development and validation of a … 008.pdf · 150 mg psa, 150 mg c18 and 900 mg mgso4 centrifugate...
TRANSCRIPT
Introduction Amphenicols are broad spectrum antibiotics that have been widely
used in food producing animals for treatments of various infections.
Chloramphenicol (CAP) is forbidden in the European Union since 1994 by the
Commission Regulation 1430/94 of 22 June 1994. Florfenicol (FF) and
Thiamphenicol (TAP) are allowed for treatment of different animal species
followed by the appropriate withdrawal period. According to the Regulation
37/2010, FF determination must include FF and Florfenicol amine (FFA)
quantitation, as a marker residue. There are different maximum residue limits
(MRLs) for many combinations of matrix/species which are shown in table 1.
Experimental
Table 1.- Regulation limits for the researched amphenicols.
Analyte Specie MRL in muscle
(µg kg-1) MRL in kidney
(µg kg-1)
Florfenicol (FF and FFA) Broiler 100 750
Bovine, ovine and caprine
200 300
Porcine 300 500
Fish 1000 -
Others (rabbit, equine…) 100 300
Tiamphenicol All species 50 50
2 g
+ 4 g of anhydrous sodium sulfate
+ 5 ml ethyl acetate with ammonia 2 % Vortex and centrifugate 5000 rpm 5 min
x 2
supernatants
Evaporate to dryness at 50 ºC
Redissolve in 5 ml of ACN/H2O (90:10, v/v)
PURIFICATION
QUECHERS DISPERSIVE SPE (EN):
150 mg PSA, 150 mg C18 and 900 mg MgSO4
Centrifugate 4000 rpm 4 min
Evaporate to dryness at 50 ºC
Redissolve in 500 µl of H2O
DETERMINATION
Analyte/concentration (µg kg-1) Trueness
(%)
Repeatability (CV%) n=35
Within-laboratory reproducibility (CV%)
n=35
CC (µg kg-1) n=35
CC (µg kg-1) n=35
CAP (0.10) 101 12 16 0.10 0.13
CAP (0.30) 98 6 7
CAP (4.00) 104 7 8
TAP (6.3) 95 13 19
TAP (50.0) 102 7 13 60.7 71.5
TAP (250.0) 98 13 16
Analyte/concentration (µg kg-1)
Trueness (%)
Repeatability (CV%) n=35
Within-laboratory
reproducibility (CV%) n=35
CC (µg kg-1) n=35
CC (µg kg-1) n=35
FF (25.0) 94 13 16
FF (100.0) 106 9 13 121.8 143.7
FF (200.0) 106 10 12 241.7 283.5
FF (300.0) 110 7 9 347.4 394.8
FF (500.0) 108 6 6 555.5 610.9
FF (1000.0) 92 9 15 1217.8 1435.6
FF (1500.0) 101 9 13
FFA (25.0) 107 13 21
FFA (100.0) 81 11 20 126.5 153.0
FFA (200.0) 96 16 19 261.0 322.0
FFA (300.0) 92 21 23 402.6 505.2
FFA (500.0) 88 13 23 663.6 827.1
FFA (1000.0) 92 14 16 1243.4 1486.7
FFA (1500.0) 105 11 14
BOVINE MUSCLE CHROMATOGRAMS PORCINE KIDNEY CHROMATOGRAMS
FLORFENICOL AMINE (m/z 248>230)
THIAMPHENICOL (m/z 354>290)
FLORFENICOL (m/z 356>336)
CHLORAMPHENICOL (m/z 321>152)
CHLORAMPHENICOL-D5 (ISTD) (m/z 326>157)
FLORFENICOL AMINE (m/z 248>230)
THIAMPHENICOL (m/z 354>290)
CHLORAMPHENICOL (m/z 321>152)
CHLORAMPHENICOL-D5 (ISTD) (m/z 326>157)
FLORFENICOL AMINE (m/z 248>230)
FLORFENICOL (m/z 356>336)
FLORFENICOL (m/z 356>336)
THIAMPHENICOL (m/z 354>290)
CHLORAMPHENICOL (m/z 321>152)
CHLORAMPHENICOL-D5 (ISTD) (m/z 326>157) CHLORAMPHENICOL-D5 (ISTD) (m/z 326>157)
CHLORAMPHENICOL (m/z 321>152)
FLORFENICOL (m/z 356>336)
THIAMPHENICOL (m/z 354>290)
FLORFENICOL AMINE (m/z 248>230)
BLANK SAMPLES
SPIKED SAMPLES at LOQ
concentration
Validation results of amphenicols in animal muscle and kidney: A)
CONFIRMATION (5.5 IPs) and CALIBRATION CURVES IN MUSCLE
CONFIRMATION (5.5 IPs) and CALIBRATION CURVES IN KIDNEY
FFA
FFA
FF
FF
TAP
TAP
CAP
CAP
Conclusions • A rapid, quantitative and confirmatory method for the analysis of amphenicols was developed. • FFA was included in FF determination in a wide range of concentrations according to council regulation 37/2010 requirements (matrix/animal specie). • The presented method provides good validation data in accordance with the EU criteria (2002/657/EC) and it was successfully accredited under the ISO/IEC 17025:2005 by the Spanish national body for the accreditation (ENAC).
B)
LC-MS/MS Triple quadrupole (details included in the proceedings)
Acknowledgements: The authors thank Carmen Navarro, Xavier Sáez, Carmen Gargallo, Mariana Lamas and Gemma Solá for their enthusiastic help in sample preparation.
DEVELOPMENT AND VALIDATION OF A CONFIRMATORY METHOD FOR THE DETERMINATION OF AMPHENICOLS IN MUSCLE AND KIDNEY OF SEVERAL ANIMAL SPECIES
Francisco Moragues1, Carmen Igualada1,2 and Nuria León1. 1Public Health Laboratory of Valencia – FISABIO. Avda. Cataluña 21, 46020-Valencia. Spain.
2Faculty of Pharmacy . University of Valencia. Av. Vicente Andrés. 46100. Burjassot (Valencia) Spain.
E-mail: [email protected], [email protected] and [email protected].