Reagents and samples

Methanol, ethanol, and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). Formic acid and ¹²C-DnsCl were purchased from J&K Scientific Ltd. (Beijing, China). Ultra-pure water was produced from a Milli-Q^® system (Millipore, Massachusetts, USA). ¹³C-DnsCl was obtained from the University of Alberta. Sodium carbonate, sodium bicarbonate, and sodium hydroxide were purchased from Sinopharm (Beijing, China). All standards were purchased from Aladdin^® (Shanghai, China).

All Baijiu samples including 5 sauce flavor, 4 strong flavor, and 6 light flavor Baijiu were obtained from the local market, and the detailed information is shown in Table S1. The blank sample was ethanol-water [53:47, volume/volume (v/v)]. QC sample was prepared by mixing equal volumes of Baijiu samples. The mixed standards labeled by ¹³C-DnsCl served as internal standards.

Sample preparation

The amine and phenol standards were dissolved in ultra-pure water or ACN; 50 mL of Baijiu or blank samples were concentrated to 2 mL by evaporating with a vacuum distillation system (70 r/min, 45℃, and –0.1 MPa), and all prepared samples were stored at –20℃ before chemical labeling.

The chemical labeling was performed as that described in previous studies with slight modifications [19, 20]. Briefly, 30 μL of the concentrated sample was freeze-dried, and 30 μL of water was used to redissolve the residues. Then, 30 μL of sodium carbonate and sodium bicarbonate buffer solution (0.25 M, pH = 9.5), 30 μL ¹³C-DnsCl (10 mg/mL) or ¹²C-DnsCl (10 mg/mL), 30 μL of ACN were added for labeling 60 min at 40℃. After that, 5 μL of sodium hydroxide (250 mM) was added and reacted for another 10 min. Subsequently, 25 μL of formic acid (425 mM) was added and shocked for 5 min. The mixture was freeze-dried, and 80 μL of ACN was added for redissolving, after centrifugation for 10 min (4℃, 10,000 g), the 60 μL of supernatant was taken out for analysis.

UHPLC-HRMS analysis

Agilent 1290 Infinity UHPLC system coupled to 6546 Q-TOF (Agilent Technologies, California, USA) was used for data acquisition. Mobile phase A was composed of 0.1% (v/v) formic acid, 5% (v/v) ACN, and 94.9% (v/v) water. Mobile phase B was 0.1% (v/v) formic acid in ACN. The chromatographic column was ACQUITY BEH C18 column (100 mm × 2.1 mm, 1.7 μm, Waters™, Massachusetts, USA), column temperature was 50℃, and the column flow rate was 0.18 mL/min. The separation started with 20% B and increased to 35% B in 3.5 min, further increased to 65% B in another 14.5 min, to 99% B in another 6 min, and held for 4 min. Lastly, the gradient was back to 20% B in 0.1 min and maintained for 2.9 min [21]. The temperature of the sample manager was 4℃, injection volume was 5 μL.

Data were acquired in positive ionization mode. In full scan mode, the scan range was mass-to-charge ratios (m/z) 200–1,500 Da. In target MS/MS mode, the scan range was m/z 30–1,500 Da. Drying gas temperature and drying gas flow rate were set as 320℃ and 8 L/min, respectively. The fragmentation voltage was 175 V, and capillary voltage (Vcap) was 4,000 V. Sheath gas flow rate and Sheath gas temperature were 11 L/min and 350℃, respectively. The secondary collision energies were 10 eV, 20 eV, and 40 eV.

Evaluation of analytical characteristics

Analytical characteristics including linear range, the lower limit of detection (LLOD), the lower limit of quantification (LLOQ), intra-day precision, inter-day precision, and recovery were evaluated to ensure the robustness of the method. The mixed standards were diluted to 8–10 calibration solutions with the blank sample. Signal to noise ratio (S/N) ≥ 3 and S/N ≥ 10 were defined as the LLOD and LLOQ, respectively [22]. The intra-day precision and inter-day precision were evaluated based on the mixed standards of low, medium, and high concentrations. The mixed standards of the above three concentrations were diluted by the blank sample and prepared in 6 replicates in one day or 8 replicate samples within 3 consecutive days. Then, the relative standard deviation (RSD) of each compound was calculated. For the recovery experiment, mixed standards with three different concentrations were added to the QC samples. Afterward, the QC samples, spiked QC samples, and mixed standards were analyzed. The recovery of each compound was calculated by the peak area ratio of spiked QC samples subtracted from the peak area ratio of QC, and the difference was divided by the peak area ratio of standard, where peak area ratio means the ratio of peak area of analyte to internal standards. Each sample of the recovery experiment was prepared in 6 replicates.

Data processing and statistical analysis

The peak table was generated by MS-DIAL (version 4.24). In-house python programming was used to pick out the amine and phenol compounds from the peak table. In-house database, MassBank of North America (MoNA), and the Yeast Metabolome Database (YMDB) were used for annotation. The elements composition of amine and phenol compounds were set as C, H, O, N, and S. The formulas of amine and phenol compounds were obtained by removing the DnsCl residue (C₁₂H₁₁NO₂S). The amine and phenol compounds were annotated at 5 different levels, Level 1: the amine and phenol compounds were positively identified based on matching the retention time (t_R), precursor ions, and MS/MS with authentic standards; Level 2: the amine and phenol compounds were putatively identified by in-silico MS/MS; Level 3: the amine and phenol compounds were putatively identified by manually annotated fragments; Level 4: the amine and phenol compounds were putatively identified by searching the databases with precursor ions; and Level 5: the amine and phenol compounds were identified with the molecular formula. One-way analysis of variance (ANOVA) was performed for amine or phenol compounds to evaluate the differences between the three flavors of Baijiu. Partial least squares-discriminant analysis (PLS-DA) was carried out on SIMCA^® P 13 software (Umetrics^®, Umea, Sweden). The heat maps were plotted based on the online website of Omicstudio (https://www.omicstudio.cn).

Improvement of chromatographic retention and detection sensitivity after DnsCl labeling

To explore whether the retention and MS response of labeled amine/phenol compounds were improved, we analyzed 30 amine/phenol standards before and after labeling. The extracted ion chromatogram (EIC) profile before and after labeling was shown in Figure 2. It can be seen from Figure 2A, 30 compounds were eluted completely in 10 min, and most of the compounds were eluted in 5 min. However, after being labeled, the t_R of 30 compounds was in the range of 26 min (Figure 2B). Therefore, since introducing hydrophobic DnsCl, the retention of labeled amine and phenol compounds on the reversed phase column was significantly enhanced. In addition, the MS response of most labeled amine/phenol compounds was greatly improved (Figure 2).

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Figure 2. 

Extracted-ion chromatograms of 30 amine or phenol compounds. (A) before DnsCl labeling; (B) after DnsCl labeling. The compound information corresponding to the numbers is shown in Table 1

Untargeted analysis of amine/phenol compounds in Baijiu

To show the DnsCl labeling derivatizing method was helpful for high coverage detection of amine/phenol compounds, 15 Baijiu were analyzed. It can be seen from Figure 3A, 261, 258, 261, 261, 249, 154, 237, 139, 232, 98, 76, 115, 83, 126, and 132 compounds were detected in S1–S15, respectively. After removing duplicates, a total of 267 amine/phenol compounds were obtained (Table S2). Among the 30 compounds unambiguously identified, pentan-1-amine, dodecan-1-amine, 4-allylsyringol, and 2-vanillin were identified in Baijiu for the first time, and the 3D structure of four newly identified were shown in Figure S1.

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Figure 3. 

The number of amine or phenol compounds in Baijiu. (A) different samples; (B) different flavor

The numbers of amine/phenol compounds in sauce flavor Baijiu, strong flavor Baijiu, and light flavor Baijiu were 266, 227, and 127, respectively (Figure 3B). The PLS-DA was plotted, and the result is shown in Figure 4A, the distributions of amine/phenol compounds in strong flavor Baijiu and light flavor Baijiu were similar, while they were greatly different from in sauce flavor Baijiu. In Figure 4B, it shows the VIP distribution of all compounds, the number of compounds with VIP greater than 1 was about half of the total number (115), suggesting amine/phenol compounds in different types of Baijiu were greatly different. The cluster analysis was performed based on amine/phenol compounds of VIP > 1.2 and P < 0.05. As shown in Figure 4C, 34 amine/phenol compounds were divided into two categories, one of which showed the highest content in sauce flavor Baijiu (23), and the other showed the highest content in strong flavor Baijiu (11). In addition, 34 amine/phenol compounds had the lowest content in light flavor Baijiu. Therefore, it can be seen that significant differences in the number and content of amine/phenol compounds contribute to the discrimination of Baijiu with different flavor from each other.

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Figure 4. 

Statistical analysis. (A) PLS-DA R² = (0.0, 0.0603), Q² = (0.0, –0.238); (B) variable importance of projection (VIP) distribution of all of the compounds; (C) cluster analysis based on compounds of VIP > 1.2 and P < 0.05. The compound information corresponding to the numbers is shown in Table S2

Quantitative analysis of amine/phenol compounds in Baijiu

In the untargeted analysis, we found that the relative content of some compounds in Baijiu with different flavor were different. To explore the differences in compound contents, 30 amine/phenol compounds confirmed with standard were quantitated by using the internal standard method.

The results of the analytical characteristic evaluation are shown in Table 1. First of all, the R² of all compounds was larger than 0.99, indicating that the established method had good linearity between the concentration of the analyte and the MS response. Secondly, the LLODs and LLOQs were in the range of 1–14,564.63 ng/L and 0.01–61.90 μg/L, respectively, hence, high sensitivity was achieved. Thirdly, the RSDs of intra-day and inter-day were smaller than 15%, indicating good stability. Finally, the recovery rates were in the range of 80–120%, suggesting high accuracy. In summary, the established method can be used for the quantification of DnsCl labeled compounds.

The quantification method was used for the quantitative analysis of 30 compounds (12 amino acids and derivatives, 6 aliphatic amines, 1 aromatic amine, 2 alkylphenols, and 9 other phenolic compounds) in different flavor of Baijiu, including 5 sauce flavor Baijiu, 4 strong flavor Baijiu, and 6 light flavor Baijiu. In Table 2 it shows that 12 compounds had significant differences among different Baijiu (P < 0.05). The concentration of tyrosine was in the range of 2,221.38–43,887.19 ug/L, and much higher than other compounds. The contents of phenol and 4-ethylphenol in sauce flavor Baijiu were higher than those in the other two types of Baijiu. Besides, the other 6 compounds including serine, 2-(methylamino) acetic acid, alanine, Ile-Val, proline, and 2-vanillin also showed the highest content in sauce flavor Baijiu, and the contents of the remaining 4 compounds were the highest in strong flavor Baijiu. However, most of the compounds in light flavor Baijiu had the lowest contents, especially 5-aminopentanoic acid, Ile-Val, peonol, 2-vanillin, and 4-ethylphenol.