There are two main methods to extract pigments from avian eggshells. There are those using strong acids (e.g. sulphuric, hydrochloric or formic), that allow for a quantification of pigment concentrations but with which samples are destroyed. Alternatively, there are methods employing weaker acids (e.g. acetic), with which samples are not destroyed, although pigment concentrations may not be quantified and only their presence is detected. In some cases, there may be a need to quantify pigments in samples in which the pigments have already been detected. Here, we assess whether the quantity of pigments extracted from eggshells using a strong acid is affected by previous treatment of eggshells with a weak acid. for this, we used eggshells of Kentish Plovers Charadrius alexandrinus and domestic Japanese Quails Coturnix japonica, and aliquots of individual eggshells treated either with both acids (first applying a weak acid and then a strong acid) or only with the strong acid. Quantities of extracted protoporphyrin and biliverdin (the main eggshell pigments) were lower when using a strong acid after application of a weak acid. However, there were significant positive correlations between the quantities of pigments extracted using the two methods, suggesting that eggshell samples used to determine pigment presence may still be usable to quantify pigment concentrations using a strong acid.
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