基于凝胶过滤色谱的β2微球蛋白标准品单体定量检测方法
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摘要
β2微球蛋白(B2M)标准品是研究透析相关淀粉样变和评估血液透析器透过效率的重要试剂。B2M易于聚集的特性导致其作为标准品的单体浓度降低,使得检测结果准确性下降。为了准确评价B2M标准品的质量,该研究建立了基于凝胶过滤色谱的B2M样品中非聚集单体的定量检测方法。使用TSKgel SuperSW2000 (30 cm×4.6 mm, 4μm)凝胶筛分色谱柱,流动相为0.01 mol/L磷酸盐缓冲液(PBS, pH 7.2～7.4),流速为0.5 mL/min,柱温为25℃。使用紫外检测器,检测波长为280 nm,外标法定量。在0.05～0.50 g/L的B2M单体质量浓度范围内线性良好,相关系数为0.994 8。定量限(信噪比为10)为0.08 g/L,添加水平为0.10～0.30 g/L时,回收率为85.0%～96.7%,相对标准偏差为1.7%～3.3%。使用该方法对实验室自制重组人B2M标准品进行了质量检测,结果显示,该方法处理简单,准确度高,稳定性好,且不受溶液中B2M二聚体的干扰,适用于B2M标准品的质量分析。
Beta-2-microglobulin(B2 M) is an important material in dialysis-related amyloidosis research. Unfortunately, the quantitative detection of the B2 M monomer is difficult during B2 M production. In this study, we established a method for the detection of the B2 M monomer and its dimer in solution via gel filtration chromatography. The B2 M powder was dissolved in 0.01 mol/L phosphate-buffered solution(PBS, pH 7.2-7.4) with a final mass concentration of 0.5 g/L. The B2 M sample was analyzed on a TSKgel SuperSW2000 column(30 cm×4.6 mm, 4 μm) by an Agilent 1200 Series HPLC using 0.01 mol/L PBS(pH 7.2-7.4) as the mobile phase at a flow rate of 0.5 mL/min. The temperature of the column was 25 ℃, and the detection wavelength was 280 nm. The purities of the B2 M monomer and the B2 M dimer were tested. A series of concentrations of B2 M monomer solutions were prepared to create a standard working curve. The standard working curve of the B2 M monomer had a good linear relationship(coefficient of correlation(r~2) was 0.994 8). The limit of quantitation for the B2 M monomer in PBS was 0.08 g/L(S/N=10). The recoveries were 85.0%-96.7% with relative standard deviations of 1.7%-3.3% at spiked levels of 0.10-0.30 g/L. The quantification of the B2 M monomer was undisturbed by the B2 M dimer, which can form during the B2 M purification process. This determination method is simple, stable, and reliable for the determination of the B2 M monomer in B2 M industrial production.
Beta-2-microglobulin(B2 M) is an important material in dialysis-related amyloidosis research. Unfortunately, the quantitative detection of the B2 M monomer is difficult during B2 M production. In this study, we established a method for the detection of the B2 M monomer and its dimer in solution via gel filtration chromatography. The B2 M powder was dissolved in 0.01 mol/L phosphate-buffered solution(PBS, pH 7.2-7.4) with a final mass concentration of 0.5 g/L. The B2 M sample was analyzed on a TSKgel SuperSW2000 column(30 cm×4.6 mm, 4 μm) by an Agilent 1200 Series HPLC using 0.01 mol/L PBS(pH 7.2-7.4) as the mobile phase at a flow rate of 0.5 mL/min. The temperature of the column was 25 ℃, and the detection wavelength was 280 nm. The purities of the B2 M monomer and the B2 M dimer were tested. A series of concentrations of B2 M monomer solutions were prepared to create a standard working curve. The standard working curve of the B2 M monomer had a good linear relationship(coefficient of correlation(r~2) was 0.994 8). The limit of quantitation for the B2 M monomer in PBS was 0.08 g/L(S/N=10). The recoveries were 85.0%-96.7% with relative standard deviations of 1.7%-3.3% at spiked levels of 0.10-0.30 g/L. The quantification of the B2 M monomer was undisturbed by the B2 M dimer, which can form during the B2 M purification process. This determination method is simple, stable, and reliable for the determination of the B2 M monomer in B2 M industrial production.
引文
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