Analysis of Amikacin in Human Serum By UHPLC With Fluorescence Detector Using Chloro-Formate Reagent With Glycine

Abstract

Background: Amikacin belongs to aminoglycosides family, commonly administered in the treatment of systemic infections due to gram negative bacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicity and ototoxicity. Objective: Optimize an ultra-high performance liquid chromatography (UHPLC) based analytical method for the determination of amikacin sulfate in human serum using derivatizaon with FMOC-Cl and glycine. Methods: Pre-column derivatization reaction of amikacin performed using fluorescence reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) at ambient temperature in the presence of borate buffer (0.2 M). Stabilizing reagent glycine (0.1 M) added into the reaction mixture solution after completion of the derivatization reaction for stabilization of fluorescent complex product. Fluorimetric detection of amikacin was performed at excitation and emission wavelength of 265 nm and 315 nm respectively, using C18 UHPLC column. The reported method was validated by performing linearity, precision, recovery and ruggedness. Results: The optimum mobile phase composition was found to be Acetonitrile:water in the ratio of 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin in serum samples ranging from 0.5-10 µg/ml was obtained, with correlation coefficient of 1.00. The limit of detection (LOD) was found to be 50 ng/ml. Both inter-and intra-day analysis coefficient values were found to be less than 10%. Conclusion: The developed UHPLC method will be useful for pre-clinical and pharmacokinetic study of amikacin in human serum.

Bioanalysis of aminoglycosides using high-performance liquid chromatography

“…Solvents that were used for PPT included acetonitrile for the extraction of netilmicin, amikacin, tobramycin, gentamicin, etimicin, neomycin, and isepamicin [6,15,21,23,32,[37][38][39][40]42,68,[73][74][75][76]78,79,82,86]; trichloroacetic acid for the extraction of geneticin, spectinomycin, tobramycin, gentamicin, kanamycin, hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin, neomycin, and paromomycin [29,34,35,57,61,72,81]; methanol for the extraction of geneticin, amikacin, tobramycin, sisomicin, and netilmicin [29,41,48]; perchloric acid for the extraction of kanamycin, dibekacin, arbekacin, and streptomycin [26,33,49,51]; formic acid for the extraction of amikacin [84,85]; and methylene chloride for the extraction of isepamicin [62]. PPT aids in reducing interference during derivatization [7], and this may have accounted for its use by 41 articles.…”

“…In this review, columns were kept at temperatures ranging from 22 °C to as high as 100 °C [41]. Overall, the most widely used column temperature was 25 °C for the separation of netilmicin, amikacin, tobramycin, gentamicin, dibekacin, sisomicin, isepamicin, geneticin, streptomycin, dihydrostreptomycin, and neomycin [6,24,[28][29][30]42,61,64,68,73]; followed by 30 °C for the separation of neomycin, streptomycin, dihydrostreptomycin, amikacin, kanamycin, paromomycin, tobramycin, spectinomycin, apramycin, hygromycin, gentamicin, netilmicin, etimicin, and isepamicin [5,16,32,72,78,81,82]; and 50 °C for the separation of sisomicin, netilmicin, astromicin, micronomicin, streptomycin, gentamicin, tobramycin, and amikacin [25,27,43,46,47,50].…”

“…4-sulfonate for the derivatization of streptomycin [49]; ninhydrin for the derivatization of streptomycin [51]; and 9-fluorenylmethylchloroformate for the derivatization of gentamicin, neomycin, netilmicin, sisomicin, isepamicin, and amikacin [16,60,63,78,82] were other derivatizing agents used in the bioanalysis of aminoglycosides.…”

“…therapeutic reference range [14,15,24,25,30,31,34,37,48,55,57,61,65,67,71,74,75,[80][81][82], which invalidates the measurements obtained from the bioanalysis.…”

Analytical Methods for the Determination of Aminoglycosides Antibiotics by Chromatrographic Technique

“…Chromatographic condition was achieved by using mobile phase composed of Acetonitrile: water in the ratio 70:30 (v/v) and Shim-Pack XR-ODS III, Shimadzu) C18 column. Fluorimetric detection at excitation and emission wavelength of 265 nm and 315 nm, respectively was used for the proposed chromatographic method [28].…”