May, 19661 SHORT PAPERS 333 SHORT PAPERS The Assay of Neomycin BY R. A. HOODLESS (Ministry of Technology, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, S.E. 1) COMMERCIAL neomycin consists of true neomycins, which are glycosides yielding ribose on hydrolysis, with some admixture of the relatively inactive neamine, which yields no ribose. Neomycin is normally assayed microbiologically. However, various methods have been described for the chemical determination.l~~*~ Most of these are based on the fact that when neomJ-cins are heated with a strong mineral acid, furfural is formed from the ribose as one of the decomposition products. method has been developed which is less time consuming than the other published methods and yet is of the same order of reproducibility.The determination is based on the reaction of ribose with phloroglucinol in a mixture of concentrated hydrochloric acid and glacial acetic acid.4 Optimum conditions for the determination of neomycin are found in heating a solution of neomycin with a mixture of 110 ml of glacial acetic acid, 4 ml of concentrated hydrochloric acid, 1 ml of a 0.8 per cent. aqueous solution of glucose and 5 ml of a 5 per cent. alcoholic solution of phloroglucinol in a boiling water-bath for 15 minutes, and measuring the extinction a t 552 mp. If a higher concentration of hydrochloric acid is used the blank becomes much darker. TYhen neomycin is heated with glacial acetic acid and concentrated hydrochloric acid in the ratio used in the reagent, the maximum amount of furfural is formed after heating for 45 minutes at 100" C.Therefore it seemed unlikely that the determination depended on the formation of furfural. This is confirmed by the fact that furfural does not react with the reagent. Hydrolysis of neomycin with 1.5 N hydrochloric acid and determination of the released ribose with the phloroglucinol reagent gave very erratic results for the ribose content. Hence it is more satisfactory to carry out the hydrolysis and development of the colour with phloroglucinol simultaneously. 5-Hydrosymethylfurfural has been used as a qualitative test for sugars, and it was thought that this might be used for determining neomycin. However, although ribose reacted with 5-hydrosymethylfurfural under the conditions outlined by Love5 to give a straight line relation- ship between extinction and concentration of ribose, neomycin did not react with 5-hydroxy- methylfurfural.METHOD REAGEXTS- Glacial acetic acid, mzalvtical-veagrnt gyacle, fvactionallv distilled. Coircentvated h?ldrorhlovic acid, analytical-reagent gvade, sP.gv. 1- 18. Glucose solittion-Prepare a 0.S per cent. w/v solution in distilled water. Phloitoglzrcinol solution-Prepare a 5 per cent. w/v solution in 95 per cent. alcohol just before use. Phlovoglztcznol reagent-bIix together in a stoppered flask 110 ml of glacial acetic acid, 4 ml of concentrated hydrochloric acid, 1 ml of glucose solution and 5 ml of phloroglucinol solution. This solution must be freshly prepared. A-eomycin standavd-The International Reference Preparation for Neomycin obtained from the National Institute for Medical Research was used.1 mg of this preparation is equivalent to 680 international units. Scorn-ycin samples-These were dried over phosphorus pentoxide in a vacuum desiccator for 24 hours. PROCEDURE- Transfer by pipette 1 ml of test solution containing about 200 pg of neomycin sulphate per ml into a glass-stoppered test-tube. Add 10 ml of the phloroglucinol reagent, mix the solutions, and place the tube in a boiling water-bath with the water level above the liquid level in the tube,334 SHORT PAPERS [A~zal.t)st, Vol. 91 for 15 minutes. against a blank prepared with water instead of test solution. sulphate, and carry them through the procedure a t tlie same time as the sample solutions. After this time cool tlie tube and immediately measure the extinction a t 552 mp Prepare a series of standard solutions containing between 80 and 400pg per ml of neomj-cin Determine the amount of neomycin sulphate in the samples by referring to the standard curve.RESULTS The results obtained from separate assays on samples of neomjcin sulphate pouder are shown in Table I. Thcse are compared with the results obtained by using the methods of Korchagin TABLE I INTERNATIONAL USITS OF SEOMYCIS PER mg OF SAMPLE 2 3 4 Sample Proposcd method 1 65 1 655 665 687 662 648 694 687 653 662 656 637 699 708 694 695 714 707 655 668 658 638 674 637 Method of Korchagin et al. 64 1 622 683 652 645 694 7 00 710 703 689 71 1 682 707 700 699 650 648 659 628 64 1 - - - - Method of Brooks, Forist and Loehr 652 734 662 642 655 829 717 637 - 856 704 683 668 802 661 805 728 - et al., and Brooks, Forist and Loehr on the same solutions.Both the latter methods take longer to carry out owing to the greater hydrolysis time required and the need to make up to a known \-olume after hydrolysis. The over-all time required to carry out an assay by the proposed method is about 1+ hours, as compared with about 2 hours by the method of Korchagin et al., and about 3 hours by the method of Brooks, Forist and T,oehr. The method as proposed is not specific for neomycin because other amino sugar antibiotics yielding ribose on hydrolysis, e.g., paramomycin, will also react with the phloroglucinol reagent. The author thanks thc Govcrnment Chemist for permission to publish this paper. REFERENCES 1. 3. 3. 4. 5. Brooks, A. X., Forist, A. A., Loehr, B. F., Analyt. Chwz., 1936, 28, 1788. Korchagin, I-. R., Iiorobitskaya, A. A,, Uruzhinina, E. N., and Semenor, S. M I Aiztibioliki, 1962, Foppiano, R., Brown, B. B., J . Pharm. Scz., 1965, 54, ( 2 ) , 206. Dische, Z., Borenfreund, E., Baochim. Biophys. Ada, 1957, 23, (3), 639. Love, R. M., Analyst, 1953, 78, 733. 7, (2), 124. Received July 1 2 h , 1965