Sub Topic | Secondary Topic: Analytical Methodology | HPLC (Biopharmaceutical Molecule)
Authors: Katrina Chan, University of Houston (Main Author, Presenting Author); Jian Zhou, University of Houston; Weiqun Wang, University of Houston; Vincent Tam, University of Houston
Presenting Author: Katrina Chan
Purpose: Amikacin, a semi-synthetic aminoglycoside derived from kanamycin, is used to treat several types of serious Gram-negative bacterial infections such as pneumonia and urinary tract infections. However, pharmacological analysis of amikacin and other aminoglycosides present multiple challenges with developing a robust method for LCMS quantification of these compounds. Amikacin cannot be detected using an ultraviolet detector, as is favored among LCMS methods in recent research, and its high polarity results in short retention time on conventional reverse-phased columns.
Methods: To improve retention time, a very basic pH of 11.0-11.2 was found to work best. For the aqueous mobile phase, LCMS-grade water and HPLC-grade ammonium hydroxide (10%) were mixed to achieve a final concentration of 20 mL 10% NH4OH / 500 mL of H20. Similarly, for the organic mobile phase, a final concentration of 20 mL 10% NH4OH / 500 mL of acetonitrile was used. Tobramycin, another aminoglycoside, was used as the internal standard, at a final concentration of 2 &[micro]g/ &[micro]L amikacin sample. The method was conducted in positive mode electrospray. The method was developed on an AB Sciex QTrap 5500 LCMS machine with the column maintained at 50o C. The column used is an Acquity UPLC BEH C18 1.7 &[micro]m, 2.1 x 150 mm column.
Results: The parent to daughter ion pairs for amikacin were m/z 586.1 &[rarr]63.4 and m/z 586.1 &[rarr]425.4 with tobramycin at m/z 486.4 &[rarr]163.3 The best results were found under gradient conditions using flow rate of 0.25 mL / minute, with a total running time of 6 minutes per sample. The linear calibration curve has been obtained for the range of 2 ng/mL to 4000 ng/mL. Both amikacin and the internal standard tobramycin showed good retention times and symmetric peak shapes.
Conclusion: This method allows for the simple and time-efficient quantitative analysis of amikacin. This will be used for in vitro and in vivo toxicology studies.
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