Tuinstra L G, Traag W A, Keukens H J, van Mazijk R J
J Chromatogr. 1983 Nov 25;279:533-42. doi: 10.1016/s0021-9673(01)93654-4.
As gas chromatography-mass spectrometry is the most conclusive confirmation technique available today for the detection of ppb levels of anabolics in the urine of cattle, the following procedure was used. The urine is hydrolysed with Helix pomatia intestinal juice, extracted, and the extract cleaned by gel-permeation chromatography or with Extrelut. In one fraction are eluted diethylstilboestrol, dienoestrol, hexoestrol, methyltestosterone, ethinyloestradiol, zeranol and trenbolone. This fraction is injected into a two-dimensional high-performance liquid chromatography system. From the first column, the fraction containing the above-mentioned compounds is transferred to the second column, where a separation into two fractions is obtained. The first fraction contains zeranol and trenbolone, the second fraction the stilbenes, methyltestosterone and ethinyloestradiol. In general, the compounds are derivatized with an N,O-bis (trimethylsilyl)trifluoroacetamide-trimethylchlorosilane mixture to a trimethylsilyl derivative. With stilbene, confirmatory derivatization into heptafluorobutyryl derivatives is necessary. In combination with a Finnigan 4000/INCOS system, a CP-Sil-5 CB and a CP-Sil-19 CB capillary are used for final confirmation. Two capillaries with different polarities are necessary to overcome problems with possible interferences from compounds extracted from the urine. Recoveries at the ppb level are better than 80%.
由于气相色谱 - 质谱联用技术是目前检测牛尿液中十亿分之一级别的合成代谢类固醇最具决定性的确认技术，因此采用了以下步骤。尿液先用缢蛏肠液进行水解，然后萃取，萃取液通过凝胶渗透色谱法或用硅酸镁载体进行净化。在一个馏分中洗脱得到己烯雌酚、双烯雌酚、己烷雌酚、甲基睾酮、炔雌醇、玉米赤霉醇和群勃龙。将该馏分注入二维高效液相色谱系统。从第一根色谱柱中，含有上述化合物的馏分被转移到第二根色谱柱，在那里得到分离成两个馏分。第一个馏分含有玉米赤霉醇和群勃龙，第二个馏分含有芪类、甲基睾酮和炔雌醇。一般来说，这些化合物用N，O - 双（三甲基硅基）三氟乙酰胺 - 三甲基氯硅烷混合物衍生化为三甲基硅基衍生物。对于芪类，需要确证衍生化为七氟丁酰基衍生物。与菲尼根4000/INCOS系统联用，使用CP - Sil - 5 CB和CP - Sil - 19 CB毛细管进行最终确认。需要两根不同极性的毛细管来克服尿液中提取的化合物可能产生干扰的问题。十亿分之一级别下的回收率优于80%。
