was removed with the precipitated proteins. DISCUSSION
Using Cellosolve in place of ethyl alcohol, the systems obtainable are more efficient for carbon-14 determinations. Cellosolve, which does not absorb moisture from the atmosphere as ethyl alcohol does, has the added advantage of two functional groups, making it 'more useful than ethyl alcohol for solubilizing a greater variety of carbon compounds. The solvent system and the counting procedure described are recommended in biological investigations involving
carbon-14- labeled materials, since the procedure provides the highest figure of merit with convenience of sample analysis. The method should be applicable to many biological tracer problems as well as other problems including contamination monitoring of human urine for carbon-14. Although carbon-14 was the only isotope studied to date, the method should be applicable to the assay of other isotopic tracers. The count rate ratio method of determining absolute activity has provided a very convenient and rapid method for accurate assay of large numbers of urine and plasma samples.
LITERATURE CITED
(1) Bruno, G. A., Christian, J. E., ANAL. C m . 33,650 (1961). (2) Bruno, G. A., Christian, J. E., J . Am. Pharm. ASSOC., Sci. Ed. 49, 560 (1960). (3) Herberg, R. J., ANAL.CEEM.32, 42 (1960). (4) Kinard, F. E., Rev. Sci. Instruments 28,293 (1957). (5) Langham, W. .T.. Eversole. W. J..
.,
M. R., Proc.'Soc. Expti. Biol.' & Med: 102,8 (1959).
RECEIVED for review December 12, 1960. Accepted June 2,1961.
Separation of Toxicologically Important Bases via Centrifugally Accelerated Paper Chromatography LEO A. DAL CORTIVO, CAROL H. WILLUMSEN, and SIDNEY 6. WEINBERG Office of fhe Medical Examiner, Suffolk County, Brookhaven Memorial Hospital, Patchogue,
N. Y.
WALTER MATUSIAK Division of laboratories, Office of the Chief Medical Examiner, New York,
b The practicability of applying centrifugal paper chromatography to problems in forensic toxicology was determined. Experiments with known reference alkaloids and organic bases indicated that separations could b e achieved in 5 to 15 minutes as opposed to the hours consumed by classical chromatographic techniques. An unknown powder was readily resolved into two components (heroin and quinine) in 10 minutes. Similarly, centrifugal paper chromatography, requiring 12 minutes, revealed the presence of morphine in extracts of hydrolyzed bile and urine specimens. Calculation of Rl's showed that values obtained by the centrifugal method are reproducible and approximate those of ascending chromatography. Recoveries and sensitivities proved adequate for all substances subjected to centrifugal chromatography.
I
N FORENSIC COLOGY, the
AND
CLINICAL
TOXI-
need frequently arises for rapid yet unequivocal separation, identification, and determination of substances contained in powders, tablets, capsules, and material extracted from tissue and biological fluids. At present, there are no methods available which adequately fulfill these requirements. Conventional ascending and descending paper chromatography procedures yield suitable separations but require long periods of time for completion.
1218
ANALYTICAL CHEMISTRY
N. Y.
Stationary radial filter paper techniques decrease time requirements appreciably but permit analysis of only a few samples per sheet. In addition, sensitivity is somewhat sacrificed because of lateral spreading of the spots. Filter paper electrophoresis affords relatively rapid migrations but the resolution achieved is not sufficient for this type of analytical problem. Williams and associates (8) described the use of agar gel media for rapid electrophoretic separation of some alkaloids. This method holds promise but still requires 25 minutes for completion of the electropherogram alone. In 1955, Caronna ( 1 ) separated some alkaloids chromatographically using rotating paper disks pressed between Plexiglas plates. The I2, values obtained closely approximated those observed in classical methods, but the time necessary for the partitions was greatly reduced. Similarly, McDonald and coworkers (6-7) used centrifugal chromatography to separate dyes and amino acids in 5 to 15 minutes as o p posed to the hours previously required. Again, R,'s were similar to those observed in conventional paper chromatography. In preliminary studies by the present authors (g), centrifugally accelerated paper chromatography appeared promising with respect to the separation of toxicologically important substances in instances where time was an essential factor.
This report embodies the results of an investigation of some of the variables of this technique and its application to actual medical examiner case material. EXPERIMENTAL
Apparatus. The Precision Scientific CO'S. explosion-proof Hi Speed chromatograph (No. 67305), equipped with a newly devised solvent feed system (No. 67300), was used. Initial experiments employing this system indicated that the solvent did not flow freely and uniformly on the paper. A l/leinch air vent was drilled in the reservoir cap to maintain atmospheric pressure within the reservoir. This modification not only permitted solvent to flow readily, but also allowed introduction of additional solvent during the operation. The motor was driven by an air pump, producing pressures of 15 p.s.i.g. for continuous service. The Perkin-Elmer Model 4000A double-beam recording spectraphotometer equipped with matched silica cells, 1 cm. in path length and 2 ml. total volume, was used for ultraviolet absorptiometry of eluted spots. Materials. Whatman No. 1 circular filter paper sheets, 32 cm. in diameter, were used. A hole punched in the center with a No. 6 cork borer affords a good fit over the shoulder on the support stand of the solvent feed assembly. Glass fiber washers (Fisher Scientific Co. No. 9-873), 3.7 cm. in diameter, were employed. A No. 4 hole punched
MACHINE DlRECTlON
MACHINE DIRECTION OF PAPER
OF PAPER L
0
\
M
Figure 1. Centrifugully accelerated paper chromatogram of an unknown powder
Figure 2. Centrifugally accelerated paper chromatogram of extracts from hydrolyzed bile and urine specimens
X = Starting points Developmenblme = 10 minutes Outermost line in shape of ellipse is sdvent front
in the center permits the washer to fit snugly over the center post. Reagents. Sorensen’s phosphate buffer, M/15, p H 5. Iodoplatinate reagent, prepared according to Goldbaum and Williams (4,was used to locate spots. Procedures. PREPARATIONOF URINE AND BILE EXTRACTS. All available bile and 100 ml. of urine (if available) are used for analysis. Sufficient sulfuric acid is added to the samples to furnish a final acid concentration of 10%. The specimens are hydrolyzed a t 15 p.s.i.g. for 30 t o 35 minutes in a pressure cooker or autoclave. After cooling, the hydrolyzed samples are made strongly alkaline with sodium hydroxide and extracted twice with 100-ml. portions of ether. The combined ether layers are labeled alkaline fraction. This fraction contains the basic drugs such as quinine, strychnine, phenothiazines, etc. The aqueous layer is reacidified and the pH is then adjusted to 8.5 to 9.0 with ammonium hydroxide. Two 100-ml. portions of chloroform containing 5 ml. of et,hyl alcohol are used to extract the amphoteric drugs such as morphine and N-allylnormorphine. The combined chloroform extracts are labelled ampholytes. Both the alkaline and ampholyte fractions are back-extracted with 25 ml. of 1 N sulfuric acid. The aqueous layers are adjusted to pH 8.5 to 9 with ammonium hydroxide and reextracted with 25 ml. of chloroform containing 2.5 ml. of ethyl alcohol. To ensure against evaporation of the semivolatile bases, one drop of glacial acetic acid is added to each extract. The chloroform is then evaporated just to dryness on a steam bath. The residues are dissolved in a minimum (0.1 to 0.2 ml.) of ethyl alcohol for application on the filter paper sheets. CHROMATOGRAPHY. As reported previously (g), initial experiments employ-
X = Starting points Development time = 12 minutes Numbers adjocent to spots are R,’s
ing 1-butanol, acetic acid, and water as the solvent system proved unsatisfactory because of severe spreading of the spots. Filter paper sheets, presaturated with p H 5 buffer solution and air-dried prior to use furnished the highest degree of separation and the most compact spots. The developing solvent consisted of 1-butanol saturated with the buffer a t room temperature. The residues are spotted on the buffered paper by conventional means. As many as 20 samples may be chromatographed simultaneously. The paper sheet and glass fiber washer are placed into position in the recommended manner. Air pressure driving the motor is adjusted to afford the desired centrifugal speed. Three milliliters of solvent are introduced into the solvent assembly and the partition is continued until the solvent front almost reaches the outer edges of the paper. If necessary, additional solvent, in 1-ml. increments, may be introduced. After completion of the migration, the paper is removed and dried with a stream of warm air. The spots are localized by spraying with the iodoplatinate reagent. R, values are measured in the usual manner. The spots are then cut out, eluted, and subjected to direct and/or differential ultraviolet spectrophotometry by established procedures (3, 4,8). RESULTS AND DISCUSSION
Figure 1 is a tracing showing a chromatogram of a confiscated powder. In this instance, i t was imperative to ascertain the type of narcotic present, if any, in the shortest possible time. The time required for completion of the chromatogram was 10 minutes. The unknown, U , consisted of two compounds, U I and U2, whose R,’s
coincided with those of heroin and quinine. Ultraviolet,spectrophotometry of the eluted spots confirmed that these two substances were present in equal proportions. Controls of morphine, M, heroin, H, quinine, Q, strychnine, S, and a mixture of heroin and quinine, H Q, were chromatographed simultaneously. Strychnine was chosen to exclude the possibility of the powder being a so-called “hot shot.” Quinine is frequently used as a diluting agent in the illicit narcotics trade. In Table I, R, values obtained by centrifugal and ascending techniques are compared. Buffered papers and butyl alcohol saturated with buffer were used for both types of chromatography. The average time required for the centrifugal chromatograms included in the table was 8 minutes. In all instances the Rf’s obtained by the
+
Table 1. Comparison of R, Values by Centrifugally Accelerated and Ascending Chromatography
Rt Valuesa CentrifAscending Substanceb ugal Morphine (8) 0.46 =k 0.02 0.39 f 0.01 Cocaine (5) 0.49 =k 0.01 0 . 4 6 f 0.01 Strychnine ( 5 ) 0.51f 0.01 0.50 f 0.01 Diacetylmorphine (8) 0 . 7 3 f 0.02 0.69 f O . O 1 Meperidine (6) 0.60 i 0.03 0.57 + 0 . 0 2 Promazine ( 5 ) 0.71 f 0.02 0.66 f 0.01 Chlorpromazine ( 5 ) 0.68 f 0.01 0 . 6 1 f 0.01 0.95 Quinine (IO) 0.97 Precision stated as average deviation. Number of determinations given in rsrentheses.
VOL. 33, NO. 9, AUGUST 1961
1219
Table II. Recoveries of Bases after Centrifugally Accelerated Paper Chromatography
Substance Morphine Quinine Diacetylmorphine Promazine Chlorprcmazine
Amount, rg. Recovered, Chromato- with av. graphed dev. 25 10
25 7 f 1 0 108f04
25 15
25 6 f 0 6 14 5 f 0 6
15
14 O f 0 5
centrifugal technique are slightly higher than those obtained in the conventional ascending method. Recoveries of alkaloids and other toxicologically important bases after centrifugal paper chromatography are presented in Table 11. Recoveries and sensitivities are adequate in all cases. Because no concomitant radial migration occurs, centrifugal chromatography yields spots rather than bands. The resulting small surface areas of the spots afford excellent over-all sensitivity. Figure 2 shows the results when extracts from eight hydrolyzed urine, U, and bile, B, specimens were sub-
jected to centrifugal chromatography. Time for development was 12 minutes. Acute narcotism was suspected in all eight cases. Ultraviolet absorptiometry of the eluted spots confirmed morphine in the seven cases where R, values (numbers adjacent to spots) indicated its presence. The ellipsoid shape of the chromatograms (Figures 1, 2) results from the grain of the filter paper. Although absolute migrational distances are affected by this elliptical pattern, RIJs are not influenced. This confirms the observations reported previously by McDonald and coworkers ( 7 ) . The Rl values appearing in Figures 1 and 2 and Table I illustrate the reproducibility attainable whether the substance migrates parallel to the major or minor axes of the ellipse. Centrifugal speeds in the range 300 to 1000 r.p.m. are reported to have no effect on R,’s (6, 7 ) . The instrument used in this investigation is not equipped with a tachometer; however, regulation of the air pressure driving the motor furnished the following speeds as measured with a hand tachometer: 6 to 7 p.s.i.g. 9 to 10 p.s.i.g. 12 p.s.i.g.
600 f 100 r.p.m. 1100 f 200 r.p.m. 2000 f 200 r.p.m.
No effects on R, values were noted at any of the above speeds. However, speeds approaching 2000 r.p.m. created
a solvent-washing action resulting in spreading of the spots and generally unsatisfactory chromatograms. Speeds of less than 600 r.p.m., on the other hand, lengthen the time required for completion of the chromatogram, thereby defeating the original purpose of the technique. The speeds yielding the best separations in the shortest time were in the range 750 to 1200 r.p.m. These speeds were used throughout this investigation. LITERATURE CITED
( 1 ) Caronna, G., C h i n . e . ind. (Milan)
37,113(1955). (2) Dal Cortivo, L. A . , Willumsen, C. H., Matusiak, W., Paper presented at 2nd
International Meeting on Forensic Medicine and Pathology, New York City, September 1960. ( 3 ) Goldbaum. L. R.. Kazvak. L.. J .
Pharmacal. ‘Exptl. Therap.
106, ’No.
4 (1952). (4) Goldbaum, L. R., Williams, M. A,, J . Forensic Sci. 4, 144 (1959). (5) McDonald, H. J., Bermes, E. W., SheDherd. H. G.. Chromatoo. Methods (6) ‘McDonald, H. J., McKendell, L. V., Bermes, E. IT., J . Chromatog. 1, 259 (1959). ( 7 ) McDonald, H. J., Ribeiro, L. P., Banaszak, L. J., ANAL. CmM. 31, 825 (1959’). ( 8 ) Williams, L. A., Brusock, Y. M., Zak, B., Zbid., 32, 1883 (1960).
RECEIVED for review December 19, 1960. Accepted May 22,1961.
Rapid Spectrophotometric Determination of Total Cholesterol in Small Amounts of Blood and Cerebrospinal Fluid YUNG S. SHIN and JAMES C. LEE Deparfmenf of Biochemistry, St. Anthony Hospital, Terre Haute, Ind.
b A simple method for the extraction of cholesterol with complete removal of color-interfering substances and a rapid ( 1 minute), sensitive ( 1 pg.), micro spectrophotometric determination is described. Total cholesterol is determined in 10 pl. of serum from skin puncture and in 1 ml. of cerebrospinal fluid in a maximum of 15 minutes. The sample is washed with water and ion exchange resin. and extracted with chloroform-methanol. Color is developed with acetic acid and ferric chloride reagent within 1 minute in a boiling water bath. Quantitative recoveries of cholesterol (99.3 f 4.7%) were obtained after extraction. Relative standard deviation of assay for cholesterol on dupli1220
ANALYTICAL CHEMISTRY
cate determinations is 1.770. Results for total cholesterol agreed with the method of Chiamori and Henry within 5.2%, and that of Bloor within 8.8%.
T
HE ACID FERRIC CHLORIDE R E A G E N T
of Zak (12) has been considered more sensitive than many other color reagents and has been used for the microdetermination of cholesterol in serum (3, 11) and cerebrospinal fluid (?‘,9). However, the color reaction with this reagent is less specific than previously reported classical methods, it gives occasional higher values when used directly on plasma (1, 6 ) , and it is troubled by interfering substances ( 3 , 8). Extraction of lipide with al-
cohol-acetone (4, 10) or chloroformmethanol (7, 9) is inadequate for the complete .removal of color-interfering substances. A modified extraction procedure with removal of color-interfering substances for accurate assay and reduction of the samples to smaller amounts than used previously is presented. EXPERIMENTAL
Reagents. Dowes 1-X8 ion exchange resin (200- to 400-meshJ Baker), chloride form. Chloroform, washed with IN hydrochloric acid twice and with distilled water twice, filtered, dried over anhydrous sodium sulfate, and distilled.
