the mean algebraic error was f0.002Z. Based on a typical sample value of 0.136%, the mean absolute errors are 5 x (GC) and 4x (ADH), relative; well within the limits normally imposed on chromatographic assays. The precision of the proposed technique was evaluated using a series of 20 specimens prepared from the same pool. The experiment was repeated a number of times on different days, and the data are summarized in Table 11. The data are reported as ratio of ethanol to internal standard peak area, The conversion to per cent ethanol would show corresponding relative deviations. The average overall precision with respect to the mean value was 3%. From the precision study, we conclude that the method is capable of a routine precision of 3-4% (relative). This figure may be compared to the reported precision for other methods. For the macro gas chromatographic procedure, precision of 5 % ( I ) and 6 % (2) was reported; for alcohol dehydrogenase techniques, 5 % (7). The distillation technique, the standard method for many blood alcohol determinations, has a precision of 4 % (7). An important general objective in some applications of the present. research is to reduce the time required for individual analyses. In actual experiments in the laboratory, samples taken on site were shaken in the Unopette, centrifuged, injected, and quantitated (using visual chart reading and slide rule) in five minutes. By proper choice of solvent system and instrumental parameters, the technique is suitable for use in monitoring certain drug or metabolite (or anesthesia) levels during actual administration. Because of the required solvent elution time, approximately 12-13 minutes are required between successive injections on the same column. By using (7) R. Bonnichsen and G. Lundgren, Acta Pharmacol. Toxicol., 10, 223-6 (1954).
Table 11. Precision of Samples Taken in Unopettea Ratiob ethanol :propanol Number mean value Std dev of data Series No. 1 0.312 0.009 20 2 0.358 0.008 20 3 0.333 0.014 18 4 0.379 0.008 20 5 0.405 0.009 20 Av std dev 0.010 (3 relative) a Sample from pool of 0.200x ethanol in blood. Experiment repeated five times on three different days. Chromatographic conditions specified in text. b Ratio of peak ar:as. ~~
the dual column instrument, with a single electrometer and single recorder, samples could be run at the rate of 2 every 15 minutes. The ethanol-propanol section of the chromatogram emerges 3 to 4 minutes after injection. ACKNOWLEDGMENT
The author acknowledges the assistance of Miss Toni Motisi who performed much of the work associated with this project and assisted in the preparation of the data for publication. H. W. Gerarde, Becton, Dickinson Co., supplied the experimental Unopet tes. RECEIVED for review July 8, 1968. Accepted August 16, 1968. Paper presented at Great Lakes Regional Meeting of the American Chemical Society, June 13-14, 1968, Milwaukee, Wis. Research supported in part by the Institutional Research Grant to the University of Wisconsin from the American Cancer Society (IN-35H-15).
Determination of Amphetamine by Ultraviolet Spectrophotometry Jack E. Wallace, John D. Biggs, and Sheldon L. Ladd Forensic Toxicology Branch, USAF Epidemiological Laboratory, Lackland Air Force Base, Tex. 78236
THEamphetamines are used principally as stimulants of the central nervous system. Side actions, overdosages, or illegal uses may occur which often result in a forensic requirement for evaluation of the drug level in biologic specimens. Paper chromatography ( I ) , thin layer chromatography (2, 3), and gas chromatography (2, 4-7) have all been used to determine the amphetamines. Although the chromatographic methods are sensitive, they often lack the ability to provide the specificity required in analyses performed by the forensic scientist. When derivative formation is employed in the gas chromatographic determination of the amphetamines, the testing of a large number of specimens during a limited time interval becomes difficult. (1) A. Wickstrom and B. Salvesen, J. Pharm. Pharmacol., 4, 631 (1952). (2) A. H. Beckett, G. T. Tucker, and A. C. Moffat, ibid., 19, 273 (1967). (3) I. Sunshine, W. W. Fike. and H. Handesman. J. Forensic Sci.. 11, 428 (1966). (4) A. H.Beckett and M. Rowland, J. Pharm. Pharmacol,, 17, 59 (1965). (5) E. Brochmann-Hanssen and A. B. Svendsen. J. Pharm. Sci.. 51, 393 (1962). (6) L. Goldbaum, Progr. Chem. Toxicol., 2,221 (1965). (7) K. D. Parker, C. R. Fontan, and P. L. Kirk, ANAL. CHEM., 34, 1345 (1962).
Many nitrogen containing organic bases which absorb in the region of 260 mp and have an aromatic ring as part of their chemical structure interfere with direct ultraviolet spectrophotometric methods (6, 8) for determining amphetamine, and give a positive reaction in several colorimetric methods (9-12). With the latter, unpredictable variations of color development are frequently observed. Amphetamine at concentrations often found in biologic specimens absorbs ultraviolet radiations weakly, and direct ultraviolet analysis of the unchanged drug in biologic materials is nonrewarding. At toxic levels of amphetamine, background absorption at 260 mp induces a wide degree of variance in the final results, unless extensive purification of the biologic extract is achieved. In this report a sensitive spectrophotometric method for quantitative analysis of amphetamine and certain related compounds is described. The procedure is applicable for screening biologic specimens for the presence of the compounds. (8) F. A. Rotandaro, J . Ass. Osfic. Agr. Chem., 40, 824 (1957). 19) G. A. Alles and B. B. Wiseaarver. Toxicol. Auul. _ . Pharmacol., .
L
-
I
3, 678 (1961). 1101 K. H. Bever. J. Amer. Chem. SOC..64.1318 (1942). iiij L. G. Chitten and L. Levi, ANAL'CHEM., 31; mi (1959). (12) E. Rathenasinkam,Analyst (London),76,115 (1951). VOL. 40, NO. 14, DECEMBER 1968
2207
Table I. Standard Curve Data for Amphetamine Reaction Product Amphetamine in sample Ilg/ml 40.0
30.0 20.0 16.0 12.0
a
Absorbance Concentration 0.022 0 022 0.023 0.024 0.024 0.024 0.024
Absorbance in n-hexane0 0.860 0.653 0.462 0.376
t
0.292
0.195 0.096
8.0
4.0 Read at 287 mp.
0.900 80-
0.70-
,-,
0
f
/
0.60-
'm
,.I K
Product
; '-,',
1
i
0.50 0.401
Antphetamlne
',
?'
// I
I
I I I
I I
!
Table 11. Compounds Yielding Absorbance at 287 mp after Cerium Oxidation. Compound Amphetamine Blank 1,2-Diamino-l-phenylpropane Benzphetamine Chlorphentermine 3,4-Dimethoxyphenylethylamine Ephedrine Hydroxyamphetamine Mephentermine Methamphetamine Methoxamine Methoxyphenamine 4-Methyl-2,5-dimethoxyamphetamine (STP)
Absorbance Absorbance in n-hexaneb Concentration 0.46 0.01
0.023
0.52
0.026
...
0.08
0.004
0.29
0.015
0.01
0.12 0.01
0.39 0.21
...
0.006 ... 0.020 0.011 0.034
0.68 0.19
0.010
1.00
0.050
Phenmetrazine 0.02 0.001 0.52 0.026 Phentermine 0.003 Phenylalanine 0.06 P-Phenylethylamine 0.39 0.020 Phenylpropanolamine 0 13 0.007 0.008 Tranylcypromine 0.16 a Each compound was determined from aqueous solution (0.8N HCI) to yield a corresponding concentration in n-hexane of 20 Pdml. * Average of three results.
Figure 1. Ultraviolet absorption spectra of amphetamine in water, and of the cerium oxidation product in n-hexane, each corresponding to an amphetamine concentration of30 pg/ml removed and read against a blank through the region 250 to 360 mp. If analysis at a single wavelength is required, determine the absorbance at 287 mp. For the most accurate results, the blank should consist of a refluxed hexane solution prepared with the aqueous extract from an equivalent amount of biologic specimen known to contain no amphetarnine. When therapeutic levels of the drug are indicated, the n-hexane containing the product should be evaporated to dryness by means of a rotary vacuum evaporator at 30 "C and the resulting residue suspended in 0.5 to 5 ml of spectro grade n-heptane depending upon the absorbancy observed for the n-hexane solution. The amphetamine content of the sample is calculated from a standard curve prepared by subjecting aqueous solutions of the drug in appropriate concentrations to the reflux system. The standard solutions of amphetamine product should be read against a blank of n-hexane. For gas chromatographic analysis, the residue from the evaporation of n-hexane is dissolved in 50 to 100 pl of n-heptane, after which an aliquot of 1-5 pl is injected into the chromatograph.
EXPERIMENTAL Apparatus. A Beckman DK-2A ratio-recording spectrophotometer with linear wavelength presentation was used for the ultraviolet absorption measurements. A Beckman IR-4 double beam infrared spectrophotometer was used for infrared spectra characterization of functional groups in the amphetamine product. A Barber-Colman Model 5000 equipped with a 10% SE 30 column (6 ft X 3.5 mm) on Gas Chrom Q (100-120 mesh) and a flame ionization detector was employed for gas chromatographic studies. Column temperature was 238 "C. The carrier gas was nitrogen flowing at a rate of 50 ml per minute. Procedure. Ten milliliter amounts of oxalated blood or serum, 10- to 50-ml amounts of urine, or 10 grams of homogenized tissue are placed in a separatory funnel to which 5 ml of 1N NaQH and 200 ml of n-hexane are added. This mixture is shaken vigorously for three minutes. The nhexane layer is removed and the recovered volume is recorded. Ten milliliters of 0.8N HCl are added to the hexane and this mixture is shaken for three minutes. After the layers have separated, 9 ml of the aqueous layer are placed in a 250-ml round-bottom flask along with 1.5 grams of anhydrous cerium sulfate (Fisher, purified grade) and 50 ml of spectro quality n-hexane. The contents are slowly refluxed with magnetic stirring for 30 minutes. After cooling, the n-hexane is 2208
.a
ANALYTICAL CHEMISTRY
RESULTS AND DISCUSSION
The reaction product of amphetamine has a well-defined ultraviolet absorption curve which has a maximum at 287 mp and a minimum at 253 mp in hexane. The absorbance at 287 mp, illustrated in Figure 1, provides the increase in sensitivity of the present method over those procedures which rely on direct ultraviolet spectrophotometric analysis of unconverted amphetamine. At the levels investigated, a linear relationship exists between the absorbance of the reaction product and the concentrations of the drug in the original sample (Table I). The absorptivitylpg ratio of 0.024 for the product represents approximately a twenty-fold increase in sensitivity over the 0.0013 absorptivity/pg ratio observed for amphetamine. Several other drugs which have the p-phenylethylamine group as part of their structure give a positive reaction with ceric sulfate and can be assayed by the proposed method (Table 11). The products from several of these compounds have ultraviolet maxima and absorption curves identical to that of the amphetamine product. The method cannot distinguish spectrophotometrically between these drugs ; however, at temperatures greater than 204 "C on the gas chromato-
~~
graph, the reaction products have retention times on SE 30 which in most cases differ extensively from that of the amphetamine product (Table 111). Of interest is that @-phenylethylamine, which interferes with amphetamine in the analysis of biologic samples that have undergone some degree of putrefaction, gives, after oxidation, a product yielding two chromatographic peaks considerably different in retention time from that observed for the amphetamine product. Amphetamine, methamphetamine, and 1,Z-diamino-1phenylpropane give products which have identical gas chromatographic retention times as well as ultraviolet spectrophoiometric spectra. Oxidation of the amino group by the conditions of this method could yield from these three compounds an identical product. The report that methamphetamine is excreted in urine as amphetamine (13) overshadows certain specificity requirements for the two drugs not met by the procedure of this report. Many basic drugs other than those containing the 8phenylethylamine group were investigated for possible interference with the determination of amphetamine. Such compounds were observed not to interfere. Table I V summarizes the recovery of amphetamine which had been added in known amounts to whole blood and urine. The average recovery was 89% from blood and 97% from urine. Hydrochloric acid was observed to be the only acid which functions as a catalytic agent. The normality of 0.8 rt 0.2 is critical to the effectiveness of the procedure. Absorbance values in the optimum normality range of acid agree with those values given in Table I. The amount of cerium sulfate should not exceed the limits of 1.5 i: 0.25 gram. Cerium salts other than the sulfate yield lesser amounts of .the desired product. With five adult humans, each of whom received 5 mg of d-amphetamine by mouth, the highest concentration of unchanged amphetamine in the urine was found in samples collected during the period 4 to 8 hours following ingestion of the drug. The amount of amphetamine in urine samples collected at designated time intervals is shown in Table V. The drug can be detected in urine 48 hours after the ingestion of a single 5-mg dose, and during this time 25 to 4 4 x of the drug was recovered. The results for this dosage level are in agreement with previous observations (14) that amphetamine excretion in urine occurs mainly in the first 24 hours and is essentially over 48 hours after administration. The procedure is specific for those compounds which have a @-phenylethylaminegroup as part of their structure. Specificity requirements are: a @-methylenecarbon, no hydroxy or methoxy substituent on the para position of the aromatic ring, and a primary or secondary amine group.
The beta carbon must exist as a methylene group presumably for oxidation to a carbonyl function. This is evidenced by the inability of tranylcypromine, ephedrine, and phenylpropanolamine to yield significant amounts of product (Thble 11). The observation that para-hydroxy-amphetamine, the primary metabolite of amphetamine, 3,4-dimethoxyphenylethylamine, and 3,4,5-trimethoxyphenylethylamine (mescaline) d o not react implies that a hydroxy or methoxy (13) L.M. Gunne, Biochem. Pharmacol., 16,863 (1967). (14) G. P. Cartoni and F. DeStefano, Itaf. J. Biochem., 12, 296 (1963).
~
~
~
-
_
_
Table 111. Relative Retention Times of Reaction Products by Gas Chromatography. Max UV absorption,b Compound mP Ratio0 Amphetamine 287 1.00 Amphetamine (from urine) 287 1.00 1,2-Diamino-1-phenylpropane 287 1.00 Chlophedianol 247 0.39 Chlorphentermine 287 0.48 Diphenhydramine 247 0.25 Mephentermine 287 0.50 Methamphetamine 287 1.00 Methoxamine 286 0.70 Methoxyphenamine 245 1.05 4-Methyl-2,5-dimethoxyamphetamine 282 0.39;0.44 (STPI Phentermine 287 0.50 8-Phenylethylamine 287 0.50;0.77 Tranylcypromine 228 . . .d a Conditions, see text. b In hexane, Retention time of product from drug divided by retention time of amphetamine product. None observed. Table IV. Recovery of Amphetamine after In Vitro Addition to Blood and Urine Amphetamine added pg/ml
NO. of
50.0 25.0 10.0
9 9 10
5.0
7
Recovery, mean Whole blooda 45.4 rt 1.6 22.1 rt 0.9
detns.
Av recovery a Ten milliliters assayed.
8.9=!= 0.2 4.3i 0.3 88.6%
+ std dev Wm1) Urinea
47.9i 1.9 24.3 i 1.0 10.1 f 0.3 4.7=IC 0.4 97.0%
Table V. Amphetamine Recovered from Human Urine" Time, hours 4 8 12 16 20 24 32 40 48
pg/ml I 1.27 2.18
11
. . .b
1.27 3.23 1.48
1.37 0.52 0.29 0.44 0.03 0.01
0.97 0.72 0.21 0.18 0.04
...
111 0.56 1.11 0.80 0.24 0.34 0.52
...
... 0.16
IV 2.49 5.73 1.07 0.58 3.21 0.55 0.08 0.02 0.11
V 2.26 3.4 1.45 1.35 1.09 0.78 0.22 0.15 0.07
Total recovered 1.25 2.02 1.64 1.86 2.21 (mg) 0 Each adult received 5 mg of d-amphetamine by mouth. Not determined.
substituent at the para position of the aromatic ring inhibits the desired reaction. Of further significance is that 4-methyl2-,5-dimethoxyamphetamine (STP) and chlorphentermine give excellent yields of product. None of the hydroxyphenyl. derivatives interfere with the analysis of amphetamine because they are insoluble in n-hexane, and are not removed from biologic materials in the initial extraction. Several of the @-phenylethylamines that are converted to ultraviolet absorbing products have one or more alkyl subVOL. 40, NQ. 14, DECEMBER 1 9 6 8
* 2209
_
I
stituent on the alpha carbon, and it is indicated that such groups at this point enhance the amount of product obtained. A carboxyl group at this same carbon inhibits the reaction. The latter is illustrated by the inability of the amino acid phenylalanine to afford a product with a measurable amount of absorption at 287 mp (Table 11). The requirement for a primary or secondary amine is indicated by the failure of benzphetamine to react. As a rule primary amines react much better than secondary amines. The requirements of the reaction establish a relatively high degree of specificity for the @phenylethylamirie structure.
The sensitivity limit of the method is approximately 0.5 pg/ml of specimen providing a 10-ml sample is used. The procedure of this report is as sensitive as the spectrophotometric methods for determining barbiturates (15). Barbiturates and amphetamines are often taken concomitantly, and now determination of both compounds at therapeutic levels in urine can be performed by spectrophotometric analysis. Received for review May 15,1968. Accepted August 21,1968. (15) L. R. Goldbauni, ANAL.CHEM., 24, 1604 (1452).
pectrophotometric etermination of Microgram mounts of Tantalum with Victoria Blue G . F. Kirkbright, M. D. Mayhew, and T. S. West Chemistry Department, Imperial College, London, S . W.7, England SEVERAL spectrophotometric methods are available for the determination of small amounts of tantalum. Hydrogen peroxide ( I ) , pyrogallol (2),gallic acid (3),and various fluorone derivatives (4), have been extensively employed as reagents for this determination. Methods for the determination of tantalum based on the formation of binary complexes in aqueous medium with reagents of this type, however, are basically relatively unselective. More selective methods have been reported for tantalum based on the formation and solvent extraction of ternary complexes of the tantalum with fluoride and methyl violet (5) and malachite green (6). This paper describes the application of the similar triphenylmethane type of dyestuff, Victoria Blue B (Basic Blue 26, Colour Index 44045), to the determination of microgram amounts of tantalum. Victoria Blue B forms a ternary complex with tantalum and fluoride in hydrofluoric-sulfuric acid medium. The complex i s extractable into benzene. The complex has a somewhat higher molar absorptivity in benzene than the corresponding malachite green and methyl violet complexes, and a selective determination of tantalum is possible under the optimum conditions established in this study. EXPERIMENTAL
Apparatus. Beckman model DB recording spectrophotometer fitted with Honeywell Electronik integrating recorder and matched I-cm glass cells were used. Victoria Blue B Reagent. The dyestuff (obtained from CIBA, Manchester, England) was purified by chromatography on an alumina column with isopropanol as solvent. An aqueous solution, 1.2 x lO-*M, of the purified material was prepared by dissolving 0,600 gram of dyestuff in one liter of distilled water. Thermogravimetric analysis of a sample of the chromatographically pure dyestuff revealed a progressive loss of weight of 4.7% between 20 and 80 "C. This corresponds to the (I) P. Klinger and W. Koch, Arch. Eisenhuttenw., 13, 127 (1939). (2) E. C. Hunt and R. A. Wells, Analyst (London), 79, 345 (1954). (3) H. Freund, K. H.Hammill, and F. C . Bissormette, U.S . Bureau of Mines, Investigation Report No. 5242, U. S . Government Printing Office, Washington D. C., 1954. (4) C. L. Luke, ANAL.CHEM., 31,904 (1959). (5) N. S. Poluetkov, L. Z. Konenenko, and R. S . Lauer, J. Anal. Chem. USSR, 13,449 (1958). (6) Y. Kakita and H.Goto, ANAL.CHEM., 34,618 (1962).
210 *
ANALYTICAL CHEMISTRY
"32
Figure 1. Victoria Blue B, C3a& N s C1 presence of 1.5 molecules of water in association with each molecule of the dyestuff. Elemental analysis gave the following results: calculated for CaaHazNaC1,C, 74.6 %; H, 6.0%; N, 7.92%; found C, 74.2%; H, 5.9%; N, 7.6x. Tantalum Stock Solution. Dissolve 0.181 gram of tantalum metal (Specpure reagent, Johnson and Matthey, London, England) in 23 ml of 40% hydrofluoric acid and warm to assist dissolution. Dilute to 100 ml with distilled water and store in a polythene bottle. This stock solution is 10-2M (1800 ppm) with respect to tantalum, and was diluted as required to give a final solution which was 2 ppm in tantalum and 0.01M with respect to hydrofluoric acid. Hydrofluoric and sulfuric acids and salts of diverse ions used in interference studies were analytical reagent grade. Preparation of Calibration Graph for Tantalum. When the tantalum complex was formed and extracted in glass separating funnels, poor reproducibility was obtained in the determination. This may possibly be attributed to the capricious adsorption and release of tantalum onto the glass surface in the dilute hydrofluoric acid solution. Polythene bottles were found satisfactory both for storage of reagents and for formation and extraction of the tantalum complex. To each of a series of six 100-ml polythene screw-top bottles, transfer 3.3 ml of 18N sulfuric acid and 1.5 ml of 5N hydrofluoric acid. Add 0, 2, 4, 6, and 8 ml aliquots of standard tantalum solution (2 ppm) to the six bottles, and sufficient distilled water to make the volume 13 ml. Transfer 2 ml of stock 1.2 X 10-aM Victoria Blue B reagent solution to each bottle. Pipet 10 ml of benzene into each bottle, shake the solutions for ea. 30 seconds, and allow to stand to ensure complete phase separation. Remove an aliquot of the benzene phase from each bottle with a pipet and measure its absorbance at 635 mg in a I-cm glass cell against the blank solution (containing no tantalum) prepared simultaneously.
