Fluorescence and Phosphorescence of Antihistamines Having the Diphenylmethane Chromophore Effect of Halide Ions on Luminescence Donald R. Wirz, David L. Wilson, 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 diphenylmethane andlor p-chlorotoluene chromophores, and two related antihistamines have been compared with the spectra of diphenylmethane and p-chlorotoluene. 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 halide ions on the fluorescence and phosphorescence of these antihistamines has been studied to evaluate the possibility of fluorescence quenching and phosphorescence enhancement. Fluorescence and phosphorescence analytical curves are linear over several orders of magnitude of concentration so that fluorometric and phosphorimetric analysis of single components is feasible. Benadryl Injection (Parke-Davis) was analyzed fluorometrically for its diphenhydramine hydrochloride content.
The antihistamines form a pharmacologically important class of compounds whose luminescence properties as a class have 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. Of interest in this study are the four antihistamines that contain the diphenylmethane chromophore with the general structure:
This group includes diphenyhydramine hydrochloride (RI = H, Rz = 0, and R3 = CHz-NH(CH3)2), bromodiphenhydramine hydrochloride (R1 = Br, Rz = 0, and R3 = CHz-NH(CH3)2, chlorocyclizine hydrochloride (R1 = C1, RZ = N(CHz)zNH, and Ra = H), and meclizine hydrochloride (RI = C1, Rz = N(CHz)zNH, and R3 = rnC ~ H ~ C H SIn) .the latter two, Rz is the piperazine moiety. Two other antihistamines that are similar enough to the above four to include in this study are phenindamine tartrate (I),and pyrrobutamine phosphate (11).
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A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 7, J U N E 1974
11
Apart from our previous study ( 1 ) of the native luminescence of antihistamines having the 2-aminopyridine chromophore, there appear to be no references to the native luminescence of antihistamines in the literature. In fact, Udenfriend et al. ( 2 ) reported that diphenhydramine did not fluoresce in a number of media, including a neutral aqueous solution. Some antihistamines have been reported to react with certain reagents to yield fluorescent products. Both cyanogen bromide ( 3 ) and hydrogen peroxide ( 4 ) gave such products only with antihistamines having the aminopyridine chromophore. Rose bengal ( 5 ) has been used for the fluorometric determination of chlorpheniramine maleate. Recent spectroscopic methods for antihistamines are entirely colorimetric (6); indeed the National Formulary (7) and the U S . Pharmacopeia (8) assay methods involve only spectrophotometry and nonaqueous titration. Thus, the luminescence spectra and methods below are apparently the first such reported for these six antihistamines.
EXPERIMENTAL Apparatus. The Turner Model 210 “Spectro” absolute spectrofluorometer equipped with a 75W Xenon source and quartz cuvettes (10.0 mm) was used to obtain the fluorescence excitation and emission spectra. This absolute spectrofluorometer is capable of recording fluorescence intensity in quanta per unit bandwidth and has been described by Turner (9). The Aminco-Bowman spectrophotofluorometer with phosphorescence attachment and 150-watt xenon source was used to obtain all of the phosphorescence excitation spectra and many of the emission spectra. Quartz sample tubes (1-mm i.d.) were used, and when the rotating can was used, it was run at medium speed (2000- to 3000-rpm range). Many of the phosphorescence emission spectra and all of the phosphorescence calibration curves were obtained by using a mercury line source (MLS) spectrometer constructed in our laboratory. This instrument was constructed from the following components: a low pressure mercury arc excitation source (power supply
Author to whom reprint requests should be sent (1) D L. Wilson, D R. Wirz. and G . H. Schenk, Anal. Chem., 45, 1447 (1973). ( 2 ) S. Undenfriend. D. E. Duggan, B. M. Vasta, and B . 8. Brodie, J. Pharmacol. Exp. Ther.. 120, 26 (1957) (3) E. J . Pearlman, J. Pharmacol. Exp. Ther., 95, 465 (1949) (4) R . E. Jensen and R. T. Pflaum, J. Pharm. Sci., 53, 835 (1964). (5) W . E. Lange. J M. Theodore, and F . J Pruyn, J. Pharm. Sci., 57, 124 (1968). (6) J W. Sutherland. D. E. Williamson, and J . G . Theivagt, Anal. Chem., 43, ( 5 ) ,206R (1971). (7) National Formulary X I I , American Pharmaceutical A s s . , Washington, D.C.. 1965 (8) United States Pharmacopeia. X V I I , The U S . Pharmacopeia1 Convention, inc. New York, N . Y . , 1970. ( 9 ) G. K . Turner. Science. 146, 183 (1964).
from the Aminco Fluoro-microphotometer, a 254-nm mercury line interference primary filter (Oriel Optics Corp.), an RCA 1P28 photomultiplier tube (power and amplification from Aminco Fluoro-microphotometer component and McKee-Pederson Housing), a housing constructed by us for the phosphoroscope containing the micro Dewar and quartz sample cell, a McKee-Pederson scanning monochromator, an ARF model AAM-10 operational amplifier, and a Beckman recorder. These have all been described in more detail ( 1 ) . Some fluorescence calibration curves and detection limits were obtained using a Turner Model 110 filter fluorometer with a low pressure mercury arc source emitting at 254 n m . The main primary filter used was the 254-nm mercury line interference filter mentioned above. Appropriate sharp cut secondary filters were used. Other fluorescence calibration curves were obtained using the MLS spectrometer described above, after removing the phosphoroscope and inserting a fluorescence cuvette holder. 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 p H meter were prepared from Coleman certified buffer tablets. Distilled water was prepared as previously described ( I ) . The solvent for all phosphorescence measurements was Rossville Gold Shield bottled ethanol (Commercial Solvents). Diphenylmethane was obtained from Aldrich Chemical Co; chlorotoluene and bromotoluene were MCB reagent grade; and quinine sulfate was recrystallized three times from water. All other reagents were ACS reagent grade and were used without purification. The phosphate buffer having a pH of 6.3 was prepared according to the Clark and Lubs formula ( I O ) . Reagent grade potassium dihydrogen phosphate and carbonate-free sodium hydroxide solution, prepared according to Vogel's "Procedure B" (11). were used for the buffer. For each 100 ml of buffer solution, 10.6 ml of 0.10N sodium hydroxide and 50 ml of 0.10Ai potassium dihydrogen phosphate are required. Cleaning of Glassuare. Nitric acid ( 1 2 ) and nitric acid-sulfuric acid mixture ( 13) have been used for cleaning glassware. For this work, volumetric flasks and pipets were soaked at least 12 hours in concentrated nitric acid, washed repeatedly with tap water, and then rinsed with redistilled water. Procedures. Quantum efficient) . Absorption and emission spectra for the determination of quantum efficiencies were obtained on the Turner 210 "Spectro." This instrument automatically records "corrected" excitation and emission spectra as well as absorption spectra. The quantum efficiencies were calculated using Turner's (9, 14) equation, used by us for acetylsalicylic acid ( 1 5 ) . The standard used was quinine bisulfate in 0.1N sulfuric acid containing oxygen; this has a quantum efficiency of 0.53 when excited at 348 nm ( 1 6 ) . Measurements were made in the presence of dissolved oxygen for analytical comparisons. Data for quantum calculations were obtained by preparing 10 -4M solutions of antihistamines and standards in dilute aqueous acid solvent. After measuring the absorbance. the solution was diluted to the 10-6-10- ' M range, and the fluorescence emission spectrum was recorded with excitation at the appropriate wavelength. All emission spectra of unknowns and standards were recorded with all variable instrumental parameters held constant. A blank spectrum was recorded on the same chart with each emission spectrum. The area under the emission spectrum was taken to be the average of at least three tracings with a planimeter. Antihistamine Calibration Curves and Fluorometric Titration Curves. To establish a fluorometric or phosphorimetric calibration curve for a particular antihistamine. a 10-3 to lO--*M stock solution was freshly prepared in distilled water, aqueous acid 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 N A . Lange, E d . , "Handbook of Chemistry." Handbook Publishers, Inc., Sandusky, Ohio, 1952, p 938. A I . Vogel. "Quantitative Inorganic Analysis," 3rd ed.. Wiley. New York, N . Y . , 1961, p 2 4 1 R . Zweidinger, L. B. Sanders, and J. D . Winefordner, Anal. Chim. Acta, 47, 558 (1969) R . E. Thiers in "Methods of Biochemical Analysis." D Glick. E d . . Vol. 5 , Interscience, New Y o r k , N . Y , 1957 G . K . Turner, "Notes on the Determination of Quantum Efficiency with the Model 210 Spectro." G . K. Turner Associates, Palo Alto, Calif., Feb. 1966 C. i . Miles and G . H . Schenk, Anal. Chem., 42,656 (1970). W H Melhuish. J. Phys. Chem., 65, 229 (1961).
Table I. Corrected Fluorescence Data and Uncorrected Phosphorescence Data for Antihistamines and Model Compounds Antihistamine and model compound
la. D i p h e n y l m e t h a n e lb. Diphenhydramine HC1 IC. Toluene 2a. p-Chlorotoluene 2b. Chlorcyclizine H C 1 3a. p-Bromotoluene 3b. Rromodiphenhydram i n e . HCI 4a. I n d e n e 4b. P h e n i n d a m i n e tar-
Fluorescence, pH 2 (0.01M acid) A,,,
brr,
Phosphorescence, ethanol X e x , Xem
260, 28Za
ca. 254,h 311
258, 285 261, 284 270, 293a 265, 292 270, 3090
237, 375 ca. 260, 346 270, 430 240, 407 270, 400
None" . . ., ca. 310 260, 313
ca. 254,h, 413 . . ., ca. 430 253, (290), 480
240, 315 265, 291
240, (265), 430 238, 410
trate 6. 6.
Pyrrobutamine phosphate Meclizine. H C 1
T h e solvent is absolute ethanol. The phosphorescence emission was obtained with the 254-nm mercury line, but 254 nm is not necessarily the excitation maximum. Photodecomposition yields fluorescence emissions bands at 350 and 400 nm.
then diluted with the appropriate solvent. These solutions were then excited at 254 nm or at the appropriate excitation wavelength (Table I) in a fluorometer or phosphorimeter. The intensity of emission was read at the appropriate wavelength (Table I) if the spectrometer was used or with an appropriate secondary filter if a filter fluorometer was used. Although chlorocyclizine hydrochloride and meclizine hydrochloride were slightly sensitive to ultraviolet radiation, calibration curves could be obtained as long as the exposure to the excitation source was minimal. All fluorescence measurements on a particular instrument were made using the same quartz cuvette. The cuvette was usually emptied with a suction device and rinsed with solvent between samples. For utmost precision, 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 flask. The Dewar flask was then placed in the instrument and partially covered. After activating the microphotometer, the Dewar flask was rotated until the maximum reading was obtained. The Dewar flask was then covered and a final reading was taken. Readings taken in this manner had a relative standard deviation of lessthan 5%. Changes of &20% in the velocity of rotation of the phosphoroscope had no effect on phosphorescence intensity readings. Fluorometric titration curves were plotted to ascertain how fluorescence intensity changes as the pH changes. 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 and the pH was measured continuously. After an initial reading. the solution was adjusted to a p H above 7' by adding a few drops of 0.lA' sodium hydroxide. The acidity of the solution was then increased by adding the minimum volume of sulfuric acid solutions of various concentrations. Samples were withdrawn and the fluorescence 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% so that fluorescence readings were not corrected for dilution. Analysis of Benadryl Injection. The procedure for assay of diphenhydramine hydrochloride in Benadryl injection (ParkeDavis) is as follows. Combine equal volumes from two vials of the 50 mg/ml injection to make a composite sample for analysis. Dilute a 1.00-ml aliquot of the composite sample to 100 ml with 0.01N sulfuric acid. Further dilute 5 ml of this solution to 100 ml with 0.01N sulfuric acid to lower the fluorescence readings to a linear range. Analyze this solution by exciting a t 254 nm in the MLS spectrometer (or at 258 n m in a spectrofluorometer) and measure the fluorescence at 285 nm. If it is desired to use a filter fluorometer, it is possible to use a 254-nm mercury line interference filter as a primary filter, and a 313-nm mercury line interference filter as a secondary filter. (The emission band of diphenhydramine hydrochloride extends to almost 350 nm.) Use the same procedure for 10 mg/ml injections but with half as much dilution. A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 7 , J U N E 1974
897
Table 11. Relative Fluorescence Q u a n t u m Efficiencies in Air and Detection L i m i t s of A n t i h i s t a m i n e s Model compound or antihistamine
+J us. quinine sulfate-
la. Diphenylmethane l b . Diphenhydramine
f(Xma,l,
0 . 16c
490 (260)
drochloride
0.011
340 480 830 480
3a. p-Bromotoluene 3b. Bromodiphenhydramine hydrochloride 4. Phenindamine tartrate 5 . Pyrrobutamine phosphate
0.003
Light sens. 0.003
(258) (270) (265) (269)
Photodec.
...
0,844 0.002
9 , 2 0 0 (264) 1 4 , 2 0 0 (240)
det. lim.b
...
79
hy-
2a. p-Chlorotoluene 2b. Chlorocyclizine hydro-
Phos.
Fluor. det. lim. lex = 254b
nm)
3.7 1.4
4
...
x 10-*M
3
x 10-7~
x 10-7~
2
x
...
...
2
1.4
.
.
I
...
... 400 28
2 2
x 10-9~ x 10-7~
...
10-7~
...
4
x
2
x 10-6M
10-7~
No emis.
The solvent used was an aqueous 0.1N sulfuric acid. Dissolved oxygen was not removed; hence, only one figure is significant. Detection limits were determined on the MLS spectrosphorimeter (the rotating can was not used for the fluorescence measurements) using 254-nm excitation and a 5-mm emission monochromator slit width. I n cyclohexane solvent (I.B. Berlman, “Handbook of Fluorescence Spectra of Aromatic Molecules,” 1st ed., Academic Press, New York, N.Y., 1965).
10 F
P
F
t
‘.
‘..’
250
300
350 WAVELENGTH,
400
450
NM
Figure 1. Left: Corrected fluorescence excitation spectra of simin ilar concentrations of diphenhydramine hydrochloride (-) 0.01N sulfuric acid and diphenylmethane (. - ) in ethanol, emission being read at 285 n m for the former and 282 for the latter. Center: Corrected fluorescence emission spectra ( F ) for the same solutions, diphenhydramine being excited at 258 n m and diphenylmethane being excited at 260 n m . Right: Uncorrected phosphorescence emission spectra ( P ) of similar concentrations of diphenhydramine hydrochloride (- - -) and diphenylmethane (- both in ethanol glass and both excited at 254 n m in the MLS spectrometer. The three curves for diphenylmethane have been displaced downward for clarity s),
Compare the fluorescence of the composite sample with that from a standard prepared as follows. Weigh 12.5 mg of USP or NF reference standard diphenhydramine hydrochloride into 500 ml of 0.01N sulfuric acid, or otherwise prepare an equivalent solution. Use this standard for assay of both 50 mg/ml and 10 mg/ml injections. Analyze by exciting the standard at 254 nm (or 258 nm) and measure intensity of the fluorescence, F, at 285 nm. Compare the F values of the two standards with the F values of the samples to calculate the diphenhydramine content. RESULTS AND DISCUSSION Antihistamine Luminescence. Table I lists the antihistamines and model compounds investigated in this study. The excitation and emission maxima for both fluorescence and phosphorescence are reported. All fluorescence data are corrected in that they were measured on the Turner “Spectro” 210 absolute spectrofluorometer. The antihistamine fluorescence spectra were recorded a t pH 2 where there is little variation in fluorescence with a change in PH. 898
ANALYTICAL CHEMISTRY. VOL. 46, NO. 7, J U N E 1974
300
350 400 WAVELENGTH, NM
450
‘.‘. ‘. ‘. .‘ ‘. ’.
’. ‘.
\.
500
Figure 2. Emission spectra of chlorcyclizine hydrochloride. Excitation wavelength, 254 n m ; fluorescence emission, before irradiation (- -): phosphorescence emission, before irradiation (. . . ) ; fluorescence emission, after irradiation ( - - - ) ; phosphorescence emission, after irradiation (- - ) ; solvent for fluorescence, 0.1N sulfuric acid; solvent for phosphorescence, ethanol
.
In the case of the antihistamines having the diphenylmethane chromophore ( l b , 2b, 3b, 6), there is a distinct similarity among the fluorescence spectra of three of the antihistamines ( l b , 2b, 6) and among the phosphorescence spectra of all four compounds. Bromodiphenhydramine hydrochloride does not exhibit any measurable fluorescence until it has been exposed to ultraviolet irradiation; the fluorescence bands that result are distinctly different than those of the other three (footnote, Table I). It is interesting that Udenfriend et al. (17) did not observe fluorescence of up to 10 ppm ( 5 x W 5 M )diphenhydramine in acid or base. Evidently our 254-nm mercury line source is a much more effective means of excitation (Table 11) than the xenon source used by Udenfriend (17). In Figure 1, the fluorescence excitation and emission spectra of diphenhydramine hydrochloride ( l b ) and diphenylmethane ( l a ) are shown together. It appears that the “diphenylmethane” chromophore is the photoactive chromophore rather than, say, benzene, which has a more complex excitation spectra and an emission maxima a t 278 nm. Although there is not much difference in the fluorescence spectra of toluene and diphenhydramine, the phosphorescence emission spectra of freshly prepared di(17) S. Undenfriend, D. E. Duggan, B. M. Vasta. 6. B. Brodie, J. Pharm. Exp. Ther.. 120, 26 (1957)
Table 111. Analytical Effect of Heavy A t o m Ions on Phosphorescence and Fluorescence Intensities of Antihistamines Compounds Antihistamine-this study
(P/Pda 0.lM
Diphenhydramine . HCl Bromodiphenhydramine .HC1 Chlorcyclizine. HC1 Meclizine.HC1 Phenindamine tartrate
c1-
0.1M Br-
1.1 1.o 1.o 1.4 1.o
(F/F~)~
-
O.1MI-
0.1M c1-
-
0 . 1 M Br-
1.2 1.1 1.o 1.3 1.3
1.4 1.o 1.o 1.3 1.8
1.o
1.o
0.91
0.75 1.3 1.5
1.o 1.2
1.o 0.99 1.oo
0.99 0.98 0.99
... ... ...
0.93
...
... ...
Aminopyridine antihistamine ( I )
0.30
Pyrilamine maleate Methapyrilene HC1 Thenyldiamine . HC1
1.2 0.25
Other'
3-Indoleacetic acid
2
10%
c1-
x
101
10% Br-
1
x
10'
2.2 107"I 2 x 101
...
a Ratio of phosphorescence intensity in halide salt/ethanol to phosphorescence intensity in ethanol. Ratio of fluorescence intensity in halide salt/O.OlM acid to fluorescence intensity in 0.01M acid. Calculated from Figure 3 of reference 17.
phenylhydramine is similar to that of diphenylmethane, but not to that of toluene. In contrast, the phosphorescence emission spectra (Figure 2) of freshly prepared chlorcyclizine (2b) and meclizine (6) are not similar to that of either diphenylmethane or p-chlorotoluene, even though both chromophores could be visualized as parts of these two antihistamines. Both the fluorescence and phosphorescence emission spectra of irradiated chlorcyclizine (2b) and meclizine (6) are quite different from the corresponding spectra of diphenylmethane, toluene, and p-chlorotoluene. As shown in Figure 2 for chlorcyclizine, there is a broadening and a definite shift to longer wavelengths of both the fluorescence and phosphorescence bands. In addition, there are two phosphorescence maxima which may indicate two different emitting chromophores. Finally, the intensities of both the fluorescence and phosphorescence emission bands do not decrease as would be expected after photodecomposition, but appear to increase somewhat. This is in definite contrast to the fluores- and phosphorescence bands of meclizine (not shown) whose intensities decrease markedly, as though photodecomposition had occurred. The meclizine fluorescence band shifts about the same as that of chlorcyclizine but its phosphorescence band does not shift nor does it split. The decrease in the fluorescence intensity of irradiated meclizine and the increase in intensity of irradiated chlorcyclizine are not rapid, so that quantitative analysis for these should be possible, assuming minimum exposure to radiation. A few comments should be made about phenindamine (4b) and pyrrobutamine ( 5 ) . The former may be thought of as having an indene or phenylindene chromophore. Its fluorescence and phosphorescence emission bands are at quite different wavelengths than those of indene (Table I, 4a), but the conjugated phenyl ring may be responsible. In any case, the major effect of this chromophore is felt in the molar absorptivity, quantum efficiency, and fluorescence detection limit (Table 11). Pyrrobutamine (5) may be viewed as having both a p chlorotoluene (2a) and a styrene chromophore. Since styrene fluoresces in the 290- to 365-nm region, it appears it is the emitting chromophore here. Quantum Efficiencies: Detection Limits. The relative quantum efficiencies of several of the antihistamines as measured on the Turner Spectro 210 in the presence of air are reported in Table 11. Because oxygen quenching can occur to varying degrees, only one significant figure is reported. (A second digit is given as a subscript only to indicate the trend.) We measured these values in air partly
because of our interest in how efficient fluorescence would be under actual analytical conditions. The quantum efficiencies are in the same range as those reported for antihistamines having the 2-aminopyridine chromophore ( I ) with the exception of phenindamine tartrate. The higher quantum efficiency for the latter is undoubtedly the result of the rigid conjugated 1-phenylindene chromophore. This contrasts with the unconjugated, less rigid diphenylmethane chromophore of diphenhydramine ( I b ) ; the quantum efficiency of the latter is only one fourth that of phenindamine. In general, the fluorescence detection limits for the four antihistamines listed in Table I1 are fairly low and in the same range as those with the aminopyridine chromophore ( I ) . The relatively large tpf product of phenindamine tartrate (4b) compared to the other products is certainly responsible for the low detection limit. This is about 75% lower than any other reported antihistamine detection limit ( I ) . Because the fluorescence excitation bands of the antihistamines listed in Table I1 are near 260 nm, they will all be fairly efficiently excited by the 254-nm mercury line. The phosphorescence detection limits for the four antihistamines listed in Table I1 are poor when compared with their fluorescence detection limits or with the other reported antihistamine phosphorescence detection limits ( I ) . This may be the result of low phosphorescence quantum efficiencies as well as low molar absorptivities. Fluorometry is certainly the preferred trace analysis method except for bromodiphenhydramine. Effect of Heavy Atom Ions. The effect of heavy atom ions on the phosphorescence and fluorescence of five antihistamines is summarized in Table 111. Also given is the effect of these ions on aminopyridine antihistamines for comparison. Since Lukasiewicz, Mousa, and Winefordner (18) had reported the enhancement of phosphorescence of certain organic molecules by sodium halide salts, the main interest was centered on possible enhancement of phosphorescence. The degree of enhancement was calculated in a manner similar to that used by Hood and Winefordner (29) for ethyl iodide heavy atom studies. The first four antihistamines in Table I11 did not exhibit the significant enhancement reported for 3-indoleacetic acid or other molecules (18). In contrast, phenindamine tartrate did exhibit some enhancement in 0.1M iodide salt medium. Because of the large aromatic ring system, the excit(18) R. J. Lukasiewtcz, J. J. Mousa, and J D. Winefordner, Anal. Chern., 44,963 (1972). (19) L. V . S. Hood and J. D Winefordner, Anal. Chern., 38, 1922 (1966). A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 7, J U N E 1974
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Table IV. Fluorometric and Phosphorimetric Analytical Curve Ranges Analytical curve ranges, M Type of fluorometer Antihistamine
MLS spectrometer*
Diphenhydramine HC1 Bromodiphenhydramine .HCl Chlorcyclizine .HCl Meclizine .HC1 Phenindamine tartrate Pyrrobutamine phosphate
x
Filterb
...
... ...
10-7-10-4 10-7-10-4 2 X 10-9-10-5 2 x 10-7-10-5
5 X 10-6-4 X lo-’ 2 x 10-7-2 x 10-8 4 x 10-7-4 x 10-5 4 x 10-7-2 x 1 0 - 6
4 2 2
x x
10-5-10-4
Phosphorimeter, MLS spectrometer‘
x 10-7-10-3 x 10-7-10-3 2 x 10-7-10-3 2 x 10-7-10-4 2 x 10-6-10-4 3
4
...
Obtained in 0.1N sulfuric acid 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 a t pH 2 using the 254-nm mercury line with the Turner filter fluorometer, the 254-nm mercury interference filter, and the 7-60 narrow band pass secondary filter except that a 465-nm sharp cut secondary filter was used for phenindamine tartrate. Obtained in an absolute ethanol glass, using the 254-nm mercury line, the 254-nm mercury interference filter, and setting the emission monochromator a t the phosphorescence emission wavelength listed in Table I. The rotating can was run a t 2000-3000 rpm. a
Table V. Analysis of Benadryl Injection. for Diphenhydramine Hydrochloride. Excitation at 254 nm; Emission at 285 n m Orig. concn of diphen.b in sample
No. aliquots of sample analyzed
Mean concn of diphen. found
Std dev, mg/ml
1 2 . 7 5 mg/500
2
ml (std) 50 mg/ml
...
...
8
50.1
10 mg/ml
3
a
Parke-Davis pharmaceutical
9.99
* (“Diphen”
=
0.44 (0.9 % rel.) 0.096 (0.96% rel.)
Diphenhydramine HC1).
ed levels of phenindamine tartrate probably interact more with the iodide ion than the other antihistamines studied. In any case, the phosphorescence of the latter does not appear to be enhanced to the same degree as 3-indoleacetic acid. (The background of Figure 3 of reference 18 was used as a very conservative estimate of a reference point to calculate enhancement.) For comparison, some of the aminopyridine-type antihistamines studied earlier ( I ) were included in this study. The results of the phosphorescence measurements of the aminopyridine-type antihistamines indicate that hydrochloric acid solutions should be avoided as solvents in phosphorimetric studies. The bromide ion does quench the fluorescence of the antihistamine slightly; chloride appears to have little effect so that hydrochloric acid can be used as solvent for pH 2 solutions. The effect of acidity on the fluorescence of these antihistamines was also studied, but only chlocyclizine exhibited an appreciable decrease in fluorescence as the p H was increased. The inflection point for the fluorometric titration curve ( I ) of chlorcyclizine was a t pH 2.2. Its fluorescence appeared to remain a t a maximum at pH 1 and below. 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 as well as in the filter fluorometer. The useful range of the analytical curves for each of the antihistamines is listed in Table IV. The lower limits for all the analytical curves obtained on the MLS spectrometer are also the detection limits. These detection limits were calculated from the standard 900
A N A L Y T I C A L CHEMISTRY, VOL. 46,
NO. 7, JUNE 1974
deviation of the solvent blank and the slope of the analytical curve. The detection limit is defined as two times the standard deviation of the blank divided by the slope of the analytical curve (20). The luminescence detection limits in Table IV are applicable only t o the MLS spectrometer because they are dependent on the characteristics of this instrument (1). (With this instrument, the blank will be affected by the small transmittance of mercury lines above 254 nm through the 254-nm mercury line interference filter used as the excitation monochromator.) Even with these limitations, the detection limits are still useful for comparisons and to indicate what other instruments may achieve. 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 diphenhydramine the slope of the fluorescence analytical curve begins t o decrease just above 2 x 10-4M, but the upper limit is listed as 10-4M. The slope of the phosphorescence analytical curve for meclizine begins to decrease just above 4 x 10- 4M, but the upper limit is listed as 10- 4M. To evaluate the effect of substituting a filter for an emission grating, some analytical curves were obtained on a filter fluorometer (Table IV). For chlorcyclizine, much of the 285-nm band (Figure 2) is not transmitted by the second filter (peaks a t 360 nm) so that the filter fluorometer will not detect as low a level as the MLS spectrometer. In contrast, more of the 315-nm emission of pyrrobutamine is transmitted by this filter, and the lower limits of the two instruments are almost equal. Analysis of Pharmaceuticals Containing One Antihistamine, Because the antihistamines all have fairly similar luminescence properties, the most feasible analysis is that of pharmaceutical preparations containing just one antihistamine. (Intensely fluorescent phenindamine could probably be determined in the presence of the others.) Benadryl injection (Parke-Davis), which contains diphenhydramine hydrochloride, was chosen for analysis. Although the analysis could have been done on the Turner filter fluorometer, the MLS spectrometer was employed. (The 313-nm mercury line interference filter could have been used t o transmit the emission shown in Figure 1.) The results of the spectrometer analyses are shown in Table V; they agree closely with the stated concentration of the antihistamine on the label. Received for review October 9, 1973. Accepted January 7 , 1974. (20) J E. Barney, Talanta, 14, 1363 (1967)
