sorption of 0 2 and Hz on Pt and Sn discloses that tin is likely to be enriched in the surface by oxygen chemisorption, whereas platinum is enriched by hydrogen chemisorption. The combined impact of both types of interaction gives rise to the expectation of tin enrichment in the surface by oxygen chemisorption at elevated temperature. For hydrogen chemisorption, such a prediction is not possible; but our experimental results show that the surface is enriched with tin also in this case, if to a smaller extent. A detailed discussion of the chemisorption-induced surface enrichment phenomena of the platinum/tin system has been published elsewhere ( 5 ) . The high-energy resolution attained in X-PS permits the observation of chemical shifts in the line position, AE Alloying as such causes the line position of the Sn 3d5/2 and Pt 4f7/2 peaks to shift as is shown in Table 11. It is interesting to note that the Sn 3d5,2 peak is found a t an ionization energy which is about 1 eV less for the alloys than for pure tin, whereas the Pt 4f712 peak is subject to much smaller effects. The excess negative charge a t the tin atoms in the alloy, indicated by the line shifts of the 3d line, is not in agreement with Knight shift and Mossbauer data (9) on Pt/Sn alloys which suggest a transfer of the Sn 5s electron to the Pt 5d band. From Table 111, it is seen that oxidation of PtSn causes a shift of approximately 1 eV of the
Sn 3d5,2 line and of approximately 3 eV of the Pt 4f7/2 line. The latter value is in close agreement with the finding of Kim et a / . (10) for the Pt/PtOz system. Oxidation of PtsSn causes a AE value of the Sn 3d line similar to that in PtSn but the Pt 4f line remains at about the same position. The above-mentioned results suggest that platinum is thoroughly oxidized when present in a diluted form in a tin-oxide matrix (see surface concentration in Table I). Our results prove that the surface enrichment effects studied by AES are detectable by X-PS as well. For this particular alloy system, the kinetic energy of the relevant photoelectrons (-1000; -1400 eV) is much greater than that of the relevant Auger electrons (-400; -250 eV). As the mean free path for inelastic scattering increases with the kinetic energy in the above energy range, X-PS probes the alloy to a greater depth than AES. This is reflected in the average surface concentrations (see Table I) which lie closer to the corresponding bulk values for the reduced alloys studied by X-PS than by AES. The difference in probing depth of the two techniques on one and the same alloy system provides the opportunity to study the concentration profile between the tin-enriched surface and the underlying bulk in more detail (8).
(9) C R Kanekar, K R P Mallikarjuna Rao, and V Udaya Shankar Rao, Phys Lett 19,95 (1965)
(10) K S. Kim, N. Winograd, and R E. Davis, J Amer Chem S o c , 93, 6296 (1971)
Received for review May 14, 1973. Accepted August 27. 1973.
Simple, Sensitive Spectrophotofluorometric Method for Hydrazine in Plasma Stanley Vickers and
E. K. Stuart
Merck lnstitute for Therapeutic Research, West Point, Pa. 19486
The use of hydrazine and its derivatives as sources of energy and as chemotherapeutic tools has prompted interest in their toxicity and metabolism, and has generated methodology for their measurement in plant and animal tissues. Watt and Chrisp ( I ) developed a spectrophotometric analytical assay based on the observation of Pesez and Petit ( 2 ) that hydrazine and dimethylaminobenzaldehyde (DMAB) produced a yellow colored derivative. Investigators utilizing the DMAB procedure have obtained information on the dose-blood level relationship of hydrazine in rats ( 3 ) and dogs (4, 5 ) a t the microgram level. This report shows how DMAB may be used to measure nanogram quantities of hydrazine in plasma. Dimethylaminobenzalazine, which is the chromophore utilized in the above spectrophotometric assay, also fluoresces intensely in chloroform solutions saturated with trichloroacetic acid. The application of this fluorometric method enabled levels of hydrazine to be measured in (1) G. W. Watt and J. D. Chrisp, Anal. Chem., 24, 2006 (1952). (2) M . Pesez and A . Petit, Bull. SOC.Chlm. France, 1947, 122. (3) 8. A . Reynolds and A . A . Thomas, Amer. lnd. Hyg. Ass. J . , 26, 527 (1965). (4) H . McKennis, J r . , J . H. Weatherby, and L. 8 . Witkin, J. Pharmacol. ~ x p Ther., . 114, 385 (1955). (5) F. B. Smith and D. A . Clark, Toxicol. Appi. Pharmacoi.. 21, 186 (1972).
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monkey plasma after administration of a much smaller dose than previously required by the colorimetric method.
EXPERIMENTAL Apparatus. Fluorescence was measured in an Aminco-Bowman spectrophotofluorometer using quartz cuvettes (10 mm lightpath) at an excitation wavelength of 466 nm and an emission wavelength of 546 nm (the instrument was calibrated by the use of quinine sulfate). Reagents. Hydrazine hydrate (99-lOO%, Matheson. Coleman & Bell), trichloroacetic acid (Baker Analyzed), chloroform (Fisher Analytical), p-dimethylaminobenzaldehyde (Fisher Certified), and anhydrous magnesium sulfate (Baker Analyzed) were used in this method. Authentic dimethylaminobenzalazine was prepared by mixing a solution of hydrazine hydrate (1.6 g) in ethanol ( 4 ml) with a solution of p-dimethylaminobenzaldehyde ( 5 g) in hot ethanol (20 ml). The yellow precipitate was filtered and recrystallized from dimethylformamide. Dimethylaminobenzalazine was identified by mass spectral data: mp 274" [lit. (6) 264-266 "C]. Anal. Calcd for C I ~ H Z ~ N C,~73.45; H, i.48; N , 19.05. Found: : C, 73.52; H, 7.85; N, 18.98. Procedure. Plasma (1 ml) was mixed with aqueous 10% trichloroacetic acid ( 3 ml) and centrifuged (15 min). The supernatant was removed as completely as possible and shaken with chloroform ( 2 ml) for 15 min; the aqueous layer was removed as completely as possible and mixed with 0.4% ethanolic p-dimethyl(6) P. R . Wood, Anal. Chem., 25, 1879 (1953).
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Table I . Reproducibility of the Fluorescence Assay for Hydrazine Hydrazine hydrate, Arbitrary fluorescence units (fstd dev) ng/ml 80 30.7 f 2.6 ( n = 4) 25.3 f 1.7 ( n = 4 ) 60 15.3 & 0.9 ( n = 4 ) 40 8.3 f 0.5 ( n = 4 ) 20
H
Tautomerism of ion pair formed between dimethylaminobenzalazine and trichloroacetic acid Figure 1 .
aminobenzaldehyde ( 2 ml). The solution was heated in a stoppered tube a t 70 "C in a water bath for 45 min, cooled and shaken with chloroform ( 3 ml) for 10 min. The chloroform layer was removed following centrifugation and anhydrous magnesium sulfate (10 mg) was added. After centrifugation the fluorescence of the solution was measured. Three male Rhesus monkeys (3.9 to 4.4 kg) were dosed with hydrazine hydrate (0.3 mg/kg i.p. in saline solution). Plasma was drawn at 0.5, 1, 2, 3 , 4, 5, and 6 hours, and levels of hydrazine were measured by the use of a calibration curve constructed by using normal plasma spiked with known amounts of hydrazine hydrate and plotting the fluorescence of the chloroform solution (after correcting for the blank value) us. the concentration of hydrazine hydrate in plasma. The fluorescence of the chloroform solution was linearly related to the concentration of hydrazine hydrate in plasma over a range of 20 to 200 ng of hydrazine hydrate per ml of plasma.
Table II. Levels of Hydrazinea in Monkey Plasma after an Intraperitoneal Dose (0.3 mg/kg hydrazine hydrate) Hydrazine in monkey plasma, time (hours) Monkey 0.5 1 2 3 4 5 6 1 2
3 a
98.5 120 78
84 100
70
41 44 31
18 26 18
15 16 16
11.5
15 16
10 10 16
Expressed as ng of NHzNHz per ml of plasma
Levels of hydrazine were measured in the plasma of monkeys dosed intraperitoneally with hydrazine hydrate RESULTS AND DISCUSSION (see Experimental). Maximum levels of 99 ng (average)/ Ten nanograms of hydrazine per ml of plasma was the ml were achieved after 0.5 hour which then declined to an detection limit by the present method. Levels of hydraaverage value of 12 ng per ml at 6 hours post dosing. The zine greater than 200 ng per ml of plasma caused deviainitial half life was approximately 1 hour (Table 11). tions from linearity in the curve, and plasma levels of this Halogenated solvents saturated with aqueous solutions magnitude have been determined by spectrophotometric of trichloroacetic acid or perchloric acid may enhance the assay ( 3 ) . The sensitivity of the method could be further fluorescence properties of organic compounds (9). Many increased by using larger volumes of plasma, since this studies have been made on the ion pair extraction of an caused no concomitant increase in background fluoresion from an aqueous solution. The studies of organic amcence, which was equivalent to 5 ng of hydrazine. To test monium ion extractions have in most cases employed the reproducibility of the method, plasma samples (1 ml) CHC13 or CHzClz (10). containing hydrazine hydrate (uit., 80 ng/ml, 60 ng/ml, In the present investigation, fluorometric analyses were 40 ng/ml, and 20 ng/ml) were analyzed. Results are remade on solutions of dimethylaminobenzalazine (both aucorded in Table I. The intensities of the fluorescence obthentic and generated in situ) in chloroform extracts satutained in the experiments such as those above were comrated with TCA. Presumably structures I1 and I11 (Figure 1) are the tautomers responsible for the fluorescence proppared to those of standard solutions of the fluorophore in chloroform saturated with TCA. It was determined that erties of dimethylaminobenzalazine. The isolation of the p-quinone structure was reported by Wood (6). the reaction and extraction of the product were essentially complete (93.5% f 4.4 std dev). A temperature of 70 "C Six hours after a 0.19 mg/kg dose (intraperitoneally) of and a 45-min period of incubation were optimal for maxihydrazine, the latter could still be detected in monkey mum intensity of fluorescence. The latter was reduced plasma. Although other methods ( 3 ) were unable to moniwhen ethyl acetate or butanol was substituted for chlorotor low levels of hydrazine in rat plasma for more than 1 form. Magnesium sulfate, unlike sodium chloride or hyhour following injection of hydrazine (0.5 mg/kg i.p.), drochloric acid, did not decrease the sensitivity of the comparison of the data obtained from rats at 1 mg/kg (i.p.) dosage levels ( 3 ) suggests that in the first 3 hours assay. Trichloroacetic acid (TCA) served a dual purpose of precipitating protein and of increasing the fluorescence the half life in both species is the same at low dose levels. The present method may find application for the deterintensity of the product. There was no advantage in using TCA concentration greater than 10%. mination of hydrazine either free, or liberated by chemical ( 1 1 ) or enzymic means from hydrazine derivatives, in No interference was produced by dopa, dopamine, phenylhydrazine, tryptamine, or tyrosine at levels of 3, 1, plant and animal tissue a t concentrations heretofore im1, 10, and 10 pg, respectively, per ml of plasma. Carbidopossible to measure. pa [~-(-)-hydrazino-3,4-dihydroxy-ol-methylhydrocinnamic acid], an inhibitor of aromatic amino acid decarboxylReceived for review May 29,1973.Accepted July 25, 1973. ase ( 7 ) , will interfere, since under these conditions carbidopa generates the same fluorophore, uiz., dimethylami(9) A . J. Glazko, W. A . Dill, L. M . Wolf, and A. Kazanko, J . Pharmacol. nobenzalazine (8). Exp. Ther., 1 1 8 , 377 (1956). ( 7 ) C . C . Porter, L. S. Watson, D. C. Titus. J. A . Totaro, and S. S. Byer, Biochem. Pharmacol., 11, 1067 (1962). (8) S. Vickers and E . K . Stuart, J . Pharm. Sci.. (in press).
(10) 6 . A. Persson and S. Eksborg, Acta Pharm. Suecica, 7, 353 (1970). (11) T. Momose, Y . Uedo, Y . Mukai, and K. Watanabe, Yakugaku Zasshi, 80, 225 (1960).
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