## 3 Results  
The hyphenated system LC-DAD-QTOF provided two independent pieces of useful information on caffeic acid derivatives. A double screening process is possible based on the typical UV absorbance (\(\lambda\) = 270 and 360 nm) and on the accurate measurements of pseudomolecular ions corresponding to the selected compounds. LC-UV was used as the reference protocol for screening caffeic acid derivatives, also giving a rough indication of the relative abundance, as the molar responses are comparable for caffeic acid derivatives. As a matter of fact, we used the MS response for its higher selectivity and sensitivity, as several compounds were co-eluted in UV and minor components were not detected, as shown in Figure 1. The presence of potentially sensitizing agents, as evaluated by [PERSON] et al. [10; 11; 12; 13] in guinea pigs or in human case studies, and relevant phenolic compounds ascribed to _Z. punctata_ were investigated in plant and propolisis extracts. Results are reported in Table 1, using the notation **s** for strong, **m** for moderate, **w** for weak, and **ne** for not established allergenic potential. For the sake of simplicity, conventional diagnostic ions are often reported in the text without decimal digits though accurate measurements were recorded. The typical MS fragmentation scheme of caffeic acid derivatives is extensively described in the literature [26; 27; 28]--caffeate esters generally produce diagnostic MS fragment ions at _m/z_ 179 (_m/z_ 179.0350) as base peak, and at _m/z_ 135 (_m/z_ 135.0452) corresponding to [179-CO\({}_{2}\)]\({}^{-}\). Their related radical ions at _m/z_ 178, 134, and 161 were also reported as base peaks when a benzyl moiety is present, such as for the compound (**s4**), as shown in Figure S1 in the supplementary material, ascribed to benzyl caffeine. _Z. punctata_ was previously characterized, and the propolis here analyzed, were demonstrated to be _Zuccagnia_-type [21]. Thus, the major biomarker of _Z. punctata_, i.e., 4'-hydroxy-2'-methoxydihydrochalcone (**ne3**), was taken as the reference compound and the abundance of caffeic acid derivatives was expressed as a relative percentage. Many of those compounds were not detected as precursor ions at the MS level. Other compounds, like vanillin (**w10**) or ferulic acid (**w17**), were detected with very low counts at the MS level so that their fragmentation was not possible. In this case the correct identification was not allowed and their presence in the samples was not considered confirmed.  
Figure 1: Chromatograms of the diode array detector (DAD) profile at 270 nm and extracted ion (accuracy 10 ppm) of the [M-H]\({}^{-}\) with the relative intensities of allergenic agents and major constituents described in Table 1, identified in Amaicha del Valle propolis. In detail: (**s2**) = phenyl caffeates; (**s4**) = benzyl caffeine; (**s7**) = geranyl caffeate; (**m3**) = cinnamyl caffeine; (**ne1**) = 1-methyl-3-(4’-hydroxyphenyl)-propyl caffe acid ester; (**ne2**) = 1-methyl-3-(3’,4’-dihydroxyphenyl)-propyl caffe acid ester; (**ne3**) = 4’-hydroxy-2’-methoxydihydrochalcone; (**ne5**) = 2’,4’-dihydroxycholcone; (**ne6**) = 7-hydroxyflavanone; (**ne7**) = 2’,4’-dihydroxy-3’-methoxychalcone; (**ne8**) = 7-hydroxy-8-methoxyflavanone, where **s** means strong, **m** moderate and **ne** not established allergenic potential. A shift of about 0.15 min occurred between UV and mass spectrometric analysis.  
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\begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c c c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**N**} & **w** & **w** & **IM-H\({}^{*}\) & \multicolumn{3}{c}{**Compounds**} & \multicolumn{3}{c}{**Fengments and Relative Abundances (\(\mathbf{c}\))**} & \multicolumn{3}{c}{**Samples