Leroy P, Decolin D, Nicolas S, Archimbault P, Nicolas A
Laboratoire de Chimie Analytique, URA CNRS 597, Faculté des Sciences Pharmaceutiques et Biologiques, Nancy, France.
J Pharm Biomed Anal. 1989;7(12):1837-46. doi: 10.1016/0731-7085(89)80201-8.
Residues of two antibacterial agents, cephalexin and colistin, co-administered by intramuscular injection to calves, were quantified in four different tissues (muscle, fat, liver and kidney) by column switching HPLC and by a microbiological method. For cephalexin assay, tissue samples with cephradin as internal standard were homogenized in a 5% trichloroacetic acid solution and filtrates were injected onto a concentration precolumn filled with LiChroprep RP-18 (25-40 microns). A clean-up step was incorporated by flowing a mobile phase (methanol-0.01 M phosphate buffer (pH 3.0); 15:85, v/v) through the enrichment column before elution on a LiChrospher RP-18e (5 microns) column with a methanol-phosphate buffer (30:70, v/v) at a flow rate of 1 ml min-1. Spectrometric detection was at 260 nm. An additional "off-line" washing step of extracts with methylene chloride was operated to achieve higher selectivity in the case of liver and kidney samples. The limit for quantitative assay was 0.045 micrograms g-1 with relative standard deviations in the range 5-8% and recoveries within 70%. For microbiological assay of colistin, samples were homogenized in 0.1 M hydrochloric acid-acetonitrile mixtures (3:1, v/v, for kidney and liver; 3:2, v/v, for fat and muscle). The supernatants were assayed by the cylinder plate method after evaporation to dryness under vacuum. Bordetella bronchiseptica ATCC 4617 was chosen as test organism. After a 3-h diffusion step at room temperature, the medium was incubated at 37 degrees C for 18 h and then the diameter of the growth inhibition zones was measured. Sensitivity reached 0.10-0.15 micrograms g-1. Results from the analysed samples over a 7-28 day period after drug administration show that no cephalexin was found at concentrations higher than the quantitation limit in the four test tissues and that colistin was found in muscle (injection site only) for 15 days and in kidney for 21 days.
通过柱切换高效液相色谱法（HPLC）和微生物学方法，对肌肉注射给予小牛的两种抗菌剂头孢氨苄和黏菌素在四种不同组织（肌肉、脂肪、肝脏和肾脏）中的残留量进行了定量分析。对于头孢氨苄的测定，以头孢拉定作为内标，将组织样品在5%的三氯乙酸溶液中匀浆，滤液注入装有LiChroprep RP - 18（25 - 40微米）的浓缩预柱。在以甲醇 - 磷酸盐缓冲液（30:70，v/v）、流速为1 ml min⁻¹在LiChrospher RP - 18e（5微米）柱上洗脱之前，通过使流动相（甲醇 - 0.01 M磷酸盐缓冲液（pH 3.0）；15:85，v/v）流经富集柱进行净化步骤。光谱检测波长为260 nm。对于肝脏和肾脏样品，还进行了额外的用二氯甲烷对提取物的“离线”洗涤步骤，以实现更高的选择性。定量测定限为0.045微克 g⁻¹，相对标准偏差在5 - 8%范围内，回收率在70%以内。对于黏菌素的微生物学测定，样品在0.1 M盐酸 - 乙腈混合物（肾脏和肝脏为3:1，v/v；脂肪和肌肉为3:2，v/v）中匀浆。真空蒸发至干后，上清液通过圆筒平板法进行测定。选择支气管败血波氏杆菌ATCC 4617作为测试菌株。在室温下扩散3小时后，将培养基在37℃下培养18小时，然后测量生长抑制圈的直径。灵敏度达到0.10 - 0.15微克 g⁻¹。给药后7 - 28天期间分析样品的结果表明，在四种测试组织中未发现浓度高于定量限的头孢氨苄，在肌肉（仅注射部位）中发现黏菌素达15天，在肾脏中发现达21天。
