Profiling and annotation of carbonyl compounds in Baijiu Daqu by chlorine isotope labeling-assisted ultrahigh-performance liquid chromatography-high resolution mass spectrometry

As a fermentation starter, Baijiu Daqu provides microorganisms, flavor compounds and precursors in the process of making Baijiu [1,2]. During the fermentation of Baijiu Daqu, the microenvironment exhibits a great influence on the microecology, which leads to diverse enzymes and metabolites in Baijiu Daqu and further affects the flavor of Bajiu [3,4]. Carbonyl compounds are among the most important flavor substances and greatly influence the taste of Baijiu [5]. Hence, developing a strategy for performing high-coverage analysis of carbonyl compounds in different Baijiu Daqu can not only help reveal why sauce-flavored Baijiu possesses a unique flavor, but also guides the manufacturing of high-quality Baijiu.

Currently, carbonyl compounds in Baijiu Daqu are mainly detected based on gas chromatography‒mass spectrometry (GC‒MS) or liquid chromatography‒mass spectrometry (LC‒MS) [6], [7], [8]. However, GC‒MS is suitable to detect nonpolar and semipolar compounds, while LC‒MS is specifically targeted at polar and semipolar compounds [9,10]. Therefore, neither GC‒MS nor LC‒MS can simultaneously detect both polar and nonpolar compounds. More importantly, most of the carbonyl compounds in Baijiu Daqu occur in trace amounts, have low MS signal responses, and exhibit diverse structures. Based on all these factors, only a small fraction of carbonyl compounds has been detected to date [11,12].

Stable isotope labeling (SIL) has been widely used to improve the MS detection sensitivity of carbonyl metabolites [13], [14], [15], [16]. Although these SIL strategies were capable of achieving high coverage detection of carbonyl metabolites, the comprehensive annotation of labeled carbonyl compounds remains a challenge. In general, the annotating of labeled carbonyl compounds involves two steps, i.e., screening and identification. Regarding screening labeled carbonyl compounds, a pair of light and heavy isotope derivatization reagents are usually needed to generate light and heavy isotope labeled features [17], [18], [19]. However, heavy isotope derivatization reagents are usually expensive and even commercially unavailable. In addition, to use a pair of isotope derivatization reagents, the light and heavy isotope derivatization reagents need to label the samples, and after the labeling is completed, the labeled products are mixed for detection. All the issues mentioned above greatly increase the cost and difficulty of the experiment. Thus, many efforts are still needed for the identification of labeled carbonyl compounds. In the literature, labeled carbonyl compounds were often unambiguously identified through the established standard databases [14,17]. However, some standards are expensive, and not all standards are commercially or synthetically available. Moreover, the retention times (tR), MS1 and MS/MS spectra of different labeled reagents are different, which restricts the wide identification of labeled carbonyl compounds. For putative identification of labeled carbonyl compounds, the usual operation is to transform MS1 into the formula and then subtract the formula of the labeling reagents. Then, the possible structures are found by searching databases with the obtained formulas [20]. As database searching with MS1 is often accompanied by higher false-positive qualification results, some other strategies are needed to improve the accuracy of identification. In our previous study, predicted tR, in silico MS/MS and diagnostic ions were integrated to enhance the identification confidence of labeled carboxyl compounds [21]. Nevertheless, as far as we know, no strategy is available to confidently identify carbonyl compounds. Therefore, developing an economical, convenient and comprehensive strategy to identify carbonyl compounds in complex samples is an urgent task.

In this study, we proposed a comprehensive strategy for profiling and annotating carbonyl compounds in sauce-flavored Baijiu Daqu using chlorine isotope labeling-assisted ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (CIL-UHPLCsingle bondHRMS). After being labeled by a new derivatization reagent named 4‑chloro-2-hydrazinylpyridine (HP-Cl), the carbonyl compounds were picked out by the characteristics of natural isotopes from chlorine; subsequently, MS1, predicted tR, in silico MS/MS and manually annotated fragments were used to identify the carbonyl compounds at 6 levels. A total of 487 carbonyl compounds were detected and annotated in Baijiu Daqu samples. Finally, the developed method was applied to study the metabolic characteristics of carbonyl compounds in different colors and different seasons of Baijiu Daqu samples.

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