TITLE: Determination of florfenicol and florfenicol amine in fish plasma (Salmo salar) through HPLC MS/MS
AUTHORS: Betty San Martín, Lisette Lapierre, Marcela Fresno, Javiera Cornejo

[STEPS]
?. [Sample preparation]
of each plasma sample were placed in  Falcon tubes and and were added.
250 µl
15 ml
500 µl
2 ml
?. [Sample preparation]
Extraction was carried out according to the method described by Li et al. (2006), with modifications.
?. [Sample preparation]
The tubes were shaken for , sonicated for  and centrifuged at 5,000 rpm for
?. [Sample preparation]
Subsequently, the resulting supernatant was transferred to another  Falcon tube containing, and then shaken, sonicated and centrifuged at 5,000 rpm for the same amount of time as before
15 ml
2 ml
?. [Sample preparation]
Then, the upper phase () was discarded and the remaining phase evaporated at to under nitrogen flow.
?. [Sample preparation]
Once completely dry, of mobile phase and  hexane were used to reconstitute each sample, which then was shaken, sonicated and centrifuged at 5,000 rpm.
1 ml
1 ml
?. [Sample preparation]
Finally, of the lower phase were transferred to a vial to be injected into the .
300 µl
?. [Sample preparation]
The limit of detection (LOD) and the limit of quantification (LOQ) established for both analytes in plasma were and ,  respectively.
0 Parts per Million (PPM)
0 Parts per Million (PPM)
?. [Instrumental Analysis]
For instrumental analysis, an LC (Agilent, 1290 infinity series) coupled to a triple quadrupole mass spectrometer (API 5500, ABSCIEX) was used.
?. [Instrumental Analysis]
A SynergiTM 4 µm fusion RP 80Â 50 x 2.0 mm analytic column was used.
?. [Instrumental Analysis]
Analyst 1.6.3 and Multiquant 3.0 software was used for equipment management and integration, respectively.
?. [Instrumental Analysis]
Chromatographic separation was performed through a mobile phase using solvent A: 0.1% of  in ; and a mobile phase solvent B: 0.1% of  in /1:9 ratio; with a gradient flow of min-1and a gradient elution from 32% up to 68% solvent A in  of 35% phase solvent A, and 75% phase solvent B.
350 µl
?. [Instrumental Analysis]
Injection volume was ; column oven temperature was set at .
2 µl
?. [Instrumental Analysis]
Parameters of the MS/MS detector  AB1IonizationElectrospray (ESI)2Scan typeMRM3Source temperature (TEM)550ºC4Nebulizer (GS1)60 psi5Turbo ion (GS2)80 psi6Curtain gas (CUR)20 psi7Collision gas (CAD)10 psi8Ion-spray voltage (IS)4500 V
AB1IonizationElectrospray (ESI)2Scan typeMRM3Source temperature (TEM)550ºC4Nebulizer (GS1)60 psi5Turbo ion (GS2)80 psi6Curtain gas (CUR)20 psi7Collision gas (CAD)10 psi8Ion-spray voltage (IS)4500 V
?. [Instrumental Analysis]
Monitored ion masses  ABCDEFG1AnalytePrecursor ion (Q1 mass) (Da)Fragment ion (Q3 mass) (Da)Time (ms)EP (V)CE (V)CXP (V)2FF1356.0336.0100.0-5,000-15,000-8,0003FF2356.0185.0100.0-5,000-17,000-12,0004FFA1248.0230.0200.05,00022,00025,0005FFA2248.0130.0200.02,00030,00010,0006CAF-d5 (IS)326.0157.0100.010,000-25,000-20,000
ABCDEFG1AnalytePrecursor ion (Q1 mass) (Da)Fragment ion (Q3 mass) (Da)Time (ms)EP (V)CE (V)CXP (V)2FF1356.0336.0100.0-5,000-15,000-8,0003FF2356.0185.0100.0-5,000-17,000-12,0004FFA1248.0230.0200.05,00022,00025,0005FFA2248.0130.0200.02,00030,00010,0006CAF-d5 (IS)326.0157.0100.010,000-25,000-20,000
?. [Validation of analytical method]
Prior to determining concentrations in plasma, the analytical method was validated by HPLC MS/MS, according to instructions from the European Commission Decision 2002/657/EC (2002).
?. [Validation of analytical method]
Precursor ions and the two product ions were identified for florfenicol and florfenicol amine, respectively.
?. [Validation of analytical method]
Values for essential parameters were estimated for the validation of the analytical method on plasma: specificity, recovery, repeatability, intralaboratory reproducibility, decision limit (CCα), detection capability (CCβ), and linearity.