In-House Validation of HPLC-MS/MS Methods for Detection and Quantification of Tetracyclines in Edible Tissues and Feathers of Broiler Chickens

Pokrant, Ekaterina V.; Maddaleno, Aldo E.; Araya, Carolina E.; Martín, Betty V. San; Cornejo, Javiera

doi:10.21577/0103-5053.20170180

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Articles • J. Braz. Chem. Soc. 29 (3) • Mar 2018 • https://doi.org/10.21577/0103-5053.20170180 copy

In-House Validation of HPLC-MS/MS Methods for Detection and Quantification of Tetracyclines in Edible Tissues and Feathers of Broiler Chickens

Authorship SCIMAGO INSTITUTIONS RANKINGS

For the detection of tetraciclines in feathers, muscle and liver, an internal protocol was designed for in-house validation of two methods, through high performance liquid chromatography with tandem mass spectrometric (HPLC-MS/MS) techniques. This protocol was based on the recommendation of the decision 2002/657/EC from the European Community as well as the food and drug administration (FDA) VICHGL49. Limit of detection (LOD) was set at 20 μg kg^-1 and the limit of quantification (LOQ) ranged from 21.5 to 24.2, 21.2 to 21.6, and 25.0 to 27.7 μg kg^-1 in feahers, muscle and liver samples, respectively, for all analytes. The calibrations curves show a coefficient of determination (R²) above 0.98, 0.99 and 0.96 for feathers, muscle and liver samples, respectively. Analyte recovery ranged from 92 to 108%. In conclusion, these methods can be deemed accurate and reliable, and their validation is a fundamental step to be performed in depletion studies on these matrices.

Keywords:
in-house validation; HPLC-MS/MS; tetracyclines; feathers; broiler chickens

Biological matrix	Analyte	LOD^a a Limit of detection of the analytical methodology; / (µg kg^-1)	Average concentration / (µg kg^-1)	CV^b b coefficient of variation; / %	LOQ^c c limit of quantification. / (µg kg^-1)
Feather	OTC 4-epi-OTC CTC 4-epi-CTC	20	20.551 14.833 18.965 22.926	7.6 17.40 4.91 8.03	22.5 24.2 21.5 22.9
Muscle	OTC 4-epi-OTC CTC 4-epi-CTC	20	21.028 19.030 20.090 20.077	3.62 1.45 3.96 4.81	21.2 21.3 21.2 21.6
Liver	OTC 4-epi-OTC CTC 4-epi-CTC	20	24.173 21.064 21.122 20.075	12.65 20.82 15.95 23.45	25.0 27.2 25.5 27.7

Biological matrix	Analyte	R² average^a a Coefficient of determination, average from three calibration curves according analyte and matrix;	SD^b b standard deviation;	CV^c c coefficient of variation. / %
Feather	OTC 4-epi-OTC CTC 4-epi-CTC	0.986 0.985 0.981 0.985	0.005 0.011 0.009 0.005	0.60 0.97 1.14 0.56
Muscle	OTC 4-epi-OTC CTC 4-epi-CTC	0.995 0.997 0.991 0.997	0.003 0.001 0.007 0.003	0.32 0.19 0.72 0.40
Liver	OTC 4-epi-OTC CTC 4-epi-CTC	0.966 0.975 0.960 0.964	0.005 0.008 0.009 0.006	0.60 0.97 0.90 0.67

Analyte	Concentration^a a Fortification concentration (levels correspond to 20, 60 and 100 µg kg-1); / (µg kg^-1)	Feather		Muscle		Liver
Analyte		Average recovery / %	CV^b b coefficient of variation. / %	Average recovery / %	CV / %	Average recovery / %	CV / %
OTC	20 60 100	96 103 99	19 12 4	104 97 100	11 7 2	93 105 99	13 8 2
4-epi-OTC	20 60 100	107 95 101	14 11 3	106 95 101	8 6 2	89 107 98	5 3 1
CTC	20 60 100	92 105 98	15 9 3	108 94 101	8 10 3	89 108 98	16 9 3
4-epi-CTC	20 60 100	103 98 101	13 9 3	104 97 100	8 6 2	96 103 99	8 5 2

Analyte	Concentration^a a Fortification concentration (levels correspond to 20, 60 and 100 µg kg-1); / (µg kg^-1)	CV^b b coefficient of variation. of repeatability / %			CV of intralaboratory reproducibility / %
Analyte		Feather	Muscle	Liver	Feather	Muscle	Liver
OTC	20 60 100	16.0 9.1 2.9	9.7 6.9 2.0	13.1 7.8 2.5	19.0 11.8 3.7	10.2 7.1 3.3	16.3 11.8 3.4
4-epi-OTC	20 60 100	13.0 9.0 2.8	7.9 5.8 1.7	15.5 8.5 2.8	15.6 9.1 4.5	10.6 7.0 2.1	13.7 9.9 2.8
CTC	20 60 100	8.1 6.4 1.8	12.9 9.8 2.7	5.1 3.1 0.9	14.0 10.5 3.0	12.2 9.3 2.6	15.1 10.4 3.0
4-epi-CTC	20 60 100	8.6 6.6 1.9	6.6 4.6 1.4	8.4 5.2 1.6	13.0 9.1 2.7	8.9 6.5 1.8	8.6 7.0 1.9

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