it is necessary to have as many relations between the p Las their number.
The Method of Lagranges Undetermined Multipliers. A powerful tool in the solution to extremum problems is the so-called Undetermined-Multiplier Method due to Lagrange. The problem is typically the following. An algebraic relationship exists among several variables PA, PB, p c , namely Equation B-4. It is desired to solve for the numerical values of these variables subject to the condition that the function F ( p , ) be minimum, where
pA
+
Aa = 0
PB
+
Ab = 0
pc
+
Ac = 0
These three equations plus the auxiliary Equation B-4 may be solved together to-yield PA, P B , pc,. and X (A, however, need not be determined). Solving by the determinant method we obtain for the denominator
2
(B-6) F ( P ~ ) = PA + PB2 + PC2 Although Equations B-4 and B-6 appear as two relations among the three unknowns, the problem can be solved in the following way [it should be pointed out that we have eliminated the rigorous mathematical details of the problem. For these, the reader may refer to several known texts on variational principles (7)]. Consider the variation 6F which would vanish when PA, PB, p c have reached the proper value:
6 F = 0:
2 p ~ 6 p+~ 2pB6pB
+
Dp
+
+
a 6p,
+
+
b6pB
C
+
La)
PA + (PB+
Ab)
(Pc
+
0
0
1
lo
:I
+
b2
+
c2
(B-11)
l o bi O
O
l
C
Id b c
01
= ad
(B- 12)
or
(B-8)
(B-13) Similar calculations for B-5.
+
~ P B
Ac) 6pc = 0
1
D p A =
Multiply Equation B-8 by an unknown X and add to Equation B-7: @A
0
a/
constants on the right hand sides of the four equations. I O 0 0 al
= 0
6pc = 0
O
The numerator for P A is a determinant D p , similar to Dp except for its first column which is replaced by the
BPCSPC = O
P C ~ P C
O
D p = a2
(B-7) Furthermore, consider the variation of Expression B-4 (called the auxiliary condition): P B ~ P B
=
I1
la b c 01 which after expansion takes the simple form
or P A ~ P ,
(B-10)
and p c will yield Equations
ACKNOWLEDGMENT
(B-9)
If this equation is to hold for all variations &PA, 6pc, one must have, for every term in the Equation B-9
PB
We thank J. V. Gilfrich and D. J. Nagel for their valued 6p~, criticism and comments on the manuscript. Received for review December 22, 1972. Accepted February 23, 1973.
( 7 ) C. Lanczos, "The Variational Principles of Mechanics," University of Toronto Press, Toronto, 1962, pp 43-49.
Fluorescence and Phosphorescence of Antihistamines Having the 2-Aminopyridine Chromophore. Effect of pH on +,Fluorescence David L. Wilson, Donald R. Wirz, and George H. Schenk' Department of Chemistry, Wayne State University, Detroit, Mich. 48202
The fluorescence and phosphorescence excitation and emission spectra of four antihistamines having the 2aminopyridine chromophore and one having the 2-aminopyrimidine chromophore have been correlated with the spectra of 2-aminopyridine and 2-aminopyrimidine. Fluorescence quantum efficiencies in air have been measured together with molar absorptivities to give a practical relative indication of the fluorescence intensities of these molecules. The effect of pH on the fluorescence and phosphorescence of these antihistamines has been 1
Author to whom reprint requests should be sent.
studied by means of titration curves; it appears that a + 1 cation is the main emitting species in a pH 6.3 aqueous buffer. Fluorescence and phosphorescence calibration curves are linear over several orders of magnitude of concentration, so that fluorometric and phosphorimetric analysis of single components is feasible. Sominex tablets were analyzed fluorometrically for their methapyrilene hydrochloride content.
The antihistamines form a pharmacologically important class of compounds whose luminescence properties have A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 8, J U L Y 1973
1447
not been studied from an analytical viewpoint. Such a study can provide the basis for analytical methods based on the native luminescence, rather than the chemically induced-luminescence, of these compounds. Many, though not all, of the antihistamines are substituted ethylamines. There are five antihistamines that contain the 2-aminopyridine moiety and have the general structure: R,
y 2
+
N-CH2-CH2-NH(CH,)2,
cy
A-
This group includes tripelennimine citrate (R = C6H5, A- = citrate), pyrilamine maleate (R = CH30C6H4, A= maleate), methapyrilene hydrochloride (R = 2-thenyl, A- = Cl-), thenyldiamine hydrochloride (R = 3-thenyl, A- = Cl-), and thonzylamine hydrochloride (R = CHsOCsH4, A- = C1-, and heterocylic ring is pyrimidine instead of pyridine). These five compounds comprise a useful group to investigate. A study of the effect of pH on the luminescence of these five compounds should also yield further information on the effect of substitution of the aminoethyl group on the 2-aminopyridine and 2-aminopyrimidine rings. Weisstuch and Testa ( I ) and Schulman et al. ( 2 ) have already reported the effect of pH on the fluorescence of the parent compounds. The present assay methods for antihistamines as described in the National Formulary (3) and the U.S. Pharmacopeia ( 4 ) involve only spectrophotometry and nonaqueous titration. Some antihistamines, however, will react with certain reagents to give fluorescent products. Pearlman reacted a number of antihistamines with cyanogen bromide ( 5 ) . He found that antihistamines which contained the aminopyridine group gave fluorescent products; the other antihistamines yielded products having little or no fluorescence. The reaction of 16 antihistamines with hydrogen peroxide was compared to their reaction with cyanogen bromide by Jensen and Pflaum (6). The results were similar to Pearlman’s work. All of the fluorescence excitation wavelengths were longer than 275 nm, most being near 350 nm. The emission maxima ranged from 352 to 467 nm with 14 of the 16 being longer than 390 nm. A fluorometric determination of chlorpheniramine maleate was based on complexation with rose bengal(7). In none of these studies do the authors refer t o previous studies of the native luminescence of antihistamines nor do they claim that their methods result in an increase in antihistamine luminescence. In fact, no reference to the native luminescence of antihistamines can be found in the literature. The methods and results reported below are apparently the first reported methods to be based on the native luminescence of any antihistamines.
EXPERIMENTAL Apparatus. The Turner Model 210 “Spectro” absolute spectrofluorometer equipped with a 75-watt Xenon source and quartz cuvettes (10.0 mm) was used to obtain the fluorescence excitation (1) A . Weisstuchand A. C. Testa, J. Phys. Chem.. 72, 1982 (1968). (2) S. G .Schulman, A . C. Capomacchia, and M. S. Rietta. Ana/. Chim. Acta, 56, 91 (1971). (3) National Formulary XI I , Amerlcan Pharmaceutical Ass., Washington, D.C., 1965. ( 4 ) United States Pharmacopeia, XVII, The U.S. Pharmacopeia1 Convention, lnc., New Y o r k , N . Y . , 1970. (5) E. J . Pearlman, J. Pharmacal. f x p . Ther., 95, 465 (1949). ( 6 ) / R. E. Jensen and R. T. Pflaum, J. Pharm. Sci., 53, 835 (1964), (7) W. E. Lange, J. M . Theodore, and F. J. P r u y n . J. Pharm. Sci. 57, 124 (1968),
1448
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 8, J U L Y 1973
and emission spectra. This absolute spectrofluorometer is capable of recording fluorescence intensity in quanta per unit bandwidth (BW) and has been described by Turner (8). The Aminco-Bowman spectrophotofluorometer with phosphorescence attachment and 150-watt xenon source was used with quartz sample tubes (1-mm i.d.) to obtain all of the phosphorescence excitation spectra and many of the emission spectra. When the rotating can was employed, it was run a t medium speed (2000- to 3000-rpm range). Many of the phosphorescence emission spectra and all of the phosphorescence calibration curves were obtained on a mercury line source (MLS) spectrometer constructed in our laboratory. This instrument was constructed from anaAminco low pressure mercury arc, an Oriel Optics 254-nm mercury line interference filter, a RCA 1P28 photomultiplier amplified by an Aminco Fluoromicrophotometer photomultiplier component, an Aminco phosphoroscope and micro Dewar, a McKee-Pederson model MP-1018 scanning monochromator, an ARF model AAM-10 operational amplifier, and a Beckman model 93506 recorder. A housing was also constructed to hold filters and the phosphorescence sample cell or cuvette for fluorescence. (The MLS spectrometer was used for most of the phosphorescence work because the Aminco-Bowman spectrofluorometer was not available to us during the later stages of the research.) Some fluorescence calibration curves and detection limits were obtained using the Turner Model 110 filter fluorometer with a low pressure mercury arc source emitting at 254 nm. The main primary filter used was the 254-nm mercury line interference filter (20% transmittance at 254 nm) mentioned above. Appropriate sharp cut secondary filters were used. A filter fluorometer was used for comparison with results obtained using the MLS spectrometer. This was also important because the Turner fluorometer is commercially available. All p H measurements were made with a Beckman “Zeromatic” pH meter equipped with a combination glass/reference electrode. Absorption spectra were obtained on a Beckman DB spectrophotrometer. This was also important because the Turner fluorometer is commercially available. Reagents. Antihistamine samples were provided by the U.S. Food and Drug Administration, Detroit District Laboratory. The samples were either USP or N F reference standards or analyzed FDA working standards. They were not further purified. Buffer solutions for standardizing the pH meter were prepared from Coleman certified buffer tablets. Distilled water was prepared by passing the laboratory distilled water through an “Illco-way” Research Model deionizing column and then redistilling in an apparatus constructed of borosilicate glass and tygon tubing. Water purified in this way had a negligible fluorescence blank. The distilled water was stored in borosilicate glass containers. The solvent for all phosphorescence measurements was Rossville Gold Shield ethanol manufactured by Commercial Solvents Corporation of Terre Haute, Ind. The ethanol did not contain fluorescent or phosphorescent impurities a t levels detectable with available instruments and was not further purified. The 2-aminopyridine was from Matheson, and the 2-aminopyrimidine and salicylamide were from Eastman. The 2-aminopyridine and 2-aminopyrimidine were purified by recrystallization twice from ligroin. They were further purified by twice resubliming under reduced pressure. Quinine sulfate was recrystallized three times from water as was acetylsalicylic acid. Eastman spectrophotometric grade ether was used in extraction procedures. All other reagents were ACS reagent grade and were used without purification. The phosphate buffer having a p H of 6.3 was prepared according to the Clark and Lubs formula (9). Reagent grade potassium dihydrogen phosphate and carbonate-free sodium hydroxide solution, prepared according to Vogel’s “Procedure B” ( I O ) , were used for the buffer solution. For each 100 ml of solution, 10.6 ml of 0.10N sodium hydroxide and 50 ml of 0.10N potassium dihydrogen phosphate are required. Cleaning of glassware: Nitric acid (11) and a nitric acid-sulfuric acid mixture (12) have been used for cleaning glassware. For G . K . Turner, Science, 146, 183 (1964) “Handbook of Chemistry.” N . A . Lange, Ed., Handbook Pubiishers. Inc., Sandusky, Ohio, 1952, p 9 3 8 . A . I . Vogel, “Quantitative Inorganic Analysis,” 3 r d ed., Wiley, New York, N . Y . , 1961, p 2 4 1 . R. Zweidinger, L. B. Sanders, and J . D . Winefordner, Ana/. Chim. Acta. 47, 558 (1969). R. E.
Thiers in “Methods of Biochemical Analysis,” D. Glick, Ed.,
Vol. 5 , Interscience, New Y o r k , N . Y . , 1957.
this work, pipets and vislumetric flasks were soaked in concentrated nitric acid. Procedures. Quantum Eificiency. Absorption and emission spectra for the determination of quantum yields were obtained on the Turner model 210 “Spectro.” This instrument automatically records “corrected” excitation and emission spectra. For recording absorption spectra, the 210 Spectro operates as a constant bandwidth dual-monochromator spectrophotometer. The use of a monochromator between the sample cell and detector eliminates any fluorescence artifact in absorbance measurements. The quant u m efficiencies were calculated using Turner’s (8, 13) equation, which we have recently used for acetylsalicylic acid (14). The standard used was quinine bisulfate in 0.1N sulfuric acid which has a quantum yield of 0.53 when excited at 348 nm. The quant u m yield for this suhstance is the subject of some controversy ( 1 5 ) . However, the problem is of little consequence for this work since comparisons to other reported quantum yields are not involved and measurements were made in the presence of dissolved oxygen for practical analytical purposes. The data for calculating quantum yield was obtained by preparing 10- 4M solutions of antihistamines, model compounds, and standards in appropriate solvents. After measuring the absorbance, the solution was diluted to the l O - 6 - l O - 7 M range, and the fluorescence emission spectrum was recorded with excita.tion at the appropriate wavelength. All emission spectra of unknowns and standards were recorded with all variable instrumental parameters held constant. A blank spectrum of the phosphate buffer alone was recorded on the same chart with each emission spectrum. The difference in these two areas was used as the corrected emission spectrum. Each area was the average of a t least three planimeter tracings. Antihistamine Analytical Curves and Fluorometric Titration Curces. To establish a fluorometric or phosphorimetric analytical curve for a particular antihistamine, a or 10-4M stock solution was freshly prepared in distilled water, aqueous acid solution, phosphate buffer solution. or ethanol. (Although most of the antihistamine solutions are stable, storage of solutions of low concentrations is not recommended.) Appropriate aliquots of the stock solution were diluted with the appropriate solvent: for example, if p H 6.3 was needed. the pH 6.3 phosphate buffer was used for dilutions. These solutions were excited a t 254 nm or a t the appropriate excitati0.n wavelength (Table I) in a fluorometer or phosphorimeter. The intensity of emission was read a t the appropriate wavelength (Table I) if the spectrometer was used or with an appropriate secondary filter if a filter fluorometer was used. All fluorescence measurements on a particular instrument were made using the same quartz cuvette. The cuvette was usually emptied with a suction device and was rinsed thoroughly with solvent between samples. An alignment mark on the cuvette was used when quantitative measurements were made. For each phosphorescence reading, the sample cell was aligned as carefully as possible in the Dewar. The Dewar was then placed in the instrument and partially covered. After activating the microphotometer, the Dewar was rotated until the maximum reading was obtained. The Dewar was then completely covered and a final reading was taken. Readings taken in this manner have a relative standard deviation of less than 5%. The phosphoroscope was operated at about 2000 to 3000 rpm. Changes of +c207~in the velocity of rotation had no effect on phosphorescence intensity readings. Fluorometric titration curves were plotted to establish the conditions under which ma.rimum fluorescence intensity and the minimum change in intensity with changing p H would be observed. Data for the curves were obtained by preparing 100 ml of a 10-5-10-6M solution of each antihistamine in distilled water. The solution was stirred with a magnetic stirrer, and the p H meter was operated continuously. After an initial reading, the solution was adjusted to a pH above 7 by adding a few drops of 0.1N sodium hydroxide. ‘The solution was then adjusted to increasingly more acidic pH values by adding the minimum number of drops of sulfuric amcid solutions of various concentrations. Samples were withdrawn and the fluorescence was read every 0.5 pH unit. The samples were returned to the 100 ml of solution after each reading. The total volume change was less than 5% and fluorescence readings were not corrected for dilution. Analysis of Sominex Tablets. The procedure for the assay of (13) G . K . Turner, “Notes or1 the Determination of Quantum Efficiency with the Model 210 Speclro,” G . K . Turner Associates, Feb., 1966. (14) C. I . Miles and G . H. Schenk. Ana/. Chem., 42, 656 (1970). (15) T. G . Scott, R. D. Spencer, N . J. Leonard, and G . Weber, J. Amer. Chem. SOC.92, 687 (1970).
Table I. Corrected Fluorescence Data and Uncorrected Phosphorescence Data for Antihistamines and Model Compoundsa Fluorescence
Antihistamine
Methapyrilene hydrochloride Pyrilamine maleate Thenyldiamine hydrochloride Thonzylamine hydrochloride Tripelennamine citrate
pH 6 . 3 bufferb b x :
A,
pH 2 ( 0 . O l M Phosphorescence
acid) At.,
ethanol
h x :
A,,
238 ( 3 0 3 ) ; 363
313; 4 2 0
2 9 2 ( 2 4 4 ) ; 445
2 4 2 ( 3 0 6 ) ; 366
300; 3 6 0
3 0 0 ( 2 5 0 ) ; 420
2 4 3 ( 3 0 5 ) ; 366
315; 4 0 0
3 0 3 ( 2 5 3 ) ; 430
2 4 5 ( 3 0 3 ) ; 383
272; 3 8 0
2 4 5 ( 3 0 0 ) ; 400
248 ( 3 0 6 ) 363
313; 420
3 0 0 ( 2 5 0 ) ; 425
~
Model Compounds
2-Aminopyridine 2-Aminopyrimidine
230(295); 363 228(295); 363
. . .; 44OC . . , ; 4OOc
a The excitation band of higher intensity is listed first: that of lower intensity is listed in parenthesis. !] The buffer is an aqueous phosphate buffer prepared from certified Coleman buffer tablets. The phosphorescence emission spectra was obtained by exciting with the 254-nrn mercury line.
methapyrilene hydrochloride in Sominex tablets is as follows. Grind the sample tablet(s) to a fine powder. Weigh a 0.2-gram sample ( = Y2 tablet) and transfer to a stoppered flask. Add 25 ml of 1N sulfuric acid and shake for 15 minutes. Filter into a 250-ml volumetric flask and dilute to volume with distilled water. Extract a 5-ml aliquot of the solution twice with ether. Heat the aqueous phase a few minutes t o expel ether. Transfer to a 1000ml volumetric flask and dilute to volume (or dilute to Y ~ O Oof the original volume in steps) with the p H 6.3 phosphate buffer solution. Read the fluorescence of the solution and compare to the fluorescence of a standard treated in the same manner. A final concentration range of 0.25-2.5 mg/l. of methapyrilene hydrochloride is recommended for samples and standards. Excite a t 254 nm (or 238 nm) and measure the fluorescence at 363 nm on the spectrofluorometer. If a filter fluorometer is used, use a 254-nm interference primary filter and 7-60 secondary filter.
RESULTS AND DISCUSSION Antihistamine Luminescence. Table I lists the antihistamines investigated in this study as well as the two model compounds. The excitation and emission maxima for both fluorescence and phosphorescence are reported. All fluorescence data are corrected for instrumental parameters in that they were measured on the Turner “Spectro” 210 absolute spectrofluorometer. Fluorescence spectra were observed a t p H 6.3 where the emission is a t a maximum, and a t p H 2 where some new, less intense, bands appear. As seen in the table, the five antihistamines exhibit similar fluorescence spectra a t p H 6.3. At pH 2 , the emission bands of the methapyrilene, thenyldiamine, and tripelennamine salts definitely shift to longer wavelengths. (The effect of p H on the fluorescence emission of all these antihistamines will be discussed in more detail below.) The phosphorescence excitation data agree fairly closely with the fluorescence excitation data, except that the lower energy phosphorescence excitation band appears to be more intense than the higher energy band in the uncorrected spectra. There appears to be more variation among the phosphorescence emission maxima than among the fluorescence emission maxima. However, the bands overlap so that phosphorescence emission would still only be useful for the analysis of a pharmaceutical preparation containing only one antihistamine. Several model compounds were chosen to determine the chromophores responsible for luminescence. The model ANALYTICAL CHEMISTRY, VOL.
45, NO. 8, J U L Y 1 9 7 3
1449
WAVELENGTH, NM Figure 1. Left: Corrected fluorescence excitation spectra of ca. 1OW5M tripelennamine citrate (-) and ca. 10-6M 2-aminopyridine in aqueous pH 6 . 3 phosphate buffer, emission being read at 363 n m for both. Right: corrected fluorescence emission spectra (F) of ca. 1 0 - 5 M tripelennamine citrate (-) and ca. 1 0 - 6 M 2-aminopyridine in aqueous pH 6 . 3 phosphate buffer, and uncorrected phosphorescence emission spectra (P) of ca. 1O-5M tripelennamine citrate (---) and ca. 10W6M 2-aminopyridine both in ethanol ( . a * )
( . * a )
(-.-e),
WAVELENGTH, N M Figure 2. Left: Corrected fluorescence excitation spectra of ca. 10 -5M thonzylamine hydrochloride (-) and ca. 10 -6M 2-aminopyrimin aqueous pH 6.3 phosphate buffer, emission being read at 383 for the former and 363 nm for the latter. Right: coridine rected fluorescence emission spectra (F) of ca. 1 0 - 5 M thonzylamine hydrochloride (-) and ca. 10 V 6 M 2-aminopyrimidine (..*) in aqueous p H 6 . 3 phosphate buffer, and uncorrected phosphorescence emission spectra ( P ) of ca. 10-5M thonzylamine hydrochloride (---) and ca. 10 -6M 2-aminopyrimidine both in ethanol glass (sa.)
(-e-),
compounds studied were 2-methylthiophene, 3-methylthiophene, 2-aminopyridine, 2-aminopyrimidine, toluene, and anisole. The methylthiophenes did not exhibit fluorescence or phosphorescence in acidic, basic, or neutral solutions. The corrected fluorescence excitation and emission spectra, and the uncorrected phosphorescence emission spectra, for 2-aminopyridine and 2-aminopyrimidine are shown in Figures 1 and 2. The analogous spectra for two of the five antihistamines in the same solvents are 1450
ANALYTiCAL CHEMISTRY, VOL. 45, NO. 8, J U L Y 1973
also shown in Figures 1 and 2. The obvious similarities in all aspects of these spectra indicate that the chromophore responsible for the luminescence of these antihistamines is the 2-aminopyridine or 2-aminopyrimidine group. From Table 11, it can be seen that the quantum efficiencies of all the antihistamines are small, the median being 0.009. Radiationless deactivation processes are therefore more important than the fluorescence radiational process for these molecules. It is likely that the
Table I I. Relative Fluorescence Quantum Efficiencies in Air and Detection Limits of Antihistamines
4 r
Antihistamine
Methapyrilene hydrochloride Pyrilamine maleate Thenyldiamine hydrochloride Thonzylamine hydrochloride Tripelennamine citrate 2-Aminopyridine 2-Arninopyrimidene
Fluor. Det. Lim.
Phos. Det.
hex= 254,bM
Lim.,b M
0.01, 0.01,
19,000 (238) 19,000 (238)
21 0 21 0
8 1
x
10-8
1 2
x x
10-8
0.009,
21,000 (238)
190
8
x
10-9
1
x
10-7
0.0005 0.0095 0.19
23,000 (240) 16,000 (246) ... ...
12 150
4
x x
10-7 10-9
6X 2 x 10-8
0.038
9
x 10-9
10-7
...
...
, . .
...
...
...
1 1 The solvent used was an aqueous phosphate buffer at pH 6.3. Dissolved oxygen was not removed: hence only one figure is significant. " Detection limits were determined on the MLS spectrophosphorimeter (the rotating can was not used for the fluorescence measurements) using 254-nm excitation and a 5-rnm emission monochromator slit width.
n , r * singlet state(s) present because of the nitrogen functions in each antihistamine dominates the fate of the excited molecule. Although the spectral features of the antihistamines can be expected, a t least in some cases, to resemble those of model compounds, the same correlation of quantum efficiencies is not found. The larger, less rigid antihistamine molecules would be expected to be more susceptible to radiationless deactivation of excited states as well as to rapid intersystem crossing. This idea is supported by comparison of quantum efficiencies for 2aminopyridine and 2-aminopyrimidene to the antihktamines containing these structures. Under the same conditions, the quantum efficiencies were 0.19 for 2-aminopyridine, 0.038 for 2-aminopyrimidene, about 0.01 for the 2aminopyridine antihistamines, and 0.0005 for thonzylamine hydrochloride (contains 2-aminopyrimidene moiety). The quantum yields, when combined with the respective molar absorptivities, provide another basis for comparison of the fluorescence of the antihistamines. Their qbj products were calculated and are listed in Table 11. This product is not dependent on instrumental parameters as is the detection limit. However, in using the information, one must consider whether or not the compounds compared can be excited a t the absorption maximum if a mercury source is used and, also, whether the excitation energy is the same a t the maximum wavelengths for compounds being compared. Another consideration is the shape of the fluorescence excitation bands. For example, pyrilamine maleate and tripelennamine citrate both have a quantum yield of about 0.01. If both were excited with the same source intensity a t their respective maxima, the fluorescence intensity of the latter would be expected to be lower since its e & product is almost 30% lower than that of pyrilamine maleate. However, if the 254-nm mercury line is used for excitation, the situation is changed because of the narrow excitation bands in this region. The 254-nm line can excite tripelennamine a t almost seveneights of the maximum intensity (see line a t 254 nm in Figure 1). In contrast for pyrilamine, the 254-nm line is located a t a point where it can excite a t only about half of the maximum intensity (not shown). I t is not surprising, therefore, that the detection limits for the two compounds are about the same (Table 11). In general, the fluorescence detection limits for the four antihistamines having the 2-aminopyridine chromophore are about the same using 254-nm excitation and the MLS spectrometer. Although thonzylamine hydrochloride has a much lower quantum efficiency, its t& is only about onetwentieth that of the other antihistamines; its fluorometric detection limit is somewhat more than twenty times
that of the others. Thonzylamine hydrochloride has a much lower phosphorescence detection limit (Table 11), and traces of it should be measured using phosphorimetry. Traces of the other antihistamines can also be measured by phosphorimetry, although fluorometry is certainly the preferred technique, Further comments should be made about pyrilamine maleate and thonzylamine hydrochloride which both contain the anisole chromophore (R = CH30-C6H4). Anisole emits intensely, & = 0.29 in cyclohexane ( 1 6 ) , and it might be hypothesized that this chromophore, rather than either of the two nitrogen heterocyclic rings, is the photoactive chromophore. However, anisole emits fluorescence centered a t 290 nm (methanol), whereas pyrilamine emits a t 366 nm and thonzylamine emits a t 383 nm. Anisole emits phosphorescence a t 380 nm compared to 420 nm for pyrilamine and 400 n m for thonzylamine. In neutral solution, the molar absorptivity of anisole a t 269 nm is only about 1500 compared to c's of 4100 and 3000 a t 292 nm for 2-aminopyridine and 2-aminopyrimidine, respectively. Two possibilities therefore exist: one, that the anisyl chromophore does absorb and transfers its excitation energy to the nitrogen chromophore, which then emits, and two, the nitrogen chromophore both absorbs and emits. The first possibility may be less important because the anisole chromophore absorbs about one third of the exciting radiation that the other chromophores absorb. This finding has important general analytical implications. First of all, it implies that antihistamines and other similar molecules having a 2-aminopyridine (or 2-aminopyrimidine) chromophore should fluoresce and phosphoresce and should be amenable to luminescence analysis. This of course may be limited by the presence of other functional groups. This finding also implies that such molecules should emit a t the same wavelengths as 2aminopyridine (or 2-aminopyrimidine) provided that other chromophores are not more photoactive. Quantum Efficiencies. The relative quantum efficiencies of the antihistamines and 2-aminopyridine as measured on the Turner Spectro 210 in the presence of air are reported in Table 11. It is important to note that oxygen was not excluded from any of the solutions so that oxygen quenching can occur in varying degrees with any of the antihistamines or model compounds. For this reason, only one significant figure is reported where quantum efficiencies are lower than 0.10. (A second digit is given as a subscript only to indicate the trend.) We measured the (16) I. E. Berlman, "Handbook of Fluorescence Spectra of Aromatic Molecules," Academic Press, New York, N . Y . , 1965, p 65. A N A L Y T I C A L C H E M I S T R Y , VOL. 45, ,NO. 8, J U L Y 1973
1451
WAVELENGTH, NM
Figure 3. Effect of acidity on the fluorescence emission spectra of three antihistamines: methapyriline hydrochloride, thenyldi-
amine hydrochloride, and tripelennamine citrate The curves are composites for all three: (1) Aqueous pH 6.3 phosphate buffer solvent (-). (2) Aqueous pH 2 (0.OlM sulfuric acid) solvent ( - - - ) , (2') Aqueous pH 2 (0.01M sulfuric acid) solvent at high instrumental sensitivity than for curve 2 ( - - - ) . (3) Dilute sulfuric acid ( H , = -4) solvent (4) Dilute sulfuricacid ( H o= -7) solvent ( a * . ) .
( - e - - - )
Table I l l . Fluorometric Titration Curve Data for Antihistamines and 2-Aminopyrimidine Relative fiuorescence intensity at 363 nm (max. = 1 .O)"
p>
Thenyldiamine
Tripelennamine
Pyrilamine
Methapyrilene
Thonzylamine
7.0 6.0
1.0 1.0
5.0
1.0
4.0 3.5 2.5
0.76 0.41
0.98 0.97 0.94 0.67 0.36 0.06 0.04 0.03 0.03
1.0 0.99 0.97 0.60 0.31 0.04 0.02
0.98 1.0 0.97 0.79 0.53
0.99 0.98 0.98 1.0 0.96 0.84
2.0 1.5
1.0
0.10 0.08 0.07 0.07
0.01
0.09 0.03 0.01
0.01
0.01
2-Amino pyrimidine
1.0
0.20
1.0 0.98 0.75 0.18 0.06 0.02 0.01
0.01
0.00
0.57
(I The fiuorescence emission of thonzylamlne (hydrochlorlde) was measured at 383 nm
quantum efficiencies in air partly because we were interested in the practical problem of how efficient fluorescence would be under actual analytical conditions; i.e., in air where oxygen quenching would occur. It is, of course, not intended that the values in Table Il be used in the usual theoretical sense. Effect of pH on Luminescence. The fluorescence and phosphorescence of these antihistamines is very sensitive to the pH of the solution. Very generally, the fluorescence intensity is a t a maximum in neutral to slightly acid solutions; it gradually falls to essentially zero in basic or highly acidic solutions. For three of the Z-aminopyridine-containing antihistamines, a weak longer wavelength fluorescence appears around a pH of 2 (Figure 3), and for all four, a strong fluorescence is again observed in very highly acidic solutions. The phosphorescence of all antihistamines is substantial in pure ethanol. It is increased slightly in 0.1N alcoholic KOH and, with one exception, decreased by the addition of acid. These observations will now be discussed in more detail. The change in fluorescence with changing p H was most carefully studied in the pH range 1-7 by means of fluorometric titration curves. These curves were obtained as described in the Experimental section. The data for fluorometric titrations for the five antihistamines and 2-aminopyrimidene are summarized in Table 111; 2-aminopyridine does not show a significant change in fluorescence over the pH range of 1-7. The inflection point for these curves occurs a t a point a t which the relative fluorescence intensity is one half the maximum intensity. With the aid of some additional data, the interpretation of the trends in Table I11 appears to be straightforward. In the third column of Table IV are listed ground state PKaz values for some antihistamines and the corresponding values (PKal) for 2-aminopyridine and 2-aminopyrimidene. These are values for the ionization of a proton from the heterocyclic ring nitrogen (PKal values would reflect ionization of a proton from the antihistamine-CH2NH(CH3)2+ side chain). The pKa2 values can also be estimated from the inflection point of the fluorometric titration curves. For the five antihistamines and 2-aminopyrimidene, the inflection point (second column in Table IV) is within h0.2 pK unit of the ground state PKa2 for the heterocyclic ring nitrogen (the first ring nitrogen in the case of thonzylamine.HC1 and 2-aminopyrimidine). The difference between ground state PKaz and the excited state pKa2 (pK*) values for the four antihistamines related to 2-aminopyridine were estimated from the change in absorption spectra with p H according to the following equation ( 2 ) :
Table IV. Comparison of pK, Values for Antihistamines and Model Compounds Ground state
-
Compound
Tripelennamine citrate Pyrilamine maleate Methapyrilene HCI Thenyldiamine HCI Thonzylamine HCI 2-Aminopyrimidine 2-Aminopyridine
(PKa2)fa
3.75 3.88 3.50 3.75 1.90 3.60
.
..
(Fa Fb)' (pK* pm-' pK,z)(' PK~z
3.92 4.02
-0.08 -0.10
3.66 3.94 2.08 3.45' 6.8O
-0.10 -0.10
-
1.7 2.1 2.1 2.1
a p K a 1 2estimated , from fluorimetric titration curves for antihistamines; pK,, for 2-amlnopyrirnidene. b These are pKa,r values for the 2-aminopyrimidene and 2-aminopyridine. Ali were determined by potentiometric titration. These values are the differences in the absorption maxima Ta for + 2 ions: V b for + 1 ions. OThese values are the differences between the excited state .DK,~ZI and the around state D K ~ [, z -~ (DK*) .
,,
1452
where c is the velocity of light, (Fa - bb) is the difference in energies of the two absorption maxima, and k is the Boltzmann constant. The conclusions which are consistent with these data are as follows: The significant fluorescence of this group of antihistamines appears to originate from the cation in which only the dimethylamine nitrogen is protonated: R\
y 2 t N-CHz-CH2-NH(CH&
I
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, J U L Y 1973
The decrease of fluorescence with increasing acidity corresponds to protonation of the ring nitrogen in the ground state:
Table V. Fluorometric and Phosphorimetric Analytical Curve Ranges Analytical curve ranges, M Type of fluorometer Antihistamine
Methapyrilene hydrochloride Pyrilamine maleate Thenyldiamine hydrochloride Thonzylamine hydrochloride Tripelennamine citrate
M LS spectrometero
Phosphorimeter Filterh
1 x 10-7-10-~ 2 x 10-8-10-5
8 X 10-9-10-5
10- 7-1
0-6
1 x 10-8-10-5
10-7-1
0-6
8 X 10-9-10-5
6 X 10-7-5 X
4 x 10-7-10-5 9 x 10-9-10-5
7
...
x 10-'-5
M LS spectrometer'
X
I x 10-7-10-5
6 x 10-9-10-5 2 x 10-8-10-5
aObtained in pH 6.3 phosphate buffer using the 254-nm mercury line, the 254-nm mercury interference filter, and setting the emission monochromator at the emission wavelength listed in the first data column of Table I. Obtained at pH 2 using the 254-nm mercury line wifh the Turner filter fluorometer, the 254-nm mercury interference filter, and the 7-60 narrow band pass secondary filter except that a 485-nm sharp cut secondary filter was used for methapyrilene, and a 465-nm sharp cut secondary filter was used for pyrilamine. Obtained in an absolute ethanol glass, using the 254-nm mercury line, the 254-nm mercury interference filter, and setting the emission monochromator at the phosphorescence emission wavelength listed in Table I . The rotatino can was run at 2000-3000 rDm.
The data also appear t o indicate that excited state prototropic equilibria are too slow to compete with fluorescence emission. The excited states of the four antihistamines are about 2 pK units more basic than the ground state with respect to protonation of a ring nitrogen in agreement with the observations of others for the excited states of heterocyclic nitrogen compounds (2). Since the excited state is more basic, the protonation of an excited +1 cation in the p H region corresponding to the ground state pK,2 to give an excited + 2 cation would be thermodynamically favored. However, it appears that this protonation is not fast enough to be important. If the excited state protonation did occur fast enough to compete with fluorescence emission, the fluorescence intensity a t that point would be lower than if the reaction did not occur. Applying this argument to all points on the curve, the result would be a curve with an inflection point a t a higher pH. Since all of the fluorimetrically estimated pK values are lower than the corresponding potentiometric values, the excited state protonation reaction does not appear to be important. Although the fluorescence of 2aminopyridine does not decrease on protonation of the ring nitrogen in the ground state, other workers did conclude that the analogous protonation reaction in the excited state does not compete effectively with fluorescence emission ( 2 ) . The effect of acidity on phosphorescence was studied by measuring the phosphorescence intensity of antihistamine solutions in pure ethanol and 1.ON HzS04, 0.01NH2S04, and 0.1N KOH in ethanol. The antihistamines showed increases of 110 t o 210970 in phosphorescence intensity in 0.1N KOH as compared to the solution in pure ethanol. The phosphorescence of all of the compounds except methapyrilene hydrochloride is decreased to about 1% of the original intensity in acid. Although the direct comparison of ethanolic media a t 77 "K and aqueous solutions a t room temperature may not be valid, it appears t h a t a cation with only the dimet hylamino nitrogen protonated may be responsible for phosphorescence while protonation of the ring nitrogen reduces the emission as it does for fluorescence in aqueous solution. The phosphorescence of methapyrilene hydrochloride in acid is in marked contrast t o the other members of this group. (This unusual behav-
ior was rechecked and found to be the same when freshly prepared antihistamine and acid solutions were used.) The behavior of methapyrilene hydrochloride is particularly interesting when compared to thenyldiamine hydrochloride to which it is similar in structure. Since the luminescence is apparently originating in the 2-aminopyridine part of the molecule, it is possible that the substantial difference in behavior is related to some kind of interaction between the thenyl group and the 2-aminopyridine group. Another effect of acid on tripelennamine citrate, methapyrilene hydrochloride and thenyldiamine hydrochloride is that all exhibit a new, but weak fluorescence band centered a t 400-410 nm in acidic solution. In very strong acid, all four antihistamines with the 2-aminopyridine group show a strong fluorescence near the location of the fluorescence in neutral solution. A representation of the average fluorescence spectra of these compounds is shown in Figure 3. Curve 1 represents the average spectra for the four antihistamines in neutral aqueous solution. As the solution is made more acidic, the fluorescence peak a t 365 nm is quenched and a new weaker peak centered around 405 nm appears (curve 2) for three of the compounds, but not for pyrilamine maleate. Curve 2' is the same as curve 2 , but the sensitivity of the instrument is higher. Curve 2' shows more clearly that the peak is more symmetrical and has a larger half-width than curve 1. The peak a t 405 nm is observed from a p H of 2 to a Hammett acidity (H,) value of -4 for tripelennamine citrate and thenyldiamine hydrochloride and from a p H of 2 to Ho of - 1 for methapyrilene-hydrochloride. In stronger acid solutions, the fluorescence is described first by curve 3 and then by curve 4. The peak a t 405 nm either disappears or is hidden beneath the tail of the new 370 nm peak. In solutions with H, of -7 or -10, all four compounds develop colors ranging from light yellow-brown to pink; this suggests that some decomposition may be occurring. Interpretation of these observations is rather difficult. Since three different fluorescence peaks are observed, they might be related to the +1 cation with the dimethylamine nitrogen protonated (curve l), the + 2 cation with the ring nitrogen also protonated (curve a), and the +3 cation with all three nitrogens protonated (curve 4). The results of studies ( I , 2 ) of the fluorescence of 2-aminopyridine suggest that, although this interpretation may be correct, there are reasons to doubt this. Further study is obviously needed, especially of the nature of the fluorescence in strongly acidic solutions. ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 8, JULY 1973
1453
Table V I . Analytical Details and Results of Analysis and Spiking of Sominex Tablets Analytical details Mgjtablet (Label)
Compound
Meth.-HCP Salicylamide Scopolamine aminoxide.H B r
25 200 0.25
Excited at 254 nm?
L n .
pH 6.3
Yes Yes
363 420
nm nm
Yes (weak)
285
nm (weak)
Analytical results Mg Sominex taken
Sample
1
Mg meth.HCI 'tabiet found
200.3 198.7 210.1
2
3
Mean Recovery for Sominex Spiked with Meth.-HCI, YO Mg meth.HCI in Pure meth.- Mg meth.analyzed HCi added HCi. finai Sample tablet (spiked) analysis
1 2 3
6.18 6.63 6.70
6.25 6.25 6.25
26.2 25.9 25.7 = 25.9
Recovery. 0%
12.62 12.85 12.98
Mean = a ("Meth.-HCI"
101.5 99.7 100.2 101%
= Methapvrilene . HCI)
Fluorometric and Phosphorimetric Determination of Antihistamines. Both spectrofluorometric and filter fluorometric measurements were used to obtain analytical curves of F, the intensity of fluorescence emission, us. the concentration of each antihistamine. The 254-nm mercury line was used for excitation in the MLS spectrometer described in the Experimental section; 5-mm monochromator slit widths were employed for measurement of emission. The useful range of the analytical curves for each of the antihistamines is listed in Table V. The lower limits for the analytical curves obtained on the MLS spectrometer for both fluorescence and phosphorescence are also the detection limits. These detection limits were calculated from the standard deviation of the solvent blank and the slope of the analytical curve. The detection limit is defned as two times the standard deviation of the blank divided by the slope of the analytical curve ( 1 7 ) . The luminescence detection limits in Table V are applicable only to the MLS spectrometer because they are dependent on the intensity of the source, the nature of the light path, the slitwidth and alignment of the monochromator, and the sensitivity of the photomultiplier tube. For the MLS instrument, the standard deviation of the blank is different a t different wavelengths due to the small transmittance of mercury lines at wavelengths longer than 254 nm. Even with these limitations, the detection limits are still useful for comparisons and for a general indication of what other instruments may attain. The upper limits for the fluorescence and phosphorescence analytical curves from MLS spectrometer are limited by the inner filter effect, and these, in general, are conservative estimates. For example, for methapyrilene hydrochloride, the slope of the fluorescence analytical curve begins to decrease just above l X 10-4M, but the upper limit is listed as l0-5M. The slope of the phosphorescence analytical curve for the same antihistamine begins to decrease just above 3 X 10-4M, but the upper limit is listed as l0-4M. TOevaluate the effect of working a t pH 2 and eliminating the need for a buffer, some analytical curves were obtained on a filter fluorometer using solutions at pH 2. ( 1 7 ) J E Barney, Taianta, 1 4 , 1363 (1967)
1454
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8 , J U L Y 1973
This involves much weaker fluorescence emission bands, except for pyrilamine maleate. As can be seen from a comparison of curves 1 and 2 in Figure 3, the fluorescence band a t pH shifts to longer wavelengths and decreases to about 15% of its intensity a t pH 6.3. It is understandable that the lower limits of these analytical curves are much higher than those of the MLS spectrometer. To check whether a filter fluorometer would, in fact, give a lower detection limit a t p H 6.3 than the MLS spectrometer, some measurements were made on solutions of methapyrilene hydrochloride a t p H 6.3 using the Aminco filter fluorometer and a 7-60 narrow band pass secondary filter (305-400 nm range). Although different photomultiplier tubes were used in the monochromator and filter fluorometers, their response was similar over the wavelength range studied. The same microphotometer was used for all readings. The sensitivity is the slope (F/M) of the working curve and the detection limits were calculated as previously described. The sensitivity of the spectrometer was 105 F/M compared to 106 F / M for the Aminco filter instrument. However, the detection limits were about the same for both instruments. The reason is the higher blank reading and the larger standard deviation of blank readings when a filter is used. These higher values are the result of higher transmittance of scattered radiation which leaks through the primary filter and the emission of solvent contaminants.
Analysis of Pharmaceuticals Containing One Antihistamine. Because the antihistamines reported in Table I all have fairly similar luminescence properties, it was decided to limit analysis to pharmaceutical preparations containing just one antihistamine. It should be noted that because thonzylamine hydrochloride fluoresces relatively weakly (see c @ f products in Table 11), any of the other four antihistamines could probably be determined in the presence of the former a t pH 6.3. In addition, any one of methapyrilene, thenyldiamine, and tripelennamine might be determined at 400-420 nm a t pH 2 in the presence of either of the remaining two antihistamines. A typical problem with pharmaceuticals is that one of the components will interfere by absorbing a t the excitation wavelength of the fluorescent component and exerting an inner filter effect. This error can be eliminated by diluting if the intensity of fluorescence is strong enough, or by removing enough of the interfering component by a separation to reduce the inner filter effect to negligible levels. The percentage of the interfering component removed by separation would not have to be as large as for other methods such as spectrophotometry for example. Sominex tablets were chosen for analysis since this preparation contained only one antihistamine as well as two other components which were thought capable of exerting an inner filter effect. Each Sominex tablet is stated to contain 25 mg of methapyrilene hydrochloride, 200 mg of salicylamide, and 0.25 mg of scopolamine aminoxide hydrobromide. The analytical details have been summarized in Table VI. As seen in Table VI, the 285-nm fluorescence band of scopolamine aminoxide hydrobromide is weak and is considerably below the 365-nm band of methapyrilene hydrochloride. In addition, its concentration is too low to cause an inner filter effect, so that it causes no interference in either respect. Salicylamide, which is present at higher concentration compared to the other components, absorbs 254 nm radiation and its fluorescence band is a t 420 nm a t a pH of 6.3. If the amount of salicylamide were smaller, methapyrilene hydrochloride could be analyzed on a spectrofluorometer without separation. In a solution of Sominex tablets, however, the overlap of the bands and the
inner filter effect make a separation necessary. Salicylamide is not too soluble in aqueous acid so that some of it is eliminated in the dissolution step. The remainder is removed by an ether extraction. Sominex tablets were analyzed in triplicate by the procedure outlined in the Experimental section. Samples spiked with standard methapyrilene hydrochloride solu-
tion were analyzed to determine the recovery. The mean of the results (Table VI) for methapyrilene was 0.9 mg higher than the stated amount of 25 mg on the label, a reasonable deviation. Received for review August 25, 1972. Accepted February 21,1973.
Correlation of Enhancement of Atomic Absorption Sensitivity for Selected Metal Ions with Physical Properties of Organic Solvents Alice J. Lemonds' and B. E. McClellan2 Department of Chemistry, Murray State University, Murray, Ky. 42071
Various alcohols, ketones, esters, and other organic compounds, as solvents for Ag, Cd, Co, Ni, and Zn ions, were studied to determine the enhancement values for each metal-solvent system and to correlate the enhancement values with the physical properties of the solvents. Optimum instrumental conditions were determined for each metal-solvent system employing two different burner-aspirator systems. Absorbance values for the metal-organic solvent systems were measured and compared with the absorbance values for the aqueous system of'the same concentration in order to calculate an enhancement value. Plots of enhancement vs. log viscosity and enhancement YS. log boiling point for each ion resulted in lines with negative slopes. Various plots involving density and surface tension showed little or no dependence of enhancement on either of the constants. However, a linear relationship existed between log (viscosity X boiling point) and enhancement.
Since the introduction of atomic-absorption spectrometry in 1955, improved detection limits have been sought for this already sensitive analytical technique. Many workers have observed that the addition of water-miscible organic solvents leads to increased sensitivity (1-6). Gains in sensitivity of up to sevenfold have been reported with water-immiscible organic solvents ( I ) . Immiscible organic solvents can serve not only as enhancing agents for a given metal ion, but they can serve to separate and/or concentrate the metal ions. Many papers have appeared describing extraction procedures for separation and/or concentration of metal ions by solvent extraction prior to determination by atomic-absorption spectrometry. Several of Present address, U n i o n C a r b i d e Corp., N u c l e a r Division, P a ducah, Ky. 42001. 2 A u t h o r t o w h o m inquiries should be addressed. (1) J. E. Allan, Spectrochim. Acta, 17, 467 (1961). (2) J. W. Robinson, Ana/. Chim. Acta, 23, 479 (1960). (3) W. T. Eiwell and J. A. F. Gidley, "Atomic Absorption Spectrophotometry," Macmiilan, New York, N . Y . , 1962, pp 26-27. (4) R . Lockyer. J. E. Scott, and S. Slade, Nature (London). 189, 830 (1961 ) . ( 5 ) I . Atsuya, Sci. Rep. Inst., Tohoku Univ.. Ser. A , 18, 65 (1966). (6) I . Atsuya, J . Chem. SOC. Jap., Pure Chem. Sect., 86, 1145 (1965); Anal. Abstr.. 14, 4434 (1967).
these describe the extraction of Ag, Cd, Ni, Co, and Zn ( I , 7-13). The increased sensitivity using organic solvents partially results from an increase in the amount of solution reaching the flame. Physical properties of the solvent such as viscosity, density, and droplet size have been reported as factors which may contribute to enhancement. Allan (I) concluded that increased sensitivity is due primarily to an increase in the amount of solution reaching the flame and suggested that this increase might be the result of an increased uptake rate, the formation of a finer aerosol, and the more rapid evaporation of solvent to produce smaller droplets. Allan also suggested that viscosity, surface tension, and vapor pressure might be controlling factors in the physical processes of atomization. Other studies ( 1 1 , 24-1 7 ) have indicated similar results. The only report of an attempt to correlate the enhancement observed for organic solvents with their physical properties came with the work of Feldman, Christian, and Bosshart ( 1 8 ) . In the study of manganese with various water-miscible solvents, they reported a linear relationship between absorbance and the product of viscosity and density. The purpose of the present work was to determine enhancement factors for a number of metal-solvent systems and to determine if any correlation existed between the enhancement factors obtained and the physical properties (7) J. B. Willis. Anal. C h e m . , 34, 614 (1962). ( 8 ) J. E. Allan, Analyst (London). 86, 530 (1961). (9) T. T. Chao, M. J. Fishman. and J. W. Bali, Anal. Chim. Acta, 47, 189 (1969). (10) B. Fleet, K. V. Liberty, and T. S. West, Anaiyst (London), 93, 701 (1968). (11) T. Takeuchi, M. Suzuki, and M . Yanagisawa, Ana/. Chim. Acta, 36, 258 (1966) (12) I . Atsuya, J. Chem. SOC.Jap.. Pure Chem. Sect.. 88, 179 (1967); Anal. Abstr., 15, 2513 (1968). (13) R. E. Barringer, H. G . King, and A. D. Condrey, U.S. At. Energy Comm.. 23, Y 1661, (1969); Chem. Abstr.. 71,357249 (1969). (14) T. P . Taskaeva and E. E. Vainshtein, I z v . Sib. Otd. Akad Nauk SSSR. Ser. Khim. Nauk, 4, 110 (1967); Chem. Abstr., 69, 15930b (1968). (15) T. Takada and K . Nakano. Nippon Kagaku Zasshf. 90, 487 (1969); Chem. Abstr.. 71, 356892 (1969) (16) K . Nakano and T. Takada, Nippon Kagaku Zasshi. 88, 575 (1967); Chem. Abstr.. 67, 50029v (1967) (17) D. W. Kohlenberger, At. Absorption Newslett.. 8 ( 5 ) , 108 (1969). (18) F. J. Feldman, R. E. Bosshart, and G. D. Christian, Anal. Chem., 39, 1175 (1967)
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 8, J U L Y 1973
1455
