Optimization and Comparisons for Separation, Detection and Quantification of 12 Aminoglycosides Using 2 Chromatographic Conditions by LC-MS/MS


Aminoglycosides are a family of antibiotics with important applications in veterinary medicine. Their ionic character, the similarity structures and the high polarity due to the presence of two or more amino and hydroxyl groups cause a difficulty in separation and make these compounds poorly retained on the reversed phase column. An analytical method for the separation and detection of 12 aminoglycosides has been optimized using two kinds of chromatographic conditions (HILIC, Ion pairing). In Hydrophilic Interaction, ZIC_HILIC column was used, by which the following parameters for the mobile phase were evaluated: concentration of ammonium acetate buffer, percentage of formic acid and effect of acid type. The maximum and adequate concentration of ammonium acetate for the majority of analytes was set to 30 mM. The percentage 0.1% of formic acid increases the response for the majority of analytes. On the other side, the use of 0.1% of trifluoroacetic acid improves the response when compared with the response obtained with 0.1% of formic acid except for Spectinomycin Dihydrostreptomycin and Streptomycin. For ion pairing chromatography, the concentration of pentafluoropropionic acid was tested and the greatest value appeared to be 9.2 mM. Therefore, the comparison between the two separation methods shows that the response area of the majority of analytes tested increases when using the ion pair mode. Also, the high value of S/N and the lower detection limit (5 - 15 μg m·L﹣1 for most aminoglycosides studied make the ion pairing method more preferable than HILIC interaction.

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Mokh, S. , Jaber, F. , Kouzayha, A. , Budzinski, H. and Al Iskandarani, M. (2014) Optimization and Comparisons for Separation, Detection and Quantification of 12 Aminoglycosides Using 2 Chromatographic Conditions by LC-MS/MS. American Journal of Analytical Chemistry, 5, 982-994. doi: 10.4236/ajac.2014.514105.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] McGlinchey, T., Rafter, P., Regan, F. and McMahon, G.A. (2008) Review of Analytical Methods for the Determination of Aminoglycoside and Macrolide Residues in Food Matrices. Analytica Chimica Acta, 624, 1-15.
[2] Stevens, R.C. and Rodman, J.H. (1998) Pharmacokinetics of Antimicrobial Therapy. Seminars in Pediatric Infectious Diseases, 9, 273-280.
[3] Turnipseed, S.B. and Long, A.R. (1998) Analytical Procedures for Drug Residues in Food of Animal Origin. Science Technology System.
[4] Shulman, E., Belakhov, V., Wei, G., Kendall, A., Meyron-Holtz, G., Ben-Shachar, D., Schacht, J. and Baasov, T. (2013) Designer Aminoglycosides That Selectively Inhibit Cytoplasmic rather than Mitochondrial Ribosomes Show Decreased Ototoxicity: A Strategy for the Treatment of Genetic Diseases. The Journal of Biological Chemistry.
[5] Zhu, W., Yang, J., Wei, W., Liu, Y. and Zhang, S. (2008) Simultaneous Determination of 13 Aminoglycoside Residues in Foods of Animal Origin by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry with Two Consecutive Solid-Phase Extraction Steps. Journal of Chromatography A, 1207, 29-37.
[6] Commission Decision 2002/657/EC (2002) Performance of Analytical Methods. Official Journal of European Commission.
[7] Oertel, R., Renner, U. and Kirch, W. (2004) Determination of Neomycin by LC-Tandem Mass Spectrometry Using Hydrophilic Interaction Chromatography. Journal of Pharmaceutical and Biomedical Analysis, 35, 633-638.
[8] Megoulas, N. and Koupparis, M. (2005) Development and Validation of a Novel HPLC/ELSD Method for the Direct Determination of Tobramycin in Pharmaceuticals, Plasma and Urine. Analytical and Bioanalytical Chemistry, 382, 290-296.
[9] Garcia-Mayor, M., Garcinuno, M., Fernandez-Hernando, P. and Durand-Alegria, J. (2006) Liquid Chromatography-UV Diode-Array Detection Method for Multi-Residue Determination of Macrolide Antibiotics in Sheep’s Milk. Journal of Chromatography A, 1122, 76-83.
[10] Serrano, M. and Silva, M. (2006) Determination of Amikacin in Body Fluid by High-Performance Liquid-Chromatography with Chemiluminescence Detection. Journal of Chromatography B, 843, 20-24.
[11] Abuin, S., Codony, R., Compano, R., Granados, M. and Prat, M.D. (2006) Analysis of Macrolide Antibiotics in River Water by Solid-Phase Extraction and Liquid Chromatography-Mass Spectrometry. Journal of Chromatography A, 1114, 73-81.
[12] Preu, M., Guyot, D. and Petz, M. (1998) Development of a Gas Chromatography-Mass Spectrometry Method for the Analysis of Aminoglycoside Antibiotics Using Experimental Design for the Optimisation of the Derivatisation Reactions. Journal of Chromatography A, 818, 95-108.
[13] Clarot, I., Chaimbault, P., Hasdenteufel, F., Netter, P. and Nicolas, A. (2004) Determination of Gentamicin Sulfate and Related Compounds by High-Performance Liquid Chromatography with Evaporative Light Scattering Detection. Journal of Chromatography A, 1031, 281-287.
[14] Tawa, R., Matsunaga, H. and Fujimoto, T. (1998) High-Performance Liquid Chromatographic Analysis of Aminoglycoside Antibiotics. Journal of Chromatography A, 812, 141-150.
[15] Posyniak, A., Zmudzki, J. and Niedzielska, J. (2001) Sample Preparation for Residue Determination of Gentamicin and Neomycin by Liquid Chromatography. Journal of Chromatography A, 914, 59-66.
[16] Kim, B., Otsuka, K. and Terabe, S. (2001) Anion Selective Exhaustive Injection-Sweep-Micellar Electrokinetic Chromatography. Journal of Chromatography A, 932, 129-137.
[17] McGlinchey, T. (2010) The Determination of Veterinary Antibiotics in Live Animals and Animal Products. School of Chemical Sciences Dublin City University, Dublin.
[18] Cai, Y.Q., Cai, Y.E., Mou, S.F. and Lu, Y.Q. (2005) Multi-Walled Carbon Nanotubes as a Solid-Phase Extraction Adsorbent for the Determination of Chlorophenols in Environmental Water Samples. Journal of Chromatography A, 1081, 245-247.
[19] Kato, Y., Iwamoto, M., Koike, T., Suzuki, F. and Takano, Y. (1988) Terminal Differentiation and Calcification in Rabbit Chondrocyte Cultures Grown in Centrifuge Tubes: Regulation by Transforming Growth Factor Beta and Serum Factors. Proceedings of the National Academy of Sciences of the United States of America, 85, 9552-9556.
[20] Sakairi, M. and Kambara, H. (1988) Characteristics of a Liquid Chromatography/Atmospheric Pressure Ionization Mass Spectrometer. Analytical Chemistry, 60, 774-780.
[21] Tao, Y., Chen, D., Yu, H., Huang, L., Liu, Z., Cao, X., Yan, C., Pan, Y., Liu, Z. and Yuan, Z. (2012) Simultaneous Determination of 15 Aminoglycoside(s) Residues in Animal Derived Foods by Automated Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry. Food Chemistry, 135, 676-683.
[22] Plozza, T., Trenerry, V., Zeglinski, P., Nguyen, H. and Johnstone, P. (2011) The Confirmation and Quantification of Selected Aminoglycoside Residues in Animal Tissue and Bovine Milk by Liquid Chromatography Tandem Mass Spectrometry. International Food Research Journal, 18, 1077-1084.
[23] Loffler, D. and Ternes, T. (2003) Analytical Method for the Determination of the Aminoglycoside Gentamicin in Hospital Wastewater via Liquid Chromatography—Electrospray-Tandem Mass Spectrometry. Journal of Chromatography A, 1000, 583-588.
[24] McLaughlin, L.G. and Henion, I.D. (1994) Multi Residue Confirmation of Aminoglycosides Antibiotics in Bovine by Ion Spray High Performance Liquid Chromatography/Tandem Mass Spectrometry. Biological Mass Spectrometry, 23, 417-429.
[25] (2008) A Practical Guide to HILIC. Se Quant, Sweden.
[26] Ikegami, T., Tomomatsu, K., Takubo, H., Horie, K. and Tanaka, N. (2008) Separation Efficiencies in Hydrophilic Interaction Chromatography. Journal of Chromatography A, 1184, 474-503.
[27] Garbis, S.D., Melse-Boonstra, A., West, C.E. and van Breemen, R.B. (2001) Determination of Folates in Human Plasma Using Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry. Analytical Chemistry, 73, 5358-5364.
[28] Alpert, A.J., Shukla, M., Shukla, A.K., Zieske, L.R., Yuen, S.W., Ferguson, M.A., Mehlert, A., Pauly, M. and Orlando, R. (1994) Hydrophilic-Interaction Chromatography of Complex Carbohydrates. Journal of Chromatography A, 676, 191-202.
[29] Oyler, A.R., Armstrong, B.L., Cha, J.Y., Zhou, M.X., Yang, Q., Robinson, R.I., Dunphy, R. and Burinsky, D.J. (1996) Hydrophilic-Interaction Chromatography on Amino-Silica Phases Complements Reversed-Phase High Performance Liquid Chromatography and Capillary Electrophoresis for Peptide Analysis. Journal of Chromatography A, 724, 378-383.
[30] Strege, M.A. (1998) Hydrophilic Interaction Chromatography-Electrospray Mass Spectrometry Analysis of Polar Compounds for Natural Product Drug Discovery. Analytical Chemistry, 70, 2439-2445.
[31] Oertel, R., Neumeister, V. and Kirch, W. (2004) Hydrophilic Interaction Chromatography Combined with Tandem-Mass Spectrometry to Determine Six Aminoglycosides in Serum. Journal of Chromatography A, 1058, 197-201.
[32] Chiaochan, C., Koesukwiwat, U., Yudthavorasit, S. and Leepipatpiboon, N. (2010) Efficient Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry for the Multiclass Analysis of Veterinary Drugs in Chicken Muscle. Analytica Chimica Acta, 682, 117-129.
[33] Hammel, Y.A., Mohamed, R., Gremaud, E., LeBreton, M. and Guy, P. (2008) Multi-Screening Approach to Monitor and Quantify 42 Antibiotic Residues in Honey by Liquid Chromatography-Tandem Mass Spectrometry. Journal of Chromatography A, 1177, 58-76.
[34] Confirmation of Aminoglycosides by HPLC-MS/MS (2011) Department of Agriculture Food Safety and Inspection Service Office of Public Health Science, USA. http://www.fsis.usda.gov/wps/wcm/
[35] De Miguel, I., Puech-Costes, E. and Samain, D. (1987) Use of Mixed Perfluorinated Ion Pairing Agents as Solvents in Ion Pair High Performance Liquid Chromatography for the Preparative Purification of Aminoglycoside Antibiotics. Journal of Chromatography A, 407, 109-119.
[36] Gustavsson, S., Samskog, J., Markides, K. and Langstrom, B. (2001) Studies of Signal Suppression in Liquid Chromatography-Electrospray Ionization Mass Spectrometry Using Volatile Ion-Pairing Reagents. Journal of Chromatography A, 937, 41-47.
[37] Kaufmann, A., Butcher, P. and Maden, K. (2011) Determination of Aminoglycoside Residues by LC-MS/MS in a Variety of Matrices. Analytica Chimica Acta, 711, 46-53.
[38] Ishii, R., Horie, M., Chan, W. and MacNeil, J. (2008) Multi-Residue Quantitation of Aminoglycoside Antibiotics in Kidney and Meat by Liquid Chromatography with Tandem Mass Spectrometry. Food Additives & Contaminants, 25, 1509-1519.

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