Analytisch laboratorium voor voedings- en residue onderzoek SOP ARO/489 v 3

Printdatum: 1-Oct-08 r

The determination of resorcyclic acid lactones in biological samples using LC-MS/MS

Document: SOP ARO/489 versie 3 Auteur: Klaas van Twillert Documentgebied: ARO Methods Autorisator: Leen van Ginkel (labhoofd) Status: Definitief

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Printdatum: 1-Oct-08 r 1 Introduction Objective This SOP describes a quantitative method of analysis of α-zearalanol (Zeralanol), β-zearalanol (Taleranol), α-zearalenol, β-zearalenol, zearalanone and zearalenone in urine. The procedure for the same analysis carried out in liver is described as well, although this part was not validated. Therefore the performance characteristics given concern the analysis in urine. The clean-up consists of the following steps: deconjugation, liquid-liquid extraction, SPE extraction with C18 and amino. Separation and quantification is performed by liquid chromatography coupled via an electro spray interface to a mass spectrometer. Scope The method is used to perform quantitative and confirmation analysis of α-zearalanol (Zeralanol), β- zearalanol (Taleranol), α-zearalenol, β-zearalenol, zearalanone and zearalenone in urine of cattle, as well as in liver of cattle. The CCß is for α-zearalanol 0.20 ng/ml, β-zearalanol 0.47 ng/ml, α-zearalenol 0.34 ng/ml, β- zearalenol 0.48 ng/ml, zearalanone 0.23 ng/ml and zearalenone 0.29 ng/ml

3 Safety and environment The RIVM have defined rules to work safe. These rules can be found on insite. Waste is disposed according to SOP ARO/487. More information how to handle waste is found on the RIVM intranet.

4 Chemicals and reagents Reference to a company and/or product is for purpose of identification and information only. All reagents must be of analytical grade, unless otherwise specified 4.1 Water; Milli-Q, Waters. 4.2 Tertiair-ButylMethylEther (TBME). 4.3 Methanol 4.3.1 Methanol/water, 40/60 v/v-%: Mix 400 ml methanol with 600 ml water. 4.3.2 Methanol/water, 50/50 v/v-%: Mix 500 ml methanol with 500 ml water. 4.3.3 Methanol/water, 80/20 v/v-%: Mix 800 ml methanol with 200 ml water. 4.4 Acetone 4.4.1 Acetone/methanol, 80/20 v/v-%: Mix 800 ml acetone with 200 ml methanol. 4.5 β-glucuronidase (from Eschericha coli), Boehringer Mannheim, art.no 0307601001. 4.6 Hydrochloric acid, 37%. 4.6.1 HCl 1 M: Add 10 ml HCl to 90 ml water (proceed with caution).

4.7 HPLC eluentia: A(weak) methanol/water 5 mM NH4Ac, 10/90 v/v-%: mix 100 ml methanol with 900 ml water and 0.346 gram NH4Ac. B(strong) methanol/water 5 mM NH4Ac, 90/10 v/v-%: mix 900 ml methanol with 100 ml water and 0.038 gram NH4Ac.

4.8 Phosphate buffer 0.1 M, pH 7.0: Prepare a solution of 14.2 g Na2HPO4 (MW 142 g/mol) in 500 ml water. Prepare a solution of 13.6 g KH2PO4 (MW 136 g/mol) in 500 ml water. Add ca 360 ml of the KH2PO4 solution to the Na2HPO4 solution. Fill up till 1000 ml with water. 4.9 Acetic acid. Sodiumacetate. 4.10 Acetate buffer, 2 mol/l, pH 5.2. Dissolve 25.2 g of acetic acid and 129,5 g of sodiumacetate in 800 ml water. Adjust the pH with a pH meter to 5.2 ± 0.1 and add water to a final volume of 1000 ml.

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Printdatum: 1-Oct-08 r 4.11 Helix Pomatia Juice, Bio Sepra SA, (β-glucuronidase > 100.000 FU/ml, Sulfatase > 1000.000 RU/ml). 4.12 Bottles, 10 ml, brown glass, with screw cap.

4.13 SPE C18 column, 1 g, 6 ml, Alltech # 205430.

4.14 SPE NH2 column, 1 g, 6 ml, Alltech # 211153. 4.15 HPLC column: UPLC BEH C18, 1,7 µ, 2.1 x 100 mm. 4.16 Plastic centrifuge tubes, 50 ml. 4.17 Injection vials, Waters, 12 x 32 mm, glass screw neck vial, Quick Thread, LectraBond cap, preslit, PTFE/silicone septa. 4.18 The LC-MSMS equipment consists of a Waters Acquity UPLC system, a Waters Acquity autosampler and a Micromass Quattro Ultima Pt mass spectrometer. Reference standards and materials 4.19 Internal standards: Relevant internal standards are α-zearalanol-d4, β-zearalanol-d4, α- zearalenol-d4, β-zearalenol-d4 (source RIVM) and zearalenone-d6 (source DVS). 4.15.1 Stock solutions, containing 1 mg/ml, are prepared by dissolving the appropriate amount of the analyte in ethanol. These solutions are stored in the dark at -20 ºC for a maximum period of 5 year. 4.15.2 Working solutions, containing 0.1 mg/ml, are prepared by 10-fold dilutions of the stock solutions with ethanol. These solutions are stored in the dark at 4 ºC (range 1 – 10 ºC) for a maximum period of 6 months. 4.16 Standards: Relevant standards are α-zearalanol, β-zearalanol, α-zearalenol, β-zearalenol, zearalanone and zearalenone. 4.16.1 Stock solutions, containing 1 mg/ml, are prepared by dissolving the appropriate amount of the analyte in ethanol. These solutions are stored in the dark at -20 ºC for a maximum period of 5 year. 4.16.2 Working solutions, containing 0.1 mg/ml, are prepared by 10-fold dilutions of the stock solutions with ethanol. These solutions are stored in the dark at 4 ºC (range 1 – 10 ºC) for a maximum period of 6 months. Relevant data of the analytes are listed in table 1.

Table 1: Information about analytes Analytes CAS # Formula Mol. Weight (g/mol)

α-zearalanol 26538-44-3 C18H26O5 322,4

β-zearalanol 42422-68-4 C18H26O5 322,4

α-zearalenol 36455-72-8 C18H24O5 320,4

β-zearalenol 71030-11-0 C18H24O5 320,4

zearalanone 17924-92 C18H24O5 320,4

zearalenone 597578-0 C18H22O5 318,4

4.17 Preparation of a mixture of standards. 4.17.1 Pipette 0.1 ml of each of the standard working solutions (4.16.2) in a 10 ml bottle and fill up with ethanol until the total volume is 10 ml. This is the 0.001 mg/ml standard mixture. 4.17.2 Dilute this mixture (4.17.1) 1:10 by mixing 0.5 ml of this standard mixture with 4.5 ml ethanol in a 10 ml bottle. Concentration is 0.1 ng/µl. This mixture of standards is used to fortify blank matrix and to make the calibration curve. 4.18 Preparation of a mixture of internal standards. 4.18.1 Pipette 0.1 ml of each of the internal standard working solutions (4.15.2) in a 10 ml bottle and fill up with ethanol until the total volume is 10 ml. This is the 0.001 mg/ml internal standard mixture.

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Printdatum: 1-Oct-08 r 4.18.2 Dilute this mixture (4.18.1) 1:10 by mixing 0.5 ml of this internal standard mixture with 4.5 ml ethanol in a 10 ml bottle. Concentration is 0.1 ng/ul. This mixture of internal standards is used with the urine samples and the calibration curve. Preparation of the calibration curve. Transfer standard mixture and internal standard mixture according to table 2 in 2 ml sample vials. After pipetting the required amounts, these standards are further processed from section 5.22 onwards.

Table 2. Calibration curve.

ID *) µl standard 0.1 ng/μl µl internal standard 0.1 ng/μl 0 0 100 0.2 10 100 0.4 20 100 1.0 50 100 2.0 100 100 4.0 200 100 10.0 500 100 *) the value of ID gives the amount of standards in ng/ml sample. This value is calculated by dividing the total amount of standard by the amount sample in ml.

5 Procedure Procedure for urine 5.1 Transfer a portion of 5 ml of the urine samples in 50 ml plastic tubes. 5.2 Transfer a portion of 5 ml blank urine in a 50 ml plastic tube and fortify this blank with 50 μl of the mixture of standards (4.17.2). 5.3 Add 100 µl of the I.S.-mix (4.18.2) to these samples. 5.4 Adjust the pH of the urines to 7 ±0.3 with 1 M HCl. 5.5 Add 1 ml phosphatebuffer, pH 7 (4.8). 5.6 Add 50 μl β-glucuronidase and deconjugate during 2 hrs at 37 ºC (4.5) 5.7 Cool the samples to room temperature. 5.8 Bring 10 ml TBME into the tubes and rotate during 10 min. 5.9 Centrifuge the tubes for 10 min with 3600 rpm. 5.10 Transfer the upper (organic) layer into a glass test tube and evaporate the TBME at 60 ºC under a gentle stream of nitrogen. 5.11 Redissolve the residue in 5 ml methanol/water 50/50 vol-% by carefully vortexing. 5.12 Add 2 ml of water to make the polarity of the solution suitable for the next step (SPE).

5.13 SPE C18: Precondition the C18 column with 5 ml of methanol followed by 5 ml water. 5.14 Pass the sample through the column and wash with 5 ml 40/60 v/v-% methanol/water. 5.15 Dry the column before the next step by applying slight vacuum. 5.16 Elute with 5 ml 80/20 v/v-% methanol/water. 5.17 Collect the eluate in a test tube by applying slight vacuum. 5.18 Dry the eluate at 50 ºC under a gentle stream of nitrogen. 5.19 Redissolve the dry residue in 5 ml of 80/20 v/v-% acetone/methanol.

5.20 SPE NH2: Precondition the NH2 column with 5 ml 80/20 v/v-% acetone/methanol. 5.21 Pass the sample through the column and collect the eluate.

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Printdatum: 1-Oct-08 r 5.22 Dry the eluate at 50 ºC under a gentle stream of nitrogen. 5.23 Redissolve the residue in 0.5 ml ethanol and transfer it into a Waters injection vial (4.18). 5.24 For calculation: Add (internal) standard mixtures to Waters injection vials according to table 2. 5.25 Evaporate the ethanol under a gentle stream of nitrogen at 50 ºC. 5.26 Redissolve the residue in 40 μl 40/60 v/v-% methanol/water. 5.27 Check the performance of the analytical system by injecting 20 μl of the lowest standard of the calibration curve. 5.28 Analyze samples and standards according to the following sequence: st 0 – st 0.2 – st 0.4 – etc.- blank –sample 1 – sample 2 – etc. – st – 10.0 Procedure for liver 5.29 Grind ca 10 g of liver with a blender. 5.30 Weight 2 g of this material in a 50 ml plastic test tube. 5.31 Weight 2 g of blank liver material in another test tube and fortify this sample with 50 µl of the mixture of standards (4.17.2) 5.32 Add 100 µl of the I.S.-mix (4.18.2) to these samples. 5.33 Add 5 ml acetate buffer, 2 M, pH 5.2 (4.11) and disintegrate the liver by use of the Bransonic 250 Sonifier equipped with a microtip under the following conditions: duty cycle 50%, output control 6, 20 – 25 cycles per sample. 5.34 Add 40 µl Suc Helix Pomatia (4.12) and vortex during 30 sec. 5.35 Check the pH and adjust when necessary to pH 5.2 with acetic acid (4.9). 5.36 Hydrolyse the mixture during 2 hrs at 37°C in an oven under slight shaking. 5.37 Bring 10 ml TBME in the tube and rotate during 10 min. 5.38 Continue the procedure as described starting from Procedure line 5.9. Analytical Method Table 3, 4 and 5 contain all information of the LC-MSMS method used.

Table 3. LC conditions.

Column temperature 40 ºC Sample temperature 5 ºC Injection volume 20 μl.

Gradient Time (min) A% (weak) B% (strong) Flow (ml/min) Curve 0 70 30 0.35 1 1 45 55 0.35 11 7.5 35 65 0.35 6 9 0 100 0.35 1 11 70 30 0.35 1

Table 4. MS conditions.

Ionization mode ES- Capillary (kV) 2.50 Cone (V) 50

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Printdatum: 1-Oct-08 r RF Lens 1 30 Aperture (V) 0.5 RF Lens 2 (V) 0.5 Exit (V) 2 Aperture (V) 0.5 Source temperature (°C) 130 Desolvation temperature (°C) 350 Cone gas flow (L/Hr) 95 Desolvation gas flow (L/Hr) 800 LM/HM 1 resolution 14.0 LM/HM 2 resolution 14.0 Ion energy 1 and 2 (V) 0 / 2 Entrance (V) -1 Multiplier (V) 650 Collision cell pressure (mbar) 3.0e-3

Table 5. Measured transitions.

Analyte MRM Dwell Cone Volt. Col. (secs) Energy Zearalenone * 317.15 > 0.075 50.0 25.0 131.15 Zearalenone ** 317.15 > 0.075 50.0 25.0 175.15 α/ß-Zearalenol * 319.15 > 0.075 50.0 25.0 160.15 Zearalanone ** & α/ß- 319.15 > 0.075 50.0 25.0 Zearalenol ** 275.15 Zearalanone * 319.15 > 0.075 50.0 25.0 205.15 α/ß-Zearalanol * 321.15 > 0.075 50.0 20.0 277.15 α/ß-Zearalanol ** 321.20 > 0.075 50.0 25.0 259.20 Zearalenon-D6 *** 323.15 > 0.075 50.0 25.0 134.15 α/ß-Zearalenol-D4 *** 323.15 > 0.075 50.0 25.0 160.15

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Printdatum: 1-Oct-08 r α/ß-Zearalanol-D4 *** 325.15 > 0.075 50.0 25.0 281.15 * Trace used for quantification ** Trace used for confirmation in combination with the quantification trace *** Internal standard

The suitability of the LC-MSMS system is checked by injection of the lowest standard of the calibration curve. If the S/N for all compounds is higher then 6 the system is considered as suitable for analysis of all the samples.

6 Calculation Areas of the selected ion of the standard and of the internal standard are determined with Masslynx. The ratio is the response variable. A calibration curve is constructed by linear curve fitting using least squares linear regression calculation. Unknown concentrations are calculated by interpolation by use of the program Calwer version 3. Criteria for compliance/non/compliance To judge if a sample is compliant or non-compliant, commission decision 2002/657 is used.

7 Validation and Measurement uncertainty The validation has been carried out according to the LC-MSMS conditions described in version 1 of this SOP (ARO SOP 489 version 1). The method described in this SOP was validated conform SOP ARO/475 The validation level used is 1.0 ng/ml. Table 6 gives an overview of the validation results for the different compounds. The complete validation report is archived by the QA-manager.

Table 6. Performance characteristics of the method

Compound transitions Internal standard CCα CCβ Measurement Uncertainty (%) α-zearalanol 321.15>277.15 α-zearalanol(d4) 0.11 0.18 0.53 321.20>259.20 0.14 0.24 0.58 β-zearalanol 321.15>277.15 β-zearalanol(d4) 0.27 0.47 5.24 321.20>259.20 0.38 0.65 2.95 α-zearalenol 319.15>275.15 α-zearalenol(d4) 0.19 0.33 1.76 319.15>160.15 0.20 0.34 4.09 β-zearalenol 319.15>275.15 β-zearalenol(d4) 0.35 0.59 1.90 319.15>160.15 0.28 0.48 1.59

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Printdatum: 1-Oct-08 r Zearalanone 319.15>275.15 α-zearalanol(d4) 0.13 0.23 3.42 319.15>205.15 0.24 0.41 6.43 Zearalenone 317.15>175.15 α-zearalanol(d4) 0.18 0.30 1.16 317.15>131.15 0.17 0.29 1.46

8 Quality control Quantification is only valid if: • The maximum of the signal originating from the analyte has an S/N ratio >3. • In the blank control samples all the internal standards are visible. • In the spiked control samples all components are visible (S/N ratio >6 for internal standards, >6 for the non-deuterated compounds). Process control Each series contains a spiked sample and all samples are spiked with deuterated internal standards, both should fulfill the criteria as stated in 7.1.

9 Responsibilities See ARO/MIS FUNC for the organization of responsibilities within ARO.

10 Relating documents Council Directive 96/22/EC of 29 April 1996 concerning the prohibition on the use in stockfarming of certain substances having hormonal or thyrostatic action and of beta-agonists and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EC. Off. J. Europ. Commun. 1996, L125, 3

M.Jonker, H.Egmond, R.Stephany, Mycotoxins in food of animal origin, CRL-document 389002 096, December 1999, 39

M Kleinova P Zöllner , H Kahlbacher, W Hochsteiner and W Lindner. Metabolic profiles of the mycotoxin Zearalenone and the growth promoter Zeranol in urine, liver and muscle of heifers. J. Agric. Food Chem. 50 (2002) 4769-4776.

FAIR-CT97-3443. An improved immunoassay for zeranol to distinguish zeranol abuse from natural contamination with Fusarium spp. toxins in food animals,

E O van Bennekom, L Brouwer, E H M Laurant, H Hooijering, M W F Nielen Confirmatory analysis method for zeranol, its metabolites and related mycotoxins in urine by liquid chromatography-negative ion electrospray tandem mass spectrometry. Anal. Chim. Acta 473 (2002) 151-160.

F M Launay, P B Young, S S Sterk, M H Blokland and Kennedy DG. Confirmatory assay for Zeranol, Taleranol and the Fusarium spp. Toxins in bovine urine using liquid chromatography-tandem mass spectrometry. Food Add. Contam. 21 (1) (2004a), 52-62

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Printdatum: 1-Oct-08 r J Jodlbauer, J Zöllner and W Lindner. Determination of Zeranol, Taleranol, Zearalenone, α- and β- Zearalenol in urine and tissue by high-performance liquid chromatography-tandem mass spectrometry. Chromatographia 51 (2000) 681-687.

F M Launay, L Ribeiro, P Alves, V Vozikis, STsitsamis, G Alfredsson, S S Sterk, M Blokland, A Iitia, T Lovgren, M Tuomola, A Gordon, D G Kennedy. Prevalence of zeranol, taleranol and Fusarium spp. toxins in urine: implications for the control of zeranol abuse in the European Union. Food Addit Contam. 21(9) (2004a) 833-9.

Commission Decision No. 2002/657/EC of 12 August 2002 implementing Council Directive 96/23 concerning the performance of analytical methods and the interpretation of results. Off. J. Europ. Comm. (2002): L221, 8

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Printdatum: 1-Oct-08 r Documentbeheer Algemeen Invoerdatum: 25 augustus 2006 Wijzigingsdatum: 29 januari 2008 Controledatum: 29 januari 2013 Publicatiedatum: 31 januari 2008 Wijzigingen ten opzichte van vorige versie: Wijziging van LC-systeem naar UPLC, toevoeging van lever als matrix

Beoordelaars Marco Blokland (Onderzoeker) Saskia Sterk (afdelingshoofd)

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