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Anal. Chem. 1981, 53, 540-542 Beam
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Block diagram of the double-beam system.
ModUatfd Laser Earn
P A Signal
Composite PA Signal I
Time
Time
Comparison of PA signals wRh single beam (A, refereme; B, sample) and the double beam (C): sample, 4 X lo-’ mol/L Amaranth solution; reference, distilled water; laser power, 300 mW; wavelength of laser, 514.5 nm; modulation frequency, 115 Hr. Flgure 3.
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SamNe Cell ( Solvent. Solute )
Principle of operation.
were placed in the airtight chamber which was grounded electrically and secured to a vibration-free stand to prevent a pressure fluctuation caused by vibrations from external sources. As the result, the noise level of our cells became less than 8 nV, and this value almost coincided with a noise of a preamplifier and a lock-in amplifier used in the present experiment. Hence, the addition of the two PA signals, that is, those caused by the sample and reference cells, affected to a lesser extent the increase of the background noise level. When an argon ion laser (Spectra Physics, Mountain View, CA, Model 164-03), operating in a single line mode of 514.5 nm, was modulated at a frequency of 115 Hz, and its power was 300 mW, the PA signal amplitude for twice distilled water was about 550 nV. A variable neutral density fiter (ND fiiter) was inserted in one of the two diffracted laser beams in order to correct the difference of the sensitivity between the two PZT cells. Filling the sample cell with a solvent rather than a sample adjusted the ND filter in such a way that the output of lock-in amplifier indicated zero. Figure 3 shows PA signals for water as a solvent (A), 4 X mol/L Amaranth solution as a sample (B), and Amaranth itself of the same concentration measured with the present apparatus, The PA signal difference between (A) and (B) agrees well with that of (C). This shows that the present system works perfectly. The calibration graph made in the concentration range of IO4 mol/L Amaranth was quite linear. And the detection limit was improved by a factor of 2 in
comparison with the previous one (7). A new double beam system presented here will be particularly useful in the measurement of a trace constituent in colored solution. In our previous paper (7), a simultaneous determination of dye mixtures in liquid was carried out by means of laser-induced PAS. However, for a mixing ratio of l:lO, the error for the small portion component of the mixture became enormously large, and the lower the concentration, the bigger the error became. The present system, however, improved the error greatly, that is, 3% or less. A prospective application of the present technique would be detection of a weak spectrum hidden in a big background spectrum. This application is currently being studied.
LITERATURE CITED (1) Rosencwaig, A. Anal. Chem. 1075, 47, 592A-604A. (2) Adams, M. J.; King, A. A.; Klrkbright. G. F. Analyst (London) 1078, 101, 73-85. (3) Oda, S.: Sawada. T.: Kamada. H. Bunsekl Kaaeku 1078.. 27.. 269-273. (4) Oda, S.; Sawada, T.; Kamada, H. Anal. Chem. 1078. 50, 865-667. (5) Sawada, T.; Oda,S.; Kamada, H. Pmc. Jpn. Acsd., Ser. B 1078, 54, 189- 193. (6) Sawada, T.; Oda, S.; Simlzu, H.; Kamada, H. Anal. Chem. 1070, 51. 688-690. (7) Oda, S.; Sawada, T.; Nomura, M.; Kamada. H. AM/. Chem. 1070, 51, 666-688.
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Tsuguo Sawada* Shohei Oda Department of Industrial Chemistry Faculty of Engineering The University of Tokyo Hongo, Bunkyo-ku, Tokyo, Japan
RECEIVED for review August 21,1980. Accepted November 14, 1980.
Electrochemical Behavior of Adriamycin at Carbon Paste Electrodes Sir: The anthracycline antibiotics adriamycin and daunorubicin (Figure 1) are highly effective antitumor agents which are currently experiencing wide clinical use in anticancer therapy (I, 2). In addition, many new analogues and derivatives are being formulated and are coming to clinical trial. The development of sensitive and efficient analytical methods 0003-2700/81/0353-0540$01.00/0
for the determination of these compounds and their metabolites in body fluids and tissue is essential for the testing and evaluation of these drugs. Previously, fluorescence (3, radio-immunoassay (4), and polarographic (5) methods which are useful for the determination of total anthracycline content have been reported. 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
0
541
OH
Flgure 1. Structural formula: adriamycin, R = COCH20H; daunorubicin, R = COCH3.
In addition, analytical methods which involve the use of liquid chromatography and fluorescence or photometric detection and are capable of differentiating between individual anthracyclines have also been reported (6, 7). An alternate approach might involve the chromatographic separation of these compounds followed by electrochemical detection. In specific instances, the combination of liquid chromatography and electrochemistry (LCEC) has been shown to compete favorably with other methods of chromatographic detection, possessing the general advantages of high sensitivity, low cost, simplicity of operation, and selectivity of response based on electrode potential (8). Previous applications of LCEC have demonstrated its utility for the determination of electroactive organics including aromatic amines, phenols, sulfhydryls, nitro compounds, and nitrosamines. The anthracycline antibiotics represent another potentially attractive class of compounds for LCEC as all these compounds contain two commonly electroactive sites: a reducible quinone group and an oxidizable hydroquinone center. In this report, we will describe the electrochemical behavior of adriamycin a t a graphite electrode in order to evaluate its suitability for LCEC. In particular, we will consider the previously unreported and chromatographically important anodic electrochemistry of the dihydroxyanthraquinone moiety.
EXPERIMENTAL SECTION Reagents. Adriamycin (or doxorubicin hydrochloride) was obtained from Adria Laboratories (Columbus, OH) in a 1:5 mixture with lactose and was used as received. Other chemicals were reagent grade and were used without further purification. All experiments were carried out in one of the following 0.2 M buffer solutions: pH 2.3, sodium phosphate/phosporic acid; pH 4.5, sodium acetate/acetic acid; pH 7.1, sodium hydrogen phosphate/citric acid. Solutions were prepared by using triply distilled water and were deoxgenated before use by degassing with prepurified nitrogen. Apparatus. Cyclic and differential pulse voltammograms were obtained with Princeton Applied Research Model 364 polarographic analyzer, a Hewlett-Packard Model 3310B function generator, and a Houston Instruments Model 2000 X-Y recorder. The working electrode was a carbon paste electrode (Princeton Applied Research Model 9326), and a fresh electrode surface was utilized for each experiment. Saturated calomel reference and platinum wire counterelectrodes were also employed. All potentials were reported with respect to the saturated calomel reference. RESULTS AND DISCUSSION The cathodic electrochemistry of adriamycin and other anthracyclines has been the subject of several previous investigations, most notably those of Rao, Lown, and Plambeck (9) and Molinier-Jumel et al. (IO). Both of these groups considered the mechanism involved in the reduction of various anthracyclines a t mercury electrodes and attempted to relate the observed behavior to the therapeutic action of the drugs. In both studies, two sets of reduction waves were reported. The first, occurring at approximately -0.6 V vs. SCE, was assigned to redox processes involving reduction of the quinone
I
+O 8
1
0
+04
E
-04
i
-08
[VVS.SCE]
Cyclic vottammogram of 1.O X lo-' M adriamycin at a carbon paste electrode at pH 4.5: scan rate, 340 mV/s.
Flgure 2.
center to the hydroquinone form and actually consisted of multiple waves resulting from the presence of adsorbed or hydrolyzed forms of adriamycin. The number of peaks observed in this region, their potential, and their apparent reversibility were found to depend strongly on the solution pH and the rate of the potential scan. The second set of waves occurred a t considerably more negative potentials and was assigned to the irreversible reduction of side chain carbonyl groups attached to the anthraquinone nucleus. In a related analytical study, Sternson and Thomas (5) described the use of differential pulse polarography for the determination of total adriamycin species in blood plasma, reporting a detection limit of 8 X lo4 M using the quinone reduction at -0.6 V for quantitation. Though a wide variety of electroanalytical techniques was employed in these studies, all experiments utilized mercury electrodes exclusively and thus were limited primarily to observations of cathodic processes occurring a t negative potentials. Most previous LCEC has been performed by using solid electrodes, owing to the generally higher sensitivity, simpler cell design, and lower background noise level that are attainable under chromatographic conditions with carbon, platinum, and gold electrodes than with dropping mercury or mercury film electrodes. The electrode material most widely employed to date has been carbon paste. Thus, this electrode was selected as most appropriate to observe the anodic electrochemistry of adriamycin in this work. A typical cyclic voltammogram observed for adriamycin at a carbon paste electrode is shown in Figure 2. As with mercury, the redox pattern observed here was also drastically dependent on pH and scan rate. At pH 4.5, two sets of waves were apparent in the accessible potential range. The first consisted of a complex and apparently irreversible group which occurred a t negative potential and was similar to that previously attributed at mercury to the reduction of the anthracycline's quinone center to the hydroquinone (9,lO). This set of waves shifted anodically by approximately 60 mV/pH unit as the pH was decreased and appeared to be reversible only under acidic conditions (pH 2.3). At pH 4.5, two separate reduction processes were distinctly evident while, under both neutral and more acidic conditions, only one wave was observed. Under the latter conditions, only one reducible species is present; or both processes occur at nearly the same potential. The second set of waves occurred at approximately +0.5 V vs. SCE and consisted of an apparently reversible anodic and cathodic pair. Again, as the pH was increased, the peak potentials of this system shifted cathodically by slightly less than 60 mV/pH unit; and the cathodic portion of the system
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
concentrations). The adsorption was sufficiently strong that, when fresh electrodes were briefly exposed to adriamycin solution and then removed, rinsed thoroughly with water, and reimmersed in a buffer solution containing no adriamycin, stable voltammograms similar to those in Figures 2 and 3 continued to be observed. Although differential pulse voltammetric analysis of adriamycin and related compounds at carbon paste electrodes appears to present a very sensitive method for the determination of these species, such an approach would probably not permit the direct determination of individual anthracyclines in physiological samples. All such compounds differ only in the nature of the substituents on the central dihydroxyanthraquinone moiety and thus are expected to exhibit similar redox behavior. Furthermore, interferences could be expected from aromatic amine and hydroxyl species which may be present in physiological samples and are known to undergo oxidation in a similar potential range. However, the oxidation of anthracyclines a t carbon paste electrodes does appear to be well-suited for quantitating these compounds following their separation by liquid chromatography. Thus, the electrochemical behavior reported here indicates the feasibility of determining a new and important family of compounds by LCEC. Actual analyses of adriamycin and other anthracyclines in physiological samples by LCEC will be forthcoming.
i 2 uA
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Flgure 3. Differential pulse voltammogram of 1.0 X 10" M adrhmycin at a carbon paste electrode at pH 4.5: initial potential, 0.0 V; pulse amplitude, 50 mV; scan rate, 2 mVls.
disappeared. These waves occur in the potential range expected for the oxidation of a hydroquinone functionality to the quinone and are probably related to the corresponding group in the parent adriamycin molecule. These positive potentials are not normally accessible by using a mercury electrode, and thus this set of waves was not observed in previous investigations. Since the anodic wave at +0.5 V was well-defined and free from background interference under all experimental conditions, this wave was chosen to evaluate the direct differential pulse determination of adriamycin. Figure 3 illustrates a typical differential pulse polarogram recorded for a 1.0 X lo-' M adriamycin sample at pH 4.5. Even a t this extremely low concentration, the oxidation peak is well-shaped and easily resolved from the background. However, standard calibration curves obtained under these conditions for adriamycin concentrations over the range lo4 to lo-' M were not linear; this was due at least in part to the tendency of adriamycin to adsorb onto the carbon paste, thereby enhancing its effective concentration a t the electrode surface (especially at low
LITERATURE CITED (1) Blum, R. H.; Carter, S. K. Ann. Intern. M. 1974, 80, 249-259. (2) Bachur, N. R. Biochem. phsrmacol. 1974, 23, 207-216. (3) Schwartz, H. S. Bhxhem. Med. 1973, 7 , 396-404. (4) Van Vunakls, H.; Langone, J. J.; Rlceberg, L. J.; Levlne, L. Cancer Res. 1974, 34, 2546-2552. (5) Sternson, L. A,; Thomas, G. Anal. Left. 1977, 10, 99-109. (6) Plerce, R. N.; Jatlow, P. I. J . Chromatogr. 1979, 164, 471-478. (7) Eksborg, S.; Ehrsson, H.; Andersson, I. J . Chromatogr. 1979, 164, 479-486. (8) Klsslnger, P. T. Anal. Chem. 1977, 49, 447A-456A. (9) Rao, G. M.; Lown, J. W.; Plambeck, J. A. J. Electrochem. SOC. 1978, 125, 534-539. (IO) MolinierJumel, C.; Malfoy, B.; Reynaud, J. A.; AubeCSadron, Q. Blochem. Blophys. Res. Commun. 1978, 84, 441-449.
Richard P. Baldwin* Dianne Packett Department of Chemistry University of Louisville Louisville, Kentucky 40292
Thomas M. Woodcock Department of Pharmacology University of Louisville Louisville, Kentucky 40292 RECEIVED for review September 16,1980. Accepted November 24,1980. This work was supported by the National Science Foundation RIAS Grant No. 77-06911.
