Determination of Propoxyphene in Human Plasma by Gas Chromatography Robert L. Wolen and Charles M. Gruber, Jr. Lilly Laboratory f o r Clinical Research, Marion County General Hospital, Indianapolis, Ind. 46202
A method for the measurement of propoxyphene (~~-d-dimethylamino-1,2-diphenyl-3 -methyl - 2 propion-
It is based
on oxybutane) in plasma is reported. solvent extraction at neutral or near neutral pH values followed by purification and concentration of the extract. The extract is analyzed by gas chromatography using a short column with a liquid phase of silicone gum rubber. Quantitation is possible through the inclusion of d-pyrroliphene hydrochloride (CY-d-2acetoxy 1,2 -diphenyl - 3 methyl 4- pyrrolidinobutane hydrochloride) as a mass internal standard which is carried through the extraction. The ratio of chromatographic peak heights of propoxyphene to pyrroliphene is compared to ratios derived from standards prepared in plasma and treated in the same manner as the sample. Patient data is presented indicating considerable patient to patient variation in maximum plasma concentrations at a fixed dose and in the rate of clearance of propoxyphene from the plasma.
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WITH THE SYNTHESIS ( I ) and introduction of propoxyphene 2 - propionoxybu(a-d-dimet hyl-amino-l,2-diphenyl-3-methyltane), its use has been widespread. A number of studies relating to the analgesic properties of d-propoxyphene have been reported (2-5). Studies relating to its metabolic fate (6), an analytical method by chromatography (7), and a study of the urinary excretion pattern (8) have also been published. Emmerson (9) recently reported the tissue concentrations of this drug in animals by using N-methyl-14Clabeled propoxyphene. Each of these studies suggests a need for determinations of plasma concentrations following the administration of therapeutic doses. This report deals with a method which accomplishes this goal. The concentration of propoxyphene in biologic fluids is so low following oral administration of the usual therapeutic doses to human volunteers that it presents a problem which has only recently been solved. The method requires the use of gas chromatography (IO)and of a mass internal standard (11) and permits the accurate determination of small amounts of material after they are carried through a series of analytical steps.
(1) A. Pohland and H. R. Sullivan, J. Amer. Chem. Soc., 7 5 , 4458
(1953). (2) E. B. Robbins, J. Amer. Pharm. Ass., Sci. Ed., 44,497 (1955). (3) C. M. Gruber, Jr., J. Amer. Med. Ass., 164, 966 (1957). (4) S. M. Chernish and C . M. Gruber, Jr., J. Amer. Geriat. Soc., 12, 249 (1964). (5) A. Baptisti, S. M. Chernish, and C. M. Gruber, Jr., Arch. Intern. Pharmacodyn., 159, 234 (1966). (6) H. M. Lee, E. G. Scott, and A. Pohland, J. Pharmacol. Expt/. Therap., 125, 14 (1959). ( 7 ) J. L. Emmerson and R. C. Anderson, J. Chromatogr., 17,495 (1 965). (8) M. E. Amundson, M. L. Johnson, and J. A. Manthey, J. Pharm. Sci., 54, 684 (1965). (9) J. L. Emmerson, J. S. Welles, and R. C . Anderson, Toxicol. Appl. Pharmacol., 11, 482 (1967). (10) L. Kazyak and E. C. Knoblock, ANAL. CHEM.,35, 1448 (1963). (11) M. Sparagana, Steroids, 5 , 7 7 3 (1965).
EXPERIMENTAL
Apparatus. The gas chromatography was carried out on an F & M Model 402 chromatograph with an all glass U-tube column system. The instrument is equipped with a hydrogen flame ionization detector. The column is one foot long with an internal diameter of 4 mm. Samples were taken to dryness on a multiple flash evaporator of the vortex type (Evap-0-Mix, Buchler Corp., Fort Lee, N. J.). Reagents. The chloroform utilized in the final extraction is spectral grade, all other solvents are reagent grade. The labeled propoxyphene used was either N-methyl-14C dpropoxyphene hydrochloride or benzyl 14Cd-propoxyphene. The column packing is 3.8% W98 silicone rubber on SOjlOO mesh Diataport S which is subsequently silicone treated by coating with 0.25 dichlorodimethylsilane or on-column exposure to Silyl-8 column conditioner (Pierce Chemical Co., Rockford, Ill.) at a column temperature of 200 "C with appropriate gas flow. Sample Preparation. An aqueous solution of the mass internal standard, pyrroliphene hydrochloride (a-d-2-acetoxy1,2-diphenyl-3-rnethyl-4-pyrrolidino-butane hydrochloride), is prepared at a concentration of 0.025 mg/ml. A uniform amount (0.1 ml) of this solution is added to each 5-ml aliquot of plasma to be assayed. The extraction is carried out in a siliconized tube, with a Teflon lined screw cap. The mixture is extracted twice, each time using 5 ml of n-butyl chloride, by mixing 5 minutes in a mechanical shaker. Emulsions are broken by freezing in a dry ice ethanol bath. Organic and aqueous layers are separated throughout the procedure by centrifugation at 2200 RPM for 5 minutes. The butyl chloride phase is collected from each extraction and pooled in a centrifuge tube with cap. The butyl chloride is then extracted with 5 ml of 0.2N HC1 by shaking for two minutes. After centrifugation the organic layer is aspirated and discarded. The acid extract is washed one time with 5 ml of n-hexane which is aspirated and discarded after centrifugation. The washed acid phase is brought to a pH of between 11 and 12 with 0.2 ml of 6N NaOH and extracted with 10 ml of chloroform by mechanical shaking for 5 minutes. The chloroform is recovered after centrifugation and placed in a 15-ml glass-stoppered centrifuge tube. The final extract is taken to dryness on a flash evaporator, tubes are rinsed with 1 ml of fresh chloroform and again taken to dryness to assure collection of the sample in the lower portion of the tube. The residue is dissolved in 12 to 16 microliters of carbon disulfide and of the solution injected into the gas chromatograph. Chromatographic Conditions. The temperatures utilized are 205 "C at the flash heater, 172 "C (isothermal) at the column, and 195 "C at the detector. Helium is the carrier gas at a flow rate of 60 ml/minute (40 psi), hydrogen flow to the detector is 25 ml/minute (20 psi), and oxygen flow (20 psi) is adjusted to give maximal detector response to propoxyphene. The electrometer response is range 10, attenuation four with attenuation changes made as required to accommodate any variation in sample size, or concentration. Data Handling. Following chromatography, the base line is drawn and peak heights, as well as width at */2 peak height, are recorded for the propoxyphene and pyrroliphene peaks. The ratio of propoxyphene peak height to that of the inVOL. 40, NO. 0, JULY 1966
* 1243
20
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0
o-c-cH$
I FW$
15I
z
x
10-
E
8
u
-
~-PROPOXYPHENE HCL
5-
d-PYRROLIPHENE BCL
Figure 2. Structural formulae of d-propoxyphene HCl and d-pyrroliphene HCI
0 3
4
5
6
7
8
9
1
0
1
1
pH-Plarma
Figure 1. Benzene extraction of benzyl-14Cd-propoxyphene from plasma
ternal standard, corrected for attenuation if necessary, is calculated. Samples showing gross differences in width at '/z peak height are reinjected and a second chromatogram is obtained. Quantitation is carried out by utilization of a standard curve constructed from values of known concentration of propoxyphene hydrochloride prepared and equilibrated in propoxyphene-free human plasma. The ratio of peak heights is plotted against concentration of propoxyphene in pg/ml of plasma. Samples containing less than 5 ml of plasma are corrected for volume after calculation of concentration based on 5 ml. All values are in terms of propoxyphene hydrochloride equivalents. Tracer Study. The use of radioisotope labeled material permits examination of the separate steps of the extraction procedure. Tracer quantities of either N-methyl- 14C dpropoxyphene hydrochloride or benzyl I4C d-propoxyphene hydrochloride were added to samples prior to processing. Samples taken at various steps in the analytical procedure were processed and counted in a dioxane based scintillation liquid (12) using conventional liquid scintillation counting equipment. Corrections for quenching were made either by external standardization, internal standardization, or channels ratios method. In Vivo Study. In vivo evaluation provided information relative to possible applications and limitations in the design of future experiments. So far only pilot studies have been undertaken. In these experiments capsules containing 32 or 65 mg of d-propoxyphene hydrochloride were administered with 200 ,ml of water to fasting, healthy subjects. Food and water were then restricted for an additional two hours. Samples of blood (10 ml) were drawn into heparinized tubes (Vacutainers) at 0, 'Iz, 1, 2, 3, 4, 5 l,I2, 7 1/2, 9, and 24 hours after drug administration. They were centrifuged within 15 minutes; the plasma was removed and then stored in the frozen state at -20 "C until assayed.
n-butyl chloride shows little or no pH dependence between pH6.1 andpH11.3. The choice of hexane for wash of the acid extract is based upon isotope data which indicates that this solvent, while providing a c adequate wash, contributes minimally to the loss of propoxyphene. Other solvents including butyl chloride, benzene, and chloroform also removed interfering materials but resulted in higher losses of propoxyphene. The chloroform, in fact, is able to remove as much as 84% of the labeled propoxyphene from 0.1N HC1. This led to the use of this solvent in the final step of sample preparation in order to assure a maximum recovery of the drug. Overall recovery determinations carried out with labeled propoxyphene vary somewhat because of differences in the volume recovered. The range of recoveries in four runs was 91.6% to 104.4% with a mean recovery value of 97.9x. The choice of an internal mass standard is based on a need for a compound which will follow the desired compound
'-1 I 90
RESULTS AND DISCUSSION Sample Preparation, The initial procedure utilized benzene for extraction from the plasma. During this extraction it became apparent that the ability of the solvent to remove propoxyphene from plasma was pH dependent. Figure 1 indicates the recovery of added 14Cpropoxyphene from plasma as it related to pH. It is obvious that at high pH values there is a marked decrease in recovery although the propoxyphene should be in the free base form. A pronounced increase in the strength of protein binding is observed at elevated pH values when the technique of gel filtration is used. On the other hand, recovery of the propoxyphene from plasma by (12) R. J. Herberg, ANAL.CHEM.,32, 42 (1960).
1244
ANALYTICAL CHEMISTRY
20
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d
0 1
0 I
2
I
I
4 TIME
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I
6 8 MINUTES
I
10
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12
d
Figure 3. Chromatogram of an extract of plasma to which 0.100 pg/ml of d-propoxyphene HCl has been added and which has been equilibrated at 37" C for 15 minutes prior to extraction
D
=
propoxyphene; S
=
internal standard (pyrroliphene)
-E
. 01
2
4
IO
6
24
Time in hours
Time in hours
Figure 4. Propoxyphene levels in patient plasma expressed in terms of propoxyphene hydrochloride
Figure 5. Propoxyphene levels in patients’ plasma
Curves represent plasma levels on the same patient at one week intervals. -- 130mgdose; . . = 65 mg dose; - - - - = 32 mg dose
during the extraction and which can be separated satisfactorily on the gas chromatograph. Further, the compound of choice should be readily available and of sufficient purity to be free from interfering peaks. Ideally an isomer or homolog is chosen for this purpose. Figure 2 presents the structures of d-propoxyphene and of d-pyrroliphene hydrochlorides. Their structural similarity is apparent with the latter having a lower molecular weight by approximately 23. Other compounds of similar structure have been tried but they lacked either the needed purity or were not separable under the chromatographic conditions employed. Partition coefficient studies of the two compounds with the appropriate organic phases and buffers have shown their behavior to be quite similar at the
Each subject received a single dose of 130 mg of d-propoxyphene HCI at time 0. * ,” . = 79.8 kg male; o . . . , o = 68 kg male; x - - - x = 68 kg female
pH values used in the assay. Recovery studies on the internal standard have not been quantitated but recovery appears constant under conditions of the assay. Gas Chromatography. The choice of carbon disulfide as the final solvent is based upon inability of the flame ionization detector to respond to this compound. Figure 3 presents a typical chromatogram. Variation in the amount of pyrroliphene added between 0 and 1.5 pg/ml shows no effect on the amount of radio propoxyphene recovered under the described conditions. It can be seen from this chromatogram that the internal standard is well separated from the propoxyphene. The paper is run at 30 inches per hour in order to facilitate measurement of peak width at l/* height. Plasma blanks obtained from subjects not receiving propoxyphene and from blood bank plasma have been free of interfering peaks with few exceptions. In those cases where an interference is noted, a correction has been used in subsequent calculations. Plasma Determinations. Standard curves are usually established in the range of 0.05 to 0.300 pg/ml range; however, linearity extends through 1.5 pg/ml. The slope of the line varies with changes in the concentration of added internal
Table I. Determination of Propoxyphene Added to Human Plasma R(diff.)
=
0.133,
t = 0.164, t(p-0.8
t(o-0.8,
Assay values
PP/l
4 1
80 80
100 100 100 140 140 150 200 250 300
df-14)
=
0.128,
= 0.258
Concentration 40 40 50 75
1 C
di-14)
Difference
40 43 52 77 78 78 99
0 $3 +2
101 102 137 145 155 195 250 295
+1 +2 -3 +5
$2 -2 -2 -1
+5 -5
0 -5
VOL. 40, NO. 8, JULY 1968
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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).
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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).
