Phorbol Diester Constituents of Croton Oil: Separation by Two-Dimensional TLC and Rapid Purification Utilizing Reversed-Phase Overpressure Layer Chromatography (RP-OPLC)

Abstract

Fourteen known phorbol diester constituents of Croton tighum L. seed oil (croton oil) have been separated by a two-dimensional thin-layer chromatographic (2D-TLC) technique using different sorbent layers on the same plate. After a preliminary purification step using low-pressure liquid chromatography, reversed-phase overpressure liquid chromatography (RP-OPLC) has been shown to be potentially useful for the rapid preparative purification of these toxins. The identity of the major croton oil phorbol diesters was established by cochromatography with authentic compounds and/or by off-line chemical ionization mass spectrometry. The standard tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate, was obtained free from impurities as a result of the application of both the 2D-TLC and liquid chromatography-RP-OPLC procedures.

Essential Oils and Waxes

“…1) being short-chain esters of phorbol and 4-deoxy-4a-phorbol, respectively. However, we have found that the individual homologs within the separated A-and B-factor groups will separate by two-dimensional TLC, if a second reversed-phase phase-bonded silica gel layer is used (Erdelmeier et al 1988). 1), which are monoesters (Marshall and Kinghorn 1984).…”

“…All of these minor constituents were found to be diesters, with the exception of compounds 22, 23, and 27 ( Fig. We have also developed a reversed-phase OPLC method suitable for the rapid separation of many of the known long-and short-chain phorbol esters of croton oil (Erdelmeier et al 1988). Phorbol esters occur in a combined yield of over 1 % w/w in croton oil, with the most abundant and also most potent biologically active compound in this series being 12-0-tetradecanoylphorbol 13-acetate (TP A, 3) (Hecker and Schmidt 1974).…”

“…Rather more detailed fragmentation pathways may be observed for these compounds when methane is used as the reactant gas, with the molecular ions of the phorbol esters typically observed via prominent [M + Ht protonated molecular ions and [M + C2Hs]+ and [M + C3H7]+ adduct ions (Erdelmeier et al 1988). An instrument on which CI-MS may be performed is the Finnigan 4510 gas chromatograph-mass spectrometer (Finnigan-MAT, Sunnyvale, California, USA), equipped with a direct exposure probe system.…”

Forced-Flow Development in Overpressured Layer Chromatography

“…In the fully off-line mode, the isolation of separated zones can be achieved by scraping bands from the adsorbent layer followed by elution of the isolated materials, similar to conventional preparative layer chromatography. Phorbol diester constituents of croton oil were isolated by off-line OPLC separation followed by extraction and chemical ionization mass spectrometry for identification [76].…”

Overpressured Layer Chromatography

“…In the off-line OPLC method, the steps of operation after development are similar to those of conventional TLC methods: drying, scraping of the sorbent layer, elution, and crystallization. Phorbol diester constituents of croton oil were identified by off-line OPLC separation followed by extraction and chemical ionization mass spectrometry (CI-MS) (90). The on-line method is more effective for preparative applications because time-consuming scraping and elution can be eliminated.…”