Lipid extraction techniques for stable isotope analysis of bird eggs: Chloroform–methanol leads to more enriched 13C values than extraction via petroleum ether

• The amount of lipid in a sample can bias its carbon stable isotope value.
• Consequently, lipids are extracted or accounted for algebraically before stable isotope analysis.
• Chloroform-methanol and petroleum ether are different solvents used for lipid extraction.
• The type of solvent affects carbon stable isotope values.
• Carbon stable isotope ratios after lipid extraction using different solvents are not comparable.

Stable isotope analysis is often used to determine dietary origin in ecological studies. Lipids are depleted in 13C compared with protein and, so, variation in lipid content can confound interpretations of diet. To avoid this issue, lipids can be extracted from samples prior to stable isotope analysis. The most common solvent used for lipid extraction, chloroform–methanol, is toxic and cannot be used in some laboratories. Here, the use of chloroform–methanol as a solvent was compared with an alternative method that uses petroleum ether as a solvent (N = 32 eggs from seven species). The δ13C values using both methods were highly correlated (R2 = 0.99) but values derived from samples that were lipid-extracted using chloroform–methanol were enriched in 13C by 0.90 ± 0.07‰ compared with samples that were lipid-extracted using petroleum ether. The C:N ratio was 3.60 ± 0.02 (s.e.) for chloroform–methanol and 4.25 ± 0.03 for petroleum ether implying that chloroform–methanol (which is more polar) more completely extracted lipids than petroleum ether. Furthermore, δ15N values derived from samples that were lipid-extracted using chloroform–methanol were enriched in 15N by 0.41 ± 0.05‰ compared with samples that were lipid-extracted using petroleum ether. In conclusion, lipid extraction by chloroform–methanol more thoroughly removes lipids than lipid extraction by petroleum ether and consequently δ13C is higher after lipid extraction by chloroform–methanol than after lipid extraction by petroleum ether. An algebraic correction formula for each method is provided.