2015
Anal. Chem. 1982, 54, 2015-2017
Table 11. Analytical Results of Inorganic Arsenic in River Water samplea
concn, ng/mL
1 2 3
0.E5 IT 0.03 0.48 = 0.02 0.42 t 0.02
% recovery
92 98 95
Samples 1, 2, and 3 were taken from different points 3,ecoveries were determined with on the Tama River. the addition of 1 ng/mI. As solution. a
highly sensitive and selective method for elemental analysis, when it is combined with hydride generation and nondispersive detection system. An attempt to apply the present method to other elements is now in progress.
ACKNOWLEDGMENT The authors are indebted to K. Fujiwara of our department for his help in the construction of the computer-controlled monochromator system. Thanks are also due to J. Takahashi of Seiko for the valuable discussion and also to M. Kurosawa of our department for his help in the analyses of real samples.
Table 111. Determination of Arsenic in NBS Coal Fly Ash and Orchard Leaf Standard Reference Materials amt certified found, value, sample rg/g Pglg coal fly ash, NBS SRM 1633a 145s 15 141 I 8 1 2 I 0.6 14 t 2 orchard leaves, NBS S R M 1571
to be extended to 5 min when real samples were analyzed. The reduction reaction seems to be retarded, when real samples are used, due to the matrix effect. However, if the reaction period is long enough, there is no problem in analyzing real samples. The analyses were carried out by the standard addition method and the results obtained were in good agreement with the certified values as shown in Tables I1 and 111. The present study demonstrates the possibility that even broad continuous molecular emission bands can be used as
LITERATURE CITED Robbins, W. B.; Caruso, J. A. Anal. Chem. 1979, 51, 889A-899A. Perkin-Eimer Corp. Reprint AA-332G “Technlque and Application of Atomic Absorption,” 1978. Thompson, K. C., Thomerson, D. R. Analyst (London) 1974, 99, 595-601. Tsunoda, K.; Fujiwara, K.; Fuwa, K. Anal. Chem. 1977, 49, 2035-2039. Tsunoda, K.; Fujiwara, K.;Fuwa, K. Anal. Chern. 1978, 50, 861-865. Everett, G. L.; West, T. S.; Williams, R. W. Anal. Chim. Acta 1974, 68, 387-394. Haraguchi, H.; Fuwa, K. Anal. Chem. 1976, 48, 784-786. Henden, E.; Pourreza, N.; Townshend, A. Prog. Anal. At. Spectrosc. 1979, 2 , 355-372. Braman, R. S.;Tompkins, M. A. Anal. Chern. 1979, 57, 12-19. Fujiwara, K.; Bower, J. N.; Bredshaw, J. D.; Winefordner, J. D. Anal. Chim. Acta 1979, 109, 229-239. Feldman, C. Anal. Chem. 1979, 50, 664-669. Braman, R. S.; Johnson, D. L.; Foreback, C. C.; Ammons, J. M.; Bricker, J. L. Anal. Chem. 1977, 49, 621-625. Smith, A. E. Analyst (London) 1975, 700, 300-306.
RECEIVED for review May 14,1982. Accepted July 16,1982.
Elucidation of Metanephrine to Normetanephrine and Epinephrine to Norepinephrine Ratios by Fluorescence Derivative Spect romet ry Robert H. Christenson * and C. Davlci McGlothlln* Department of Chemistry, Eiorlda State University, Tallahassee, Florida 32306
Flrst and second derlvatlve fluorescence spectroscopy was carrled out over the 325-380-nm excltatlon reglon on fluorescent products of the metanephrlnss and catecholamlnes. A constant 505-nm emission wavelength was used. Determlnatlon of methanephrlnle to normetanephrine and eplnephrlne to norplnephrlne ratlos was done by calculatlons using flrst and second derlvatlvra fluorometrlc spectra. A catlonexchange material was used to separate splked aqueous and urine solutlons of the metanephrlnes and catecholamlnes; subsequent spectra analysis yielded ratlos wlthln 5 % experlmental error.
Urinary determinations of epinephrine and norepinephrine (the catecholamines), and their 0-methylated metabolites metanephrine and normetanephrine (the metanephrines), are Present address: Departments of Laboratory Services, Duke University, and Durham Veterans Administration Medical Centers, 508 Fulton Street, Durham, bTC 27705. 2Present address: P.O. BOK13193, Tallahassee, FL 32308. 0003-2700/82/0354-2015$01.25/0
of considerable interest in the laboratory-assisted diagnosis of pheochromocytoma: a catecholamine secreting tumor of the adrenal medulla. “High-pressure” liquid chromatographic methods are available to separate the individual metanephrines (1) and catecholamines (2). However, the clinical information offered by differing proportions of metanephrine to normetanephrine and epinephrine to norepinephrine is incompletely understood. Success in isolating the catecholamines and methanephrines from urine was demonstrated by Sandhu and Freed (3)using a weakly acidic cation-exchange material. A modification of their method (3),using prepacked ion-exchange columns, is available through Bio-Rad laboratories (Richmond, CA) (4). Fluorescence spectroscopy has been used for detection and quantitation of total metanephrines (5-7) and total catecholamines (4). Two of these methods (4, 7) use the Bio-Rad ion-exchange columns to accomplish separation of the catecholamines and methanephrines from a urine matrix. Derivative spectroscopy was used as early as 1955 for resolving spectral lines of nearly equal wavelength (8). The technique has since found use in diverse applications including detection of erbium in the presence of cerium (9) and mea0 1982 American Chemical Society
2016
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982 METANEPHRINES
surement of bilirubin in the presence of albumin (10). Although most studies have dealt with derivative treatment of the absorption phenomenon, derivative signal processing has also been used with fluorometry (11,12). Hager (13),Cahill (14), and O'Haver (15) have published excellent reviews dealing with the theory and applications of derivative spectroscopy. Here we use previously described methods (4,")to separate and detect fluorescent products of the metanephrines and catecholamines. Subtle differences are shown to exist in the spectra of these compounds; first and second derivative spectroscopy is used to examine these differences and elucidate metanephrine to normetanephrine and epinephrine to norepinephrine ratios.
AF
410
500
M
EXPERIMENTAL SECTION Apparatus. Fluorometric measurements were made with a Model 204A fluorescence spectrometer equipped with a Model 150 xenon lamp and power supply (all from Perkin-Elmer Corp., Nonvalk CT). The derivative circuitry used was slightly modified from that described by Green and OHaver (11) to include more modern operational amplifiers and a derivative selection switch. A Model SRG recorder was used to record all data. Reagents. Crystalline metanephrine, normetanephrine, epinephrine, and norepinephrine were used as received (Sigma Chemical Co., St. Louis, MO). Working standards of all four compounds were prepared just prior to use. Reagent grade disodium EDTA, sodium hydroxide, HEPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonicacid), iodine, formic acid, boric acid, ascorbic acid, potassium ferricyanide,and zinc sulfate were used. Ion-exchange columns containing weaky acidic cation-exchange resin were used as received (Bio-Rad Laboratories, Richmond, CA). Urine used was freshly obtained from ostensibly normal human volunteers. Distilled deionized water was used unless otherwise specified. Procedures. Catecholamines and metanephrines were eluted from weakly acidic ion-exchange columns using 40 g/L boric acid and 1.0 mol/L formic acid solutions, respectively (4, 7). Fluorescent hydroxyindole products of the metanephrines were formed with iodine and alkaline ascorbate as previously described (7,16). Solutions and column fractions containing catecholamines were reacted with zinc sulfate, potassium ferricyanide,and then ascorbic acid to form fluorescent compounds (4). First and second derivative spectroscopy was done by scanning the 325-380-nm excitation wavelenghts at 1 nm/s; a constant emission wavelength of 505 nm was used. Each sample was scanned three times and quantitated to yield a proportion value (P),indicative of compound proportion. Ratio determinations of the metanephrines and catecholamines were done by use of a curve method.
RESULTS AND DISCUSSION Zeroeth derivative fluorescence excitation and emission spectra for the metanephrines (top) and catecholamines (bottom) are shown in Figure 1. The excitation spectra of the N-methylated compounds (metanephrine and epinephrine) are nearly identical. These spectra are similar to those of the primary amines (normetanephrine and norpinephrine) with the exception of the 325-380-nm excitation region. The emission spectra of all four compounds are essentially identical. All zeroeth, first, and second derivative spectra were obtained at a constant 505 nm emission wavelength. Figure 2 (top) shows the 325-380-nm excitation region for five metanephrine to normethanephrine and epinephrine to norepinephrine ratios in the zeroeth derivative mode. The appearance in this wavelength region is clearly proportion dependent. The center representation of Figure 2 shows a fiist derivative mode scan of the 325-380-nm excitation region for the same five proportions. Metanephrine and epinephrine show a large curve maximum and a slightly larger minimum consistent with features of the zeroeth derivative spectra. Normetanephrine and norepinephrine on the other hand, show a much different appearance also consistent with the zeroeth
L
1
EXCITATION
A
EMISSION
Flgure 1. Fluorescence excitation and emission spectra of metanephrine (M), normetanephrine (NM), epinephrine (E) and norepinephrine
(NE). M,E NM,NE
I 0
2 I
1
I
I 2
0 I
Figure 2. Zeroeth, first, and second derivative scans of the 325-
380-nm excitation reglon for five compound proportions. derivative curve. The second derivative spectra of these same five ratios is shown at the bottom of Figure 2 for the 325380-nm excitation wavelengths. When the slope of the first derivative curve is positive, the value (magnitude) of the second derivative is positive. When the slope of the first derivative curve is negative, the value (magnitude) of the second derivative is negative. The minima and maxima of the first derivative curve (350 and 360 nm, respectively) result in second derivative values of zero. The second derivative spectra are as expected based on first derivative curves. Quantitation of first and second derivative curves was done as illustrated in Figure 3. Due to the similarity in the zeroeth, first, and second derivative spectra of metanephrine and epinephrine, the proportion values ( P ) resulting from these compounds were equal when calculated as shown in Figure 3. Normetanephrine and norepinephrine yielded P values which were also equal. The solid line of Figure 4 shows a plot of first derivative P value on the ordinate vs. ratio of the metanephrines or catecholamines on the absissa. The dashed line of Figure 4 indicates a similar plot of second derivative data. Both first and second derivative P vs. ratio plots are independent of total metanephrines or catecholamines concentration.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
2017
Table I. Mean P Valucirbetween-Day CV and within-Day CV for First and Second Derivative Data 1st derivative 2nd derivative proportion mean mean M:NM or mean P between-day within-assay mean P between-day within-assay E:NEa nb value cv, % cv,c % value nb cv, % cv,c %
l:o
10
2: 1 1:1 1:2 0: 1
9
4.16 5.60 7.80 7.63 17.5
0.916 1.35 1.58 2.05 5.16
10 8 8
1.38 2.61 2.70 3.65 10.5
0.868 1.15 1.36 1.67 3.16
8 8 8 7 9
2.20 8.04 4.15 10.3 26
5.63 7.14 6.20 7.19 11.2
n is the number of days on which a M = metanephrine, PJM = normetanephrine, E = epinephrine, NE = norepinephrine. Each sample run was scanned three times. The mean within assay CV is the average of CV of n triplithe assay was run. cate runs.
Flgure 3. Calculation of piruportion value (PI. 1 s t D e r i v a t we 2nd Derivalive
50
b
T I
,A
20 1
cholamines and metanephrines by eluting with boric acid and formic acid, respectively (7). This material was used to separate solutions of known metanephrine to normetanephrine and epinephrine to norepinephrine proportion. First and second derivative P values from 325 to 380 nm excitation spectra were calculated from boric acid (containing the catecholamines) and formic acid (containing the metanephrines) eluents. These P values were used to obtain corresponding metanephrine to normetanephrine and epinephrine to norepinephrine proportions from plots like Figure 4. The P values calculated from the known spiked solutions were within 5 % of the corresponding value shown in Table I; the proportions read from plots agreed with the known values. In addition, urine samples from ostensibly healthy human volunteers were spiked to contain the metanephrines and catecholamines in similar proportions as above. The first and second derivative data were quantitated and proportions of the individual metanephrines and catecholamines determined. Variation again was within 5% from corresponding P values; the ratios from plots like Figure 4 agreed well with known spiked proportions.
ACKNOWLEDGMENT lk-2 ' 1
1'1
'2
0'1
RATIO
Flgure 4. Proportion value vs. ratio of eplnephrine to norepinephrine or metanephrine to normetanephrine.
Table I shows the mean P values and CV's (standard deviation divided by the mean X 100%) for five metanephrines and catecholamines proportions. The first and second derivative data showed between-day CV's of approximately 8% and lo%, respectively, for four of the five proportions examined. The exception (sdutions containing exclusively normetanephrine or norepinlephrine) possibly reflects an artifact of the quantitation technique used. Table I also shows the mean within-assay CV's for the first and second derivatives. Briefly, this term is defined as the mean CV of n triplicate sample measurements and is indicative of experimental reproducibility. Second derivative spectra are more susceptible to purturbations such as noise or signal fluctuations thsm are those on the first derivative. Mean within-assay CV's for the second derivative P values reflect this; they are 2-3 times greater than those of the first derivative. As expected, the data of Table I show the first derivative experiment to be more reproducible overall. A commercially available weakly acidic cation-exchange column material (4)achieves good separation of the cate-
Thanks to Lawrence M. Silverman for his excellent comments. Deep appreciation goes to C. K. Mann, T. J. Vickers, and the Florida State Chemistry Department for support while eompleting this work.
LITERATURE CITED Shoup, R. E.; Klsslnger, P. T. Clin. Chem. (Winston-Salem, N.C.) 1977, 23, 1288-1274. Moyer, T.; Jiang, N.; Tyce, 0.; Sheps, S. Clin. Chem. (Winston-Sa/em. N . C . ) 1979. 25. 256-262. Sandhu, R: S.; Freed, R. M. Stand. Methods Ciln. Chem. 1972, 7 , 231-246. Technical Bulletin No. 4020; Blo-Rad Laboratories, Clln. Dlv.: Rlchmond, CA, 1974. Weil-Malherbe, H.; Smith, E. R. B. Pharmacol. Rev. 1966, 18, 331-343. Elgelow, L. B.; Well-Malherbe, H. Anal. Biochem. 1968, 26, 92-103. Christenson, R. H.; McGlothlin, C. D; Hedrlck, R.; Cate, J. C. Clln. Chem. (Winston-Salem, N.C.)1982, 28, 1204-1207. Griese, A.; French, C. Appl. Spectrosc. 1955, 9 , 78-96. Pero, T. J. Anal. Chem. 1972, 4 4 , 93A-103A. Cook, T. E.; Santini, R. E.; Pardue, H. L. Anal. Chem. 1977, 49, 87 1-877. Green, G. L.; O'Haver, T. C. Anal. Chem. 1974, 46, 2191-2196. Fox, M. A.; Staley, S. W. Anal. Chem. 1976, 48, 992-998. Hager, R. N. Anal. Chem. 1973, 45, 1131A-1138A. Cahill, J. E. Am. Lab. (fairfield, Conn.) 1979, 11, 79-84. O'Haver, T. C. Anal. Chem. 1979, 57, 91A-100A. Smlth, E. R. B.; Well-Malherbe, H. J. Lab. Clln. Med. 1962, 6 0 , 212-222.
RECEIVED for review May 13, 1982. Accepted July 2, 1982.
