Gas-Chromatographic-Mass Spectrometric Assay of Tissue Malondialdehyde, 4-Hydroxynonenal, and Other Aldehydes after Their Reduction to Stable Alcohols

Abstract

Measurement of specific aldehydes such as malondialdehyde, hexanal, or 4-hydroxynonenal provides a method to evaluate the extent of lipid peroxidation. However, assay of aldehydes is complicated by their high reactivity toward other molecules and their chemical instability. In the proposed method, aldehydes are reduced to stable alcohols. Except for malondialdehyde, this is readily accomplished under neutral conditions at room temperature or at 4°C with either sodium borodeuteride (NaB 2H 4) or sodium horohydride (NaBH 4). Complete reduction of malondialdehyde requires a more powerful reducing agent, borane trimethylamine. After ether extraction and evaporation of the organic phase, the alcohols are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry in the positive chemical ionization mode for malondialdehyde and 4-hydroxynonenal and electron impact mode for all other aldehydes. Quantitation is achieved using internal standards of 1,3-[ 2H 8]propanediol for MDA, of 4-[2- 2H]hydroxynonenal for 4-hydroxynonenal, and of the corresponding saturated per-deuterated alcohols for all saturated aldehydes, for example and [ 2H 13]hexanol for hexanal. Saturated per-deuterated alcohols also serve as external standards for their unsaturated aldehydes with same chain length. The detection limit for individual aldehydes is 0.5 nmol in a given sample. In addition to being highly sensitive and selective, the present method allows one to verify the identity of each aldehyde by observing a mass shift when aldehydes are reduced with either NaB 2H 4 or NaBH 4. The usefulness of our method is demonstrated by measuring aldehyde accumulation in autoxidized arachidonic acid solutions and in heart homogenates before and after induction of peroxidation.