Cottingham L S, Smallidge R L
Purdue University, Department of Biochemistry, West Lafayette, IN 47907.
J Assoc Off Anal Chem. 1988 Sep-Oct;71(5):1012-6.
A sample portion is hydrolyzed with 6N HCl for 23 h and cooled, the pH is adjusted to 7.7 with NaOH, and the solution is diluted with pH 7.7 borate buffer. An aliquot of the sample extract is derivatized with 9-fluorenylmethyl chloroformate (9-FMC). Lysine is separated from other amino acids by isocratic reverse-phase liquid chromatography (LC) using fluorescence detection: 260 nm excitation and 313 nm emission. The mobile phase is acetonitrile-0.1M acetic acid (pH 4.2) buffer (53 + 47). Linearity is satisfactory over a range of 0.4-24 micrograms/mL. Results from 9 feed samples (1.1-2.7% lysine) analyzed by both the LC method and an amino acid analyzer were not significantly different statistically. Recovery of standard lysine, spiked just before derivatization on these same 9 samples (in duplicate), was 100.9% with a coefficient of variation (CV) of 2.4%. A study of within-day and between-day method precision resulted in CVs of 1.1 and 1.8%, respectively. The variation of results was negligible when the method was tested for ruggedness on 7 factors.
取一份样品用6N盐酸水解23小时后冷却，用氢氧化钠将pH值调至7.7，并用pH 7.7的硼酸盐缓冲液稀释溶液。取一份样品提取物用9-芴甲基氯甲酸酯（9-FMC）进行衍生化。通过等度反相液相色谱（LC）并采用荧光检测（激发波长260nm，发射波长313nm）将赖氨酸与其他氨基酸分离。流动相为乙腈-0.1M乙酸（pH 4.2）缓冲液（53 + 47）。在0.4 - 24微克/毫升范围内线性良好。用LC法和氨基酸分析仪分析的9份饲料样品（赖氨酸含量为1.1 - 2.7%）的结果在统计学上无显著差异。在这相同的9份样品（一式两份）衍生化前加入标准赖氨酸，回收率为100.9%，变异系数（CV）为2.4%。日内和日间方法精密度研究的变异系数分别为1.1%和1.8%。当对该方法进行7个因素的耐用性测试时，结果的差异可忽略不计。
