3.4.2.1. 20-hydroxyecdysone (20E) standard

Prepare a stock solution of unlabelled 20-hydroxyecdysone (20E) from a commercial source (Sigma) diluted to a concentration of 50 pg/µl in methanol. As commercial ecdysteroids usually are not sufficiently pure and there is degradation over time, it is recommended that an aliquot of commercial 20E is first purified by thin layer chromatography (TLC) or, if available by HPLC. The steps for TLC purification are essentially the same ones as described for cleaning up commercial JH (see section 3.3.2.1), the only differences are:

-  the solvent mix used for the chromatography run, which is dichloromethane : methanol (85:15) in steps 2 and 10.

- the solvent used to elute TLC-separated 20E from the silica scraps is methanol (step 16).

For preparing the 20E standard for preparation of standard curves:

1. Take a 5 µl aliquot of this purified 20E and dilute 1:100 in methanol for quantification of 20E by spectroscopy at λ = 245 nm.

2. Calculate the 20E concentration in mg/ml as: (0.48 x absorption x dilution factor)/12.6.

3. Correcting for dilution, dilute an appropriate aliquot of the purified 20E in methanol to a final concentration of 50 pg/µl.

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