Analysis of prohormones in animal feed by GC-MS

Transcription

1 Analysis of prohormones in animal feed by GC-MS Document: SOP ARO/514 versie 1 Auteur: Klaas van Twillert Documentgebied: ARO Methods Autorisator: Leen van Ginkel (labhoofd) Status: Definitief Pagina 1 van 10

2 1 Introduction This SOP describes the method of analysis for prohormones, listed in table 1, in animal feed by GC- MS. The prohormones are extracted from the animal feed samples by liquid-liquid extraction with tertiair-butylmethylether (TBME). The TBME is evaporated and the product is redissolved in 40/60 v/v- % methanol/water. This solution is washed with heptane to remove fatty compounds. Subsequently the prohormones are extracted by using a SPE C18 column and a NH SPE column. After derivatisation with heptafluorobutyric anhydride the prohormones are analysed with GC-MS. The method is a validated as a quantitative screening method (table 1). TABLE 1 OVERVIEW OF PROHORMONES nr Name trivial name molecular formula 1 4-estren-3β,17β-diol 19-nor-4-androsten- 3β,17β-diol C18H8O 5(10)-estren-3β,17βdiol 19-nor-5-androsten- C18H8O 3β,17β-diol 3 4-estren-3,17-dione 19-nor-4-androsten- C18H4O 3,17-dione 4 4-estren-17β-ol-3-one (β) 19-nortestosterone C18H6O 5 4-androsten-17β-ol-3- (β) testosterone C19H8O one 6 4-androsten-3β, 17βdiol C19H30O 7 5-androsten-3β, 17βdiol C19H30O 8 4-androsten-3, 17- C19H6O dione 9 5β-androstan-17β-ol- 5βdihydrotestosterone C19H30O 3-one 10 5-androsten-3β-ol-17- DHEA C19H8O one 11 4-estren-17α-ol-3-one (α) 19-nortestosterone C18H6O 1 4-androsten-17α-ol-3- (α) testosterone C19H8O one 13 5α-androstan-3β-ol- epiandrosterone C19H30O 17-one 14 1, 4-androstadien-3. C19H4O 17-dione 15 5-androsten-3, 17- C19H6O dione 17 5α-androstan-17β-ol- C19H30O 3-one 18 4-estren-4-chloro- androstanolone (5αdihydrotestosterone) C19H30O 17β-ol, 3-one 19 5-androsten-3β-ol-7, 7-keto DHEA C19H6O 17-dione 3 0 1, (5α)-androsten- 1- C19H8O 17β-ol-3-one dehydroandrostanolon mol weight (g/mol) CCa (ng/g ) CCβ (ng/g ) U (%) Pagina van 10

3 1 1, (5α)-androsten-3, 17-dione e 1-androstenedione C19H6O Apparatus Reference to a company and/or product is for purpose of identification and information only..1 Bottles, 10 ml, brown glass, with screw cap.. SPE C18 column, 1 g, 6 ml, Alltech # SPE NH column, 1 g, 6 ml, Alltech # Aspec XL, an automatic sample preparation system, Gilson. The Aspec consists of a model 401 dilutor, a sample processor and racks to handle SPE columns..5 GC-MS equipment;.5.1 A Hewlett Packard 6890 GasChromatograph equipped with a 6890 injector and a Mass Selective Detector (MSD 5973)..5. Injector: injection vol. : μl. injection mode : pulsed splitless injector temp : 50 C pressure : 14.0 psi pulse time : 1.10 min purge flow : 40.0 ml/min. purge time : 1.00 min. total flow : 43.8 ml/min. gas saver : On saver flow : 0.0 ml/min. saver time : 6.00 min..5.3 Column: Factor Four capillary column, VF-1MS, 60 m x 0.5 mm i.d., DF 0.5 μm, Varian # CP Max. temp.: 345 C Mode: constant flow Initial flow: 0.7 ml/min. Temperature program: initial temp: 100 C, initial time min. Rate ( C/min.) Final temp. Final time (Off) Run time: 37.1 min. Pagina 3 van 10

4 .5.4 Detector: ions are recorded in SIM mode..6 Plastic centrifuge tubes, 50 ml. 3 Safety and environment The RIVM has defined rules to work safe. These rules can be found on the intranet. 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. Tris(hydroxymethyl)aminomethane 4..1 Tris buffer, 0.1 molair, ph 9.5. Dissolve 1.1 g Tris(hydroxymethyl)aminomethane in 800 ml water. Adjust the ph to 9.5 ± 0.1 with hydrochloric acid and add water to a volume of 1000 ml. 4.3 Tertiair-ButylMethylEther (TBME). 4.4 Methanol Methanol/water, 40/60 v/v-%. Mix 400 ml methanol with 600 ml water. 4.5 Heptane. 4.6 Ethanol. 4.7 Acetone. 4.8 Heptafluorobutyric Anhydride (HFBA); Pierce # /75 v/v-% HFBA acetone mixture: mix 50 μl HFBA and 750 μl acetone. 4.9,, 4-Trimethylpentane (iso-octane) Stock solution: Weight 3 (=x) mg prohormone in a 10 ml bottle. Dissolve the prohormone in x ml ethanol. This is the 1 mg/ml stock solution and is stored in the dark at -180C. Working solution: Dilute the stock solution 1:10 with ethanol. This is the 100 μg/ml working solution and is stored at 40C Prohormone enrichment mixture. Bring 100 μl of all the prohormone working solutions in a 10 ml bottle and fill up to 10 ml with ethanol. This is the 1 μg/ml (1 ng/μl) enrichment mixture Internal standard mixture. A mixture of internal standards is used in this experiment: Prepare as described in.10 and.10.1 working solutions of Testosteron-d and Nortestosteron-d3. Pagina 4 van 10

5 Bring 100 μl of these working solutions in a 10 ml bottle and fill up to 10 ml with ethanol. This is the 1 ng/μl internal standard mixture. 5 Procedure Sometimes the animal feed samples, suspensed in the Trisbuffer, form a gel when they are shaken with TBME. It concerns the samples with a white, milkpowder like appearance. These samples are handled in an alternative way. The lines hereunder are then replaced with lines Normal clean up 5.1 Samples are stored at -0 C until analysis. 5. Weight a gram portion of the sample in a 50 ml plastic tube. 5.3 Weight 6 portions of blank animal feed in plastic tubes to prepare the calibration line. 5.4 Spike these samples animal feed with a mixture of prohormones standards according to table. TABLE PREPARATION OF CALIBRATION CURVE Code of Standard Volume (μl) enrichment mixture (.10.) concentration in ng/g Add 40 μl internal standard mixture (.11) to all tubes. 5.6 Add 5 ml Trisbuffer to all tubes and shake the tubes. 5.7 Add 10 ml Tertiair-ButylMethylEther (TBME) to the tubes. 5.8 Extract the prohormones into the TBME-layer by rotating the tubes during 5 min. 5.9 Centrifuge the tubes for 10 min with 900 g Transfer the upper (organic) layer into a glass testtube and evaporate the TBME at 60 C under a gentle stream of nitrogen Bring another 5 ml of TBME in the plastic tubes and repeat the extraction procedure ( ). 5.1 Bring this TBME layer too in the glass testtube and evaporate Dissolve the dry residue in 5 ml 40/60 v/v-% methanol/water Add 1 ml heptane and shake vigorously on a Vortexmixer to remove fatty components Centrifuge the testtubes during 10 min with 900 g Discard the heptane layer Filter the remaining methanol water fraction via a 5 ml pipette tip filled with a small wad of cotton wool into another glass testtube (small, 75 x 1 mm) Clean this solution by use of SPE columns (C18, NH). The SPE clean up is performed by using the Aspec XL. The complete method is described in the ASPEC-method METHPROHORMONEN. See appendix 1. A short description of the method: a C18 SPE column is activated with methanol and flushed with water. The sample is passed through the column. The column is washed with water, followed by a 40/60 v/v-% methanol/water wash. The components are eluted from the C18 column into a testtube with acetone. The NH column is preconditioned with acetone. The sample is passed through this column and collected by air push in a testtube Evaporate the acetone at 60 C under a gentle stream of nitrogen. Pagina 5 van 10

6 5.0 Dissolve the residue in 1 ml ethanol and transfer this into a ml derivatisation vial. 5.1 Evaporate the ethanol under N flow at 60 C. 5. Derivatise the prohormones with 5 μl of 5/75 v/v-% HFBA acetone mixture during 1 hour at 60 C. 5.3 Remove the HFBA acetone mixture under N flow at 60 C. 5.4 Redissolve the product in 5 μl iso-octane and transfer it into an insert which is thereupon placed in this vial and mount a new screwcap. 5.5 Place the vials in the GC-MS autosampler and inject μl on the GC-MS. 5.6 Detect the components according to m/z values in table Add another 50 μl iso-octane in the insert and reinject the sample when the peaks have a bad shape (e.g. broader, tailing) compared to the standards. Alternative clean up 5.8 Add 10 ml Tertiair-ButylMethylEther (TBME) to the tubes. 5.9 Rotate these tubes during 90 min head over head Centrifuge the tubes during 10 min with 900 g Transfer the upper TBME layer through a paper Schleicher & Schüll filter (S&S whiteband, diam. 110 mm, ref.# ) into a 10 ml pipette tip equipped with a clad cotton wool in the tip. 5.3 Collect the TBME fraction in a glass test tube Evaporate the TBME fraction at 60 C under a gentle stream of nitrogen 6 Calculation Check The first step is a performance check of the GC-MS system after tuning. This is done by injecting a 10 ng/g standard (table ). The S/N ratio of the components is determined. This must be >100. Calculation Areas of the selected ion of the standard and of the internal standard are calculated. 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. TABLE 3 DIAGNOSTIC IONS HFBA DERIVATIVES nr name m/z prohormone Other ions Internal standard M/z int. stand. 1 4-estren-3β,17β-diol nortestosteron(d3) (10)-estren-3β,17β-diol nortestosteron(d3) estren-3,17-dione nortestosteron(d3) estren-17β-ol-3-one nortestosteron(d3) androsten-17β-ol-3-one testosteron(d) androsten-3β, 17β-diol testosteron(d) androsten-3β, 17β-diol testosteron(d) androsten-3, 17-dione testosteron(d) β-androstan-17β-ol-3-one testosteron(d) androsten-3β-ol-17-one testosteron(d) estren-17α-ol-3-one nortestosteron(d3) androsten-17α-ol-3-one testosteron(d) α-androstan-3β-ol-17-one testosteron(d) 68. Pagina 6 van 10

7 14 1,4-androstadien dione testosteron(d) androsten-3, 17-dione testosteron(d) α-androstan-17β-ol-3-one testosteron(d) estren-4-chloro-17β-ol, 3-one testosteron(d) androsten-3β-ol-7, 17-dione testosteron(d) , (5α)-androsten-17β-ol-3-one testosteron(d) , (5α)-androsten-3, 17-dione testosteron(d) 68. Note: other ions can be used for confirmation purposes to fulfil the confirmation criteria as described in commission decision 00/ Validation and Measurement uncertainty The method described in this SOP was validated during routine control experiments. Samples were spiked at 0, 5, 10, 0, 5 and 50 μg kg-1. The additions of three separate experiments were combined and further evaluated by use of weighted regression and the model Y=aX+b. From these data is conform CD 00/657 the CCα and CCβ calculated according the following formulas. See table 1 for an overview of the CCa and CCß. The reproducibility is determined by combining the results from the three separate series and also combining the results of the different spiking levels (the data is normalized). In table 4 is the results for the determination of the reproducibility shown. TABLE 4 RESULT DETEMINATION REPRODUCIBILITY Concentration (ng/g) 4-estren-3b,17b-diol 5(10)-estren-3b,17b-diol 4-estren-3,17-dione * 4-estren-17b-ol-3-one 4-estren-17a-ol-3-one 4-androsten-17b-ol-3-one 4-androsten-3b,17b-diol 5-androsten-3b,17b-diol 4-androsten-3,17-dione/5- androsten-3,17-dione 5b-androstan-17b-ol-3-one 5 16,6 13,8 103,4 116,6 116, 18, 138,8 117,0 111,8 103, ,5 115,0 94,8 98,9 95,0 105,4 103,4 98,0 99,6 98,9 0 16,5 10,5 100,7 106,7 106,7 11,0 131,1 101,9 106,3 98,8 5 79,3 88,4 8,9 89, 88,5 9,0 77,9 86,1 84,9 83, ,8 105,8 91,1 101,9 104,6 101,3 103,5 99,0 90,0 104,0 Pagina 7 van 10

8 5 90,6 113, 96,4 10,8 10,8 114,8 91,0 100, 99,0 98, ,9 10,8 89,5 100, 104,6 104,9 104,1 103,4 9,3 98, ,8 97,3 87,7 97,4 10,6 98,5 114,5 9, 87,9 88,1 5 93,0 101,4 93,0 100,4 101,3 100,5 96,1 99,4 9, 91, , 97,4 78,6 88, 88, 101,6 97,4 111,8 90,0 95, 10 87,3 107,0 90,6 99,6 99,5 110,6 10,1 101,0 97,9 109,7 0 89,1 106,6 88,0 100, 100,9 104, 89,7 99,9 95,5 97, , 103,8 91,6 97,7 99, 100,9 95,8 103,1 95,1 109, ,1 101,8 90,8 98,8 99,1 98,7 9,9 100,9 9,1 104,1 average 11,1 104,8 91,4 99,9 100,7 105,3 10,7 101,0 95,3 98,8 CV (%) 3,4 8, 7,0 6,8 7,1 8,5 15,7 7,3 7,6 7,6 U 64,8 16,4 14,1 13,6 14,1 16,9 31,4 14,5 15, 15,1 Concentration (ng/g) 5-estrene-3a--ol-17-one 4-androsten-17a-ol-3-one 5a-androstan-3b-ol-17-one 1,4-androstadien-3,17-dione* 5a-androstan-17b-ol-3-one 4-estren-4-chloro-17b-ol,3-one 5-androsten-3b-ol-7,17-dione 1,(5a)-androsten-17b-ol-3-one 1,(5a)-androsten-3,17-dione 5 18,8 1,4 11,0 15,8 113,4 8,8 93,6 18, 11, ,1 91,4 107, 115,0 97,8 76,1 13,9 13,0 103, ,1 103,6 101,7 11,4 101,4 74,4 88,6 116,8 98,7 5 78,7 88, 84,4 90,5 83, 63,1 77,7 95,8 84, ,9 99,6 10,3 94,7 10,7 57,9 61,9 106,9 101, , 105, 107,6 11,0 86,0 60,8 118,0 14,4 78, ,9 109,3 9,1 99,8 90,3 54,9 78,6 14,7 80,5 0 9,7 10,5 86,0 94,5 87,7 54,0 61,3 1,8 78,7 5 90,0 101,3 93,4 94,6 91, 59,8 67,8 103,7 74, ,6 10,6 91,6 10, 98,6 65,4 11, 114, 97, ,5 114,7 96,4 111,0 108,1 67,5 90,1 118,0 99, ,4 108,9 10,6 114,1 103,8 65, 7,6 104,0 103,9 5 96, 108, 98,7 10,1 99,4 71,8 78, 116,9 100, ,0 103,6 97,8 98,0 100,5 67, 7,7 110,0 99,8 average 101,4 104,4 98,1 104,8 97,4 65,8 86, 115,6 93,7 CV (%) 1,9 8, 8,3 9,8 8,9 1,7 4,6 9,1 1,8 U 5,9 16,5 16,5 19,7 17,9 5,3 49, 18,1 5,5 * Compounds are not separated on the GC and share therefore the validation results 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 >10 for internal standards, >6 for the non-deuterated compounds). 9 Responsibilities See ARO/MIS FUNC for the organization of responsibilities within ARO. 10 Relating documents Pagina 8 van 10

9 10.1 SOP ARO/475, Method validation using ResVal, revision, 1 april 004, RIVM. 10. Multi residue analysis anabolic agents, SOP ARO/113, revision 4, 1 january 1997, RIVM Commission Decision 00/657/EC of 1 August 00 implementing Council Directive 96/3/EC concerning the performance of analytical methods and the interpretation of results Annex The cleanup method with the Aspec: Methprohormonen. Pagina 9 van 10

10 Documentbeheer Algemeen Invoerdatum: 0 februari 007 Wijzigingsdatum: 14 augustus 007 Controledatum: 31 december 01 Publicatiedatum: januari 008 Wijzigingen ten opzichte van vorige versie: Omzetting naar HTML vervangt SOP ARO/481 Beoordelaars Marco Blokland (Onderzoeker) Saskia Sterk (afdelingshoofd) Hyperlinks Pagina 10 van 10