An HPLC method has been developed for the determination of the cephalosporin antibiotic Ro 13-9904 in plasma, urine, and bile of dogs and of human volunteers using the technique of ionpairchromatographywith a LiChrosorbRP-18 column.The three mobile phases employed contained tetrapentyl-, tetraoctyl-and hexadecyltrimethyl-ammonium bromide, respectively, as lipophilic counterions. The chromatographic conditions chosen allowed simple and rapid sample preparation. Plasma was deproteinired with ethanol and the supernatant was directly injected onto the column; urine and bile were diluted with mobile phase and injected without any purification. The detection limit for the cephalosporin was about 0.5 pg/ml for plasma samples and approximately 5 pg/ml for bile and urine.

With the help of radioactive dilution analysis allowing the qualitative and quantitative deter­mination of all three presently known juvenile hormones (JH-1 to 3) the following seven species of four orders were investigated in the adult stage: Coleoptera: Tenebrio molitor, Leptinotarsa decemlineata; Orthoptera: Schistocerca gregaria; Blattodea: Blatta orientalis, Leucophaea maderae, Nauphoeta cinerea; Hymenoptera: Apis mellifera. In all these species of the 3 known juvenile hormones only m ethyl (2E ,6E)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate (JH-3) was found, in an amount of 0.5 to 11 ng per gram of body weight. The results of the chemical analyses were confirmed biologically by the Galleria wax test. The results demonstrate the wide spread occurrence of JH-3 in insects of different orders.

A method is presented permitting the qualitative and quantitative determination of all three presently known hormones (JH1-3). The determination is based on the method of radioactive isotope dilution, whereby a very small known amount of tritium-labelled JH-1 is added to the ether extract of the particular species. The addition of radioactive JH-1 permits the isolation of all three hormones, because of their similar behaviour during the chosen work up. The quantitative determination was carried out by gas chromatography and the identification was confirmed with the help of retention-times and GC-MS combination. The method was checked by using an extract of Hyalophora cecropia. For the first time methyl 10,11-epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecadienoate (JH-3) could also be identified as the juvenile hormone of Melo­lontha melolontha. In Vanessa io larvae, Tenebrio molitor larvae and adults and in Musca domestica larvae none of the three known hormones could be detected. The preparation of JH-1 labelled with tritium in the methyl group of the ester was accomplished with very high specific activity (4.34 Ci/mmol) of the tritiated acid with diazomethane.

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