system for conventional atomic fluorescence spectrometry. Additionally, in many applications, especially those involving significant amounts of impulse noise, a further advantage will be realized. Impulse noise is generated by such sources as spark generators, lightning flashes, and the start-up of large equipment (e.g, motors) and is characterized by a short, powerful pulse. This pulse is easily picked up by electronic instrumentation where it appears as noise. In this study, impulse noise was simulated by striking a brief spark from a Tesla coil to the grounded photomultiplier housing. Sufficient transmission occurs through the housing to cause a brief noise pulse on the measured photocurrent while the grounding serves to protect the preamplifier from an excessive current surge. The effects of the impulse on the readout from the correlator and the lock-in amplifier are shown in Figures 5 and 6, respectively. The traces in Figures 5 and 6 represent the readout signal obtained from a 100-ppm rhodium solution, in the presence of an impulse noise source. In Figure 6, it is seen that large excursions are produced in the output of the lock-in amplifier whenever an impulse (marked by arrow) occurs. When several impulses occur, it becomes rather difficult to establish a “true” level for the output. This situation, which often occurs when impulse noise is present, is seen to greatly increase the readout noise and can lead to considerable error in the recorded signal value. The correlator output, displayed in Figure 5 , is considerably less susceptible to impulse noise. The impulses, which still cause deviation in the output signal, can now be simply discarded as being obviously deviant from the otherwise smooth sinusoidally varying waveform. Using simple testing criteria, even large amounts of impulse noise need cause no error in this type of readout, thereby effectively eliminating its influence. Even with the advantages presented above, it is questionable that many flame spectrometric applications will justify the additional expenditure necessary to purchase a correlator over a lock-in amplifier. However, with the increasing introduction and use of small digital computers into the analytical
laboratory, it is likely that most flame spectrometers will eventually be interfaced for purposes of sample handling and data collection and processing. With the availability of such systems, the purchase of a hardware correlator will no longer be necessary. Auto- and cross-correlation, unlike lock-in amplification, can be conveniently performed numerically on a digital computer. If such a digital system is already available, adoption of a correlation technique for signal processing could even result in a savings over the cost necessary for a lock-in readout system. For the calculation of a cross-correlogram, the necessary reference waveform could even be stored digitally in the computer memory, to obviate the need for repeated sampling of the reference waveform. This approach, while requiring a very stable source modulation frequency, is typicai of the advantages which can be derived from a software approach to correlation. Whether a hardware or software system is employed, correlation has been shown in this study to be a realistic and often advantageous alternative to conventional readout systems. In particular, cross-correlation can provide increased signalto-noise ratios and lower detection limits than a commercial lock-in amplifier. The additional advantage of freedom from the effects of impulse noise also makes cross-correlation the signal processing technique of choice in many applications. Freedom from drift, direct digital compatibility, and the ability to handle nonsinusoidal signals, all combine to encourage further use of this technique in other studies in flame spectrometry and other methods of chemical analysis.
RECEIVED for review August 28, 1972. Accepted October 11, 1972. One of the authors (GMH) wishes to acknowledge the partial support of this work through National Institutes of Health Grant GM 17904-01. Work performed under the auspices of the Atomic Energy Commission. Reference to a company or product name does not imply approval or recommendation of the product to the exclusion of others that may be suitable.
Electrochemical Characteristics of the Gold Micromesh Electrode W. J. Blaedel and S. L. Boyer Department of Chemistry, University of Wisconsin, Madison, Wis. 53706 The design and construction of a flow-through gold micromesh electrode are described. Current-voltage curves are reported for various flow rates. Measured limiting currents are shown to be directly proportional to the number of screens ( N ) in the electrode, to the concentration of electroactive material (c), and to the cube root of the volume flow rate (V,) of solution through the electrode. Various mesh sizes are examined. Application is made to the measurement of su bmicromolar concentrations.
electrodes, such as higher sensitivity, simplicity, and versatility (3). The platinum rotating electrode ( I , 4) and the platinum tubular electrode (PtTE) (3, 5 ) have been utilized for quantitation purposes. Fundamental studies of these two electrodes have shown that the limiting current for a reversible reaction controlled only by mass transfer may be represented by the following equations: iL = 1.88
x
105 nDZl3 v-l’B r2
wlI2
C (rotating electrode) (1)
FORCED-CONVECTION ELECTRODES have been reviewed by Adams ( I ) and Nicholson (2). Convective transport gives these electrodes several analytical advantages over stationary
iL = 5.306 x l o 5 nD213 X213 C (PtTE) (2) Each hydrodynamic relationship contains a dependence upon electrode velocity (a) or solution flow rate (V,), electrode
( I ) R. N. Adams, “Electrochemistry at Solid Electrodes,” Marcel Dekker, New York, N.Y., 1969. (2) R. S. Nicholson, ANAL.CHEM., 44 ( 5 ) , 478R (1972).
(3) W. J. Rlaedeland S. L. Boyer, ANAL.CHEM., 43,1538 (1971). (4) V. G. Levich, “Physiochemical Hydrodynamics,” PrenticeHall, Englewood Cliffs, N.J., 1962. ( 5 ) W. J. Blaedel and L. Klatt, ANAL.CHEM., 38,879 (1966).
258
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SAMPLE IM KCI
Table I. Micromesh Screen Dimensions" Mesh size, openings/ Thickness, inch a , inches b , inches inches 200 0 0012 0.00081 0.00029 1000 ( 1 I k l 1: 0.00056 0.00033 2000 (1 OI,L2 i 0,00030 0.00021 a See Figure 2.
INLET
SOLUTION OUTLET
radius ( r ) or length (X), and the concentration (C) and diffusion coefficient ( D )of the electroactive species. A flow-through electrode consisting of a micromesh screen greatly increases the ratio of electrode surface to solution volume, which results in increased sensitivity. For this reason, a flow-through gold micromesh electrode (AuMME) was constructed and its properties were examined. SAMPLE SOLUTION INLET
EXPERIMENTAL
Apparatus. The cell design is shown schematically in Figure 1 . The body consisted of two 1-inch thick X 2-inch diameter Plexiglas blocks (Rohm and Haas Co., Philadelphia, Pa.), held together with four bolts (not shown). The silversilver chloride reference electrode (SSCE) was coiled around a core of Plexiglas (0.35-inch long, 0.375-in. o.d., 0.0938-in. i.d.) extending from the upper block into a cavity (1.078-in. diameter, 0.42341. deep) in the lower block. The gold micromesh (Buckbee-Mears, St. Paul, Minn.) was similar to that used by investigators of optically transparent electrodes (6). Three mesh sizes were used, with 200, 1000, and 2000 openings per inch. Microscopic examination revealed the high degree of uniformity reported previously (6), with the dimensions shown in Figure 2 and Table I. A mechanical comparator (Sigmatic by Pratt & Whitney, West Hartford, Conn.) was used to measure the thickness. Unless otherwise stated, all data were obtained with the 1000mesh screen. The gold micromesh electrode (AuMME) consisted of a disk of gold micromesh (0.281-in. diameter) sandwiched between two Parafilm washers (0.004411. thick, 0.281-in. o.d., 0.0938-in. i.d., Marathon Corp., Neenah, Wis.). Two notches were cut from opposite sides of one parafilm washer to provide electrical contact with two partially exposed gold contact plates sealed into the lower Plexiglas block. When the AuMME contained more than one micromesh disk, one notched parafilm washer separated adjacent disks. Bolting the two blocks together exerted pressure by the top block upon the AuMME through two cation exchange membrane washers (0.0938-in. i.d., 1.070-in. o.d., C-60-46 Series, American Machine and Foundry Co., Springfield, Conn.) and two rubber washers (both 0.0938-in. i d . , 0.0625-in. thick; top, 1.078in. o.d., bottom, 0.281-in.0.d.). Valspar Clear Casting Resin (Laboratory Supplies Co., Inc., Hicksville, N.Y.) was used to seal the silver electrode lead into the upper block, and also to seal the two gold contact plates, with a gold wire lead spot-welded to the edges, into the lower block. The sample inlet tube (0.0625-in. i d . ) was machined to contain the upper half of a millipore filter holder, the bottom half being of mated dimensions, but consisting of glass (a No. 12 O-ring joint ground flat) and attached to the solution delivery apparatus. All other inlets and outlets were drilled to receive Tygon tubing (U.S. Stoneware Co., Akron, Ohio), press fitted, and sealed with cyclohexanone. Solutions to be passed through the cell were stored in a 1-liter glass reservoir above the cell. Deaeration was provided
through a fritted glass bubbler by a stream of nitrogen that had passed through a column of amalgamated zinc and acidic vanadous sulfate solution, a column packed with glass wool, a column of alkaline permanganate solution, and a column of supporting electrolyte. The permanganate solution was necessary to remove traces of hydrocarbons from the nitrogen. Solution flowed from the reservoir to the cell through capillary tubing (2-mm i d . ) and a millipore filter (Versapor Type 6424,5-p pore size, Gelman Instrument Co., Ann Arbor, Mich.). Flow rates were measured with a calibrated flowmeter placed downstream from the cell. The large cavity in the lower Plexiglas block containing the Ag-AgC1 reference electrode was flushed continuously with 1M KC1 saturated with AgCl. All voltages in this paper are given with respect to the SSCE in 1 M KCl. Current-voltage measurements were made with a Sargent Model XV Polarograph. The resistance of the cell containing 1MKCl in both electrode compartments was 1100 ohms, measured with a conductance bridge (Model RCM 15B1, A. H. Thomas Co., Philadelphia, Pa.). Reagents. All chemicals required further purification to remove traces of heavy metal impurities. Potassium ferricyanide was recrystallized twice from triply distilled water, which was deionized water redistilled first from alkaline permanganate, and then from 3mM H2S04. Analytical Reagent Grade NaH2P04and Na2HP04were used to prepare a 0.25M sodium phosphate pH 7.5 buffer solution, which was purified using a I-liter electrolysis cell to remove the impurities by electrodeposition (7). The electrolysis cell contained a
(6) M. Petek, T. E. Neal, and R. W. Murray, ANAL.CHEM., 43,1069 (1971).
(7) S. L. Boyer, Ph.D. Thesis, University of Wisconsin, Madison, Wis., 1972.
Figure 1. Flow-through cell with gold micromesh electrode and silver-silver chloride reference electrode
Figure 2. Micromesh screen dimensions
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
259
1.5 -
g\ 1.0w
E e 3
u
0.5
-
I
I
I
I
I
,
0.4 0.2 0 -0.2 APPLIED VOLTAGE V, VS. SSCE IN IM KCI
Figure 3. Effect of anodic pretreatment on current-voltage curves obtained with the AuMME Number on each curve indicates applied voltage used for 5-minute pretreatment. Solution contained 9.9pM KaFe(CN)6, and flow rate was 1.7 ml/min. Voltage scan rate was -0.3 V/min
3.O
5
+‘ 2.0
z w
0.25
U
020
OJ
APPLIED VOLTAGE V, VS. SSCE
LL 3
u
Figure 6. Log (iL - i ) / i plots for various volume flow rates
I.o
Data taken from Figure 4. Number on each curve represents flow rate in ml/min 0.4 0.2 0 -0.2 APPLIED VOLTAGE 4‘, VS. SSCE IN IM KCI
Figure 4. Current-voltage various flow rates
curves for
Number on each curve represents flow rate in ml/min. Voltage scan rate was -0.3 V/min. K3Fe(CNI8was 12.51M. Curve for blank solution was independent of flow rate
4-in. long, 4-in. diameter platinum gauze cathode and a 1’14in. long, l1I4-in. diameter platinum gauze anode, and was capable of lowering the Cu(I1) content of 1 liter of deaerated 0.25M sodium phosphate from 0.18pM to about 0.004pM in 24 hours. All solutions were prepared from triply distilled water. Two stock solutions were used. The blank buffer solution, containing 0.25M Na2HP04adjusted to pH 7.5 by addition of a solution containing 0.25M NaH2P04,was purified as above and used as supporting electrolyte. A ferricyanide stock solution of lmMK3Fe(CN)6in the purified pH 7.5 phosphate buffer was diluted to ‘prepare solutions having K3Fe(CN)6 concentrations in the range 0-12.5pM. 260
RESULTS AND DISCUSSION Study of Electrode Pretreatment. Contamination of the AuMME surface was caused by substances at ultratrace levels in the supporting electrolyte solution. From other experiments (7), it was observed that all chemicals contained Cu(I1) and Pb(I1) which deposited onto the electrode at voltages in the region where ferricyanide was reduced. Also, in the same voltage region, traces of acetylene (less than 0.5 ppm) from the nitrogen supply appeared to adsorb on the gold electrode surface. Considerable effort was expended in the purification of reagents and nitrogen, and although the impurities were successfully removed, enough still remained to be perceptible in measurements on solutions in the micromolar concentration range. These impurities were deposited and accumulated on the AuMME during each polarographic scan. Therefore, the AuMME was cleaned by applying 1.35 V for about 5 minutes before each cathodic scan. Figure 3 shows the effect of such contamination on the shape of the current-voltage curve. Each curve is a replot of a polarogram obtained at a voltage scan rate of -0.3 V / m h Before each curve was obtained, the electrode was “cleaned”
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
Table 11. Half-Wave Potentials and Slopes from Log (iL - i)/i Plots at Various Flow Rates’ Vf,ml/min E1/2, volts Slope, volts/decade 0.94 1.74 3.44 5.85 8.89 13.6 5
0.206 0.210 0.206 0.198 0. I98 0.194
0.068 0.068 0.065 0.071 0,074 0.081
Data from Figure 4.
for about 5 minutes at various applied voltages. Voltages more positive than 1.35 V were not applied because they resulted in an oxide dissolution peak so large that the initial portion of the ferricyanide wave was obscured. Unless the electrode was cleaned before each cathodic scan, surface coverage increased, current diminution occurred, and data reproducibility disappeared. For this reason, the electrochemical cleaning procedure described above was used to obtain all data for this paper. Dependence of Current upon Applied Voltage and Flow Rate. The current-voltage curves for a solution of 1 2 . 5 ~ M K3Fe(CN)Gat various volume flow rates are shown in Figure 4. Current-flow rate data are usually taken in the voltage region where the current is limited by mass transfer-i.e., where the current is independent of voltage. For most of the current-voltage curves in this paper, a strictly constant limiting current region was not achieved with the AuMME, probably because of the previously-mentioned contamination, so limiting currents were read at -0.4 V, where the slopes of the current-voltage curves were a minimum (uide infra, Figure 8). Data obtained from Figure 4 yielded a linear log iL - log V , plot with a slope of 0.321 over a 15-fold range of flow rates (Figure 5 ) . Calculation of the Reynolds number for flow through a screen (8) at 1 ml/min gave a value around 0.2, far below the critical value of 85 suggested for incipient eddy flow currents (9). Flow through the screen is therefore apparently laminar. The observed cube root dependence of current upon flow rate through the micromesh electrode must be regarded as empirical because the electrode has not been treated theoretically. This is in some contrast to the tubular electrode, for which the cube root dependence of current upon the flow rate under laminar conditions has been supported both theoretically and experimentally (5). A similar dependence on slow rate has been observed by Sioda (IO) at much higher ferricyanide concentrations for flow through an 80-mesh platinum wire screen. Figure 6 presents the log (iL - i)/i us. E plots for various flow rates, from data read at IO-mV intervals on the curves of Figure 4. For clarity in presenting the data, each curve is offset vertically from the next. Curvature of the plots for applied voltages more cathodic than about 0.22 V was ascribed to electrode contamination. Frumkin (11) ascribed similar effects on electrode kinetics to adsorption on the electrode. However, the plots do approach straight lines above 0.22 V at all flow rates indicating that electrode contamination is not significant for this part of the current-voltage curve. This straight line portion yielded half-wave poten(8) J. H. Perry, Ed., “Chemical Engineers’ Handbook,” 4th ed., McGraw-Hill, New York, N.Y., 1963, pp 5-35. (9) K. E. G. Wieghardt, Aerormut. Quart., 4, 186 (Feb. 1953). (10) R. E. Sioda, Electrochim. Acta., 15, 783 (1970). ( I 1 ) A. N. Frumkin. E/ecfrcchin?.Ac/u, 9,465 (1964).
Figure 7. Dependence of analytical current upon number of micromesh disks K3Fe(CN)6 was 9.9pM. Flow rate was 3.4 ml/min. Constant applied voltage was -0.3 V
tials and slopes summarized in Table 11. The observed value of 0.20 V (in pH 7.5, 0.25M phosphate buffer) for the hdlfwave potential is in fairly good agreement with values of 0.228 V in 1MKCI (5)and 0.20 V in 0.1NHzS04(12) reported earlier. The small departures from constancy of the halfwave potential and slope toward the high flow rates may be explained by the fact that contamination occurs more rapidly at higher flow rates. The dependence of current upon voltage and volume flow rate was also obtained for a 6-disk AuMME. The 6-disk AuMME gave data like those of Figures 3-5, in excellent agreement with that found for the I-disk AuMME, but with currents that were about 6-fold greater (uide infra). Dependence of Current upon Number of Gold Micromesh Disks. The stopped-flow technique (3) was used to obtain analytical currents for AuMME’s consisting of 1, 2, 4, or 6 micromesh disks. Figure 7 indicates the linear relationship between current and number of micromesh disks. The directly proportional dependence of current upon number of disks, in conjunction with the cube root dependence of current upon volume flow rate observed with both the 1-disk and 6disk electrodes, indicates identical flow regimes at all screens. The laminar nature of the flow through a screen was previously indicated and seems to remain through the 6-disk AuMME, in spite of the non-alignment of the openings (a conclusion based upon its zero transmittance of light). The direct proportionality between limiting current and number of disks may be interpreted as a directly proportional dependence of limiting current upon total electrode surface area for screens of a given mesh size. An attempt was made to extend this to different mesh sizes by hypothesizing that the limiting current is proportional to total electrode surface area, even for screens of different mesh sizes and geometry. This hypothesis was tested by obtaining data like those of Figure 4 with 200- and 2000-mesh screens. For four flow rates ranging from 0.87 to 12.5 ml/min, the 200- and the 2000-(12) P. Zurnan. Co//ect. Czech. Cliem. Commun., 19, 602 (1954).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
261
0.01 .
os
0.6
Figure 9. Dependence of analytical current upon ferricyanide concentration
-0.3 -0.6 APPLIED VOLTAGE V, VS.SSCE IN IM KCI
0.3
0
Flow rate was 3.4 mlimin. Constant applied voltage was -0.3 V
Figure 8. Current-voltage curves for various sizes of micromesh Number on each curve indicates mesh size in openings per inch. Voltage scan rate was -0.3 Vjmin. K3Fe(CN)6was 12.5pM. Volume flow rate for all curves was 3.4 ml/min. Curves for blank were similar for all flow rates and mesh sizes
I I.o IO KJF~CCN)~ CONC.,pM
each case. Sensitivity is remarkably high, corresponding to about 0.2 aud 1 pAJpM for the 1-disk and 6-disk electrodes, respectively. CONCLUSIONS
mesh AuMME gave current-voltage curves similar to Figure 4. Limiting current ratios were 9.2 :6.0: 1.0, independent of flow rate (Figure 8), for the 2000-, 1000-, and 200-mesh, respectively. However, the total surface area ratios were calculated as 3.0:3.0:1 from the dimensions of Table I, showing that the limiting current was not simply proportional to total surface area of the electrode, Wieghardt (9) has pointed out that the axial solution velocity in the openings of a screen is greater than that preceding the screen, and is a function of the open area of the screen. Therefore, it is interesting to consider the inside surface area of these fine-meshed screens as a more effective collector of electroactive species than the upper or lower surface. However, because of the considerable complexities of the shape of the wires and the flow patterns, any attempt to correlate this inside surface area with the observed limiting current ratio must await a more extensive experimental treatment, supported by a better understanding of the flow regime than now exists. Dependence of Current upon Concentration. Figure 9 shows stopped-flow analytical currents for ferricyanide concentrations ranging from 0.1 to lOpM, for 1-disk and 6-disk electrodes. Log-log plots are used to accommodate current data ranging over 3 orders of magnitude. Linearity between current and concentration is shown by slopes very close to unity. Least squares analyses of the plots indicate concentrations to be measurable with a standard deviation of about 0.44% relative, over the entire concentration range. The blank faradaic currents (not shown) amounted to less than 0.003 p A for the 1-disk electrode, and 0.025 pA for the 6-disk electrode, corresponding to concentrations around 0.02pM in 262
*
It has been shown that the limiting current for an AuMME of a particular mesh size is directly proportional to the number of disks (N), to the concentration of electroactive material (C),and to the cube root of the volume flow rate (V,) of the solution through the electrode, (3)
Experiments with different mesh sizes failed to reveal any simple relationship of limiting current to electrode surface area. No work was done to elucidate the dependence of limiting current upon the diffusion coefficient of the electroactive species. Several applications of the AuMME have been indicated, such as stopped-flow measurements and stripping analysis. Although the lower limit of detection for the AuMME was not determined, the measurement of 177 nA for 1pM ferrricyanide is an order of magnitude more sensitive than the PtTE (3). The quantitation of heavy metal impurities in water and reagents using stripping analysis with the AuMME is currently under investigation. In addition, the generation of high yields of electrochemical products in short times is possible when several disks are used in the electrode. For example, it was calculated from the preceding data that a lOpM ferricyanide solution flowing at 3.4 ml/min through a 6-disk electrode gives an 18 yield for its reduction to ferrocyanide, and occurs over a residence time of about 40 milliseconds in the electrode. The use of solid electrodes for quantitation purposes is complicated by surface phenomena. Taking the limiting current to be a measure of the sought-for substance can re-
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
sult in error if the available surface area is changing, because this causes a change in sensitivity. Since only a few per cent of the electroactive material is transformed during solution passage, the concentration in close proximity to the AuMME remains nearly that of the bulk, so a coulometric procedure is suggested for quantitation of the electroactive species. With the stopped-flow technique, integration of the current from
the time the flow is stopped to its steady state value should be a measure of the concentration of electroactive material. RECEIVED for review September 1, 1972. Accepted October 3, 1972. The authors gratefully acknowledge support of this work by funds from the Atomic Energy Commission (Grant No. AT(11-1)-1082) and from the Upjohn Company.
Determination of 2,4-Diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (Trimethoprim) in Blood and Urine by Differential Pulse Polarography M. A. Brooks, J. A. F. de Silva, and L. M. D’Arconte Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, N. J . 07110 A sensitive differential pulse polarographic assay was developed for the determination of trimethoprim (2,4diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (I) in blood, and modified for the determination of the compound in urine. The intact drug (I) is selectively extracted from blood buffered to pH 11.5 into chloroform, is then back-extracted into 0.1N HzS04,and analyzed by differential pulse polarography. The overall recovery of I added to blood is 81.7% i. 6.3 (std dev). No biotransformation products of I were observed in the blood of dog and man. The urine assay involves the selective extraction of the intact drug (I) into chloroform from urine buffered to pH 11.5 followed by thin layer chromatographic separation, elution, and analysis by differential pulse polarography. The overall recovery of the I added to urine is 75.0% 7.2 (std dev). The sensitivity limits of detection from blood and urine are of the order of 0.5-0.75 pg of I per ml of blood or urine. The method was applied to the determination of blood levels and the urinary excretion of I in dog and man following oral administration of Bactrim (Hoffmann-LaRoche) tablets containing both I and sulfamethoxazole.
*
TRIMETHOPRIM [2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine] (I) was synthesized and reported to have in vivo antibacterial activity by Roth et al. ( I ) . Dosage of I is usually in combination with sulfonamides, e.g. Bactrim (Trimethoprim and sulfamethoxazole are the active compounds of the antibacterial combination Bactrim marketed by F. Hoffmann-La Roche and Company, Basle, Switzerland.), because of the POtentiation of the sulfonamide by I (2, 3). The pharmacokinetic profiles of I in man and dog ( 4 ) and of 1 in combination with sulfamethoxazole in man ( 5 ) have been reported. Studies by Schwartz et al. (6) on the metabolism of I in man, dog, and rat showed that all three species excreted four biotransformation products in the urine, with each species show( I ) B. Roth. E. A. Falco, G. H. Hitchings, and S. R. M. Bushby, J . Med. Pliurm. Chem., 5,1103 (1962). (2) E. Grunberg and W. F. DeLorenzo, Antimicrob. Ag. Cliemoflier., 1966,430. (3) S. R. M. Bushby and G. H. Hitchings, Brit. J. Plzarmacol., 33, 72 (1968). (4) S. A. Kaplan, R. E. Weinfeld, S. Cotler, C. W. Abruzzo, and K. Alexander, J . Plzurm. Sci., 59. 358 (1970). (5) D. E. Schwartz and J. Rieder, Cliemotlzerapy,15,337 (1970). (6) D. E. Schwartz, W. Vetter, and G. Engleit, Arz/wim.-Forscli., 20,1867(1970).
ing its own characteristic metabolic pattern. The four metabolites, the Nl-oxide (11), the hydroxymethyl derivative (111), and the two isomeric phenols (IV) and (V), are shown in Figure 1. Metabolites IV and V were reported to be almost totally conjugated with glucuronic acid (6). Synthesis of metabolites I1 and V by Rey-Bellet et al. (7) and metabolites I11 and 1V by Brossi et al. (8)have been reported. Analytical methodology reported for the determination of I includes the use of UV spectrophotometry ( 3 ) ,microbiological techniques ( 3 , 9 ) ,and a spectrofluorometric method (10) based on the alkaline permanganate oxidation of I to the fluorescent trimethoxybenzoic acid. Of the above mentioned techniques, only the fluorometric assay is capable of measuring blood levels of I in patients given therapeutic doses of Bactrim with a desired degree of reproducibility and specificity (IO). Direct-current polarographic techniques have been reported for the investigation of pyrimidines (11-13). Measurement at concentration levels lower than lO+M has been greatly hindered because of the difficulty of distinguishing the reduction wave of the pyrimidine from that of the decomposition of the supporting electrolyte. Differential pulse polarography is a more sensitive technique in which the resultant current us. potential plot is presented as a peak rather than a wave, and it can measure concentrations down to 10-6M(14). The assay developed for the determination of I in blood and urine employs selective extraction into chloroform of the compound from blood and urine buffered to pH 11.5. For the assay of blood, I is back-extracted into 0.1N H2S04 and subjected to polarography. Urinary I, however, must first be separated by thin layer chromatography (TLC). The urinary I separated by TLC is eluted with ethanol, the residue of which is dissolved in 0.1NH2SO4for polarography. (7) G. Rey-Bellet and R. Reiner, Helu. Chim. Acta, 53.945 (1970). (8) A. Brossi, E. Grunberg, M. Hoffer, and S. Teitel, J . Med. Clzem., 14,58 (1971). (9) H. Beck and J. C. Pechere, 6th International Congress of Chemotherapy, Tokyo, 1969. (IO) D. E. Schwartz, B. A. Koechlin, and R. E. Weinfeld, Clzemotlzerupy Supfil., 14,22 (1969). (11) B. Janik and P. J. Elving, Clzem. Reo., 68,295 (1968). (12) L. F. Cavalieri and B. A. Lowy, Arch. Bioclzem. Biophys., 35, 83 (1952). (13) D. L. Smith and P. J. Elving, J . Amer. Chem. Soc., 84, 2741 (1962). (14) J. A. F. de Silva and M. R. Hackman, ANAL.CHEM., 44, 1145 (1972).
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