Liu Ang, Coleman Stuart P
Matrix BioAnalytical Laboratories, 25 Science Park at Yale, New Haven, CT 06511, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Nov 1;877(29):3695-700. doi: 10.1016/j.jchromb.2009.09.020. Epub 2009 Sep 17.
A sensitive, simple and rapid assay based on hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry was developed and validated for the quantitative analysis of metformin in human plasma using protein precipitation. Plasma samples were prepared using a protein precipitation solution containing acetonitrile, 0.5% formic acid and the internal standard, metformin-D(6). The analytes were separated on a GL Sciences Inertsil HILIC column using an isocratic mobile phase consisting of water/acetonitrile (30:70, v/v) and 0.1% formic acid. Metformin and internal standard were recorded using multiple reaction monitoring in positive ion electrospray mode with transitions of m/z 130-71 and m/z 136-77, respectively. No endogenous components in plasma were found to interfere with metformin measurements. The lower limit of quantification (LLOQ) was 0.5 ng/mL (0.1 pg on-column). The linear range was 0.5-500 ng/mL with an average correlation coefficient of 0.999 using weighted (1/x(2)) linear least-squares regression. Dilutional linearity was evaluated up to 5000-fold dilution and the results indicate no influence on the accuracy of analysis. The absolute extraction recovery was 81% for metformin. Intra-day and inter-day precision (CV, %) ranged from 0.73% to 7.18%, and accuracy within +/-10.98% from nominal. The analyte was found to be stable for at least 38 days at -20 and -80 degrees C, 24 h at room temperature, and stable for four freeze-thaw cycles. The processed extracts were stable for 88 h at 4 degrees C.
建立了一种基于亲水作用液相色谱（HILIC）与串联质谱联用的灵敏、简单且快速的检测方法，并通过蛋白质沉淀法对其进行验证，用于定量分析人血浆中的二甲双胍。使用含有乙腈、0.5%甲酸和内标二甲双胍-D(6)的蛋白质沉淀溶液制备血浆样品。在GL Sciences Inertsil HILIC柱上，使用由水/乙腈（30:70，v/v）和0.1%甲酸组成的等度流动相分离分析物。采用正离子电喷雾模式下的多反应监测，分别记录二甲双胍和内标的m/z 130-71和m/z 136-77的跃迁。未发现血浆中的内源性成分干扰二甲双胍的测定。定量下限（LLOQ）为0.5 ng/mL（柱上0.1 pg）。线性范围为0.5-500 ng/mL，采用加权（1/x(2)）线性最小二乘回归，平均相关系数为0.999。评估了高达5000倍稀释的稀释线性，结果表明对分析准确性无影响。二甲双胍的绝对提取回收率为81%。日内和日间精密度（CV，%）范围为0.73%至7.18%，准确度在名义值的±10.98%以内。发现分析物在-20和-80℃下至少稳定38天，在室温下稳定24小时，并且在四个冻融循环中稳定。处理后的提取物在4℃下稳定88小时。
