The two juvenile hormones from the cecropia silk moth.

Some years ago we reviewed the status of the insect juvenile hormone (JH) and its isolation.1 Since then two noteworthy contributions toward purification of the hormone have appeared.2' In all these purification attempts adult male Cecropia silk moths4 proved to be an invaluable source material. The first nearly pure hormone preparation was isolated in 1965.1 We obtained it from Cecropia oil (second batch) and characterized it by means of a gas-liquid chromatographic (GLC) analysis in conjunction with an ultrasensitive argon ionization systemA The hormonal activity was located exclusively in the two major peaks B and E. Both these substances had a specific activity of the same magnitude as the original preparation. They were retained on neopentyl glycol adipate (NGA) columns at ratios of 1.60 and 1.29 relative to methyl stearate. Only one contamination, peak H, was apparent, which amounted to less than 10 per cent of the total peak area. Thus our 3 X 105-fold enriched preparation was at least 90 per cent pure. A host of subsidiary gas chromatographic peaks (A,D, etc.) were artifacts that resulted from reactions of the active compounds in the analytical system. These substances were produced even though the hormonal preparations had been injected with a Hamilton syringe directly onto the packed glass columns, but they were more plentiful in the fractions that had been previously submitted to a GLC separation and collected after passage through the brass detector cell. Since the same pyrolytic products were formed whether they were derived from compound B or compound E, the two hormonally active compounds had to be closely related structurally. We have now processed a new (third) batch of Cecropia oil by a five-step purification sequence similar to that applied to the second batch. These steps w-ere: (I) molecular distillation at -1 mTorr and 1000C; (II) separation from acidic products formed; (III) leaching with cold aqueous methanol; (IV) SiO2 chromatography; and (V) A1203 chromatography. One modification had been made in step IV in which an improved silica gel system was substituted for the zinc carbonate sy-stem6 previously used. This resulted in eliminating compound H. In the Galleria N-lX test, fractions of the final preparation again exhibited a potrency of some 3 X 105 JH units per milligram (Table 1). Our GLC unit wsas perfected by construction of a metal-free injector and a glass effluent splitter. As a result, the hormone preparation could be processed through the system without contacting any metal. From then on, much simplified chromatograms have been recorded (Fig. 1). The GLC analysis of the biologically most active fractions of the pure preparation revealed that they were composed of only two substances: compounds B and E (Table 1). When the