Fluorescence and phosphorescence of phenylethylamines and

Fluorescence and Phosphorescence of Phenylethylamines and Barbiturates Analysis of Amphetamine and Barbiturate Preparations Corbin I. Miles1 US.Food and Drug Administration, 1560 E. Jefferson Avenue, Detroit, Mich. 48207 George H. Schenk Department of Chemistry, Wayne State University, Detroit, Mich. 48202 The fluorescence and phosphorescence excitation spectra of seven phenylethylamines, ten barbiturates, and certain model compounds have been examined and correlated. 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 case of phenobarbital is unusual in that its phosphorescence emission maximum at 370 nm is at higher energies than its fluorescence emission maximum at 415 nm. It is shown that each emission arises from a different chromophore in phenobarbital. Fluorometric analysis was used to assay pharmaceutical preparations for single components and for amphetamine plus barbiturate. The results compare favorably with those obtained by conventional analyses.

THEBARBITURATES and the so-called phenylethylamines are two pharmacologically important classes of compounds whose luminescence properties have not been studied in detail. Such a study can provide the basis for analytical methods based on the native luminescence, rather than the chemicallyinduced luminescence, of these compounds. The so-called phenylethylamines comprise compounds having the following general structure:

The correct nomenclature for this general structure would thus in most cases be “1-amino-2-phenylethane.” Because the phenylethylamine label is so commonly used, we will employ it throughout. Included in the phenylethylamine class are such important compounds as amphetamine (R1 = R, = R 3 = R j = H ; R i = -CH3), and epinephrine (R1 = Ri = R3 = OH; Ri = H ; R 5 = CH3),andephedrine(R1 = Ra= H ; R3 = OH; R J R j = CHy). A study of the phenylethylamines should also shed light on the effect of substitution of the aminoethyl group o n the benzene ring. Benzene itself is weakly fluorescent (1) in solution (of = 0.04-0.07). The substitution of a methyl group o n benzene to give toluene results in a more intense ( I ) fluorescence (of = 0.17). The substitution of a n ethyl group on benzene also results ( I ) in intense fluorescence ( q f = 0.18). Such a study hopefully should indicate the possible influence of the amino substituent since this is the 1

Present address, U.S. Food and Drug Administration, Bureau of Foods, Petitions Processing. 200 C St., S.W., Washington, D.C. 20204. ( I ) C . E. White and R. J . Argauer. “Fluorescence Analysis.” Marcel Dekker, New York, N.Y., 1970. pp 186-7, 200-209. 130

major difference between ethylbenzene and the phenylethylamines. In the case of epinephrine, the situation is more complex because of the two hydroxyl substituents on the benzene ring. It appears that the luminescence of this compound will be dominated by the pyrocatechol moiety rather than by the phenylethylamino moiety. While numerous analytical fluorescence methods ( I , 2 ) are available for naturally occurring epinephrine and norepinephrine, scarcely any methods based on measurement of native luminescence have been reported for synthetic phenylethylamines. Some methods based on chemically-induced fluorescence have been devised for amphetamines (3, 4 ) and other P-phenylethylamines ( 5 ) . Barbiturates are derived from barbituric acid and vary as t o the nature of the R group substituted in the five position of these tautomers: H

H

oJNZo J+H 0

0

I

II

Tautomer I is the keto tautomer and tautomer I1 is the enol tautomer. A complete study of the fluorescence of the barbiturates was thought desirable because it would again reveal the effect of substitution on a very small conjugated a system. This a system is that found across carbons 2 , 3, and 4 of the anion of the enol tautomer. We have reported preliminary analytical fluorescence data only for most barbiturates (6), although analysis by direct fluorescence of amobarbital (7) and thiopental (8) in biological materials was reported previous to our work. Most of the analytical utility of chemically-induced or native fluorescence of barbiturates has been in TLC or paper chromatography (Y-11). (2) C. E. White and A. Weissler, ANAL.CHEM.. 42, 57R (1970). (3) J. T. Stewart and D. M. Lotte, J . P h r m . Sei., 60, 461 (1971). (4) W. Rusiecki and J. Brezezenski. Diss. Pliurm. Pl~onnucol.,19, 315 (1967). ( 5 ) N. Seiler and M. Weichmann. Hoppe-Segler’s 2. Pliysiol. Cliem., 337, 229 (1964). (6) C. I. Miles and G. H. Schenk, A m / . Lerr., 4, 71 (1971). (7) J. F. Swagzdis and T. L. Flanagan, A m / . Biocliem.. 7, 145 (1964). (8) P. G . Dayton, J. M. Pevel. M. A. Langran. L. Braend. and L. C. Mark, Biocliem. Phurmacol., 16, 2321 (1967). (9) B. Kaempe, Actrr Phurmaco/.. 23, 15 (1965). (10) J. Bogan, F. Rentoul, and H. Smith. Foremic Sei. Soc. J., 4, 147 (1964). (11) A. V, Gudaitis and R. M. Donaur. C/i/l. C l ~ e m . 13, , Abstr. No. 21 (1957).

ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973

The luminescence studies and analytical methods described below should be valuable t o pharmaceutical chemistry as well as to law enforcement since both phenylethylamines and barbiturates are important prescription items which have experienced considerable drug abuse. 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 initial 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 (12). The Aminco-Bowman spectrophotofluorometer with phosphorescence attachment and a 150-watt Xenon source was used to obtain the phosphorescence excitation and emission spectra. Quartz sample tubes (1-mm i.d.) were used, and when the rotating can was used, it was run a t medium speed (2000- to 3000-rpm range). Reagents. Analytical data for the phenylethylamines were obtained using 0.1N sulfuric acid. This acid was made by direct dilution of the concentrated acid and was stored in glass containers until used. The 0.1N sodium hydroxide used in the assay of barbiturates was prepared and stored in similar fashion. Phosphorescence spectra were determined in Gold Shield absolute ethanol containing sufficient concentrated sulfuric acid or potassium hydroxide pellets to produce 0.1N acid or base solution. The same ethanol solutions were also used for fluorometric determination of the phenylethylamines and barbiturates on the AmincoBowman spectrofluorometer. This was done to check for solvent or instrumental effects caused by the two sets of conditions. Phenylethylamine and barbiturate standards used were either USP or NF reference materials. When these purity standards were not available, analyzed laboratory drug standards of minimum 98.5 2 purity were used. Procedures. QUANTUM EFFICIENCY.The quantum efficiency of the fluorescence of phenylethylamines, barbiturates, and certain structurally related compounds was calculated using the method described by Turner (13) for the Model 210 “Spectro.” Quinine bisulfate and fluorescein were used as standards. Mean literature quantum efficiency values of = 517 nm) for fluorescein (14-16), 0.85 (Aex = 323 nm, ,,X, and 0.53 (Aex = 348 nm, Tern = 457) for quinine bisulfate (17, 18) were used. The calculation was made using the formula :

where the subscript u denotes the unknown and the subscript s denotes the standard o r reference values. Other symbols are defined as follows:

4 F A X

= = = =

Quantum efficiency of fluorescence Mode (sensitivity) setting Area of emission band Exciting wavelength

2) G. K . Turner, Science, 146, 153 (1964). 3) G. K. Turner, “Notes on the Determination of Quantum Efficiency With the Model 210 Spectro,” G. K. Turner Associates, Palo Alto, Calif. 94303, Feb., 1966. (14) G. Weber and F. W. Teale, Tram. Furuduy SOC.,53, 646 (1957). (15) C. A. Parker and W. R. Rees, Analyst (Lojidon), 85, 587 (1960). (16) L. S. Forster and R. Livingston, J . Chem. Phys., 20, 1315 ( 195 2). (17) W. H. Melhuish, N . Z .J . Sci. Techid., Sect. B , 37, 142 (1955). (18) W. H. Melhuish, J. Pliys. Cliem., 65, 229 (1961).

Table I. Corrected Fluorescence Data and Uncorrected Phosphorescence Data for Phenylethylamines Fluores- Range, Phosphorescence mg/lOO - cence Phenylethylamine ,A, ,X, ml A, hem If : I p b Amphet aminea 260 282 0.2-20 265 368 1:3 Desoxyephedrine 260 282 0.2-30 212 375 1:2 Ephedrine” 260 282 0.2-30 268 372 1:3 Phenylpropanolamine 260 282 0.2-30 268 375 1:2 Methoxyphenamine 274 296 10-3-0.5 280 386 3:l Phenylephrine 276 300 10-3-0.5 282 380 5:1 Epinephrine 284 313 10-3-0.5 285 425 25:l Measured as the bisulfate salt. Others measured as chloride salt. b Intensities of fluorescence and phosphorescence were both measured on the Aminco-Bowman SPF spectrofluorometer. The phosphorescence was measured in a 1-mm cell.

D

=

d =

Absorbance of a convenient, but not necessarily known concentration, measured o n the “Spectro” Dilution factor from absorbance measurement to fluorescence measurement

Absorbance values for approximately 10-4M reference solutions of quinine bisulfate and fluorescein were measured in aqueous 0.1N sulfuric acid and 0.1N sodium hydroxide, respectively. Concentrations for fluorescence measurement were precise dilutions of the 10-4Msolutions, and were generally lO-7M solutions of the reference materials. The unknown solutions were measured a t concentrations suitable for adequate absorbance, and were diluted t o represent absorbance values of less than 0.04 for fluorescence mode operation, Concentrations used for fluorescence measurement were also determined to be within the region of linear response for fluorescence, as discussed previously. Although quantum efficiency values of unknown us. quinine and fluorescein were in general agreement, some variation in fluorescence response of fluorescein was observed. For this reason, quantum efficiency values cs. quinine sulfate were considered most reliable and are subsequently used in quantum efficiency comparisons between compounds. Other precautions described by Turner (13) were also closely adhered to. Additionally, unknowns and quantum efficiency reference material solutions were measured a t identical mode or sensitivity settings. As much as was possible with established linearity data, dilutions were chosen so that the areas of fluorescence bands of the reference and compound whose is unknown would be approximately the same. This resulted in planimeter measurements of comparable accuracy. PHENYLETHYLAMINE PROCEDURE. To establish a calibration curve for a particular phenylethylamine, a stock solution having the same concentration, in mg/100 ml, as the upper limit of the linear range listed in Table I was prepared from the monochloride (or bisulfate) salt in 0.1N sulfuric acid. Appropriate aliquots of the stock solution were diluted with 0.1N sulfuric acid to cover the linear range listed in Table I. These solutions were then excited a t the appropriate excitation wavelength (Table I) in the Turner spectrofluorometer using a 3 x , 10 x , or 30 x mode (photomultiplier) sensitivity setting (30 X = highest sensitivity). Calibration curves were constructed by plotting fluorescence intensity cs. concentration in mg/100 rnl. The analysis of phenylethylamine tablets and capsules was performed by grinding a representative sample to a fine powder, weighing a portion of the powder equivalent to approximately 3 mg of phenylethylamine for most phenyethylamines, and placing the portion in a 100-ml volumetric flask. This portion was shaken with approximately 50 ml of 0.1N sulfuric


131

Table 11. Corrected Fluorescence Data and Uncorrected Phosphorescence Data for Barbiturates in Base Fluores- Range. Phosphorescence mg/i’&j cence Barbiturates“ A,, A,, ml A,, ,A, If:I$ Amobarbital 255 412 0.05-1.5 255 425 1oO:l Aprobarbital 255 412 0.02-1.2 NO phosphorescence Barbital 255 412 0.02-1.7 248 418 6:l Butabarbital 253 420 0.01-2 .o NO phosphorescence Cyclopentallyl 255 410 0.04-1.4 NO phosbarbituric acid phorescence Diallylbarbit uric 255 415 0.04-1.5 NO phosacid phorescence Pentobarbital 253 415 0.02-2.0 NO phosphorescence Phenobarbital 255 415 0.03-1 .O 262 370 1:lO 255 405 0.02-0.3 NO phosSecobarbital phorescence Thiopental 305 534 0.01-0.6 NO phosphorescence a Most were used in the molecular form, but some sodium salts had to be used. Intensities of fluorescence and phosphorescence were both measured on the Aminco-Bowman spectrofluorometer. The phosphorescence was measured in a 1-mm cell.

acid for 5 minutes (also heated on a steam bath for 5 minutes for problem samples) and the flask then brought to volume with 0.1N sulfuric acid. The solution was filtered and the filtrate examined directly on the Turner spectrofluorometer at the appropriate excitation wavelength (Table I). If necessary, the filtrate was diluted to provide a suitable concentration for linear response for the phenylethylamine. The concentration of phenylethylamine was determined from the calibration curve or by comparison of the fluorescence intensity of the sample with that of a standard solution of roughly the same concentration of the particular phenylethylamine involved. BARBITURATE PROCEDURE.To establish a calibration curve for a particular barbiturate, a stock solution having the same concentration as the upper limit of the linear range listed in Table I1 was prepared from the pure barbiturate, or sodium salt, in 0.1N sodium hydroxide. Appropriate aliquots of the stock solution were diluted with 0.1N sodium hydroxide to cover the linear range listed in Table 11. These solutions were then excited a t 255 nm, or at the wavelength listed in Table 11, in the Turner spectrofluorometer using a 3 X , lox, or 30X mode (photomultiplier) sensitivity setting (30 X = highest sensitivity). Calibration curves were constructed by plotting fluorescence intensity cs. concentration in mg/100 ml. Barbiturate tablets were analyzed by weighing a portion of the sample tablet equivalent to 10 mg of barbiturate into about 50 ml of 0.1N sodium hydroxide in a 100-ml volumetric flask. After five-minute shaking to dissolve the sample, the solution was diluted to 100 ml with 0.1N sodium hydroxide and filtered through a fast-tine porosity filler paper. Where necessary, the filtrate was diluted to achieve a concentration within the linear range listed in Table 11. The fluorescence was read on the Turner spectrofluorometer using the wavelengths listed in Table 11. The concentration was read from the calibration curve or was found by comparing the fluorescence intensity to that of a solution of the same barbiturate of approximately the same concentration. PHENYLETHYL~MINE-BARBITURATE MIXTUREPROCEDURES. Combination phenylethylamine-barbiturate capsules were analyzed using a sample containing at least six mg of phenylethylamine. The sample capsule was dissolved in O.1N sodium hydroxide as directed above for the barbiturates, 132

0

Table 111. Relative Quantum Efficiencies in Air of Phenylethylamines and Model Compounds - Relative $ j c fS.

CS.

Phenylethylamines tb Q.S. Fluor. Amphetamineo 361 0.01s 0.02 Desoxyephedrine 144 0.01r 0.02 Ephedrine” 285 0.01 0.014 Phenylpropanolamine 142 0.01 0.016 Methoxyphenamine 2080 0.12 0.16 Phenylephrine 2110 0.08 0.11 Epinephrine 3090 0.08 0.11 Model compounds (Aem) Benzene (282 nm) 0.012 Benzylamine (284 nm) 0.004 y-Phenylpropylamine (285 nm) 0.01, Catechol (309 nm) 0.21 0,001 N-Methylbenzylamine (280 nm) ri-Propylbenzene (282 nm) 0.02; a Determined as bisulfate salt. Others determined as monochloride salt. Molar absorptivities determined on “Spectro” 210 with IO-nm bandwidths at the fluorescence excitation wavelength. Dissolved oxygen was not removed for these quantum efficiency measurements; hence only one figure is significant. The solvent used for the phenylethylamines was 0.1N aqueous sulfuric acid; the solvent used for the model compounds was usually absolute ethanol.

except that 50 ml of the filtrate was reserved for the phenylethylamine analysis. The remaining portion was analyzed for barbiturates according to the barbiturate procedure above. The 50-ml portion of the filtrate was analyzed for phenylethylamines according to the following procedure: the 50ml portion was poured into a separatory funnel and acidified with approximately ten drops of concentrated sulfuric acid. The acidified solution was extracted with a single 25-ml portion of chloroform, the chloroform discarded, and the remaining aqueous layer diluted to an appropriate volume for linear response with 0.1N sulfuric acid. The fluorescence of the solution was measured according to the phenylethylamine procedure and compared with a standard solution of approximately the same concentration, for quantitation. RESULTS AND DISCUSSION

Phenylethylamine Luminescence. Table I lists the phcnylethylamines investigated in this study. The excitation and emission maxima for both fluorescence and phosphorescencc and the corresponding linear ranges of fluorescence response observed o n the “Spectro” 210 are reported. As seen in the table, many of the phenylethylamines exhibit similar fluorescence characteristics, being excited at 260 nm. The last three phenylethylamines are excited a t slightly longer wavelengths, although their spectra exhibit the same excitation and emission band forms as the others. This shift to longer wavelengths is the result of a substitution on the phenyl ring of one oxygen (phenylephrine and methoxyphenarnine) or two oxygens (epinephrine). The phosphorescence excitation data reported in Table I agree fairly closely with the fluorescence excitation data for each compound. There appears t o be more differentiation among the phosphorescence emission maxima than among the fluorescence emission maxima. However. the bands still overlap so that phosphorescence emission is only ~iseftilfor identification of pure compounds, aside from an analysis where only one phenylethylamine is present. For purposes of rough comparison, Table I also gives the approximate relative intensities of the fluorescence and phos-


>

5z W

k

tXClTATlON AND ’, ABSORPTION

Z I

Figure 1. Fluorescence (-) and phosphorescence (- - -) excitation (left) and emission (right) spectra of epinephrine (Ri = R? = R:j = OH; Rq = H , R , = CHI) Concentration is 7.8 mg per 100 ml of 0.1N sulfuric acid in Gold Shield absolute ethanol. Fluorometer meter multiplier settings: fluorescence,0.1 ; phosphorescence,0.01

phorescence maxima of the same solution measured on the Aminco-Bowman SPF. Although these relative intensity values have no significant impact related to analysis, it is significant that many compounds exhibit apparent enhanced sensitivity by one or the other luminescence technique. It is important to note that these relative intensities are measured using 10.0-mm fluorescence cells compared t o 1.0-mm phosphorescence cells and the rotating can. Thus, the ratio is important for practical analytical purposes but it does not necessarily give any true picture of the ratio of dr to q p . As a n illustration of the relationships of the fluorescence and phosphorescence spectra of phenylethylamines, the uncorrected excitation and emission spectra of epinephrine are given in Figure 1. The main fluorescence excitation and phosphorescence excitation bands overlap as d o the weak shoulders of these bands. The fluorescence emission band a t 313 nm is at higher energy than the broad phosphorescence emission band a t 425 nm. Quantum Efficiencies. The relative quantum efficiencies of the phenylethylamines and certain model compounds as measured o n the Turner Spectro 210 in the presence of air are reported in Table 111. 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 phenylethylamines 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.) The maximum effect of oxygen quenching is estimated a t less than 50% on the basis of a few measurements under nitrogen. Values for benzene and propylbenzene in the absence of oxygen in cyclohexane (19) a r e higher. The quantum efficiencies in the table are both lower but pJ for propylbenzene is still larger than that for benzene. We measured the quantum efficiencies in air partly because we were interested in the practical problem of how efficient fluorescence would be under actual analytical condi(19) I. R . Berlman. “Handbook of Fluorescence Spectra of Aromatic Molecules.” Academic Press, New York, N.Y., 1965, pp 42-55.

250

350

“.\ ..

450

WAVELENGTH, nm

550

Figure 2. Luminescence spectra of phenobarbital in 0.1N alcoholic sodium hydroxide (- fluorescence spectra; - phosphorescence speetra; .- absorption spectrum)

- -

tions-i.e., in air where oxygen quenching would occur. It is of course not intended that the values in Table I11 be used in the usual theoretical sense. From Table 111 it can be seen that the quantum efficiencies of amphetamine, desoxyephedrine, ephedrine, and phenylpropanolamine are very similar, and small relative to the other phenylethylamines. The quantum efficiencies in air of these molecules are closer to that of benzene rather than npropylbenzene even though these phenylethylamines have three-carbon side chains. It is apparent that oxygen quenching and/or intersystem crossing far outweigh fluorescence emission of these phenylethylamines, thus partly accounting for the higher fluorescence linearity ranges listed in Table I. Of course the lower molar absorptivities (Table 111) of these molecules also are responsible for the higher ranges as well. It is interesting to note that oxygen quenching and/or intersystem crossing reduce the fluorescence efficiency of amines such as benzylamine and N-methylbenzylamine far more than in the phenylethylamines. When the amino group is substituted on the second o r third carbon (as in y-phenylpropylamine) away from the ring, the molecule is apparently able to fluoresce somewhat more efficiently in the presence of oxygen. The last three phenylethylamines in Table I11 have much higher quantum efficiencies and molar absorptivities than the first four. It is obvious that both of these factors are responsible for the greater fluorescence emission and lower fluorescence linearity ranges listed in Table I. The higher quantum efficiencies of the molecules apparently reflect the favorable effect of oxygen substitution on the rate of fluorescence emission compared to the rates of oxygen quenching and intersystem crossing. It does appear that the aminocontaining side chain of epinephrine does somehow reduce its quantum efficiency. One might expect that epinephrine would have a q I closer to that for catechol (pyrocatechol) since it contains the catechol moiety. Instead, p, for epinephrine is less than half that of catechol. Barbiturate Luminescence. Table I1 lists the barbiturates investigated in this study. The excitation and emission maxima for both fluorescence and phosphorescence as well as the corresponding linear concentration ranges of fluo-


133

I

>

Table IV. Relative Quantum Efficiencies in Air of Barbiturates Relative + f b Barbiturate us. Q.S. us. Fluor. 6920 0.0034 0.0048 Amobarbital 6010 0.0020 0.0021 Aprobarbital 7100 0.0038 0.0054 Barbital 6920 0.0032 0.004e Butabarbital Cyclopentallyl barbituric 7290 0.001s 0.001, acid Diallylbarbituric acid 6180 0.0007 0.001o 7370 0.003, Pentobarbital 0.0044 7140 0.0011 0.0012 Phenobarbital 6480 0.0026 0.0026 Secobarbital 25,440 0.003o 0.0042 Thiopental a Molar absorptivities determined on “Spectro” 210 with 10-nm bandwidths. b Because dissolved oxygen was not removed for these quantum efficiency measurements, it is intended that only the first figure is significant. The second figure is intended only to show the trend and is thus written as a subscript. The solvent used was 0.1N aqueous sodium hydroxide. €6

Figure 3. Luminescence spectra of phenobarbital in 0.1N sulfuric acid (- fluorescence phosphorescence spectra) spectra;

-----

rescence response observed o n the Turner “Spectro” 210 are reported. With the exception of phenobarbital, the luminescence of all the compounds studied is consistent with established luminescence theory-Le., phosphorescence from the lowest energy triplet state occurs a t longer wavelengths than fluorescence from the lowest energy singlet state. Phenobarbital, however, is a n apparent violation of this theory and is worthy of further consideration. The phenobarbital absorption maximum, and fluorescence and phosphorescence excitation maxima (Figure 2 and Table II), are all observed near 255 nrn. The phosphorescence emission maximum occurs a t 370 nm whereas the fluorescence maximum is at 415 nm. If the probable structures of the emitting species in each case are considered, two hypotheses may be proposed t o resolve this anomaly. The following facts are relevant to both hypotheses. The phenobarbital structure (Figure 2) exhibits two unconjugated chromophores--i.e., the “enol” barbiturate chromophore and the benzene ring chromophore. Excitation of phenobarbital at 255 nm should result in excitation of the “enol” barbiturate chromophore, , A,( = 255 nm) and/or the benzene chromophore, ,A,( = 254 nm). In addition, the absolute excitation maxima of benzene is a t 255 nm, its fluorescence maximum is a t 282 nm, and its phosphorescence maximum is a t approximately 370 nm (close to the phosphorescence maximum observed for phenobarbital). The fluorescence of phenobarbital at 415 nm is additionally substantiated by the characteristic fluorescence of other barbiturate enol structures, without benzene substituents, as observed in Table 11. Finally, by preparing a n acidified solution of phenobarbital (0.1N H2S04 in absolute ethanol), it is observed that the fluorescence and phosphorescence spectra of fully protonated phenobarbital (Figure 3) coincide identically with the corresponding benzene spectra. One hypothesis consistent with the above facts is that the “enol” barbiturate chromophore fluoresces and the “keto” barbiturate chromophore phosphoresces. Since the “keto” tautomer is non-absorbing a t the excitation wavelengths (20), this requires that energy transfer from the excited benzene chromophore to the keto tautomer be postulated (21). This is likely as long as the thermally equilibrated lowest excited singlet partially involves the “keto” tautomer. The keto tautomer then undergoes intersystem crossing and phosphoresces. (20) W. B. Furman, J . Ass. OBc. Anal. Cliem., 51, 1111 (1968). (21) S. G. Schulman, University of Florida, Gainesville, Fla., personal communication, 1972. 134

A second hypothesis, which is appealing to us, is that the “enol” barbiturate chromophore fluoresces and the benzene chromophore phosphoresces. This is appealing simply because of the very close match between the phosphorescence spectra of fully protonated phenobarbital and benzene. Without a detailed solvent and/or p H study, it is not possible t o make a definite choice between these hypotheses at this time. In summary then, all of the barbiturates in Table I1 fluoresce only in alkaline solution when excited in the 250-nm region. This fluorescence must arise from excitation of the barbiturate “enol” anion, which must exist for significant absorption t o occur in this region (20). The only barbiturates not found to fluoresce in 0.1N base were mephobarbital (5-phenyl-5-ethyl-3-methylbarbituricacid) and metharbital (5,5-diethyl-3-methylbarbituric acid). These obviously cannot form the enol tautomer (tautomer 11) because neither has a proton on the nitrogen in the three position. One other interesting point is that the fluorescence bands of the barbiturates are at much lower energies than would be predicted from the location of the excitation bands (Table 11). It appears as though some of the excitation energy of the lowest excited singlet state is lost through a process such as molecular reorganization, before emission occurs. This point is under further study. Barbiturate Quantum Efficiencies. The relative quantum efficiencies of the barbiturates as measured on the Turner Spectro 210 in the presence of air are reported in Table IV. As for the phenylethylamines, oxygen was not excluded from any of the solutions so that oxygen quenching can occur in varying degrees. For this reason, only one significant figure is reported for the quantum efficiencies. (A second digit is given as a subscript only t o indicate the trend.) It is difficult to evaluate the degree of oxygen quenching since we were not able to discover any literature values of quantum efficiencies measured in the absence of oxygen. [No significant photodecomposition has been found for irradiation a t 250 nm (611. The quantum efficiencies were measured in air partly because we were interested in how efficient fluorescence would be under common analytical conditions--i.e., in air where oxygen quenching would occur. Because it was felt that most routine work would not involve removal of oxygen,


it was decided to obtain values which would have practical, rather than theoretical, use. From Table IV, it can be seen that the quantum efficiencies of all the barbiturates are all small, averaging 0.0025 and being less than 0.005 in all but one case. Oxygen quenching and/or intersystem crossing far outweigh the rate of fluorescence emission for all of these molecules. It is likely that the n, T * singlet state@)present because of the carbonyl function in each barbiturate dominates the fate of the excited barbiturate. Fortunately, the intensity of fluorescence emission also depends on the magnitude of the molar absorptivity as well as that of the quantum efficiency. Except for thiopental, the molar absorptivities of the barbiturates studied average about 6,800 cm-’M-l. The average product for the barbiturates is thus about 17 as compared t o a n average tpf of 3 for the first four phenylethylamines in Table 111. It is not surprising that the lower end of the linear concentration range for the barbiturates (Table 11) is about 1 X 10+M (0.02 mg’l00 ml) as compared t o the corresponding figure for the first four phenylethylamines (Table I) of 1 X 10-jM (0.2 mg/lOOml). Quantitative Analysis for Barbiturates and Amphetamine. Pharmaceutical preparations were analyzed for barbiturates and amphetamine using the procedures described in the Experimental section. The phenylethylamine procedure was used for the amphetamine preparations; the barbiturate procedure was used for the analysis of phenobarbital tablets. The mixture procedures were used for the analysis of the combination time-release capsules. The analytical results are given in Table V and are compared with results obtained by conventional assay. Only one analysis of a simple barbiturate preparation is included because a number of analyses of barbiturate preparations have been previously described (6). As shown in Table V, single ingredient amphetamine sulfate tablets were analyzed with good agreement between the USP and fluorescence methods. Although the fluorescence method does not give the complete identification desired for this compound, conventional methods with an ultraviolet spectrophotometric measurement step cannot provide complete identification either. The advantage of fluorescence analysis is its simplicity. This assay can be performed in less than one hour compared t o the USP and other methods which require four t o eight hours. For regulatory work, identification can be completed as described in the USP, by microchemical identification (22),or by other methods. Because it was more important to establish the accuracy of the fluorescence method, its precision was not evaluated a t length. However, the relative values of individual deviations between duplicate runs was of the order of = 1 so that a rough estimate of the relative precision could be stated as 1 %. The assay of the time-release capsules reported in Table V involved special problem samples. The fluorometric analysis of these products was performed with the same speed and simplicity as reported above for the single component preparations. However, the conventional methods required elaborate cleanup procedures before ultraviolet spectrophotometric determination could be performed. For instance, the USP XVIII assay for amphetamine (23) utilized column chromatography and extraction while the Rotondaro

Table V. Comparison of Fluorometric and Conventional Methods of Analysis for Amphetamines and Barbiturates Amphetamine or barbiturate found, Commercial tablet or mg/tablet capsule analyzed Fluorometric Conventional Amphetamine sulfate tabs., 4.80 4.W No. 1 (yellow) Amphetamine sulfate tabs., 4.78 4.80 No. 2 (yellow) Amphetamine sulfate time18.4, 17.4b 17.1, 17.7c release caps Phenobarbital tabs., No. 2 0.477,O.481 0.483 (green) Combination time-release caps. : 60.2d 60.6, 60.4 Amobarbital Amphetamine HCl 14.0, 14.1 13.1-14. 3e a USP XVIII method. b Lower results obtained after one chloroform extraction. XVIII method first and modified USP XVIII method second. Rotondaro method (24). e Range of results found by Rotondaro method, distillation, Soxhlet extraction, and other methods.

methods (24) involved liquid-liquid extraction for barbiturates and phenylethylamines. The steam distillation (25) and Soxhlet extraction required time-consuming steps, and all of these methods were plagued by incomplete recovery of phenylethylamines in time-release capsules. Whether fluorescence assay can always yield improved analytical results for this type problem sample cannot be definitely concluded here. It is encouraging to note however that fluorescence analysis provides comparatively higher assay results from amphetamine with apparent greater precision than the conventional methods which all involve ultraviolet spectrophotometric measurement. As noted in the procedure for mixtures, phenylethylarnines cannot be analyzed by direct fluorescence in the presence of large amounts of barbiturates. Although both exhibit characteristic fluorescence when dissolved in basic solution, the barbiturates, with their larger molar absorptivities, will absorb the exciting radiation preferentially so that the phenylethylamines will not be excited (inner filter effect). This situation requires a single extraction t o remove most of the interfering barbiturates present in common pharmaceutical ratios of these ingredients. The presence of phenylethylamine does not cause any apparent interference in the barbiturate fluorometric measurements when dilutions are made t o ranges of linear response for barbiturates.

x

(22) “Methods of Analqsis of AOAC.” 11th ed , Association of Official Anal) tical Chemists, Washington, D.C.. 1970. (23) United States Pharmacopeia. XVIlI ed.. p 179.

CONCLUSIONS

Fluorometric measurement of pharmaceutical preparations of phenylethylamines and barbiturates is observed t o have numerous advantages over conventional spectrophotometric methods preceded by a cleanup procedure. The absence of fluorescence interference in pharmaceutical solution matrices makes it possible t o quantitate these products without conventional cleanup procedures. The sensitivity of fluorescence is generally 10-1000 times greater than the spectrophotometric determination, and excitation and emission spectra generally provide better identification of compounds than does a single (24) F. A. Rotondaro, J. Ass. Oflc. Anal. Cliem., 41, 509 (1958). (25) R. W. McCullough, ibid.,52, 3 (1969).


135

ultraviolet spectrum. Although it may not always be superior, fluorescence assay may also be the preferred method for analysis of special problem dosages. In addition t o the factors mentioned above, the fluorescence intensities of the barbiturates may be increased considerably by using filter fluorometry with the intense 254-nm mercury line. In addition to the wider emission bandwidths possible, the more intense excitation eeergy corresponding to the wavelengths of excitation of the barbiturates, would considerably increase response. These same factors also make filter fluo-

rometry attractive for detection of the phenylethylamines, except that it may be more difficult t o isolate the 280-310 nm emission using a filter.

RECEIVED for review July 7, 1972. Accepted September 25, 1972. Presented a t the 19th Annual Detroit Anachem Conference, October, 1971, Dearborn, Mich. Taken in part from the Ph.D. Thesis submitted by C. I. M. t o Wayne State University, November, 1971.

On the Spectrophotometric Determination of Dissolved Silica in Natural Waters Kent A. Fanning and Michael E. Q. Pilson Graduate School of Oceanography, University of Rhode Island, Kingston, R.I. 02881 The precision and accuracy of methods for the determination of dissolved silica in natural waters can be greatly improved by taking account of the time courses of some of the reactions involved. The kinetics of these reactions are dependent on the concentrations of electrolytes present in the samples. Based on results from a metol-sulfite reduction method, the molar absorptivity of a reduced mixture of the 0-and 8-isomers of molybdosilicic acid in sea water is apparently affected only by the ionic strength but not by the nature of the component salts of the solution.

DISSOLVED SILICA in natural waters is usually determined from the absorbance of colored species of rnolybdosilicic acid (H4SiMoi?040), and all methods for the analysis involve the synthesis of a n unreduced form of this heteropoly acid-at least as a first step. Under the reaction conditions usually used, two isomers of the unreduced acid can exist: a-molybdosilicic acid which is the principal product in solutions acidified with up to 1.5 equivalents H+ per gram-atom of Mo and 8-niolybdosilicic acid which predominates when 2 or more equivalents H+ are added per gram-atom of Mo (1-3). Many analytical methods have been devised in which unreduced or reduced forms of one or both isomers are used ( e x . , 4-12). In solution, the unreduced p-isomer decays rapidly to the unreduced a-isomer (10% per hour initially, a t room temperature), and the decay rate is increased by dissolved electrolytes ( 2 ) . Morrison and Wilson (13) found that the decay rate of

the absorbance of unreduced P-molybdosilicic acid at 430 nm was increased 20% in 0.34M NaCl. Strickland (3) reported that this decay was catalyzed in a solution of N a S O , at the same rate as in a solution of (“&SO, of the same molarity. I n contrast to the unreduced isomer, the reduced p-isomer appears to decay very slowly under the reaction conditions usually employed (8). Thus, for practical purposes, reduction with any one of many reducing reagents may be considered to stop the decay. Unreduced P-molybdosilicic acid has a greater absorbance than unreduced a-rnolybdosilicic acid from 335-500 nm, and, between 800 and 850 nm, reduced 6-niolybdosilicic acid has a n absorbance peak which is greater than any absorbance peak of the reduced a-isomer (2, 10). Thus many analytical methods use conditions favoring the p-isomer to increase sensitivity and are affected by the decay of its unreduced form. Methods which use the unreduced p-isomer are affected from the time that the sample and the molybdate reagent are mixed until the absorbance is measured. Most methods which use the reduced p-isomer are affected only during the time that its unreduced form exists. This paper presents a n evaluation of the eRects of dissolved electrolytes and of timing on “unreduced” and “reduced” methods for dissolved silica and provides an improved version of the reduced procedure given by Strickland and Parsons (14). EXPERIMENTAL

(1) J. D. H. Strickland, J . Amer. Chem. Soc., 74, 862 (1952). (2) Ibid., p 868. (3) Ibid., p 872. (4) F. Dienert and F. Wandenbulcke, C. R. Acud. Sci., 166, 1478 ( 192 3). ( 5 ) R. J. Robinson and H. J. Spoor. IND. ENG. CHEM., ANAL.ED., 8, 455 (1936). (6) R. .1. Robinson and T. G. Thompson, J. Mar. Res., 1,118 (1946). ( 7 ) D. T. Chow and R. J. Robinson, ANAL.CHEM., 25, 646 (1953). (8) J. B. Mullin and J. P. Riley, AIINI.Chirn. .4ctu, 12, 162 (1955). (9) J. R. Morrison and A . L. Wilson, A / l d y s t (Londou), 88, 100 ( 1 963). (10) K. Grasshoff, Deep-Sei, Res., 11,597 (1964). (1 1) A. L. Wilson. ,411ulysr(Lotido~i), 90, 270 (1965). ( 1 2) D. R. Schink. AKAL.CHEM., 37,764 (1965). (13) J. R . Morrison and A. L. Wilson, A d y s f (London), 88, 88 (1963).

136

Apparatus. All measurements of absorbance were made with a Beckman DU spectrophotometer using 1-cm cells. Standards. Concentrated silica standards (20mM S O , ) were prepared by fusing 0.6008 gram of pure SiO? with 4 grams of sodium carbonate and dissolving the pellet in 500 milliliters of silica-free deionized water. Sodium hydroxide pellets were added (3 to each 500 milliliters) to guarantee stability. Sodium hexafluorosilicate has been suggested as a standard (14), but was not used because it was found t o give a lower color yield than fused SiO,. Dilute working standards (0 pM to 200 p M SiOJ were prepared by diluting acidified (14) J. D. H. Strickland and T. R. Parsons, “A Practical Handbook of Sea Water Analysis,” BuN. Fish. R e , . Bd., C U I I ~ LNo. L 167, 1968, p 65.


Fluorescence and phosphorescence of phenylethylamines and

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