Lord Heather L, Rosenfeld Jack, Raha Sandeep, Hamadeh Mazen J
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada.
J Sep Sci. 2008 Feb;31(2):387-401. doi: 10.1002/jssc.200700390.
Analyte derivatization is advantageous for the analysis of malondialdehyde (MDA) as a biomarker of oxidative stress in biological samples. Conventionally, however, derivatization is time consuming, error-prone and has limited options for automation. We have addressed these challenges for the solid phase analytical derivatization of MDA from small volume tissue homogenate samples. A manual derivatization method was first developed using Amberlite XAD-2 (12 mg) as the solid phase. Subsequently an automated column switching process was developed that provided simultaneous derivatization and extraction of the MDA-DH hydrazone product on a cartridge packed with XAD-2, followed by quantitative elution of the product to an analytical LC column (Waters NovoPak C18, 3.9 x 150 mm). The LOD was 0.02 microg/mL and recovery was quantitative. The method was linear (r(2) >0.999) with precision < 5% from the LOQ (0.06 microg/mL) to at least 35 microg/mL. The method was successfully applied to the analysis of small volume (30 microL) mouse tissue homogenate samples. Endogenous levels of MDA in the tissues ranged from 20 to 40 nmol/g tissue (ca. 0.1-0.2 microg/mL homogenate). Compared to conventional MDA analyses, the current method has advantages in automation, selectivity, precision and sensitivity for analysis from very small sample volumes.
分析物衍生化对于分析生物样品中作为氧化应激生物标志物的丙二醛（MDA）具有优势。然而，传统上，衍生化过程耗时、容易出错且自动化选项有限。我们已针对从小体积组织匀浆样品中固相分析衍生化MDA的这些挑战进行了应对。首先开发了一种使用Amberlite XAD - 2（12毫克）作为固相的手动衍生化方法。随后开发了一种自动柱切换过程，该过程可在填充有XAD - 2的柱上同时对MDA - DH腙产物进行衍生化和萃取，然后将产物定量洗脱至分析型液相色谱柱（沃特世NovoPak C18，3.9×150毫米）。检测限为0.02微克/毫升，回收率为定量。该方法呈线性（r(2)>0.999），从定量限（0.06微克/毫升）到至少35微克/毫升的精密度<5%。该方法已成功应用于小体积（30微升）小鼠组织匀浆样品的分析。组织中MDA内源性水平范围为20至40纳摩尔/克组织（约0.1 - 0.2微克/毫升匀浆）。与传统的MDA分析相比，当前方法在从非常小的样品体积进行分析的自动化、选择性、精密度和灵敏度方面具有优势。
