Abstract
A HPLC-DAD method for the determination of cysteamine supplementation in commercial animal feeds was developed. Samples were extracted with a mixture of 0.5 % hydrochloric acid – acetonitrile (90:10, v/v), matrix interferences were removed with a C18 cartridge, cysteamine was derivatized using 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) as Ellman's reagent targeting to the thiol group in the molecule. Quantification of cysteamine was performed on a c18 column with DAD at 323 nm. The developed method had LOD of 1.1 mg/l, good linearity of the calibration curve (R2 ≥ 0.9998), high recoveries (> 92 %), and high reproducibility (RSD < 2.0%).
Highlights
A high performance liquid chromatography with diode-array detection (HPLC-DAD) method for the determination of cysteamine supplementation in commercial animal feeds was developed
It was found that the optimal pH range was between [8,9] where there was a compromise between thiolate formation and the stability of dithiobis-(2-nitrobenzoic) acid (DTNB) (Figure 1 b)
We focus on optimizing the derivatization reaction with 5,5’-dithiobis-(2nitrobenzoic) acid (DTNB), i.e. Ellman’s reagent, and analyzing by HPLC with DAD detection at 323 nm
Summary
A high performance liquid chromatography with diode-array detection (HPLC-DAD) method for the determination of cysteamine supplementation in commercial animal feeds was developed. Samples were extracted with a mixture of 0.5 % hydrochloric acid (HCl) – acetonitrile (ACN) (90:10, v/v), matrix interferences were removed with a C18 cartridge, and cysteamine was derivatized using 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) as Ellman's reagent targeting to the thiol group in the molecule. Quantification of cysteamine was performed on a C18 column with DAD at 323 nm. The developed method had a limit of detection (LOD) of 1.1 mg/l, good linearity of the calibration curve (R2≥ 0.9998), high recoveries (> 92 %), and high reproducibility (RSD < 2.0%).
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