Abstract
The use of thyreostatic drugs, like thiouracil (TU), in animal production has been banned for over three decades by the European Union, due to potential teratogenic and carcinogenic effects of its residues upon human consumption. Besides, thyreostats induce water retention in livestock, causing fallacious weight gain and deterioration of meat quality. Development of more competent analytical methods gave rise to sporadic TU detection in urine of untreated animals, questioning the actual synthetic origin TU. Research showed that TU can be formed upon digestion of Brassicaceae feeds in vivo and in vitro, which called for a means of differentiation between endogenous formation of TU and illicit administration. Therefore, in the present study, a routinely applicable liquid chromatography (LC) ion trap multiple mass spectrometry (MS2) method for TU analysis in animal feeds was optimised and validated, according to CD 2002/657/EC. A fractional factorial Plackett-Burman design was used to optimise the extraction procedure for TU from Brassicaceae and non-Brassicaceae feeds. This resulted in the discrimination of five influential factors (amount of feed, myrosinase, pH 7 buffer, 3-iodobenzyl bromide and elution solvent), for which the most optimal conditions were perfected. The limit of quantification for TU amounted 0.5 ng g−1. The individual recoveries for TU ranged between 90.9 and 99.7 %. Good results for repeatability and intra-laboratory reproducibility (RSD%) were observed, i.e. ≤6.0 and ≤5.2 %, respectively, for TU. Excellent linearity was proven based on determination coefficient (R 2 ≥ 0.99) and lack-of-fit test (F test, α = 0.05). Subsequently, a selection of feeds sampled during European national monitoring campaigns were evaluated with the present method showing concentrations ranging from 0.32 to 20.60 ng g−1, demonstrating the relevance of the method in the analysis of TU from animal feeds.
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References
Khera KS (1973) Ethylenethiourea—teratogenicity study in rats and rabbits. Teratology 7(3):243–252
Martinez-Frias ML, Cereijo A, Rodriguezpinilla E, Urioste M (1992) Methimazole in animal feed and congenital aplasia cutis. Lancet 339(8795):742–743
Eghbalian F (2007) Aplasia cutis congenita after methimazole exposure in utero; a case report and literature review. Iran J Pediatr 17(3):293–296
International Agency for Research on Cancer (2010) IARC monographs on the evaluation of carcinogenic risks to humans. Available online on: http://monographs.iarc.fr/ENG/Classification/index.php
European Community (1981) Council Directive 81/602/EC. Off J Euro Comm L 222:32–33
Pinel G, Maume D, Deceuninck Y, Andre F, Le Bizec B (2006) Unambiguous identification of thiouracil residue in urine collected in non-treated bovine by tandem and high-resolution mass spectrometry. Rapid Comm Mass Spec 20(21):3183–3187
Vanden Bussche J, Vanhaecke L, Deceuninck Y, Wille K, Bekaert K, Le Bizec B, De Brabander HF (2011) Ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry detection of naturally occurring thiouracil in urine of untreated livestock, domesticated animals and humans. Food Add Contam 28:166–172
European Food Safety Authority (2010) Report for 2008 on the results from the monitoring of veterinary medicinal product residues and other substances in food of animal origin in the Member States. EFSA J 8:1559. doi:10.2903/j.efsa.2010.1559
Pinel G, Bichon E, Pouponneau K, Maume D, Andre F, Le Bizec B (2005) Multi-residue method for the determination of thyreostats in urine samples using liquid chromatography coupled to tandem mass spectrometry after derivatisation with 3-iodobenzylbromide. J Chrom A 1085(2):247–252
Lõhmus M, Kallaste K, Le Bizec B (2009) Determination of thyreostats in urine and thyroid gland by ultra high performance liquid chromatography tandem mass spectrometry. J Chrom A 1216(46):8080–8089
Vanden Bussche J, Vanhaecke L, Deceuninck Y, Verheyden K, Wille K, Bekaert K, Le Bizec B, De Brabander HF (2010) Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation. J Chrom A 1217(26):4285–4293
Pinel G, Mathieu S, Cesbron N, Maume D, De Brabander HF, Andre F, Le Bizec B (2006) Evidence that urinary excretion of thiouracil in adult bovine submitted to a cruciferous diet can give erroneous indications of the possible illegal use of thyrostats in meat production. Food Add Contam 23(10):974–980
European Union Reference Laboratories (2007) EURL guidance document. Available online on: http://www.rivm.nl/bibliotheek/digitaaldepot/crlguidance2007.pdf
Zhou CH, Tokuhisa JG, Bevan DR, Esen A (2012) Properties of beta-thioglucoside hydrolases (TGG1 and TGG2) from leaves of Arabidopsis thaliana. Plant Sci 191:82–92
Vanden Bussche J, Kiebooms JAL, De Clercq N, Deceuninck Y, Le Bizec B, De Brabander HF, Vanhaecke L (2011) Feed or food responsible for the presence of low-level thiouracil in urine of livestock and humans? J Agr Food Chem 59(10):5786–5792
Krul C, Humblot C, Philippe C, Vermeulen M, van Nuenen M, Havenaar R, Rabot S (2002) Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model. Carcinogenesis 23(6):1009–1016
Cheng DL, Hashimoto K, Uda Y (2004) In vitro digestion of sinigrin and glucotropaeolin by single strains of Bifidobacterium and identification of the digestive products. Food Chem Toxicol 42(3):351–357
Tripathi MK, Mishra AS (2007) Glucosinolates in animal nutrition: a review. Anim Feed Sci Tech 132(1–2):1–27
Kiebooms JAL, Vanden Bussche J, Hemeryck LY, Fievez V, Vanhaecke L (2012) Intestinal microbiota contribute to the endogenous formation of thiouracil in livestock. J Agr Food Chem 60(32):7769–7776
Bones AM, Rossiter JT (1996) The myrosinase-glucosinolate system, its organisation and biochemistry. Physiol Plant 97(1):194–208
Rosa EAS, Rodrigues AS (2001) Total and individual glucosinolate content in 11 broccoli cultivars grown in early and late seasons. Hortscience 36(1):56–59
Wallig MA, Belyea RL, Tumbleson ME (2002) Effect of pelleting on glucosinolate content of Crambe meal. Anim Feed Sci Tech 99(1–4):205–214
Van Doorn HE, van der Kruk GC, van Holst GJ, Raaijmakers-Ruijs NCME, Postma E, Groeneweg B, Jongen WHF (1998) The glucosinolates sinigrin and progoitrin are important determinants for taste preference and bitterness of Brussels sprouts. Anim Feed Sci Tech 78(1):30–38
Mithen RF, Dekker M, Verkerk R, Rabot S, Johnson IT (2000) The nutritional significance, biosynthesis and bioavailability of glucosinolates in human foods. J Sci Food Agr 80(7):967–984
Tanii H, Takayasu T, Higashi T, Leng S, Saijoh K (2004) Allylnitrile: generation from cruciferous vegetables and behavioral effects on mice of repeated exposure. Food Chem Toxicol 42(3):453–458
Schöne F, Winnefeld K, Kirchner E, Grun M, Ludke H, Hennig A (1990) Copper and iodine in pig diets with high glucosinolate rapeseed meal. 3. Treatment of rapeseed meal with copper, and the effect of iodine supplementation on trace-element status and some related blood (serum) parameters. Anim Feed Sci Tech 30(1–2):143–154
Burel C, Boujard T, Escaffre AM, Kaushik SJ, Boeuf G, Mol KA, Van der Geyten S, Kuhn ER (2000) Dietary low-glucosinolate rapeseed meal affects thyroid status and nutrient utilization in rainbow trout (Oncorhynchus mykiss). Brit J Nutr 83(6):653–664
Tripathi MK, Agrawal IS, Sharma SD, Mishra DP (2001) Effect of substitution of soybean meal with treated or untreated high glucosinolate mustard (Brassica juncea) meal on intake, digestibility, growth performance and body composition of calves. Anim Feed Sci Tech 94(3–4):137–146
Zang XP, Tanii H, Kobayashi K, Higashi T, Oka R, Koshino Y, Saijoh K (1999) Behavioral abnormalities and apoptotic changes in neurons in mice brain following a single administration of allylnitrile. Arch Toxicol 73(1):22–32
Ahlin KA, Emanuelson M, Wiktorsson H (1994) Rapeseed products from double-low cultivars as feed for dairy cows—effects of long-term feeding on thyroid-function, fertility and animal health. Acta Vet Scand 35(1):37–53
Zukalova H, Vasak J (2002) The role and effects of glucosinolates of Brassica species—a review. Rost Vyroba 48(4):175–180
European Community (2003) Commission Regulation (EC) No 1035/2003. Off J Euro Comm L 150:24–25
European Community (2002) Commissions Decision 2002/657/EC. Off J Euro Comm L 221:8–36
Box GE, Hunter JS (1961) 2 k-P fractional factorial designs part I. Technometrics 3(3):311–351
Yu GYF, Murby EJ, Wells RJ (1997) Gas chromatographic determination of residues of thyreostatic drugs in bovine muscle tissue using combined resin mediated methylation and extraction. J Chrom B 703(1–2):159–166
Blanchflower WJ, Hughes PJ, Cannavan A, McCoy MA, Kennedy DG (1997) Determination of thyreostats in thyroid and urine using high-performance liquid chromatography atmospheric pressure chemical ionisation mass spectrometry. Analyst 122(9):967–972
Buick RK, Barry C, Traynor IM, McCaughey WJ, Elliott CT (1998) Determination of thyreostat residues from bovine matrices using high-performance liquid chromatography. J Chrom B 720(1–2):71–79
Pensabene JW, Lehotay SJ, Fiddler W (2001) Method for the analysis of thyreostats in meat tissue using gas chromatography with nitrogen phosphorus detection and tandem mass spectrometric confirmation. J Chromatogr Sci 39(5):195–199
Asea PE, MacNeil JD, Boison JO (2006) An analytical method to screen for six thyreostatic drug residues in the thyroid gland and muscle tissues of food producing animals by liquid chromatography with ultraviolet absorption detection and liquid chromatography/mass spectrometry. J AOAC Int 89(2):567–575
Broekaert N, Van Peteghem C, Daeseleire E, Sticker D, Van Poucke C (2011) Development and validation of an UPLC-MS/MS method for the determination of ionophoric and synthetic coccidiostats in vegetables. Anal Bioanal Chem 401(10):3335–3344
Wrede F (1941) Die Methoden der Fermentforschung, Georg Thieme, Leipzig, Bd2, 1835
De Brabander HF, Verbeke R (1982) Determination of oxazolidine-2-thiones in biological fluids in the ppb range. J Chrom A 252:225–239
Pollet B, Vanhaecke L, Dambre P, Lootens P, Steppe K (2011) Low night temperature acclimation of Phalaenopsis. Plant Cell Rep 30(6):1125–1134
Palmieri S, Leoni O, Iori R (1982) A steady-state kinetics study of myrosinase with direct ultraviolet spectrophotometric assay. Anal Biochem 123(2):320–324
Shen JZ, Zhang SX, Wu CM, Jiang HY, Wang ZH, Cheng LL (2010) Determination of six resorcylic acid lactones in feed by GC-MS. Chromatographia 71(1–2):163–165
Wang WY, Zhang YL, Wang JY, Shi X, Ye JN (2010) Determination of beta-agonists in pig feed, pig urine and pig liver using capillary electrophoresis with electrochemical detection. Meat Sci 85(2):302–305
Ying YF, Wu YL, Wen Y, Yang T, Xu XQ, Wang YZ (2013) Simultaneous determination of six resorcylic acid lactones in feed using liquid chromatography-tandem mass spectrometry and multi-walled carbon nanotubes as a dispersive solid phase extraction sorbent. J Chrom A 1307:41–48
Karnes HT, March C (1991) Calibration and validation of linearity in chromatographic biopharmaceutical analysis. J Pharm Biomed 9(10–12):911–918
Youssef YA, Beauchamp Y, Thomas M (1994) Comparison of a full factorial experiment to fractional and Taguchi designs in a lathe dry turning operation. Comput Ind Eng 27(1–4):59–62
Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325
Eriksson L, Johansson E, Kettaneh-Wold N, Wold S (2008) Multi- and megavariate data analysis. Principles and applications. Umetrics Academy, Umea
Beres DL, Hawkins DM (2001) Plackett-Burman techniques for sensitivity analysis of many-parametered models. Ecol Model 141(1–3):171–183
Jia JS, Deng HB, Duan J, Zhao JZ (2009) Analysis of the major drivers of the ecological footprint using the STIRPAT model and the PLS method—a case study in Henan Province. China Ecol Econ 68(11):2818–2824
Esteve-Romero J, Escrig-Tena I, Simo-Alfonso EF, Ramis-Ramos G (1999) Determination of thyreostatics in animal feed by micellar electrokinetic chromatography. Analyst 124(2):125–128
Perez-Ruiz T, Martinez-Lozano C, Sanz A, Galera R (2005) An ultrasensitive method for the determination of thiouracil and phenylthiouracil using capillary zone electrophoresis and laser-induced fluorescence detection. Electrophoresis 26(12):2384–2390
Kong DX, Chi YW, Chen LC, Dong YQ, Zhang L, Chen GN (2009) Determination of thyreostatics in animal feeds by CE with electrochemical detector. Electrophoresis 30(19):3489–3495
Woźniak B, Witek S, Matraszek-Żuchowska I, Żmudzki J (2014) Determination of the thyreostats in animal feeding stuffs using liquid chromatography-tandem mass spectrometry. Bull Vet Inst Pulawy 58(3):413–419
Arioli F, Gavinelli MP, Fracchiolla ML, Casati A, Fidani M, Ferrer E, Pompa G (2008) Evaluation of boldenone formation and related steroids transformations in veal faeces by liquid chromatography/tandem mass spectrometry. Rapid Comm Mass Spec 22(2):217–223
McCarrison R (1933) The goitrogenic action of soybean and groundnut. Indian J Med Res 21:179
Velasco P, Soengas P, Vilar M, Cartea ME, del Rio M (2008) Comparison of glucosinolate profiles in leaf and seed tissues of different Brassica napus crops. J Am Soc Horticult Sci 133(4):551–558
European Union Reference Laboratories (2014) EURL reflection paper. Not yet published online (http://www.wageningenur.nl/nl/Publicatie-details.htm?publicationId=publication-way-343536373039)
Acknowledgments
The authors would like to thank J. Goedgebueur and D. Stockx for their technical support as well as Tierenteyn (Deinze, Belgium) for the supplying of mustard seeds. We are also thankful to the Irish Department of Agriculture, Food and the Marine, Veterinary Medicines division, Backweston Campus (Celbridge, Co. Kildare, Ireland), for their collaboration and supply of animal feeds.
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Published in the topical collection on Hormone and Veterinary Drug Residue Analysis with guest editors Siska Croubels, Els Daeseleire, Sarah De Saeger, Peter Van Eenoo, and Lynn Vanhaecke.
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Kiebooms, J.A.L., Wauters, J., Vanden Bussche, J. et al. Validation of a quantitative method using liquid chromatography coupled to multiple mass spectrometry for thiouracil in feedstuffs used in animal husbandry. Anal Bioanal Chem 407, 4373–4384 (2015). https://doi.org/10.1007/s00216-014-8347-x
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DOI: https://doi.org/10.1007/s00216-014-8347-x