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).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
263
Figure 1. Postulated metabolic pathways of I in man, dog and rat as reported by Schwartz et a/.
(6) The polarographic blood assay has the advantage of being able to measure I without the time-consuming conversion of I to the trimethoxybenzoic acid and subsequent extraction required in the fluorescence assay (IO). Although the fluorometric assay is more sensitive, this polarographic assay will ?:.able the analyst to measure I in approximately twice the number of samples as that accommodated by the fluorometric assay in the same amount of time. This technique was employed for the quantitation of blood and urine levels of I following the administration of Bactrim in dog and man. EXPERIMENTAL
Apparatus. A Princeton Applied Research Model 171 Polarographic Analyzer equipped with a P.A.R. Model 172 Drop Timer was used for analysis. A three-electrode cell containing a dropping mercury electrode (DME; Capillary tube of inside diameter = 0.05-0.08 mm from E. H. Sargent No. S-29417) as the indicator electrode, a saturated calomel electrode (SCE; fiber junction calomel from Beckman, No. 39178) as the reference electrode, and platinum wire as the auxiliary electrode (14). The drop mass was 2.32 mg/sec.: [M2'3 x t 1 I 6 = 1.56131, using a 0.5second drop time. The instrumental parameters (15) included the derivative pulse mode of operation (i.e.,constant amplitude pulse superimposed on a linear dc ramp; sampled for the last 16 msec during the period of pulse application), the differential current readout (ix., the signal displayed is due to the difference in current prior to the pulse application and 16 msec before drop dislodgment), and a pulse amplitude of 50 mV peak-topeak (applied 56 msec before drop dislodgment). Other parameters include scanning from -0.700 V to -1.200 V GS. SCE; scan rate, 5 mV/second; scan range, 1.5 volts; and current range, 1 pA full scale deflection. (15) Princeton Applied ReFearch Corporation, Instruction Manual for PAR Model 171 (IV-31), Princeton, N. J. (1970). 264
Reagents. All reagents used were of analytical grade purity (>99 %) and were used without any further purification. All aqueous reagents were made in double distilled water. Reagents used included 1M sodium carbonate (aqueous), 0.1N sulfuric acid (aqueous), chloroform (reagent grade), and ethanol (reagent grade). A standard solution of I is prepared by dissolving 10.0 mg of authentic I (Cl4H18N4O3, mol wt of 290.33, mp of 199 "C) in 100 ml of absolute ethanol to give a stock solution containing 100 pg/ml. Dilute 1.0 ml of this solution to 10 ml with ethanol to give a working solution containing 10.0 pg/ml. Suitable aliquots of this solution are added to blood or urine as internal standards. OF BLOOD AND URINE. Analytical Procedure. EXTRACTION Into a 50-ml glass-stoppered centrifuge tube, add 1 ml of blood or urine and 9 ml of distilled water. Then add 1.O ml of 1M Na2C03solution and mix on a Vortex super-mixer. Add 11.O ml of reagent grade CHC13,shake on a reciprocating mechanical shaker for 10 minutes, and centrifuge 5 minutes at 2000 rpm to separate the layers. Along with the samples, run a 1-ml specimen of control blood or urine. Duplicate 1-ml specimens of control blood or urine containing 2.5 and 5.0 pg of I, prepared by evaporating 0.25 ml and 0.5 ml, respectively, of the working solution of I in a nitrogen stream at 60 "C and then adding 1 ml of the control specimen, are also analyzed at the same time. Similarly residues of 2.5 and 5.0 pg of I are dissolved in 0.1N H2S04as external standards for the determination of per cent recovery. Aspirate the upper aqueous phase as completely as possible taking care not to aspirate any of the lower CHCL layer. BLOODASSAY. Backwash the CHC13 with 5 ml of distilled water by shaking for 5 minutes. Centrifuge the samples for 5 minutes at 2000 rpm to effect a complete separation of the phases. Aspirate the upper aqueous layer. Transfer an 8-ml aliquot of the CHCl, extract into a 15-ml glass stoppered conical centrifuge tube and back-extract with 4 ml of 0.1N HzS04. Shake 10 minutes and centrifuge for 5 minutes as above. Transfer as much of the 0.1N HzS04supernatant as possible (usually 3.5-3.8 ml) into a clean 15-ml conical centrifuge tube.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
CURRENT
7CURRENT 1PA FULL S C A L E
D [c
I
-0800
I
I
1
I
I I
-0900
-1000 -1"oo -'200 POTENTIA, (VOLTS vs S C E )
Figure 3. Effect of pH on polarographic peaks of I
-
---
Y 100
-0900
-1000
POTEHTIAL,
-1 100
E
-1
200
(VOLTS v s 5 C E )
Figure 2. Polarograms and sample calculation of resultant current for Trimethoprim(I) (1) Reagent blank, (2) Blood control, (3) External standard (5 pg), (4) Internal standard (10 pg), and (5) Dog blood-1 minute post I.V.
administration
De-aerate the sample in the 15-1111 centrifuge tube for 5 minutes with NBflowing through a fritted glass tube. Transfer the contents into the polarographic cell and analyze by differential pulse polarography using the operational parameters described above. URINE ASSAY. Evaporate a 10-ml aliquot of the CHC1, extract to dryness under a stream of nitrogen at 60 "C. Dissolve the residue in 100 p1 of CHC13 and transfer quantitaSilica gel G chromatotively onto a 20 x 20 Brinkmann (FZs4) plate. Develop the plate in a vapor saturated chamber using ethyl acetate :methanol : concentrated ammonium hydroxide (4: 1 :0.1) until the solvent front has ascended 15 cm. The plate is examined under short wave UV light, and the areas on the silica gel corresponding to I are identified by comparison with the R, of 10 pg of authentic I (R, = 0.45) chromatographed with the sample extracts. These areas are scraped off and transferred into a 15-ml centrifuge tube to which 5 ml of ethanol are added. The tubes are slurried on a Vortex super-mixer for approximately 30 seconds, then extracted on a reciprocating mechanical shaker for 10 minutes, and finally centrifuged 5 minutes at 2000 rpm to spin down the silica gel. The ethanol supernatant is transferred to another 15-ml tube, evaporated to dryness, and the resulting residue is dissolved in 4 ml of 0.1N H2SO4. These solutions are then de-aerated and assayed by differential pulse polarography.
0.1N H2SOc 0.01N H2S01
Calculations. Calculate the resultant current due to the polarographic reduction of I on the basis of peak heights measured at -1.070 V us. SCE, as shown in Figure 2. The determination of the resultant current is easily accomplished thus : d (in cm) X current scale (PA) = Sample (PA) D (in cm) where d = sample peak height and D = full scale deflection. The concentration of I is calculated on the basis of the resultant current of the internal standard thus : Concn. of Int. Std. (pg) (PA) Sample ml of sample (PA) Int. Std. pg I/ml of blood or urine
The current (PA) per pg of the internal standards corrected for the aliquot taken for analysis is then compared directly to that of the external standards to obtain per cent recovery thus : (pA/pg/ml): Int. Std. x (Aliquot Factor) X 100 = (pA/pg/ml): Ext. Std. Recovery Aliquot Factor: l l / S = 1.375 (Blood assay); 11/10 = 1.10 (Urinary assay). Alternately, the per cent recovery can be determined from the slope values (pA/pg) of standard curves. The per cent recovery of the internal standards should be calculated routinely as a check on analytical precision. RESULTS AND DISCUSSION
The polarographic peak noted at -1,070 V us. SCE for I is due to the reduction of one of the azomethine bonds in the
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
265
Table I. Blood and Urine Levels in the Dog Following Oral Doses of I Total dose: 200 mg (16 mg/kg) of I. Administration: 2l/2 Bactrim tablets-wal (each tablet contains 400 mg sulfamethoxazole and SO mg trimethoprim) Blood level, pg I/ml Polarographic Fluorometric Sample time assay assay 10 min n.m. n.m. 20 min n.m. 0.2 35 min 0.5 0.6 45 min 2.7 2.4 1 hr 4.2 4.5 1 . 5 hr 4.5 5.0 2 hr 4.9 5.8 3 hr 4.6 5.3 4 hr 3.9 4.1 6 hr 4.0 4.7 7 . 5 hr 12 hr 24 hr 48 hr
3.7 1.8 n.m. n.m.
3.9 2.7 0.8 n.m.
Urine levels (polarographicmethod) I/ml Total mg I Dose
pg
0-24 hr 24-48 hr
235.5 42.0
74.1 26.9
37.0 13.5 Sensitivity limits: Fluorometric = 0.1 pg I/ml using a 1-ml sample per assay. Polarographic = 0.5 pg I/ml using a 1-ml sample
per assay. Table 11. Blood and Urine Levels in Man Following Oral Doses of I Total dose: 400 mg (6 mg/kg) of I. Administration: 5 Bactrim tablets-oral (each tablet contains 400 mg sulfamethoxazole and 80 mg trimethoprim) Blood level, pg I/ml Polarographic Fluorometric Sample time assay assay 1 hr 3.2 3.7 2 hr 4.4 4.9 3 hr 4 hr 6 hr 8 hr 12 hr 24 hr
4.3 3.9 3.4 3.2 2.2 1.4
4.8 4.4 4.2 3.6 2.8 1.4
Urine levels Polarographic assay I Fluorometric assay time, Total I Total hr pgI/ml mgI %Dose pgI/ml mgI %Dose SamPle
0-24 24-48 48-72
80 1 29 7 7 0 Sensitivity limits:
165 4 52 4 145
41 4 74 8 154 5 38 6 13 1 29 4 51 8 13 0 200 5 0 3 6 1 9 7 Fluorometric = 0.1 pg I/ml using a 1-ml sample per assay. Polarographic = 0.5 pg I/ml using a 1-ml sample
’
per assay.
pyrimidine ring (11-13). The peak is pH dependent in that it shifts to more negative potentials with decreased acidity and was not observed in basic solution. The choice of 0.1N HzSOa as the supporting electrolyte is dictated by the fact that the reduction of the >C=N-group gave a sufficiently sensitive signal (pA/pg) in this medium for measurement and the compound is quantitatively back-extracted from CHC13 into this medium, thus providing a useful means of removing biological interferences prior to analysis. 266
The overall recovery of I from blood and urine is 8 1 . 7 x =+ 6.3 (std dev) and 75.0% f 7.2 (std dev), respectively. The sensitivity limit of detection from blood and urine is of the order of 0.5-0.75 pg of I per ml of blood or urine. The correlation coefficient (16) between the spectrofluorometric and polarographic assays is 0.90. It should be noted that all samples showed a secondary peak at - 1.OOO V us. (SCE). This peak is usually larger in biological samples and is probably due to a co-extracted impurity, see Figure 2. This component is usually present in a reproducible manner, and it has negligible effect on the measurement of the analytical peak of I at - 1.070 V us. SCE. Further studies indicated that 0.01N H2S04 could be substituted, if desired, for the 0.1N H2S04 without any loss in recovery, but with a greater ease of measurement of the analytical peak due to the resolution gained between the analytical and the interfering peak, see Figure 3. Specificity of the Assay. BLOOD. The specificity of the blood assay was vertified by TLC which showed the absence of 11, 111, IV, and V in the blood of dog following the oral administration of 16 mg/kg of I or in the blood of a human subject given 6 mg/kg of I. The hydroxy analog (111) was not recovered using the blood assay procedure described. However, chronic administration of I may result in the presence of some of the above metabolites. In this event, the TLC separation employed in the urine assay must be included in the blood assay to ensure specificity. Sulfamethoxazole, the second active ingredient of Bactrim, is not extractable under the assay conditions and does not have polarographic activity under the conditions employed. URINE.The TLC separation step employed effectively resolves I from co-extracted biological impurities and from the reported metabolites, thus establishing the specificity of the urine assay. Application of the Method to Biological Specimens. DOG. Blood and urine levels were obtained in a dog following oral administration of 16 mg/kg of I in 2’iZ Bactrim tablets. Each tablet contained 400 mg of sulfamethoxazole and 80 mg of trimethoprim for a total dose of 1000 mg of sulfamethoxazole and 200 mg of trimethoprim. In Table I, the blood levels measured by the polarographic assay were compared to those obtained by the fluorometric assay (10). Both methods show analytically acceptable data with the fluorometric assay yielding greater sensitivity. Using the polarographic method to quantitate I in the urine, approximately 50% of the dose administered was found to be excreted as unchanged I in the 0-48 hour collection period (Table I). MAN. Blood and urine levels were obtained in a man following oral administration of 6 mg/kg of I in 5 Bactrim tablets. Each tablet contained 400 mg of sulfamethoxazole and 80 mg of trimethoprim for a total dose of 2000 mg sulfamethoxazole and 400 mg of trimethoprim. In Table 11, the results obtained by the polarographic and the fluorometric assay (IO) showed good agreement for both blood and urine. Approximately 50% of the dose was recovered as I in the 0-72 hour collection period. ACKNOWLEDGMENT
The authors wish to extend thanks to Robert Weinfeld for providing the fluorometric data presented in this manuscript. RECEIVED for review July 20,1972. Accepted October 2,1972. (16) M. G. Kendall and A. Stuart, “Advanced Theory of Statistics,” Vol. 2, Charles Griffin and Company Ltd., New York, N.Y., 1961, pp 375-415.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
