Civitareale C, Fiori M, Ballerini A, Brambilla G
Istituto Superiore di Sanità, Laboratorio Medicina Veterinaria, Viale Regina Elena, 299 I-00161 Rome, Italy.
J Pharm Biomed Anal. 2004 Oct 29;36(2):317-25. doi: 10.1016/j.jpba.2004.06.010.
The use of the two macrolides antibiotics Spiramycin (S) and Tylosin (T) as growth promoters in animal feeding has been recently withdrawn in the European Union due to a concern about the outbreaks of farmacoresistance fenomena as a possible hazard for humans. For feed additives monitoring purposes, an analytical method has been developed for their extraction, purification and identification in different animal feedingstuffs (pelleted beef, pig, poultry feeds and calves milk replacer) at a minimum performance required limit (MRPL) of 1 microg g(-1) (ppm). Such limit has been established according to the lowest dosage of additives still able to elicit an appreciable growth promoting effect. Blank feeds were spiked at two concentration levels, 1.0 and 2.5 ppm in six replicates. After methanolic extraction, samples were cleaned up on SPE CN columns and extracts analysed in HPLC-UV/DAD, using a gradient elution. Detection limits, calculated as the tree time mean noise of 20 blank feeds, were 176 and 118 ng g(-1) for S and T, respectively. Results show good repeatability (CV% not exceeding the value of 15) and mean recovery in the range of 99-74% and 81-53% for S and T, respectively, at 1 ppm. When the standards were injected up to 250 ng the chromatographic method can resolve the components of analytes (Spiramycin I, II and III; Tylosin A and B) but can not resolve the components on real feed samples at the spiked levels considered. For this reason the identification and quantification of analytes on matrix were carried out considering the main compound of the drugs (Spiramycin I and Tylosin A). As a verification, the overlapping of UV spectra in the range 220-350 nm between analytical standards and the compounds in the matrix were considered.
由于担心大环内酯类抗生素耐药现象的爆发可能对人类构成危害,欧盟最近已停止在动物饲养中使用两种大环内酯类抗生素——螺旋霉素(S)和泰乐菌素(T)作为生长促进剂。为了进行饲料添加剂监测,已开发出一种分析方法,用于在最低性能要求限度(MRPL)为1微克/克(ppm)的情况下,从不同动物饲料(牛肉颗粒饲料、猪饲料、家禽饲料和犊牛代乳粉)中提取、纯化和鉴定这两种抗生素。该限度是根据仍能产生明显生长促进作用的添加剂最低剂量确定的。空白饲料以1.0 ppm和2.5 ppm这两个浓度水平进行加标,每个浓度水平重复6次。经过甲醇提取后,样品在固相萃取CN柱上进行净化,提取物采用梯度洗脱,在高效液相色谱-紫外/二极管阵列检测器(HPLC-UV/DAD)上进行分析。以20份空白饲料的三倍时间平均噪声计算,S和T的检测限分别为176纳克/克和118纳克/克。结果表明,在1 ppm时,重复性良好(变异系数不超过15%),S和T的平均回收率分别在99%-74%和81%-53%之间。当进样标准品高达250纳克时,色谱方法可以分离分析物的组分(螺旋霉素I、II和III;泰乐菌素A和B),但在考虑的加标水平下,无法分离实际饲料样品中的组分。因此,在基质上对分析物进行鉴定和定量时,考虑的是药物的主要化合物(螺旋霉素I和泰乐菌素A)。作为验证,还考虑了分析标准品与基质中化合物在220-350纳米范围内紫外光谱的重叠情况。