12 through 15 the sample solutions were adjusted to 1M in NaCI, NaN03, (NH4):!S04,and NaH2P04, respectively, so that large quantities of .foreign common ions are present along with thiocyanate ions. As can be seen, the presence of the common anions has no effect on the separation. The standard deviations (lexpressed in per cent) were: 1.55 for 0.906 mg of Re(VII:), 1.32 for 9.06 mg of Re(VII), 1.15 for 1.01 mg. of Mo(VI), and 1.82 for 1.00 mg of W(VI), respectively. Recovery of leach metal ion is also satisfactory:
average deviations (expressed in per cent) 1.1 for Re(VII), 1.6 for Mo(VI), and 2.6 for W(V1) were obtained based on all results listed in Table I. In summary, the method presented allows widely varying proportions of the chemically-similar metals to be separated with a simple eluant on a column containing only 1 gram of DEAE. RECEIVED for review August 12,1966. Accepted November 6, 1966.
Quantitative Determination of the Components of Neomycin by Paper Chrornatography Mrinal K. Majumdar and Saroj K. Majumdar Department of Food Tei-hnology and Biochemical Engineering, Jadavpur University, Calcutta, India
I n a paper chromatographic method for quantitative estimation of neomycins B and C and neamine (neomycin A) from a miixture or fermentation broth the neomycins from fermentation broth or a commercial sample are adsorbed on Amberlite IRC-50 (NH4+form) Eluate is spotted on Whatand eluted with 1N “,OH. man No. 4 paper and N-acetylated by dipping the papers for 12 hour!; in methanol-acetic anhydridecarbon tetrachloride! (3:2:95 v/v) reagent. N-Acetyl neomycins are separated by developing the paper in 1-butanol-water-piperidine (84:16:2 v/v) and then converted to N-chloro derivatives by dipping in chlorine-carbon tetrachloride reagent. The neomycin spots are cut from the untreated paper with the guidance of parallel paper strips (sprayed previously with starch-iodide-pyridine reagent) and then treated with starch-iodide-IHCI to develop color. The absorbances of the solutions are measured at 570 mp imn a spectrophotometer. The quantitative aspect of this method and the role of interfering substances are discussed in detail. This method might be applicable to the estimation of other amino sugar antibiotic complexes and amino sugars which are negative to MorganElson test.
THETERM “NEOMYCIN” usually refers to the amino sugar antibiotic complex, composed of two stereoisomers, neomycin B and neomycin C, and their degradation product, neamine (neomycin A). Commercial preparations contain neomycin B as the major constituent. Sokolski et al. (1,2)have reported methods for quantitatively determining neomycins B and C in mixtures by microbiological assay. Our attempts to carry out these procedures failed to give satisfactory results because of the interfering effects of salts in the fermentation broth. In the past a considerable amount of work was carried out to separate and quantitatively estimate components of the (1) W. T. Sokolski, C. G. Chidester, 0. S. Carpenter, and W. M. Kaneshiro, J. Pharm. 6ci., 53, 826 (1964). (2) W. T. Sokolski and 0. S. Carpenter, “Antibiotics Annual 1955-56,” p. 383, Medical Encyclopedia, New York, 1955.
neomycin complex by chemical and chromatographic assay methods (3-7). The paper chromatographic methods employed by Leach et al. (8,9) and Saito and Schaffner (10) for the separation of neomycins B and C and neamine proved unsatisfactory. Pan and Dutcher (11) developed a semiquantitative method for the estimation of neomycins B andC by paper chromatography of their N-acetyl derivatives, but neomycin B and neamine were not effectively separated by this method. Kaiser (12) described a quantitative radioisotopic method for determining neomycins B and C and neamine after chromatographic separation of the carbon-14 N-acetyl derivatives. The purpose of this investigation is to develop a simple and convenient colorimetric method for the estimation of neomycins B and C and neamine after resolution of their N-acetyl derivatives by paper chromatography. EXPERIMENTAL
Antibiotic Preparations and Standards. The neomycin samples used for analysis consisted of neamine hydrochloride and sulfates of neomycins B and C. The materials used as standards were the free bases of neomycins B and C (Upjohn Co., Kalamazoo, Mich.). Neamine base was prepared from commercial neomycin (3) T. F. Brodasky, ANAL.CHEM.,35, 343 (1963). (4) J. D. Dutcher, N. Hosansky, M. Donin, and 0. Wintersteiner, J . Am. Chem. SOC.,73, 1384(1951). ( 5 ) J. H. Ford, M. E. Bergy, A. A. Brooks, E. R. Garrett, J. Alberti, J. R. Dyer, andH. E. Carter, J. Am. Chem. SOC.,77, 53P1
(1955). (6) H. Maehr and C. P. Schaffner, ANAL.CHEM., 36, 104 (1964). (7) K. L. Rinehart, Jr., A. D. Argoudelis, W. A. Goss, A. Sohler, and C. P. Schaffner, J. Am. Chem. SOC.,82, 3938 (1960). (8) B. E. Leach and C. M. Teeters, J. Am. Chem. SOC.,73, 2794 (1951). (9) B. E. Leach, W. H. DeVries, H. A. Nelson, W. G . Jackson, and J. S . Evans, J. Am. Chem. SOC.,73, 2797 (1951). (10) A. Saito and C. P. Schaffner, Angew. Chem., 67, 666 (1955). (11) S. C. Pan and J. D. Dutcher, ANAL.CHEM., 28, 836 (1956). 35, 552 (1963). (12) D. G. Kaiser, ANAL.CHEM., VOL 39,
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sulfate (Unichem Laboratory, Bombay, India) by methanolysis (5) and recrystallized by the method of Leach and Teeters (8). Its purity was tested by paper chromatography. Reagents. Commercial carbon tetrachloride was washed with sodium hydroxide solution, layer-separated, treated with fused CaCI,, and distilled. The methanol-acetic anhydride-carbon tetrachloride reagent, composed of 3 ml of methanol, 2 ml of acetic anhvdride. and 95 ml of carbon tetrachloride. was . DreDared . j i s t before use. CHLORINE-CARSON TETRACHLQRIDE REAGENT(13). Chlorine was prepared from 100 ml of concentrated hydrochloric acid and 50 grams of potassium permanganate, then passed through water and concentrated sulfuric acid and absorbed in 1 liter of carbon tetrachloride. About 5 grams of barium carbonate and 5 grams of fused calcium chloride were added. It was stored in a glass-stoppered amber colored bottle. STARCH-IODIDE-PYRIDINE REAGENT.One gram of starch and 0.25 gram of potassium iodide were dissolved by heating in 3.5 ml of water and then 0.5 ml of this solution was added quickly to 50 ml of pyridine. Freshly prepared reagent was used. STARCH-IODIDE-BARIUM CARBONATE REAGENT.One gram of starch and 0.25 gram of potassium iodide were dissolved by heating in 100 ml. of water. Two grams of barium carbonate were then added to the cold solution and thoroughly shaken. After the solid particles settled, the upper turbid portion was used for spraying the chromatograms. This could be used for 5 to 6 days. STARCH-IODIDE-HCI REAGENT (13) was prepared freshly hy boiling 1 gram of starch, 0.25 gram of potassium iodide, and 1 m l of 5N HCI in 100 ml of water. Preparation of Resin. Amherlite IRC-SO, 200- to 400mesh (Rohm & Haas), was converted to the NH4+ form by shaking with 1 N ",OH. The resin was suspended in water and allowed to settle and the upper turbid portion was decanted. This process was repeated until all finer particles were removed. The resin was then suspended in an equal volume of water. Two milliliters of the suspension were transferred to a funnel with stem (i.d. 8 nun; column volume 1 ml) plugged with glass wool and then washed with water until the washing was neutral. Such a column could be used several times. After each use it was washed with 1N HCI and then converted to the ammonium form. Procedure. PREPARATION OF N-ACETYL NfoMYcMs. The fermentation broth or the solution of the neomycin sample (containing about 4 mg of neomycins) was passed through an Amherlite IRC-50 (NH,+ form) column. The column was washed with 5 ml of water and eluted with 5 ml of 1 N ",OH. This eluate was spotted at points 20 nun apart along the base line (7.5 cm from the end) of Whatman No. 4 paper (43 X 16 cm) in a volume to contain 4 to 12 fig of neomycin. Standard solutions of neomycins B and C and neamine free bases were applied in the same concentration range. For N-acetylation, the papers were rolled and soaked in the methanol-acetic anhydride-carhon tetrachloride reagent for at least 12 hours, then air-dried for 1 hour to remove acetic anhydride. PAPERCHROMATOGRAPHIC DETERMINATION OF N - A m NEOMYCIN. The papers were developed for 24 to 36 hours at 28" C with the solvent system 1-butanol-water-pipxidine (84: 16:2 v/v) by descending chromatography. The chromatograms were air-dried to remove the solvent. The papers were then placed in a chamber with water-saturated atmosphere a t 37" C for humidification, which was completed within 1 to 2 hours. This treatment was necessary for the complete removal of piperidine (also positive to the color reaction) from the papers by the solvent used in the subsequent step. The papers were next rolled inside the (13) R. F. Pawning and H. Irzykiewicz, J. Chromatog., 17, 621 (1965).
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CHEMISTRY
Figure 1. Paper chromatogram (") of spot (9N-acetyl neomycins A. Eluate from IRC-50 after adsorption of neomycins Band C and neamine salts R. Neomycin C free base C. Neomycin B free base D. Neamine free base E. Eluate from IRC-50 after adsorption of neomyerns limn reimentation broth (synthetic medium) F. Eluate from IRC-50 after adsorption of neomycins from fermentation broth (complex medium). '. 1-Butanol-wster-pipridine (84:16:2 viv), =-hour development at 34" C. '. After application of neomycin solutions on the paper, spots N-acetylated chamber and quickly transferred to a cylinder and the filtered chlorine-carhon tetrachloride reagent was poured in. The whole was kept in the dark for 20 minutes a t room temperature. The chromatograms were air-dried for 0.5 hour at 4" to 6" C in the dark to remove excess of chlorine. The papers were divided into a number of parallel strips 20 nun wide. One of a pair of strips was sprayed with the colorproducing reagent for the development of a guide chromatogram to locate the spots in the other. Bluish pink spots were obtained on a completely white background by spraying chromatograms with starch-iodide-pyridine reagent and transferring it to a water-saturated atmosphere. For rapid work, detection by starch-iodidebarium carbonate reagent and marking of wet spots by ball pen were preferred. But when chromatograms were sprayed with the starch-iodideHCI (13) reagent, spots quickly spread throughout the paper and vanished. Rectangular areas corresponding to N-acetyl neomycins B and C and neamine were removed from the untreated paper strip with the help of the guide chromatogram and then cut into small pieces. Rectangles were cut from spot-free areas for blanks. The spots were then extracted for 30 minutes with 4 ml of water and 1 ml of starch-iodideHCI reagent in clean test tubes. The absorbances of the colored solutions were measured a t 570 mfi in a Universal spectrophotometer, VSU-1 Model, Carl Zeiss, Jena, using 1-cm cells. The amount of neomycins was determined by reference to standard curves. IDENTITYOF N-ACETYLNEOMYCINS.Free bases of neomycins B and C and neamine were applied on the papers and N-acetylated by our method. After air-drying, the papers were spotted at different points on the same base line with a solution of N-acetyl neomycins prepared by the method of Rinehart et al. (7). The R,.I (migration value relative to N-acetyl neamine) values of N-acetyl neomycins prepared by the two methods were compared. The spots of N-acetyl neomycins B and C obtained from neomycins of fermentation broths were tested for homogeneity. The eluate from IRG50 after adsorption of neomycins of the broth was spotted closely on a Whatman No. 4 paper, then N-acetylated and developed in l-hutanol-water-piperidine (84:16:2 v/v) solvent system. Matched with the guide chromatogram the hands corresponding to N-acetyl neomycins B and C were cut out, eluted with water, and concentrated in vacuo. This concentrate was chromatographed in different solvent systems to determine the homogeneity
after color development by the present method. Antibiotic activity was regenerated by deacetylation of N-acetyl derivatives from the concentrate in a sealed tube by the method of Wagman and Weinstein (14). RESULFS AND DISCUSSION
The paper chromatographic method described was used for the separation and quantitative estimation of free bases of neomycins B and C and neamine (neomycin A). Because commercial samples are mixtures of neomycin salts and fermentation broth contains many interfering substances, it is necessary to pass these solutions over an ion exchange resin. In general, 1 ml of Amberlite IRC-50 was sufficient for complete adsorption of 4 mg of neomycins, and 5 ml of 1N ",OH were required for complete elution. Commonly occurring biological materials like amino sugars and basic amino acids would be adsorbed on this cation exchange resin. Experiments showed incomplete adsorption of histidine that could not be eluted even by 3N NHIOH. Arginine, lysine, and glucosamine were adsorbed completely and eluted with 1N "*OH. Because it would be necessary to dry the eluate from IRC50 before carrying out the N-acetylation reactions according to the method of Rinehart et a[. (7), an attempt was made to simplify the step by N-acetylating the spots of neomycin bases on paper. This was successful only when carbon tetrachloride was used as the main solvent along with methanol. But in the presence of benzene or ether, unknown substances besides the N-acetyl derivative were formed. N-Acetyl neomycins B and C and neamine gave a negative test for ninhydrin reaction arid antibiotic activity. N-Acetylated derivatives were compared with compounds prepared by the method of Rinehart et al. ( 7 ) ; the results in Table I indicate the indentity of two preparations. Because glucosamine, lysine, and arginine might be present in the fermentation broth, their effects on the assay were studied. Although these compounds after acetylation gave a negative ninhydrin test, only glucosamine exhibited a spot with a Rrel value of 1.95 in Ihe solvent 1-butanol-water-piperidine (84 :16 :2 v/v) under the experimental conditions described. Figure 1 shows that N-acetylated neomycins are completely separated by paper chromatography. Table I shows the identity and purity of N-acetyl neomycins B and C prepared from neomycins of fermentation broths. The antibiotic activity of neomycins B and C could be regenerated by deacetylating the N-acetyl derivatives. Table I1 shows that mixtures of neomycins B and C and neamine can be completely resolved and individual components quantitatively determined by tb: present method. Rydon and Smith (15) observed that the chlorination of the acetyl amino group (with at least one free hydrogen attached to acylated nitrogen) rlzsulted in the formation of N-chloro derivatives. Our unpublished data showed that N-chloro derivatives of N-acetyl neomycins were very unstable in diffuse sunlight at room temperature and followed the order: neomycin B > neomycin C > neamine. A 45-pg spot of neomycin B gave a reading equivalent to 30 bg in 0.5 hour under the stated conditions. The standard curve ('3 straight line) for neomycins B and C as obtained by plotting absorbance against concentration was
Table I. Identity and Homogeneity of N-Acetyl Neomycins RI.f values in solvent systems6 C A B N-Acetyl neomycin B, IC 0.69 1.11 1.09 N-Acetyl neomycin B, I1 0.70 1.09 1.07 N-Acetyl neomycin B, 111 0.70 1.10 1.09 N-Acetyl neomycin C, I 0.35 0.73 0.84 N-Acetyl neomycin C, I1 0.35 0.73 0.84 N-Acetyl neomycin C, 111 0.35 0.74 0.85 N-Acetyl neamine, I 1.o 1.0 1.o N-Acetyl neamine I1 0.99 0.98 1.01 Rre1Migration of the substance relative to N-acetyl neamine. * I Systems: A. 1-Butanol-water-piperidine (84: 16 :2 v/v), Whatman No. 4, descending, 34" C., 24-hr. development. B. 1Butanol-water-pyridine (6 :3 :4 v/v). C. Isoamyl alcohol-waterpyridine (1 :0.8:1 v/v); Whatman No. 1, ascending, 28' C. I. Standard preparation by Rinehart's method. 11. Stand preparation by present method. 111. Preparation from neomycins of fermentation broth. Table 11. Determination of the Components of Neomycin in a Mixture NeNeoNeomycin mycin amine, B, Error, C, Error, 2 Error, Sample % % % % % Known mixture I 41.5 3.7 30.8 2.6 31 3.3 I1 92.5 2.8 2.8 6.6 6.7 4.3 Fermentation broth I= 88 12 0 115 17.4 75.6 7 Unichem neomycin sulfate 63.7 36.3 0 a 0.51 sodium nitrate, 1.5 % maltose, mineral mixture (16). 0.88 glutamic acid, 1.5% maltose, mineral mixture (16).
different from that for neamine. This may be due to the fact that there are six NH2groups in each molecule of neomycins B and C (mol wt 614) and four NH2 groups in the neamine molecule (322) and so the percentage of chlorine in the N-chloro derivative of N-acetyl neamine is comparatively higher. Theoretically, 2.56 pg of neomycin B or C will be equivalent to 2 01 pg of neamine. Calculation from the calibration curve shows 2.56 pg of neomycin B or C to be equivalent to 1.93 to 1.96 pg of neamine, which is very near to theoretical. So, all three neomycins could be determined by reference to a single standard curve. The error by this 4 z . As little method with 4 to 12 pg is of the order of as 1 fig of neomycin base can be estimated with an accuracy of =k7Z and a 60-pg sample can be analyzed if the minor component is very low. This method might be applicable to the analysis of amino sugars and amino sugar antibiotic complexes like catenulins, kanamycins, paromomycins, etc., using different solvent systems where necessary.
*
ACKNOWLEDGMENT
Thanks are due to the Director, Unichem Laboratories, Bombay, India, for the supply of neomycin sulfate and to B. K. Bhuyan of the Upjohn Co., Kalamazoo, Mich., for the supply of neomycins B and C.
(14) G . H. Wagman and M. J. Weinstein, J. Med. Chem., 7, 800 (1 964).
(15) H. N. Rydon and P. W. G. Smith, Nature. 169. 922 (1952). (16) M. K. Majumdar and S. K. Majumdar, Appl..Microbiol.; 13, 190 (1 965).
Received for review August 15, 1966. Accepted October 19, 1966. Work supported in part by a Council of Scientific and Industrial Research Fellowship (M.K.M.) VOL. 39, NO. 2, FEBRUARY 1967
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