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Another important finding in this work is that the gradient elution must be very fast if the separation starts from around 20-30% of acetonitrile with 1% of formic acid. Otherwise, broad and tailing peaks were normally obtained, which may be due to sample solubility and ionic interaction issues. We found 50% mobile phase A as a starting condition gave best peak shape and chromatographic resolution.
Conclusion
Aminoglycosides exhibited good retention and separation efficiency on a zwitterionic HILIC stationary phase. The separation is based on a mixed mode separation mechanism depending on both hydrophilic partitioning and electrostatic interaction. The addition of formic acid and high buffer concentration in the mobile phases, in combination with fast gradient elution, were key factors influencing the chromatographic performance. Mass spectrometry had excellent compatibility with these HILIC separations and provided good detection sensitivity.
References 1. Wikipedia: Aminoglycoside,
http://en.wikipedia.org/wiki/Aminoglycosides.
2. D.A. Stead, J. Chromatogr. B, 747 (2000) 69-93. 3. T.A. McGlinchey, P.A. Rafter, F. Regan, and G.P. McMahon, Anal. Chim. Acta, 624 (2008) 1-15.
4. S. He, Q.Y. Chen, Y. Sun, Y.C. Zhu, L.X. Luo, J.Q. Li, and Y.S. Cao, J. Chromatogr. B, 879 (2011) 901-907.
5. X.M. Li, Y.Q. Chen, S.S. Tang, J. HK. He, Y.H. Shang, and X.L. Xiao, J. Agric. Food Chem., 57 (2009) 7356-7362.
Figure 3: Chromatograms of the three aminoglycosides (5 µg/mL) analysed with a zwitterionic HILIC stationary phase and ESI- MS detection.
especially at sample concentration below 5 µg/mL. The peak shape of all three model aminoglycosides became better when much faster gradient elution, from 5% B to 95% B within one minute, was used. However, the resolution between neomycin and apramycin/kanamycin tended to be poorer. After investigation with a combination of different mobile phase and gradient profiles, best separation was obtained with acetonitrile plus 1 w% formic acid as mobile phase A and 150 mM ammonium acetate plus 1.5 w% formic acid as mobile phase B and a gradient starting from 50/50 A and B. Although many attempts were undertaken in order to reduce the ammonium acetate in mobile phase B and formic acid concentration in both A and B, the conditions described above still gave best
results. Figure 4 shows chromatograms of the separation of the three aminoglycosides under optimal conditions as described in the experimental section.
Practical Aspects
Since the aminoglycosides have strong electrostatic interaction with the zwitterionic stationary phases, it is mandatory to use relatively high concentrations of ammonium acetate and formic acid to achieve good separation efficiency and peak shape. Formic acid may act as a weak ion-pair reagent with the amino groups to reduce the electrostatic interaction between analytes and stationary phase. This is especially important for the analysis of aminoglycosides at lower sample concentration.
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W.Kirch, J. Chromatogr. A, 1058 (2004) 197-201.
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14. A. Shen, L. Morgan, M.L. Barroso, X. Zhang, and T. Nguyen, Application Note form Tandem Labs,
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15.Y. Du, H.Y. Yang, W.D. Xu, Chin. J. Antibiot., 34 (2009) 669-677.
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