Alternating current polarography in the harmonic multiplex mode

subsequent acquisition by the minicomputer system. The fidelity of the concept is established by the model electrode reactions associated with the sys...
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Alternating Current Polarography in the Harmonic Multiplex Mode Simultaneous Acquisition of Direct Current, Fundamental Harmonic Alternating Current,and Second Harmonic AIternating Current Polarographic Responses Donald E. Glover and Donald E. Smith’ Department of Chemistry, Northwestern University, Euanston, Ill. 60201

The literature points out convincingly that dc, fundamental harmonic ac, and second harmonic ac polarographic responses represent highly complementary data forms for electrochemical kinetic-mechanistic investigations. These data usually are acquired in a series of separate experiments with different instruments. The present work describes one approach whereby a small on-line laboratory computer (minicomputer) can be utilized for simultaneous, automated acquisition of each type of polarogram, including both amplitude and phase characteristics of the ac responses. The procedure relies heavily on operational amplifier analog circuitry to condition signals for subsequent acquisition by the minicomputer system. The fidelity of the concept is established by the model electrode reactions associated with the systems; Cr(CN)6*-/Cr(CN)e4- in 1M KCN and Cd*+/Cd(Hg) in 1M Na2S04.

AMONGTHE MANY ADVANTAGES attributed to small, on-line digital computers (“minicomputers”) in electrochemical measurements (1-14), their multi-channel data acquisition capability has been mentioned, but has received relatively little implementation. The typical minicomputer data acquisition system includes an analog-to-digital (A/D) converter and multiplexer combination which enables computer-controlled monitoring of large numbers of data channels on a relatively low cost per channel basis. When a set of data channels must be interrogated at effectively the same point in time, sampleand-hold (S/H) amplifiers (IS)can be used in combination with the A/D converter-multiplexer system to achieve this end. 1

To whom correspondence should be addressed.

(1) G. Lauer, R. Abel, and F. C. Anson, ANAL.CHEM.,39, 765 (1967). (2) G. Lauer and R. A. Osteryoung, ibid., 40(10), 30A (1968). (3) S.P. Perone, J. E. Harrar, F. B. Stephens, and R. F. Anderson, ibid., 40,899 (1968). (4) S. P. Perone, D. 0. Jones, and W . F. Gutknecht, ibid., 41,1154 (1969). (5) F.B. Stephens, F. Jakob, L. P. Rigdon, and J. E. Harrar, ibid., 42, 764 (1970). (6) W . F.Gutknecht and S . P. Perone, Ibid., p 906. (7) D. 0.Jones and S.P. Perone, ibid., p 1151. ( 8 ) L. B. Sybrandt and S . P. Perone, ibid., 43, 382 (1971). (9) H. E.Keller and R. A. Osteryoung, ibid., p 342. (10)J. Lawrence and D. M. Mohilner, J. Electrochem. SOC.,118, 259 (1971). (1 1) “Applications of Computers in Analytical Chemistry,” Vol. 2, H. B. Mark, Jr., J. S. Mattson, and J. C. MacDonald, Ed.,

M. Dekker, Inc., New York, N.Y., all articles, in press. (12) S. P. Perone, J. W. Frazer, and A. Kray, ANAL.CHEM.,43, 1485 (1971). (13) H. Kojima and S . Fujiwara, Bull. Chem. SOC.Jap., 44, 2158 (1 971). (14) B. J. Huebert and D. E. Smith, ANAL.CHEM., 44,1179 (1972). (15) D. E. Smith, in “Applications of Computers in Analytical Chemistry,” Vol. 2, H. B. Mark, Jr., J. S. Mattson, and J. C.

MacDonald, Ed., M. Dekker, Inc., New York, N.Y., in press. 1140

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We are aware of one recent report (13) in which non-routine (>2 channels) implemntation of the on-line computer multichannel data acquisition capability was a key factor in determining the feasibility of an experiment. The case in question involved a 9-channel measurement scheme in which the fundamental harmonic ac polarographic responses (amplitude and phase) to four applied input frequencies were simultaneously measured, along with the dc polarographic response. The present report describes a less extreme, but rather useful, departure from conventional ac polarographic experimental procedures in which the on-line computer multi-channel data acquistion feature again plays a key role. Direct current polarography, fundamental harmonic ac polarography, and second harmonic ac polarography are three measurement concepts which have been regarded in the classical context as separate, distinct experiments and are often implemented with separate instruments. Each of these measurement schemes has been quite successful in its own right, and many electrochemical processes have been elucidated adequately with just one of them. ‘However, electrochemists are becoming increasingly aware of the fact that kinetic-mechanistic conclusions about electrode processescan bereached with greater fidelity and/or convenience when one invokes a set of complementary measurement procedures, rather than a single technique. Convincing theoretical (16,17) arguments, as well as experimental data (It?), have been provided which demonstrate that the kinetic-mechanistic information provided by the various current harmonics in question is frequently complementary. Consequently, significant stimulus exists in fundamental electrochemical measurements for obtaining all of the faradaic admittance response characteristics provided by dc, fundamental harmonic ac, and second harmonic ac polarography. In an effort to supplement this stimulus with convenience, we are investigating several means of effecting these measurements simultaneously through computerized data acquisition and processing procedures. We describe here one successful approach which involves the use of a set of parallel analog signal conditioning circuits similar to those employed when the signals of interest are observed individually, as in conventional instruments, The outputs of the signal conditioning array provide the desired dc, fundamental harmonic ac, and second harmonic ac responses to a pure sinusoidal input signal. We refer to the measurement concept as “A.C. Polarography in the Harmonic Multiplex Mode.” The term “harmonic” is introduced in an effort to avoid ambiguity with a technique referred to as “A.C. Polarography in the Frequency Multiplex Mode” which involves measurement of the fundamental harmonic responses arising from a multiple frequency input signal (14). (16) T.G.McCord and D. E. Smith, ANAL.CHEM., 40,289 (1968). (17) T. G. McCord, H. L. Hung, and D. E. Smith, J . Elecrrourral. Chem., 21, 5 (1969). (18) B.J. Huebert and D. E. Smith, ibid., 3 1 , 3 3 3 (1971).

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non-faradaic contributions was valid. Except for the use of Zeltex Model 145L operational amplifiers as control and current amplifiers, the potentiostats were identical to those described previously (22). Front panel switches enable convenient switching of the potentiostats between chemical cells and dummy cells. The dc potential applied to the potentiostat was derived from a conventional initial voltage source (20) and a computer-controlled incremental dc “sweep” source whose nature and mode of action were discussed previously (14). The sinusoidal test signal was derived from a Hewlett-Packard Model 241A sinusoidal oscillator whose output was passed through a tuned amplifier (14, 15) and operational amplifier attenuator to obtain a signal amplitude of 10 mV peak-to-peak. The faradaic current signal from the subtractor is coupled to the inputs of five signal conditioning networks whose purpose is to separate, amplify, and, when necessary, transform into an amplitude proportional dc format the cell current components required to fully characterize the dc, fundamental harmonic ac, and second harmonic ac responses. Amplification factors and output polarities of these signal conditioning circuits were designed to match the 0 to -10 volt input range of the A/D converter. The dc signal detector was comprised of a low-pass filter of fourth-order

EXPERIMENTAL

Instrumentation. Figure 1 provides a schematic of the instrument employed in this work. Although the particular combination of analog circuitry is unique, the individual building blocks are all comprised of standard operational amplifier circuits which have been described in the literature (14, 15, 19-23). A dual potentiostat system is employed to effect subtractive compensation of double-layer charging current (20,21). The potentiostats are provided with positive feedback iR compensation which permits elimination of significant ohmic potential drop effects at the frequencies of interest (21,22). Thus, in all work reported here the assumption that the subtractor circuit output contained negligible (19) D. E. Glover and D. E. Smith, ANAL.CHEM., 43,775 (1971). (20) E, R. Brown, T. G. McCord, D. E. Smith, and D. D. DeFord, ibkf., 38, 1119 (1966). (21) E, R. Brown, H. L. Hung, T. G., McCord, D. E. Smith, and G . L. Boornan, ibid., 40, 1424 (1968). (22) E, R. Brown, D. E. Smith, and G. L. Boornan, ibid., p 1411. (23) “Operational Amplifiers: Design and Applications,” J. G. Graeme, G. E. Tobey, and L. P. Huelsman, Ed., Burr-Brown Research Corp., Tucson, Ark, 1971.

ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972

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System: 1.97 X 10-3MCr(CN)6'- in 1.00MKCN at 25 "C Applied: 10.0 mV peak-to-peak, 228 Hz sine wave: computer-controlled incremental dc scan (14 Measured: direct current, total and in-phase alternating currents at 228 Hz in-phase and quadrature currents at 456 Hz 0 = experimentaldata, average of four replicate measurements _ -- theoretical responses for 01 = 0.55, k , = 0.40 cm sec-', t = 5.00 sec, D o = D R = 8.20 X cmz sec-l, A 25 "C, A E = 5.00 mV, n = 1, C,* = 1.97 X 10-3M,w = 1432 rad sec-l Butterworth response (23, 24) with a cut-off frequency of about 2 Hz and an inverter amplifier (23). The fundamental harmonic amplitude and phase response was acquired by (24) Philbrick Researches, Inc., "The Lightning Empiricist," vel. 13, Nos. 1 and 2, Philbrick Researches, Inc., Dedham, Mass., 1965. 1142

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measuring the total and in-phase ac components with the aid of tuned preamplification, followed by full-wave rectification and phase-sensitive detection (14). Direct current components proportional to the in-phase and quadrature second harmonic Signals were generated by tuned preamplification followed by phase-sensitive detection (19). Because the resulting dc signal levels normally will exhibit both positive

It provides for signal averaging in which the measurement cycle is repeated at the same dc potential for the desired number of times before it is incremented to a new level. It acquires and stores digital cell current data when a pulse from the external timing circuit indicates that data have been sampled by the S/H amplifiers. It calculates from calibration factors the magnitude (in microamps) of each cell current component provided by analog signal conditioning channels (dc, total fundamental harmonic, in-phase fundamental harmonic, in-phase second harmonic and quadrature second harmonic), as well as the phase angle cotangents for the fundamental and second harmonic components. All of these data are printed out in real-time at each dc potential, thus keeping the operator informed of the experiment status. It provides for analog point plotting of the various polarograms defined by the stored data (conventional dc polarograms, total and phase selective ac polarograms, complex plane ac polarograms). Electrode Processes. Model redox systems used to test the instrument for ac polarography in the harmonic multiplex mode were: C ~ ( C N ) G ~ - / C ~ ( C Nin) G1M ~ - KCN and Cd*+/ Cd(Hg) in 1 M Na2S04. The polarographic cell contained a dropping mercury working electrode (Sargent S-29417 capillary), a platinum wire auxiliary electrode, and a saturated silver-silver chloride reference electrode. Measurements were performed at 25.0 f 0.1 “C. Methods of compound and solution preparation, purification of nitrogen for cell degassing and solvent (HzO) purification, as well as peripheral supporting equipment employed in these measurements have been described elsewhere (20,21).

and negative polarities as the second harmonic polarogram is generated (dc potential scanned), it was necessary to further condition these signals with an absolute value-polarity indicator circuit (AVPIC) to generate signals compatible with the aforementioned A/D converter requirements (15, 25). The AVPIC provides a negative output equal to the input magnitude, together with a second output (polarity indicator) from a trigger circuit whose output state indicates the actual sign of the original input signal (15, 25) S/H amplifiers enable acquisition of the signal conditioning circuit outputs at a precise point in the life of the dropping mercury working electrode. This operation, together with mechanical drop dislodging and synchronization of the computer data acquisition cycle with these events is controlled by an external timing circuit ( 1 5 , 2 0 ) . The timing circuit control pulse is monitored by the computer via a multiplexer input channel. The digital electronics shown in Figure 1 are comprised of a Digital Equipment Corp. (DEC) Model PDP-8/S central processor with an 8K, 12-bit word core memory, a DEC Type A121 20-channel multiplexer, a DEC Type AFOlA 12-bit A/D converter, two DEC Type AAOlA 12-bit D/A converters, two DEC Type 34D 10-bit D/A converters, and a Teletype Corp. Model ASR-33 teletypewriter. Performance characteristics of this equipment have been described (25, 26). Provision for obtaining analog point plots of digital data arrays was obtained by interfacing an Electro Instruments Model 480 X-YY’ recorder (with Model 468 and 470 plug-ins) to the computer via the Type AAOlA D/A converters. A Hewlett-Packard Model 141B oscilloscope with Model 1400A and 1402A plug-ins and a Hewlett-Packard Model 5243L electronic counter with a Model 5265A digital voltmeter accessory aided in system testing, trouble-shooting, etc. Programmed Experiment Sequence. The program employed to implement the harmonic multiplexing experiment was written in the PAL-I11 assembly language (27). The program listing and the source and binary tapes (punched paper tapes) are available from the authors on request. The major aspects of the program’s functions are almost identical to one developed for ac polarography in the non-coherent wave frequency multiplex mode which was described in detail (14). Differences in the number of analog signal channels monitored and the manner in which certain input channels are interpreted comprise the only noteworthy distinctions between the previously described program (14) and the one applied to this work. Briefly, the program for the harmonic multiplexing experiment performs the following functions : It asks the operator for information concerning the experiment, such as the dc potential range to be scanned, the frequency employed, whether signal averaging is to be used and, if so, how many data sets per average, etc. It calibrates the five analog signal channels which provide voltage proportional to cell current, relating each voltage to the corresponding cell current component by a constant calibration factor. The basic calibration procedure and rationale have been described (14,19). It informs the operator of unacceptable input, such as an overloaded A/D channel or unacceptable operator input. Diagnostic messages are printed out via Teletype which help the operator correct the problem. It applies an incremental dc ramp potential to the cells. The potential is held constant over the life of a mercury drop as data are acquired, then incremented to take data at the next dc potential, etc.

RESULTS AND DISCUSSION

(25) S. C. Creason, R. J. Loyd, and D. E. Smith, ANAL.CHEM., 44, 1159 (1972).

(26) Digital Equipment Corp., “Small Computer Handbook,” Digital Equipment Corp., Maynard, Mass., 1966. (27) Digital Equipment Corp., “Introduction to Programming,” Digital Equipment Corp., Maynard, Mass., 1969.

Figures 2 and 3 depict some typical results obtained in this work. In all cases the data observed for the various current components were self-consistent and in good agreement with expectations based on previous studies of the model electrochemical systems employed (14, 19-21, 28, 29). The k , values which gave the best fit to the experimental data were slightly higher than those given in previous reports for both systems studied. These slight differences may be associated with a more lengthy activated charcoal treatment utilized in the present work (overnight or longer rather than a few hours or less). This apparent effect is under investigation. The disparity in question is not crucial for purposes of this report because it is small and, more important, because the various ac data forms self-consistently lead to the same k , value, which is the key requirement for validating the harmonic multiplex concept. It should be noted that, for the systems investigated, the dc response shows negligible contributions of the heterogeneous charge transfer rate process and obeys the equations for a pure diffusion-controlled (“reversible”) process, as one would expect for the k, values characterizing these systems. In no instance did we observe evidence for loss in data quality attending the implementation of the harmonic multiplex scheme, either with respect to precision or quantitative accuracy. In fact, expected improvements in data precision associated with digital data acquisition, digital data processing, and signal averaging were realized in this work. Data scatter was normally lower than previously observed in our laboratory when strictly analog data acquisition was utilized. Figure 3 illustrates that this applies also when one combines data from several separate runs at different frequencies. Of course, with __

(28) T. G . McCord and D. E. Smith, ANAL.CHEM.. 41,131 (1969). (29) J. E. B. Randles and K. W. Somerton, Trans. Faraduy SOC., 48, 937 (1952). ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972

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the exception of the dc polarograms, there is no fundamental theoretical basis for anticipating any difficulty in performing measurements in the harmonic multiplex mode. From the viewpoint of ac measurements, the difference between the simultaneous (parallel) data acquisition mode employed here and the conventional sequential (serial) approach involves no difference in significant experimental parameters, such as the applied potential signal. On the other hand, the existence of the applied ac potential does represent a perturbation on the dc experimental in that this input is normally absent and, because of faradaic nonlinearity, it is expected to give rise to an “extra” direct current contribution known as the faradaic rectification component (30, 31). However, theoretical guidelines (31) indicate that the faradaic rectification contribution to the total dc signal normally will be negligible (less than 0.1 Z of the total dc signal) if applied ac potential amplitudes do not exceed significantly the levels employed in this work. The data in Figure 2A seem to confirm this prediction. The approach to harmonic multiplexing invoked in the present work (Figure 1) relies heavily on analog signal processing to separate the various current harmonics of interest. A very appealing alternative is the use of digital signal conditioning whereby the functions of the five analog signal conditioning circuits (Figure 1) are replaced by digital Fourier analysis. The computational efficiency and minicomputer-compatibility associated with the Fast Fourier Transform (FFT) algorithm (32, 33) makes digital Fourier analysis quite feasible (32), provided A/D conversion of the cell current and applied ac potential waveforms (subtractor output and oscillator output, respectively, in Figure 1) can be effected with adequate speed. In principle, the FFT requires at least two data points per cycle of the highest ac frequency of interest (34,but unless the signals under-going analysis are essentially noise-free, it is recommended that one substantially exceed this minimum data acquisition rate. In addition to this, it is necessary to sample the current and potential waveforms simultaneously with S/H amplifiers prior to A/D conversion in order to define the phase angle between them. The fact that the PDP-8/S computer system available for this work does not provide an adequate A/D conversion rate to meet the foregoing requirements, except at rather low frequencies, is the primary reason for our selection of the analog signal conditioningapproach for the present work. The fidelity of digital Fourier analysis as a means of simultaneously acquiring the fundamental and second harmonic ac polarographic responses (amplitude and phase) has been demonstrated quite convincingly by Kojima and Fujiwara (13), aL though their observations were confined to low frequencies by the digital data acquisition rate. However, minicomputer systems are available (32) which do provide the necessary data acquisition speed to implement digital signal conditioning over the frequency range normally considered in ac polarography (frequencies less than 50-100 kHz.) If such fast minicomputer systems are used in a dedicated manner (rather than timesharing), digital signal conditioning is the recommended ap-

(30) P. Delahay, in “Advances in Electrochemistry and Electrochemical Engineering,” P. Delahay, Ed., Vol. 1, Chap. 5, WileyInterscience, New York, N.Y., 1963. (31) D. E. Smith, in “Electroanalytical Chemistry,” A. J. Bard, Ed,, M. Dekker, New York, N.Y., Vol. 1, 1966, pp 21, 34. (32) Bulletin SP-360, Raytheon Computer Corp., Santa Ana, Calif., 1970. (33) V. W. Cooley and J. W. Tukey, Math. Comput., 19,297 (1965). (34) M. Schwartz, “Information Transmission, Modulation, and Noise,” McGraw-Hill, New York, N.Y., 2nd ed., 1970, pp 116--22.

computer or when applied signals of very high frequency are employed.

proach to ac polarography in the harmonic multiplex mode because it replaces numerous analog circuits, which require tuning, offset adjusts, etc., by a drift-free, highly automated digital program. On the other hand, because it places minimal demands on data acquisition capability, an analog conditioning approach such as the one used in this work is favored whenever digital data acquisition rates are too slow to meet the demands of digital signal conditioning-e.g., whenever one has to operate with a slob dedicated minicomputer system, or with a low-priority time shared terminal to a large

ACKNOWLEDGMENT

The authors are indebted to Barry J. Huebert for helpful discussions and programming assistance. RECEIVED for review January 5 , 1972. Accepted February 9, 1972. D. E. G .is an NASA Fellow 1971-72. Work supported by National Science Foundations Grant GP-16281.

Determination of Glibornuride (a Tolylsulfonyl Urea Hypoglycemic Agent) in Blood by Differential Pulse Polarography J. Arthur F. de Silva and Martin R. Hackman Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche lnc., Nutley, N.J. 07110 A sensitive and specific differential pulse polarographic assay was developed for the determination of glibornuride (a tolylsulfonylurea hypoglycemic a ent) in blood. It involves the selective extraction the compound from whole blood into diethyl ether and back-extraction into 1.ON “,OH. Following suitable “clean-up” of the sample, the compound is nitrated in 10% KNOa/H2S04at 105 O C for 2 hours. The nitro derivative is extracted into ethyl acetate, the residue is dissolved in 0.1N NaOH, deoxygenated, and analyzed by differential pulse polarography. The overall recovery of the assay is 80.7% i 8.0 (Std dev) from blood and the sensitivity limit is 0.05-0.10 pg/ml of blood (using a 2-ml sample per assay). The method was applied to the determination of blood levels of the intact drug in man following single oral doses of 50 and 100 mg of glibornuride.

09

GLIBORNURIDE; 1-[(1R)-d-endo-hydroxy-3-endo-bornyl]-3-(ptolylsulfony1)urea (Ro 6-4563/00) is a new member of the tolylsulfonylurea class of compounds being tested as hypoglycemic agents (1). The chemistry (2), pharmacology (3-6), and toxicology (7) of the drug in several animal species have been reported. The compound has been extensively tested in man as a oral hypoglycemic agent (8-11) and has been reported to be more effective at lower doses than either tolbutamide or chlorpropamide (12,13). (1) “Recent Hypoglycemic Sulfonylureas-Mechanisms of Action and Clinical Indications,” U. C. Dubach and A. Biickert, Ed., Hans Huber, Bern, Switzerland, 1971, pp 1-327. (2) H. Bretschneider, ibid., pp 22-33. (3) 0. Oelz and E. R. Froesch, ibid., pp 36-48. (4) E. Lorch and K. F. Gey, ibid., pp 49-55. (5) J. Beyer, U. Cordes, H. Krall, W. Ewald, and K. Schoffling, ibid., pp 56-63. ( 6 ) U. Cordes, J. Beyer, H. Krall, E. Haupt, C. Rosak, and K . Schoffling, ibid., pp 6 4 8 . (7) K. Scharer and H . Hummler, ihid., pp 163-70. (8) D. Pometta, W. Stauffacher, G. Zahnd, H. Micheli, and A. E. Renold, ibid., pp 182-8. (9) U. C. Dubach, A. Biickert, and I. Forg6, ibid., pp 187-99. (10) E. Haupt, J. Beyer, W. Koberich, K. M. Bartelt, U. Cordes, C . Rosak, and K. Schoffling, ibid., pp 200-205. (11) N. 0. Lunell, B. Persson, and J. Thorell, ibid., pp 234-40. (12) W. Ewald, W. Kunkel, M. D. Saenger, and K . Schoffling, ibid., pp 278-82. (13) A. Biickert and E. Schweda, ibid., pp 283-8.

Studies on the absorption, metabolism, and elimination of the drug in man, dogs, and rats by. Bigler et al. (14-16) indicated that it was well absorbed, extensively metabolized (Figure l), and eliminated in man with a mean half-life of 8 hours (range 4.7 to 11.5 hours). Following oral administration, intact drug was the major component in the blood but only trace amounts of the intact drug could be found in the urine. The compound undergoes oxidation at the methyl group in the phenylsulfonamide portion of the molecule to produce the alcohol (M- l), which in man is further oxidized to the carboxylic acid (M-2). Oxidation and hydroxylation also took place in the borneol moiety of the compound resulting in the introduction of hydroxyl groups in several positions producing the metabolites (M - 3 to M -6.) The chemical structures of the metabolites (M-1 to M-6) were verified by thin layer chromatography (TLC) of their respective dinitrophenyl (DNP)aminoborneol derivatives ( 1 9 , by pyrolysis gas chromatography, and mass spectrometry of the respective trimethylsilyl (TMS)- aminoborneol derivatives (16). The chemical synthesis of the respective authentic reference compounds has also been reported (17). The enzymatic hydrolysis of glibornuride to 3-endo-aminoborneol and p-tolylsulfonamide has not been established although chemical hydrolysis of the compound can be effected (Figure 1). Spectrophotometric methods for the determination of a similar compound, tolbutamide, in plasma based on the formation of a 2,4-dinitrophenyl (DNP) derivative (18) and a p-N-dimethylaminobenzaldehyde derivative (Ehrlich’s Reagent) (19)have been reported. The strong UV absorption of tolbutamide at 228 nm was also used in the determination of the drug in plasma (20-22). (14) F. Bigler, G. Rentsch, and J. Rieder, ibid., pp 171-80. (15) F. Bigler, J. Rieder, and G. Rentsch, F. Hoffmann-La Roche & Co., Ltd., Basle, Switzerland, unpublished data, 1969. (16) F. Bigler, P. Quitt, M. Vecchi, and W. Vetter, Arz/teim.-Forsc/i., in press. (17) P. Quitt, Cliirnia, 24,452 (1970). (18) H. Spingler, K/in. Woc/ie/isc/ir.,35, 533 (1965). (19) T. Chulski, J . Lab. C/i/i.Med., 53,490 (1959). (20) H . Spingler and F. Kaiser, Armeim.-Forsc/i, 6 , 700 (1956). (21) A. A. Forist, Proc. Soc. Ex/J.Biol. Med., 96, 180, (1957). (22) E. Rladh and A. Norden, Acta Pharmacol Toxicol., 14, 188 (1958).

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