Identification of bound alcohols in soil humic acids by gas chromatography-mass spectrometry

Humic acids are complex, partly macromolecular, yellow-brownish substances occurring in soils, waters and sediments. In order to bring some light on their molecular structure, crop humic acids were cleaved by alkaline hydrolysis (KOH). The products were fractionated by thin layer chromatography to give mono-alcohols which were analysed as acetate derivatives by gas chromatography coupled to mass spectrometry. Linear alcohols, sterols, stanols and plant-derived triterpenoid alcohols were identified by co-injection of pure standards and by comparison with literature data. These findings imply that alcohols could have been incorporated into the humic matrix by esterification with carboxylic acids. Furtehrmore, the presence of stanols as hydrogenated counterparts of sterols suggests that a process of hydrogenation is operating in soils. INTRODUCTION Humic substances represent a major pool of organic carbon occurring in soil, sediment and waters1-2. It is generally accepted that humic acids are characterised as polydispersed, acidic, amorphous substances ranging in molecular mass from a few hundred to several thousand daltons. However, the pathways of formation of humic acids remains an enigma mainly because of the lack of structural identification of its network at the molecular level. To unravel the molecular structure of complex, high molecular weight natural organic substances such as kerogen, asphaltene, coal and humic substances, various chemical and physical methods have been applied3-5. In particular, the breakdown of humic substances by chemical and enzymatic degradation can release substantial amounts of compounds amenable by gas chromatography coupled to mass spectrometry5-7. In a recent investigations, we identified alkane and alkene biomarkers released by pyrolysis of humin8-12. Here, we report the identification of n-alkanols, stanols, sterols and plant-derived triterpenoid alcohols released by mild alkaline degradation of soil humic acids from crop soils. EXPERIMENTAL Labware decontamination procedures are described elsewhere13. Soils cultivated for 23 years with maize (Zea mays) or wheat (Triticum aestivum) were sampled at Boigneville, France, then dried and sieved to 2 mm. 2 g of humic acids were extracted with 0.1 M sodium hydroxide under nitrogen to prevent oxidation of organic matter, precipitated at pH 1.5 with HCl, dialysed against water to remove molecules smaller than 2000 daltons, freeze-dried, extracted with diethyl ether to remove free organic compounds then refluxed 16 h. in 5 M potassium hydroxide-methanol under nitrogen. After addition of water, alcohols were extracted with diethyl ether (3 x), Na2SO4-dried then fractionated by thin layer chromatography (TLC) on silica-gel eluting methylene chloride with cholesterol as reference (RF ~ 0.13) to yield a mono-alcohol fraction which was then derivatised with an excess of acetic anhydride and pyridine (50°C, 1 h). The