*2.3. FAs Profile of Avocado Oils*

Except for the most abundant compounds of TG in avocado oil, the derived FA composition can also be exploited as a peculiar fingerprint indicative of the oil's quality and authenticity [37]. FAs are a group of very complex compounds, including monounsaturated FAs, polyunsaturated FAs and saturated FAs. In this study, the selected HP-88 with (88% cyanopropy)aryl-polysiloxane stationary phase GC capillary column is a high-polarity column designed for the separation of FAMEs including those positional cis/trans isomers. All the authenticated and commercial oils were transesterified and analyzed by GC/MS. The FA profile comprised a total of seven FAs in avocado oils, viz. palmitic acid, palmitoleic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid and linolenic acid. Oleic acid was the major FA (~60%), followed by palmitic acid (~15%), linoleic acid (~10%), palmitoleic acid (~7%) and vaccenic acid (~6%). This agreed with the individual FA moiety identified in the TGs by UHPLC/ESI-MS. The quantification results are summarized in Table 4. Again, the FA profiles of soybean and sesame oils showed significant differences from the avocado oils. Oleic acid was the most abundant FA (~44%) in sesame oil, followed by linoleic acid (~40%), palmitic acid (~9%) and

stearic acid (~5%). In soybean oil, linoleic acid (~57%) was the major FA, followed by oleic acid (~20%), palmitoleic acid (~11%) and linolenic acid (~7%). Interestingly, avocado oils contain much less stearic acid (~0.3%) compared to soybean oil (~4%) and sesame oil (~5%). The FA compositions of the commercial samples were evaluated. Similar to the TGs analysis, S4, S6, S9, S16 and S17 contained a relatively high concentration of stearic acid (3.0%–4.8%), like sesame and soybean oils, and the FA profiles were significantly different with the authenticated avocado oil. Therefore, these five samples are likely adulterated.


**Table 4.** Concentrations (mg/g) of FAs quantified in authenticated and commercial oils.

\*: The formula is expressed as CN (carbon number): DB (double bond) with the position of double bond.

To verify the precision and accuracy of the developed UHPLC/ESI-MS method, the composition data of TG and FA obtained from UHPLC and GC were compared to the authenticated avocado peel and pulp oils. The concentration of individual FA was calculated for all the identified TGs. Oleic acid and vaccenic acid are structurally similar and only differed in double bond positions and these two FAs could not be separated with UHPLC/MS. Thus, they were combined when compared with the TG data. The comparison of TG and FA data is summarized in Table 5. The measurement of TGs by the UHPLC/MS method might be affected by several factors, such as: i) one FA may be distributed among many different combinations in TGs, resulting in a TG concentration below the detection limit [31]; ii) the trace amount of mono/diacylglycerols were not calculated in the TGs method; and iii) the coelution of TGs may complicate the identification of trace FAs. All these factors may result in making the total compositions of TGs slightly lower than FAs. Taking all the factors into account, the data from LC and GC methods were consistent and within an acceptable experimental error range.

#### *2.4. Identification of Adulteration Using Chemometric Method*

To further identify possible adulteration, both UHPLC/MS and GC/MS data were subjected to multivariate data reduction chemometric analysis consisting of the principal component analysis (PCA). The PCA score plots are shown in Figure 7A,B for the UHPLC/MS and GC/MS data, respectively. Distinctive groups were observed in both techniques. In each plot, the avocado peel and pulp oils

were grouped together, whereas the avocado seed oil was much further away from the peel and pulp oil (Figure 7A,B). None of the samples claimed as avocado seed oils (S4, S9, S10 and S19) were clustered with the seed oil. Instead, S10 and S19 were grouped with pulp and peel oils, and S4 and S9 were grouped closely with soybean oil. S6 and S16 were labeled as avocado oil, but PCA indicated soybean oil as a possible adulterant. S17 might be a mixture of avocado oil with soybean or sesame oil. The adulteration identified by PCA further confirmed the results from both UHPLC/MS and GC/MS analyses.

**Table 5.** Comparison of concentrations (weight %) of individual FA calculated from UHPLC/MS and GC/MS.


**Figure 7.** PCA score plots of authenticated and commercial avocado oils, sesame oil and soybean oil. (**A**) UHPLC/MS TG data and (**B**) GC/MS FA data. Each sample was prepared in duplicate.
