OFERTA DE TRABAJO Y MAXIMIZACIÓN DE LA UTILIDAD

The HPLC system used consisted of two pumps by, Altex model 1 lOA, an Altex controller (supplied by Altex, USA) and Waters 991 photodiode-array detector (supplied by Millipore UK). The analytical HPLC was performed on an 250 mm long Apex Prepsil silica column, 4.6 mm in internal diameter and particle size of 5pm (supplied by Jones, UK). The semi-preparative HPLC column was an 250 mm long Apex Prepsil 10mm in internal diameter and particle size of 8 pm (supplied by Jones, UK). Samples were injected manually using a micro-syringe into a 2 0pl loop.

Solvents were Super Purity HPLC-grade ( supplied by Romil Chemicals Ltd, UK) degassed prior to use by sonicating (sonicator supplied by Dawe) each litre for twenty minutes. Samples were eluted using a mixture of chlorofom:methanol at a ratio of 99.2:0.8 and at a flow rate of Iml/min. Absorbencies were detected between 220 and 400 nm. HPLC procedures were carried out at temperature between 20-25 °C and elutes were collected manually. Data was processed using PDA software version 6.22. Chromatograms were printed using Waters 5200 printer plotter (supplied by Waters Association).

6.3 Results and Discussion

Analysis of pure daphnane and tigliane derivatives on analytical TLC silica gel plates revealed that each group has a characteristic colour reaction when the plate was sprayed with 60% sulphuric acid and heated for 15 minutes, for Rf values and visualization refer to Table 6.1, pp 196. The analysed tigliane derivatives fall into three groups: the phorbol, the 12-deoxy phorbol and the 4-deoxy phorbol derivatives (refer to Figure 6. La., pp. 205 for chemical structures).

Examining the TLC chromatograms in day light revealed that the analysed tigliane derivatives produced orange-brown colour however, each group had characteristic fluorescence under the 356nm ultraviolet lamp. The most noticeable was the blue ultraviolet fluorescence produced by the nitrogen-containing phorbols known as sapintoxins. This fluorescence was clearly visible under the ultraviolet light even before spraying with the reagent. Daphnanes produced black spots on TLC chromatograms when

viewed in day light. Under the 365nm ultraviolet light 12-hydroxy daphentoxin derivatives (mezerein and thymelaeatoxin-A) (Figure 6.1.b, pp. 206) produced bright yellow fluorescence while the resiniferonol derivatives (resiniferatoxin and resiniferonol- 9,13,14-orthophenylacetate) produced bright orange fluorescence (Table 6.1, pp. 196).

The migration of the analysed diterpene esters on TLC plates appeared to be a function of the ester and the oxygen moieties at carbons 4, 12, 13 and 20. Compounds bearing an acetyl or a methyl group at C-20 have higher Rf values than those bearing a primary hydroxy group. The presence of a hydroxy group on C-4 (as in the case of sapintoxin-D and phorbol) and the cis-link between ring A and ring B (as in the case of 4a-deoxy-5-hydroxyphorbol-5,13,20-triacetate) decreases the migration distance and hence increases the retention time of the phorbol derivatives. Increasing the length of the unsaturated alkene at C l 2 decreases the migration distance of both tiglianes and daphnanes, demonstrated by the Rf and R, values of thymelaeatoxin-A, mezerein and resiniferonol-9,13,14-orthophenylacetate, resiniferatoxin, phorbol tributyrate and phorbol-12,20-dibenzyl-13-acetate. These observations indicated that the tertiary hydroxy group at C-4 has a role in the adsorption of these diterpenes on silica gel. For example the Rf value of the phorbol derivative sapintoxin-D is lower than that of the 4-deoxy phorbol derivative sapintoxin-A. The highly oxygenated phorbol was strongly adsorbed on silica gel and consequently lower Rf and high R, values were observed (Table 6.1, pp.

196).

Optimum Rf values less than or equal to 0.3 for HPLC analysis (Hostettman, Hostettman and Marston 1986) were achieved with chloroform:methanol as eluents m ixed at a ratio of 99.8:0.2. The purity of the compounds was evaluated on analytical HPLC column coupled with photodiode array (PDA) detector. Although this detector shares many elements with a conventional UV/VIS detector, the essential difference is that it can record the entire spectral range (190-800 nm) during analysis, thus monitoring the chromatogram at'selected wavelengths whilst recording the spectra of the eluates simultaneously.

o f data generated during the HPLC separation. Post-run analysis of the chromatogram provided extremely sophisticated methods for visualizing the data. The spectrum index option provided information on the composition of the sample under investigation and the spectra of each component (Figure 6.2.A, pp.207, Figure 6.2 E, pp. 211) which enabled the detection of contaminants. The contour plot (refer to Figure 6.2.C, pp. 209) and the three-dimensional topographical plot (Figure 6.2.B, pp. 208) provided information on the purity of each component of the analysed sample. The library feature enabled comparison o f experimental spectra with standards. Contaminant! were recognised and quantified by comparing peak areas hence q uan tification of purity. Integration and q u an tification o f elution peaks can then be achieved, once peak area has been calibrated with standard amount of the references. Increasing the sensitivity of the detector to 0.001 AUFS enabled the detection of nanogrammes of the test samples, which was not feasible on TLC, despite the difficulties from the signal to noise ratios. The method was scaled up to a semi-prepaiative column to analyse and detect irritant diterpene esters (refer to Table 6.1, pp. 196 for analytical data).

In addition to the characteristic UV blue fluorescence produced by nitrogen- containing phorbol derivatives on TLC the HPLC spectrum exhibited a unique UV profile, showing two UV maxima on chromatogram analysis and spectrum index (Figure 6.2 D, pp. 210 & Figure E, pp. 211). Investigation of the purity of 12-deoxy phorbol-13- phenyl acetate (DOFF) and 12-deoxy phorbol -13-phenyl acetate-20-acetate (DOFFA), stored at -20°C for five years, suggested that the latter compound had partially disintegrated into the de-acetylated derivative. Both compounds were purified on the semi-preparative column. Froton-NMR and mass spectra were used to confirm the identity of the isolates (Table 6.2, pp. 197 & Figure 6.3, pp. 198).

Data generated by TLC and HFLC analysis was used to isolate compounds from semi-pure biologically active fractions. Semi-preparative HFLC was used to purify 400pg resiniferatoxin from 800pg impure fraction. The identity of the isolate was confirmed by running FAB and E l mass spectra (Table 6.4, pp. 199).

hydrolysed croton oil. The isolate produced brown-orange colour on TLC, indicating the presence of a phorbol derivative. The NMR and mass spectral data (Table 6.5, pp. 199) identified the isolate to be phorbol. Data from ‘H-NMR and mass spectra confirmed that its acétylation reaction product was phorbol-12,13,20-triacetate (Table 6.6, pp. 202). TPA was purified from solutions stored under nitrogen at -20 °C for more than 2 years. The identity of the isolate was confirmed from FABMS and ’H-NMR spectral data (Table 6.7, pp. 203).

Optimum separation of the diterpene esters was achieved by injecting 1 - 2 mg of the sample into the semi-preparative column. Application of more than 2mg resulted in poor resolution. Analytical R, values were reproduced on the semi-preparative column using a flow rate of 4ml/min. However, the optimum semi-preparative separation was achieved at a flow rate of i m l / m i n higher rates resulted in tailing of the peaks’ down

slopes and overlapping of diterpenes with polar contaminants.

The use of HPLC in analysing and purifying the diterpene esters proved to be reproducible, rapid and a highly sensitive chromatographic technique. The high yield and resolution of the analytical and the semi-preparative HPLC compared to other chromatographic methods suggested the use ofasim ilar method in biochemical, pharmacological and toxicological studies (Ryves et al 1994). Colour reaction on TLC plates could be used as a guide for classification of the iditerpene under investigation. However, the complexity of different systems containing diterpene esters whether plant extracts or biological extracts suggests that the use of a single purification or analytical method is not possible. Additional advantages for using the HPLC include reduction of toxicity, usually associated with the exposure of the chemists to the samples, and minimizing the decomposition of the sensitive compounds during purification procedures.

Although the cost of the high performance liquid chromatography and the diode- array detector could be considered as a disadvantage, their use greatly simplified the analysis and the purification of the tigliane and daphnane derivatives. It is more likely that this method described here would be increasingly used in the future to isolate new members of the irritant diterpene esters.

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