, Volume 76, Issue 21–22, pp 1505–1512 | Cite as

Assay of Kanamycin A by HPLC with Direct UV Detection

  • B. Blanchaert
  • E. Poderós Jorge
  • P. Jankovics
  • E. Adams
  • Ann Van SchepdaelEmail author


The development of a simple reversed phase ion pair liquid chromatographic method for the assay of kanamycin A has been described. Because of the lack of a UV chromophore in the structure of kanamycin A, borate complexation was used to allow direct UV detection at 205 nm. Three columns were evaluated in this study: Zorbax Extend C18 (4.6 mm × 250 mm; 5 μm), XBridge C18 (4.6 mm × 250 mm; 5 μm) and apHera C18 (4.6 mm × 250 mm; 5 μm). The mobile phase was a mixture of 0.1 M disodium tetraborate (pH 9.0) and water (20:80, v/v) supplemented with 0.5 g L−1 sodium octanesulphonate. Final chromatographic conditions were achieved on the XBridge column at 50 °C. The method was validated according to ICH guidelines and applied to a commercially available sample. It is much faster and more specific than the current microbiological assay prescribed in the European Pharmacopoeia. No expensive equipment is necessary to perform this assay making it a viable replacement.


HPLC–UV Ion pair liquid chromatography Borate complexation Kanamycin 



This paper was supported by the TÁMOP 4.2.1 B-09/1/KMR tender of the Hungarian Government (P. Jankovics).


  1. 1.
    Hermann T (2007) Aminoglycoside antibiotics: old drugs and new therapeutic approaches. Cell Mol Life Sci 64:1841–1852CrossRefGoogle Scholar
  2. 2.
    Zembower T, Noskin G, Postelnick MJ, Nguyen C, Peterson LR (1998) The utility of aminoglycosides in an era of emerging drug resistance. Int J Antimicrob Ag 10:95–105CrossRefGoogle Scholar
  3. 3.
    Durante-Mangoni E, Grammatikos A, Utili R, Falagas ME (2009) Do we still need the aminoglycosides? Int J Antimicrob Ag 33:201–205CrossRefGoogle Scholar
  4. 4.
    Li H, Steyger P (2009) Synergistic ototoxicity due to noise exposure and aminoglycoside antibiotics. Noise Health 11:26–32CrossRefGoogle Scholar
  5. 5.
    Tzovaras V, Tsimihodimos V, Kostara C, Mitrogianni Z, Elisaf M (2011) Aminoglycoside-induced nephrotoxicity studied by proton magnetic resonance spectroscopy of urine. Nephrol Dial Transplant 26:3219–3224CrossRefGoogle Scholar
  6. 6.
    Umezawa H, Ueda M, Maeda K, Yagashita K, Kondo S, Okami Y, Utahara R, Osato Y, Nitta K, Takeuchi T (1957) Production and isolation of a new antibiotic. Kanamycin. J Antibiot 10(5):181–188Google Scholar
  7. 7.
    Gikalo MB, Nosova EY, Krylova LY, Moroz AM (2012) The role of eis mutations in the development of kanamycin resistance in Mycobacterium tuberculosis isolates from the Moscow region. J Antimicrob Chemother 67:2107–2109CrossRefGoogle Scholar
  8. 8.
    Maneke E, Pridmore A, Goby L, Lang I (2010) Kill rate of mastitis pathogens by a combination of cefalexin and kanamycin. J Appl Microbiol 110:184–190CrossRefGoogle Scholar
  9. 9.
    Ishii R, Horie M, Chan W, MacNeil J (2008) Multi-residue quantitation of aminoglycoside antibiotics in kidney and meat by liquid chromatography with tandem mass spectrometry. Food Addit Contam Part A-Chem 25:1509–1519CrossRefGoogle Scholar
  10. 10.
    Vakulenko SB, Mobashery S (2003) Versatility of aminoglycosides and prospects for their future. Clin Microbiol Rev 16:430–450CrossRefGoogle Scholar
  11. 11.
    Rothrock JW, Guegelman RT, Wolf J (1958) A resin chromatographic analysis for kanamycin mixtures. Antibiot Annu 6:796–803Google Scholar
  12. 12.
    Claes P, Vanderhaeghe H, Compernolle F (1973) Isolation and identification of minor components of commercial kanamycin. Antimicrob Agents Chemother 4:560–563CrossRefGoogle Scholar
  13. 13.
    Manyanga V, Dhulipalla RL, Hoogmartens J, Adams E (2010) Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of kanamycin. J Chromatogr A 1217:3748–3753CrossRefGoogle Scholar
  14. 14.
    Stead DA (2000) Current methodologies for the analysis of aminoglycosides. J Chromatogr B Biomed Sci Appl 747:69–93CrossRefGoogle Scholar
  15. 15.
    El-Attug MN, Adams E, Hoogmartens J, Van Schepdael A (2011) Capacitively coupled contactless conductivity detection as an alternative detection mode in CE for the analysis of kanamycin sulphate and its related substances. J Sep Sci 34:2448–2454CrossRefGoogle Scholar
  16. 16.
    Kumar P, Rubies A, Companyó R, Centrich F (2011) Hydrophilic interaction chromatography for the analysis of aminoglycosides. J Sep Sci 35:498–504CrossRefGoogle Scholar
  17. 17.
    Flurer CL (1995) The analysis of aminoglycoside antibiotics by capillary electrophoresis. J Pharm Biomed Anal 13:809–816CrossRefGoogle Scholar
  18. 18.
    El Rassi Z (2002) Carbohydrate analysis by modern chromatography and electrophoresis. Elsevier, AmsterdamGoogle Scholar
  19. 19.
    De Muynck C, Beauprez J, Soetaert W, Vandamme EJ (2006) Boric acid as a mobile phase additive for high performance liquid chromatography separation of ribose, arabinose and ribulose. J Chromatogr A 1101:115–121CrossRefGoogle Scholar
  20. 20.
    European Pharmacopoeia (2012) 7th edn. The Council of Europe, Strasbourg Cedex, France, pp 2313–2314Google Scholar
  21. 21.
    Fabre H (1996) Robustness testing in liquid chromatography and capillary electrophoresis. J Pharm Biomed Anal 14:1125–1132CrossRefGoogle Scholar
  22. 22.
    Adams E, Dalle J, De Bie E, De Smedt I, Roets E, Hoogmartens J (1997) Analysis of kanamycin sulfate by liquid chromatography with pulsed electrochemical detection. J Chromatogr A 766:133–139CrossRefGoogle Scholar
  23. 23.
    Kaale E, Van Schepdael A, Roets E, Hoogmartens J (2001) Development and validation of a simple capillary zone electrophoresis method for the analysis of kanamycin sulfate with UV detection after pre-capillary derivatization. J Chromatogr A 924:451–458CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • B. Blanchaert
    • 1
  • E. Poderós Jorge
    • 1
  • P. Jankovics
    • 2
    • 3
  • E. Adams
    • 1
  • Ann Van Schepdael
    • 1
    Email author
  1. 1.Laboratory for Pharmaceutical AnalysisKU LeuvenLeuvenBelgium
  2. 2.Department of Pharmaceutical ChemistrySemmelweis UniversityBudapestHungary
  3. 3.National Institute of PharmacyBudapestHungary

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