retention indices of nine standard perfluorocarbons are listed in Table IV. Figure 2 is a typical 980 "C pyrogram of n-C5F12 on the 3 0 z D F H A column. C2F6, CzF4, C&, n-C86, and n-C4Flo were identified as n-CSF12 pyrolysis products by comparing their retention indices with those of standard perfluorocarbons on the two different columns. Separation of the n-C5F12pyrolysis products required less than 15 minutes on each column. Rogers and Cady ( I ) suggested that if CZF,is formed during n-C5Flz'pyrolysis, it disappears because of rapid polymerization to yield one or more forms of C4Fs and (CFJ. The detection of C2F4 and n-C4Flo in our study lends support to their suggestion. Rogers and Cady ( I ) reported small amounts of CF4present at higher pyrolysis temperatures. Steunenberg and Cady ( 2 ) did not list CF4as a n-CsF12pyrolysis product. CF4was not detected in our study; however, this may be a result of the
~
~
~~~
~~
very low response of the flame ionization detector to this perfluorocarbon (9). Thermistor and electron capture detectors give less varied responses to perfluoroalkanes than flame ionization detectors. However, concurrent investigations and performance characteristics warranted the use of the flame ionization detector for our studies. RECEIVED for review July 27, 1972. Accepted November 14, 1972. This investigation was conducted under the auspices, of the Institute for Environmental Health, Purdue University, and was supported in part by PHS Training Grant No. 5T 0 1 - E S 0 0 0 7 1 from the National Institute of Environmental Health Sciences. (9) W. C. Askew and K. D. Maduskar, J. Chromatogr. Sci., 9, 702 (1971).
~
Deuterium Substituted Amphetamine as an Internal Standard in a Gas Chromatographic/Mass Spectrometric (GC/MS) Assay for Amphetamine Arthur K. Cho, Bjorn Lindeke, Barbara J. Hodshon, and Donald J. Jenden Department of Pharmacology, UCLA School of Medicine, Los Angeles, Calif, 90024
GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS) is a technique that couples the high resolving power of gas chromatography with the sensitivity and specific ion detecting capability of the mass spectrometer. These features are especially important when small quantities of drugs and metabolites are to be measured in biological fluids, and the technique has found numerous applications in the qualitative identification of drugs ( I ) . An additional modification has been described by Gaffney et a!. (2) in which stable isotope substituted compounds are used as internal standards for the quantitative analysis of drugs. Similar methods have been described for prostaglandins (3), homovanillic acid (59, and acetylcholine (5). This paper describes a GC/MS assay for the quantitative analysis of amphetamine in biological fluids in which three deuterium substituted amphetamines have been synthesized and evaluated as internal standards. A preliminary account of this research has been presented (6). The criteria used to evaluate stable isotope labeled internal standards for GC/MS analysis are somewhat different from those for the more conventional gas chromatography technique. Quantitation is achieved by comparing the response of the mass spectrometer at two nominal masses, one char~~
~
( I ) N. C. Law, V. Aandahl, H. M. Fales, and G. W. A. Milne, C h i . Cliitn. Acta. 32, 221 (1971). (2) T. E. Gaffney, G. C. Hammar, B. Holmstedt, and R. E. McMahon, ANAL.CHEM., 43,307 (1971). (3) 9.Samuelsson. M. Hamberg, and C. C. Sweeley. Anal. Biocliem., 38, 301 (19:O). (4) 9.Sjoqvist, E. Anggdrd, B. Fyro, and C. T. Sedual, Proc. I t i t . Pliurmacol. Cotigr.. J t h , 1972,1291. (5) D. J. Jenden, Fed. Proc., Fed. Amer. So?. Exp. Biol. 31, 5 1 5 (1972). (6) A. K Cho, B. Lindeke, R J. Hodshon, and D. J. Jenden, Proc. I t i t . Pliarmacol. Cotigr.. 5th, 1972, 41.
570
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
acteristic of the internal standard and the other characteristic of the substance to be analyzed. Since the internal standard is an isotopic variant of the compound analyzed, the fragmentation patterns and retention times will be the same. However, some fragments will contain one o r more atoms of an isotope such as deuterium rather than the more abundant natural isotope, and will therefore appear at masses shifted by one or more mass units from the compound to be analyzed. These different mje values are the basis for the assay and should be chosen with care. An ion of high relative abundance should be selected to maximize sensitivity, and should be isolated on the mass scale from other ionic fragments SO that each of the two masses selected is as far as possible representative of one isotopic variant, and is not produced by the other. Since any unlabeled variant present in the internal standard will appear as a blank, it is likely to limit the sensitivity of the method by providing a statistically variable background, and the internal standard should therefore have the maximum possible isotopic purity. This is particularly important because with picomole levels of amphetamine the internal standard also serves as a carrier to minimize mechanical losses and may be present in 100- to 1000-fold excess. Resolution of the mass spectrometer will generally not be limiting even with a low resolution instrument such a s a quadrupole, because the need for low contamination of the internal standard with the unlabeled variant makes it desirable to prepare a standard with more than one labeled atom, and the masses of the fragment ions selected for measurement would thus differ by 2-3 mass units. The isotopically substituted compounds used in this work are shown below. The synthesis (7) and mass spectral analy(7) B. Lindeke and A. K. Cho. Acta Pliurm. Suecicn, 9,363 (1972).
sis (8) of these compounds have been described. The compounds differ in their deuterium isotope content, isotopic purity, and site of labeling. The analysis utilizes the trifluoroacetic acid amide derivative because of its desirable gas chromatographic and mass spectral properties.
EXPERIMENTAL
Apparatus. The instrument used in this study was an EA1 Quad 300 quadrupole mass spectrometer coupled to a Varian 1400 gas chromatograph with a glass frit separator. The ionizing energy was set at 70 eV with the emission current ranging from 100 to 250 FA. For specific ion detection, the mass spectrometer was programmed with a multiple specific ion detector developed in this department (9). The output of the specific ion detector for each mass was monitored with a Rikadenki Series KA Multi Pen recorder. A 2 m x 2 mm (i.d,) silanized glass column was packed with 3 z OV 17 on Gas Chrom Q (80-100 mesh) with helium (20 mljmin) as the carrier gas. Temperatures of the injection port, column, and molecule separator were maintained at 180,130,and 185 "C,respectively. Extractions were carried out on an International Bottle Shaker and the layers separated by centrifugation in an International Centrifuge at 110 x g. Reagents. Trifluoroacetic anhydride (Aldrich Chemical Co.) was redistilled (39.540 "C) in 25-gram batches and stored at 0 "C in a dark bottle. The compound becomes colored after storage for several months and was not used without prior purification. The compounds used as internal standards had the minimum isotopic purities: Adl 9 7 . 7 z , Ads 9 7 . 9 z , Ad3 99.9%. The isotopic purity of the TFA derivatives of the labeled amines was determined from the peak height ratio of the base peak for the deuterium enriched analog and the peak present (in the labeled analog) at the mje value for the corresponding fragment in the unlabeled compound ( 7 ) . For example, the isotopic purity of Ada was determined by comparing the mass spectrometer response at masses 143 [C2H3CHNHCOCF,I+ and 140 [CH5CHNHCOCF3]+. The response at mle 140 in the spectrum of Ada was 0.1 of mje 143, and this was taken to be the maximal impurity present in the isotopic analog. The plasma used for the standard curve was Plasmanate (Cutter Laboratories), a 5 % solution of human plasma protein available in 50-ml vials. The solution was stored at -80 "C after o1)eningbut was not used after 7 days storage. Procedure. The initial extraction was carried out in a 20-ml screw cap test tube. A 2-ml volume was used for the analysis and samples of plasma less than 2 ml were adjusted to 2 ml with 0.9 NaCI. Five hundred pmoles of deuterated amphetamine (Ad?) was added to each tube as the internal standard. Next, 0.5 ml of 20% NaOH and 6 ml of benzene (analytical grade) was added to each tube and the mixture extracted by shaking for 20 minutes on a mechanical shaker. After centrifugation for 15 minutes, 5 ml of the benzene was transferred to a 12-ml conical screw cap centrifuge tube containing 0.6 ml of 1N HCI. The tubes were shaken for 15 minutes and centrifuged for 5 minutes. The benzene was aspirated and discarded. One 500-pl aliquot of the lower
z
(8) Ibid.,10 (1973)in press. (9) D. J. Jenden and R. W. Silverman, in Proc. Meet. Use Stable Isotopes Clin. Phurmucol. ; University of Chicago, Chicago, Ill.,
Nov. 1971,
acid phase was removed with a micropipet and transferred to another conical centrifuge tube containing 1.2 ml of benzene (spectrograde) and 0.2 ml of 2 0 z NaOH. After shaking for 15 minutes and centrifuging 5 minutes, 1.0 ml of benzene (upper) phase was removed by two transfers with a 500-p1 micropipet to a microcentrifuge tube (Kontes, Microflex tube). Next, 20 p1 of trifluoroacetic anhydride was added and immediately after addition, the tubes were capped and mixed on a Vortex Mixer. The tubes were left overnight in a 4 "C refrigerator and evaporated the next morning under a stream of Nz at room temperature. Each tube was removed immediately after evaporation of solvent to avoid physical loss. The amphetamine trifluoroacetamide residue was dissolved in 20 pl of acetonitrile and a 2-1.11 aliquot was injected into the gas chromatograph. A standard curve was prepared by adding 10, 50, 100, 200, 400, and 500 pmoles of amphetarnine standard (unlabeled, Ado) to tubes containing 2 ml of Plasmanate which were then carried through the procedure. The ratio of the peak height at mass 140 (Ado) to the peak height at mass 143 (Adl) was determined for each sample. The peak height ratios were quantitated by comparison with the standard curve which was run with each assay. RESULTS AND DISCUSSION
The trifluoroacetyl (TFA) derivative was superior to the amphetamine base in this analysis for two reasons. The free base was found to tail on several columns tested on which the amide gave sharp peaks with very little evidence of adsorption. Furthermore, the base peak for amphetamine is at mje 44 with no major peaks at mje values greater than 91 (IO). Therefore, in spite of an additional step in the analytical procedure, the TFA derivatives were used. The mass spectra of the TFA derivative of amphetamine and the three isotopic variants used in this study are shown in Figure 1. The peak chosen for GCjMS analysis was the mje 140 peak of amphetamine which represents the fragment
1
\CFICO-N-CH resulting from ,&cleavage between C1and C2 of the side chain ( 7 ) . The corresponding fragment for Adl, Ads, and Ad3 gives peaks at 141, 141, and 143, respectively. This peak is the base peak of the TFA derivatives and is isolated in the spectrum. The corresponding peaks in the Adl and Ads derivatives have small (-273 peaks at 140 while the Ad3 derivative has a small peak at 142 with very little response (0.1 %) at 140. Although the presence of a 140 peak in the mass spectrum of Adi and Ads does not prevent their use as internal standards, the absence of the 140 peak in Ad3 together with its higher isotopic purity made the latter compound the standard of choice. When there is peak overlap as in the Adl and Ad? derivatives, the specific ion detector (9) can be adjusted to subtract out contribution from the internal standard, but the contribution is statistically variable and would require careful adjustment each clay in addition to increasing the noise level. Since the theoretical "blank" is the ratio of ion current response of masses 140 to 141 or to 143 when no amphetamine has been added, it should be lower with higher isotopic purity in the internal standard. The two-step extraction procedure provides substantial purification of the extract and since the final step is an evaporation, maximal preliminary purification is essential. The extraction removes neutral and acidic substances and will not (IO) A. H. Beckett, G. T. Tucker, and A. C. Moffat, J . Pharm. Phurnzacol., 19,273 (1967).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
571
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
I14O’I143 II
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The response to mie 143 ( - - - - ) and m/e 140 (--) is shown after injection of an equimolar (IIA) mixture of unlabeled amphetamine (Ado), and labeled amphetamine Ada and of Ads alone (IIB). The initial shoulders on each tracing are due to pressure change in the mass spectrometer that result from the introduction of the GC effluent. The retention time for the amphetamine derivative was 3.5 minutes
extract hydroxylated amphetamine derivatives. These latter compounds are extracted by more polar organic solvents (11). The recoveries in these extractions were found to be 90 to 100% when corrections were made for aliquots. The specificity of the analytical procedure was also evaluated by comparing intensities of 4 major mass peaks in the mass spectrum of authentic amphetamine and of the gas chromatographic peak corresponding to amphetamine obtained from plasma extract. If compounds other than amphetamine were present in the plasma GC peak, the relative intensities of the 4 mass peaks should be different from the internal standard and the plasma extract. The results of this experiment are shown in Table I. The deuterium substituted internal standard is of particular value in this multistep extraction procedure because it is added a t levels of 10 to 100 times higher than the unknown and thereby acts as a “carrier” to minimize losses by adsorption onto glass etc. in the extraction procedure. The deuterium compound has essentially identical physical properties to the unsubstituted compound. O’Brien et al. (12) have found it necessary to add triethylamine as a scavenger to minimize losses of this type in a GC assay. (11) G. A. Clay, A. K. Cho, and M. Roberfroid. Biochern. Pliurmacol.,20, 1821 (1971). (12) J . E. O’Brien. W. Zazulak, V. Abbey, and 0. Hinsvark, J . Cliromafogr.Sci., 10, 336 ( 1972).
,
0 50
Figure 3. Standard curve of amphetamine extracted from plasma Varying amounts of amphetamine were added to 2 ml of Plasmanate containing 500 pmoles of deuterium labeled amphetamine (Ad,) and the resulting mixtures carried through the procedure Table I. Relative Peak Intensities of Major Fragments‘ Source of m / e value amphetamine 69 91 118 140 Standard 0.29 0.56 0.74 1 .oo Plasma extract 0.30 0.55 0.74 1 .oo aThe multiple ion detector described in Experimental was focused on the mass numbers indicated and the ion current at each mass was integrated over the chromatographic peak for standard amphetamine and the extract from plasma. Table 11. Plasma Levels o f Amphetamine in Rats’ Dose, mg/kg Plasma levels, ng/ml 0.25 0.5 I.o
7.5, 13.4 17.8, 17.4 26.0, 32.4 144.0, 139 0 302.0, 276,O 630.0. 960.0
2.5 5.0 10.0 a Sets of 2 rats (male, 180-200 grams) were given intraperitoneal injections of amphetamine at the indicated doses. One hour later the animals were anesthesized with ether and blood was drawn by
cardiac puncture and the plasma amphetamine determined. An example of the type of peaks that are analyzed in the procedure is shown in Figure 2. Trace A shows the specific ion detector response to a mixture of 5 x lo-’* mole each of unlabeled amphetamine and Ads. Trace B shows the response to an injection of 10-10 mole of Ada alone and constitutes a “blank” for the assay. A series of 5 standard curves werr: linear from 0 to 1 nmole and the slopes had a
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
573
standard dwiation of 0.3 %. Data from individual standard curves, one of which is shown in Figure 3, indicate that 95% confidence h i t s of the assay are 5-10 pmoles/2 ml sample. The results of a dose plasma level study in rats are shown in Table 11. Two-ml aliquots of plasma'taken from rats given 0.25 to 10 mg/kg amphetamine were extracted and analyzed as described.
ACKNOWLEDGMEW
The advice and assistance of Margareth Roch and R. W. Silverman are gratefully acknowledged. RECEIVED for review September 25, 1972. Accepted November 16, 1972. This work was supported by USPHS Grants: No. MH20473 and MH17691.
Rapid Determination of Barbiturates by Gas Chromatography- Mass Spectrometry Ronald F. Skinner,*t2 Edward G . Gallaher, and David B. Predmore Washington State Toxicology Laboratory, Unicersity of Washington, Seattle, Wash. 98195
THEWIDESPREAD AVAILABILITY of barbiturates obtained both by prescription and from illicit sources has caused a rapid increase in accidental and deliberate overdosages from these drugs. Over 15,000 persons are hospitalized each year for treatment of acute barbiturate poisoning ( I ) . In order to properly treat these overdose cases, it is necessary to rapidly establish the identity and amount of the barbiturate present. In cases of lethal intoxication, this information is essential for establishing the cause of death, since the different barbiturate derivatives have a wide range of lethal levels. Gas chromatography and thin-layer chromatography have been used in most laboratories to provide identification of the particular barbiturate involved in a n overdose case. Neither of these techniques can unquestionably identify the barbiturate or barbiturates present in a sample, because of the large number of structurally and chromatographically similar barbiturates. Other compounds in the sample may occasionally give rise to results which could be misinterpreted as evidence of barbiturates; therefore, a back-up method such as ultraviolet analysis must be used to verify the presence of the barbiturate. The recent advent of less expensive and less complex gas chromatograph-mass spectrometer combinations (GC-MS) has made this powerful tool a reasonable one for use in laboratories doing routine analyses. The GC-MS provides rapid, accurate qualitative and quantitative identification of barbituric acid derivatives. The advantages of and conditions for chromatographing the barbiturates as their 1,3-dimethyl derivatives have been discussed by Brochmann-Hansen and Oke (2) and others (3-6). The procedure presented here is a modification of the Brochmann-Hansen and Oke methodology and makes use of triPresent address, Finnigan Instrument Corp., 595 N. Pastoria
Ave., Sunnyvale. Calif. 94086. Mass spectra of the methylated barbiturate derivatives are available from this author.
( I ) N. M.Simon and F. A. Krumlovsky, Ratioiiul Drug Tllerupy, 5, l(1971). (2) E. Brochmann-Hansen and T. 0. Oke, J . PIiurrn. Sci., 58, 370
(1969). (3) G. W. Stevenson. ANAL.CHEM., 38, 1948 (1966). (4) J. G. H. Cook, C. Riley, R . F. Nunn, and D. E. Budgen, J . Clirornurogr., 6, 182(1961). (5) H. F. Martin and J. L. Driscoll, ANAL.CHEM., 38, 345 (1966). (6) G. A. Neville, ibid.. 42, 347 (1970). 574
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
2
3
5
4
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6
7
8
9
10
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Figure 1. Chromatogram of 200 ng each of (1) barbital, (2) probarbital, (3) aprobarbital, (4) butabarbital, (5) amobarbital, (6) pentobarbital, (7) secobarbital, (8) hexethal, (9) glutethimide, (10) phenobarbital, (11) heptabarbital, (12) diphenylhydantoin
methylanilinium hydroxide both to extract the barbiturate from the organic phase and to methylate it. This considerably shortens the procedure necessary for the preparation of an injectable fraction for GC-MS ideritification. EXPERIMENTAL Apparatus. A Beckman GC-5 gas chromatograph equipped with a hydrogen flame detector, a n optical follower temperature programmer, and a Beckman 10-inch strip chart recorder was used for the initial scanning of samples. Mass spectrometry was done with a Finnigan Model 3000 gas chromatograph-mass spectrometer equipped with a glass jet separator, a Heath Model EU-205-11 recorder, and a Honeywell Visicorder. The electron ionization energy was 70 eV. Both gas chromatographs were equipped with 6-ft x '/,-inch stainless steel columns packed with 3% OV-1 on 1001120 mesh Gas Chrom Q. During analysis, the column temperature was programmed from 120-220" C at 10' C,/minute. The flow rate of the helium carrier gas was 30 m l h i n u t e . The column was kept at 240 "C when not being used for analysis, as this resulted in less column background.
