical recoveries obtained by addition of different histamine amounts to each sample are summarized in Table 111. The results found have been compared with those obtained by the Shore method (14). For samples with higher histamine levels, the results are very similar, but those obtained for samples with low histamine levels are lower in the conventional method than in our method, possibly owing to losses in the different extraction steps. Canned Tuna Samples. The conventional fluorometric method used to determine histamine in these samples, which involves a separation step, also requires the prior homogenization of the sample with methanol. When second-derivative synchronous fluorometry is applied, only the homogenization step is required. Several canned tuna samples have been analyzed for histamine (Table IV). Analytical recoveries were calculated by comparing the results obtained before and after addition of standard histamine solutions. The results found by the proposed method were also compared with those obtained by the conventional method (14) and, as with the wine samples, there was good correlation between the two methods in samples with high histamine levels, but the second-derivative synchronous method provides higher results than the conventional method when applied to samples with low histamine levels.
LITERATURE CITED Lloyd, J. B. F. Nature (London) 1971, 237. 64. Vo-Dinh, T. Anal. Chem. 1978, 50, 396. Green, G. L.; O'Haver, T. C. Anal. Chem. 1974, 4 6 , 2191. Cahill, J. E. Am. Lab. (FairfleM, Conn.) 1979, 7 7 , 79. O'Haver, T. C. Anal. Proc. (London) 1982, 79, 22. John, P.; Soutar, I. Anal. Chem. 1976, 48, 520. Fell, A. F.; Smith, G. Anal. Proc. (London) 1982, 19, 28. Miller, J. N.; Ahmad, T. A.; Fell, A. F. Anal. Proc (London) 1982, 79, 37. Rublo, S.; Gbmez-Hens, A.; Valcbrcei, M. Anal. Chem. 1985, 57, 1101. Rubio, S.; G6mez-Hens, A.; ValcBrcel, M. Clin. Chem. ( Wiston-Sa/em, N . C . ) 1985, 3 7 , 1790. Petidler, A.; Rubio, S.; Gbmez-Hens, A,; ValcBrcel, M. Anal. Blochem. 1988, 757, 212. AOAC Offlckl Methods of Analysis, 12th ed.; Association of Official Analytical Chemists: Washington, D.C., 1975; p 316. Lerke, P. A.; Bell, L. D.J . FoodSci. 1976. 4 7 , 1282. Shore, P. A. Methods fnzymol. 1971, 77(R B), 842. Hakanson, R.; Ronnberg, A.L.; Sjolund, K. Anal. Blochem. 1972, 4 7 , 356. Hakanson, R.; Ronnberg, A. L. Anal. Biochem. 1974, 60. 560. Taylor, S. L.; Lieber, E. R. J . Food Sci. 1977, 4 2 , 1584. O'Haver, T. C. Clin. Chem. (Wlnston-Salem, N . C . ) 1979, 25, 1548. Shore, P. A.; Burkhalter, A.; Cohn. V. H. J . Pharmacol. fxo. Ther. 1959, 727, 182. Wlnefordner, J. D.; Schulman S. G.; O'Haver, T. C. Luminescence Spectroscopy in Analyflcal Chemistry, 1st ed.; Wiley: New York 1972: n 215. r St. John, P. A.; McCarthy, W. J.; Winefordner, J. D. Anal. Chem. 1967, 39, 1495. Long, G. L.; Winefordner, J. D. Anal. Chem. 1983, 5 5 , 712A. . - . - 1
RECEIVED for review June 16, 1986. Resubmitted October 24, 1986. Accepted November 3, 1986.
