1490
Anal. Chem. 1985, 57, 1490-1492
1
~ 1 7 5
11503
IO
14 18 ACtIlIf B U l l f R m M
22
Figure 1. The relationship between concentration of acetate buffer wash for the Chelex resin and pH and total alkalinity (as CaCO,) of sewage samples equilibrated (a) for 30 min (dashed curve equilibrated for 18 h) or (b) for 20 rnin with washed Chelex. Deionized water (DW) wash in (a) for comparison.
tration, the curve for pH alone was considered suitable as a standard for determining the concentration of wash buffer to be used for maintenance of those parameters once the pH of the original sample was known. The influence of equilibration time on pH and alkalinity of sewage and river water samples was examined further by construction of standard curves (15-min wash and 20-min equilibration) for determination of the concentration of wash buffer needed (1.7 and 1.6 mM for sewage and river water, respectively, determined from curves much like that for pH in Figure lb), and then equilibration of the samples with the washed resin for 20 min, 4 h, or 24 h. The results, shown in Table I, indicate that the original pH and alkalinity of the sewage samples were maintained for the full 24-h period. Over the same time period, the pH of the less-well-buffered river water increased slightly, from 7.38 to 7.60. When unwashed Chelex was used, both pH and alkalinity values increased substantially over control values.
The influence of wash duration on the pH of river water after a 24-h equilibration was studied by selecting a concentration of wash buffer (1.7 mM) from a standard curve and then washing the Chelex for periods of 15 min to 5 h before equilibration. The results, in Table 11, indicate that increasing duration of wash to 25 (by graphical interpolation) to 30 min would be effective in maintaining pH to approximately that of the control sample over the 24-h equilibration and would eliminate the slight increase in pH observed for the river water sample, in Table I. The procedure outlined above for maintaining the pH and alkalinity at ambient levels during batch treatment with Chelex-100 was tested at three values of pH in the range 6.5-8.1 of natural waters and sewage. Before each test the pH of the water sample (-7.3) was adjusted with HNOB(6.5) or NaOH (8.1) and then a standard curve of sample pH vs. concentration of wash buffer (represented by Figure l b for samples of pH 7.3 and by Figure l a for sample of pH 6.5 and 8.1) was graphed from data obtained with a 15-min wash of the resin followed by a 20-min equilibration with the water sample. The results, in Table 111, show that washing the resin with a predetermined concentration of buffer was effective in maintaining pH and alkalinity very close to that of the control sample. The results presented here indicate that substantial increases in pH can occur in samples of river water and sewage undergoing batch equilibration with Chelex-100 and that the method presented here is useful in maintaining ambient levels of pH as well as alkalinity of these samples during equilibration. Hegistry No. H20,7732-18-5;sodium acetate, 127-09-3;acetic acid, 64-19-7; Chelex-100, 11139-85-8.
LITERATURE CITED (1) Stiff, M. J. Water Res. 1971, 5 , 585-599. (2) Gardiner, J. Water Res. 1974, 8 , 23-30. (3) Giesy, J. P.; Briese. L. A,; Leversee, G. J. Environ. G e o / ( N . V . ) 1978, 2 , 257-268. (4) Shephard, B. K.: McIntosh, A. W.; Atchison, G. J.; Nelson, D. W. WaterRes. 1980, 7 4 , 1061-1066. (5) Sposito, G. Environ. Sci. Technoi. 1981, 75, 396-403. (6) Florence, T. M.; Batley, G. E. Talanta 1975, 22, 201-204. (7) Figura. P.: McDuffie, B. Anal. Chem. 1977, 4 9 , 1950-1953. (8) Hart, B. T.; Davies, S. H. R . Aust. J . Mar. FreshwaterRes. 1977. 28, 397-402. (9) Pakalns, P.; Batley, G. E.; Cameron, A. J. Anal. Chlm. Acta 1978, 9 9 , 333-342. (10) "Cheiex 100 Chelating Ion Exchange Resin for Analysis, Removal or Recovery of Trace Metals"; Bulletin 2020; Bio-Rad Lab.: Richmond, CA, 1983. (11) "Standard Methods for the Examination of Water and Wastewater," 15th ed.; American Public Health Association, American Water Works Association and Water Pollution Control Federation: Washington, DC, 1980.
RECEIVED for review October 29, 1.984. Resubmitted and accepted February 25, 1985.
Determination of Iodine-I 31 at Low Concentrations in Formaldehyde-Preserved Milk Kar-Chun To* Radiation Laboratory, Kansas Department of Health and Environment, Office of Laboratory Services and Research, Topeka, Kansas 66620
Edward P. Rack Department of Chemistry, University of Nebraska--Lincoln, Lincoln, Nebraska 68588-0304 This paper describes a method for the analysis of total
l3II
(tljz= 8.04 days) at low concentrations in milk in the presence 0003-2700/85/0357-1490$01.50/0
of formaldehyde which serves as a preservative. It has been found that when milk is preserved with formaldehyde, the 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
latter enhances the binding of the iodine to milk protein. Consequently any separation schemes such as ion-exchange chromatography will result in low recovery yields. For relatively high 1311levels in milk, the 1311concentration can be measured directly by y-ray spectrometry ( I ) , with the obvious advantage that no preliminary sample preparation or chemical separation is required. For milk samples containing low 1311levels, the direct counting method is unreliable because of the low counting efficiency of y-ray detectors and/or the presence of other y-ray radionuclides whose Compton continuum can interfere with the measurement of 1311photopeaks. Therefore, in order to measure low-level 1311 activity in milk, the 1311must be isolated from the sample and counted. Ion-exchange chromatography (2-9) is generally employed to isolate 1311from milk. I t is found or assumed that 90% of the 1311exists in the iodide form. The 1311can be separated from other radionuclides present by employing, for example, a Dowex-1-X 8 or Dowex 2-X 8 resin, as well as other resins. Radioassay for 1311can be performed either by direct counting of the resin (IO) or as a solid precipitate of silver or palladous iodide after elution off the resin by a suitable oxidizing agent such as sodium hypochlorite. Total 13'1 concentration is calculated with the assumption that 90% of the total iodine exists in milk as the iodide. However, for formaldehydepreserved milk ( 1 I , I 2 ) ,a large fraction of 1311can be protein bound and will escape separation. In lieu of an ion-exchange separation procedure, separation and concentration of all 1311, regardless of the extent of protein binding, are accomplished in the following manner: (a) precipitation of milk protein; (b) oxidation of filtrate iodide to diiodine with subsequent carbon tetrachloride solvent extraction; (c) reduction and concentration of diiodine to the iodide by aqueous bisulfite; and (d) acid digestion of the milk protein with subsequent oxidationsolvent extraction and reduction-solvent extraction concentrating the 1311as the iodide. The iodide-131 may then be precipitated as palladous iodide and radioassayed for 1311.
EXPERIMENTAL SECTION Apparatus. A Canberra Model 2201 low-background a/@ counting system equipped with gas-flow detector and anticoincidence guard detector was used to count palladous iodide. The background of the a/@ counting system is less than 2 counts per minute. Iodine-131 concentrations in liquid samples were determined by counting the samples with a Princeton Gamma-Tech Ge (Li) detector interfaced to a Nuclear Data ND6600 computer-based data acquisition system. The counting efficiency of the Ge (Li) detector is 20% at 25-cm source-to-detector distance relative to a 3 X 3 in. NaI (Tl) detector. Chemical yields of palladous iodide were determined gravimetrically by using a Mettler Model AE 160 analytical balance with a 0.1-mg readability and fO.l-mg precision. Reagents and Solvents. National Bureau of Standards 1311 standard solutions with an uncertainty of less than 2% were used. Palladous chloride, trichloroacetic acid, hydrochloric acid, sodium iodide, sodium bisulfite, carbon tetrachloride, diethyl ether, and 30% hydrogen peroxide solutions were all reagent-grade quality. Standard iodide carrier solutions were prepared by dissolving 5.9 g of sodium iodide in 100 mL of distilled water. The iodide carrier solution was standardized by precipitating iodine as palladous iodide. Sodium bisulfite solutions were prepared prior to the extraction of iodine were carbon tetrachloride. Fresh milk was used for all the experiments. Pasteurized milk could also be used for the analysis. Radioassay Procedure. The counter efficiency of the low background counter was determined by counting PdlSIIa t different masses. Pd1311was prepared by adding a known quantity of standard 1311solution with various amounts of iodide carrier to a test tube containing 20 mL of 2 M HCl. Two milliliters of 0.2 M palladous chloride in 1M HCl was added to the solution. The mixture was heated in a water bath for about 10 min to allow palladous iodide to precipitate. Palladous iodide was filtered,
1491
Table I. Percent Iodine-131 Recovered after a 3-h Protein Digestion HC1 concn, M
temp, "C
recovered
8 8 8 6 3 2
97 120
92 & 3 90 f 2
55 97 97 97
27 3z 1 66 f 2 32 & 1 16 f 1
1
97
none
%
1311
dried, weighed, and counted for iodine-131 activity. The counter efficiencies over a range of palladous iodide masses were determined after yield and decay corrections. Sample Analysis. A 1-L milk sample was used for an analysis. The milk sample was spiked with a known quantity of standard iodine-131 solution and preserved with 10 mL of 37% formaldehyde solution. After the sample was allowed to stand at room temperature for 3-4 days, 50 mg of iodide carrier was added and stirred for 1 h. One hundred milliliters of 24% trichloroacetic acid was added to the sample after 1h, and the resulting solution was stirred for an additional 30 min to precipitate milk protein. Separation of milk protein was achieved by suction filtration. The separation can take approximately 10 h to complete. At the end of the separation, milk protein was transferred to a 1-L beaker. The filtrate was poured into a 2-L beaker and saved. Fifty milliliters of 30% hydrogen peroxide was added to the filtrate and the solution stirred for 2 min. The filtrate was then transferred to a separatory funnel and extracted gently with three 150-mL portions of carbon tetrachloride. A small quantity of deforming agent can be used here t o separate the aqueous layer from the carbon tetrachloride layer. The carbon tetrachloride portions were combined and saved. Iodine was extracted from carbon tetrachloride with two 50-mL portions of 0.2 M sodium bisulfite solution. The bisulfite portions were combined and saved for further analysis. Digestion of Milk Protein. Two hundred and fifty milliliters of 8 M HC1 was added to the beaker containing the milk protein. The protein was broken into small chunks with a glass rod, and a magnetic stirring bar was placed on the bottom of the beaker. The beaker was then covered with a watch glass and the protein digested at 97 "C on a hot plate for 3 h with constant stirring in a fume hood. At the end of the digestion, the mixture was allowed to cool to room temperature and filtered. The filter was washed with 20 mL of distilled water, and the wash was combined with the filtrate. The filtrate was extracted gently with two 100-mL portions of diethyl ether. The ether layers were discarded after extraction. Thirty milliliters of 30% hydrogen peroxide was added to the aqueous layer, and the mixture was extracted gently with two 100-mL portions of carbon tetrachloride. Again a small quantity of deforming agent was used to separate the aqueous layer from carbon tetrachloride. The carbon tetrachloride portions were combined and extracted twice with 50 mL of 0.2 M sodium bisulfite solution. These bisulfite solutions were combined with the 100-mLbisulfite solution obtained from the previous section. The combined bisulfite solution was slowly evaporated to a volume of 20 mL. A few milliliters of concentrated hydrochloric acid was added to the bisulfite solution to liberate sulfur dioxide. The mixture was transferred to a test tube, and 2 mL of 0.2 M palladous chloride solution was added. The resulting solution was digested in a hot water bath for about 10 min. Palladous iodide was then filtered, dried, weighed, and P-counted for 1311 activity. The concentration of 1311was calculated from the net count rate, counter efficiency, chemical yield, and the decay correction.
RESULTS AND DISCUSSION In order to optimize the acid digestion of milk protein, 1311 recovery yields were studied a t different HC1 concentrations and digestion bath temperatures. Presented in Table I are recovery yields from milk protein with protein-bound 1311. At the end of the digestion, the residue was separated by filtration. Iodine-131 activity in the filtrate was counted with
1492
Anal. Chem. 1985, 57, 1492-1494
Table 11. Determination of Total Formaldehyde-Preserved Milk" 1311
added 21.3 nCi/L 162.0 pCi/L
81.2 pCi/L 26.2 pCi/L 17.7 pCi/L
1311
in
activitv foundb 20.5 f 1.5 nCi/L 164.7 f 11.8 pCi/L 85.9 f 7.7 pCi/L 27.3 f 2.0 pCi/L 19.2 f 1.4 pCi/L
% error
3.8 1.7 5.8 4.2
8.5
1311 activity was radioassayed as the palladous iodide precipitate. *Six separate determinations were performed.
the y-counting system. The iodine-131 in the filtrate was extractable with carbon tetrachloride after oxidation with 30% hydrogen peroxide solution. The percent iodine-131recovered from the milk protein is the percent iodine-131 in the filtrate. As shown in Table I, the percent I3lI recovered from the milk protein is greater than 90% at both 97 and 120 OC when 8 M HCl was used for the digestion. However, when the same HC1 concentration was used and the protein digested a t 55 OC, only 27% iodine-131 was recovered. With the digestion temperature remaining at 97 "C, iodine-131recovery decreased with decreasing HC1 concentration. At 1M HC1, all iodine-131 remained protein-bound after 3 h of digestion at 97 "C. These results indicate that the liberation of iodine-131 from milk protein depends on the digest temperature and the HCl concentration. The mechanism for the enhancement of protein-bound iodine in milk protein in the presence of formaldehyde is not clear. I t may be due to the change in net protein charge and/or protein conformational changes as a result of the reaction of formaldehyde with the primary amino groups of the milk protein which influence the binding of iodine to the protein (13). Iodine is bound to the tyrosine residue of the milk protein; when the milk protein is digested with concentrated HC1 at high temperature, the more labile amino acid
groups such as tyrosine, threonine, and serine are easier attacked by the acid than the more stable amino acid groups. This may explain the relatively short time needed in the digestion step to liberate iodine from milk protein. The accuracy of the method reported here was tested by analyzing 1-L milk samples spiked with iodine-131 activity which ranged from 21 nCi/L to 17.7 pCi/L. Table I1 shows the experimental results compared to the known iodine-131 concentration in the milk samples. The experimental results are in good agreement, within experimental error, with the known iodine-131. concentration. Palladous iodide obtained from samples with picocurie iodine-131 concentrations were P-counted for 200 min to minimize the counting errors. Registry No. 1311,10043-66-0;formaldehyde, 50-00-0. LITERATURE C I T E D (1) Hagee, G. R.: Goldln, A. S. Environmental Health Series, Public Health Service, No. 999-R-2, 1963, p 16. (2) Bori, A. L. Analyst (London) 1963, 88, 64. (3) Kahn, B. J. J . Agric. FoodChem. 1965, 73, 21. (4) Murthy, G. K.; Gilchrist, J. E.: Campbell, J . E. J . Dairy Sci. 1962, 45 (9), 1066. (5) Smith, H.; Whitehead, E. L. Nature (London) 1963, 799, 503. (6) Murthy, G. K. Environmental Health Series, Public Health Service, No. 999-R-2, 1963, p 7. (7) Iwaskima, K.; Yamagata, N . Koshu Eiseiin Kenkyu Hokoku 1964, 2 , 126. (8) Walker, J. P.: Reknberg, B. F.; Brooks, I . 6.J . Dairy Sci. 1968, 57 (9), 1373. (9) Fairman, W. E.; Sedlet, J . Anal. Chem. 1966, 38, 1171. (10) Willis, C.; Parker, D. Radiological and Environmental Science Laboratories, Idaho Falls, ID, private communication, 1984. (11) Montgomery, D. M.; Gibson, J. E. Health Phys. 1977, 3 2 , 562. (12) Gabay, J. J.; Paperiello, C. J.; Goodyear, S.; Daiy, J. C.; Matuszek, J. M. Health Phys. 1974, 2 6 , 89. (13) Holt, C.; Muir, D. D.;Sweetsur, A. W. M. J . Dairy Res. 1978, 4 5 , 47.
RECEIVED for review December 17, 1984. Accepted February 8,1985. This research was partially supported by the Division of Chemical Sciences of the U.S. Department of Energy and a University of (Contract DE-A502-76ER01617.OAOO5) Nebraska Research Council NIH Biomedical Research Support grant.
Analysis of Urine for 11-Nor-As-tetrahydrocannabinol-9-carboxylicAcid Using Sep-PAK Cartridges for Sample Cleanup George R. Nakamura,* Walter J. Stall, Ronnie G. Masters, a n d Victor A. Folenl
United States A r m y Criminal Investigation Laboratory-Pacific, The widespread use of marihuana requires a rapid and reliable procedure for its detection in urine for routine forensic use. The major active principal of marihuana, Ag-tetrahydrocannabinol, rapidly disappears from human blood. It is metabolized mostly to 11-nor- Ag-tetrahydrocannabinol-9carboxylic acid (9-THC-COOH) and excreted in the urine. Most of the metabolite is found in urine as a conjugated form of a glucuronide (1). Urine is generally the preferred specimen for detecting the wrongful use of drugs. The collection is convenient, and the concentrations of drugs and their metabolites are much higher in urine than in the blood. In the case of cannabinoids, the relatively low concentration of 9-THC-COOH requires large amounts of urine as sample. This necessitates an efficient clean-up procedure. I
Present address: Naval Hospital, Jacksonville Naval Air Station,
Jacksonville, FL 32202.
APO S a n Francisco, California 96343 A number of methods have been reported in the literature for the detection of 9-THC-COOH in urine. These methods include thin-layer chromatography (TLC), enzyme multiplied immunoassay technique (EMIT), radioimmunoassay (RIA), gas chromatography (GC), and gas chromatograhy/mass spectrometry (GC/MS). EMIT and RIA do no require an extraction of samples for analysis, but they do not provide a specific identification of 9-THC-COOH. GC/MS is the preferred technique for forensic identification of drugs such as 9-THC-COOH ( 2 ) . Double liquid-to-liquid extraction procedures are used to prepare samples for GC (3) and GC/MS ( 4 , 5 ) . While TLC provides cleaner samples for GC/MS (6), this approach is time-consuming. Bond Elute and high-pressure liquid chromatography (HPLC) (7) and Bond Elute and TLC (8)in combination are used for sample cleanup and detection of 9-THC-COOH. A TLC method (9) is used to detect 9-THC-COOH after a liquid-to-liquid extraction of urine specimens.
This article not subject to U.S. Copyright. Published 1985 by the American Chemical Society
