C-17 methyl ketone (not completely characterized) were identified in this fraction. There was no evidence of compounds such as phytol in this fraction, and none of the carbonyl compounds were apparent in the gas chromatogram of the hexane extract containing the phytol. Until both hexane fractions are completely characterized, however, we cannot state with certainty the degree of separation. In conclusion, many Girard T hydrazones are susceptible to hydrolysis even under mild conditions. This problem limits the use of the Girard reagent for many separations of carbonyl from noncarbonyl compounds. A nonaqueous system has been developed to overcome this problem. Al-
though poor partitioning factors may arise in the nonaqueous system these can be minimized by increasing the number of extractions. ACKNOWLEDGMENT
The authors thank J. Showell and E. Rothman for their helpful discussions during the course of this work. RECEIVED for review September 27, 1966. Accepted January 18, 1967. Work supported by the Cigar Manufacturers Association of America.
Determination of Ephedrine and Certain Related Compounds by Ultraviolet Spectrophotometry Jack E. Wallace Forensic Toxicology Branch, USAF Epidemiological Laboratory, Lackland Air Force Base, Texas
THEALKALOID EPHEDRINE (1-phenyl-2-methylamino-propanol) and the related compounds pseudoephedrine and phenylpropanolamine have similar medical uses. These drugs have a low toxicity but their identification and quantitative determination in biologic specimens is, nevertheless, frequently required. Each of the compounds has a structure that includes a benzyl alcohol group and this functional group is utilized as a basis for their determination by a spectrophotometric method described in this report. Many pharmacologic and physiologic investigations have been concerned with the occurrence and biochemical action of this chemical class of compounds and the literature contains numerous descriptions of isolation and determination procedures. The many methods attest not only to the importance of the drugs but also to the difficulty in achieving analytical methods which are sensitive and selective. For example, many alkaloids give a positive reaction with the colorimetric procedures which have been described (1-3) for determining ephedrine. Fluorometric techniques ( 4 ) are extremely sensitive but these procedures are often time-consuming. Nonaqueous titration systems (5, 6) exhibit a low order of sensitivity and specific;ty when applied to biologic specimens. The ion exchange chromatographic methods (7, 8) are excellent for separation (of closely related compounds but they often fail to yield reproducible quantitative results. Paper (9, IO) and gas (11-13) chromatographic procedures are (1) L. G . Chatten and L, Levi, ANAL.CHEM., 31,1581 (1959). (2) F. Feigl and E. Silva, J. Am. Pharm. Assoc., 47,460 (1958). (3) G . N. Thomis and A. Z . Kotionis, Anal. Chim. Acta, 16, 201 (1957). (4) P. Laugel, Compt. Rend.,225,692 (1962). (5) R. Reiss, 2.Anal. Chem., 167, 16 (1959). (6) M. Rink and R. Lux,Arch. Pharm., 294,117 (1961). (7) S. Blaug and L. Zopl', J. Am. Pharm. Assoc., 45, 9 (1956). (8) L. Sanders, P. H. Elworthy, and R. Fleming, J . Pharm. Pharmacol., 6, 32 (1954). (9) K. Hiller, Phapmazie, 16, 600 (1961). (10) E. Vidic and J. Schuette, Arch. Pharm., 295, 342 (1962). 34, 730 (1962). (11) M. Anders and G. Mannering, ANAL.CHEM., (12) L. Kazyak and E. Knoblock, Ibid.,35,1448 (1963). (13) K. Parker, C. Fontan, and P. Kirk, Zbid.,34, 1345 (1962).
sensitive, but they lack the ability to provide the positive identification which is required by the forensic scientist. Ephedrine and its related compounds have a low order of molar absorptivity; therefore, direct ultraviolet spectrophotometric assays do not exhibit sufficient sensitivity for determination of therapeutic concentrations of these drugs in biologic materials (14). Chafetz (15) described a sensitive ultraviolR spectrophotometric method utilizing periodate oxidation for determining several phenethanolamine compounds in pharmaceutical preparations but the procedure is not applicable to biologic specimens. The need for a specific and sensitive spectrophotometric method for determining ephedrine in biologic fluids is apparent. The method described in this report is rapid, sensitive, and is group specific for ephedrine and those related compounds which extract as a base and have a benzyl alcohol functional group. Preliminary separation from other alkaline drugs is not required. EXPERIMENTAL
Apparatus. A Beckman DK-2A ratio-recording spectrophotometer with linear presentation, settings and operation routine, was used for the ultraviolet absorption measurements. The sample path length was 10 mm throughout. Infrared spectrograms were prepared on a Beckman IR-4 spectrophotometer. A Barber-Colman Model 5000 gas chromatograph with a coiled 6-foot 1 S.E. 30 column was used for gas chromatographic analysis. Procedure. Ten-milliliter amounts of blood, serum, or urine are placed in a 250-ml separatory funnel to which 1 ml of 1N NaOH and 100 ml of ether are added. This mixture is shaken vigorously for 3 minutes. The ether is removed and filtered through Whatman No. 541 filter paper. Complete recovery of the ether should not be attempted, but the volume of ether recovered is recorded and is included in the final calculations. Five milliliters of 1M acetic acid are added to the filtered ether and the mixture is shaken for 5 minutes. Four milliliters of the aqueous layer are transferred to a dry 1000-ml round-bottom flask, and 10 ml of (14) H. Thies and Z . Oezbilici, Arch. Pharm., 295, 715 (1962). (15) L. Chafetz, J . Pharm. Sci., 52, 1193 (1963). VOL. 39, NO. 4, APRIL 1967
531
.BO
-
.70
-
.90
1
I
'7
SEMICARBAZONE OF EPHEDRINE PRODUCT
Figure 2. Infrared absorption spectrum of the hydrochloride of the steam-distilled ephedrine reaction product, 2 mg in 400 mg of potassium bromide -1
I
210 220 230 240 250 260 270 280290 300 310 320 330 340 350 360
MILLIMICRONS
Figure 1. Ultraviolet absorption spectra of ephedrine in water, of the steam-distilled ephedrine reaction product, and of the semicarbazone of the reaction product, each corresponding to an ephedrine concentration of 8 pg/ml
0.2N NazCrOI in glacial acetic acid are added. The contents of the flask are refluxed for 15 minutes, cooled, and neutralized with 200 ml of 1M sodium carbonate. The mixture is then steam-distilled at a rate of 5 to 10 ml per minute. Fifty milliliters of distillate are collected and read against a water blank at 250 mp. The sensitivity and specificity of the procedure are greatly enhanced by reacting, at 100' C in boiling water for 10 minutes, 4 ml of the distillate with 1 ml of 0.5M semicarbazide hydrochloride buffer to pH 3.5 with sodium acetate. This reaction is conveniently done in 10-ml tubes fitted with Teflon-lined screw caps. The semicarbazone is read at 282 mp against a blank prepared by the addition of 1 ml of water to 4 ml of the steam distillate. If the steam distillate contains less than 2 pg/ml of ephedrine as the reaction product, the following concentration procedure should be accomplished : Fifty milliliters of distillate are extracted by vigorous shaking with 10 ml of methylene chloride. Eight milliliters of
Table I. Standard Curve of Ephedrine Reaction Product Absorbance of semicarbazone Ephedrine Absorbance of of in reaction Absorbance reaction Absorbance sample product Concn Concn product d m l 0.059
1.17 0.90 0.59 0.30
20.0 15.0 10.0 5.0
0.060 0.059 0.060
2.22 1.70 1.09 0.57
0.111 0.113 0.109 0.114
Table II. Recovery Studies of Ephedrine Recovery, mean i std. dev. (pglml) Ephedrine added Whole blood Urine pk?/ml 89.3& 2.5 96.5 1.5 100.0 50.0 25.0 10.0 5.0
Av recovery
45.8 f. 1.5 21.2 f 0.8 8.4f 0.3 3.9& 0.1 85.1 %
-~
532
ANALYTICAL CHEMISTRY
48.5 f 1.8 23.4 f 0.7 9.3 & 0.4 4.5 f 0.1 93.6%
Figure 3. Infrared absorption spectrum of ephedrine hydro= chloride, 2 mg in 400 mg of potassium bromide the methylene chloride are placed in a 125-ml flask to which 0.05 ml of 4M acetic acid are added. The solvent is evaporated at room temperature while the flask rotates on a rotary vacuum evaporator. The residue is dissolved in 4, 8, or 16 ml of water, depending on the absorbance at 250 mp previously observed for the unextracted distillate. At this point the semicarbazide reaction described above is performed, The ephedrine content of the sample is calculated from a standard curve prepared from aqueous solutions in appropriate concentrations carried in identical fashion through the procedures outlined, including the concentration technique for solutions containing only small amounts of ephedrine. RESULTS AND DISCUSSION
Figure 1 shows the ultraviolet absorption spectrum of ephedrine, of its steam-distilled reaction product, and of the semicarbazone of the reaction product. Similar data are observed for pseudoephedrine and for phenylpropanolamine. Each reaction product affords a well-defined absorption curve with a maximum at 250 mp and a minimum at 222 mp. The semicarbazones have absorption maxima at 282 mp and minima at 245 mp. Ultraviolet absorption by the ephedrine reaction product and its semicarbazone adheres to the BeerLambert law at least over the range 0 to 20 pg/ml (Table I). The semicarbazone of the ephedrine reaction product represents an increase in sensitivity of approximately 56-fold over the 0.002 absorbance unit per pg obtained by the ultraviolet determination of unconverted ephedrine. Table I1 summarizes the recovery of ephedrine from whole blood and urine to which the drug had been added. Average recovery from blood was approximately 85 and from urine, approximately 93%. To determine tissue levels of the drug additional purification of the extract is required. This can be accomplished by performing at least two ether extractions. Best results are obtained if the first aqueous acid extraction from ether is performed with 0.5N hydrochloric acid instead of dilute acetic acid. Many alkaline drugs which might be given to patients concomitantly with ephedrine or which are similar in chemical structure were investigated for possible interference (Table 111). Pipradol and antazoline are the only non-alphahydroxy, alpha-phenyl drugs which gave significant absorb-
Table 111. Compounds Investigated for Interference with the Determination of Ephedrine6 Absorbance Absorbance of product of productb semicarbazonee Compound Ephedrine 1.17 2.22 0.88 1.34 Phenylpropanolamine 2.30 1.20 Pseudoephedrine 0.01 0.04 Blank 0.13 Amitriptyline 0.04 0.04 0.10 Amphetamine 0.87 (247) 1.36 Antazoline 0.01 0.06 Atropine 0.07 0.06 Bromodiphenhydramine 0.06 0.01 Buclizine 0.01 0.05 Caffeine 0.04 0.04 Carbinoxamine 0.01 0.08 Chlorocyclizine 0.06 0.04 Chlorothen 0.03 0.04 Chlorpheniramine 0.02 0.07 Ch emizo1e 0.01 0.05 Prochlorperazine 0.01 0.05 Covatin 0.04 0.06 Cyclizine 0.07 0.01 Meperidine 0.05 0.20 (257) Diphenylpyraline 0.11 0.10 Diphenhydramine 0.06 0.01 Doxylamine 0.11 0.12 Hydroxyzine 0.01 0.04 Lidocaine 0.01 0.08 Meclizine 0.29 0.06 Methapyriline 0.06 0.08 Met hoxamine 0.02 0.04 Morphine 0.02 0.04 Phenaglycodol 0.01 0.07 Phenindamine 0.03 0.06 Pheniramine 0.01 0.04 Phenylephrine 0.06 0.30 Phenyramidol 0.02 0.04 Pilocarpine 0.97 (257) 0.08 Pipradol 0.04 0.04 Propoxyphene 0.06 0.20 Pyrilamine 0.19 0.43 Thenyldiamine 0.03 0.15 Thonzylamine 0.20 0.40 Tripelennamine 0.03 0.04 Triprolidine a Each compound was determined from whole blood. The level corresponds to a concentration of 20 p g of the compound per ml of distillate. Each value i:; the average of three determinations. Read at 250 mp unless otherwise indicated. c Read at 282 mu.
ance at 250 mp. PipradoI is easily identified as a n interfering component by observing that its reaction product peaks at 257 mp and does not form a semicarbazone. The chemical structures of antazoline and ephedrine suggest that these compounds yield nearly identical reaction products. If through unusual circumstances there is a question of the
presence of antazoline, prior qualitative identification should be accomplished (16). Four adult human males, each of whom received 100 mg of ephedrine orally, were examined periodically to establish blood and urine levels of the drug. Only trace levels of ephedrine were demonstrated in venous blood samples drawn at 30-minute intervals from 15 minutes to 8 hours after administration of the 100-mg dose. The concentration of ephedrine in the urine was found to range from 84.8 to 248.0 (average 139.9) pg/ml in samples collected at the end of the first 4 hours following ingestion of the drug. Samples collected at the end of the second 4-hour interval contained from 101.3 to 158.2 (average 131.2) pg/ml and those from the third 4-hour interval, 17.7 to 96.5 (average 66.5) pg/ml. Samples in the 12-24 hour interval after taking ephedrine contained from 5.5 to 45.0 (average 18.7) pg/ml. While the ephedrine concentration in the individual samples varied from person to person, the total amount excreted in the urine by the four subjects during the first 24 hours was less variable: 52.1 to 70.2 (average 61.7) mg. Gas chromatographic and infrared spectrophotometric analyses of the excreted ephedrine indicated it to be unchanged. These results are in agreement with previous observations that man excretes ephedrine practically 100% in unchanged form in 48 hours, more than half of it in the first 24 hours (17). Infrared spectra show a strong carbonyl absorption band at 1720-1750 cm-I in the product (Figure 2) but not in the parent drug. In contrast, ephedrine shows a strong hydroxyl absorption at 35OC-3600 cm-' (Figure 3) which is absent in the infrared spectrum of the product. The reaction, therefore, appears to convert the hydroxyl group of ephedrine to a carbonyl group whose conjugation with the aromatic ring accounts for the increased ultraviolet absorptivity of the steam-distilled product. Dichromate is applicable to the determination of ephedrine only if glacial acetic acid is used as the reaction medium. Hydrochloric and sulfuric acids appear to oxidize the drug to degradation products which have little ability to absorb ultraviolet radiations. The aryl-alkyl ketone reacts rapidly with semicarbazide at pH 3.5 to form a derivative more sensitive to ultraviolet radiations. ACKNOWLEDGMENT
The author is indebted to Elmer V. Dah1 for helpful discussions during portions of this work. The technical assistance of John Biggs, Raymond Sumners, and Richard Walker is also deeply appreciated.
RECEIVED for review November 17, 1966. Accepted January 9,1967. (16) W. W. Fike and I. Sunshine, ANAL.CHEM., 37, 127 (1965). (17) H. Beckman, "Pharmacology, the Nature, Action, and Use of Drugs," W. B. Saunders Co., Philadelphia, 1961, p. 435.
VOL. 39, NO. 4, APRIL 1967
533
