Phenolic profile by HPLC-ESI-MS/MS-SIR mode
26 phenolic compounds were investigated in two commercial radlers (R1 and R2, different brand), made with Italian malts and lemon juice, according to the label. Other citrus fruits besides lemon were labelled for R2. One beer (B, same brand of R1) and one lemonade soft drink (L), containing Sicilian lemon juice, according to the label, were analyzed for a comparison (Table 1).
10 Hydroxybenzoic acids (HBAs), 4 hydroxycinnamic acids (HCAs), 2 caffeoylquinic esters (CQAs), 8 flavonoids (Fs) and 2 prenylflavonoids (pFs) were included in the standards pool. In detail, numbered according to the elution order (tR, Table 3): 3,4,5-trihydroxybenzoic acid 1 (gallic acid, GA), 3,5-dihydroxybenzoic acid 2 (3,5-DHBA), 3,4-dihydroxybenzoic acid 3 (protocatecuic acid, PCA), 5-caffeoylquinic acid 4 (5CQA), 2,5-dihydroxybenzoic acid 5 (2,5-DHBA, gentisic acid), catechin 6 (Cat), 4-caffeoylquinic acid 7 (4CQA), p-hydroxybenzoic acid 8 (pHBA), vanillic acid 9 (VA), caffeic acid 10 (CA), syringic acid 11 (SyA), m-hydroxybenzoic acid 12 (mHBA), 2,6-dihydroxybenzoic acid 13 (2,6-DHBA), coumaric acid 14 (CuA), sinapic acid 15 (SA), ferulic acid 16 (FA), rutin 17 (Ru), myricitrin 18 (My), quercetin-3-O-glucoside 19 (Q3G), kampferol-3-O-rutinoside 20 (K3R), salicylic acid 21 (SaA), hesperidin 22 (He), quercetin 23 (Q), kampferol 24 (K), isoxanthohumol 25 (IsoX), xanthohumol 26 (X). The analysis was carried out by an HPLC-ESI-MS/MS in SIR mode method previously developed for 14 compounds (1, 3, 4, 8–12, 14–17, 23, 24) (Petrucci et al. 2020b, 2021) and herein slightly modified to include 12 more compounds (2, 5–7, 13, 18–22, 25, 26), whose quality-of-analysis parameters are resumed in Table 2. Briefly, all calibration curves showed a good linearity in the investigated concentration range 10 ÷ 80 µg/L, as evidenced by the R2 values ranging within 0.9715 ÷ 1 (the minimum value 0.9715 was found for X), reported in Table 2; LOD and LOQ values were in the concentration ranges 0.070 ÷ 10.23 µg/L and 0.22 ÷ 31.01 µg/L, respectively (the higher LOD and LOQ values were found for SaA). Satisfactory data were obtained for accuracy (%, range − 9.59 ÷ 6.27) and precision (intraday, RSD% range 2.68 ÷ 18.80; interday, RSD% range 0.27 ÷ 19.33), the worst data found for My and 2,5-DHBA. The overall recovery percentages had RSD% in the range 9.03 ÷ 26.38, the worst values found for K3R and Q3G. ME varied between − 40 and + 41, but except My and 2,5-DHBA, a matrix effect from weak to medium (Zhang et al. 2019) was found for most of compounds. The improved method was confirmed suitable for fast analysis of complex matrices (Di Matteo P. et al., 2021).
Among the searched 26 compounds, 20 were identified in at least one sample and quantitated in most cases. Results, expressed as µg/L in the original sample, are reported in Table 3.
CQAs 4 and 7, and the hydroxybenzoic acids (HBAs) 1, 2, 5 and 12 were not detected in any of the samples; conversely, HBAs 8, 9, 11 and 21 were identified and quantitated in B and R1, 13 was identified in B, R1 and R2, 3 was found in trace only in L. Therefore, the investigated HBAs seemed not to be characteristic components of the Sicilian lemon, used for L. Noteworthy, the phenolic acids profile of lemon has been sparingly reported and the few papers are aligned with our results (Alu’datt et al. 2017; Gorinstein et al. 2001; Singh et al. 2020; Xi et al. 2017). Some differences between R1 and R2 (different brand) are likely due to different starting materials (malts and/or hops) used for the beers (Cheiran et al. 2019; Cortese et al. 2020; Gouvinhas et al. 2021; Petrucci et al. 2020b, 2021). Conversely, similarity between B and R1 might be expected, since R1 is likely prepared with B (same brand): in fact, B and R1 were not significantly different (p < 0.05) for HBAs, except for VA 9.
The hydroxycinnamic acids (HCAs) were identified in all samples, except CA 10, found only in B and R1 (same brand). FA 16 was predominant in all alcoholic samples, as expected since it is typical of beer, while SA 15 was more abundant in L. R1 and R2 resulted significantly different (p < 0.05) for HCAs, R1 in general richer than R2, analogously to what observed for HBAs, described above. B and R1 resulted significantly different for HCAs, except for CuA 14: the general decrease observed in R1 compared to B was likely due to the dilution step occurring during the radler production, that prevails over the possible intake from the lemon juice.
CuA, SA and FA (14–16) might be typical of the Italian lemon, used to prepare L, R1 and R2. Noteworthy, some authors reported the presence of HCAs 10 and 14–16 in fresh lemon, mainly in the peel (Gorinstein et al. 2001); others reported the presence of CA 10 in different fruit parts of lemon cultivars, juice included though in lower amounts, while very low content of FA 16 was found only in the peel, not in the juice (Xi et al. 2017).
Flavonoids were differently distributed among the analyzed samples.
Prenylflavonoids (pFs) from hops X 26 and IsoX 25 are characteristic of beer and were absent in L. Similar content of 25 was found in the alcoholic samples; conversely, a low quantity of 26 was found in R2, suggesting, once more, differences in the beer recipes used for R1 and R2, likely regarding hop, thermic treatments or pH (Zambrzycka-Szelewa et al. 2020).
Among flavonoids (Fs),Cat 6 was the most abundant in B, R1 and R2, in agreement with literature reporting 6 as the typical flavonoid of beer (Cheiran et al. 2019; Gouvinhas et al. 2021), and it was absent in L.
Q 23 and K 24 are typical of beer too (Di Matteo P. et al., 2021; Gouvinhas et al. 2021; Petrucci et al. 2020b, 2021; Quifer-Rada et al. 2015), mainly coming from hops. Quercetin was found in the free form (23) and in the bonded form, identified as Q3G 19; conversely, kampferol was present mainly as the bonded form, identified as K3R 20, though it was detected also in the free form (24) in B and R1. B, R1 and R2 resulted not significantly different (p < 0.05) with regard to 20. Noteworthy, K3R 20 was reported as a discriminant compound for lager beer (Cheiran et al. 2019).
My 18 was found in trace in B and L, and it was quantitated in R1, in which the higher content was likely due to the intake from both B and the lemon juice. It was not detected in R2.
Conversely, Ru 17 and He 22 were found typical of the Italian lemon juice; particularly, He 22 was present in L in very high amount, in agreement with literature (Mcharek and Hanchi 2017; Singh et al. 2020; Xi et al. 2017). Ru 17 and He 22 were absent in B, consequentially their presence in R1 and R2 was due to the Italian lemon juice.
He 22, Ru 17, Q3G 19 and My 18 were found in L, with a content following the order 22 > > 17 > 19 > 18. These data agree with literature reporting the biosynthesis and the accumulation of high level of flavonoid glycosides in citrus (Owens and McIntosh 2011). Free quercetin 23 was not detected in L, the same occurring for K 24 and its rutinoside derivative 20. He 22 and Ru 17 were reported in the literature as the main representative flavonoids of lemon (Alu’datt et al. 2017; Mchareck and Hanchi, 2017; Xi et al. 2017).
Resuming, a different phenolic profile was observed for R1 and R2, mainly regarding the absence of HBAs in R2 and a different distribution of IsoX (25) and X (26). These results may be ascribed to differences in malt, hop, and brewing process for the production of the beers used to prepare R1 and R2. This is confirmed by the correlation found between R1 and B, same brand, that had substantially the same phenolic profile, except for hesperidin.
Conversely, R2 was found much richer than R1 in hesperidin 22: since similar content of the concentrate Italian lemon juice is labelled for R1 and R2 (3.2% vs. 2.7%, respectively, see Table 1), such a higher content of He 22 in R2 was likely due to the presence of the other citrus fruits, orange and lime, reported on the label (see Table 1).
Summing up, the targeted phenolic profile of the Italian lemon juice has been firstly investigated, at least up to our knowledge, including HBAs, HCAs, Fs and pFs. Coumaric, sinapic and ferulic acids (14–16, respectively), rutin, myricitrin, quercetin-3-O-glucoside (17–19, respectively) and hesperidin 22, were identified and quantitated where possible.
The impact of the lemon juice on the phenolic profile of beer was evaluated for the analyzed samples. The total content of the phenolic compounds of B, R1, R2 and L were summarized for classes (hydroxybenzoic acids HBAs, hydroxycinnamic acids HCAs, flavonoids Fs and prenylflavonoids pFs), and comparative results are plotted in Fig. 1. Radlers resulted strongly strengthened in antioxidants content respect to beer from both qualitative and quantitative aspects, with high level of hesperidin.
GC-MS fingerprinting of the volatile fraction
The volatile fraction profile of R1, R2, B and L was investigated by GC-MS analysis (Di Matteo G. et al. 2021; Di Matteo P. et al. 2021) of the extracts obtained by a first stir bar extraction followed by a back-extraction in dichloromethane (Horák et al. 2007).
The untargeted analysis evidenced 23 peaks (27–49 in Table 4), tentatively assigned by comparison of the fragmentation spectra with NIST libraries: 8 compounds resulted typical of B, 13 compounds resulted typical of L, and 2 compounds were found in both B and L. Most of the 23 compounds were found in the volatile profile of R1 and R2.
Table 4 Compounds tentatively identified in the volatile fraction of B, R1, R2 and L, by GC-MS analysis and comparision of fragmentation spectra with NIST libraries. d: detected; nd: not detected
Peaks evidenced in B were assigned to isoamyl alchohol 28, isoamyl acetate 31, ethyl caproate 33, ethyl caprylate 48, and phenylethyl acetate 49, in agreement with literature, reporting them generally present in lager beers (Di Matteo P. et al., 2021; Horák et al. 2007; Nešpor et al. 2019); further, isoamyl n-eptanoate 32, N-hydroxymethyl-2-phenylacetamide 40 and 4-amino-1-pentanol 42 were not previously reported, up to our knowledge. All compounds were confirmed in R1 and R2, except 40 and 42.
Peaks evidenced in L were assigned to β-cimene 36, D-limonene 37, γ-terpinene 38, β-fenchol 41, terpinen-4-ol 46, and α-terpineol 47, in agreement with literature, reporting them in Italian lemon (Giuffrè et al. 2019; Scurria et al. 2021; Trovato et al. 2021); further, diacetone alchohol 29, α-pinene 34, isocineole 35, 4-carene 39, neodihydro carveol 43, cosmene 44 and vinyl-o-xylene 45 were tentatively assigned. All compounds were confirmed in R1 and R2, except for α-pinene 34 and cosmene 44, and vinyl-o-xylene 45, detected in R2 but not in R1.
Heptane 27 and m-xylene 30 were found in all the samples.
A major impact of lemon aromas was found on the radlers, D-limonene being the dominant peak, as shown in the total ion chromatograms (TIC) in Fig. 2. Qualitative data are resumed in Table 4.