高效液相色谱分析土壤氨基糖的质量控制方法

Quality Control for Determining Amino Sugar Contents in Soil by High Performance Liquid Chromatography

利用邻苯二甲醛(OPA)柱前在线衍生–高效液相色谱法测定了土壤中4种氨基糖,即胞壁酸(MurN)、甘露糖(ManN)、氨基半乳糖(GalN)和氨基葡萄糖(GluN)的含量,并从样品出峰、理论塔板数、信噪比、检出限、定量限、样品的重复性、标准样品的线性和加标回收率方面评估了检测过程中仪器和方法的灵敏度、精密度和准确度。结果表明:检测过程中,4种氨基糖的出峰先后顺序为胞壁酸、甘露糖、氨基半乳糖和氨基葡萄糖;4种氨基糖的色谱峰峰形好,相邻两峰的分离度>1.5,相邻两峰能够完全分离;理论塔板数>2000,色谱柱的柱效高;胞壁酸、甘露糖、氨基半乳糖和氨基葡萄糖的仪器检出限分别为0.030、0.300、0.150和0.150μmol/L,仪器定量限分别为0.100、1.000、0.500和0.500μmol/L,方法检出限分别为0.010、1.500、0.100和0.100μmol/L,方法定量限分别为0.030、5.000、0.300和0.300μmol/L;除0~10 cm以下土层中的甘露糖外,其他土壤样品中氨基糖的信噪比均>10,仪器和方法的灵敏度高;氨基糖样品含量的重复性(标准样品平行样、土壤样品平行样和土壤样品重复样的相对标准偏差均<5%)较好,仪器和方法的精密度较高;标准样品的线性(R^(2)>0.9970)良好,空白加标回收率和土壤基质加标回收率为80%~94%,仪器和方法的准确度较高。总之,利用OPA柱前在线衍生–高效液相色谱法测定土壤氨基糖含量的分析方法操作简单、灵敏度高,能够精确地测定土壤中的氨基糖含量。

Four amino sugars,namely,muramic acid(MurN),mannosamine(ManN),galactosamine(GalN)and glucosamine(GluN),in soil were determined by ortho-phthalaldehyde(OPA)pre-column derivatisation-high performance liquid chromatography(HPLC).The sensitivity,precision and accuracy of the instrument and method were tested from chromatographic peaks,theoretical plates,signal-noise(S/N)ratio,limits of detection and quantification,sample content repeatability,standard linearity,and recovery.The results showed that the chromatographic peak orders of the four amino sugars were MurN,ManN,GalN and GluN.The chromatographic peak shapes were good and the resolutions of each two adjacent peaks were>1.5,indicating that the two adjacent peaks were completely separated.Theoretical plates were>2000,indicating high chromatographic column efficiency.The instrument detection limits of MurN,ManN,GalN and GluN were 0.030,0.300,0.150 and 0.150μmol/L,the instrument quantitative limits were 0.100,1.000,0.500 and 0.500μmol/L,the method detection limits were 0.010,1.500,0.100 and 0.100μmol/L,and the method quantitative limits were 0.030,5.000,0.300 and 0.300μmol/L,respectively.In addition to ManN in the soil below 10 cm,S/N ratios of the amino sugars amount were all>10,indicating high sensitivity of the instrument and method.The relative standard deviations of the amino sugar amount in the parallel standard samples,parallel soil samples and repeated soil samples were all<5%,indicating high precision of the instrument and method.The R^(2) values of the standard curve were>0.9970,and the recovery rates of blank and soil substrate were between 80%and 94%,indicating high accuracy of the instrument and method.In conclusion,the method for the determination of soil amino sugar content by OPA pre-column derivatisation-HPLC,which is simple in operation and high in sensitivity,could accurately determine the contents of amino sugar in soils.