Determination of Carbon-14 in Aqueous Bicarbonare .. Solutions by Liquid Scintillation Counting Techniques Application to Biological Fluids GERALD A. BRUNO1 and JOHN E. CHRISTIAN Bionucleonics Department, Purdue University, lafayette, Ind.
b Using Cellosolve as a solubilizing additive, up to 5.84 ml. of aqueous bicarbonate solutions and essentially equivalent amounts of urine and plasma containing carbon-1 4-sodium carbonate, can be assayed by liquid scintillation counting techniques. Of particular significance is the realization of determining carbon-1 4 activity with relatively high efficiency in the important biological fluids.
A
GREAT MANY PAPERS have appeared
in recent literature describing methods of determining carbon-14 and tritium activity in aqueous solution by liquid scintillation counting techniques. The majority of these papers has been concerned with the determination of tritiated water in the various biological fluids. Of the methods described, the most outstanding seems to be that proposed by Werbin et a?. l7), in which 20% tritiated watcr solution is counted with 8.6% efficiency, with a corresponding figure of merit of 172. The method described involves the azeotropic distillation of tritiated water from the biological fluids into a pure dioxane scintillator system. I n this procedure, as in others employed for determination of tritiated water in biological fluids, no provision is made for assay of any other form of tritium or for assay of carbon-14. Procedures for counting tritiated water in urine involve either decolorizing with activated charcoal (6)or precipitation of proteins and color bodies with trichloroacetic acid ( 5 ) . Obviously these procedures are useful for increasing efficiency in tritiated watcr determinations, but are generally not permissible when one wishes to determine the activity of tritium or carbon-14-labeled metabolites in these biological fluids. The primary purpose of this investigation was to develop a system for counting carbon-14 in the maximum possible volume of biological fluids with Present address, Research & Development Division, National Dairy Products Corp., Glenview, Ill. 1216
ANALYTICAL CHEMISTRY
maximum efficiency and a minimum difficulty in determining absolute efficiency. EXPERIMENTAL
Apparatus. Counting was done in t h e Tri-Carb liquid scintillation spectrometer (Model 314S, Packard Instrument Co., LaGrange, Ill.). The counting vials were of the low potassium, tin foil-lined screw cap variety. The freezer was operated a t 5" C. The carbon-14-standard used was carbon-14-sodium carbonate (National Bureau of Standards). All chemicals were reagent grade and all phosphors were scintillation grade. I n most situations involving the assay of carbon-14 in aqueous solution, ethyl alcohol is employed as a solubilizing additive. Since use of absolute e t h j l alcohol presents handling problems which limit its practicality, future mention made to ethyl alcohol in this paper will refer to 95% ethyl alcohol. Since ethyl alcohol has enjoyed broad usage as a solvent in small volume liquid scintillation counting procedures, the first objective of this research was to demonstrate the applicability of the procedure being proposed in relation to existing procedures employing ethyl alcohol for counting of pure aqueous samples. Comparison of Ethyl Alcohol and Cellosolve for Pure Aqueous Systems.
Ethylene glycol monoethyl ether, hereafter referred to as Cellosolve, has been recognized in this laboratory as being far superior to ethyl alcohol in the majority of its applications t o liquid scintillation counting. T o demonstrate the advantages of Cellosolve over ethyl alcohol, comparison of the masimum water concentration, absolute efficiency, and figure of merit was made between the two solvents in systems commonly employed for aqueous sample counting. The comparison was made on the basis of figure proposed for evaluating of merit (4, solvent systems for tritiated water. The same procedure was applied for evaluating the carbon-14 systems with the exception that the figure of merit obtained was based on the efficiency for carbon-14sodium carbonate. Figure of merit is the product of per cent water
concentration and per cent efficiency of detection for carbon-14. Using this screening procedure it is possible to determine the solvent system capable of detecting the greatest total activity in a given sample. A simple technique for counting aqueous samples involves solution of the sample in a system consisting of 0.4% PPO (2,5-diphenyloxazole) and 0.0 1% POPOP [ 1,4-bis-2-( 5-phenyloxazoly1)-benzene] in equal parts of toluene and ethyl alcohol (6). Comparison of this system with a similar one employing Cellosolve in place of ethyl alcohol, indicated that both efficiency of detection for carbon-14 a d water-holding capacity of the system were greater when Cellosolve was employed as the solubilizing additive. The results based on figure of merit for various water concentrations are listed in Table I. A second system employing ethyl alcohol is that described by Kinard (4) consisting of a 5 : 5:3 xylene-dioxaneethyl alcohol mixture with 8.0% naphthalene, 0.5% PPO, and 0.005% a-NPO 12-(1-naphthyl) - 5 - phenyloxazole] added (XDE system). The maximum figure of merit for this system was greatly improved upon by systematically varying the concentrations of the components of the mixture and replacing ethyl alcohol with Cellosolve. A system consisting of 1 part xylene, 3 parts dioxane, and 3 parts Cellosolve, with 1.0% PPO, 0.05% POPOP, and 8.0% naphthalene (I) (XDC system) yields a figure of merit almost twice that attainable with the system proposed by Kinard. Table I lists the figure of merit for the two systems a t maximum water concentration. Systems for Large Volumes of Water. A dioxane system recommended by Werbin et al. ('7) permits a
maximum water concentration of 20% with an cfficiency of detection for carbon-I4 of 60%. The figure of merit for this eystem based on C14-sodium carbonate was determined to be 1200. By employing Cellosolve in combination with the dioxane and systematically varying the concentrations of the
various components of the system, it was possible to obtain a system consisting of 5 parts of dioxane and 1 part of Cellosolve with 1.0% PPO, 0.05% POPOP, and 5.0yc naphthalene (11) which was capable of holding 29.2% water (5.84 ml. in 20.0 ml. of solvent a t 5" C.) with an efficiency of detection for carbon-14 of 46.57;. The figure of merit for this system was 1358 (Table 11). The diosane-Cellosolve (11) system is recommended for large volumes of aqueous materials; hence, when i t is necessary to count smaller volumes, up to 3 ml., the 1:3:3 xylene-dioxaneCellosolve (I) system would yield a higher figure of merit. Since the dioxane-Cellosolve (11) provided the highest figure of merit for carbon-14 in aqueous solution, thir system was studied for its application to the important and practical problem of determining levels of carbon1.2 activity in urine and plasma. Determination of Carbon-14 in Urine. Steer urine could be mixed 1% ith t h e dioxane-Cellosolve scintillator system and counted directly, b u t efficiency was poor and precipitation was a problem. Using hydrogen peroxide to decolorize ( 3 ) the urine, a significant increase in counting efficiency was noted. The procedure developed consisted of mixing 5.3 ml. of urine with 0.2 ml. of 3oy0 hydrogen peroxide and heating the mixture in a tightly closed counting vial a t 80' C. for 6 hours. Repeated experimentation with this volume and larger volumes of hydrogen peroxide showed t h a t there was no hazard involved when it was handled in this manner. The samples were then rooled and 14.5 nil. of the dioxaneCellosolve (11) scintillator was added. Since the dioxane-Cellosolve scintillator system displayed phosphorescence until it was cooled, it was necessary to precool the samples to 5" C. before counting. The concentration of urine in the scintillator was purposely kept below the maximum concentration of water the system could handle to protect against possible precipitation of solids dissolved in the aqueous phase. The 27.5% aqueous concentration was arbitrarily chosen for convenience of sample preparation. The small amount of finely divided precipitate encountered was not of a n y significance in the analysis. To determine absolute activity for samples counted in this system, a series of calibration curves was prepared based on the count rate ratio method worked out in this laboratory (1). The diosane-Cellosolve (11) scintillator system containing 2i'.570 water was quenched to varying degrees with small increments of aniline. The ratio of the count rate for a 10-100 volt window to the count rate of a 35-100 volt window was plotted us. a correction factor (reciprocal of efficiency) on semilog paper for each increment of quencher added. The typical curves representing the high voltage t a p settings used are displayed
Table 1. Comparison of Toluene-Ethyl Alcohol, Toluene-Cellosolve, and XDE, XDC Systems for Water-Holding Capacity and Efficiency for Carbon-1 4 Determinations
Toluene-Ethyl Alcohol H20 concn., Efficiency, Fig. of 72 %b merit 66.6 44.5 1.5 144.6 48.2 3.0 165.1 36.7 4.5 235.8 39.3 6.OC XDE
7.7c
56.5
Toluene-Cellosolve HzO concn., Efficiency, 704
1.5 3.0 4.5 6.0 7.5 9.cc
435.0
Fig. of merit
%b
44.7 47.7 48.9 51.1 47.5 50.6 XDC 55.0
16.lC
67.0 143 .O 220.0 306.6 356.2 455.4 885.0
In a total volume of 20.0 ml. of scintillator solution. Determined by addition of NBS C-14 sodium carbonate; figure recorded is average of triplicate analyses. c Maximum water concentration attainable. 5
b
in Figure 1. Unknowns can be analyzed by experimentally determining the ratio for a given sample and determining graphically the corresponding C.F. (correction factor) value from the calibration curve of the high voltage t a p setting being used. Absolute activity is the product of the count rate for the 10-100 volt window and the C.F. value. Table I1 lists the average figure of merit for a series of such urine determinations carried out in the manner described above. The figure of merit for these determinations, although considerably less than that observed for pure water counted in this system, was still higher than that attainable with pure water counted in toluene-ethyl alcohol or XDE scintillator systems. Determination ef Carbon-14 in Plasma. The procedure for analyzing steer plasma consisted of mixing 4.3 ml. of t h e plasma with 3.8 ml. of 6% trichloroacetic acid. After centrifugation, to remove precipitated proteins, the supernatant which totaled 5.5 ml. was dissolved in 14.5 ml. of t h e dioxane-Cellosolve (11) scintillator. The resulting solution, which totaled 20.0 ml., was a 27.5% aqueous solution. If it is desirable to determine the presence of activity in the precipitated
Table 11. Determination of Carbon-14 in Pure Water, Urine, and Plasma Using a Dioxane-Cellosolve Scintillator System
Concn.
of Aa. EffiMaterial, ciency,
Pure water Urine Plasma
70"
yob
Fig. of Merit
29 2 27 5 27 5
46 5 24 8 26 3
1358 683 723
a In a total volume of 20.0 ml. of scintillator solution. b Determined by count rate ratio method ( I ) . Results are based on average of 5 replicate samples with a range of &1.5% from reported value.
protein, it can be dissolved readily in 1 . O M hyamine base ( 2 ) and also analyzed by liquid scintillation means. Also listed in Table I1 is the average figure of merit for a series of such plasma determinations, carried out in the manner described above. The greater figure of merit for plasma over that of urine is attributable to the fact that most of the color in the plasma
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Figure 1. Calibration curves for determination of absolute activity of carbon-1 4 aqueous solutions Or
High-voltage tap setting
VOL 33. NO. 9, AUGUST 1961
e
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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 t h a t 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
