Vicente Faye B, Vespa Gina K, Carrara Fabiola, Gaspari Flavio, Haymond Shannon
Department of Pathology, Northwestern University Feinberg School of Medicine, Ann & Robert H. Lurie Children's Hospital of Chicago, USA.
Laboratory of Pharmacokinetics Clinical Chemistry, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Clinical Research Center for Rare Diseases "Aldo e Cele Daccò", Ranica, BG, Italy.
Clin Biochem. 2015 Jul;48(10-11):679-85. doi: 10.1016/j.clinbiochem.2015.03.017. Epub 2015 Mar 31.
Measured glomerular filtration rate (mGFR) is the best indicator of renal function in children and adolescents. GFR determination using iohexol clearance has been increasingly accepted and applied in clinical practice because it is accurate, readily available, non-radioactive, safe and is used intravenously even in the presence of renal disease. This study describes the development and evaluation of a semi-automated method for determination of iohexol in human serum using liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS).
Iohexol was extracted from serum using a MICROLAB® NIMBUS4 automation robot and supernatant was dried under nitrogen gas and reconstituted in mobile phase. Ioversol was used as the internal standard. Chromatography was performed using a C-8 analytical column (Phenomenex, 3 μm, 50 × 3.0 mm I.D.) at room temperature and a gradient LC method on a Waters 2795 Alliance HT HPLC system. The flow rate was 0.5 mL/min and the retention times were 2.36 min and 2.14 min for iohexol and ioversol, respectively. Detection by MS/MS was achieved using a (Micromass Quattro Micro) tandem mass spectrometer operated in the ESI-positive mode. The multiple-reaction monitoring (MRM) method used ion transitions m/z 821.9 to 803.7 for iohexol and m/z 807.9 to 588.7 for ioversol. Method validation studies were conducted to determine the linearity, accuracy, precision, matrix effects and stability. A method comparison of blinded, residual patient samples was conducted with a well-established method.
The method was linear from 7.7 μg/mL to 2000.0 μg/mL. The low limit of quantification and the detection limit were established at 7.7 and 3.0 μg/mL, respectively. Within-run and between-run precisions were found to be <6% CV and measured values deviated no more than 5% from target concentrations. Carryover and matrix effects were not significant. Comparison to a well-established method showed very good agreement with correlation coefficient of 0.996 for iohexol and 0.993 for GFR/1.73 m(2).
This method accurately and precisely quantifies iohexol in 50 μL of serum, enabling determination of mGFR by iohexol clearance. The method is highly correlated to a reference method. Use of an automated liquid handler reduces labor-intensive, manual sample preparation steps. The stability of this analyte and the robustness of this assay fit well within our clinical workflow and we have successfully applied this method to determine mGFR in pediatric patients.
实测肾小球滤过率（mGFR）是儿童和青少年肾功能的最佳指标。使用碘海醇清除率测定肾小球滤过率（GFR）已在临床实践中得到越来越多的认可和应用，因为它准确、易于实现、无放射性、安全，甚至在存在肾脏疾病的情况下也可静脉使用。本研究描述了一种使用液相色谱-电喷雾电离（ESI）串联质谱（LC-MS/MS）测定人血清中碘海醇的半自动方法的开发与评估。
使用MICROLAB® NIMBUS4自动化机器人从血清中提取碘海醇，上清液在氮气下干燥并重新溶解于流动相中。碘佛醇用作内标。在室温下使用C-8分析柱（Phenomenex，3μm，50×3.0mm内径），并在Waters 2795 Alliance HT HPLC系统上采用梯度液相色谱法进行色谱分析。流速为0.5 mL/min，碘海醇和碘佛醇的保留时间分别为2.36分钟和2.14分钟。使用在ESI正模式下运行的（Micromass Quattro Micro）串联质谱仪进行MS/MS检测。多反应监测（MRM）方法用于碘海醇的离子跃迁m/z 821.9至803.7和碘佛醇的离子跃迁m/z 807.9至588.7。进行方法验证研究以确定线性、准确性、精密度、基质效应和稳定性。对盲法残留患者样本与一种成熟方法进行方法比较。
该方法在7.7μg/mL至2000.0μg/mL范围内呈线性。定量下限和检测限分别设定为7.7μg/mL和3.0μg/mL。批内和批间精密度均<6%CV，测量值与目标浓度的偏差不超过5%。残留和基质效应不显著。与一种成熟方法的比较显示一致性非常好，碘海醇的相关系数为0.996，GFR/1.73 m(2)的相关系数为0.993。
该方法准确、精确地定量50μL血清中的碘海醇，能够通过碘海醇清除率测定mGFR。该方法与参考方法高度相关。使用自动液体处理仪减少了劳动强度大的手工样品制备步骤。该分析物的稳定性和该检测方法的稳健性非常适合我们的临床工作流程，并且我们已成功应用该方法测定儿科患者的mGFR。
