2552
Anal. Chem. 1982, 54, 2552-2556
Table 111. Determination of Isoniazid and 4-Amionsalicylic Acid in Drugs
drug
amt, makers’ specification, mg 4-aminosalicylic isoniazid acid I I1
INH-PAS 25 Pazide 15 PASopizyd 30 Isocadipas G 150 IsocadipasT 33.4 PAS with 17 INH Inapas 25
I
amt found? mg cv I1
601.2 362.2 768.3 3622.0 724.4 384.1
22.4 17.9 26.4 158.9 30.2 15.6
0.6 0.4 0.5 0.6 0.4 0.4
604.2
26.8
0.6
593 356 773 3615 701 376
cv 1.0 0.8 0.9 1.0
0.8 0.6
615 0.6
a Average of six determinations. The amount of cab cium and sodium salts converted to that of free acid.
The electrode potentials of the 2-iodoxy-/2-iodosobenzoate system a t 25O are 1.33, 0.61 and 0.56 V at pH 1, 4 and 7 respectively. At the corresponding pH, the 2-iodoso-/2iodobenzoate system has electrode potentials 1.21, 0.53 and 0.48 V. Large amounts of glucose, maltose, sucrose, lactose, sodium formate, glycine, and alanine do not interfere with the determination of isoniazid; other materials that do not affect the analysis appear in the footnote of Table 11.
LITERATURE CITED (1) Garratt, D. C. “The Quantltative Analysis of Drugs”; Chapman & Hall: London, 1964; pp 360, 561.
Baika, M.; Bode, D.; Rhodes, H. J. J. fharm. Sci. 1974, 63, 1303. Pszonicka, M.; Skwara, W. Chem. Anal. (Warsaw) 1970, 5 , 175. Rao, P. V. K.; Rao, G. B. B. Analyst (London) 1971, 96, 712. Pinazautl, S.; Dal Piaz, V.; La Porta, E. Farmaco, Ed. f r a t . 1974, 2 9 , 136. Eremlna, 2 . I.; Antonlchlk, J. A. Farm. Zh. (Kiev) 1972, 27, 79. Rao, K. S.; Rao, G. B. 6.; Rao, P. V. K. Chem. Anal. (Warsaw) 1974, 19, 927. Sollman, R.; Belai, S. A. J . Drug. Res. 1974, 6, 7. Nair, V. R.; Nair, C. G. R. Anal. Chlm. Acta 1971, 57, 429. Chateau-Gosselin, M.; Patnlarche, G. J. Anal. Chlm. Acta 1978, 102, 215. Nair, C. 0. R.; Kumari, L. Indian J. Chem., Secf. A 1978, 1 4 A , 115. Abramo, M. K. Farmatsiya (Moscow) 1975, 24, 80. Urbanyi, T.; Wlnchurch, R. J. Assoc. Off. Anal. Chem. 1973, 56, 1464. Bark, L. S.; Prachuabpaibul, P. Anal. Chim. Acta 1976, 87, 505. Barakat, M. 2.; Shaker, M. Analyst (London) 1986, 9 1 , 466. Sarwar, M.; Yaqub, M.; Naeem, I.; Kazl, A. A. 2.Anal. Chem. 1977, 285 269. Kuhni, E.; Jacob, M.; Grossglauser, H. Pharm. Acta Helv. 1954, 29, 233. Devani, M. 6.; Shishoo, C. J. J. fharm. Sci. 1970, 5 9 , 90. Pavlyuchenkova, L. P. Farmatsiya (Moscow) 1978, 25, 73. Das, A.; Boparai, K. S. Talanta 1982, 29, 57. Verma, K. K., Mikrochim. Acta 1980, 211. Verma, K. K.; Guiatl, A. K. Anal. Chem. 1980, 5 2 , 2336. Verma, K. K.; Gulati, A. K. Analusls 1981, 9 , 506. Verma, K. K.; Gupta, A. K. Talanta 1981, 28, 849. Verma, K. K. Talanta 1982, 2 9 , 41. Verma, K. K.; Gupta, A. K. Anal. Chem. 1982, 54, 249. Banerjee, A.; Banerjee, G. C.; Bhattacharya, S.; Banerjee, S.; Samaddar, H. J. Indian Chem. SOC. 1981, 58, 606. Verma, K. K.; Bose, S. Mlkrochim. Acta 1976, 591. Verma, K. K. Talanta 1979, 26, 257.
RECEIVED for review April 1,1982. Accepted June 11,1982. Thanks are due to the Council of Scientific and Industrial Research, New Delhi, for a Senior Research Fellowship to A.K.G.
Separation of Tervalent Lanthanides from Actinides by Extraction Chromatography Don B. Martin* and Dennis G. Pope Radlological and Environmental Sciences Laboratory, U.S. Department of Energy,
A procedure Is described for the removal of milligram amounts of lanthanldes from the tervalent actlnldes. The routlne appllcatlon of an earller procedure resulted In low americium ylelds and poor resolutlon of the a spectrum, malnly due to Inadequate removal of neodymlum. The reported procedure offers many slgnlflcant Improvements and Involves prepurlflcation of the lanthanlde-actinide fractlon by precipltatlon as a hydroxide, removal of the lanthanides from the tervalent actlnldes by extraction on a column of bls(2-ethylhexy1)phosphoric acld sorbed on Teflon powder, and carrylng the actlnldes dlrectly from the eluate on about 50 hg of cerium fluoride for a spectrometry. The actlnlde recovery from a mlxture of 5 mg of cerlum, 3 mg of lanthanum, and 2 mg of neodymlum was 86% for callfornlum and 96% for amerlclum and curium, with less than 10 pg of the neodymlum remalnlng. The average overall recovery of amerlclum from routlne analysls of 131 sol1 samples was 84 f 6 % .
The separation of the tervalent actinides from the lanthanides by elution with a buffered diethylenetriamine-
550 Second Street, Idaho Falls, I d a h o 8340 1
pentaacetate (DTPA) solution through a column of bis(2ethylhexy1)phosphoric acid (HDEHP) sorbed on Teflon powder has been previously described by Filer (1). The routine application of this method to the americium fraction obtained from the analysis of 10-g soil samples (2) at the Radiological and Environmental Sciences Laboratory (RESL) resulted in frequent low yields and poor resolution of the a spectra of the subsequently electrodeposited americium (3). The poor americium yields and resolution of the a spectra were due to excess neodymium eluted with the americium. Filer studied other lanthanides but not neodymium. His conditions, though adequate for cerium and lanthanum, were not adequate for the separation of tervalent actinides from the amount of neodymium found in many soils (4). Also, other problems were encountered involving decreasing column efficiency with use, pH measurements, amounts of lanthanides greater than the 2 mg limit, initially impure lanthanide-actinide fractions, and incomplete dissolution of these fractions prior to loading on the column. In the present method these problems have been reduced significantly. Most of the conditions affecting the column chromatographic separation have been studied and are re-
This article not subject to U S . Copyright. Publlshed 1982 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
ported below. Also, an improved technique for preparing the HDEHP-Teflon powder (HT-6) by evaporation (5) and ,a simpler and faster deposition of the tervalent actinides on cerium fluoride (6-8) as an alternative to electrodeposition are reported. The improved method provides a simple, fast, and excellent separation of the tervallent actinides from up to 10 mg of lanthanides after their initial separation from up to 50 g of soil as well as other large environmental and biological samples (2, 9, 10). EXPERIMENTAL SECTION Apparatus. A scintillation counter with a 2 by Z1/2 in. well in a 3 by 3 in. NaI crystal was used for y counting. The instrumentation artd scintillation technique used for gross a counting have been deticribed previously (11). The a spectrometer using a 450-mm2surface barrier detector and 1024-channel analyzer has been described previously (2). An ion-exchange column, 1by 15 cm, with a 50-mL reservoir at the top, a small glass wool plug at the bottom, and a microadjusting stopcock was wed for the column. Tracers. Neodymium-1.47 y emitting tracer was produced at the Idaho National Engineering Laboratory by neutron activation of 99.9% pure neodymium-146 (ORNL, Oak Ridge, TN). y-emitting cerium-144 and americium-241and a-emitting curium-244 and californium-252 were aLo used to defiie separation conditions and to determine tervaleint actinide yields. Reagents. The 0.45 M bis(2-ethylhexy1)phosphoric acid (HDEHP) was purified b y diluting 150 mL of technical grade HDEHP (J.T. Baker Chemical Co., Phillipsburg, NJ) to 1L with n-heptane in a 2-L separatory funnel and continuing the purification as described previously (12). Some commercially available cerium salts may need to be purified to remove a and (Iactivity present from impurities of thorium-232 and its daughters. Cerium purification has been described previously (12). The DTPA eluant wa13prepared by dissolving 10 g of diethylenetriaminepentaaceiic acid (DTPA) and 100 g of monochloroacetic acid in 800 ml, of distilled water and 25 mL of fresh, concentrated ammonium hydroxide and diluting to 1 L. Adjust the pH to 2.80 0.05. The sodium bisulfate solution was prepared by dissolving 13 g of Na2S04in 500 mL of 9 M H2S04. The m-cresol purple indicator (MCP) was prepared by dissolving 0.1 g of m-cresokulfonephthalein in 26 mL of 0.01 N sodium hydroxide and diluting to 250 mL with distilled water. Column Preparation. Prepare the HT-6 by adding 35 mL of reagent grade acetone and 9.7 mL or 7.0 E:of purified 0.45 M HDEHP in n-heptane to 28 g of Araport T-6, poly(tetrafluoroethylene) powder, 70-80 rnesh (Analabs, Inc., Hamden, CT), in a 100-mL beaker. Slurry the mixture and carefully evaporate to dryness at low heat on a hot plate. Place the beaker under a bell jar and evacuate the jar with a water aspirator for 2 h to evaporate the last traces of heptane. Add 7.0 g of the dry HT-6 to the columns containing a small glass wool plug ai, the bottom. Wash the HT-6 with 25 mL of 0.1 N hydrochloric acid. Connect a distilled water line to the stem of the column and force the wet powder into the column reservoir. Close the stopcock and slurry the powder with 25 mL of 0.1 N hydrochloric acid. While slurrying the mixture, open the sto~ocockand chase the wet powder into the column with a squirt bottle of 0.1 N hydrochloric acid. Place a small glass wool plug over the powder to keep it in place and run about 100 mL of deaerated 0.1 N HC1 through the column to remove entrapped air. Wash and preequilibrate the column as described in the separation procedure. Separation Procedurei. The following procedure is used for the separation of tervalent, transplutonium actinides from the tervalent actinidelanthanide strip fraction from various reported procedures (2,9,10). Add 10 mL of concentrated hydrochloric acid and 3 mL of concentrated perchloric acid but no sodium sulfate or sulfuric acid (2,99 to the tervalent actinide-lanthanide strip fraction. Evaporate the solution to fuming perchloric acid. (Perchloric acid should be used with proper precautions to avoid possible explosive mixtures (13).) After cooling, transfer the solution to a 50-mL long stem conical centrifuge tube with about
*
2553
15 mL of 0.1 M nitric acid. If the solution contains observable insoluble material, centrifuge the solution for 5 min at 2200 rpm and decant the supernate into a clean 50-mL centrifuge tube. Add 0.1 mL of 20 mg/mL cerium carrier. Cerium Oxalate Precipitation. If the sample is to be carried on cerium fluoride instead of electrodeposited, skip to the cerium peroxyhydroxide step. Otherwise add 5 mL of 0.4 M oxalic acid, 2 drops of MCP indicator, and concentrated ammonium hydroxide dropwise to the appearance of the salmon pink color of the red to yellow end point of MCP (pH of 1.5). Heat the solution in a bath of boiling water and cool in a bath of running water for 5 min each. Centrifuge as before and decant and discard the supernate. Add 2 mL of concentrated HC104and place the tip of the centrifuge tube on a hot plate using an appropriate rack to hold the tube vertical. Heat the tube carefully to oxidize the oxalate and until about 1 mL of perchloric acid remains. Allow the solution to cool to room temperature and disregard any reprecipitation that may occur. Add 15 mL of 0.1 N HNOB. Cerium Peroxyhydroxide Precipitation. Add 1drop of 30% hydrogen peroxide and concentrated ammonium hydroxide dropwise until the brownish yellow cerium peroxyhydroxide precipitates. Add 5 more drops of concentrated ammonium hydroxide. Heat for 3 min, cool, centrifuge the solution as before, and carefully decant and discard the supernate. Add 6 drops (about 1/4 mL) of 2 M nitric acid around the walls of the centrifuge tube and heat in a bath of boiling water until the precipitate dissolves. Roll the hot solution around the sides of the centrifuge tube and add 10 mL of DTPA eluant. Add 2 drops of 1 M hydroxylamine hydrochloride solution and heat for a few minutes to reduce the cerium. Column Separation. Load the solution onto a 7-g HT-6 column which has been preequilibrated with two 10-mL portions of eluant. The loading and eluting flow rate should not exceed 0.3 mL/min. Rinse the centrifuge tube with a 5-mL portion of eluant and pass this through the column. Complete the actinide elution with an additional 15 mL of eluant. Collect the eluate in a 250-mL Erlenmeyer flask for electrodepositionor in a 50-mL polycarbonate centrifuge tube for carrying on cerium fluoride. The columns are washed at the maximum flow rate of the column with two 25-mL portions of 6 N hydrochloric acid and two 25-mL portions of 0.1 N hydrochloric acid. The columns should not be allowed to run dry. However, if the columns run dry,passing deaerated 0.1 N hydrochloric acid through the column may remove the entrapped air. A last resort is to reslurry the columns as described in the column preparation section. Preparation for Electrodeposition. Add 1 mL of sodium bisulfate solution, 2 mL of concentrated sulfuric acid, and 35 mL of aqua regia to the eluate in the 250-mL Erlenmeyer flask and heat on a covered hotplate to fuming sulfuric acid. Cool the solution and add 1mL of aqua regia and heat to a char. Add 2 mL of 1:lnitric:perchloric acid mixture and continue heating until the solution is colorless. Additional nitric:perchloric acid mixture can be added if necessary. Heat the flask carefully over a blast burner until all the sulfuric acid has been fumed off. Electrodeposit the sample as previously described (3). Carrying the Actinides on Cerium Fluoride. Add 0.1 mL of 0.5 mg/mL cerium carrier and 2 mL of 48% hydrofluoric acid to the 30 mL of eluate in the 50-mL polycarbonate centrifuge tube. Let the sample stand for 30 min (7). Filter the cerium fluoride onto a 25-mm HT-100 Gelman filter previously treated with cerium fluoride substrate as previously described (8). RESULTS AND DISCUSSION The advantages of the extraction column chromatographic system of HDEHP on Teflon powder have been discussed by Moore and Jurriaanse (14). The main advantage continues to be its simplicity. More recent advances in extraction column chromatography using HDEHP on various forms of silica supports give excellent separation but require much more elaborate techniques and apparatus (15, 16). The large enhancement in the separation of the tervalent actinides from the lanthanides was shown by Weaver and Kappelman (17)by using the chelating agent, DTPA, in the aqueous phase and a single solvent extraction with HDEHP. They also showed with the single HDEHP-DTPA system that
2554
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
x
t PH
Figure 1. The extraction of (0)*“Am and ( 0 )14’Nd as function of PH.
praseodymium, neodymium, promethium, and samarium are the most difficult lanthanides to separate from the transplutonium actinides and neodymium-californium are the most difficult lanthanide-actinide pair to separate from one another. Almost the same separation difficulties were found in the multistage extraction column chromatographic system reported here. Filer (1) applied the HDEHP-DTPA column extraction chromatographic system to soil samples using americium-241 and cerium-144 tracers. He showed excellent separation of the americium-241 from the cerium-144 and the cerium indigenous to the soil. However, neodymium is much more difficult to separate from the actinides than cerium and is second only to cerium in its abundance in the average soil (4). If Filer had electrodeposited the final actinide fraction he may have obtained low americium yields and degraded (Y spectra due to the presence of neodymium. In fact, routine application of this method did result in low yields and a poor resolution of spectra on some samples. Also, an emission spectrographic analysis of the americium electrodeposit detected neodymium as the major component. Reinvestigation of the extraction conditions using higher capacity 7-g HT-6 columns resulted in significant improvement in the actinide separation from the lanthanides. Solutions used to define the extraction conditions contained 5 mg of cerium, 3 mg of lanthanum, 2 mg of neodymium, and either americium-241 or neodymium-147 y-emitting tracer. The pH is the most critical condition of the separation and must be properly adjusted and controlled. A pH of 2.80 f 0.05 was selected from Figure 1 to be the optimum separation pH. Figure 1also shows an inadequate separation of neodymium at the pH of 3 recommended by Filer (1). Monochloroacetic acid with a pK, of 2.86 and half neutralized with ammonium hydroxide serves as a precise buffer at p H 2.8. However, maintaining any pH to f0.05 pH units is difficult and care in the measurement and adjustment of the pH should be exercised (18). Extraction column failure was detected first by poor americium yields. Later an iron test was devised to determine if and how far the HDEHP had moved down the column. The “iron test” involved loading about 5 mg of iron onto the column from a few milliliters of 0.1 N HC1, eluting with 10 mL of 0.1 N HC1, and then producing a yellow band of the ferric chloro complex with 3 N HC1 at the point on the column where the HDEHP was still sorbed on the T-6 powder. The “iron test” indicated the HDEHP had moved half way down or more on some of the older columns leaving organic free T-6 at the top section of the column. Obviously, the effectively shortened HT-6 columns were not adequate for most of the lanthanide-actinide separations. The various column eluates were wet ashed and analyzed for their total phosphate content to determine which eluant was dissolving the HDEHP from the T-6.
Nd imgl
Figure 2. The effect of neodymium concentratlon on the extraction of (0)241Am and (0)I4’Nd at pH 2.80. The results of the phosphate analyses in terms of HDEHP are shown in Table I. Obviously, substitution of 0.1 N hydrochloric acid washes for the 50-mL water washes used by Filer (1)greatly increases the life of the columns. Calculations using the phosphate data indicate 300 rather than 10 runs could be made before column failure. The initial method of preparing the HT-6 involved mixing the T-6 powder with HDEHP in n-heptane and drawing off the excess organic by filtration. This method gave nonreproducible amounts of diluted HDEHP on the T-6 powder. The improved method of preparation by evaporating the diluent after mixing the diluted HDEHP and the T-6 gives reproducible and known concentrations of undiluted HDEHP on the T-6 powder. The percentage of HDEHP on the powder can even be verified gravimetrically. The tervalent lanthanide-actinide fraction from the analysis of soil samples frequently contained traces of impurities such as Fe, Al, Ti, Zr, and Ca which would not be separated from the actinides by the HT-6 system and would interfere with the electrodeposition. An oxalate followed by a hydroxide precipitation purified the lanthanide-actinide fraction prior to column separation. Also the rare earth hydroxide precipitate is easily dissolved in a few drops of 2 M HN03 eliminating a difficult solubility problem that had occurred prior to column loading in the past. The cerium fluoride deposition of the actinides is much more tolerant to interferences than is electrodeposition and cerium hydroxide (8) because impurities such as Al, Ti, Zr, and Fe do not precipitate as fluorides. Therefore, the cerium oxalate precipitation step is not necessary when cerium fluoride deposition is used. The capacity of the 5% HT-6 column for neodymium was determined by loading 60 mg of I4’Nd traced neodymium onto the column from pH 2.8 DTPA eluant and washing the column with neodymium-free pH 2.8 eluant until the excess neodymium broke through and formed a small elution peak. The neodymium left on the column was assumed to be the column capacity, The capacity of a column of 7 g of 5% HT-6 for neodymium under the optimum conditions is about 55 mg with a mole ratio of HDEHP to neodymium of about 3. Unfortunately, an unusual effect of increased extraction of americium due to milligram amounts of neodymium was found and is shown in Figure 2. The effect could be due to the americium distribution coefficient being higher in the neodymium-HDEHP salt than in the HDEHP alone (19). However, the loss of americium is not severe at the less than milligram levels or even the about 2 mg levels of neodymium found in the average 10- or 50-g soil samples, respectively (4). One attempt to reduce the effect of neodymium on the americium is shown in Figure 3. The loading volume was increased to effectively reduce the concentration of the added 10 mg of neodymium in the presence of the americium-241.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
Table I. HDEHP Content in Various Column Eluates type of eluate
amt of HDEHP in 20 mL of eluate, f i g
DTPA water 0.1 N HCI
180 1740 14 24
6 N HCl
2555
Table 11. Recovery of the Tervalent Actinides and Neodymium in 30 mL of DTPA Eluate at pH 2.8 recovery, % no stable 5 mg of Ce, lanthanides 3 mg of La, and 2 mg of Nd added nuclide added I4'Nd 241Am "Cm ZQCf
0.5
0.4
99.9 99.3 92.8
96.1 95.5 86.0
8ol - - -0
I 10 15 ILoading volume ImL)
20
25
Figure 3. The effect of loading volume on the extraction of (0)241Am and ( 0 )14'Nd from 10 mg of neodymium. Increasing the loading volume up to 10 mL increased the amount of the americium eluted, but further increases in loading volume approaching the total 30-mL eluant volume were not of further help. The effects of flow rate, temperature, and percent HDEHP on the T-6 powder were ritudied and found not to be critical. Doubling the 0.3 mL/mm optimum flow rate gave only a 1 pg increase in neodymium in the americium fraction. The temperature was varied kietween 0 and 40 "C with essentially no effect on the column separation. A column containing 7 g of 10% HT-6 required a 0.1 unit increase in pH to give the same elution characteriekics as the 5% HT-6 column. Although the 10% HT-6 column has a higher capacity for lanthanides, the limiting factor is still the effect the macrolanthanides, particularly neodymium, has on decreasing americium yields. Therefore, increasing the percentage of HDEHP on the T-6 to more than 5% has little practical value in the HT-6-DTPA system. The elution characteri,etics of americium and neodymium on 5% HT-6 columns prepared from HDEHP purified by the copper salt method (20) or by the citrate method (12) were essentially the same. The simpler citrate purification method was used. The column firee volume was determined to be 5 mL with cesium-137 which is not extracted by HDEHP from 6 N HCl. Recently we have been using cerium fluoride to carry the actinides (7,8)as a simple alternative to electrodeposition. The 5 0 - ~ gof cerium precipitated as a fluoride carries the actinides almost quantitatively with little degradation of the a spectrum. The cerium fluoride carries the tervalent actinides directly from the DTPA eluant without thie wet-ashing step needed prior to electrodeposition. Also, the column separation of lanthanides is much less critical with the cerium fluoride technique because as much as 100 pg of lanthanides could elute with the actinides without significantly affecting the actinide yields or resolution. The excellent perormance of the improved method is shown by the results in Table 11. The actinide recoveries obtained on samples containing 5 mg of cerium, 3 mg of lanthanum, and 2 mg of neodymium (7 to 10 times the lanthanides expected in an average soil (4))are of particular interest, because
I 1
0.20
6.60
1
Energy (MeV)
Figure 4. a spectrum of (A) "'Am, (B) *&Cm,and (C) 252Cfon cerium fluoride after separation from 10 mg of mixed lanthanides. less than 10 pg of neodymium was found in the final actinide fraction. The a spectrum of this actinide fraction shown in Figure 4 indicates the excellent resolution that can be obtained. The resolution is about 80 keV fwhm in each case. Because neodymium is the worse case of the three lanthanides, the percentage of cerium and lanthanum in the final fraction is much smaller than that shown for neodymium.
ACKNOWLEDGMENT The authors gratefully thank J. S. Morton for the preparation of the neodymium-147 tracer and R. L. Williams for the a spectrometric determinations.
LITERATURE CITED (1) Filer, T. D. Anal. Chem. 1974, 4 6 , 608-610. (2) Sill, C. W.; Puphal, K. W.; Hindman, F. D. Anal Chem. 1974, 4 6 ,
1725-1737. (3) Puphal, K. W.; Olson, D. R. Anal Chem 1972, 4 4 , 284-289. (4) Vlnogradov, A. P. "The Geochemistry of Rare and Dlspersed Chemical
Elements in Soils", 2nd ed., Engl. trans.; Chapman and Hall: London, 1959. (5) Cerrai, E.; Testa. C. J . Inorg. Nucl. Chem. 1963, 25, 1045-1050. (6) Lleberman, R.; Mohlssl, A. A. Healfh Phys. 1968, 15,362-363. (7) Hindman, F. D.; RESL, USDOE, Idaho Falls, ID, 1981,personal communications. (8) Sill, C. W.; Willlams, R. L. Anal. Chem. 1981, 53,412-415. (9) Sill, C. W.; Hindman, F. D.; Anderson, J. I. Anal. Chem. 1979, 51,
1307-1314. (IO) Bernabee, R. P.;Percival, D. P.; Hlndman, F. D. Anal Chem. 1980, 52,2351-2353. (11) Sill, C. W. Healfh Phys. 1989, 17,89-107. (12) Perclval, D. R.; Martin, D. B. Anal. Chem. 1974, 46, 1742-1749. (13) Schllt, A. A. "Perchloric Acld and Perchlorates"; G. Frederick Smlth Chemical Co.: Columbus, OH, 1979;p 155. (14) Moore, F. L.; Jurriaanse, A. Anal. Chem. 1987, 39, 733-736.
2556
Anal. Chem. 1982, 5 4 , 2556-2560
(15) Schadel, M.; Trautmann, N.; Herrmann, (3. Radiochim. Acta 1977, 24, 27-3 1. (16) Horwitz, E. P.; Bioomquist, C. A. A.; Deiphin, W. H. J . Chromafogr. Sci. 1974, 12, 11-22. (17) Weaver, B.; Kappeimann, F. A. J. Inorg. Nud. Chem. 1967, 30, 263-272. (18) Bates, R. G. "Determination Of pH, Theory and Practice", 2nd ed.; Wiiey: New York, 1973; pp 422-425. ~~ v.;, Vasilev, V. Ya, Rad/oM(19) Erin, E. A.; Vityutev, v. M.; ~ o p y t v. miya 1979, 21, 100-103.
(20) Partridge, J. A.; Jensen, R. C. J . Inorg. Nucl. Chem. 1969, 31, 2587-2589.
RECEIVED for review May 14, 1982. Accepted September 20, 1982. Use of commercial product names is for accuracy in technical reporting and does not constitute endorsement of the product by the United States Government.
Electrochemical Determination of Adriamycin Compounds in Urine by Preconcentration at Carbon Paste Electrodes Edward N. Chaney, Jr., and Richard P. Baidwln" Deparfment of Chemlstry, Unlversity of Louisvllle, Loulsvllle, Kentucky 40292
Preconcentratlon and quantltatlve determlnatlon of the anllcancer chemotherapy agent adrlamycln are accomplished by adsorptlon of the compound at a carbon paste electrode and dlfferentlal pulse voltammetrlc analysls of the resultlng surface. By simply Immersing the electrode In the adrlamyclncontalnlng sample for a 3-mln period and then rlnslng the electrode and placlng I! In a pH 4.5 buffer solutlon, sensltlvltles well below IO-' M are readlly achleved. The llnear concentratlon range was found to occur from to M wlth the average relative standard devlatlon for replicate measurements approxlmately 10 %. Adriamycln aglycone, one of the drug's prlnclpal metabolltes, also Is strongly adsorbed and Is preconcentrated as well; however, other common chemotherapy agents lncludlng clsplatln, mltoxanthrone, mllomycln C, and vlncrlstlne do not adsorb strongly. The adsorption is sufflclently selectlve that accurate and reproduclble preconcentratlon of adrlamycln specles dlrectly from urine samples Is possible without any prellmlnary treatment of the urine speclmens. The analysls method Is demonstrated by using a urlne sample obtalned from a cancer patient followlng Intravenous admlnlstratlon of adrlamycln. The total analysls tlme required was less than 10 mln.
Adriamycin (Figure 1) is the most prominent member of a family of anthracycline antibiotics which have gained widespread clinical use in the chemotherapeutic treatment of a variety of human cancers. In fact, adriamycin has probably become the single most generally effective broadspectrum antitumor agent available today, possessing major clinical utility for breast, ovarian, bladder, and thyroid carcinomas, acute lymphoblastic and myeloblastic leukemias, Hodgkin's and nomHodgkin's lymphomas, Wilm's tumor, neuroblastoma, and soft tissue and bone sarcomas (1). In addition, many new analogues and derivatives of adriamycin are being formulated and are coming to clinical trial. The development of sensitive and efficient analytical methods for the routine determination of these compounds and their metabolites in physiological fluids and tissues is essential for the evaluation and optimized administration of these drugs. Previously, fluorescence (2-4) and radioimmunoassay ( 4 , 5 ) methods which are applicable for the determination of the total amount of adriamycin-related species have been reported. In addition, analytical methods which utilize thin-layer or liquid chromatography with spectrophotometric or fluores0003-2700/82/0354-2556$01.25/0
cence detection in order to differentiate between adriamycin and its metabolites have also been developed (6-12). Finally, in several polarographic studies (13-15), the electrochemical reduction of adriamycin at mercury electrodes has been reported. Both Rao, Lown, and Plambeck (13) and MolinierJumel et al. (14) observed two sets of reduction waves at negative potentials, corresponding to the reductions of the quinone center and of the carbonyl side chain, respectively. In a related analytical study, Sternson and Thomas (15) described the use of differential pulse polarography for the determination of total adriamycin species in blood plasma, reporting a detection limit of 8 X lo* M using the quinone reduction current for quantitation. Finally, our laboratory (16) has recently investigated the electrochemistry of adriamycin at carbon paste electrodes and reported a set of redox waves which occurs at positive potentials and is related presumably to the oxidation of the hydroquinone group of the dihydroxyanthraquinone moiety. In addition, adriamycin was found to adsorb very strongly and irreversibly onto carbon paste, retaining its characteristic electroactivity in the adsorbed state. The adsorption was sufficiently pronounced that immersion of the electrodes in an adriamycin solution for a few minutes allowed sufficient surface preconcentration to extend the detection limit of differential pulse voltammetry M. (DPV) for the compounds to less than Two potentially valuable analytical methods for adriamycin could follow directly from these electrochemical observations. First, it seems highly likely that electrochemical detection of these compounds following their separation by liquid chromatography should provide a sensitive new approach for determining individually adriamycin and its metabolites (17, 18). Second, the adsorptive preconcentration of these species onto carbon paste and their subsequent quantitation by conventional DPV offers a potentially simple means for quantitating total adriamycin content rapidly and routinely in various physiological matrices. In this work, we will concentrate on the latter objective and will demonstrate the selectivity and sensitivity of the adsorption approach for the direct electroanalysis of adriamycin-containing urine samples. The potential of the approach to aid in the clinical administration of these drugs with patients actively undergoing cancer therapy will be demonstrated.
EXPERIMENTAL SECTION Reagents. Adriamycin (or doxorubicin hydrochloride) was obtained from Adria Laboratories (Columbus, OH) and from Sigma Chemical Co. (St. Louis, MO) both in reagent form and 0 1982 American Chemical Society
