Automated derivatization and analysis of malondialdehyde using column switching sample preparation HPLC with fluorescence detection.

ABSTRACT

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.