Streptomycin and Mannosidostreptomycin in Fermentation Broths Ion Exchange Resin Separation and Spectrophotometric Determination C.
V.
ST.
JOHN, D. E. FLICK,
AND
J. B. TEPE, Eli Lilly and Co., Indianapolis, Ind.
During the streptomycin fermentation period it is necessary to know the potency of the broths and, many times, the amount of mannosidostreptomycin present. Existing microbiological and paper chromatographic techniques are too slow to be of immediate value. The cation exchange resin Amberlite IRC-50 is used for the separation of streptomycin and mannosidostreptomycin from fermentation broths. After separation, the streptomycin and mannosidostreptomycin are determined by the maltol and anthrone colorimetric methods, respectively. The maltol value is corrected for the amount of mannosidostreptomycin; therefore, the final result gives both potency and per cent mannosidostreptomycin.
D
URING recent years, various methods have been developed for the separation and chemical determination of streptomycin after its separation from fermentation broths. As early as 1945, Schenck and Spielman (11) suggested that the formation of maltol from the streptose portion of the molecule could be used as a chemical test for streptomycin. Later Boxer, Jelinek, and Leghorn ( 1 ) adapted this observation to the determination of streptomycin in broths and urine by using a solvent extraction separation to isolate the maltol formed on heating with alkali. Eisenman and Bricker ( 4 )formed maltol from the streptomycin in the broth, steam-distilled it, and determined the maltol in the distillate. Perlman (9) determined the two streptomycins in relatively pure solutions by the carbazole method. In 1948, Schenck, Shaw, and Hargie (10) employed a cation exchanger for separating the streptomycin from fermentation broths. They assayed the eluate by the maltol procedure and compared it to the microbiological assay to determine per cent mannosidostreptomycin. Recently Doery, Mason, and Weiss ( 2 ) used a cation exchange resin for separation and determined the total streptomycine present by converting the streptomycins to maltol and measuring the ultraviolet absorption a t 322 mp. This method gives only total streptomycins present as determined by the maltol procedure, making no distinction between streptomycin and mannosidostreptomycin. As there is a molecular weight difference and a marked difference in microbiological potency, agreement with the microbiological assay can be expected only if there is little or no mannosidostreptomycin. The method proposed here employs an ion exchange resin separation of the streptomycin from the broth. From the iinal analyses, it is possible to calculate the potency expressed in microbiological units and the per cent mannosidostreptomycin. The method requires filtering the mold from the sample, adsorbing the streptomycin on the cation exchange resin Amberlite IRC-50 (in the sodium form), eluting with acid, and assaying the eluate by the maltol procedure. The result obtained is a measure of the total streptomycins present. If the sample contains mannosidostreptomycin, another purification over the resin must be made. On this second eluate total streptomycin is determined by the maltol method, and an anthrone test ( 3 , 5 ,7 , 8 ) is run to determine the amount of mannose present, from which the per cent mannosidostreptomycin is calculated. From these data and the known microbiological response of the two s t r e p tomycins, a value is calculated which can be compared to the potency, expressed in units, obtained by the microbiological assay.
APPARATUS AND SOLUTIONS
Mechanical agitation is carried out on a Burrell wrist-action shaker, and absorbancy measurements are made on a Beckman Model DU spectrophotometer or a Coleman Model 9 Nephlocolorimeter against the blanks specified. All pH measurements are made with a Beckman Model G pH meter. Solutions are prepared from reagent-grade salts in volumetric glassware but need not be standardized. Sulfuric acid, 2.0% by volume. Sodium hydroxide, 2.5 N . Ferric ammonium sulfate, 5.oyOin 5.0 N sulfuric acid. Anthrone 0.2% (weightlvolume) in 95% sulfuric acid. (This anthrone should be of the best grade. If not, the material should be recrystallized.) The solution is made up and allowed to stand for 1 hour before use. Amberlite IRC-50 (regenerated in the sodium form). The desired quantity of resin is stirred for 1 hour with sufficient 4% sodium hydrovide to bring the pH of the supernatant to 9 to 10. The resin is then washed by decantation until the tvash solution is approximately neutral (pH 7 to 8). This washing procedure tends to remove some of the fine particles. The resin is then filtered on a Buchner funnel and airdried overnight a t room temperature. The drying is carried out by spreading a layer approximately 0.25 inch deep in a suitable container. After drying, the resin is screened, and particles passing a 20-mesh but not a 40-mesh screen are used. The dried resin is stored in glass jars t o prevent further loss of moisture. In order to keep acid and alkali concentrations proportional throughout the procedure, it is sometimes necessary to adjust the amount of resin used. Each batch of resin must be checked to determine its “capacity” by treating 1.5 grams of the resin with 15 ml. of 2.0% sulfuric acid (used in the test), filtering off the resin, and washing with water. The filtrate and washings are titrated with 1 N sodium hydroxide to a phenolphthalein end point. This should take approximately 6.5 me. of the sodium hydroxide. If this titration varies more than *3%, the weight of resin used for the adsorption should be adjusted accordingly. As a final check, a broth sample should be run with the old and new resin for comparison. RECOMMENDED PROCEDURE
Samples of broth containing the mold are adjusted to a pH of 2.0 to 2.3 and filtered through Whatman No. 1 filter paper. About 1.5 grams of regenerated resin are placed in a 50-ml. Erlenmeyer flask and washed by decantation several times with distilled water to remove “floaters.” A IO-ml. aliquot of the filtered broth sample is then transferred to the flask containing the resin, distilled water is added to bring the volume to approximately 20 ml., and the sample is shaken on the shaker for 10 minutes. The flask is then removed, and the supernatant is carefully decanted. The resin is washed by decantation four or five times with distilled water to remove all traces of color and suspended matter. 1289
1290 The importance of care in these operations is obvious, because the streptomycins are adsorbed on the resin, and any significant loss of resin is a loss of sample. A415-ml. aliquot of 2.0% sulfuric acid is added to the flask for elution. The sample is shaken for 10 minutes as before, and then quantitatively filtered into a 50-ml. volumetric flask. The resin on the filter paper (Whatman S o . 1) is ryashed several times with distilled water to ensure recovery of the acid solution. Sufficient 1 N alkali (usually about 6.5 ml.) is added to the flask to bring the pH of the solution to 6 to 9 or to a phenolphthalein end point, and the contents are diluted to volume. This solution is labeled eluate I. A maltol determination on this eluate represents a total maltol value for the streptomycin and niannosidostreptomycin in the sample. If there is no niannosidostreptomycin in the sample, no further calculation need be made, but if present, the following procedure should be applied. A 35-ml. aliquot of eluate I is added to 1.5 grams of the washed IRC-50 resin in a 50-ml. Erlenmeyer flask. The sample is shaken, washed with distilled water, and eluted 1 2 3 4 5 6 7 with 15 ml. of 2.0% sulfuric Figure 1. Comparison of acid as before. At least 10ml. Chemical and Paper Chroof the eluate are transferred matographic Determinato a beaker, the pK is adtion of Streptomycin justed to 6 to 9 as previously described, and the solution is Chemical, % E labeled eluate 11. The second 1. 70 (standard) 5. 2.7 2. 15.9 6. 3.4 pass over the resin is a 16.3 7. 3.0 3. “cleanup” step and is not 4. 12.7 quantitative. When strept,omycin and mannosidostreptomycin were added to broth samples, recoveries by this procedure exceded 97% indicat,ing that there is little or no fractionation. In order to adsorb completely all the streptomycin from broth solutions that may contain other constituents which may adsorb on the resin, not more than 10 ml. of broth sample should be used unless the potency is very low. This sample should not contain more than 15,000 units of streptomycin. It lvas found, when adsorbing pure solutions of the streptomycins, t,hat 5 minutes’ shaking is usually sufficient for complete adsorption. If the sample is shaken for 10 minutes, complete 4 pH of 1.6 to adsorption from fermentation broths is ensured. . 1.8 is required for complet,e elution. The 15 ml. of 2.0y0 sulfuric acid will normally give an eluate of the desired pH. This has given satisfactory and reproducible results. Ten minutes’ shaking is always more than sufficient for elution. Determination of Total Streptomycin and Mannosidostreptomycin in Eluate. A maItoI test is run on eluates I and I1 and the anthrone test on eluate 11. From these data, it is possible to calculat,e the per cent of niannosidostreptomycin and units per milliliter of streptomycin (corrected for mannosidostreptomycin) in the original sample. Five milliliters of the eluate to be XfALTOL DETERMINATION. tested are transferred to a 10-ml. volumetric flask, the sample is diluted to about 8 ml., 0.4 ml. of 2.5 N sodium hydroxide is added, and the flask is suspended in a steam bath for 3 minutes. Aft,er heating the required time, the sample is cooled, 0.4 ml. of the 5.0% ferric ammonium sulfate solution is added, the sample is diluted to volume, and its absorbancy is read a t 540 mF in 1-cm. Corex cells against a water blank. The units per milliliter are calculated from an extinction coefficient determined on a standard streptomycin sample (free of mannosidostreptomycin) adsorbed and eluted from the resin in the same manner as an unknown. This assay can also be conducted according to the procedure of Doery et al. ( 2 ) .
ANALYTICAL CHEMISTRY ANTHROXE DETERMINATION. A 5-ml. aliquot of eluate I1 i b transferred to a 25 X 105 mm. test tube cuvette and 10 ml. of the 0.2% anthrone solution are added with swirling. After thorough mixing, the solution is allowed to stand 15 minutes, and its ahsorbancy is measured with a Coleman Model 9 Kephlocolorimeter using filter 8-215 (650 mp) against a blank prepared in the same manner. The anthrone procedure is standardized with mannose on the assumption that the molecular extinction coefficient for mannose and mannosidostreptomycin is identical ( 5 ) . A standard mannose sample is assayed with each group of samples to minimize such effects as time, temperature, and impurities in reagents and glassware. Perlman (9) implies that the anthrone procedure gives a small but definite blank even with pure streptomycin. In this laboratory this blank has been less than 0.5yoand has been considered negligible. Emery and Walker (5) indicate that this blank is small. CALCULATION
From these data it is possible to calculate the results as d o sired. Emery and Walker ( 5 ) calculated the molecular proportion of the two streptomycins; however, the authors have found it more convenient to calculate per cent mannosidostreptomycin on a microbiological unit basis. I n the following calculations, streptomycin base is assumed to be 1000 microbiological units per mg. and mannosidostreptomycin base 240 microbiological units per mg. as assayed by the Food and Drug Administration procedure (6). Results from eluate I1 are used to calculate the per cent mannosidostreptomycin in the solution. This percentage is then used to correct the value from eluate I to give a figure corresponding to the microbiological potency of the solution.
Table I. Comparison of Chemical and Biological Assay Sample
a
% Ea
Chemical Uncorrected, Units/Ml.
Chemical Corrected, Units/MI.
Biological Actual. Units/Ml.
Mannosidostreptomycin.
Because of the difference in the molecular weights of the two streptomycins, equal weight of the two will not give the same maltol value. One milligram of mannosidostreptomycin base will 581 give 1000 X - = 232 units per mg. by the maltol test if based on 743 streptomycin. This same material will show 240 units per mg. by microbiological assay; therefore, the maltol value on mannosidostreptomycin is ’ E =o 2.26 times greater than micro240
biological. AB the mannosidostreptomycin is calculated on the basis of microbiological units, it will be necessary to multiply the units of mannosidostreptomycin by 2.26 and subtract this from the maltol figure to obtain the corrected maltol result. The equation would be as follows when Jf2 is equal to the inaltol value per milliliter from eluate 11. Mg. of mannose/ml. X
743 X 240 180
743
M2 - 2.26 (mg. of mannoselml. X X 240) 180
x
100 =
per cent mannosidostreptomycin
Because the second treatment with resin is not quantitative, it is now necessary to use this figure for per cent mannosidostreptomycin to correct the value obtained on eluate I. The following equation shows the type of calculation applied.
V O L U M E 2 3 , NO. 9, S E P T E M B E R 1 9 5 1 where .If, = maltol units per ml. calculated on original broth X = cheniical value that would compare to microbiological potency %*oB = per cent mannosidostreptomycin in sample
1291
graphic Laboratory and Mary Jane Campbell, L4V011 EIuhnke, Mary M. Jack, and Dorothy J. Polk of the Antibiotics Plant Control Laboratory.
DISCUSSION
Table I give3 a comparison between chemical and microbiological assay. As can be seen without correction for the niannosidostreptomycin large errors could result. Corrected results are in good agreement, and an average of results accumulated over a period of several months agrees ne11 within &5y0of the biological result,s. Occasionally, results erceed this limit, for which no explanation can be given. In assaying duplicate samples by this method, a standard deviation of i 2 % is obtained even by different operators. Figure 1 shows the paper strip chromatographs (12) of the eamplcs listed in Table I. The upper zone represents the mannosidostreptomycin and the lower zone streptomycin. The total area and relative biological activity of the two zones are compared to estimate per cent mannosidostreptomycin.
LITERATURE CITED ( l j Boxer, G.
E., Jelinek. T. C., and Leghorn, P. M., J . Bzoi Chem.,
169, 153 (194i). ( 2 ) Doery, H., Mason. E . , and Weiss, D., ANAL.CHEY.,22, 1038 (1950). (3) Dreywood, R., I b i d . , 18, 499 (1946). (4) Eisenman, IT., and Bricker. C., Ibid., 21, 1507 (1949). (5) Emery, W.B., and Walker, A . D . , I n a l y s t , 74, 455 (1949). (6) Federal Security Agency. Food and Drug A4dministration, “Compilation of Regulations for Tests and Methods of Ass a y , ” Vol. I, p. 48, 1950. (7) Kowald, J., a n d hlcCormack, R., A s a ~CHEM., . 21, 1383 (1949). (8) Morris, D. L., Science, 107, 254 (1918). (9) Prrlnian. D., J . B i d . Chem.. 179, 1147 (1949). (10) Schenck. J. R . , Shaw, ,J. L., and Hargie, hI. P., .Ihstracts of 113th Meeting h 1 . CHEM. SO^., p. 8C. Chicago. .ipril 1948. (11) Schenck, J. R., and Spielman, .\I. J.. J . Am. Ciiem. Soc.. 67, 2276 11945). (12) K i n s t o n , \ T ~A . , and Eigen, E., Ibid., 70, 3333 (1948).
ACKNOWLEDGMENT
The authors wish to acknowledge the technical assistance of Xorman Davis, Gerald Johns, and Charles Pugh of the Chromato-
RECEIVEO February 9, 1 R S l . Presented before the Division of Analytic81 ChiChemistry a t the 118th Ileeting of the .L\IERICAS C H E ! m c A L SOCIXTI., cago,
Ill.
Determination of Lead as Chloride Separation f r o m Bismuth and Other Elements SILVE KALLMANN, Ledoux & Co., 155 S i x t h Ave., New York, N. Y . Further work with the Willard and Smith reagent showed that lead chloride is insoluble in butyl alcohol containing a small quantity of hydrogen chloride. Bismuth chloride and the chlorides of other elements with which lead is associated in metals are very soluble in the same medium. In the method proposed for the quantitative separation of lead from bismuth, the chlorides are treated with a 2% solution of hydrogen chloride in butyl alcohol. Lead
H
YDROGEN chloride, as a 20% solution in n-butyl alcohol, has been used to precipitate the chlorides of several el+ ments. Willard and Smith (6) first proposed this reagent for the quantitative separation of sodium from lithium by precipitating sodium chloride from butyl alcohol solution of the two elements. Kallmann introduced the name “Willard and Smith reagent” and ehowed that it could be used in other methods (1-4). -4s would be expected, lead perchlorate is very soluble in n-butyl alcohol; the Willard and Smith reagent precipitates lead chloride. In the early part of the investigation reported here, solutions of lead nitrate and lead chloride were fumed to near dryness with perchloric acid. The lead perchlorate was dissolved in butyl alcohol and lead chloride was precipitated by addition of the Willard and Smith reagent. At this stage of the investigation it was noted that bismuth perchlorate is also very soluble in butyl alcohol, but is not affected by the Willard and Smith reagent. The value of the reagent in the separation of lead from bismuth was then investigated (Table I), The same manipulations and reagents were used as in the earlier work on the precipitation of barium and strontium ( I ) , except that a final 5 to 6% concentration of hydrogen chloride in butyl alcohol was preferred. The results for lead were corrected
chloride is collected on a Gooch crucible, dried, and weighed. Bismuth is recovered in the filtrate as oxychloride. Metals and alloys are dissolved in hydrochloric acid and hydrogen peroxide or dilute nitric acid. Antimony, tin, and arsenic are expelled as chlorides by three evaporations with hydrochloric acid. The residual chlorides are treated with the 2Yo hydrogen chloride solution and the lead chloride is collected on a Gooch crucible.
for the solubility of lead chloride in the medium chosen, arid bi.c muth was recovered in the filtrate and finally determined as the oxychloride. While determining the solubility of lead chloride in butyl alcohol containing various concentrations of the Willard and Smith reagent, it was noted (Table 11) that, unlike sodium, barium, and particularly strontium chloride, lead chloride becomes less soluble with decreasing concentrations of the Willard and Smith reagent arid is practically insoluble in n-butyl alcohol containing
Table I.
Separation of Lead from Bismuth by Precipitation Method
Lead Used
Lead Found
Bismuth Used
Bismuth Found
Gram
Gram
Gram
Gram
0 1000
0 0998 0 0999 0 2504 0.2.503 0 2497 0.2506 0,4999 0.5004 0.5003
0 2300
0 2503
0.1000 0,2500 0.5000 0.1000 0,2500 0.5000
0.0998 0.2507 0,5006 0 0997 0.2500 0.5007
0 0 0 0 0 0 0 0
1000
2500 2300 2500 2500 5000 5000 ,5000
....
....
