Amikacin, a broad spectrum aminoglycoside antibiotic derived from kanamycin A, is commonly used for treating severe, hospital – acquired infections caused by Gram-negative bacteria. The analysis of this molecule has always been challenging because it does not contain a UV chromophore.

Fig. 1. HPLC stacked plot (top): Detector response as a function of retention time overlay of the 0.05, 0.1, 0.2, 0.5, 0.8, and 1 mg/mL of amikacin peak. Gradient conditions: 0-1 min (5% B), 4.5 min (95% B), 4.51-7.00 (5% B). F = 1.5 mL/min. T = 55oC. Vinj = 3 µL. 3D view of Amikacin (bottom).

A robust HPLC method for amikacin detection and aerosol particle size distribution (aPSD) determination, with performance comparable to conventional UV absorbance detection, was developed using a charged aerosol detector (CAD). HPLC-CAD chromatograms are shown in Figure 1. To select the optimal power function value (PFV) to linearize the signal, the slope and relative standard deviation (S_rel) of the response factor as a function of concentration were evaluated across a range of PFVs (Fig. 2).

Fig. 2. Change in absolute value of the slope of response factor versus concentration plots and in Srel of the response factors as a function of PFV. The numbers reported in boxes are the correlation coefficients of the area versus concentration at each power function value. Chromatography conditions: 0 min (5% B), 8 min (40% B), 9 min (90% B), 9.1-13 (5% B). F = 1.0 mL/min. T = 55°C.

The HPLC system dwell volume inclusion in the gradient program setup reduced the variability in retention time, peak width, signal-to-noise, and resolution between amikacin and kanamycin. The incorporation of a 1.0 min isocratic hold of the initial condition allowed the method to be run on HPLC systems with 2.65 mL dwell volumes or less.

During method optimization, a power function value was carefully chosen to yield the best linearity by targeting three requirements: 1) minimum variability in response factor over concentration range (i.e., lowest S_rel), 2) slope of response factor versus concentration as close to zero as possible, and 3) correlation coefficient of area versus concentration close to unity. Figure 2 illustrates the optimum value of the PFV in the range of 1.28 – 1.35. The influence of mobile phase grade and glassware binding of amikacin during sample preparation were also addressed. A weighed (1/X²) least square regression was used for the calibration curve. The limit of quantitation (LOQ) and limit of detection (LOD) for this method were determined as 5 µg/mL and 2 µg/mL, respectively. The method was validated over a concentration range of 0.05 to 2 mg/mL. The correlation coefficient for the peak area versus concentration was 1.00 and the y-intercept was 0.2%. The recovery accuracies of triplicate preparations at 0.05, 1.0, and 2.0 mg/mL were in the range of 100 – 101%. The S_rel of six replicates at 1.0 mg/mL was 1%, and S_rel of five injections at the limit of quantitation was 4%. The method is able to support routine throughput of 200 samples per day.

Andrei Blasko, Imad A. Haidar Ahmad, James Tam, Nani Kadrichu
Novartis Pharmaceuticals Corporation, San Carlos, CA, USA

Publication

A simplified guide for charged aerosol detection of non-chromophoric compounds-Analytical method development and validation for the HPLC assay of aerosol particle size distribution for amikacin.
Soliven A, Haidar Ahmad IA, Tam J, Kadrichu N, Challoner P, Markovich R, Blasko A
J Pharm Biomed Anal. 2017 Sep 5

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