temperature = 82ºC and time = 40 min. A volume of 40 mL
was adopted.
Microwave-assisted extraction.—A set of 18 experiments
using the limits of Table 2 was used for optimization. A second
order equation was fitted to nicotine concentration (Table
6). The goodness of fit was assessed through ANOVA after
normality and homogeneity tests (Table 7). The full model
was significant at 90% confidence with R2 = 0.78 and
RAdj
2 = 0.53. These results reveal a rather low adjustment of
the model which may be due to the inclusion of some insignificant
terms. However, the model can be considered as acceptable
for a preliminary study, having R2 > 60% (20). Pressure
and time linear effects were significant at 99 and 95% confidence,
respectively. None of the quadratic effects were significant,
which allows us to conclude that neither a maximum nor
a minimum of the response function was reached. Little curvature
may signify that operating conditions are far from the optimum.
Analyzing response surfaces (Figure 2), we can state that
more pressure and time were needed to reach the optimum;
however, the lack of temperature control and practical constraints
made the safe use of other limits impossible. In spite of
the low significance of power input, the graphs suggest that an
intermediate value should give the best result. Consequently,
the method was developed with maximum equipment security
at pressure of 75 psi for 40 min and with 60% maximum magnetron
power.
Comparison of Methods
To assess method adequacy for the analysis of high and
low alkaloid levels, 2 tobacco samples were chosen. Five replicate
experiments by each method were simultaneously developed.
Significance of differences between alkaloid mean
concentration values and their deviations (ts/ n) were evaluated
using an LSD test with 95% significance (Table 8).
For low level alkaloid tobaccos, all the methods extracted
similarly precise amounts of the different alkaloids except
anatabine. For high level alkaloid tobaccos, the Soxhlet
method extracted significantly less nicotine with lower precision,
and the microwave method was less efficient in the extraction
of secondary alkaloids. Considering the practicability
of the studied methods, the solvent purification extraction
method (AOAC modified method) is perhaps more laborious;
however, several experiments can be performed simultaneously.
In spite of using benzene, this method has the advantage
of producing cleaner extracts, which generated