js not under vacuum, but the arguments are not so clear cut nor the effects so great in magnitude. APPENDIX
Figure 1 can be used t o demonstrate the principle that while a band may broaden when it encounters a velocity increase, the time interval between front and rear remains essentially constant except for the slight amount of increase due t o normal band-spreading processes. The figure shows a column containing an incompressible fluid which is speeded up when forced into a column of smaller cross-sectional area, A?. The final velocity of the band is larger than the original velocity in the ratio v2/v1 = A1/A2. The volume of the band is constant because the fluid is incompressible, and the final band length divided by its initial value is &/l1 = Al/ilz. From the above expression we have lz/ll = v2/vl, showing that the
band has broadened considerably. The passage time of the band before the transition is Atl = ll/Rvl and after the transition i t is &/Rv2. The expression just preceding this shows that ll/vl = 12/v2or Atl = At?, and consequently that the zone spreading in time units is constant. This argument, applicable to columns linked in series, may be extended to include gas compression effects in gas chromatography. If a transition occurs from a region where the pressure is p l to a region of pressure p?, and p1 > p?, the entire mass of gas, along with the contained band, expands in volume by the ratio p1/p2. The velocity also increases by this ratio. Because the cross-sectional area is constant, this means that the band increases in length, 1, such that 12/11 = p l / p z = vz/vl. The passage time of the band is first At, = ll/Rvl and after the transistion is Atz = 12/Rv2. Because 12/11 =
vz/vl, these passage times are once again equal to one another. LITERATURE CITED
(1) Bohemen, J., Purnell, J. H., J . Chem. SOC.1961,360.
(2) Desty, D. H., Goldup, *4.,Swanton, W. T., 1961 International Gas Chromatography Symposium, East Lansing, Mich., June 1961, p. 83 of preprints. 1 3 ) Giddings. J. C.. AKAL.CHEW.34. 722 (1962). (4) Giddings, J. C., hlallik, K. L., Eikelburger, M., Ibid., 1026. ( 5 ) Giddings, J. C., Seager, S. L., Stucki, L. R., Stewart, G. H., I b i d , 32, 867 (1960). (6) Kkselbach, R.,Ibid., 3 3 , 2 3 (1961). i'ii, Littlewood. A. B.. "Gas Chromatography, 19p8," D. H. Desty, ed., p. 35, Academic Press, K e x York, 1958. (8) Sterart, G. H., Seager, S. L., Giddings, J. C., h A L . CHERT. 31,1738 (1959). RECEIVEDfor review October 8, 1962. Accepted January 14, 1963. Kork supported by a Research Grant S o . GM 10851-06 from the National Institutes of Health, Public Health Service. Y
l
~
\
Rapid Gas Chromatographic Method for Screening of Toxicological Extracts for Alkaloids, Barbiturates, Sympathomimetic Amines, and Tranquilizers KENNETH D. PARKER, CHARLES
R. FONTAN, and PAUL L. KIRK
School of Criminology, University of California, Berkeley, Calif.
b A gas chromatographic method is described for the screening of toxicological extraction residues for alkaloids, barbiturates, sympathomimetic amines, and tranquilizers. Retention data are given for 41 alkaloids chromatographed on an SE-30 column a t five temperatures. Representative members of other groups of compounds chromatographed on the same column are listed. With a sufficient elevation of temperature these groups emerge under the same conditions, enabling the toxicologist to examine a tissue extract rapidly for the presence of microgram quantities of a large number of acidic, basic, and neutral organic compounds. Two Carbowax 20M (alkaline and nonalkaline) columns are compared to determine their usefulness in the screening of toxicological extracts. A solid injector which allows concentration of the extractives and elimination of the solvent is described.
T
toxicological extracts have been screened for nonvolatile organic compounds by the tedious process of systematic color and crystal RADITIONALLY
356
ANALYTICAL CHEMISTRY
tests (17). Ultraviolet absorption and its change with pH have been used extensively as a systematic approach to the presumptive identification of compounds to which they are applicable (6, 16). Serious limitations are imposed, however, on the use of ultraviolet absorption for the examination of mixtures, because a composite spectrum of all absorbing compounds is obtained. Paper chromatography has been used most successfully to screen tissue extracts (17). The resulting fractions, located and characterized by an array of chromogenic reagents, may, depending upon previous treatment, be eluted for further examination. Many different chromatographic solvent systems are required, however, for adequate separation within the acidic, basic, and neutral fractions that result from toxicological extractions. Brackett (4) used a single generally applicable solvent system and attained unique characterization of many compounds by chromatographing simultaneously the parent compound and various derivatives formed from it on the chromatogram at the origin by the addition of oxidizing, reducing, and other reagents. Anders and h h n n e r -
ing (1) illustrated a similar approach applicable to gas chromatography. Derivatives were formed on the column from a number of alkaloids and steroids. As with paper chromatography, gas chromatography of alkaloids (9), barbiturates (6, 7 , 13, 1.4)) sympathomimetic amines ( l a ) ,and tranquilizers (11) requires very different operating parameters for adequate separations within the different groups. This paper presents a single gas chromatographic system of general applicability to the screening of toxicological extraction residues for the groups of compounds specified above. An injector designed for the introduction of dry samples is also presented. EXPERIMENTAL
Apparatus. Figure 1 shows t h e construction of t h e solid injector. T h e components were chosen t o make t h e injector gas-tight when assembled. The sample carrier was adapted from a cleaning wire supplied with t h e B-D Yale hypodermic needle, 22 G, 3 inches. The plunger handle was formed by inserting one end of t h e sampling wire into a B-D Yale 24 G, I-inch, hypodermic needle and crimping
::
B
A Figure 1.
C
E
D
D B
Exploded view of solid injector A. B. C. D.
E.
Plunger Sampling-wire guide Silicone rubber disks Sampling wire Injection needle
the needle to secure the wire. A guide for the sampling wire was made by cutting off one end of a doublepointed blood-collecting needle (B-D Yale 4-042-240) and shortening the other. Two small disks were cut from a silicone rubber injection gasket, one being slipped onto the sampling wire and the other onto the wire guide, to assure a hermetic seal. -4B-D Yale 22 G, 11/2inch hypodermic needle was slipped over the sampling wire to provide a penetration tip. The sampling end of the central wire was reduced in diameter to prevent the sample from being scraped from the wire as it was drawn into the injection needle. After assembly the components were secured with electrician's tape. The Hy-Fi gas chromatograph (Wilkens Instrument and Research Co., Walnut Creek, Calif.) with hydrogen flame ionization detector, Aerograph Model 600, and the Leeds and Northrup Speedomax H, 0- to 1-mv. recorder, Model S, equipped with a Disc chart integrator Model 207, were employed. The chromatographic columns were stainless steel tubes of l/s-inch o.d., 0.093-inch i d . , 5 feet in length. They were packed with 60- to 80-mesh Chromosorb W, acid-washed, coated with either SE-30 (5y0 by weight) or Carbowax 2033 (1% by weight). I n preparing one of the Carbowax 20M columns the Chromosorb SV was coated with KOH (5y0 by weight) before the liquid phase was added. The column filling was prepared by mixing the solid support with a chloroform solution of the liquid phase and evaporating the solvent immediately by warming the mixture under vacuum, with occasional stirring. The operating conditions were: injector temperature 30' C. above oven temperature; oven temperatures 190°, 210', 230°, 250', and 270' C.; flow rate of carrier gas (nitrogen) 30.7 cc. per minute; flow rate of hydrogen 22 cc. per minute; attenuation x 4 . Procedure. Liquid injections containing 1 to 10 pg. of pure compound, dissolved in ethyl alcohol or acetone, were made with a 1-p1. Hamilton syringe. Corresponding solid injections were made with the instrument described above. A measured volume of solution was applied to the tip of the central wire, and the solvent was allowed to evaporate, leaving the residue. Compounds which are volatile a t room temperature-e.g., some sympathomimetic amines-were converted on the central wire to the salt by the addition of
F
E
n
D
Q
a
c 0 n a Q
a
Q
a
0
1
2
3
Retention Time
Figure 2.
4
5
6
7
8
(minutes)
Separation of mixture of alkaloids A.
Acetone Procaine C. Homatropine D. Cocaine E. Codeine F. Morphine G. Diacetylmorphine H. N-Allylnormorphine Samples 1 to 10 pg. in acetone
B.
alcoholic hydrochloric acid (HC1 50 pg. per pl.), to prevent loss from premature vaporization. A drop of drug solution and a drop of acid solution were added to the wire tip and allowed to dry together. To introduce the solid sample into the gas chromatograph, the samplebearing wire is retracted into the injection needle, the needle is inserted through the injection diaphragm, and the sampling wire is extruded into the injection port by pressing on the plunger After about 4 seconds the solid injector is withdrawn. I n this study the injection port was maintained a t least 30' C. above the column temperature. Since this work was directed toward the development of a screening procedure, rather than toward quantitation, variations resulting from altering the duration of the solid injection were not investigated. Volumetric alcohol or acetone solutions were made from the toxicological extraction residues, aliquots then being subjected to dry injection by the method described for pure compounds. RESULTS
Table I presents the retention times in minutes. and the retention values relative to codeine, for 1- t o 10-pg. samples of 41 alkaloids chromatographed on the 5% SE-30 column a t five different temperatures. For convenience the Merck Index page number for each compound is listed. Figure 2 shows the partial separation of seven alkaloids on a newly prepared 5% SE-30 column a t 250' C., and at a
nitrogen flow rate of 14.5 cc. per minute, these conditions having been found to produce the best separation of the largest number of alkaloids in the shortest time. A linearity study (weight of injected sample us. peak height and area) was made with codeine and cocaine, both chromatographed a t 230" and 250" C. under the conditions described above. The response to these two alkaloids was nonlinear but fairly reproducible. An experimental attempt was made to elucidate the cause of this nonlinearity. After the chromatograph had stood unused for 2 hours a t 230' C. the response to 5 pg. of codeine was determined. Then 11 successive 10-pg. samples of codeine were injected into the instrument at 5-minute intervals. The response to 5 pg. of codeine was again determined, and was found to be four to five times the previous value. The chromatograph was again allowed to stand for 2 hours a t 230' C., and the 5-pg. injection of codeine was repeated. The response corresponded with that obtained a t the beginning of the experiment. Response variability influenced by the history of the column may be attributable to the absorption of the codeine by the solid phase, Chromosorb
w.
Table I1 lists representative barbiturates, sympathomimetic amines, and tranquilizers which were chromatographed under the same conditions VOL. 35,
NO. 3, MARCH 1963
e
357
fi9
Alkaloids Tronquilizers
270' C.
Temperatures
3, Ranges of retention times
Figure
Table 1.
250'
230'
210'
190.
Column
Barbiturates Sympathomimetic Amines
Retention Data on Some Alkaloids and Related Compounds
Retention data Compound (free base) Aconitine Apomorphine Atropine (d-hyoscyamine) Berberine Betaine Caffeine Cinchonine (d-cinchonine) Cinchonidine (2-cinchonine) . Cocaine Codeine Colchicine Cotarnine Diacetylmorphine Dibucaine Dihydrocodeinone Dihydrohydroxycodeinone Dihydromorphinone Emetine Ethylmorphine Gelsemine Homatropine Hydrastine 2-Hyoscyamine Meperidine Mescaline d,l-Methadone Morphine N-Allylnormorphine Nicotine Papaverine Pilocarpine Piperine Prqcftine Quinine Scopolamine Sparteine Strychnine Thebaine Theobromine d-Tubocurarine Yohimbine
M.I., P." 15 94
c
10.6 1.08 5.1 1.06 3.0 1.00 ii.3 i.38 12.3 1.28 5.1 1.06 3.2 1.07
9.2 0.60 111 143 2i.O i.56 147 187 6.0 3.4 0.22
... ...
262 261 273 275 277 291 333 342 360
...
9.2 15.4 .._ 3.1 32.0
361 362 25.0 401 433 1'9.5 474 522 7.3 528 ... 547 9.4 646 657 ... 2.3 662 691 17.0 701 719 5.0 2.6 771 818 ... 823 6.1 854 888 13.6 925 2.8 971 986 25.6 1028 1029 24.3 1075 ... 1111
...
...
5.7 3.9 11.3 2.1
0.59 0.41 1.18 0.22
3.0 2.1 5.2 1.2
0.63 2.1 0.70 0.44 ... 1.08 3.1 i.03 0.25 ... ...
13.9 1.45
7.9 1.65 5.3 1.77
13.9 5.5 9.6 11.0 2.2 13.3 20.3 11.3
1.45 0.57 1.00 1.15 0.23 1.39 2.12 1.18
7.9 2.8 4.8 5.2 1.2 6.3 9.4 5.4
1.65 0.58 1.00 1.08 0.25 1.31 1.96 1.12
1.8 1.62 14.1 11.6 i.27 11.6 27.0 0.47 4.2 11.3 0.61 5.7 1.7 0.15 2.0 4.7 1.10 11.6 17.8 0.17 0.8 35.6 ... 7.0 28.5 0.40 3.5 27.0 0.88 8.1 0.18 1.9
0.19 1.47 1.21 1.21 2.81 0.44 1.18 0.59 0.18 0.21 0.49 1.21 1.85 0.08 3.71 0.73 2.97 0.36 2.81 0.84 0.20
1.1 6.1 5.1 5.0 10.0 2.4 5.2 2.9 1.1 1 .o
0.23 1.27 1.06 1.04 2.08 0.50 1.08 0.60 0.23 0.21
0.60 1.00 ._. 0.20 2.08
* R T retention time (minutes);
358
*
ANALYTICAL CHEMISTRY
1.73 0.63 1.00 1.00
3.9 1.30 5.5 1.83
1.2 0.40
3:i 6.1 1.6 3.2 2.1
i.07 2.04 0.53 1.07 0.70
... . . .
5.8 1.19 4.2 1.40 8.0 1.67 5.0 1.67
...
13.2 2.3 13.6 2.0 13.0 3.8 1.6 ... . . . 26.0 6.0 1.44 i.66 13.8 1.58 10.4 1.08 5.0 . . . 11.2 1.17 5 . 0 ... 3.6 0.38 1.9
...
2.75 0.48 2.84 0.42 2.70 0.79 0.33 5.40 1.25 1.04 1.04 0.40
Page references to Merck Index (IO). r.RT retention time relative to codeine. 6 Compound injected but no response observed. a
5.2 1.9 3.0 3.0
7.8 2.60 1.7 0.57 7.8 2.60 1.4 0.47 8.2 2.74 2.6 0.87
. . . ...
3.8 1.27 3.2 1.06 .,.
...
...
*..
as those employed for the alkaloids, summarized in Table I. Specific retention data are not given because the column was not ideal for these compounds, though it served well for screening purposes. Early- and late-emerging compounds were included in each group. Figure 3 shows the approximate ranges of retention times of these groups of compounds at the fire column temperatures employed. Because of the wide structural variety of the compounds included in this screening study, the choice of column involved a compromise. The graph represents general trends rather than the limits of specific retention times, the latter being influenced by sample size on this less-than-ideal column. A more complete list of phenothiazines has been chromatographed on a silanized SE-30 column by Anders and Mannering (2). The bar graph for the alkaloids chromatographed a t 210' C. shows an unexpectedly short retention time range. This is attributable to the fact that a t lower temperatures compounds with long retention times do not produce noticeable responses, because of absorption and diffusion. At a flow rate of 30 cc. per minute suitable operating temperatures for the two columns utilizing Carbowax 20M (IQj,) as the liquid phase and Chromosorb W (60- to 80-mesh) as the solid support are as follows: alkaloids, 210" t o 260" C.; barbiturates, 210" to 230" C.; sympathomimetic amines, 150" to 180" C.; tranquilizers, 180" to 250' C. The peaks were more symmetrical than those given by the SE-30 column, and the order of emergence of the barbiturates was different. Details of a Carbowax 20M column for the gas chromatography of the barbiturates have been described ( I S ) . The linearity of both alkaline and nonalkaline columns was tested using benzphetamine, n-octadecane serving as an internal standard. Both columns gave linear responses. Caffeine was tested for linearity only on the alkaline column: The response was linear. Morphine failed to emerge on the Carbowax 2019 columns. Most of the alkaloids, sympathomimetic amines, and tranquilizers gave symmetrical peaks on these columns. The barbiturates, as expected, failed to emerge on the alkaline column. The solid injector was used on many of the compounds listed in Table 11. It was found most useful for compounds whose retention times are so short that their emergence would be obscured by a large solvent peak. Retention times were not affected by solid injection. DISCUSSION
The retention data in Table I and Figure 3 have been found useful in rapid screening for alkaloids, bar-
Table II.
Representatives of Three Groups of Drugs Chromatographed
Barbiturates Sympathomimetic amines Free acids Hydrochlorides hfetharbital Metamphetamine Probarbital Methoxyphenamine Allylbarbituric acid Pseudoephedrine Rutabarbital Ephedrine Butalbital Phenylpropanolamine Cyclopal Benzphetamine Phenobarbital Arranged in order of increasing retention times.
Tranquilizers Free bases Phenyltoloxamine Diphenylpyraline Trifluopromazine Promazine Chlorpromazine Methoxypromazine
Although the SE-30 (5%) coliinin shows absorptive properties toward polar compounds with which the toxicologist must deal, it is useful in the screening of toxicological extracts primarily because of its general applicability to acid, basic, and neutral compounds. Siliconizing the solid support of the SE-30 column may offer a partial answer to the problem of absorptivity. I n laboratories where temperature programming is ayailable, i t may be usefully applied to screening. ACKNOWLEDGMENT
biturates, sympathomimetic amines, and tranquilizers in quantities of from 1 t o 10 fig. extracted from tissue in c a w of toxicological interest. dcidic and basic chloroform and ether extractions have been made of urine, blood, and liver. Concentrates of the tissue extractives produced only minor responses, as compared with these foreign compounds in microgram quantities, and were not found t o interfere with the chromatographic method. Extraction residues representing as much as 4 grams of liver or kidney, or 5 t o 10 ml. of urine or blood, were chromatographed using the solid injector to evaluate the blank response obtained from normal tissue extractives. The magnitude of the response from the blank was dependent upon the type and quality of the extraction used or required, and the amount of starting tissue represented by the injection. Obviously extracts of known normal tissue should be run parallel t o all determinations until the operator is familiar with the blank responses t o be expected. If 250” C. were chosen to screen acidic or basic extracts by liquid injection on the SE-30 (5%) column and no significant peaks emerged within 14 minutes, all of the alkaloids in Table I would be excluded except nicotine (Khich emerges with the solvent) and strychnine (retention time 26 minutes), However, at this temperature the presence of many of the sympathomimetic amines and barbiturates may be obscured by the solvent peak. I n this situation the solid injector was found very useful, facilitating the detection of both of these groups of compounds. Lower temperatures and other columns could then be used to make tentative identification of these substances. A graph similar t o Figure 3, derived
from d a t a collected under specified chromatographic conditions, can save much time in the screening of toxicological extracts. For example, a n unknown which gave a peak at 250’ C. between 10 and 30 minutes after injection would probably prove to be a n alkaloid. If, on the other hand, the retention time lay between about 10 and 4 minutes, one should test the material for both alkaloids and tranquilizers. A very short retention time at this temperature would indicate t h a t any of the four groups of drugs might be represented. Many of these compounds are difficult t o chromatograph, so that chromatography on several different columns is often required for the total exclusion upon which a firm identification may be based. The absorption of basic compounds b y the solid support has continued to be a problem in this study. In the past this difficulty has been dealt with by coating the solid support with KOH (15) or with a low percentage of polyethylene glycol (3) (Carbowax) and by the use of inert solid supports such as glass microbeads (8).However, from the toxicological point of view this problem has not been completely solved, as each of the above columns has serious disadvantages. The alkaline column prevents acidic compounds as well as basic compounds with acidic functional groups-e.g., morphine and epinephrine-from emerging. The temperature limitation of Carbowax 20M (excessive loss of liquid phase above 250” C.) as well as its polarity and possible interaction with phenolic groups-e.g., those on morphine-restricts its usefulness. The use of glass microbeads as a solid support does not entirely eliminate absorption ( I I ) , and t h e efficiency of such a column is comparatively low.
The authors are indebted to Darrell B. McComas, University of California hledical Center, San Francisco, for an initial design of the solid injector. The technical assistance of Marjorie Doyle is gratefully acknowledged. LITERATURE CITED
(1) Anders, ?*I. W., Mannering, G. J., ANAL.CHEY.34,730 (1962). (2) Anders. M. W.. Mannering, G. J., ’ 3. Chrokatog. 7, 258 (1962).
(3) Bohemen, J., Langer, S. H., Perrett, R. H., Purnell, J. H., J . Am. Chem. SOC. 82,2444 (1960). (4) Brackett, J. W.,San Mateo County
Coroner’s Laboratorv. Redwood Citv. Calif., unpublished dktal1962; (5) Bradford, L. W., Brackett, J. W., Mzkrochzm. Acta 3,353 (1958). (6) Brochmann-Hanssen, E., BaerheimSvendsen, A.. J . Pharm. Sci. 50. 804 d l
I
,
(1961). (7)’ Cook, J. G. H., Riley, C., Piunn,
R. F., Budgen, D. E., J . Chromatog.
6, 182 (1961). (8) . . Hishta,. C.,. ANAL. CHEM. 32, 880
(1960). (9) Lloyd, H. A., Fales, H. M., Highet,
P. F., Vanden Heuvel, W. J. A., Wildman, W. C., J . Am. Chem. SOC.82,3791
(1960). (10) Merck Index, 7th ed., 1960. (11) Parker, K . D., Fontan, C. R., Kirk, P. L., ANAL.CHEM.34, 757 (1962). (12) Ibid.. D. 1345. (13) Zbid.; 35, 418 (1963). (14) Parker, K. D., Kirk, P. L., Ibid., 33, 1378 (1961). (15) Smith, E. D., Radford, R. D., Zbid., 33, 1160 (1961). (16) Smith, H. F., MacDougal, J. R.,
Roy. Can. Mounted Police Seminar,
No. 1, 117 (1954). (17) Stewart, C. P., Stolman, A., eds.,
“Toxicology,” Vol. 2, Chap. 7 , Academic Press, New York, 1961. RECEIVED for review August 6, 1962. Accepted December 26, 1962. Work supported by grants from the National Institutes of Health, E.S. Public Health Service [EF 21 (C3)], and the Research Committee, University of California. Spring Meeting, California Association of Criminalists, San Diego, Calif., May 1962.
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