Determination of apramycin in oral soluble powder by a HPLC method using pre-column derivatization with o-phthalaldehyde and UV detection

Abstract

A high-performance liquid chromatographic method employing pre-column derivatization with o-phthalaldehyde (OPA) and 2-mercaptoacetic acid was developed for the determination of apramycin, an aminoglycoside antibiotic used in veterinary medicine, in the oral soluble powder form. The chromatographic separation was done by ion-pair HPLC using a C18 reversed-phase column, Synergy Hydro (150 mm x 4.6 mm x 4 μm) and mobile phase composed of 0.005 mol/L sodium octanosulfonate in a mixture of acetonitrile: water: acetic acid (45:55:2) (v/v/v) with a flow rate of 1.0 mL/min; the UV detector was operated at 332 nm. The developed method was validated according to official compendia guidelines, having demonstrated robustness, selectivity and linearity for the concentration range of 0.02 to 0.05 mg/mL, precision (with RSD < 2.0% both for intra and inter-day precision) accuracy (average recuperation of 99.33%) and detectivity (quantification and detection limits of 0.08 and 0.02 μg/mL, respectively). Three batches of commercial apramycin oral soluble powder were analyzed by both the proposed method and the official microbiological method, where all the results obtained were in the acceptable range (95% to 105% of labeled value of apramycin). Both methods were statistically compared by the t test, which yielded no significant differences (α = 0.05) thereby confirming the equivalence of the methods.Uniterms: Apramycin. HPLC. Microbiological Assay. Pre-column Derivatization. OPA.Foi desenvolvido um método por cromatografia líquida alta eficiência empregando derivatização pré-coluna com o-ftalaldeído (OPA) e ácido mercaptoacético para determinação de apramicina, um antibiótico aminoglicosídeo de uso veterinário, em pó oral solúvel. A separação cromatográfica foi feita por fase reversa com pareamento iônico utilizando-se coluna Synergy Hydro C18 (150 x 4,6 mm x 4 µm) e fase móvel composta por octanossulfonato de sódio 0,005 mol/L em mistura de acetonitrila:água:ácido acético nas proporções 45:55:2 (v/v/v), numa vazão de 1.0 mL/min; efetuou-se detecção por UV a 332 nm. O método foi validado de acordo com os compêndios oficiais e demonstrou robustez, seletividade, linearidade na faixa de 0,02 a 0,05 mg/mL, precisão (com DPR < 2,0% tanto para a precisão intra-dia quanto para a precisão inter-dia), exatidão (recuperação média de 99,33%) e detectabilidade (limite de quantificação e de detecção iguais a 0,08 e 0,02 µg/mL, respectivamente). Analisaram-se 3 lotes de apramicina pó oral solúvel pelo método proposto e pelo método microbiológico oficial e todos os resultados obtidos estavam dentro do limite de aceitação (95%-105% valor rotulado de apramicina). Ambos os métodos foram comparados estatisticamente pelo teste t, não sendo encontradas diferenças significativas entre eles para α=0,05, sendo os dois equivalentes.Unitermos: Apramicina. CLAE. Doseamento Microbiológico. Derivatização pré-coluna. OPA.

Characterization of Resistance Patterns and Detection of Apramycin Resistance Genes in Escherichia coli Isolated from Chicken Feces and Houseflies after Apramycin Administration

“…It is still used in several European countries and it was approved for use in China in 1999 ( Zhang et al, 2009 ). It is used to treat or prevent infections caused by Gram-negative bacteria such as colibacillosis, salmonellosis, and bacterial enteritis in poultry, swine, and calves ( Antunes et al, 2011 ). Epidemiological investigations of apramycin-resistant bacteria from food producing animals showed differential prevalence of apramycin resistance in different animals ( Choi et al, 2011 ).…”

Antibiotic microbial assay using kinetic-reading microplate system

“…The results also verified that the ideal proportion of OPA-apramycin is 5:1. The HPLC method employing OPA-mercaptoacetic acid derivatization showed good correlation to the conventional turbidimetric assay (Antunes et al 2011).…”

Development and Validation of Microbiological Assay for Ceftriaxone and its Application in Photo-stability Study

“…Besides this, microbiological assay can reveal subtle alterations that are not demonstrable by convention chemical methods [16][17]. However, the low cost and simple procedures of bioassays have allowed them to become an alternative method for drug assessment in pharmaceutical formulations.…”