Bagnati R, Fanelli R
Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
J Chromatogr. 1991 Jun 28;547(1-2):325-34. doi: 10.1016/s0021-9673(01)88656-8.
The known reaction of 3-ketosteroids with carboxymethoxylamine (to form the corresponding carboxymethoximes), followed by esterification of the carboxyl group with pentafluorobenzyl bromide, has been used to obtain derivatives of 19-nortestosterone, testosterone and trenbolone suitable for high-sensitivity detection with gas chromatography-negative-ion chemical ionization mass spectrometry. These derivatives, after further silylation of the alcoholic groups of the steroids, showed excellent chromatographic and spectrometric characteristics and were detectable in the low picogram range. The derivatization gave rise to the formation of two isomers which were distinguishable by gas chromatography. The existence of the two isomers was also confirmed by high-performance liquid chromatography. Examples of the usefulness of this derivatization procedure are given for the analysis of 19-nortestosterone, testosterone and trenbolone in meat and urine samples. By the use of immunoaffinity extraction and addition of deuterated internal standards (synthesized by isotopic exchange), the new derivatization procedure allowed a correct identification and quantitation of the steroids and reached very low detection limits [0.02 ppb (10(9] for 19-nortestosterone and testosterone, 0.06 ppb for trenbolone].
已知3-酮甾体与羧甲氧基胺反应（形成相应的羧甲氧肟），然后用五氟苄基溴将羧基酯化，已被用于获得适合用气相色谱-负离子化学电离质谱进行高灵敏度检测的19-去甲睾酮、睾酮和群勃龙的衍生物。这些衍生物在甾体的醇基进一步硅烷化后，表现出优异的色谱和光谱特性，并且在低皮克范围内可检测到。衍生化产生了两种异构体，它们可通过气相色谱区分。这两种异构体的存在也通过高效液相色谱得到了证实。给出了该衍生化程序在分析肉类和尿液样品中的19-去甲睾酮、睾酮和群勃龙方面的有用性示例。通过使用免疫亲和萃取并添加氘代内标（通过同位素交换合成），新的衍生化程序能够对甾体进行正确的鉴定和定量，并达到非常低的检测限[19-去甲睾酮和睾酮为0.02 ppb（10⁻⁹），群勃龙为0.06 ppb]。
