College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, People's Republic of China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Aug 1;879(23):2351-4. doi: 10.1016/j.jchromb.2011.06.027. Epub 2011 Jun 22.
A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.
建立了一种同时测定鸡蛋中氯霉素（TAP）、氟苯尼考（FF）和氟苯尼考胺（FFA）的反相高效液相色谱荧光检测（RP-HPLC-FLD）法。样品用乙酸乙酯-乙腈-氨水（49:49:2，v/v）提取，用正己烷脱脂，然后进行 RP-HPLC-FLD 测定。采用 5μm LiChrospher C(18)柱，流动相为乙腈（A）、0.01 M 磷酸二氢钠中含 0.005 M 十二烷基硫酸钠和 0.1%三乙胺，用 85%磷酸调 pH 至 4.8（A:B，35:65 v/v），流速为 1.0mL/min。HPLC 的荧光检测器激发波长为 224nm，发射波长为 290nm。TAP 和 FF 在鸡蛋中的检测限（LOD）为 1.5μg/kg，FFA 的检测限（LOD）为 0.5μg/kg；TAP 和 FF 在鸡蛋中的定量限（LOQ）为 5μg/kg，FFA 的定量限（LOQ）为 2μg/kg。TAP 的浓度在 0.025-5.0μg/mL 范围内，FF 的浓度在 0.01-10.0μg/mL 范围内，FFA 的浓度在 0.0025-2.50μg/mL 范围内，校准曲线的线性相关系数分别为 0.9997、0.9997 和 0.9998。TAP 的回收率为 86.4%-93.8%，FF 的回收率为 87.4%-92.3%，FFA 的回收率为 89.0%-95.2%。日内和日间变异（相对标准偏差，R.S.D.）分别小于 6.7%和 10.8%。
