Anal. Chem. 1980, 52, 558-561
558
Differential Pulse Polarographic Determination of Adenine, Cytosine, and Their Nucleosides Timothy E. Cummings Department of Chemistry, University of Miami, Coral Gables, Florida 33 124
James R. Fraser and Philip J. Elving" Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48 109
Instrumentation. A Princeton Applied Research Model 174 polarographic unit was used for potential control and current measurement; current-potential data were displayed on a Hewlett-Packard Model 7005B x-y recorder. Potentials were monitored with a Hewlett-Packard Model 3430A digital voltmeter. Experiments were run in a jacketed three-compartment cell, thermostated at 25 O C . All potentials are reported vs. the aqueous saturated calomel electrode (SCE). Procedures. A 15.0-mLaliquot of buffer solution was pipetted into the working electrode chamber; the counter and reference electrode compartments were filled with buffer to the same level as in the working chamber. The working chamber solution was deoxygenated for 15 min with purified nitrogen, which was kept flowing over the test solution during measurement. Polarograms were first run on each background solution. Sequential additions of millimolar stock solutions of the pyrimidine or purine species of interest were used to obtain calibration curve and test mixture data; for total addition of less than 50 pL, a Unimetric Model 1010 10-pL syringe was used; for additions of 50 pL or more, a B-D Tuberculin 1.00-mLall-glass syringe was used. A t each concentration, one to five replicate responses were obtained. For examination of the effects of pH, drop time, pulse amplitude and scan rate on adenine and cytosine response, the buffer (initially 0.50 M NaOAc and 0.36 M HOAc) was adjusted to pH 4.9 with HOAc. The pH was decreased by additions of HOAc to the working electrode chamber. For calibration curves, a 0.10 M XaOAc, pH 4.20 acetate buffer was used. Background Subtraction. All compounds studied shifted the background discharge t o more positive potential; the magnitude of this shift was dependent on both nature and concentration of the compound. To perform background subtraction, the blank response was shifted along the potential axis until background currents for both blank and test solution matched a t potentials negative of -1.55 V; i, was taken as the difference between total current at E , and the corresponding blank current after alignment. Statistical Analysis. A statistical analysis is included on calibration curve parameters and observed concentrations; uncertainties on slopes, intercepts, etc., are one standard deviation in each case. Since the calibration curve is of the form i, = b + me, the uncertainty in concentration is based on
The analytical utility of differential pulse polarography for the determination of adenine, cytosine, adenosine, and cytidine alone and in mixtures has been examined. Best resolution of the base compounds' peak potentials, E,, is in acetate buffer at pH 4.2. Linear peak current-concentration calibration curves were observed for all compounds. Detection limits are: adenine, 0.05 pM; adenosine, 0.04 pM; cytosine, 0.4 pM; cytidine, 0.1 pM. E , for adenine and adenosine (-1.31 and -1.34 V vs. SCE, respectively) preclude analysis of this mixture; mixtures of cytosine and cytidine ( E , = -1.44 and -1.32 V, respectively) can be analyzed. Results of analysis of mixtures of adenine and cytosine, and of cytosine and cytidine are reported.
Analytical determination of pyrimidines, purines, and their nucleosides and nucleotides is important in biochemistry and for assay purposes in commercial samples. Adenine, cytosine, and their nucleosides and nucleotides are essential components in DNA and RNA as well as in other biologically significant species, e.g., ATP. Although these compounds are frequently determined spectrophotometrically, determination a t the micromolar level is difficult, since the molar absorptivities are ca. I O 4 M-' cm-'. Polarographic and voltammetric determinations of pyrimidines and purines have been described (1-3); the detection limit for dc polarography usually is ca. or M, b u t may be higher under non-optimum conditions. Determination of adenine and other purines, based on the anodic peak current, i,, obtained by linear scan voltammetry at carbon electrodes, requires use of nonlinear calibration curves. T h e nonlinear dependence of i, on concentration, which has been observed for wax-impregnated carbon ( I ) , pyrolytic graphite ( 2 ) ,and the glassy carbon ( 3 ) electrodes, is postulated to be caused by adsorption on the carbon-based surface ( 2 , 3 ) . Detection limits for adenine a t carbon electrodes appear to be no better than that obtainable by dc polarography, which yields linear calibration curves and a reproducible surface area in contrast to the carbon electrodes. Since pulse polarography should improve sensitivity at trace level concentrations, the use of pulse polarographic techniques for determination of some adenine and cytosine species has been investigated. Because of reported instrumental artifacts with the employed instrumentation ( 4 ) ,differential pulse polarographic i, dependence on scan rate, drop time, and pulse amplitude were studied.
s Z C=
(1)
DETERMINATION OF OPTIMUM C O N D I T I O N S pH. T h e p H dependence of dc polarographic Id and E1,2 for adenine have been investigated ( 5 ) . In McIlvaine buffer, Idfor adenine is sensitive to p H between 4 to 5, whereas in acetate buffer over the same p H range it is relatively constant and nearly '70% larger than in McIlvaine. For these reasons, acetate buffer was chosen for the present study. Because cytosine's Iddecreases linearly with decreasing p H (6). the p H should be as high as possible for maximum sensitivity; however, with increasing pH, the cytosine wave shifts negatively ( 6 ) ,which increases the interference by background discharge. Additionally, since the difference in El for adenine
EXPERIMENTAL Chemicals. Cytosine (Calbiochem),cytidine (Calbiochem), adenine (P-L Biochemicals), and adenosine (Calbiochem) were satisfactory for use without further purification. The buffer; supporting electrol>te system was prepared from reagent grade sodium acetate and acetic acid. 0003-2700/80/0352-0558$01.00/0
+ s 2 , ( a c / a b ) * + s2,(ac/arn)2
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1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
-1.2
-1.5 POTENTIAL, V
-1.1
-1.3 POTENTIAL, V
559
-1.5
Figure 1. Effect of scan rate on differential pulse polarographic response of adenine and cytosine. Conditions: 10 p M adenine and 20 pM cytosine in pH 4.2 acetate buffer; 5-rnV pulse amplitude: I-s drop time. Scan-rates: (A) 1 mV/s, (B) 5 m V l s . The cytosine peak ( E P= -1.43 V) is buried in background discharge
Figure 2. Effect of pulse amplitude on differential pulse polarographic response of adenine. Conditions: 10 p M adenine in pH 4.2 acetate buffer: 0 5 s drop time; 1 mV/s scan rate. Pulse amplitudes and current axis sensitivities per major division: (A) 25 mV and 40 nA, (8) 50 m V and 100 nA, (C) 100 m V and 200 nA
and cytosine increases with decreasing pH, low p H affords better resolution of the adenine and cytosine peaks from each other in mixtures of the two compounds. For the aforementioned reasons, optimum analysis of solutions containing adenine, cytosine, or both should result a t low pH. T h e p H of a solution 0.1 m M in both adenine and cytosine was systematically lowered from p H 4.9. For a 2-s drop time and 10-mV pulse amplitude, resolution of the adenine peak from background discharge improved with decreasing p H to p H 4.2 but then decreased below p H 4.2. The cytosine peak is not completely resolved from background even a t p H 4.2. Over much of the p H range studied, both the adenine and cytosine peaks are buried in background near the region of very rapidly rising background. Because of the catalytic effects of these compounds on the background reduction and the peaks' location, accurate background subtraction at the higher p H values could not be effected except a t current sensitivities too low for precise i, evaluation. Any increase in the compounds' concentration induces additional catalytic shift of background toward more positive potential; hence, the p H dependence of the peak currents and positions could not be accurately determined, but are probably similar to the pH dependence of the dc polarographic Id and E , 1 2 ( 5 , 6). Based on the fact t h a t optimum resolution from background for adenine occurs a t p H 4.2 and t h a t the resolution between adenine and cytosine peaks improves with decreasing p H (cf. previous discussion). subsequent data were obtained in p H 4.2 acetate buffer. Drop Time. Because of the catalytic effect of pyrimidine and purine reduction products on hydrogen ion reduction, the shortest feasible drop time, t d ,is desirable for optimum resolution of analytical peaks from background; however, electronic noise and stirring effects induced by mechanical drop
dislodgment ( 7 , 8) result in a decreased signal-to-noise ratio as t d is decreased. T h e latter fact becomes significant for a 0 . 5 s td. Although resolution of the adenine peak from background is not a problem a t t d of 2 s, cyt.osine resolution from background is a problem; hence, a 1-s t d was subsequently used. Scan Rate. Although an increased scan rate, L', is analytically advantageous because of reduced analysis time, a 5-mV/s L' distorts the adenine peak, decreases resolution and reduces sensitivity (Figure 1). The valley between adenine peak and background is 7,5% of the peak-height a t 5 m V / s but only 54% at 1 mV/s; additionally, the peak-height itself a t 5 mV/s is only 80% of that a t I mV/s. T h e distortion and decreased sensitivity at the larger L' is instrumentally induced ( 4 ) . T h e large time constant of the sample-and-hold amplifier, ca. 100 ms, which samples for only 16 ms, prevents accurate response t o a large change in signal from drop to drop, as is observed with L' of 5 mV/s; hence, on the rising side of the peak, the ohserved signal is less than the true signal. On the decreasing side of the true peak, the observed signal continues to rise with each drop until it equals the true signal at a given potential, which will be the observed E,. Beyond the latter, the signal, again, cannot change as rapidly as the true signal and is, therefore, larger than the true signal. Because response for a 2-m'kr/s u was very similar t o that a t 1 mV/s, the former L' was used. P u l s e Amplitude. The influence of pulse amplitude, AE, on i, peak width a t half-height, Wl and E , has been discussed ( 4 , 9). The effect of 1 E on lidenine peak response is Although a.denine resolution from shown in Figure 2. background is sufficient at 1E of 25 to 100 mV, the broadened WIj2for 1E exceeding 25 mV interferes with the cytosine assay in adenine-cytosine mixtures. For 1E < 25 mV, W1,'* is
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
560
200
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1.6
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0 6 ADENINE, p M
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12
Calibration curve for adenine. Solid line (for both A and B) is a linear least-squares fit (cf. text) to data from 0.053 to 13.1 p M . (A) Concentrations below 2.2 pM, (B) entire concentration range Flgure 3.
virtually independent of .1E; hence, a 25-mV 1E was used in subsequent studies. ANALYTICAL RESULTS AND DISCUSSION A d e n i n e . At p H 4.2, E , for adenine is -1.305 V; W1 is 0.080 V. A 17-point calibration curve (Figure 3), generated over t h e concentration range of 0.053 t o 13.1 p M , was linear with a slope of 16.9 f 0.1 nA/pM and an intercept of 0.6 h 0.4 nA; the standard deviation of the f i t was 0.9 nA. At 0.053 p M , t h e mean for five replicate measurements was 1.52 nA with a standard deviation of 0.08 nA. T h e large standard deviation of the fit is partially due to t h e nature of t h e background subtraction, which requires translation of t h e blank response along the potential axis to correct for t h e catalytically induced shift in hydrogen ion response when adenine is present. An error of h5 mV in blank alignment results in a f0.4-nA error in measured adenine response. Additionally, peak-to-peak noise on both sample a n d background solutions was 0.4 nA. Because the background discharge potential a t very low adenine concentration is not shifted relative t o the blank, t h e signal's standard deviation near t h e detection limit is smaller than the 0.9-nA standard deviation of t h e calibration curve's fit. Assuming t h a t the detection limit signal's standard deviation is equal to t h a t of the intercept, Le., 0.4 nA, the detection limit signal for S / N = 2 is 1.4 nA (based on a noise level of one standard deviation) a n d the adenine detection limit is 0.05 pM. Cytosine. Figure 4 shows representative responses a t 3 and 20 pM cytosine concentration. Point-by-point background subtraction shows a peak (E, = -1.43 V), which returns to base line a t -1.55 V. Because of the large background current a t -1.55 V, a low sensitivity (50 nA/in.) is required for measurement of cytosine-containing solutions to permit blank alignment; this significantly increases the detection limit for cytosine, as compared to adenine. A 10-point calibration curve (1.04 to 15.4 pM) showed a slope and intercept of 5.01 f 0.05 n.4/pM and 0.0 f 0.4 nA.
-1.4 POTENTIAL,V
-1.6
Figure 4. Differential ~ pulse polarographic response of cytosine. Conditions: pH 4.2 acetate buffer; 25-mV pulse amplitude; 1-s drop time; 2-mV/s scan rate. Cytosine concentrations: (A) 0.0 pM, (B) 2.77 pM, (C) 20.4 pM. The arrow at -1.43 V on curve C indicates the point at which the peak current was measured
respectively; standard deviation of the fit was 0.70 nA. T h e standard deviation for five replicates a t 1.04 p M was 1.1 nA. This standard deviation corresponds t o a 0.03-in. signal on t h e recorder and is the minimum reasonable basis for calculating a detection limit for S / N = 2, since recorder repeatability is 0.01 in. and mechanical measurement uncertainty is a t least 0.01 in.; based on a 1.1-na standard deviation, t h e detection limit is 0.4 pM. Adenosine. At p H 4.2, E , for adenosine is -1.334 V. A 14-point calibration curve (0.049 t o 4.93 p M ) had a slope of 21.0 f 0.1 nA/pM and an intercept of 1.0 f 0.2 nA; standard deviation of t h e fit was 0.57 nA. T h e 25% larger slope, as compared to that of adenine, is partially explicable on the basis of the 10 7" difference in electrode area due t o different capillaries; it is probably also partially due to a difference in rate of carbanion protonation ( I O ) . It is reasonable to assume that t h e standard deviation of the adenosine signal near the detection limit is similar t o t h a t of adenine, Le., 0.4 nA; hence, the adenosine detection limit is estimated t o be 0.04 p M . Cytidine. At pH 4.2, E, for cytidine is -1.315 V. A 20-point calibration curve (0.138 t o 20.2 p M had a slope of 6.49 0.05 nA/pM and an intercept of -0.2 0.4 nA; standard deviation of the fit was 1.4 nA. At 6.85 pM, the standard deviation of five replicates was 0.38 nA. Since t h e cytidine peak is not buried in background discharge, its peak height uncertainty is less than that for cytosine. For a standard deviation of 0.4 nA, the detection limit is estimated t o be 0.12 p M . A n a l y s i s of M i x t u r e s . Because E , for adenine and adenosine differ by only 0.03 V, these compounds cannot be determined in the presence of each other except by the solution of simultaneous equations analogous to those used in spectrophotometric analysis of mixtures. T h e greater difference in E,, for t h e cytosine-cytidine pair, which permits analysis of cytosine-cytidine mixtures, is due to the closer proximity in cytidine t h a n in adenosine of the electronwithdrawing ribose group to the initial electroactive N=C site. T h e smaller effect for adenosine is counterbalanced by adsorption and association effects ( 1 1 ) . ( I ) A d e n i n e and C y t o s i n e . Table I shows t h e results of analysis of mixtures of adenine and cytosine below the 10-pM
*
*
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
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Table I. Analysis of Mixtures of Adenine and Cytosine by Differential Pulse Polarography solu-
tioii 1
2 3
4
5 6 7 8
concentration taken, p M adenine cytosine 0.27 1.07 1.07 1.07 4.55 4.55 8.80 8.80
1.04 1.04 2.08 3.10 3.10
5.17 5.17 7.23
concentration observed, p M n adenine cy tosine 0.42 (0.08) 0.94 ( 0 . 1 0 ) 1.25(0.12) 1.15 ( 0 . 1 1 ) 4.57 (0.35) 4.54 ( 0 . 3 5 ) 8.77 (0.66) 8.31 (0.63)
1.00(0.16) 1.00 (0.16) 2.40 (0.20) 3.00 ( 0 . 2 2 ) 2.60 (0.20) 5.20 ( 0 . 3 0 ) 5.69 ( 0 . 3 2 ) 6.99 ( 0 . 3 8 )
difference, p M adenine cytosine 0.15 -0.13 0.18
0.08 0.02 0.01
-0.03 0 49
difference, "Gb adenine cytosine 56 12
--0.04
-0.04 0.32 -0.10 -0.50 0.03 0.52 -0.24
17
4
4 5
7
3
0 0 0 6
16
mean: 12"
1 10
3 6
Values in parentheses represent uncertainty of one standard deviation, based o n standard deviations of calibration curve If the result at 0.27 p M adenine is omitted, the slope and intercept, and calibration curve fit. Based on amount taken. mean is 6%. Table 11. Analysis of Mixtures of Cytosine and Cytidine by Differential Pulse Polarography solu-
tion
concentration taken, p M cytosine cytidine
1 1 1
1.04 1.04 1.04
2 3 4 5 5
1.04
3.80 3.80
6.55 6.55
1.15 1.15 1.15 1.61 1.61 5.72 5.72 5.72
concentration observed, p M a cytosine cytidine 1.00 (0.16)
1.00(0.16) 1.00 (0.16) 1.00 ( 0 . 1 6 ) 3.80 ( 0 . 2 5 ) 4.20 (0.26) 6.29 ( 0 . 3 5 ) 6.49 ( 0 . 3 6 )
0.71 ( 0 . 2 3 ) 1 . 1 3 (0.24) 1 . 2 1 (0.24) 1.75 ( 0 . 2 5 ) 1.98 (0.26) 4.43 ( 0 . 3 3 ) 5.93 ( 0 . 4 0 ) 3.69 ( 0 . 3 9 )
difference, p.M cytosine cytidine
difference, % b cytosine cytidine
0.04
-0.44
4
0.04
0.02 0.06 0.14 0.37 -1.29 0.21 -0.03
4 4 4 0 11 4 1
0.04
0.04 0.00
0.40 0.26 0.06
mean:
4
38 2 5 9 23 23 4 1
13
Values in parentheses represent uncertainty of one standard deviation, based on standard deviations of calibration curve slope and intercept, and calibration curve fit. Based on amount taken. a
level. Observed concentrations are based on comparison of the measured i, with the previously discussed calibration curves. Uncertainties are based on the standard deviations of the appropriate calibration curve's slope, intercept, and fit. If the result a t 0.27-pM adenine is omitted, the mean adenine difference is 6%. (2)Cytosine a n d Cytidine. Table I1 shows the results for the analysis of mixtures of cytosine and cytidine below the 7-gM level. T h e cytosine peak slightly overlaps the cytidine peak, e.g., the current due to cytosine a t E, for cytidine is 7.0% of cytosine's i,; corrections for this interference, which is reflected in the Table I1 data, decrease the observed cytidine concentration by less than 0.2 p M in all cases except that for the highest cytosine concentration, for which the decrease is 0.35 p M . Intkrference by cytosine on adenine determination in mixtures of the two was not observed because adenine's E , is 0.01 V more positive than that of cytidine and because the adenine response is much larger than t h a t of either cytosine or cytidine. CONCLUSIONS Adenine and adenosine can be determined a t submicromolar concentrations by differential pulse polarography; linear calibration curves with detection limits of ca. 0.05 g M are obtained for both compounds. For cytosine and cytidine, linear calibration curves are obtained; however, due to the sloping base line, the shift in background and the lower sen-
sitivity compared to the purines, the detection limits are ca. 0.5 g M . Simultaneous determination of adenine and adenosine is not easily achievable a t the mercury electrode; hence, mixtures of these two compounds should be analyzed using a carbon electrode (1-3) when the concentrations are within the detectable range. T h e other common nucleic acid bases-guanine, thymine, and uracil-would not interfere in the procedures described, because of their nonfaradaic activity in the potential range involved ( I , I I ) . LITERATURE CITED (1) Smith, D. L.; Elving, P . J. Anal. Chem. 1962, 3 4 , 930-936. (2) Dryhurst, G. Talanta 1972, 19. 769-786. (3) Yao. T.: Wasa. T.: Musha. S. Bull. Chem. SOC. Jon. 1977. 50.
2917-2920 (4) Christie, J H , Osteryoung, J , Osteryoung, R A Anal Chem 1973, 45, -3in-3 . - - i. -5 . (5) Smith, D. L.; Elving, P. J. J . Am. Chern. SOC. 1962, 8 4 , 1412-1420. ( 6 ) Smith, D. L.; Elving, P. J. J . Am. Chem. SOC. 1962, 8 4 , 2741-2747. (7) Cummings, T. E.; Elving, P. J. Anal. Chem. 1978, 5 0 , 480-488. (8) Cummings, T. E.; Elving. P. J. Anal. Chem. 1978, 50, 1980-1988. (9) Parry, E. P.; Osteryoung, R. A . Anal. Chem. 1965, 37, 1634-1637. (10) Webb, J. W.; Janik. E.: Elving, P. J. J . A m . Chem. SOC. 1973, 9 5 , 8495-8505. (11) Elving, P. J. "Topics in Bioelectrochemistry and Bioenergetics", Vol. I, Milazzo, G., Ed.: John Wiley: London, 1976; pp 179-286.
for review August 13, 1979. Accepted November 28, 1979. The authors thank the National Science Foundation, which helped support the work described. RECEIVED
