Quantification of ecdysteroids and retinoic acids in whole daphnids by liquid chromatography-triple quadrupole mass...

Quantification of ecdysteroids and retinoic acids at picograms per individual is typically achieved with radioimmunoassay methods. However, those methods cannot identify individual types of ecdysteroids or provide an absolute concentration, which poses problems for comparative assays such as the metabolic profiling approach for toxicity testing. The method described in the present paper, based on liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry, was developed to allow the quantification in whole daphnids extracts of ecdysteroids (20-hydroxyecdysone, ecdysone, ponasterone A) and retinoic acid (sum of isomers). This approach avoids having to perform the difficult task of sampling the haemolymph on small organism (<5mm). Recoveries, evaluated at three concentrations in matrix blank fortified samples, ranged from 83 to 119% for ecdysteroids and from 144 to 155% for retinoic acids. Precision (2.4-14.2%) and accuracy (-41.7 to 14.5%) were reproducible and stable over three quality control concentrations. The described liquid chromatography-triple quadrupole mass spectrometry method achieved quantification limits ranging from 210 to 380 pg mL(-1) for ecdysteroids and 5 ng mL(-1) for retinoic acids in spiked matrix blanks. 20-hydroxyecdysone was quantified in Daphnia magna adults (19 ± 8 pg ind(-1)) and juveniles (3.6 ± 1.0 pg ind(-1)), but was below the limit of quantification in neonates (≈ 0.19 pg ind(-1)). Ecdysone was also detected in adult specimens (≈ 1.8 pg ind(-1)).