Croubels, Siska

De Backer, Patrick

De Baere, Siegrid

Devreese, Mathias

Maes, An

English

Ghent University Academic Bibliography

DETECTION AND QUANTIFICATION OF THE EU MARKER RESIDUE OF TIAMULIN IN ANIMAL TISSUESS. De Baere*, A. Maes, M. Devreese, P. De Backer and S. CroubelsDepartment of Pharmacology, Toxicology and Biochemistry, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium, *corresponding author, e-mail: siegrid.debaere@ugent.beIntroduction and Aim− HPLC: Surveyor autosampler Plus and MS Pump Plus,autosampler t°: 5 °C (Thermo Scientific)− Column: Hypersil Gold, 50 mm x 2.1 mm, dp: 1.9 µm, 50 °C + pre-column of the same type (Thermo Scientific)− Gradient elution: see Table 1Table 1. Gradient programme⇒ 8-α-hydroxymutilin eluted at retention time (Tr) of 3.10 min (seeFigure 1B)Sample preparation MS/MS conditionsHPLC conditionsTiamulin is a diterpene antimicrobial agent with a pleuromutilin chemical structure, which is mainly active against Gram-positive micro-organisms and Mycoplasma. In rabbits, tiamulin isused for the treatment of Epizootic Rabbit Enteropathy. Maximum Residue Levels (MRLs) for rabbit tissues have been established by the European Union for the marker residue, i.e. thesum of metabolites that may be hydrolysed to 8-α-hydroxymutilin, at 100 µg kg-1 for muscle and 500 µg kg-1 for liver.1In the literature, only one method has been reported for the determination of the marker residue of tiamulin in animal tissues. This method, based on gas chromatography (GC) withelectron capture detection (ECD), was very time-consuming, resulting in a sample throughput of 3 to 5 samples per person per day.2The aim of this study was to develop and validate an LC-MS/MS method for the quantitative determination of 8-α-hydroxymutilin in rabbit tissues.Table 2. MS/MS conditionsTime (min)MF A(%)MF B(%)Flow rate (µl/min)0.0 75 25 3004.3 75 25 3005.0 10 90 3007.3 10 90 3008.0 75 25 30012.0 75 25 300− Instrument: TSQ Quantum Ultra (Thermo Scientific)− Ionization mode: positive electrospray ionization− Pseudo MS/MS: 8-α-hydroxymutilin could not be fragmented andtherefore the mass spectrometer was operated in the pseudoMS/MS mode (see Table 2)Parent  ion(m/z)Product ion(m/z)Scan width (sec)Scan time (sec)Collision energy (%)Tube lens(V)337.25 337.25 0.01 0.30 5 85− Method A, with alkaline hydrolysis ⇒ to quantitate the sumof tiamulin and metabolites that may be hydrolysed to8-α-hydroxymutilin in rabbit tissues− Method B, without alkaline hydrolysis ⇒ to investigate thestability of 8-α-hydroxymutilin (in matrix, freeze-thaw stability)+ 5 mL of 0.01 N HCl in water/aceton (50/50, v/v)extraction, 20 mincentrifugation, 10 min, 4000 rpm1 g of minced tissue (muscle or liver)upper liquid phasedefatting using 5 mL of n-hexaneevaporation of 4 mL of lower phase (50 °C, N2) to 2 mL+ 3 mL of 7 N NaOHValidationValidation was performed in rabbit muscle and liver according to European Guidelines3,4.−Linearity: 50 – 2000 µg kg-1, r ≥ 0.99, g ≤ 10 %−Accuracy: muscle (50 – 100 – 200 µg kg-1), liver (250 – 500 – 1000 µg kg-1) → within -20 % to +10 % oftheorethical concentration−Within-run precision: muscle (50 – 100 – 200 µg kg-1), liver (250 – 500 – 1000 µg kg-1); relative standarddeviation (RSD) < RSDmax = 15 % for conc. ≥ 10 µg kg-1 < 100 µg kg-1; RSDmax = 10 % forconc. ≥ 100 µg kg-1−Between-run precision: muscle (50 – 100 – 200 µg kg-1), liver (250 – 500 – 1000 µg kg-1); relative standarddeviation (RSD) < RSDmax = 2(1-0.5logC)−LOQ: 50 µg kg-1 for both muscle and liver tissue−LOD: S/N ≥ 3, method A: muscle, 11.9 µg kg-1; liver, 20.6 µg kg-1; method B: muscle, 27.8 µg kg-1; liver,21.9 µg kg-1−Specificity: no interferences from endogenous compounds (see Figure 1A)−Stability: in acetonitrile during storage at ≤ - 15 °C (stock solution, at least 189 days) or at 2 – 8 °C (workingsolutions, at least 20 days) in matrix during storage at ≤ - 15 °C: at least 9 months in extract during storage at 2 – 8 °C: at least 1 day during three freeze-thaw cycles at ≤ - 15 °C Sample preparation: extraction recovery: 66.2 % (muscle) – 75.5 % (liver); signal enhancement/suppression: 51.7 % (muscle) – 43.3 %(liver); apparent recovery: 34.2 % (muscle) – 32.5 % (liver) up to 50 samples can be analysed per person and per day (= advantage compared to reported GC-ECD mehtod2) Alkaline hydrolysis: conversion of tiamulin and related metabolites to 8-α-hydroxymutilin wasoptimal at a temperature of 45 °C and a hydrolysis time of 20 min Quantitation: no suitable internal standard could be found and therefore the method of external standardization was applied⇒ importance of working with exact volumes during the sample preparation procedure Method validation: results fell within the ranges specified Applicability of the method: was demonstrated by the analysis of 8-α-hydroxymutilin in rabbit liverand muscle tissues that were taken during a residue study with tiamuline in rabbits after theadministration via the drinking water (dose: 16 mg tiamulin base/kg BW/day, 14 consecutive days)Developed LC-MS/MS method:straightforward → up to 50 samples can be analysed per person per dayin-house validatedcan be used for the analysis of 8-α-hydroxymutilin, the EU marker residue fortiamulin, in animal tissuesReferences. 1Commission Regulation (EU) No 37/2010; 2Markus J.R. and Sherma J., J. AOAC Int., 1993, 76(2) 451 – 458; 3EMEA/CVMP/573/00; 4EMA/CVMP/VICH/463199/2009Results and discussionFigure 1. LC-MS/MS chromatogram of (A) a blank rabbit liver sample, (B) a blank rabbit liver sample spiked with 500 µg kg-1 8-α-hydroxymutilin (= MRLlevel) and (C) a liver sample of a rabbit that received 16 mg tiamulin base/kg BW/day via the drinking water for 14 consecutive days and that wasslaughtered at 1 h after withdrawal of the medicated drinking water (concentration of the marker residue 8-α-hydroxymutilin: 454 µg kg-1).Conclusionsvortex mixingevaporation of 9 mL of organic phase until dryness (45 °C, N2)alkaline hydrolysis, 45 °C, 20 min+ 2 mL concentrated HClvortex mixing, cooling+ 10 mL ethylacetateextraction, 20 mincentrifugation, 10 min, 4000 rpmfor calibrator and QC samples:spiking with 8-α-hydroxymutilin+ 250 µL 0.1 % formic acid in watervortex mixingfiltration using Millex Nylon 0.22 µm syringe filterin vial → LC-MS/MS analysis(A) (B) (C)

Detection and quantification of the EU marker residue of tiamulin in animal tissues

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Detection and quantification of the EU marker residue of tiamulin in animal tissues

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