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Abstract

A COMPARATIVE STUDY IN THE CONFIRMATORY METHOD DEVELOPMENT AND VALIDATION OF FLUBENDAZOLE IN FISH AND SHRIMP MUSCLES BETWEEN HPLC-MS/MS (Q MICRO) AND UPLC-MS/MS (TQD)

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

ABSTRACT

A confirmatory method for determination of flubendazole was
developed in ultra performance liquid chromatography–tandem
quadrupole mass spectrometry (AQUITY UPLC–TQD) and in HPLCMS/
MS (Quattro micro) at same time. The method was validated in
Bagda shrimp (Penaeus monodon) and Tilapia (Oreochromis niloticus)
fish. The liquid chromatographic separation was done with gradient
elution by using (Atlantis® dC18, 3μm, 4.6x100 column in case of
HPLC system whereas AQUITY UPLC BEH C18, 2.1x50mm, 1.7μm,
Column in UPLC system) same mobile phase of 0.1% formic acid in
water and 0.1% formic acid in acetonitrile. Mass spectral acquisition
was done using electrospray ionization in the positive ion mode applying multiple reaction
monitoring (MRM) of two diagnostic transition reactions for flubendazole and for
flubendazole_d3. Shrimp and fish samples were extracted with ethyl acetate and evaporated
to dryness and finally reconstituted with acetonitrile. 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 1 to 20 ng/g with the correlation
coefficient of >0.996 in all cases. In case of shrimp samples the decision limit (CCα) and the
detection capability (CCβ) were 5.8μg/kg and 6.5μg/kg respectively in system1 and 5.6μg/kg
and 6.2μg/kg in system2. In case of fish sample CCα values were 6.00μg/kg and 5.4μg/kg in
system1 and system2 and CCβ values were 7.1μg/kg and 5.8μg/kg respectively for flubendazole. The precision of the method, expressed as RSD values for the within laboratory
reproducibility at the three levels of fortification (2.5, 5.0 and 7.5) μg/kg was less than 15%.
The mean recoveries were in the range of 90–107% for shrimp and that of 90-109% for fish.

Keywords: UPLC-TQD, HPLC-Quattro micro, Flubendazole, Shrimp, Fish and Validation.


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