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Abstract

A COMPARISON OF CONFIRMATORY METHOD DEVELOPMENT AND VALIDATION OF ANTIBIOTIC: NITROFURAN METABOLITES (AMOZ, AOZ, AHD and SEM) IN FISH AND SHRIMP MATRIX AMONG SOME LC-MS/MS SYSTEMS (HPLC-QUATTRO MICRO API, UPLC-QUATTRO PREMIER XE AND UPLC-TQD)

Md. Ashraful Alam*, Akter Mst Yeasmin, Talukdar Muhammad Waliullah, Saleh Ahmed and Md. Manik Mia

ABSTRACT

Confirmatory methods for determination of banned antibiotic: nitrofuran metabolites (AMOZ, AOZ AHD and SEM) were developed in HPLC-MS/MS (Quattro micro), AQUITY UPLC–TQD and AQUITY UPLC-Quattro Premier XE at the same time. The method were validated in Bagda shrimp (Penaeus monodon) and Tilapia (Oreochromis niloticus) fish. The liquid chromatographic separations were done with gradient elution by using XTerra® MS C18, 3.5μm, 2.1x150mm column in case of HPLC system whereas AQUITY UPLC BEH C18, 2.1x50mm, 1.7μm, Column in UPLC systems) same mobile phase 5mM ammonium acetate in water and methanol and same extraction protocol. Mass spectral acquisition was done using electrospray ionization in the positive ion mode applying MRM of two diagnostic transition reactions for AMOZ, AOZ AHD, SEM and AMOZ_D5 in derivatized condition. Shrimp and fish samples were extracted with ethylacetate and evaporated to dryness and finally reconstituted with 50% methanol. The method validation was carried out according to the criteria of Commission decision 2002/657/EC. The calibration curve showed a good linearity in the concentration range from 0.25 to 5.0 ng/g with the correlation coefficient of >0.997 in all cases. The decision limit (CCα) was in the range of 0.12-0.23ng/g whereas detection capability (CCβ) was in the range of 0.21-0.38 ng/g respectively. The precision of the method, expressed as recovery values for the within laboratory reproducibility at the three levels of fortification (0.5, 1.00, 1.5 2.0) ng/g were less than 15%. The mean recoveries were in the range of 92–113%.

Keywords: HPLC-QM, UPLC-TQD, QP-XE, Nitrofuran metabolites, Shrimp, Fish and Validation.


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