Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC—Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma

Abstract

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C18 column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min−1. A fluorescence detector (λex 390 and λem 480 nm) was used. The method was linear over the concentration range 20–200 ng mL−1. The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference.

Purity determination of amphotericin B, colistin sulfate and tobramycin sulfate in a hydrophilic suspension by HPLC

“…Analytical methods for this substance are described in both the EP [12] and United States Pharmacopoeia (USP) [13] as well as in the literature. Derivatization and subsequent UV or fluorescence detection is a well-known approach for analyzing aminoglycosides [12,14,15,13,16,17]. There are, however, some major drawbacks with this approach including unstable products and/or incomplete derivatization.…”

“…Frequently named ion-pair-reagents are sodium octanesulfonate [10] and fluorinated carboxylic acids like trifluoroacetic acid [21] or heptafluorobutyric acid (HFBA) [22,23]. Detection strategies include diode-array, fluorescence [12,14,15,13,16,17], refractometry [9], evaporative light scattering (ELSD) [21,23], mass spectrometry (MS) [24] and pulsed electrochemical detection (PED) [11,25]. Each of these detection methods has its own advantages and disadvantages.…”

Bioanalysis of aminoglycosides using high-performance liquid chromatography

“…The most common derivatizing agent used in the bioanalysis of aminoglycosides was o-phthalaldehyde; reported by 24 articles for the derivatization of gentamicin, amikacin, kanamycin, netilmicin, isepamicin, sisomicin, neomycin, tobramycin, dibekacin, astromicin, and micronomicin [6,14,20,[22][23][24][25][26]28,36,37,43,44,48,50,52,53,55,[57][58][59]61,62,66]. Dansyl chloride for the derivatization of netilmicin [21]; 1-fluoro-2,4-dinitrobenzene for the derivatization of geneticin, gentamicin, and amikacin [29,38,41,45,56,64]; 7-fluoro-4-nitrobenz-2-oxa-1,3diazole for the derivatization of amikacin [74]; benzene sulphonyl chloride for the derivatization of gentamicin [39]; 6-aminoquinolyl-N-hydroxysucciminidylcarbamate for the derivatization of isepamicin [76]; fluorescamine for the derivatization of gentamicin, and tobramycin [40,80]; 1-naphthyl isothiocyanate for the derivatization of amikacin, and tobramycin [65,67]; fluorescein isothiocyanate for the derivatization of tobramycin [77]; o-phthalicdicarboxaldehyde for the derivatization of gentamicin [42]; 2,4,6trinitrobenzene sulfonic a...…”

“…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.…”

“…Intermediate precision also refers to laboratory variations such as different days, instruments, and analyses. Out of the 71 reviewed articles, 56 reported intermediate precision values ranging between 0.331 % [80] to 19.76 % [52], which is within the acceptable limit of 20 % [111]. Source, [113]; d) source, [114]; e) source, [115]; f) source, [116] ; g) source, [117]; h) source, [118].…”