Determination of etimicin in rat plasma using 9-fluorenylmethyl chloroformate precolumn derivatization by HPLC with fluorescence detection

Abstract

Um método novo e sensível foi desenvolvido para a determinação de etimicina (ETM) utilizando pré-coluna de derivatização (PCD) com 9-fluorenilmetilcloroformiato (FMOC-Cl) por separação de fase reversa e subsequente detecção por fluorescência. As condições PCD foram todas otimizadas para o menor limite de detecção. A separação cromatográfica foi efetuada em uma coluna Agilent XDB-C8 a 25 o C, velocidade de fluxo de 1,0 mL min -1 e fase móvel acetonitrila/água (87:13, v/v). O derivativo etimicina-FMOC-Cl foi monitorado por detecção de fluorescência a λ excitação 265 nm e λ emissão 315 nm. Neomicina, um composto de estrutura similar ao ETM foi usado como padrão interno. A avaliação estatística do método foi examinada, mostrando-se preciso e exato com um intervalo de linearidade de 0,038-9,69 µg mL -1 (r > 0,9994). Os estudos de precisão intra-e inter-dia mostraram boa reprodutividade com desvio padrão médio (R.S.D.) menor que 5%, e recuperação de 97,80-100,09 % (n = 3). O limite de detecção (LOD) e o limite inferior de quantificação (LLOQ) em plasma foram 0,01 e 0,02 µg mL -1 , respectivamente. Um volume de 50 µL de plasma foi suficiente para detecção de etimicina. O método estabelecido fornece um método bionalítico confiável para a farmacocinética de etimicina em plasma de rato.A new and sensitive method was developed for the determination of the etimicin (ETM) using precolumn derivatization (PCD) with 9-fluorenylmethyl chloroformate (FMOC-Cl) by a reversed phase separation and subsequent fluorescence detection. The PCD conditions were fully optimized towards the lowest limit of detection. The chromatographic separation was carried out on an Agilent XDB-C8 column at 25 o C using a constant flow rate of 1.0 mL min -1 and mobile phase of acetonitrile/water (87:13, v/v). The etimicin-FMOC-Cl derivative was monitored by fluorescent detection at λ excitation 265 nm and λ emission 315 nm. Neomycin (NMC), a similar base structure compound with ETM, was used as an internal standard. The statistical evaluation of the method was examined and the method was found to be precise and accurate with a linearity range of 0.038-9.69 µg mL -1 (r > 0.9994). The intra-and inter-day precision studies showed good reproducibility with relative standard deviation (R.S.D.) less than 5%, and the relative recovery was 97.80-100.09 % (n = 3). The limit of detection (LOD) and lower limit of quantification (LLOQ) in plasma were 0.01 and 0.02 µg mL -1 , respectively. A volume of 50 µL of plasma was sufficient for the determination of etimicin. The established method provides a reliable bioanalytical method to carry out etimicin pharmacokinetics in rat plasma.Keywords: high-performance liquid chromatography, 9-fluorenylmethyl chloroformate, derivatization, etimicin, pharmacokinetic IntroductionEtimicin (ETM) is mainly used as the sulfate and the fourth-generation aminoglycoside antibiotics, which is all water soluble aminoglycoside derived from gentamycin C 1a (Figure 1).1 It is widely used in human and veterinary medicine against both Gram-posit...

Challenges in the determination of aminoglycoside antibiotics, a review

“…mL -1 [85]. Similar FMOC-Cl derivatization and detection was performed for the determination etimicin in rat plasma using an RP C8 column [52].…”

Development of an ultra fast liquid chromatography–tandem mass spectrometry method for simultaneous determination of cefazedone and etimicin in beagle dog plasma: Application to the pharmacokinetic study of the combination of cefazedone and etimicin injections

“…For single group, they were decided according to the guidance for preclinical pharmacokinetics and some reported references [20,21]. As for the combined group, we took both the time points for the two analytes and different start times of each analyte into consideration.…”

“…For the determination of etimicin for pharmacokinetics, most of them were carried out by bioassay methods [6]. A new chromatographic method of precolumn derivatization with HPLC and fluorescence detection for determination of etimicin in rat plasma was reported recently, which was sensitive but the sample pre-treatment procedures were technically demanding and time consuming [21]. Some LC-MS/MS methods were developed for the impurity profiling of etimicin sulfate or determination of etimicin in some biological samples [22,23], but none of them was deal with the pharmacokinetic application.…”