significant change of the limit of detection nor of the absolute phosphorescence signal can be definitely attributed to the influence of the solvent. The minor variations observed in the case of vitamin K3, extensively studied in six pure or mixed solvents, could be due to changes in the signal level of phosphorescence related to the differences in the matrix structure. In effect, the matrix structure is varying in our studies from a clear, rigid glass (ethanol) to a cracked glass (methanol) and a snow (hexane, methanol-water mixtures). The possible application of the phosphorimetric method reported in this paper for the determination of the K vitamins can be compared with previously used analytical procedures. That the phosphorimetric method compares quite favorably with different analytical techniques for the quantitative determination of vitamins KI and K3 is shown by the data in Table 111, (up until now, no literature data were available for quantitative determination of vitamin K 5 ; however, qualitative studies of vitamin K, were performed by polarography (2, 33)). For vitamin K1, while colorimetric and chromatographic methods result in limits of detection no smaller than 25 pg/ml, the present phosphorimetric method results in a value of 1 pgiml. Similarly, the phosphorimetric limit of detection of vitamin K; is 0.07 pg/ml; whereas, only 2 pg/ml can be detected by colorimetry. Also, the phosphorimetric analytical curves are linear over about three orders of magnitude in concentration, while linearity for other methods is generally over a range of concentration of less than 2 orders of magnitude. Finally, the absolute minimal detectable amounts of vitamins K 3 and K1 determined by phosphorimetry are as low as 2 and 20 ng, respectively, which compare well with the corresponding values obtained by chromatographic methods (respectively, 4 and 500 ng). The reduction of the size of the sample used by means of the capillary tube (31) and the improvement of the instrumental sensitivity (32) are major reasons for recom-
Io-+
-J
a z 2
lo+-
v)
W
V
z w
10-10-
V v)
W
a:
9a
10-11-
v)
0
I
a
I-
IO+
I
10-1
IO
CONCENTRATION
IO=
103
(pg/ml)
Figure 7. Analytical curves for vitamin K3 in methanol/water vjv l0/90 ( A ) , methanol/water vjv 20/80 (B), and methanoljwater v/v 30/70 ( C ) , and for Vitamin K5 in methanol/water v/v l0/90 (D), and methanol/water v,’v 20180 (E), at 77 “K; Phosphorescence signal, in A, us. concentration, in jg/ml. The ordinate of curve ( A ) was lowered by 0.3 logarithm unit to clarify the representation mending the quantitative determination of the K vitamins by phosphorimetry. RECEIVED for review March 16, 1972. Accepted June 15, 1972. Research was carried out as a part of a study on the phosphorimetric analysis of drugs in blood and urine, supported by U.S. Public Health Grant No. GM-I 1373-09.
Analytical Study of the Phosphorescence of Purines in Aqueous Solution at 77 O K J. J. Aaron’ and J. D. Winefordner* Department of Chemistry, Uniaersity of Florida, Gainesaille, Fla. 32601 Phosphorescence excitation and emission spectra, lifetimes, and phosphorimetric analytical curves and limits of detection have been determined at 77 O K in methanol/water solution for purine and 8 of its derivatives. The substituted purines studied include amino, methyl, rnethylmercapto, benzylamino, chloro, and bromo substituents in either the 6- or the 2,6-positions on the pyrimidine ring. Fine structure of phosphorescence spectra is valuable for the identification of purine derivatives. Analytical curves are linear over large concentrations ranges (lo3 to 10; concentration units). Because of the high phosphorescence yields of purines, very low limits of detection between 0.1 and 0.0002pg/rnl are obtained. Absolute limiting detectable quantities of purine and the 8 derivatives are in the picogram range (3 x 10-9-4 x gram).
PURINE DERIVATIVES are currently the object of great biochemical interest, primarily because of the fundamental imOn leave from Laboratoire de Chimie Organique Physique, Paris, France. * Author to whom reprint requests should be sent.
portance of adenine and guanine in the biochemistry of naturally-occurring nucleic acids, and also because of the use of sulfur-containing purines in the chemotherapy of various types of cancer. To better understand the photochemical processes of nucleic acids, the investigation of the excited states of their constituents is of interest. Extensive studies have been undertaken on the fluorescence and phosphorescence of purine, adenine, and other derivatives ( I ) and concerned mainly with identification of the electronic transitions involved in lowest excited singlet and triplet states (2-4, solvent and temperature effects on the emission character~~
~~
(1) S . Udenfriend, “Fluorescence Assay in Biology and Medicine,” Vol. 11, p 361, Academic Press, New York, N. Y., 1969. (2) J. Drobnik and L. Augenstein, Photochem. Pltotobiol., 5, 13 ( 1966). (3) Zbid., p 83. (4) B. J. Cohen and L. Goodman, J . Amer. Cltem. Soc., 87, 5487 ( 1965).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
2127
Figure 1. Phosphorescence emission spectra of adenine at 77 "K
6-substituted purines is not well-resolved in water containing glasses (2, 3), while others obtained apparently good resolution of the vibrational structure of phosphorescence spectra for adenine (6-aminopurine) and its derivatives in ethyleneglycol/water 50/50 matrices (6). This dispute is of importance not only from a spectrometric point of view, but also from an analytical point of view as far as the identification of the purine derivatives may be helped by the resolution of fine structure of the phosphorescence spectra. Another analytical application of the phosphorescence of substituted purines would be their quantitative determination by phosphorimetry, as very few methods of quantitative analysis are available for these compounds (7-9). In this laboratory, considerable instrumental improvements of phosphorimetry have permitted minimization of the problems encountered in the previous phosphorescence analytical studies of biologically-important compounds in aqueous, snowed, rigid media (10-12). Therefore, in this paper, the phosphorescence characteristics for a series of purines in rigid aqueous solution are shown to be useful for their identification, and phosphorimetry is shown to be useful for the quantitative measurement of these compounds.
A . In ethylene glycol/water, v/v SO/SO (excitation wavelength 278 nm) B. In methanol/water, v/v, l0/90(excitation wavelength 278 nm)
EXPERIMENTAL
6
60
s
50-
iij
e W
c
40-
n
v)
p'
30
-
e
.-L
-
20-
0
a 10
-
0 300
350
400
450
500
Wavelength
0 x)O
350
550
600
650
700
750
(nrn)
I
I
I
I
I
1
L
I
400
450
500
550
600
650
700
750
Wavelength
(nm)
Figure 2. Phosphorescence emission spectra of 6-methylmercaptopurine (curve A , excitation wavelength 291 nm) and 2-aminomethylmercaptopurine(curve B, excitation wavelength 321 nm) at 77 OK in methanol/water, v/v, l0/90 istics and quantum yields (2, 3, 5 ) and the pH effects on luminescence intensity and spectra (6). However, most of the studies of luminescence of purine derivatives at low temperature were performed in nonaqueous, polar or nonpolar, rigid glasses (2-4). There are some controversial results about the use of aqueous solutions-the medium for biological systems-as a rigid matrix for the study of the phosphorescence of purines; some authors found that the vibrational structure of phosphorescence bands of (5) J. W. Eastman and E. J. Rosa, Photochem. Photobiol., 7, 189
(1968). (6) J. W. Longworth, R. 0. Rahn, and R. G. Shulman, J. Chem. Phys., 45,2930 (1966), and reference therein. 2128
Apparatus. Phosphorescence spectra were obtained on a n Aminco-Bowman spectrophotofluorometer (SPF) with an Aminco-Keirs phosphorescence attachment (American Instrument Co., Silver Spring, Md.). All phosphorescence measurements were performed at 77 OK. A Harrison (Model 6268 A, Hewlett-Packard, Palo Alto, Calif.) constant current dc power supply was used to power the 150 W xenon arc lamp. The starter circuit for xenon lamp was described previously (13). Signals from a RCA-1P28 multiplier phototube (American Instruments Co.) powered with a Keithley Model 244 high voltage supply (Keithley Instruments, Cleveland, Ohio) were measured with a low-noise nanoammeter (14) and recorded on an X-Y recorder (No. 1620-827, American Instrument Co.). A rotating capillary tube, approximately 1-mm i.d. and 6-mm 0.d. made of synthetic, high purity, optical grade quartz (Quartz Scientific Inc., Eastlake, Ohio) was used as the sample cell (10). Excitation light was polarized to decrease the phosphorescence background of the quartz tube ( I O ) , by means of a thin-film, quartz plate UV transmitting polarizer (Polacoat, Inc., Cincinnati, Ohio) mounted in the excitation beam. Phosphorescence lifetimes were measured by recording the nanoammeter output as a function of time, after cornplete termination of the exciting radiation by a guillotinetype shutter. UV absorption spectra were measured at room temperature (298 OK) with a Beckman DB-G grating spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). Reagents. Purine, adenine (6-aminopurine), 6-chloropurine, 6-bromopurine, 6-methylpurine, 6-benzylaminopurine, (7) B. E. Bonnelycke, K. Dus,and S. L. Miller, Anal. Biochem., 27, 262 (1969). (8) J. M. Finkel, ibid., 21, 362 (1967). (9) S. P. Kelernens and E. T. Degens, Nature (London), 211, 857 (1966). (10)R. Lukasiewicz, P. Rozynes, L. B. Sanders, and J. D. Winefordner, ANAL.CHEM., 44,237 (1972). (11) R. J. Lukasiewicz, J. J. Mousa, and J. D. Winefordner, ibid., p 963. (12) J. J. Aaron and J. D. Winefordner, ibid., p 2122. (13) R. Zweidinger and J. D. Winefordner, ibid., 42, 639 (1970). (14) T. C . O'Haver and J. D. Winefordner, J. Chetn. Educ., 46, 241 (1969).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
6-methylmercaptopurine: 2-amino-6-methylmercaptopurine and 2,6-diaminopurine hemisulfate were purchased from Nutritional Biochemical Corp., Cleveland, Ohio, and used without further purification; their UV absorption spectra were found to be very close to the available literature data (15).
Solvents used were methanol (Matheson, Coleman and Bell, Manufacturing Chemists, Norwood, Ohio, “spectroquality” grade), ethylene glycol (Fisher Scientific Co., Fair Lawn, N.J.) and deionized water. Procedure. Stock solutions of purines (concentrations of about 10-3M) were prepared in 10 volume per cent of mixture methanol-water. Adenine and purine stock solutions were also prepared in pure water and in different mixtures of methanol-water (v/v, 1/99 through v/v, 40/60). Lower concentrations of purines were obtained by successive dilutions. The slit arrangement used for the high resolution of phosphorescence spectra was 4, 0.5, 0.5, 4, 0.5, 4 (in mm corresponding to about 2.5-nm spectral half-band pass). The slit arrangement used for the quantitative analytical studies was 4, 3, 3, 4, 3, 3 (in mm corresponding to about 14-nm spectral half-band pass). RESULTS AND DISCUSSION
Spectrometric Characteristics. Phosphorescence excitation and emission wavelengths and lifetimes of phosphorescence of purine and the substituted purines are reported in Table I. The phosphorescence spectra of all substituted purines obtained in methanol/water mixture (v/v, 10/90), have fine structure (see for example Figures 1 and 2); this demonstrates the usefulness of predominately aqueous snowed matrices (at 77 OK) for enhancing the vibrational fine structure of phosphorescence assuming optimal instrumental conditions are used, such as narrow spectral band pass and randomization of the frozen matrix heterogeneities. Our observations agree well with the conclusions of Longworth (6, 16) o n phosphorescence of adenine, of derivatives of adenine, and of native D N A . It is also of interest to notice that the data of Longworth ( 6 , 16) were obtained in an ethylene glycol/water 50/50 glass, while ours were obtained in a methanol/water (v/v, l0/90) snowed matrix. For direct comparison, the adenine phosphorescence spectrum was also obtained using a n ethylene glycol/water, v/v, 50/50 solution. As shown by Figure 1, there is a dramatic similarity between phosphorescence spectra of adenine in these two partially aqueous media, which indicates that the variation of the structure and composition of a n aqueous matrix has n o important effect o n the vibrational fine structure of phosphorescence. Phosphorescence lifetimes for purine, adenine, 6-methylpurine, 6-benzylaminopurine, and 2,6-diaminopurine, are greater than one second ; lifetimes, however, are shorter for the other derivatives (see Table I). For the former compounds, lifetimes longer than 1 second indicate that phosphorescence originates from a K,T* triplet state (18). As the latter purines include a bromo, chloro, o r methylmercapto substituent in the 6-positio11, it is probable that the marked decrease of phosphorescence lifetime is due to the internal heavy atom effect of bromine, chlorine, or sulfur which increases the probability of the singlet-triplet interSadtler Standard Spectra, Sadtler Research Laboratories, Philadelphia, Pa. (16) J. W. Longworth, Photochem. Photobiol., 8, 589 (1968). (17) P. I. Honnas and H. B. Steen, ibid., 11, 67 (1970). ( 1 8) R. S. Becker, “Theory and Interpretation of Fluorescence and Phosphorescence,” J. Wiley and Sons, New York, N.Y., 1969, (1 5)
p 155.
Table I.
Phosphorescence Properties of Substituted Purines in Methanol/Water, l0/90 v/v at 77 O K a Peak wavelengths, nmt Excitation Emission Lifetimes, sece Purine maximumc maximad 2.21 Purine 272 370,387,405,415 2.90 Adenine (6-amino278 368,377,386,396, 406,417,425 purine) 3.2 6-Methylpur ine 272 368,390,405,425 0.6 6-Methylmer291 398,410,420,435, captopurine 459 6-Benzylamino286 371,391, (407),413, 2.8 purine (423), (503) 0.64 6-Chloropurine 273 397,419, (434) 0.5” 6-Bromopurine 273 394,420, (434) 0.66 2-Amino-6-methyl321 440,456,461, (477) mercaptopurine 2.7 2,6-Diaminopurine 288 389,410,430, (445) All concentrations hpproximately IO-‘M. Spectra uncorrected for instrumental response. Peak wavelength error: s 2 nm. Phosphorescence spectra are recorded with this excitation wavelength. d Wavelengths of the main peaks are italic-wavelengths of shoulders are given in parentheses. e Precision of the lifetime values: 1 3 % . f Previous literature value was 2.1 sec in water matrix (3). 0 Previous literature values were 2.2 sec (6) and 2.5 sec (17) in ethylene-glycol!water 50/50 matrix. This value may be even smaller, due to the limiting factor of the response time of the recorder (0.5 sec). Table 11. Singlet-Triplet Splitting of Substituted Purines AbsorpPhospho- SI - TI, rescence energy tion maximum, maximum, difference, Purine cm-la cm-l cm-l 13100 Purine 37800 24700 13700 24600 6-Aminopurine 38300 13500 6-methyl purine 38200 24700 11500 34500 23000 6-Methylmercaptopurine 13000 24200 6-Benzylaminopurine 37200 13700 23900 6-Chloropurine 37600 13700 23800 6-Bromopurine 37500 2-Amino-6-methyl10300 32200 21900 mercaptopurine 10100 24400 34500 2,&Diaminopurine Measured in methanol/water. vjv, l0/90 at room temperature (298°K).
‘
system crossing transition. It is worthwhile to observe that sulfur-containing derivatives (like 6-methylmercaptopurine) and chlorine containing derivatives (like 6-ch1oropurine)compounds including sulfur and chlorine heavy atoms of nearly equal atomic number-have practically the same phosphorescence lifetimes. In the case of the bromo, chloro, and methylmercapto derivatives as well as for the other purines, the excited singlet-triplet energy differences calculated from the difference between the absorption and phosphorescence maxima, are around or greater than 10,000 cm-’ (Table 11), which are also typical of a x , ~ *phosphorescence (18). Our assignments confirm those made by several authors (2-4, 19, 20) from polarization studies, lifetimes measurements, and singlet-triplet separation studies for the phosphoresence of some purines. (19) V. Kleinwachter, J. Drobnik, and L. Augenstein, Pliotochem. Pliotobiol., 6 , 133 (1967). (20) J. Drobnik, V. Kleinwachter, and L. Augenstein, ibid., p 147.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
2129
u
I+'
lo"
Io-'
lo''
Concentration
I
IO'
I02
IO'
(pg/rnl)
Figure 5. Analytical curves in methanol/water, v/v, 10/90at77 O K (Phosphorescence signal, in A, cs. concentration in rg/ml)
o 3
Purine
CAminopurine
A 2,CDiaminopurine
IO-H
I
I
I
10
20
30
I 40
1 50
Methanol /Water
V/V%
Figure 3. Phosphorescence signal as function of solvent composition (methanol/water solutions) I: Purine (lO-4M) 0 Adenine (lO-'M)
lo." 10.4
10'~
I
I
I
I
IO-'
I
IO'
IO'
Concentration
0'
(pq/ml)
Figure 4. Analytical curves in methanol/water, v/v, l0/90 a t 77 OK (Phosphorescence signal, in A, cs. concentration in rg/rnl) 0 6-Methylmercaptopurine I
A
2-Amino-6-methylmercaptopurine
6-Chloropurine
Qualitative Analytical Applications. As is apparent from the data previously discussea, the fine structure of phosphorescence spectra obtained with narrow band pass is of interest for the identification and qualitative analysis of the substituted purines in aqueous medium. For example, adenine could be easily determined by its well-resolved phosphorescence spectrum (Figure l), after separation from the nucleotide in the 2130
acid hydrolyzate of nucleic acid. Also 6methylmercapto derivative could be differentiated from 2-amino-6-methylmercaptopurine by the wavelength shift and change in shape of the phosphorescence spectrum (see Table I and Figure 2). With the exception of 6-chloro- and 6-bromopurine which have very similar vibrational structure, all purines of the present study have phosphorescence emission spectra shifted or resolved sufficiently to be useful as a fingerprint of a n unknown purine sample. Phosphorescence lifetimes, which are quite different from one purine to another, as seen previously, might also be of value to confirm the structure identification. Matrix Effect Studies. To determine the solvent dependency of phosphorescence of purines in aqueous solutions a t 77 O K , the phosphorescence signal was measured at close intervals of solvent composition over the range from OjlOO t o 45/55, v/v, methanol/water. This quantitative study was necessary t o determine the optimal matrix composition for phosphorimetry of the series of compounds. The results obtained for purine and adenine are shown in Figure 3. Both compounds result in the same solvent dependency: in pure water, very little phosphorescence is observed and a region between 0/100 and 2/98, v/v, methanol/water is marked by a sharp increase in signal (1 to 2 orders of magnitude), followed by a less rapid increase, in a curvature region between about 2/98 to 5/95 v/v and by a plateau region between about 5/95 to 40/60 v/v. For solution, with more than 40/60, v/v, methanol/water, the phosphorescence signal decreases and becomes less reproducible, While the signal magnitude is changing with solvent composition, a visible change in the physical aspect of the matrix occurs from a cracked glass (pure water solution) to a frozen, snowed matrix (greater than 5/95, v/v, methanol/water solution). Because of the relatively high homogeneity of the snowed matrix and because of the constancy of phosphorescence signals for significant changes in solvent composition, a lOi90, v h , methanol/ water solution was chosen for the present phosphorimetric study of the substituted purines. A more detailed study of the nature of the matrix effect on the phosphorescence signal is reported in another paper (11). Quantitative Analytical Studies. All substituted purines have phosphorescence signals sufficiently intense to be useful
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
Table 111.
Phosphorescence Analytical Characteristics of Substituted Purinesa Concentration Slope of Linear Limit of linear Standard correlation detection, range ( M ) of near linearityb portion deviationc coefficient rg/mld 2 x 103 0.88 0.04 0.999 0.01 3 x 103 0.76 0.05 0.998 0.02 3 x 103 0.96 0.02 1.ooo 0.01 104 0.97 0.04 0.999 0.0006 2 x 103 0.98 0.02 1 ,000 0.02 104 0.91 0.07 0.999 0.0015 104 0.89 0.06 0.999 0.002
Minimal detectable amount, nge 0.2
Purine Purine 6-Aminopurine 0.4 6-Methylpurine 0.2 6-Methylmercaptopurine 0.012 6-Benzylaminopurinef 0.4 6-Chloropurine 0.03 6-Bromopurine 0.04 2-Amino-6rnethylmercaptopurinef 106 0.94 0.03 1 ,000 0.0002 0.004 2,6-Diaminopurine 103 0.93 0.08 0.998 0.15 3.0 a In neutral methanol/water, lO/W solution, v/v except otherwise noticed. * Near linearity means region over which slope of analytical curve is within 1 of the values designated in column 3. Standard deviation of phosphorescence signals taken on the linear portion of the analytical curve. Limit of detection is defined as the concentration giving a phosphorescence signal (located on the linear part of the analytical curve) two times greater than the background noise. The background signal was subtracted from the observed signal value or suppressed by means of the bias adjustment of the nanoammeter readout. e Absolute limiting quantity of compound detected by the method-calculated from the limit of detection with a volume of sample of 20 MI. f Analytical curves obtained in H2SOI0.1N MeOH/H?O l0/90 v/v solution.
for quantitative analysis. A preliminary study of the p H effect o n the magnitude of phosphorescence signals of purines indicates that neutral medium is satisfactory for all purines, with the exception of 6-bemylamino and 2-amino-6-methylmercapto derivatives which have greater signals in acidic medium (pH -2), and so analytical curves for the later two species were determined a t p H -2. The phosphorescence analytical characteristics of purine and several purine derivatives are reported in the Table 111. The linear portion of the analytical curves is accurately determined ; relative standard deviations of phosphorescence signals are around or less than 8 %, and the linear correlation coefficients are close to unity, which indicates good reproducibility. Slopes of the analytical curves are close to unity as expected, and the ranges of linearity are especially large ( l o 3 to lo9 concentration units). Limits of detection are particularly low, due to the high phosphorescence signals and low noise levels of most of the purines, and range between 0.0002 and 0.1 pgjml. Lowest concentrational limits of detection are given by mercapto, bromo, and chloro derivatives (see also Figure 4), which may be attributed to the influence of the internal heavy atoms, increasing the transition probability from the lowest excited singlet state to the lowest triplet state and therefore increasing the quantum yield of phosphorescence. On the other hand, introduction of amino substituents in positions 2 and 6 of purine decrease the phosphorescence yields, as shown by the higher limits of detection and lower phosphorescence signals observed for 6-amino and particularly for
2,6-diamino derivatives, compared to unsubstituted purine (Figure 5 ) . As a consequence of the small volume (about 20 pl), of sample required in the'capillary tube and of the high native phosphorescence intensity of purines, picogram quantities of these compounds can be measured by phosphorimetry (see Table 111). According to the nature of the ring substituents, limiting detectable quantities are between 3 x 10-9 and 4 x gram. It must be pointed out that analytical methods described until now for the determination of purines have principally stressed the separation of some purine derivatives from other biologically important molecules like pyrimidines and amino acids (7, 9). Limits of detection have been reported to be 0.01 pg/ml by a fluorometric method for the assay of 6-mercaptopurine in serum (8) and in the range of lo-' gram by a spectrophotometric method for adenine and other derivatives (7). These values of detection limits are larger than ours by at least one or two orders of magnitude. Therefore, because of the excellent precision, sensitivity, and broad range of linear response, phosphorimetry can be considered as the most versatile analytical method to date for the direct quantitative determination of purines.
RECEIVED for review May 1, 1972. Accepted July 10, 1972. Research was carried out as part of a study o n the phosphorimetric analysis of drugs in blood and urine supported by a U.S. Public Health Service Grant, GM-11373-09.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
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