ANALYTICAL CHEMISTRY
1208
zirconium-thorium were prepared and titrated. The results given in Table I11 confirm the quantitative nature of the thorium and hafnium interferences. It is probable that zirconiumiron mixtures can also be titrated quantitatively. LITERATURE CITED
Dhar. S. K..and Das GuDta. *4.K.. J . Sct. I n d . Research ( I n d i a ) , 11B, 500 (1952). (2) Fritz, J. s.,and Ford, J. J., Anal. Chem., in press. (3) Fritz, J. s., Ford, J. J., and Lane, W. J., Iowa State College report, in press. (1) .
I
-
1
CHEM., . 22, (4) Goetz, C. A., Loomis, T. C., and Diehl, H., - 4 ~ . 4 ~ 798 (1950).
(5) Green, ‘D.-E’.,Ibid., 20, 370 (1948). Hahn, R., Ibid., 21, 1579 (1949). (7) Kolthoff, I. M.,and Johnson, R. A., J . Electrochem. Soc., 98, 138 (1951). Kumins, C. A., ANAL.CHEM.,19, 376 (1947). Oesper, R. E.,and Klingenberg, J. J., Ibid., 21, 1509 (1949). (IO) White, J. C., Paper A-21, Anal. Information Meeting, Oak Ridge National Laboratory, May 1953. (6)
RECEIVEDfor review August 14, 1953. Accepted March 29, 1954. Contribution No. 314 from the Institute for Atomic Research and Department of Chemistry, Iowa State College, Ames. Iowa. Work performed in the Ames Laboratory of the Atomic Energy Commission.
An Isotope Dilution Assay for Total Penicillins MAXWELL GORDON, ANGEL0 J. VIRGONA, and PAUL NUMEROF The Squib6 lnstitute for M e d i c a l Research, N e w Brunswick,
A
N ISOTOPE dilution assay for total penicillins in broth is
described which uses inexpensive sulfur-35 labeled penicillin. No degradation of penicillin is required and penicilloic acid does not interfere. The 95% confidence limits are A 5 9% on the mean of duplicate assays. Samples containing a total of 10,000 units in a convenient volume can be assayed by this procedure. Levy et al. (6) have published a gravimetric method for penicillin (8) depending on precipitation as the N-ethylpiperidine salt. This method requires quantitative isolation of penicillin from broth, which is unnecessary in the isotope dilution procedure. Craig et al. ( 8 ) have published an isotope-dilution assay for penicillin using phenylacetic acid-1-carbon-14. The precision of their method was not reported. The Craig assay is specific for penicillin G, but its disadvantage is that the penicillin must be degraded to phenylacetic acid. I n the method reported no degradations or combustions of penicillin samples are required and samples are prepared for counting by plating out aliquots of solutions of known concentration. Penicillin of high specific activity can readily be produced by biosynthesis from inorganic sulfur-35 sulfate in yields up to 30% based on sulfur-35 ( 5 , 7 , 10, 1.2). The penicillin can readily be purified as the iV-ethylpiperidine salt (11). T o test the possibility of contamination of penicillin with penicilloic acid in the isolation of penicillin from broth, a 1 to 1mixture of penicillin-penicilloic acid as the N-ethylpiperidine salt was crystallized. With only the penicillin labeled, there was no decrease in specific activity of the penicillin isolated from the mixture. Hence, the penicilloic acid, the most likely contaminant, is not an interfering substance in this assay. Fourteen replicate analyses of a lyophilized broth sample were carried out and the results are in substantial agreement with those obtained by bioassay or chemical assay. EXPERIMENTAL
Microbiological Preparation of Sulfur-35 Benzylpenicillin. Two flasks containing 100 ml. of a modified synthetic medium were inoculated with a penicillin producing strain of P. chrysogenum, and 1 mc. of sulfur-35, as potassium sulfate, was added to each flask through a bacterial filter. The medium consisted of the following in grams per liter: potassium phosphate, monobasic, 1.0; magnesium sulfate heptahydrate, 0.5; zinc sulfate heptahydrate, 0.02; manganese sulfate monohydrate] 0.02; ferrous ammonium sulfate hexahydrate, 0.10; cupric sulfate pentahydrate, 0.005; ammonium sulfate, 3.0; glucose, 5.0; lactose, 35.0; calcium carbonate, 10.0. The sugars and calcium carbonate were sterilized separately. Washed mycelia were used
N. J.
for the inoculum. These fermentation flasks each contained only about 10 mg. of sulfur-32 (the weight of sulfur-35 is negligible), most of the usual ammonium sulfate being replaced by ammonium chloride in one case, and ammonium nitrate in the other (about 6 mg. of sulfur was added per flask as magnesium sulfate and the remainder was estimated to come from the inoculum). Phenylacetic acid was added in four equal portions a t 24-hour intervals after the start of the fermentation. The broth was harvested after 6 days. Paper chromatographic assay of the broth showed that 96% of the antibiotic was present as penicillin G ( 4 ) . The penicillin yield was about 250 units per ml. from ammonium nitrate supplemented medium and about half that from the ammonium chloride medium.
T a b l e I.
Specific Activities of Penicillin Sdmples
Sample
NEP
K salt, Crop 1 K salt, Crop 2 K salt, Crop 3
Weight, Mg. 172Q
381 186
Specific Activity, Dis./Min./Mg.
359,000 360,000:369,000~ 343,OOOb 367,OOOb
141 a 1.0 gram of cold benzylpenicillin added t o this sample in potassium salt crystallization. Only the first 3 fractions are shown. b Corrected for dilution.
Isolation of Sulfur-35 Benzylpenicillin as N-Ethylpiperidme Salt. Potassium benzylpenicillin (0.5 gram) was added initially to the broth as carrier. The broth (100 ml.) was extracted with four 50-ml. portions of amyl acetate in a Kutscher-Steudel extractor (each portion was recycled five times). The amyl acetate was dried over anhydrous sodium sulfate, evaporated in vacuo a t 30” C. to 1 to 2 ml., and the concentrate was filtered through a layer of anhydrous sodium sulfate. An equal volume of N-ethylpiperidine was added through the filter and the precipitate was allowed to form overnight in the refrigerator. The recovery of penicillin was about 50%, I n preparing the potassium salt, 172 mg. of the N-ethylpiperidine salt was dissolved in 1 ml. of water and 1.0 gram of potassium benzylpenicillin and 0.09 ml. of 4.4,V potassium hydroxide were added. This aqueous solution was extracted with two 0.2-ml. portions of amyl acetate to remove S-ethylpiperidine. Ten milliliters of n-butyl alcohol were added to the aqueous phase and the solution evaporated in vacuo a t 25 C. to induce crystallization. Successive crops of potassium benzylpenicillin crystals were obtained for which specific activities and weights are given in Table I. The potassium salt was used for broth assays 1 to 6. Higher specific activity N-ethylpiperidine salt was used for assays 7 to 14. O
1209
V O L U M E 26, NO. 7, J U L Y 1 9 5 4
The specific activity of the potassium b enz y l p e n i c i lli n added in the isotope-dilution 1st Recrystal2nd Recrystal3rd RecrystalAssay Sample Crude Weighta, lization, Weighta, lization, Weight'", lization, assays 1 to 6 was 54,000 disNo. No. Units/Ml. Mg. Units/Ml. Ivlg. Units/Ml. hlg. Units/Ml. integrations per minute per 1 2140 127.2 2280 102.4 2297 80.7 2 69.6 2265 2160 111.6 2437 90.4 2393 milligram. Determinations on 2172 2259 6 6 . 2 104.3 2209 83.6 3 subsequent days gave, after 2233 2309 74.1 2284 4 91.6 58.6 2227 2231 83.6 2196 106.5 53.6 5 correction for decay, 55,000 2410 2113 78.9 2195 99.4 55.2 6 81.3 2320 2251 2275 129.9 107.0 7 and 54,000 disintegrations/ 2323 71.1 2235 2387 116.0 95.8 8 minute/mg. The p e n i c i l l i n 2340 2254 2258 121.1 50.1 74.4 9 2227 40.8 2365 2287 117.6 97.3 10 used in assays 7 to 14 (as N2255 69.9 11 2301 2300 119.3 96.6 12b ethylpiperidine salt) had a 19,l 13 7 2335 53.2 2349 38.6 2361 77.2 specific activity of 178,000. 129.3 2366 2404 93.2 14 2333 Av. = 2274 Av. = 2293 Av. = 2285 i 2 0 6 / 4 / 1 2 = 2285 It 54 Table I11 gives results on Av. bioassay = 2165 i 200 units/ml. some preliminary isotope-dilu0 10-mg. samples withdrawn from each for counting before recrystallization. tion assays on different penib Data from this sample was omitted from this table. cillin broth samples. Sample pairs 3, 4 and 5 , 6 are duplicates, the latter pair having Analysis of Penicillin Samples. A plant sample of filtered penibeen fortified with 136 units per ml. of added penicillin to estimate cillin broth was divided into 14 portions of exactly 250 ml. the limits of detection. each. These samples were freeze-dried and kept a t 0" C. Two Table IV shows the results of crystallization of a mixture of flasks were reconstituted daily for assay purposes. The conequal parts of penicillin and penicilloic acid as the N-ethylpiperitents of each pair of reconstituted flasks were combined. Two 100-ml. samples were withdrawn for isotope-dilution assay, and dine salt, Only the penicillin was labeled. the balance of the material was submitted for bioassay. Determination of Radioactivity and Calculation of Results. In a typical isotope-dilution assay labeled penicillin (10 to 20 All radioactivity determinations were made by dissolving about mg.) was added to a 100-ml. portion of broth in a 250-ml. centri10 mg. of the penicillin samples in 2 ml. of water, evaporating fuge bottle. The p H was brought to 2.0 t o 2.5 with phosphoric acid. The solution was cooled to 0" C. and one 100-ml. portion of 25-microliter aliquots of these solutions on 1.75-inch glass plates amyl acetate was added. The mixture was shaken for 5 minutes (d), and counting the plates in a gas-flow counter. More reproand the emulsion broken by 5 minutes of centrifugation. The ducible plates are obtained if they are wiped with a trace of chilled amyl acetate layer wa5 extracted with three 30-ml. porTergitol (Union Carbide & Carbon Corp., New York, S . Y.) tions of cold phosphate buffer, 0.1M sodium dihydrogen phosphate-0.1M sodium hydroxide, of p H 7. The combined buffer prior to use. Each radioactivity determination represents an solution was adjusted to pH 2.0 to 2.5 with phosphoric acid at average of 8 plates. Use of an end-window counter gave identi0" C. and extracted with three 20-ml. portions of chloroform. cal results (within counting error), but longer counting periods The combined chloroform layers were dried over anhydrous were required to obtain the same statistical validity (&2%), An sodium sulfate for 1 hour and evaporated a t room temperature to a volume of 1 ml. in a rotary film evaporator (9). The chloroassay is calculated from the following formula: form concentrate was treated with 2 ml. of N-ethylpiperidine and b = a ( A / B -1) allowed to crystallize in the refrigerator. The ,V-ethylpiperidine salt was removed by centrifugation, washed n-ith dry acetone, and where a = weight of labeled penicillin added dried in vacuo. It was recrystallized by dissolving it in a miniA = specific activity of added penicillin mum of dry chloroform (about 0.6 ml. for 100 mg. of N-ethylpiperidine salt) followed by addition of 2 ml. of dry acetone. b = weight of penicillin in broth Yields averaged about 80% in the recrystallization. The idenB = specific activity of isolated penicillin tity of the N-ethylpiperidine salt of penicillin was established by chemical analysis ( 1 ) and melting point. If penicillin is added as the potassium salt and isolated as the Crystallization of Mixture of Penicillin and Penicilloic Acid. AT-ethylpiperidinesalt, a correction must be made for the molecA mixture of 48.6 mg. of potassium benzylpenicillin and 51.8 mg. ular weight ratio in order to use the isotope dilution formula. of the monosodium salt of penicilloic acid was dissolved in 1 ml. of water. The solution was adjusted to p H 2 with phosphoric acid and extracted with three 1-ml. portions of amyl acetate in a centrifuge tube. The amyl acetate extracts were combined, Table IV. Penicillin-Penicilloic Acid Experiment dried, and treated with 1 ml. of N-ethylpiperidine, -4total of Specific Activity, 32.9 mg. of N-ethylpiperidine was ohtained. The recrystallizaDis./Min./Mg. tion procedure is described in the previous section. Starting penicillin, N-ethylpiperidine 3860 Table 11. Penicillin Assay Results On Replicate Broth Samples
Isolated N-ethylpiperidine, crude Recrystallized isolated N-ethylpiperidine Twice recrystallized N-ethylpiperidine
3760 3590 3860
Table 111. Penicillin Assay Results Sample No.
Crude, Units/Ml.
First Recrystallization
Bioassay, Av. 6 Detn.
...
1250' ... 1350 1269 1525 1965, 2180 2160 2085 2180 2370 2289 ... 6 2369 2233 ... a Synthetic broth made up to contain 1250 units/ml. Insufficient material for a 2nd recrystallization. Constant specific activity not achieved. 1 2 3 4 5
RESULTS
Table I1 shows penicillin crystallization yields and assay results for the 7 reconstituted broth samples. Each isotope-dilution assay was done in duplicate and each penicillin-&l=ethylpiperidine salt was recrystallized twice.
DISCUSSION
I t is appreciated that although isotope dilution methods are customarily used for the determination of a single component in a mixture, in this case the penicillin G and other penicillins represent essentially a chemical entity-namely, material precipitated by AT-ethylpiperidine under specific conditions. This same principle has also been applied to chemical assays for penicillin where the total quantity of material possessing an intact 8-lactam ring is determined. The assay procedure described permits assay of broth samples of high benzylpenicillin content in a single day (4 t o 6 man-hours). With the commercial type high-penicillin broths used in this work, fractionation of penicillins on precipitation with N-ethylpiperidine is not a factor, for it has been reported (13)that penicil-
1210
ANALYTICAL CHEMISTRY
lin G is not preferentially precipitated by N-ethylpiperidine from samples of high benzylpenicillin content. The isotopedilution method for penicillin cannot compete with chemical and biological methods of assay on a cost basis. The advantage of the isotope-dilution assay lies in the fact that i t most nearly approaches an absolute measure of total penicillins. Furthermore, penicilloic acid, the most likely contaminant of penicillin in broth, is not an interfering substance in this analysis. Statistical evaluation of the 14 replicate assays shown in Table I1 assigns a 95% confidence value of f 5 4 units or &2.4% to these data. I n addition, for a single sample assayed in duplicate, the 95% confidence limits are &5.9% on the mean of the duplicate assays. There are no significant differences in results between the made and recrystallized samples. Therefore, the Y-ethylpiperidine precipitation gives isotopically homogeneous material in one crystallization and the only limitations to its precision are those inherent in radioactive-counting procedures. ACKNOWLEDGMENT
The authors are indebted to Mavis Carroll for statistical evaluation, to J. F. Alicino for chemical assays on penicillin samples, to
S. C. Pan for providing a low-sulfur medium for penicillin production, and to Harry Hulit for technical assistance. LITERATURE CITED
(1) Nicino, J. F., IND. ENG.CHEX.,ANAL.ED.,18, 619 (1946). (2) Calvin, M., et al., ‘‘Isotopic Carbon,” pp. 113-20, New York, John Wiley & Sons, Inc., 1949. (3) Craig, J. T., Tindall, J. B., and Senkus, M . , ANAL.CHEM.,23, 332 (1951). (4) Goodall, R. R., and Levi, .4.A , , .Vatwe, 158, 675 (1946). (5) Howell, S. F., Science, 107, 299 (1948). (6) Levy, G. B., et al., ANAL. CHEM.,21, 664 (1949). (7) Rfaas, E. A., and Johnson, h1. J., J.Bactereol., 57, 415 (1949). (8) Mader, W. J., and Buck, R. R., ANAL.CHEM.,20, 284 (1948). (9) Numerof, P., and Reinhardt, C., Ihid., 25, 364 (1953). (10) Rowley, D., et al., Nature, 161, 1009 (1948); 163, 480 (1949); J . Chem. Soc., 1949, 5-405; Biochem. J., 46, 157 (1950). (11) Sheehan, J. C., et al., J . Am. Chem. Soc., 68, 2407 (1946). (12) Smith, E. L., Brit. Med. Bull., 8 , 203 (1952). (13) Subcommittee, Ministry of Health Conference on Differential Assay of Penicillin, Analyst, 74, 79 (1949). RECBIIVZID for review July 8, 1953. Accepted April 3, 1954.
Elution Chromatography with Thick Filter Paper W. J. FRIERSON’, P. F. THOMASON,
and
HELEN P. RAAEN
O a k Ridge National Laboratory, O a k Ridge, Tenn.
T
ECHNIQUE using thick filter paper was developed for the separation of milligram quantities of uranium from other
elements by elution chromatography. The technique has the advantages of chromatography with thin filter paper, which was first applied to inorganic analysis by Arden et al. ( I ) , and to the separation of uranium by Arden, Burstall, and Linstead ( 2 ) . I n addition, it is suitable for amounts of inorganic substances larger than the approximately 1-mg. maximum that can be handled successfully on thin filter paper ( 3 ) . The technique is more simple, reproducible, and adaptable than the use of cellulose pulp columns, which are often employed for chromatographic separations on the milligram scale. With a thick filter-paper strip, a single elution can effect simultaneous separations for more than one test solution. The apparatus is similar to that of Strain and Sullivan (6) for analysis by electromigration and chromatography.
the strip, cut two V-shaped sections to serve m small cells for holding excess solvent, thus preventing it from running down either side of the glass plates. If a separation is to be effected on a single volume of sample taper the other end of the strip to a point. If two volumes o/ solution are to be chromatographed simultaneously in the same assembly, cut a 0.25 6 inch section out of the center of the strip from the tip to within 2 inches of the top (Figure 2). Taper the ends of the two legs thus formed. For either type of strip, enrlose the outside (longest) edges of the
+
t I Id I
I I
I
EXPERIMENTAL
Apparatus. The assembly for elution chromatography with thick filter paper, (Figure 1) consists of: Eaton-Dikeman No. 320 filter paper, 0.1 inch thick, rapid filtration rate; two platinum foil strips, 2 mils thick; two glass plates approximately 6.5 X 6.5 X 0.25 inches; four stainless steel strips, approximately 1 X 12 X 1/16 inches; two plywood strips approximately 3 X 14 X 0.25 inches, and screws with wing nuts. Sargent polarograph, modified. Precision Scientific Co., automatic titrator, modified. Reagents. All reagents were analytical reagent grade. Diethyl ether, anhydrous. Sitric acid, concentrated. Perchloric acid, 857,. Cranyl nitrate. Standard uranium solutions that were prepared from this reagent were standardized gravimetrically by ignition to uranyl uranate. Cupric nitrate, added directly to the synthetic sample solutions. Aluminum nitrate, added directly to the synthetic sample solutions. Standard ceric sulfate solution, prepared from ceric ammonium sulfate and standardized against arsenious oxide. PROCEDURE
From a sheet of 0.1-inch-thick Eaton-Dikeman No. 320 filter paper, cut a strip 2 X 8 inches. Near each edge of an@end of 1
Preaend address, Agnes Scott College, Decatur, Ga.
I I
Id I
I I I
I
Id
C
1 E
I I
I Figure 1. Assembly for Elution Chromatography with Thick Paper A-. Snlvmt. back one screwed to plywood - -- . - ..- added
.
B . Solution spotted C. Plywood p. Edges of paper covered with platinum foil
E . Two stainless steel strips,
17
strip Front one tightened on glass with wing nuts Glass plates with strip snndwiched between
