Validation d’un procédé automatisé d’extraction sur phase solide de glandes digestives de moules pour l’identification et la quantification des dinophysistoxines en LC/ESI/SM2 par piégeage d’ions quadripolaire

This report presents a new extraction method of the dinophysistoxins (DTXs), confirmed by quantification using high-performance liquid chromatography coupled to mass spectrometry with an ion trap and electro spray interface (HPLC/ESI/MS2). The method originality consists on the adaptation of DTXs basic extraction procedure (liquid/ liquid) to a solid phase extraction (SPE) via a robotic station: ASPEC XLi The parameters of the automatization procedure were optimized to obtain the best DTXs recovery rate. These improvements were loaded with digestive gland mussel homogenat realized on a silica cartridge SPE, activated in hexane/chloroform (50:50), washed with hexane/chloroform (50:50) and extracted by an elution gradient (chloroform methanol (65:35) and methanol (100%)). This method was validated according to two normative referentials (linearity, detection quantification limits and accuracy…) : - The Guide of the Pharmacy industry: Analytical Validation, report of the commission SFSTP 1992 (French Corporation of the Sciences and Technical Pharmaceutical), - - The Procedure of validation of an alternative method in compare to a reference method. (AFNOR, 1998. NF V 03-110). Comparison with the classical liquid/liquid extraction and the automated method present clear advantages. In an analytical method the extraction is generally considered to be the most labor-intensive and error-prone step. This new procedure allowed us to increase throughput, to improve the reproducibility and to reduce the error risks due to the individual manual treatments.

Age estimation in common sole Solea solea larvae: validation of daily increments and evaluation of a pattern recognition technique

Close similarities have been found between the otoliths of sea-caught and laboratory-reared larvae of the common sole Solea solea (L.), given appropriate temperatures and nourishment of the latter. But from hatching to mouth formation. and during metamorphosis, sole otoliths have proven difficult to read because the increments may be less regular and low contrast. In this study, the growth increments in otoliths of larvae reared at 12 degrees C were counted by light microscopy to test the hypothesis of daily deposition, with some results verified using scanning electron microscopy (SEM), and by image analysis in order to compare the reliability of the 2 methods in age estimation. Age was first estimated (in days posthatch) from light micrographs of whole mounted otoliths. Counts were initiated from the increment formed at the time of month opening (Day 4). The average incremental deposition rate was consistent with the daily hypothesis. However, the light-micrograph readings tended to underestimate the mean ages of the larvae. Errors were probably associated with the low-contrast increments: those deposited after the mouth formation during the transition to first feeding, and those deposited from the onset of eye migration (about 20 d posthatch) during metamorphosis. SEM failed to resolve these low-contrast areas accurately because of poor etching. A method using image analysis was applied to a subsample of micrograph-counted otoliths. The image analysis was supported by an algorithm of pattern recognition (Growth Demodulation Algorithm, GDA). On each otolith, the GDA method integrated the growth pattern of these larval otoliths to averaged data from different radial profiles, in order to demodulate the exponential trend of the signal before spectral analysis (Fast Fourier Transformation, FFT). This second method both allowed more precise designation of increments, particularly for low-contrast areas, and more accurate readings but increased error in mean age estimation. The variability is probably due to a still rough perception of otolith increments by the GDA method, counting being achieved through a theoretical exponential pattern and mean estimates being given by FFT. Although this error variability was greater than expected, the method provides for improvement in both speed and accuracy in otolith readings.

Validation of age determination using otoliths of the European anchovy (Engraulis encrasicolus L.) in the Bay of Biscay

Validation of the age determination procedure using otoliths of European anchovy in the Bay of Biscay was achieved by monitoring very strong year-classes in successive spring catches and surveys, as well as the seasonal occurrence of edge types. Historical corroboration of the ageing method was obtained by cross-correlation between successive age groups by year-classes in catches and surveys (1987–2013). Summary annual growth in length is also presented. Yearly annuli consist of a hyaline zone (either single or composite) and a wide opaque zone, disrupted occasionally by some typical checks (mainly at age-0 and age-1 at peak spawning time). Age determination, given a date of capture, requires knowledge of the typical annual growth pattern of otoliths, their seasonal edge formation by ages and the most typical checks. Most opaque growth occurs in summer and is minimal (translucent) in winter. Opaque zone formation begins earlier in younger fish (in spring), and this helps distinguish age-1 from age-2+.