Method development for the determination of sulphonamide residues in chicken by liquid chromatography ion trap tandem mass spectrometry
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Date
2006
Authors
Ahmad, Sidek
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Abstract
A simple, sensitive and reliable method for the determination of five sulphonamide
residues (sulphadiazine, sulphamethazine, sulphaquinoxaline and sulphadimethoxine)
in chicken was developed using a combination of high performance liquid
chromatography (HPLC) with ion trap tandem mass spectrometry. Sample extraction
involvd extraction with acetonitrile, removal of fat with n-hexane followed by
purification of the extract with Strata X polymeric sorbent Solid Phase Eextraction
cartridge after reconstitution with 0.2 M phosphoric acid. The extract was eluted with
methanol and evaporated to dryness in a water bath under constant flow of nitrogen
gas. The residue was again reconstituted with a solution mixture of 0.1 % acetic acid
in ultra pure water and acetonitrile (1:1). A liquid chromatograph with an electrospray
ionization interface to the ion trap tandem mass spectrometrer (LC-MS-MS) was used
for simultaneous confirmation and quantitation of the sulphonamide residues. A
narrow bore HPLC column, Genesis C18 120 (Å, 3 m, 5 cm x 2.1 mm) and a
solution of 0.1 % acetic acid in ultra pure water and acetonitrile (65:35) with a flow
rate 60 μl/min was used to separate the sulphonamides. The analytical procedure for
the detection of sulphonamide residues was validated and the measurement of
uncertainty was determined for the compliance of the ISO/IEC 17025 quality system
requirement. During validation, specificity, linearity, limit of detection (LOD), limit
of quantitation (LOQ), precision and accuracy of the method was determined. New
product ions that could be used for confirmation and quantitation at m/z 174 for
sulphadiazine, sulphamethazine and sulphaquinoxaline, at m/z 204 for
sulphamethazine and m/z 226 for sulphaquinoxaline were observed. A linear plot was
obtained for a concentration range between 20 ppb and 400 ppb for sulphadiazine,
sulphaquinoxaline and sulphadimethoxine and 10 ppb to 400 ppb for sulphamethazin,
respectively, where the regression coefficient for each calibration range obtained was
0.999. The limit of detection (LOD) was 2 ppb for sulphamethazine and 5 ppb for
sulphadiazine, sulphaquinoxaline and sulphadimethoxine, respectively. The limit of
quantification (LOQ) was 10ppb for sulphamethazine and 20 ppb for sulphadiazine,
sulphaquinoxaline and sulphadimethoxine, respectively. The extraction recovery for
spiked samples at the LOQ level was 51, 54, 68 and 83 % with coefficient of variation
of 5, 13, 9, and 7 % for sulphadiazine, sulphamethazine, sulphaquinoxaline and
sulphadimethoxine, respectively and the expanded uncertainty values at concentration
of 100 ppb for sulphadiazine, sulphamethazine, sulphaquinoxaline and
sulphadimethoxine were 6, 9, 10 and 4 ppb, respectively. Therefore from the
performance characteristic obtained the developed method could be reliably used for
routine analytical work.
Description
Master
Keywords
Chemical science , Sulphonamide residues , Chromatography ion