Comparison of three pretreatment methods for the analysis of distributions and hydrogen isotopic compositions of leaf wax n ‐fatty acids in natural samples

RATIONALE The hydrogen isotopic compositions (δ 2 H values) of leaf wax n ‐fatty acids ( n ‐FAs) are widely used as powerful biomarkers in biogeochemical and paleoenvironmental studies. However, the use of a variety of pretreatment methods to isolate n ‐FAs from natural samples for the measurement of δ 2 H values potentially causes discrepancies in the measured δ 2 H values, thereby confounding their use as a biomarker. METHODS We tested three typical pretreatment methods for the analysis of the distributions and δ 2 H values of n ‐FAs from terrestrial higher plants ( Carex siderosticta Hance and Ilex chinensis Sim ), surface soil, and lacustrine sediment. Method 1 employed an aminopropyl‐bonded solid phase to selectively separate free n ‐FAs from extractable lipids, while Methods 2 and 3 involved alkali‐ and acid‐catalysed chemical reactions in extractable lipids prior to isolation of the n ‐FAs. RESULTS Methods 2 and 3 yielded higher amounts of C 16 ‐C 24 n ‐FAs and of certain long‐chain n ‐FAs than Method 1 due to the release of the corresponding ester‐bound n ‐FAs by saponification and transesterification reactions, respectively. Although additional ester‐bound n ‐FAs were recovered by Methods 2 and 3, there were no significant differences in the δ 2 H results for all homologous n ‐FAs from the two plants and for mid‐ and long‐chain n ‐FAs from the soil and sediment among the three pretreatment methods. CONCLUSIONS Our study indicates that the three different pretreatment methods might not seriously influence the interpretations of δ 2 H values of sedimentary long‐chain n ‐FAs as a terrestrial paleohydrological proxy; however, the release of ester‐bound n ‐FAs by alkali‐ and acid‐catalysed chemical reactions means that users should be cautious when interpreting and comparing distribution data of n ‐FAs from natural samples with different pretreatment protocols. Copyright © 2015 John Wiley & Sons, Ltd.