A common protocol for reliable comparison of pollen fatty acid profiles: highlighting pitfalls and proposing a methodology for ecological research

Introduction Pollen is an important tissue in plants that plays a vital role plant reproduction as it carries male gametes and occasionally also serves pollinator reward. There has been increasing interest pollen chemistry due to the impact of chemical variation on choices well-being, especially bees. The fat content lipid-to-protein ratio have shown play crucial regulating intake, some bee species avoid overconsumption fatty acids while specific acid ratios are essential for cognition. Therefore, knowledge composition understanding plant-pollinator interactions. However, existing methods analyses not always acids, non-pollen-derived can easily contaminate samples, making comparison between different impossible. Hence, objectives our study were highlight common mistakes pitfalls made during extraction analysis propose protocol reliable comparisons samples. Methods proposed method, developed two labs using gas chromatograph/mass spectrometers chromatograph/flame ionization detectors, involved manually homogenizing pollen, extracting with chloroform:methanol (2:1), analyzing chromatography (GC) mass spectrometry (MS) flame detector (FID) identification quantification. Results We found many present plastic materials solvents commonly used labs, cautioning against use recommending blank samples determine level contamination. suggest adding internal standard checking MS FID’s saturation limit before starting homogenization. Discussion Our method generated profiles from analyzed we hope blueprint achieving methodology characterizing comparing ecological research.