Callipo Luciano, Foglia Patrizia, Gubbiotti Riccardo, Samperi Roberto, Laganà Aldo
Department of Chemistry, Sapienza University of Rome, Box n masculine 34, Roma 62, Piazzale Aldo Moro 5, 00185, Rome, Italy.
Anal Bioanal Chem. 2009 Jun;394(3):811-20. doi: 10.1007/s00216-009-2752-6. Epub 2009 Mar 24.
A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 +/- 12%, while analytical recovery was 81 +/- 10%. For the eight samples analyzed, the matrix effect was found to be only -14 +/- 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%).
本文介绍了一种用于人血清中碳酸酐酶II（CA II）绝对定量的方法。该方法基于配备纳米电喷雾源并与三重四极杆质谱仪相连的高效液相色谱（HPLC）芯片微流控装置。通过制备型反相HPLC分离含有CA II的馏分，并用HPLC芯片系统分离蛋白质混合物胰蛋白酶消化产物得到的肽段。对选定的合适CA II肽段和肽段内标采用多反应监测采集模式，可实现对CA II的选择性和灵敏测定。该方法的绝对回收率为52±12%，分析回收率为81±10%。对于所分析的八个样品，基质效应仅为-14±6%。分别通过外标法、基质匹配校准法和标准加入法得到的三种回归线类型的比较表明，第一种方法足以获得良好的准确度和精密度。血清中CA II的方法定量限估计为2 fmol/mL。发现八名健康受试者血清中CA II的平均浓度为56 pmol/mL（相对标准偏差24%）。
