Purity of Radiolabeled Chemicals Edward J. Merrill and Arnold
D. Lewis
Department ot AnalyticallPhysical Chemistry, Warner-lambert Research Institute, Morris Plains, N.J . 07950
During the past few years, several reports (1-8) have described various problems encountered in the purchasing of radiolabeled chemicals from commercial suppliers. They all state that considerable time, energy, and money have been lost because of these problems. They also imply that considerably more problems have existed but were not detected. Little evidence exists on this subject. In an attempt to shed some light on this topic, the following is a report on the problems and experiences which this laboratory encountered with radiochemicals. A study (4,covering 63 laboratories in 18 countries, reported that only 17 of these laboratories analyzed all shipments of radiochemicals, 36 occasionally, and 10 never. This article concluded with the recommendation that users of radioactive compounds should analyze these compounds to establish their purity. This, of course, is absolutely necessary if correct interpretations of data are to be made. In tracer experiments, very little sample weight is required because radiolabeled compounds are easily detected a t very low concentrations. In addition, radiochemicals with very high specific activities (amount of radioactivity per unit of weight) are now commercially available. The important question for the user is not how pure the material is but whether the substance is sufficiently pure for the intended purpose. Thus, the requirements of purity will vary with each investigator. A few years ago, after several unfortunate experiences, a routine program of assay for most of the purchased radiolabeled compounds was begun. Specifications were selected from one or more of those chemical or physical tests, which were reasonable for each radiochemical purchased. Purchases ranged from a few micrograms up to two grams. As many of the following items as possible were determined for each radiochemical: total radioactivity, radiochemical purity, chemical purity, and radioisotopic purity.
EXPERIMENTAL Apparatus and Procedures. The ultraviolet spectra were run on a Beckman DK-1. When suitably purified solvents were used (9), the sample could be recovered. The infrared spectra were run on a Perkin-Elmer 621, equipped with ordinate expansion and a beam condensing unit, as 0.1-0.570 KBr micro pellets using chloroform or methylene chloride to deposit the sample onto the KBr. A Barnes pellet press with a paper insert was used to prepare the pellets. It should be kept in mind that the IR spectra of a high specific activity 3H or 14C labeled compound may differ from its nonlabeled counterpart, depending on the location of the label (10). (1) I . Goldman, Science. 167, 237 (1970). ( 2 ) D. M . Prescott. Science, 168, 1285 (1970). (3) E . J Merrill. Science, 169, 719 ( 1 9 7 0 ) . ( 4 ) A . Broido. Science, 1 7 0 , 1037 (1970). (5) S A Reynolds, Science, 171,955 (1970). ( 6 ) 8. E. Gordon, Chem. Eng. News, A u g . 23, 1971, p 3. ( 7 ) "Symposium on Problems in the Purity of Biochemical Compounds and Reagents-Labeled Compounds," Abstracts 164th National Meeting of the American Chemical Society, New York, N . Y , August 1972, Papers No 68-73, Analytical Chemistry Division. (8) Pure Appl. Chem., 21, 87 (1970). (9) E. J. Merriil and G . G Vernice, d. Label. Compounds, 6, 269 (1 9 7 0 ) . (10) E. A. Evans, "Tritium and Its Compounds," Van Nostrand Co., Princeton, N . J . , 1966, p 255.
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The total radioactivity from a beta-emitting radionuclide was determined by liquid scintillation spectrometry using a Packard Model 3310 spectrometer, in a toluene or dioxane based cocktail utilizing the automatic external standard method for quench correction. The gain and window width were selected for the maximum figure of merit for each radionuclide. Dimethylformamide was found to be an excellent solvent for most organic compounds yet caused little or no quenching. The total radioactivity from a gamma-emitting radionuclide was determined by scintillation spectrometry using a Packard Auto-Gamma Model 5022 sample changer [with a 2-in. X 2-in. well-type NaI(T1) detector] coupled to a Packard Model 115 fourhundred channel, multichannel analyzer calibrated with a 13'Cs source. The beta particle spectrum was obtained by interfacing the liquid scintillation spectrometer to the multichannel analyzer (MCA) via a Packard Model 5014 mate box. At a gain setting of 15% on the spectrometer, 3H and 14C spectra peaked in channels 48 and 252, respectively. The presence of 14C in a tritiated sample was easily detected a t the 1% level if, after collection of the sample spectrum, the sensitivity of the MCA was increased by 50fold. Alternatively, a standard 3H spectrum was subtracted from the sample spectrum, but the former method was preferred. The presence of 2% of 3H in I4C was detected by subtracting a standard 14C spectrum from the sample spectrum and then the sensitivity of the MCA was increased 10-fold. A Packard Model 7200 radiochromatogram scanner was used to detect radioactivity on the TLC plates. The TLC determinations were done on precoated silica gel GF 5-cm X 20-cm plates (Analtech) using the following solvent systems: System A: n-PrOHlconcd NH40H (7:3); visualized by spraying with bromcresol green, System B: C&6; yellow visible spot, System C: C&/dioxane/glacial HOAc (90:25:4); visualized by spraying with bromcresol green.
RESULTS AND DISCUSSION Total Radioactivity. One of several examples, which illustrate typical deviations from the label claim for total radioactivity, was the receipt of four millicuries of 86RbCl when eight millicuries had been ordered. Similarly, five millicuries of glycer01-1,3-~~C trinitrate were ordered but only two millicuries were received. A sample of estrone-6,73H was supplied in benzene-methanol solution in a screw-cap bottle. When received, about 15% of the total volume had leaked and was lost in the packing. Another type of mislabeling was misrepresentation of the specific activity. A sample of proge~terone-4-~~C was ordered for a particular purpose, and a specific activity of 3 mCi/mM was specified. Although the total amount of radioactivity was correct, the sample that was received exhibited a specific activity of 1.5 mCi/mM which was unusable. A sample of methyl b r o m o a ~ e t a t e - 2 - ~simi~C larly exhibited a lower specific activity than stated. Chemical Purity. A quantitative chromatographic method rather than a spectral method was the method of choice when the suspect impurities were known. The melting point of the sample was used also, but in some cases it required too large a sample. C h l ~ r o a c e t i c - l - ~acid ~ C was the only purchased sample observed to be chemically impure. This compound will be discussed later. Other workers, however, have experienced this problem. Chemical impurities in a number of amino acids (11) and discrepancies in the molecular weight range ( 1 1 ) P Peyser. "Advances in Tracer Methodology ' Vol 4, S Ed , Plenum Press, New York, N Y , 1968, p 42
Rothchild,
II
l a
Origin
S o l v e n t Front
Figure 1. Radioscan of impure chlor~acetic-l-'~C acid
Origin
Solvent Front
Figure 3. Radioscan of impure benzoi~-7-'~C acid
Origin
Solvent F r o n t
Figure 2. Radioscan of impure f~rmaIdehyde-'~C as 2,4-DNPH
of commercial inulin-14C (12)have been reported. Radiochemical Purity. Although a radiolabeled compound may be chemically pure, this does not guarantee that the compound will be radiochemically pure. The radiochemical purity deals with the relative amounts of radioactivity in each of the chemical components in any mixture. The separation of these components was most easily done via a chromatographic procedure. The determination of the amount of radioactivity in each of the separated components was accomplished by any number of available methods, such as direct scan of a developed chromatogram using an open window Geiger-Muller detector or a spark chamber; scraping or cutting increments of a chromatogram into vials for liquid scintillation counting; gas chromatography interfaced with a radioactivity detector; or autoradiography. The radiochemical purity was then expressed as a percentage of the total. Two excellent pamphlets (13, 1 4 ) describe several classical examples of radiochemical impurities. One problem encountered,was the previously mentioned c h l ~ r o a c e t i c - l - ~acid. ~ C The chromatogram shown in Figure 1 was obtained using System A and exhibited two acidic spots and three radioactive spots. The spot which has (12) G Levi, Anal. B(ochem., 32, 348 (1969). (13)J R . Catch, "Purity and Analysis of Labelled Compounds," The Radiochemical Centre, Amersham, Bucks, England. (14) G . Sheppard, "The Radiochromatography of Labelled Compounds," The Radiochemical Centre, Amersham, Bucks, England.
Channel Number-
Figure 4. Spectral rhethod for detection of carbon-14 in tritium ( a ) = Beta spectrum of carbon-14, ( b ) = Beta spectrum of tritium, (c) and ( d ) = Beta spectrum of tritium with 1 % carbon-14
la
C h a n n e l Number-
Figure 5. Spectral method for detection of tritium in carbon-14 ( a ) = Beta spectrum of carbon-14 with 2% tritium, ( b ) and ( c ) = Remaining spectrum after subtraction of carbon-14
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the same Rf value as an authentic sample of chloroacetic acid contained only 25% of the total amount of radioactivity on the plate. A thin-layer chromatogram (TLC) of the 2,4-dinitrophenylhydrazone derivative of low specific activity formaldehyde-14C exhibited a single yellow spot which migrated at the correct Rf value in System B. However, this spot when scanned was almost devoid of radioactivity. The radioactivity had remained a t the origin as shown in Figure 2. Apparently the supplier had added unlabeled formaldehyde to reduce the specific activity. A re-synthesis produced an equally unsatisfactory sample. A sample of benz 0 i c - 7 - ~ ~acid C which exhibited the correct melting point and total radioactivity, demonstrated on TLC in System C that only 65% of the radioactivity migrated at the correct Rf value while the rest of the radioactivity migrated as a nonacidic area at a higher Rf value. The radioscan is shown in Figure 3. Several commercial samples of lysineJ4C were examined (15) and found to contain as much as 18.5% of radiochemical impurities. Braddock (16) was unable to repeat published work using sodium thiopental-35s. Using both 14C- and 35S-labeled drug, he confirmed his results and concluded that the earlier work was done with sodium
(15) W. S. Chow, L . (1970). (16) L . I . Braddock
Kesner, and H . Ghadimi, Anal. Eiochem.. 37, 276
and H L. Price, "Organic Scintillatlon and Liquid Scintillation Counting," D . L . Horrocks and C . T. Peng, E d . . Academic Press, New York, N . Y . . 1971, p 599.
t h i o ~ e n t a l - ~containing ~s a radiochemical impurity. The chemical and radiochemical impurities found in a number of hydrocarbons labeled with I4C have been reported (17 ) . Radioisotopic Purity. Routinely, the spectra of betaemitting radioisotopes are examined to establish their radioisotopic purity. This is an unusual test to run, but is considered necessary after receiving a mislabeled commerical sample of cyclopentanol-l-14C. This sample gave an unusually large number of counts in the window used to count tritium. The high count rate could not be explained since only 14C should have been present. This problem remained unresolved until examination of the beta-ray spectrum of this sample demonstrated the presence of both tritium and 14C,as illustrated in Figures 4 and 5. The emphasis that individual commercial suppliers have placed on quality control has varied over the years. It would seem that these suppliers are stressing better quality control by the appearance of literature to this effect by all of the major suppliers over the last several years. The excellence of any quality control program must be a continuous effort, and cannot be allowed to deteriorate if customer confidence is to be maintained. Received for review February 23, 1973. Resubmitted ,January 31, 1974. Accepted January 31, 1974. This manuscript was condensed from a paper presented before the Medicinal Chemistry Section, Metrochem '71, San Juan, Puerto Rico, April 30, 1971. (17) M A lsotop
Muhs, E L Basttn and B E Gordon l n f J Appi Radiat 16, 537 (1965)
Studies on Several Uranyl Organophosphorus Compounds in a Poly(Viny1Chloride)(PVC)Matrix as Ion Sensors for Uranium D. L. Manning, J. R. Stokely, and D. W. Magouyrk Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
The field of ion selective electrodes is probably one of the most active and flourishing branches of electrochemistry. The preponderance of material covered in the more recent reviews (1-5) certainly confirms this interest. Notably absent, however, are ion electrodes which are responsive to the uranyl ion. To our knowledge, the work of Dietrich (6), who utilized poly(viny1 chloride) (PVC) membranes of uranium in 2-ethylhexyl phosphate, is among the first in this area. Uranyl ion sensors should find wide applications in many areas. The electrode measurements are rapid and nondestructive. Such an electrode senses ionic activity rather than total concentration; therefore, it would be useful for analytical determinations of free ions by emf measurements as well as total concentration through such methods as titrations, standard additions, etc. Sensors which are responsive to uranyl ion activity should also prove useful for investigating equilibra and kinetics of various reactions. In view of the increasing demands for uranium coupled with expanding uranium (1) G . J MoodyandJ D . R.Thomas, Taianfa, 19,623 (1971). ( 2 ) R . P. Buck, Anal. Chem., 44, 270R (1972). (3) J Koryta. A m i . Chim. Acta, 61, 329 (1972). ( 4 ) E. Pungor and K . Toth, Analyst, (London). 95, 625 (1970). ( 5 ) E . Pungor and K. Toth, Pure Appl. Chem., 34. 105 (1973). (6) W . C Dietrich, Tech. Prog. Repf., No. Y1174D, Y-12 opment Division, Aug.-Oct. 1971
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purification programs, it is evident that uranyl ion sensitive electrodes could play a useful role as uranium monitors under favorable analytical conditions. In this paper, we present the results of a survey of several uranyl organophosphorus complexes incorporated in a poly(viny1 chloride) matrix as possible uranyl ion sensors. We chose the more favorable membranes for additional studies such as Nernstian response, pH effects. useful lifetimes, and evaluation of selectivity coefficients for some interferences.
EXPERIMENTAL All chemicals were reagent grade. The uranyl organophosphorus complexes were prepared by dissolving 1.00 gram of UOz(N03)2* 6Hz0 in 2 ml of the organophosphorus acid. The aqueous phase was then removed from the yellow viscous exchanger by centrifuging and then the exchanger was dried with two 100-mg portions of anhydrous NaZS04. The exchanger was next separated by again centrifuging and stored in a dry stoppered tube. Preparation of the uranyl ion exchanger-PVC membrane was accomplished by weighing into a clean, dry, 50-ml beaker, 45 mg of the uranyl organophosphorus complex and 450 mg of the organophosphorus solvent in the optimum weight ratio of 1 : l O (7l. TO this solution was then added 6 ml of a solution of poly(viny1 chloride) which was prepared by dissolving 2.75 grams of PVC in 60 ml of tetrahydrofuran. The beaker was covered with two to three sheets of filter paper held in place by rubber bands and set aside
