For the volume of reaction zone in this study the incident flux is therefore 7.36 x 10'8 photons/min. This is a reasonably large value for a low-pressure lamp and demonstrates the benefits of the long length and close proximity of the reaction zone and source; the aluminum reflector also increases the intensity. Figure 2 also demonstrates the good agreement between theory and experiment. By taking the value of 10calculated from the initial slope of Figure 2,using the molar absorptivity at 254 nm (Table I), and assuming that the i.d. of the tubing, 0.6 cm, is b, one calculates that the exponential term becomes significant at an iron(III)-oxalate concentration of 2.65 mM. For the concentration of generator used here, 4.18mM, deviation from linearity should occur at about 65 photoconversion. This is the case as can be seen in Figure 2. It would be possible to construct a sliding shutter with calibration marks so that a variety of titrant concentrations could be
selected with a single flow rate, thus eliminating the need for a variable pump. Some conclusions can be drawn regarding the practical value of external photochemical generation. First, it is clear that reproducible flow rates and lamp intensity can be obtained with reasonable care. Second, it is possible to obtain a sufficient intensity from a low-power lamp to generate useful titrant concentrations, and the intensity is constant over many months of continuous operation. It should be noted that in situ titrations employ medium-pressure lamps which must be turned off when not in use ( I , 2). Finally, the titrant concentration can be easily changed by changing the length of the photolytic reaction zone. RECEIVED for review December 9,1968. Accepted March 21, 1969. Taken in part from Ph.D. Thesis of H. D. Drew; presented at the 156th National ACS Meeting, September 1968,Atlantic City, N. J.
Determination of Amphetamine and Related Amines in Blood by Gas Chromatography Robert B. Bruce and William R. Maynard, Jr. A . H.Robins Co. Inc., 1211 Sherwood Avenue, Richmond,Vu. 23220 A NUMBER OF METHODS have been presented during recent years for the determination and identification of amphetamine and its derivatives in urine. These methods are based primarily on GLC (1-3) and TLC (4-7) or specific color reactions (8, 9). However, none of these procedures are applicable to the determination of these drugs in blood because of their lower limits of detection. Axelrod (IO)adapted a nonspecific colorimetric procedure to the determination of amphetamine in plasma and tissues of animals that received relatively large doses. The method described below is based on the formation of the heptafluorobutryl derivatives of four of these amines and their subsequent determination by GLC using an electroncapture detector. Halogenated amide and ester derivatives prepared from amines and alcohols usually give high sensitivity with the electron-capture detector. A number of these were prepared and from the results obtained it appeared that the heptafluorobutyramides gave the best results.
(1) A. H. Beckett and M. Rowland, J. Phurm. Phurmacol., 17, 59 (1965). (2) H. Brandenberger and E. Hellback, Helv. Chim. Acta, 50, 958 (1967). (3) C. R. Hall, V. Cordova, and F. Rieders, Pharmucologist, 7, 148 (1965). (4)B. Davidow, Psychopharmucol. Bull., 3, 30 (1966). ( 5 ) H. Eberhardt and M. Debackere, Armeim.-Forsch., 15, 929 (1965). (6) M. Debackere and A. M. Massart k e n , Arch. Intern. Pharmucodyn., 15, 459 (1965). (7) M. L. Weischer and K. Opitz, Arzneim.-Forsch., 17, 625 (1967). ( 8 ) M. S. Karawya, M. A. El-Kiey, S. K. Wahba, and A. R. Kozman, J. Pharm. Sci., 56, lo05 (1967). (9) R. D. Eastman and P. A. G. Cox, Brit. Med. J., 1965,(5439) 924. (10) J. Axelrod, J. Phurmacol. Exp. Therap., 110, 315 (1954).
EXPERIMENTAL
Reagent. Heptafluorobutyric anhydridepierce Chemical Co., Rockford, Ill. Keep refrigerated. Method. An internal standard is used in each determination. Any of the other three amines may be used as the standard for the amine being determined. The internal standard is added to the blood sample before the initial extraction. Chlorphentermine (0.5-2 y) was used as the internal standard for amphetamine, methamphetamine, and fenfluramine. Amphetamine was the internal standard when chlorphentermine was being determined. The procedure for the analysis of a blood sample is as follows: Add the internal standard to 5.0ml of blood, mix and add 1.0 ml of 2 N NaOH and 5.0 ml of water. Extract the sample with 10.0 ml, then 5.0 ml of redistilled, chromatoquality pentane. Centrifuge the sample after each extraction and pass the pentane through a layer of anhydrous Na2SO4. After passing both extracts through the NatSOa, wash the Na2S04 with an additional two ml of pentane. Evaporate the pentane to dryness under a gentle stream of nitrogen and add 100 pl of heptafluorobutyric anhydride. Allow the reaction to proceed for 30 minutes with occasional mixing. Evaporate the excess heptafluorobutyric anhydride with a gentle stream of nitrogen and cool the tube in an ice bath. Add 0.5 ml of 2 N NaOH, keeping the mixture cold, mix and extract with 4, 3, and 3 ml portions of pentane, filtering each portion through anhydrous Na2S04 after each extraction. Wash the sodium sulfate with an additional 2 ml of pentane. Allow the sample to stand overnight at this point. Evaporate the pentane under a gentle stream of nitrogen. Dissolve the residue in 0.20 ml of pentane and inject 2 to 4 p1 into the gas chromatograph. A Barber-Colman gas chromatograph with a saNi detector was used in this study. The column was stainless steel, 6 feet long by l / d r in o.d., with 5% OV-1 on Gas-Chrom Q as the stationary phase. The temperature of the oven was 110 "C, injection port 250 "C, detector 250 "C, and the nitrogen flow rate 30 ml/minute. The retention times found under VOL. 41, NO. 7, JUNE 1969
977
h A
C
t
Figure 2. Comparative responses given by equal amounts (2 mg) of the heptafluorobutyramide derivatives of (A) fenfluramine, (B) methamphetamine, and (C) ethamphetamine
A
B C
D
Figure 1. The electron-capture response from the injection of 400 picograms, each of the heptafluorobutyramide derivatives of (A) amphetamine, ( B ) methamphetamine, (C) fenfluramine, and (0) chlorphentermine these conditions were : amphetamine-3 minutes, methamphetamine-5 minutes, fenfluramine-6 minutes, and chlorphentermine-8.8 minutes. It was found that pre-conditioning of the column with a pentane solution of heptafluorobutyric anhydride was necessary in order for the column to respond. A 1 to 4 mixture of heptafluorobutyric anhydride in pentane was used in this preconditioning. Five pl of this mixture was injected at 110 "C. The injection was repeated four times, and after 30 minutes the temperature was raised to 200 "C and left for 2 hours. The column exit, of course, was not connected to the detector during the preconditioning. One such conditioning for each column appeared to be sufficient. After connecting the column exit to the detector, column saturation with the derivative desired (3 injections of 20 y) aids in the overall response. It has been found to be necessary to heat the column to about 270 "C, after each four determinations to drive off accumulated extraneous material. The 08Ni detector should be cleaned at intervals in order to maintain the necessary sensitivity.
The relative responses of the four heptafluorobutyramides are shown in Figure 1. This curve represents 1 pl injection of a mixture of 400 picograms of each of the amines. The response to the fenfluramine amide is seen to be much greater than that of the other three. Fenfluramine does not give a significant response to the electron-capture detector unless a derivative is made. It has formerly been determined in blood (11)by flame ionization. The greater response of the fenfluramine derivative to electron capture could be due to either the trifluoromethyl substitutions on the aromatic ring or to the replacement of the N-methyl by the N-ethyl group in methamphetamine. That it is due to the former is seen from the response to the ethamphetamine amide (Figure 2) whose response is in the same order of magnitude as that of the amphetamine or metham(11) R. B. Bruce and W. R. Maynard, Jr., J . Pharm. Sci., 57
1173 (1968).
RESULTS AND DISCUSSION
Preconditioning of the column with heptafluorobutyric anhydride appears to be essential. Two columns prepared in the usual manner failed to give a response to the heptafluorobutyramides, even after repeated injections of large amounts. The columns were then preconditioned as described above under Method. Following this, the response was satisfactory. Silanizing alone is not sufficient. The anhydride appears to react with some sites on the packing material so that these sites can no longer tie up the amides as they pass through. 978
ANALYTICAL CHEMISTRY
A
B
Figure 3. Comparative responses given by 400 picograms of fenfluramine (A) and p-fenfluramine ( B ) heptafluorobutyramide
Table I. Recovery from Blood Added, 12 20
Found, ng/d 12 20
Methamphetamine
100 60
103 62
103 103
Chlorphentermine
20
21
105
30 40 50
29 40 51
100 102
60
62
103
80
78 99
98 99
Compound Fenfluramine
\ \ \
i 0'
P
\\
\
\ \ \ \ \ \
h
B
\ \ \
c
D 1
Amphetamine \
.
-*------3 HOURS
2
100
\
\
\-. \
+-,
rigid
----------4 A
Figure 4. Average blood levels of four subjects following the oral administration of a solution of 5 mg of each of the following drugs: A-methamphetamine, &hetamine, Cchlorphentermine, and P-fenfluramine
phetamine amides. Chlorphentermine is substituted in the para position with a chlorine atom but this does not appear to
contribute significantly to the molecule's response. It seemed possible that the meta substitution in fenfluramine might be affecting the response in some way. The amide was prepared of the corresponding compound that has the trifluoromethyl group in the para position. This amide gave a response (Figure 3) comparable to that of the fenfluramine amide. The substitution of the benzene ring of amphetamine with a trifluoromethyl group contributes significantly to the electroncapture response of the heptafluoroamide even though the trifluoromethyl and heptafluorobutyryl groups are not close to each other. In earlier studies ether was used for the extraction of the amines from blood but interferences were experienced from the control samples. Pentane was found to give satisfactory recovery with little interference. In order to confirm that the peaks obtained in gas chroma-
Recovery, 100 100
97
tography were actually due to the heptafluorobutyramides, larger (500 mg) amounts were carried through the procedure as described above using ether instead of pentane as the solvent. The resulting compounds were taken up in chloroform and their structures confirmed by their NMR and IR spectra. Injection of these compounds into the gas chromatograph gave retention times and responses corresponding to those found by carrying submicroquantities through the procedure. The rationale for the steps in the procedure are apparent except for that of allowing the pentane solution to stand overnight. It has been found from experience that the chromatograms thus obtained show greater response than when this is not done. The reaction tube is cooled after evaporation of the excess heptafluorobutyric anhydride to prevent possible losses due to volatilization from the heat of neutralization with alkali. The recovery of the amines after adding known amounts to blood are shown in Table I. These results indicate that the procedure is satisfactory. Analysis of control samples of blood did not show interference. Blood levels of the four drugs were determined in four normal male subjects in a cross-over designed study. Each subject received 5 mg of each of the drugs. This quantity is considerably below the recommended dose for fenfluramine and chlorphentermine. However, it appeared to be of interest to determine blood levels at the same dosage in this preliminary study. Samples were taken at 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 6.0 hours. The average results are shown in Figure 4. These results show that the method is satisfactory for the determination of these drugs even when administered below the recommended doses. RECEIVED for review February 13, 1969. Accepted March 13, 1969.
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