standard but remains linear. It is possible to increase the concentration of the internal standard to twice that used in this study and to decrease the sensitivity of the electrometer accordingly during the elution of the standard still obtaining essentially the same standard curve. The concentration of internal standard has been altered in sequential experiments without difficulty in analysis. Table I presents typical values for determinations of propoxyphene added to plasma. In this series of 15 determinations, the t for the difference between known and determined concentrations was 0.164. All samples were equilibrated with the plasma at 37 "C before extraction. The storage of plasma samples has been examined. No significant changes take place in plasma values when stored for one month in the frozen state at -20 "C. If storage is at 5 "C, there is a significant decrease in assayed values after three days of storage. Patient Data. The concentration in the plasma of one subject following the administration of different doses of the medication is shown in Figure 4. It is apparent from this figure that the maximum concentration obtained is proportional to the dose and that the shape of the curve does not change appreciably. Figure 5 presents the variation in plasma concentration when the same dose is given to different subjects. This varia-
tion does not appear to be related to dosage on a mg/kg basis. It seems to be related to variation in factors such as absorption, metabolism, and/or excretion rates. The data presented in the semilogarithmic plot (Figure 6) corresponds to that reported in Figure 5. It demonstrates apparent differences in the rate of clearance of propoxyphene from the plasma. The half life periods of the drug vary from 5.1 hours for Patient A to 1.1 for Patient C. The half life periods are in the order of decreasing value with decreasing maximum plasma values. The half life for Patient B when given 65 mg of propoxyphene (data reported in Figure 4) was 2.5 hours as compared to 2.7 hours with the 130-mg dose. In this patient, the response to 32 mg did not provide data which could be used to determine a half life value. ACKNOWLEDGMENT
The authors thank R. E. McMahon and F. J. Marshall who prepared the labeled propoxyphene and D. L. Clapp who carried out much of the experimental work. RECEIVED for review February 12, 1968. Accepted April 29, 1968. d-Propoxyphene HCl is marketed as Darvon by Eli Lilly and Co., compound #31518.
Determination of Brompheniramine in Blood and Urine by Gas-Liquid Chromatography Robert B. Bruce, Jefferson E. Pitts, and Franklin M. Pinchbeck A . H . Robins Co., Inc., Research Laboratories, Richmond, Va.
A method has been developed for the determination of the submicrogram quantities of brompheniramine that occur in blood following normal dosage. The procedure involves separation of brompheniramine by extraction and partition chromatography, followed by oxidation to p-bromophenyl-2-pyridyl ketone. The ketone i s determined by GLC using an electron-capture detector and an SE-30 column. The method is sensitive to less than 10 ng of brompheniramine per ml of blood. THEPHENIRAMINES are used in many pharmaceutical preparations as antihistamines. The purpose of this paper is to present a method which has been satisfactory for the determination of bromphenirarnine in blood and in urine. This method has the necessary sensitivity to measure less than 10 ng of brompheniramine, which is required if blood levels are to be determined following the normal human dose of 4 to 8 mg of drug. This method could be easily adapted to the determination of pheniramine or chlorpheniramine. Lapidus and Lordi (1) have used an ultraviolet method for measuring the release of chlorpheniramine from tablet p r e p arations. A fluorescent method reported by Jensen and Pflaum (2) offers another means of quantitating these compounds. These two methods, however, did not provide the sensitivity required, and naturally occurring amines in bio(1) H. Lapidus and N. G . Lordi, J. Pharm. Sci., 55 (8), 8@3 (1966). (2) R. E. Jensen and R. J. Pflaum,ibid., 53 (7), 835 (1964).
1246
ANALYTICAL CHEMISTRY
logical material interfered. Several methods have been reported (3, 4 ) which utilized gas chromatography as a tool for the separation of antihistamines. Beckett and Wilkinson (5) determined chlorpheniramine in urine using gas-liquid chromatography. This method was stated to provide the desired specificity and accuracy but no details were given. Initially, we sought to determine brompheniramine by GLC using a flame ionization detector. This procedure was effective for determining the compound in urine extracts, but did not provide enough sensitivity for blood concentrations and, in addition, the blood extracts contained some naturally occurring basic materials which interfered. While the bromine in the molecule appeared to possess electron capturing properties, the tritium foil detector did not permit high enough operating temperatures to investigate the unaltered molecule. Previous work in this laboratory (6), as well as some later work by others (7, 8), involving the oxidation of compounds containing a diphenyl methyl moiety to benzophenone led to (3) Alexander MacDonald, Jr. and Ronald T. Pflaum, J . Phnrm. Sci., 52, 816 (1963). ( 4 ) Leo Kazyak and Edward C. Knoblock, ANAL. CHEM.,35, 1448 (1963). (5) A. H. Beckett and G . R. Wilkinson, J. Pharm. Pliarmacol., 17,256 (1963). (6) R. B. Bruce, J. H. Newman, J. E. Pitts, and F. M. Pinchbeck, J . Med. Chem., 8, 157 (1965). (7) Jack E. Wallace, J. Forensic Sci., 11 (4), 552 (1966). (8) J. E. Wallace, J. D. Biggs, and E. V. Dahl, ANAL.CHEM., 38, 831 (1966).
the development of the method described below. This method involves the oxidation ofbrompheniramine to the c o p responding ketone and its subsequent determination by GLC. A different pheniramine, which is oxidized in the Same way, serves as an internal standard. In addition, partition chromatography is used to separate the parent compound from its two amine metabolites (9) prior to oxidation. Thus, unchanged brompheniramine as well as brompheniramine in combination with its basic metabolites may be determined in biological material.
Brompheniramine
Q
CH-CH2-CH2-N
Reagents. Reagent grade ethyl ether (B and A, 1700) after redistillation was used for the extraction of blood and urine. The isooctane was purified by passing 5 liters of technical grade isooctane through a silica gel column 4.5 cm x 35 cm, and distilling the eluate. Acid-washed Celite 545 was used as the solid support for the liquid-liquid partition chromatography, and contained 2 ml M p~ 5.4 phosphate buffer per 3 grams of Celite. Chlorpheniramine was used as the internal Method. standard for the determination of brompheniramine. As
/cH3
C ' H3
Br I Br I
I
50mm
T
65rnni
i
25 rnm
t
60mm
1 Figure 1. Oxidation tube and air condenser EXPERIMENTAL Apparatus. The gas chromatograph used in these studies was a Wilkens Aerograph Hy-Fi, Model 600-D, equipped with a tritium foil electron capture detector. The column wa 4-foot x 1/8-inch stainless steel packed with 5 x SE-30 on Gas Chrom Q (100- to 120-mesh). The column temperature was 182 "C., nitrogen flow 12 ml/min, injector temperature 300 "C, and the detector temperature 190 "C. A range setting of 1 and attenuation 8 (8-mV full scale deflection) was used for all samples run. A special oxidation tube was constructed which consisted of a 150- X 15-mm tube with a 14/20 joint and a center constriction 25 mm. long and 6 mm. in outside diameter (Figure 1). This tube permits oxidation under an air condensor (150 X 6 mm), extraction into 0.2 ml of solvent without transfer, and easy removal of the solvent layer. (9) R. B. Bruce, L. B. Turnbull, J. H. Newman, and J. E. Pitts, J. Med. Chem., in press (1968).
the internal standard is added to the initial blood sample, it is not necessary to recover total extracts in the procedure. A sample of 5.0 ml of blood and one of 0.5 ml of a solution containing 100 ng of chlorpheniramine were mixed thoroughly on a Vortex mixer in a 100-ml glass-stoppered centrifuge tube. While the contents were being mixed, 0.5 ml of 10N KOH was added. The tube was heated in boiling water for 20 minutes. After cooling, the sample was extracted by shaking gently for 10 to 15 minutes on a mechanical shaker with 95 to 100 ml of ether. After being centrifuged, the ether was transferred to another 100-ml centrifuge tube and 1 ml of 0.1N H3P04 added. The volume of ether was reduced under a gentle stream of nitrogen to approximately 10 ml and extraction into the acid completed by mixing on the Vortex mixer for 1 minute. The acid layer was transferred with a pipet to a test tube and the ether washed with an additional 0.5 ml of 0.1N H3P04 and the acid layers combined. The extracted amines were then returned to ether by making the solution alkaline with 3 to 4 drops of ION KOH, saturating with VOL. 40, NO. 8, JULY 1968
1247
Table I. Recovery of Known Amounts of Brompheniramine Maleate Added to 5.0 ml of Control Blood Brompheniramine, ng Recovery, Added Found 58.5 117 117 117 176
PH 2 3 4 5 6 7 8 9 10 5
59.5 111 123 110 179
101 95 105 94 102
Table 11. Partition. of Amines between Ether and 1M Buffer Solutions BromphenPrimary Secondary iramine amine amine 0 0.025 0.067 0.122 1.25 7.45 16.6 27.4 51.4
0 0 0 0 0.038 0.104 1.74 5.62 12.8
0 0 0 0 0.028 0.227 4.29 13.8 22.2
Ratio of absorbance of ether layer t o aqueous layer.
NaC1, and extracting three times with 2 ml, 1 ml, and 1 ml of ether, The ether extracts were placed on a buffered Celite column (1.3 gram in a 7-mm diameter tube) and the unchanged brompheniramine was eluted with 15 ml of ether (equilibrated with M pH 5.4 phosphate buffer). The brompheniramine was extracted into 1 ml of 0.1N H3P04and the acid was transferred to an oxidation tube and washed twice with ether. The ether was discarded and the acid heated in boiling water to remove the final traces of ether with aspiration of the vapors. After cooling, 0.25 ml of 2N KOH and 3 ml of 1% KMn04 were added. The tube was heated, under a n air condenser, in boiling water for 15 minutes. The tube was cooled, the condenser washed with 1 to 2 ml of water, and the solution extracted with 0.20 ml of isooctane. After the layers had separated, additional water was added so that the isooctane layer was in the constriction. Aliquots of 10 to 20 p1 were injected into the chromatograph. Brompheniramine together with its basic metabolites was determined by omitting the partition chromatography. In the determination of brompheniramine in urine, the same procedures are followed. However, because the drug concentration is so high, it is necessary to dilute the urine before analysis. In addition, total excreted drug-related material can be determined without extraction by direct oxidation of diluted urine. RESULTS AND DISCUSSION
A considerable portion of the preliminary experiments to investigate the feasibility of this method was carried out using UV spectrophotometry. This entailed the use of larger amounts of brompheniramine but quantitation was easier and more rapid. Brompheniramine has a n absorption maximum at 265 mp in acid solutions and a maximum at 258 mp in nonpolar solvents. The ketone resulting from brompheniramine oxidation shows a maximum at 270 mp in nonpolar solvents. Extraction. Cochin and Daly (10) extracted antihistamines, including the pheniramines, from blood and urine (10) Joseph Cochin and John W. Daly, J. Pharm. Exptl. Therp., 139, 160 (1963).
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ANALYTICAL CHEMISTRY
with methylene chloride-isoamyl alcohol. This solvent was not tried because the high sensitivity of the electron capture detector t o chlorinated solvents could lead t o contamination problems. Brompheniramine is fairly easily extracted from aqueous solutions with ether. Approximately 88 % is partitioned from H20 into a n equal volume of ether at pH 7. Such recoveries could not be obtained from addition of known amounts of brompheniramine t o blood. By increasing the alkalinity of the blood and by using a large volume ratio of ether, most of it could be extracted. The extraction properties of chlorpheniramine and brompheniramine are identical under these conditions. Evaporation of ether solutions of brompheniramine apparently leads t o its adsorption to the walls of the vessel and it cannot be removed with ether. Thus, a solution of brompheniramine in 25 ml of ether in a 50-ml glass centrifuge tube was evaporated to dryness in a stream of nitrogen. The tube was washed three times with 5-ml portions of ether and these extracts were evaporated in an oxidation tube. No brompheniramine was recovered. The washing was repeated with 0.1N H3P04, and the wash analyzed. The brompheniramine was now recovered. Ethanol was also effective for removing the drug from the glassware. Because this adsorption could cause appreciable contamination of the equipment used in these studies, all glassware was rinsed with ethanol and dried immediately before use. The addition of alkali to blood without dilution gave a gel which was almost impossible to wet with ether for extraction. Heating the alkaline blood in the steam bath gave a fluid mixture which was easily extracted. Such treatment did not affect the pheniramines present and it was possible t o use a large volume ratio of ether to blood. The recovery of brompheniramine from blood without the partition chromatography was satisfactory, as can be seen from Table I. Partition Chromatography. The extraction described above not only removed brompheniramine from blood but also removed basic metabolites which would also be determined as brompheniramine by the procedure for quantitation. Two such metabolites have been found: the mono- and didemethylated derivatives. A third basic metabolite is watersoluble and is not extracted into ether. Methods were investigated for the separation of brompheniramine from these metabolites. Acetylation has been used for similar separations but is not applicable in this case because the pyridine ring still gives a basic molecule. The reaction with 3-nitrophthalic anhydride (11) was also investigated and although this offered more promise, separation was still not complete. The partition characteristics between buffered solutions and ether were investigated (Table 11) and the results indicated that at below pH 6 separation should occur. A partition column was prepared from 3 grams of acid-washed Celite 545 and 2 ml of 1M phosphate buffer (pH 5.4). A mixture of brompheniramine and its two amine metabolites was applied to the column and eluted with ether. The maximum elution peaks (obtained by UV spectrophotometry) for brompheniramine, its secondary amine, and the primary amine were 15 ml, 125 ml, and 320 ml, respectively, indicating good separation. Chlorpheniramine was eluted in the same region as brompheniramine. This procedure was scaled down for nanogram quantities and recoveries were studied by GLC. (11) J. W. Alexander and S . M. McElvain, J. Am. Chem. Soc., 60, 2285 (1938).
Table 111. Separation of Brompheniramine from Its Metabolites by Partition Chromatography Brompheniramine Brompheniramine, Secondary amine, Primary amine, Brompheniramine, recovered, ng recovery ng placed on column ng placed on column ng placed on column 0 0 0 0 355 355 355 355 355
0 0 335 335 0 0 335 335 335
88 88 0 0 0 0 125 70 35
86.7 91.5 0 0 0 0 132 72 37
98.5 104 0 0 0 0 105 103 105
Table IV. Recovery of Brompheniraminefrom Urine Brompheniramine, n Added Found 46.8 351 468
42.7 ( + 0 . 5 ) 368 (&7) 473 (*4)
g
No. of
detn. 5
8 5
Re1 std dev, Z 1.9 3.1 3.2
A
The results are shown in Table I11 and show complete separation of brompheniramine from its metabolites. GLC. A number of column materials were tried for GLC separations but the one described above proved most satisfactory for the ketones. Several 5 % SE-30 columns have been prepared and used in these studies. They all proved satisfactory, but the retention times varied somewhat with different columns using the same temperature and flow rates. These parameters are adjusted so that a retention time for p-bromophenyl-2-pyridyl ketone is 4.5 minutes and that of the chloro derivative is 3 minutes. Variations in the temperature of the column oven have been from 170 to 190 "C and the nitrogen flow rate from 12 to 24 ml/minute. A typical chromatogram of a mixture of phenyl2-pyridyl ketone, the bromo and chloro derivatives, is shown in Figure 1. The order of elution is in order of increasing molecular weight, as is usual with SE-30 columns. The response to the halogenated derivative is considerably greater than that of phenyl-2-pyridyl ketone, as might be expected. However, phenyl-2-pyridyl ketone shows sufficient response so that pheniramine could be determined at higher concentrations. The relative peak height responses can be seen from Figure 2, Typical chromatograms obtained in a blood level study are shown in Figure 3. Figure 3a shows the re-
Figure 2. Gas chromatogram of oxidation products of A , pheniramine, 42 ng; B, chlorpheniramine, 4.9 ng; C, brompheniramine, 5.6 ng. 10 /*I of solution injected
Table v . Blood Levels of Total BrompheniramineAmines and Unchanged Brompheniramine Following Administration of 8 Mg to Two Subjects F J Total brompheniramine Total brompheniramine Hours after dose amines, ng/ml Brompheniramine, ng/ml amines, ng/ml Brompheniramine, n g / d 0.5 1 2 3 5 7 9 24
0 0 8.3 14.4 14.1 16.1 15.5 10.0
0 0
7.7 12.3 11.8 9.2 9.1 3.8
0 10.5 21.8
...
18.5 20.8 18.8 14.9
0 10.3 18.8 17.0 15.9 15.3 12.4 6.7
VOL. 40, NO. 8, JULY 1968
1249
C
d a
1
U
b
Figure 3. Gas chromatograms obtained for ( a ) control blood to which chlorpheniramine had been added and (6) from a subject 6.5 hours after receiving 8 mg of brompheniramine Volume injected, 10 ~1 in each case
sponse obtained from control blood with chlorpheniramine added, and Figure 36 the response 6.5 hours after the subject had received an oral dose of brompheniramine maleate. Oxidation. The oxidation of the pheniramines to their corresponding ketones was studied under a number of conditions. Oxidation could be carried out using chromic acid but those conditions described above appeared to give the most consistent results. The yield of ketone is not quantitative but is consistent. That the oxidation product is the corresponding ketone has been shown by obtaining the same elution time for authentic samples of p-bromophenyl-2pyridyl ketone and phenyl-2-pyridyl ketone as is obtained in the method. In addition, a larger quantity was prepared by the analytical method and its IR and NMR spectra were identical with those obtained from authentic samples. Accuracy and Precision. The accuracy and precision of the method are indicated by the results shown in Tables I, 111, and IV. The relative standard deviation for the recovery of brompheniramine from blood was *.5.2% at the 88-ng level with a mean error of 0 . 3 z from 10 determinations. The recovery from urine is shown in Table IV for replicate determinations. Blood Levels. The results of a study of brompheniramine blood levels is shown in Table V. These two male subjects
1250
e
ANALYTICAL CHEMISTRY
each received a single oral dose of 8 mg of brompheniramine maleate (two Dimetane tablets). Aliquots of blood were taken at intervals and analyzed for both total drug related amines and for unchanged drug. These results would indicate that the method of analysis is satisfactory. A detailed study of blood levels of brompheniramine will be presented in a future publication. RECEIVED for review February 9, 1968. Accepted April 30, 1968.
Correction Modified Molecular Sieve Reflux Extractor for Efficient Dust-Free Dehydrations In this article by David S. Rulison, Paul Arthur, and K. Darrell Berlin [ANAL.CHEM., 40, 1015 (1968)], several errors appeared. The Zip Code on line 3 should read 74074. On page 1015, column 2, line 9 should read “edges of the tube which fits around the shaft of part D.” On page 1016, column 1, the third line from the bottom should read “molecular sieve dust which is washed down into the flask.”
