3.1. Selection of the type of BX3
Different observations have been reported when BF3
are used as catalysts for fatty acid methylation. In a study aiming at comparing six widely used methods for the determination of fatty acid composition in tuna, the presence of an artefact was observed when BF3
was used [13
]. Unidentified peaks have been also recorded when a methanolic solution of BCl3
was employed in a study intended for comparing BF3
]. Lipids from a commercial omega 3 ethyl ester capsule formulation were extracted and methylated by the conventional method using BF3
as catalysts. The results of these experiments () expressed as percentage of the total fatty acid content and as mg FAME/g of sample revealed no significant differences between the concentrations of the fatty acids when either BF3
were employed. The percentage of unknown peaks averaged at 3.2 % and 3.0 % of the total fatty acid content when BCl3
were used respectively. These results were confirmed by an independent laboratory.
Comparison of BCl3 and BF3 as catalysts for FAME preparation using the conventional method (n = 6)
Although there were no significant differences between the two halides tested, it was decided to use BF3
in the subsequent experiments in accordance with the Union of Pure and Applied Chemistry (IUPAC) [14
], the International Association of Official Analytical Comunnities (AOAC) [15
], the American Oil Chemists’ Society (AOCS) [16
] and the British Standards Institution (BSI) [17
], which recommend this reagent for the preparation of FAME.
3.2. Convetional and simplified method comparison
There was no significant difference between the conventional and simplified method, the coefficients of variation were comparable for both methods. The average percentages of total variance for the former and the latter were 2.7 and 3.2 % respectively. Only 2.9 % of the variation in individual fatty acid content was due to differences between the methods which was comparable with the variation between replicate separations (2.1 %) and between repeat injections onto the column (1.7 %).
The concentrations of the major fatty acids in human red blood cells and human serum (), in salmon muscle, salmon liver and cod plasma (), and in a reference material and milk powder () prepared by the simplified and the conventional method and analysed by GC under the same chromatographic conditions, are expressed as mg FAME/g of sample. – showed that the analytical data obtained with the conventional and the simplified methods were only slightly different. In all instances, the results from the conventional and simplified method did not reveal any statistical differences at the 95 % confidence level. It is important to mention that sample homogeneity is a crucial factor to the success of the simplified method. Experiments on whole salmon samples (results not shown) revealed differences between the conventional and the simplified method at the level of particular fatty acids such as C18:2n-6, C20:5n-3 and C20:1n-9, which could be attributed to the variable amounts of bones and skin that remain in the reactors after the methylation.
Comparison of fatty acid composition in red blood cells (RBC) and serum using the conventional and the simplified method (n = 6)
Comparison of fatty acid composition in fish samples using the conventional and the simplified method (n = 6)
Comparison of fatty acid composition in food samples using the conventional and the simplified method (n = 6)
Although the chromatographic analysis time of the proposed simplified method is 3 times longer than the conventional method, it does not require time consuming operations such as extraction, solvent transfer, evaporation, centrifugation, etc, in addition to the bulk of glass material required to perform all these operations.
3.3. Effect of the variables NaOH and hexane on the validated simplified method
The effect of the selected variables (NaOH and hexane) on the major fatty acids found in a human serum sample (C16:0, C18:0, C18:1n-9 and C18:2n-6) are shown in . The results revealed clearly that the addition of hexane after the methylation process (bottom quadrants in ) increases dramatically the FAME concentrations when compared to the addition of hexane before the methylation process (top quadrants in ). The observed increments were higher than 50 %. Comparison of the left and right top quadrants in (representing the addition of hexane before the methylation step) demonstrated that the absence or presence of NaOH does not affect the FAME concentrations. The slight FAME changes observed when comparing the two top quadrants were not statistically significant at a confidence level of 95 %. This outcome was also observed when the right and left bottom quadrants (adding hexane after the formation of the FAME) were compared, giving further evidence that NaOH at the concentration level used in this study (0.5 M) does not have any influence on the methylation process. The considerable increase in FAME concentrations (> 50 %) when hexane is added after the methylation and regardless of the NaOH presence was consistently observed when the factorial design was applied on food reference material and cod liver oil (results not shown). It is important to mention that the FAME concentrations of these samples (when hexane was added after the methylation) were in accordance with the conventional method.
Effect of the variables NaOH and hexane on the major fatty acids (mg/g) in human serum and human red blood cells. The values are expressed as mean ± standard deviation of the main fatty acids (n = 6)
In contrast, the application of the factorial design on human red blood cells () and rat brain samples (results not shown) revealed that neither hexane (added before or after the methylation) nor NaOH (absence or presence) have an impact on FAME concentrations. shows that the four quadrants generate the same information. The small differences observed between quadrants were not statistically significant at the 95 % confidence level. The red blood cell FAME concentrations in the four quadrants were in accordance with those estimated by the conventional method.
3.4. Underlying mechanisms: effective methylation area model
In summary, our results have demonstrated that addition of hexane before the methylation step reduces dramatically the FAME concentrations while adding hexane after methylation brings about concentrations close to that with the conventional method. However, in some instances adding hexane before or after the methylation step does not affect the FAME concentrations. Based on these contrasting results and considering that triacylglycerols (TAG) are poorly soluble in methanol and that TAG are the major sources of fatty acids, it could be hypothesised that a priori addition of hexane promotes the dissolution of TAG in the hexane phase. The boundary between the hexane phase containing solubilised TAG (top layer) and the methanol phase containing solubilised BF3 catalyst (bottom layer) define an “effective methylation area” where the actual methylation process is taking place (double head arrows in ). A high concentration of TAG in the sample will generate FAME through the “effective methylation area”. As the concentration of FAME starts increasing in the hexane phase the efficiency of the “effective methylation area” begins to diminish, probably because the FAME begin to saturate this particular area or perhaps because the generated FAME protect the remaining TAG, hence preventing the direct contact of TAG with the reagents responsible for the methylation (). Conversely, a very low concentration of TAG in the sample cannot produce enough FAME to saturate the “effective methylation area” or shield the remaining TAG () hence leading to a more effective conversion of TAG into FAME. The a posteriori addition of hexane, independent of the TAG concentration, precludes the formation of the so-called “effective methylation area” and consequently maximizes the interaction of TAG with the methylation reagents, leading to higher FAME concentrations comparable to those obtained with the conventional method.
Underlying mechanism for samples with high levels of triacylglycerol (TAG).
Underlying mechanism for samples with very low levels of triacylglycerol (TAG).
To assess whether or not the contact area between methanol and hexane, which defined the hypothesized “effective methylation area” has an effect on the FAME yield and consequently is an important factor in the methylation process of samples rich in TAG, some experiments were performed using the simplified method to methylate a cod liver oil sample, using reaction tubes with different diameters (1.5, 2.5 and 5 cm) and keeping constant the volume of the reactants (2 ml of BF3/MeOH and 1 ml of hexane added a priori). The results () demonstrated that a decrease in the volume/area ratios of the phases brings about an increase in the FAME yield. The results obtained with the 5 cm diameter reactor were in accordance with those obtained with the conventional method. These results confirmed the importance of the proposed “effective methylation area” in the methylation process of samples rich in TAG. The model described in allows to explain why the a priori addition of hexane to human serum, with a relative high content of TAG, (top quadrants in ) reduces dramatically the FAME concentrations compared to the a posteriori addition of hexane (bottom quadrants in ). The results for food reference material and cod liver which contain high levels of TAG were also explained by .
Effect of the reaction tube diameter on FAME prepared by the simplified method and with a priori addition of hexane. The reactant volumes were kept constant (n = 3)
The results for human red blood cells () and rat brain samples (rich in phospholipids) containing low levels of TAG and showing minimal changes in FAME concentration despite of the order of hexane addition can be explained by the model described in .
The chromatogram for an unmethylated sample free of TAG such as the ethyl ester omega-3 capsules containing mainly ethyl-eicosapentaenoic acid (ethyl-EPA) and ethyl-docosapentaenoic acid (ethyl-DHA) is presented in . Addition of hexane before methylation causes a reduction of the FAME signals as a result of the incomplete conversion of the ethyl esters into methyl ester (). The levels of unreacted ethyl-EPA and ethyl-DHA were 13.17 and 18.02 % respectively. The results shown in can be explained in terms of the model depicted in but using the ethyl form instead of the TAG form. On the other hand, shows clearly that a complete conversion of the mixture of ethyl-EPA and ethyl-DHA into methyl-EPA and methyl-DHA respectively, occurs when the ethyl esters are exposed to the methylation reagents and hexane is added only after the formation of the FAME in the methylation reactor.
GC chromatograms for an omega-3 commercial capsule. a) unmethylated sample; b) addition of hexane before methylation; c) addition of hexane after methylation.
The proposed models (–) provide a rational explanation for the observed changes in FAME concentrations estimated by GC when hexane is added a priori in systems with high or very low TAG content. Nevertheless, it is necessary to obtain evidence regarding the presence or absence of TAG predicted by the effective methylation area model after the methylation process is completed. To accomplish this aim, cod liver oil (high TAG content) and red blood cells (low TAG content) samples were treated using the simplified method without NaOH and adding hexane before or after the methylation step. The methylation products in the hexane phase were analysed by HPTLC instead of GC to check whether the main product in some instances is a mixture of TAG and FAME (as is predicted in ) or mainly FAME (as is predicted in ). The HPTLC chromatograms for cod liver oil show that an a priori addition of hexane to the reactor containing methanol, BF3 and the sample with a high TAG level, yields a mixture of TAG and FAME at the end of the methylation process (). The a posteriori addition of hexane when the methylation process of the sample with a high TAG content is completed generates mainly FAME (). The red blood cells HPTLC chromatograms (not shown) were similar to that showed in , indicating that samples with a very low TAG content generates mainly FAME independent of the order (a priori or a posteriori) of addition of hexane in the methylation reactor. The HPTLC results demonstrated unequivocally that the proposed effective methylation area model (–) offers a comprehensive explanation for the observed changes in FAME concentrations by GC when hexane is added either a priori or a posteriori in systems with high or very low TAG content.
HPTLC chromatograms for a cod liver oil sample, used to study the effective methylation area model. a) a priori addition of hexane; b) a posteriori addition of hexane.