Forskolin, a labdane diterpenoid, was isolated from the roots of Coleus forskohlii Briq. (Lamiaceae)' as the activator of adenylate cyclase,2 loading to an increase of c-AMP, used widely as an important reagent. Recently, immunoassay systems using monoclonal antibodies (MAbs) against drugs and naturally occurring biologically active compounds having smaller molecular masses like forskolin have become an important tool3 For production of monoclonal antibodies, the synthesis of hapten which is derived from immuno antigen and linker bridge, and carrier protein conjugates is necessary. Hapten is 7-deacetylforskolin 7-semisuccinyl residue. The specificity of an immunoassay method is generally dependent on the site of linkage between hapten and protein moieties, and also enumeration of hapten in the immunogen conjugate. There are no direct and appropriate methods for the analysis of hapten conjugated with carrier protein without differential UV analysis, radiochemical or chemical methods. Recently, Wengatz et al. determined the hapten density of immunoconjugates by matrix-assisted UV laser desorption/ionization mass ~pectrometry.~ Herein we wish to present an improvement for the direct analytical method of hapten and carrier protein conjugates by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry using an internal standard, and by the production of antibodies against forskolin. For the first experiment, a 33 PM 7-deacetyl forskolin 7-~emisuccinate~ was conjugated with 0.65 p~ bovine serum albumin (BSA) as explained inf Ref. 6.A small amount (1-10 pmol) of the antigen conjugate was mixed with a 103-fold molar excess of sinapinic acid in an aqueous solution containing 10% trifluoroacetic acid. The mixture was placed inside a JMS-LDI 1700 time-of-flight (TOF) mass monitor and irradiated with a N, laser (337 nm, 3 ns pulse). The ions formed by each pulse were accelerated by a 30 kV potential into a 1.7 m evacuated tube and detected using a microchannel plate detector with an IBM PC compatible computer. This method allows for rapid analysis of protein samples as small as 1 p-M. Figure 1 shows the co-mass spectra of the antigen conjugate of forskolin and BSA which is added as an internal standard. The relative molecular mass of BSA is 66 928 (average for triplicates, C.V. 0.09%) in agreement with a previous report' of which the calculated relative molecular mass of BSA was 66267. Figure 1 also shows only the singly, doubly, and triply protonated molecules of the intact conjugate which can be used for determination of the molecular mass of the hapten conjugate. Fragmentation is not an artefact of the ionization technique. A sharp peak coinciding with the conjugate appeared at m/z 72 160 (average for triplicates, C.V. 0.049%) resulting in the calculated relative molecular mass of 71 447. Using a relative molecular mass of 66 267 for BSA, the calculated mass of hapten, the 7-deacetyl forskolin 7-semisuccinyl residues (RMM 451), is 5 180 indicating 11.5 molecules of forskolin conjugated with BSA.In order to c...

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A multistage liquefaction scheme consisting of coal pretreatment, solvent-mediated hydrogentransfer dissolution, and catalytic hydrocracking steps was proposed in order to achieve the highest oil yields with complete conversion of organic components in coal and to enable the recovery and repeated use of the catalyst with its least deactivation. The deashing pretreatment enhanced the depolymerization of coal macromolecules through the removal of bridging ion-exchangeable cations as well as the better contact of donor solvent with coal macromolecules. The highest oil plus asphaltene yield of ca. 75% was obtained through the two-step liquefaction of acid-treated Morwell coal by the reactions of noncatalytic hydrogen transfer (donor solvent: tetrahydrofluoranthene, solvent/coal = 1) at 430 °C-2 min in the first step and the following catalytic hydrogenation with a pyrite catalyst at 400 °C-20 min. The multistep liquefaction scheme is discussed in terms of the highest efficiency of utilization of solvent and catalyst at the respective step. Two types of recoverable catalysts were investigated to allow repeated use of the catalyst in the primary liquefaction. The first type was an acid-proof iron catalyst which was recoverable from a mixture with residual carbonates and chlorides that are soluble in acids. Such a catalyst was applicable to particular coals such as an Australian brown coal which is completely liquefied, leaving calcium and magnesium carbonates as the major residual minerals after the primary liquefaction. The second type was characterized by its sulfur-proof ferromagnetism for the recovery from the minerals and carbons by gradient magnetic field. FesAl powder and carbon/ferrite composite catalysts were found to maintain their ferromagnetism after the sulfiding to be fairly active in the liquefaction of the brown coal. Procedures for activation, recovery, and repeated use of these recoverable catalysts were preliminarily examined. cidate the effects of thermal/catalytic and catalytic/ (3)

The effective range of the competitive ELISA test for detection of forskolin content in clonally propagated plant organs of Coleus forskohlii using monoclonal antibodies extends from 5ng to 5pg. A correlation between the forskolin accumulation and the growth rate was investigated using the clonally propagated shoots. An increase of forskolin content was noted, beginning at week 6. Flowers, rachises, leaves, stems, tuberous roots, and roots were analyzed. Tuberous roots and the stem base contained higher amounts of forskolin than other organs. The forskolin content in the stem decreased gradually towards the top of the shoot.

The liquefaction ability of a mixed donor of 4HFL (tetrahydrofluoranthene) and 8HAn (octahydroanthracene) was examined to reveal the roles of donors with different qualities and reactivities of transferable hydrogens, the components being applied in the same pot at once (single stage) or consecutively (two stages). Two donors competed for the radical fragments from the coal when mixed in the same pot (solvent/coal = 2,450 OC/lO min), and the yields of oil and asphaltene were 37 and 2070, respectively, which were much the same as the averages of the values obtained by their single use. A series use of a donor with eight donable hydrogens of less reactivity (8HAn) in the first stage (8HAn/coal= 1/1,450 OC/lO min) and another donor with four hydrogens of higher reactivity (4HFL) provided high yields of oil and asphaltene (41 and 19%, respectively) at less consumption of donors. 4HFL converted a larger amount of the heavier fraction in HI (hexane-insoluble fraction), which was obtained in the first-stage reaction of 450 OC/lO min using 8HAn (8HAn/coal = l / l ) , into the lighter fractions (oil 26%) in the second-stage liquefaction of 450 OC/lO min at 4HFL/HI = 1.5/1, while 8HAn provided a lesser amount (oil 13%) with larger amounts of residue and preasphaltene. Thus, two donors of different reactivities can share the roles in the depolymerization of coal molecules in the two-stage reaction according to their reactivities, giving high yields of oil and asphaltene. The mechanisms of fragment stabilization and hydrogen-assisting bond fission may explain these results which contribute according to the reactivities of donors as well as coal molecules.