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Differentiation of qualified tea beverages from spoiled ones by the LC-MS–based analysis of their polycarboxylic acids

Differentiation of qualified tea beverages from spoiled ones by the LC-MS–based analysis of their... Polycarboxylic acids (PACs) are important metabolic products in almost all living bodies, yet current analytical methods for PACs detection in tea beverages are still unsatisfactory due to their complex matrix and physicochemical properties of PACs. In this work, a rapid method has been developed for the simultaneous determination of 7 PACs, including tartaric acid, α-ketoglutaric acid, malic acid, malonic acid, cis-aconitic acid, succinic acid and fumaric acid, in beverages, based on selective removal of the matrix in combination with liquid chromatography-mass spectrometry (LC-MS) analysis. By stirring with activated carbon and the Na CO solution, the matrix in beverages was selectively removed, and PACs 2 3 were almost retained in the supernatant of diluted Na CO solution. Under optimized parameters, the limit 2 3 of quantitation for the PACs was in the range of 1-50 ng/mL, and the content of the PACs in 8 beverages was determined with the recovery range of 72.2–122.5%. The content of malic acid, malonic acid, and succinic acid in tea beverages was found to be more than that in non-tea beverages, respectively. Moreover, the concentration of these PACs in beverages was found to be multiplied many times in their deterioration period, especially for fumaric acid and α-ketoglutaric acid. These results indicated that PACs can be selected as a criterion to differentiate the qualified tea beverages from the spoiled ones. Keywords: tea beverage, deterioration, polycarboxylic acid, simultaneous determination, LC-MS Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 1. Introduction Beverages include alcoholic beverages, dairy products, tea drinks, and mineral water, which can be consumed directly or after dilution (Duan, N. et al., 2021). The consumption scale of China's beverage industry has gradually expanded, and it is expected to exceed 1.3 trillion in 2024. Tea has been cultivated in Asia for thousands of years. Tea beverages are made from tea extracts as the main raw materials and processed through several processing steps such as filtration, blending, sterilization and packaging (Dai, J. X. et al., 2021). They have a thirst-quenching function, as well as many nutritional and health benefits (Fu, Y. Q. et al., 2020). Tea refers to one of the most common drinks in a human regular diet., due to their unique flavor and texture (Hung, W. L. et al., 2018; Ziyatdinova, G. et al., 2011; Wu, L. J. et al., 2015). The organoleptic properties of fruits, foods and beverages are strongly affected by their inherent polycarboxylic acids (PACs), which are the metabolites of living things. Some of these PACs are the important intermediates in the key metabolic pathway of the tricarboxylic acid (TCA) cycle in most organisms(Zhang, S. Y. et al., 2011; Kumar, V. et al., 2017; Huang, X. Y. et al., 2021). The concentration of PACs and their changes are commended as important indicators that reflect the ripeness, decay or fermentation of fruit and food (Qiu, X. et al., 2021). They can indicate the spoilage of fruit derivatives and can be employed as acidifiers in the food and beverage industry (Restuccia. D. et al., 2017). For example, Malic acid provides a pleasant taste that plays an important role in improving muscle performance, reducing fatigue, and more (Carocho, M. et al., 2013; Campo, G. D. et al., 2006). Fumaric acid has a strong, tart, fruity taste (i.e., tumors or acute kidney disease). Cis-aconitic acid is an intermediate in the conversion of citric acid to isocitrate by aconitase activity. Compared to citric acid, cis- aconitic acid has a pleasant and moderate sour taste in the mouth. Tartaric acid has a stronger, sharper taste than citric acid, and it is a common acid in several fruits such as grapes and bananas. Malic acid has the huge market prospect as food acid seasoning (Dai, Z. X. et al., 2018; Sun, L. et al., 2020). These PACs are also widely used as food additives in the manufacture of beverages, wine and juices (Ivanova- Petropulos, V. et al., 2022), for the purpose of acidification or oxidation resistance. Meanwhile, the spoilage of beverages will also bring about a change in the content of some of their PACs, due to the bacterial metabolism. There are also reports on Aspergillus niger infestation on grapes and citrus (Kong, Q. J. et al., 2020; Qi, J. R. et al., 2018). And the spoilage of foods and beverages imposes Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 significant annual global revenue losses for the food and beverage industries. Therefore, it is of great importance to determine the polycarboxylic acid profile in beverages for the purpose of quality control. Chromatography is currently one of the most versatile analytical techniques (Martins, V. D. C. et al., 2018), the analytical methods for the determination of PACs include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS) (Mato, I. et al., 2005). GC (or GC-MS) is the earliest chromatographic technique for the determination of PACs (Barden, T. J. et al., 1997; Bartolozzi, F. et al., 1997), while PACs can not be directly analyzed by GC or GC-MS, due to its high polarity and non-volatility (Giumanini, A. G. et al., 2001; Saraji, M. et al., 2006). Derivatization needs to be performed before GC analysis, which increases the time for sample processing and cumbersome pretreatment. LC, especially LC-MS, is currently the popular method for the determination of PACs (Chen, Q. Y. et al., 2006; Chinnici, F. et al., 2005; Gamoh, K. et al., 2003; Suto, M. et al., 2020). However, it remains to be very difficult to determine target components in the tea samples, due to the serious interference of tea polyphenols in these samples (Hu, S. P. et al., 2019; Cladière, M. et al., 2018; Jiao, W. T. et al., 2016; Rahman, M. M. et al., 2015). For example, catechins are the major tea polyphenols in green tea accounting for 16–30% of dry green tea leaves (Graham, H. N. et al., 1992). High amounts of polyphenols and tea pigments, such as theaflavins and thearubigins, will be coextracted with target components in the sample pretreatment step, and these bioactive components become the complex matrix in the extractant of the tea samples. Additionally, the test sample rich in caffeine and polyphenols always causes great contamination to MS, and further increases the trouble and costs of the maintenance of equipment. Therefore, a reasonable pretreatment process to remove these tea polyphenols is indispensable for the determination of PACs in tea beverages. The aim of this work was to develop a proper and simple pretreatment method to effectively remove the complex matrix in the tea beverages, and to establish an LC-MS method for the Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 simultaneous determination of the PACs in these samples. And the contents of PACs were compared between the commercial tea beverages and the bad ones. 2. Materials and methods 2.1. Chemicals and reagents Tartaric acid standard (≥99.5%, SCR), α-ketoglutaric acid standard (≥99%, Scien Max), malic acid standard (≥99%, Urchem), malonic acid standard (≥98%, SCR), cis-aconitic acid standard (90%, HWRK Chem.), succinic acid standard (≥99.5%, Macklin), fumaric acid standard (99%, Jiuding Chem.), acetonitrile (HPLC grade, ≥99.9%, Sigma-Aldrich), formic acid (HPLC, ≥98%, Aladdin) and activated carbon (Meryer) were obtained from commercial sources and used without further purification (Table 1). Deionized water was prepared in-house with a water polisher to a resistivity of 18.2 MΩ · cm or greater. The beverage samples analyzed in this work were all obtained from a local supermarket, and their detailed information can be obtained in Table S1 in the supplementary information. 2.2. Instrumental analysis All LC-MS experiments were carried out on an Agilent 1290 ultra-performance liquid chromatography unit combined with an Agilent 6495 tandem triple quadrupole mass spectrometer (Agilent, USA, CA). HPLC analysis was performed on a Zorbax Eclipse XDB-C18 column (250 m × 4.6 mm, 5 μm, Agilent, USA) at a temperature of 30 C. Analyte separation was achieved by gradient elution with a mobile phase consisting of 0.1% formic acid in water (A): acetonitrile (B) at a flow rate of 0.6 mL/min. The procedure of gradient elution was set as: 0 min, 4% (B); 5 min, 4% (B);12 min, 8% (B). Mass detection was carried out through electrospray ionization (ESI) in the negative mode using the following optimized parameters: capillary voltage 3000 V, nozzle voltage 1500V, atomizing gas (N ) pressure 20 psi; sheath gas flow rate 11 L/min; gas temp 200 C, sheath gas temperature 230 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 C, multi-reaction monitoring mode, activation energy 20 eV (Table 1). The quantification of individual compounds in the present study was calculated with a calibration curve of the standard compound purchased (Kelebek, H. et al., 2017). 2.3. Sample preparation The stock solution of 7 PACs was prepared by adding 100 mg of PACs standards into a volumetric flask and diluted to 10.0 mL with water-acetonitrile (V: V = 9: 1). The stock solutions were stored at −5 C before usage. Working standard solutions with different concentration levels of PACs were prepared daily by appropriate dilutions of stock solution with water-acetonitrile (V: V = 9: 1). For sample analysis, 0.5 mL beverage was mixed with 1 mL Na CO solution (0.2 mmol/L) and 2 3 0.04 g activated carbon. The mixture was stirred for 2 h and centrifuged at 3000 rpm for 30 min. The corresponding supernatant was then filtered through a 0.45 μm pore size membrane (Nylon, Dikma Technologies Co., Ltd) before HPLC-MS analysis. 3. Results and discussion 3.1. Method development for the determination of PACs by LC-MS/MS Due to its chemical structure, all PACs can be easily detected as the deprotonated molecule [M- - - H] by LC-MS in the negative mode. Upon collisional activation, [M-H] of PACs is facile to undergo fragmentation by the loss of CO or H O in the MS/MS analysis. For example, [M-H] of α- 2 2 ketoglutaric acid at m/z 145 undergoes the successive elimination of CO , to produce the fragment ions at m/z 101 and m/z 57, respectively. Fragmentation of the deprotonated malic acid generates the product ion at m/z 145, which undergoes the subsequent elimination of CO to give the product ion at 71 (Fig. 1). The MS/MS spectra of other PACs and the corresponding potential fragmentation pathway were available in the supplementary information (Fig. S1 ). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 A study of the optimal selective reaction monitoring (SRM) transitions for each organic acid was carried out using several fragmentation voltages (380 V) and collision energies (from 5 to 20 V). Table 1 shows the MS conditions selected for quantification of the selected compounds. Using these selective reaction transitions, any possible interference was mostly avoided for the determination these PACs even with similar retention times. To improve the chromatographic peak shape and to optimize the ionization efficiency in the MS detection, the optimized HPLC mobile phase usually contains an ionization boosting agent, such as formic acid, trifluoroacetic acid (TFA) or ammonium acetate. TFA is reported to be the worst additive for ESI in both the negative- and the positive-ion modes, whereas formic acid is the best choice (Temesi, D. and Law, B., 1999). Herein, different concentrations of formic acid between 0.05% and 0.5% (V/V) were investigated as the mobile phase of formic acid solution (A) and acetonitrile (B) to obtain the optimal response and separation of PACs. The optimized results were achieved by using the gradient eluted mobile phase containing 0.1% (V/V) formic acid. The gradient program of HPLC was modified according to the content of acetonitrile in the mobile phase, and all studied PACs were eluted within 10 min under the described chromatographic conditions (the supplementary Fig. S2). 3.2 Optimization of pretreament efficiency However, the determination of target components in tea samples is seriously deteriorated by the interference of tea polyphenols (Cladière, M. et al., 2018; Jiao, W. T. et al., 2016). Effective and selective extraction of PACs is essential for the analysis of tea beverages. Herein, several pretreatment parameters, including the amount of activated carbon, the pretreatment system, and pretreatment time were optimized. There are a lot of tea polyphenols and other additives in the tea beverages, and direct analysis of these samples will seriously deteriorate the detection sensitivity and cause great pollution to the ion source of the mass spectrometer. Activated carbon can absorb tea polyphenols and organic additives, and the amount of activated carbon has a significant effect on the removal efficiency of these matrices Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 in beverage samples. Fig. 2 compares the UV spectra of tea beverages treated with different amounts of activated carbon (see Section 2.3). As can be seen, the untreated tea beverage shows much UV absorption in the wavelength range of 230-360 nm, with a maximum wavelength of 270 nm. When treated with only 0.01g activated carbon, there is a distinctive drop in the UV absorption. The UV absorption decreases with increasing the amount of the activated carbon treated. There is almost no UV absorption in the tea beverage when treated with 0.04 g activated carbon. Thus, the dosage of the activated carbon for the treatment of tea beverages was selected as 0.04 g in the following experiments. Pretreatment time also significantly affects the removal efficiency of the matrix in the tea beverages. Improvement in the removal efficiency of the matrix can improve the ionization efficiency and the MS signal of PACs. Fig. 3 shows the sum MS signal of PACs in the same tea beverage with different stirring times. As shown in the figure, the sum of MS area of PACs increases with extending the stirring time, and reaches an equilibrium when it is over 2 h. In consideration of the overall experimental efficiency, the stirring time was selected as 2 h in the following experiments. Besides the matrix, activated carbon can also absorb the target PACs in the samples. Thus, it is essential to develop a pretreatment method for selective absorption of tea polyphenols rather than PACs. To our interest, PACs exist in two forms in aqueous solutions, the neutral molecule and the anion, where the anionic form can not be effectively absorbed by the non-polar activated carbon. Reaction with Na CO contributes to the transformation of the neutral PACs into the anionic ones. 2 3 The acidity of PACs is stronger than that of carbonic acid. Fig. 4 compares the sum MS signal of PACs in 0.5 mL tea beverage treated with a series of Na CO solutions with different concentrations. 2 3 As can be seen, the sum MS signal rises with increasing the concentration of Na CO from 0 to 0.2 2 3 mM, indicating that the addition of Na CO facilitates activated carbon’s selective absorption of tea 2 3 polyphenols. Then, the sum MS signal decreases with continuously increasing the concentration of Na CO , because it inhibited ionization efficiency. Thus, the concentration of Na CO in the 2 3 2 3 pretreatment process was selected as 0.2 mM in the following experiments. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 3.3 Determination of PCAs in beverages Under the optimized conditions described above, a series of mixed standard solutions of the seven PACs were analyzed by LC-MS/MS, and the corresponding calibration graphs were constructed by plotting peak area vs concentration (Table 2). Good linearity was achieved in the range studied for each organic acid with the correlation coefficient (R ) between 0.9907 and 0.9999 (Table 2). The limit of detection (LOD, S/N=3) ranged from 1 to 50 ng/mL, and the limit of quantification (LOQ, S/N=10) were measured to be in the range of 1-50 ng/mL. Intraday reproducibility (RSD) was determined to be among 1.51% and 3.01% for every PAC at 100 ng/mL, and the interday reproducibility (RSD) was 2.37% and3.54% . Then, the developed method was applied for the determination of the PACs in 8 beverage samples (Table 3), and the results had a good reproducibility with RSD (N=3) ranging from 0.9 % to 7.6% (Table S2 in the supplementary information). The content of each PAC ranged from 10.5 to 3387.3 ng/mL, and the corresponding standard recovery ranged from 72.2 % to 110.0 % (Table S3 in the supplementary information), indicating the validity of the results. Analysis of Table 3 indicated that tartaric acid (10.5 - 32.8 ng/mL), α-ketoglutaric acid (34.9 - 92.4 ng/mL), cis-aconitic acid (210.5 - 359.6 ng/mL) and fumaric acid (42.5 - 130.6 ng/mL) show similar content in both tea beverages and non-tea beverages. Interestingly, the concentration of malic acid (1140.6 - 3387.3 ng/mL), malonic acid (215.0 - 270.4 ng/mL), and succinic acid (92.9 - 275.9 ng/mL) in tea beverages was found to be significantly more than that (88.7 - 573.7 ng/mL, 11.8 - 16.7 ng/mL, and 15.3 - 22.5 ng/mL) in non- tea beverages, respectively. The difference in the concentration might be due to the characteristic metabolic pathways in tea. Finally, the content of PACs was compared among the qualified and the spoiled ones (Table S4). From the determined results, the content of PACs in the beverages increased greatly during the spoilage period. Take jasmine tea (Fig. 5) as an example, the content of tartaric acid increased from 15.9 ng/mL in the qualified one, to 42.3 ng/mL in the 4 days’ open one at room temperature (about 15 C), and to 105.2 ng/mL in the spoiled one. The content of succinic acid increased 75 times in during the spoilage period. The increasement was 2, 5, 7, 14 and 15 times for cis-aconitic acid, malonic acid, Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 malic acid, fumaric acid and α-ketoglutaric acid, respectively. Among them, the increasement is particularly obvious for succinic acid and α-ketoglutaric acid. Similar results were obtained for other beverages (Table S4 in the supplementary information). Also, analytical methods were developed to determine PACs in various food substrates, and the results indicated that the amount of PACs varied in different substrates or during fermentation and storage period. A liquid chromatographic method was developed for the investigation of tartaric, malic, ascorbic and citric acids in fruit juices, and their content was found to in the range of 8.42-228 mg/mL (Scherer, R. et al., 2012). Moreover, a simple and rapid method was developed for the determination of 5 PACs (gluconic acid, tartaric acid, malic acid, citric acid, and succinic acid) in honey by liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Suto, M. et al., 2020). The corresponding limit of detection was ranged from 0.005 to 0.70 mg/kg. Six targeted organic acids (tartaric, malic, shikimic, lactic, citric, and succinic) were determined in Chardonnay and Merlot wines by high-performance liquid chromatography method coupled with diode array detector (Ivanova-Petropulos, V. et al., 2020). During fermentation and storage, the content of malic acid decreases and the content of lactic acid increases in wine. In general, the sum of all determined organic acids was higher in white wines (mean 6.18 g/L) compared with red wines (mean 5.62 g/L). It has been reported that the concentration levels of fumaric acid in apple juice could be important indicators of microbial spoilage of juices such as fumaric acid produced by moulds (Tricard, C. et al., 1986 ). The TCA cycle is a metabolic pathway utilized by aerobic organisms to generate cellular energy and intermediates for biosynthetic pathways (Eniafe, J. and Jiang, S., 2021). Thereby, the increase of polycarboxylic acids’ content in tea beverages, such as succinic acid and α-ketoglutaric acid, is likely to be produced in the tricarboxylic acid cycle of microorganisms during the deterioration of beverages. Thus, the acidification in the spoiled beverages is partly attributed to the formed PACs, besides acetic acid. Therefore, the above PACs can be selected as a criteria to differentiate the qualified tea beverages from the spoiled ones. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 4. Conclusion In this work, we have developed a proper and simple pretreatment method to effectively remove the complex matrix in the tea beverages, and to establish an LC-MS method for the simultaneous determination of the PACs in these samples. According to the validation study, the sensitivity, linearity, repeatability and recovery of the method are satisfactory. The method has been applied for the determination of PACs in 8 commercially available beverage samples among the qualified and the spoiled ones, and the results showed that the content of PACs in the beverages increased greatly during the spoilage period. And the contents of PACs were compared between the commercial tea beverages and non-tea beverages, the results showed that some PACs content varied greatly among different beverages. The described method can be used in the routine analysis for the determination of PACs in tea beverages, and may be easily extended to other matrices and PACs. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Credit authorship contribution statement Yuting Kang: Investigation, Writing-original draft, Writing - review & editing. Chenghua Li: Formal analysis. Huiru Li: Formal analysis. Jing Li: Formal analysis. Kezhi Jiang: Initiantion, Supervision, Writing - review & editing, Project administration, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement The authors gratefully acknowledge the financial support from the Analysis and Detection Foundation of Science and Technology Department in Zhejiang Province, China (grant numbers: LGC21B050009), Hangzhou Normal University Innovation Practice and Service Local plan "diligent, careful research and innovation" scientific research project, China (Item No. YJS2022055). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 References Barden, T. J., Croft, M. Y., Murby, E. J., Wells, R. J. (1997). Gas chromatographic determination of organic acids from fruit juices by combined resin mediated methylation and extraction in supercritical carbon dioxide. Journal of Chromatography A, 785(1-2): 251–261. Bartolozzi, F., Bertazza, G., Bassi, D., Cristoferi, G. (1997). 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Single-drop microextraction followed by insyringe derivatization and gas chromatography-mass spectrometric detection for determination of organic acids in fruits and fruit juices. Journal of Separation Science, 29(9): 1223–1229. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Scherer, R., Rybka, A. C. P., Ballus, C. A., Meinhart, A. D., Filho, J. T., Godoy, H. T. (2012). Validation of a HPLC method for simultaneous determination of main organic acids in fruits and juices. Food Chemistry, 135(1): 150-154. Sun, L., Gong, M. Y., Lv, X. Q., Huang, Z. Y., Gu, Y., Li, J. H., Du, G. H., Liu, L. (2020). Current advance in biological production of short-chain organic acid. Applied Microbiology and Biotechnology, 104: 9109–9124. Suto, M., Kawashima, H., Nakamura, Y. (2020). Determination of Organic Acids in Honey by Liquid Chromatography with Tandem Mass Spectrometry. Food Analytical Methods, 13: 2249–2257. Temesi, D. and Law, B. (1999). The effect of LC eluent composition on MS responses using electrospray ionization. Lc Gc North America, 17(7): 626–632. Tricard, C., Salagoity, M. H., Sudraud, P. (1986). Fumaric acid: an indicator of changes in fruits decayed by Rhizopus stolonifer. Annales des Falsifications, de l’ Expertise Chimique et Toxicologigue, 79: 303–310. Wu, L. J., Song, C. M., Zhao, Y., He, Z. J., Zhou, G. Y., Lu, W., Wang, B. X. (2015). Determination of Organochlorine Pesticides in Tea Beverage by Directly Suspended Droplet Microextraction Combined with GC-ECD. Food Analytical Methods, 8(1): 147–153. Zhang, S. Y. and Bryant, D. A. (2011). The Tricarboxylic Acid Cycle in Cyanobacteria. Science, 334(6062): 1551–1553. Ziyatdinova, G., Nizamova, A., Budnikov, H. (2011). Novel Coulometric Approach to Evaluation of Total Free Polyphenols in Tea and Coffee Beverages in Presence of Milk Proteins. Food Analytical Methods, 4(3): 334–340. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Captions of Tables and Figures: Table 1 Retention times (RT) and SRM transitions for the studied PACs Table 2: Linear ranges (μg/mL), linear equations and correlation coefficients (R ) for the target polybasic carboxylic acid Table 3 The determined content of 7 PACs in tea beverages Fig. 1 MS/MS of [M-H] of α-ketoglutaric acid (A) and malic acid (B) Fig. 2 UV image of a certain tea beverage treated with different dosages of activated carbon (A, treated by 0.01g activated carbon; B, treated by 0.02g activated carbon; C, treated by 0.03g activated carbon; D, treated by 0.04g activated carbon) Fig. 3 The effect of the stirring time on the response of PCAs in LC-MS. Fig. 4 The effect of the Na CO concentration (mM) on the response of PACs in LC-MS. 2 3 Fig. 5 Comparison of the content of PACs in the qualified, 4 day’s open and the spoiled beverage. (A) Tartaric acid. (B) Aconitic acid. (C) α-Ketoglutaric acid, Malonic acid and Fumaric acid. (D) Succinic acid and Malic acid. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Table 1 Retention times (RT) and SRM transitions for the studied PACs Compound Molecular RT Transition Fragmentor Collision formula (min) (m/z) voltage (V) energy (V) Tartaric acid C H O 4.099 149→88 380 10 4 6 6 α-Ketoglutaric acid C H O 4.844 145→101 380 10 5 6 5 Malic acid C H O 4.696 133→115 380 10 4 6 5 Malonic acid C H O 4.851 103→59 380 10 3 4 4 Cis-aconitic acid C H O 6.232 173→85 380 14 6 6 6 Succinic acid C H O 7.743 117→73 380 10 4 6 4 Fumaric acid C H O 7.036 115→71 380 10 4 4 4 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Table 2: Linear ranges (μg/mL), linear equations and correlation coefficients (R ) for the target polybasic carboxylic acid Intra-day Inter-day Range Compound Equation R RSD RSD (ng/mL) (100ng/mL) (100ng/mL) Tartaric Acid y = 11.5x + 4.28 0.9907 1~200 1.51% 2.69% Malonic Acid y = 8.04x + 9.11 0.9971 5~500 2.00% 3.36% α-Ketoglutaric acid y = 13.34x - 190.97 0.9999 50~5000 2.87% 2.99% Succinic Acid y = 99.64x - 271 0.9976 5~500 2.58% 2.37% Malic Acid y = 66.93x - 558.95 0.9991 10~1000 1.62% 3.42% Fumaric Acid y = 2.80x - 89.64 0.9999 50~5000 1.57% 3.72% Cis-aconitic Acid y = 25.313x - 163.45 0.9994 50~5000 3.01% 3.54% Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Table 3 The determined content of 7 PACs in tea beverages Concentration (ng/mL) beverage α- Cis- Tartaric Malic Malonic Succinic Fumaric Ketoglutaric aconitic acid acid acid acid acid acid acid Jasmine tea 15.9 69.7 1848.9 215.0 236.0 115.9 89.9 Jasmine grapefruit tea 32.8 34.9 1140.6 243.4 224.6 92.9 70.2 Jasmine honey tea 16.2 71.4 2399.1 270.4 259.5 191.0 99.2 oolong tea 20.8 92.4 2815.8 226.5 237.2 253.6 130.6 peach oolong 32.8 48.9 2334.4 259.1 359.6 236.1 82.7 cold brew tea 25.9 44.1 3387.3 261.3 269.0 259.9 54.2 peach juice 10.5 65.2 573.7 16.7 210.5 22.5 50.2 Fanta Orange Juice 21.8 43.3 88.7 11.8 285.4 15.3 42.5 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 1 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 2 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 3 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 4 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 5 Accepted Manuscript http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food Quality and Safety Oxford University Press

Differentiation of qualified tea beverages from spoiled ones by the LC-MS–based analysis of their polycarboxylic acids

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Food Quality and Safety , Volume 7: 1 – Nov 14, 2022

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Oxford University Press
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© The Author(s) 2022. Published by Oxford University Press on behalf of Zhejiang University Press.
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2399-1399
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2399-1402
DOI
10.1093/fqsafe/fyac067
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Abstract

Polycarboxylic acids (PACs) are important metabolic products in almost all living bodies, yet current analytical methods for PACs detection in tea beverages are still unsatisfactory due to their complex matrix and physicochemical properties of PACs. In this work, a rapid method has been developed for the simultaneous determination of 7 PACs, including tartaric acid, α-ketoglutaric acid, malic acid, malonic acid, cis-aconitic acid, succinic acid and fumaric acid, in beverages, based on selective removal of the matrix in combination with liquid chromatography-mass spectrometry (LC-MS) analysis. By stirring with activated carbon and the Na CO solution, the matrix in beverages was selectively removed, and PACs 2 3 were almost retained in the supernatant of diluted Na CO solution. Under optimized parameters, the limit 2 3 of quantitation for the PACs was in the range of 1-50 ng/mL, and the content of the PACs in 8 beverages was determined with the recovery range of 72.2–122.5%. The content of malic acid, malonic acid, and succinic acid in tea beverages was found to be more than that in non-tea beverages, respectively. Moreover, the concentration of these PACs in beverages was found to be multiplied many times in their deterioration period, especially for fumaric acid and α-ketoglutaric acid. These results indicated that PACs can be selected as a criterion to differentiate the qualified tea beverages from the spoiled ones. Keywords: tea beverage, deterioration, polycarboxylic acid, simultaneous determination, LC-MS Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 1. Introduction Beverages include alcoholic beverages, dairy products, tea drinks, and mineral water, which can be consumed directly or after dilution (Duan, N. et al., 2021). The consumption scale of China's beverage industry has gradually expanded, and it is expected to exceed 1.3 trillion in 2024. Tea has been cultivated in Asia for thousands of years. Tea beverages are made from tea extracts as the main raw materials and processed through several processing steps such as filtration, blending, sterilization and packaging (Dai, J. X. et al., 2021). They have a thirst-quenching function, as well as many nutritional and health benefits (Fu, Y. Q. et al., 2020). Tea refers to one of the most common drinks in a human regular diet., due to their unique flavor and texture (Hung, W. L. et al., 2018; Ziyatdinova, G. et al., 2011; Wu, L. J. et al., 2015). The organoleptic properties of fruits, foods and beverages are strongly affected by their inherent polycarboxylic acids (PACs), which are the metabolites of living things. Some of these PACs are the important intermediates in the key metabolic pathway of the tricarboxylic acid (TCA) cycle in most organisms(Zhang, S. Y. et al., 2011; Kumar, V. et al., 2017; Huang, X. Y. et al., 2021). The concentration of PACs and their changes are commended as important indicators that reflect the ripeness, decay or fermentation of fruit and food (Qiu, X. et al., 2021). They can indicate the spoilage of fruit derivatives and can be employed as acidifiers in the food and beverage industry (Restuccia. D. et al., 2017). For example, Malic acid provides a pleasant taste that plays an important role in improving muscle performance, reducing fatigue, and more (Carocho, M. et al., 2013; Campo, G. D. et al., 2006). Fumaric acid has a strong, tart, fruity taste (i.e., tumors or acute kidney disease). Cis-aconitic acid is an intermediate in the conversion of citric acid to isocitrate by aconitase activity. Compared to citric acid, cis- aconitic acid has a pleasant and moderate sour taste in the mouth. Tartaric acid has a stronger, sharper taste than citric acid, and it is a common acid in several fruits such as grapes and bananas. Malic acid has the huge market prospect as food acid seasoning (Dai, Z. X. et al., 2018; Sun, L. et al., 2020). These PACs are also widely used as food additives in the manufacture of beverages, wine and juices (Ivanova- Petropulos, V. et al., 2022), for the purpose of acidification or oxidation resistance. Meanwhile, the spoilage of beverages will also bring about a change in the content of some of their PACs, due to the bacterial metabolism. There are also reports on Aspergillus niger infestation on grapes and citrus (Kong, Q. J. et al., 2020; Qi, J. R. et al., 2018). And the spoilage of foods and beverages imposes Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 significant annual global revenue losses for the food and beverage industries. Therefore, it is of great importance to determine the polycarboxylic acid profile in beverages for the purpose of quality control. Chromatography is currently one of the most versatile analytical techniques (Martins, V. D. C. et al., 2018), the analytical methods for the determination of PACs include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS) (Mato, I. et al., 2005). GC (or GC-MS) is the earliest chromatographic technique for the determination of PACs (Barden, T. J. et al., 1997; Bartolozzi, F. et al., 1997), while PACs can not be directly analyzed by GC or GC-MS, due to its high polarity and non-volatility (Giumanini, A. G. et al., 2001; Saraji, M. et al., 2006). Derivatization needs to be performed before GC analysis, which increases the time for sample processing and cumbersome pretreatment. LC, especially LC-MS, is currently the popular method for the determination of PACs (Chen, Q. Y. et al., 2006; Chinnici, F. et al., 2005; Gamoh, K. et al., 2003; Suto, M. et al., 2020). However, it remains to be very difficult to determine target components in the tea samples, due to the serious interference of tea polyphenols in these samples (Hu, S. P. et al., 2019; Cladière, M. et al., 2018; Jiao, W. T. et al., 2016; Rahman, M. M. et al., 2015). For example, catechins are the major tea polyphenols in green tea accounting for 16–30% of dry green tea leaves (Graham, H. N. et al., 1992). High amounts of polyphenols and tea pigments, such as theaflavins and thearubigins, will be coextracted with target components in the sample pretreatment step, and these bioactive components become the complex matrix in the extractant of the tea samples. Additionally, the test sample rich in caffeine and polyphenols always causes great contamination to MS, and further increases the trouble and costs of the maintenance of equipment. Therefore, a reasonable pretreatment process to remove these tea polyphenols is indispensable for the determination of PACs in tea beverages. The aim of this work was to develop a proper and simple pretreatment method to effectively remove the complex matrix in the tea beverages, and to establish an LC-MS method for the Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 simultaneous determination of the PACs in these samples. And the contents of PACs were compared between the commercial tea beverages and the bad ones. 2. Materials and methods 2.1. Chemicals and reagents Tartaric acid standard (≥99.5%, SCR), α-ketoglutaric acid standard (≥99%, Scien Max), malic acid standard (≥99%, Urchem), malonic acid standard (≥98%, SCR), cis-aconitic acid standard (90%, HWRK Chem.), succinic acid standard (≥99.5%, Macklin), fumaric acid standard (99%, Jiuding Chem.), acetonitrile (HPLC grade, ≥99.9%, Sigma-Aldrich), formic acid (HPLC, ≥98%, Aladdin) and activated carbon (Meryer) were obtained from commercial sources and used without further purification (Table 1). Deionized water was prepared in-house with a water polisher to a resistivity of 18.2 MΩ · cm or greater. The beverage samples analyzed in this work were all obtained from a local supermarket, and their detailed information can be obtained in Table S1 in the supplementary information. 2.2. Instrumental analysis All LC-MS experiments were carried out on an Agilent 1290 ultra-performance liquid chromatography unit combined with an Agilent 6495 tandem triple quadrupole mass spectrometer (Agilent, USA, CA). HPLC analysis was performed on a Zorbax Eclipse XDB-C18 column (250 m × 4.6 mm, 5 μm, Agilent, USA) at a temperature of 30 C. Analyte separation was achieved by gradient elution with a mobile phase consisting of 0.1% formic acid in water (A): acetonitrile (B) at a flow rate of 0.6 mL/min. The procedure of gradient elution was set as: 0 min, 4% (B); 5 min, 4% (B);12 min, 8% (B). Mass detection was carried out through electrospray ionization (ESI) in the negative mode using the following optimized parameters: capillary voltage 3000 V, nozzle voltage 1500V, atomizing gas (N ) pressure 20 psi; sheath gas flow rate 11 L/min; gas temp 200 C, sheath gas temperature 230 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 C, multi-reaction monitoring mode, activation energy 20 eV (Table 1). The quantification of individual compounds in the present study was calculated with a calibration curve of the standard compound purchased (Kelebek, H. et al., 2017). 2.3. Sample preparation The stock solution of 7 PACs was prepared by adding 100 mg of PACs standards into a volumetric flask and diluted to 10.0 mL with water-acetonitrile (V: V = 9: 1). The stock solutions were stored at −5 C before usage. Working standard solutions with different concentration levels of PACs were prepared daily by appropriate dilutions of stock solution with water-acetonitrile (V: V = 9: 1). For sample analysis, 0.5 mL beverage was mixed with 1 mL Na CO solution (0.2 mmol/L) and 2 3 0.04 g activated carbon. The mixture was stirred for 2 h and centrifuged at 3000 rpm for 30 min. The corresponding supernatant was then filtered through a 0.45 μm pore size membrane (Nylon, Dikma Technologies Co., Ltd) before HPLC-MS analysis. 3. Results and discussion 3.1. Method development for the determination of PACs by LC-MS/MS Due to its chemical structure, all PACs can be easily detected as the deprotonated molecule [M- - - H] by LC-MS in the negative mode. Upon collisional activation, [M-H] of PACs is facile to undergo fragmentation by the loss of CO or H O in the MS/MS analysis. For example, [M-H] of α- 2 2 ketoglutaric acid at m/z 145 undergoes the successive elimination of CO , to produce the fragment ions at m/z 101 and m/z 57, respectively. Fragmentation of the deprotonated malic acid generates the product ion at m/z 145, which undergoes the subsequent elimination of CO to give the product ion at 71 (Fig. 1). The MS/MS spectra of other PACs and the corresponding potential fragmentation pathway were available in the supplementary information (Fig. S1 ). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 A study of the optimal selective reaction monitoring (SRM) transitions for each organic acid was carried out using several fragmentation voltages (380 V) and collision energies (from 5 to 20 V). Table 1 shows the MS conditions selected for quantification of the selected compounds. Using these selective reaction transitions, any possible interference was mostly avoided for the determination these PACs even with similar retention times. To improve the chromatographic peak shape and to optimize the ionization efficiency in the MS detection, the optimized HPLC mobile phase usually contains an ionization boosting agent, such as formic acid, trifluoroacetic acid (TFA) or ammonium acetate. TFA is reported to be the worst additive for ESI in both the negative- and the positive-ion modes, whereas formic acid is the best choice (Temesi, D. and Law, B., 1999). Herein, different concentrations of formic acid between 0.05% and 0.5% (V/V) were investigated as the mobile phase of formic acid solution (A) and acetonitrile (B) to obtain the optimal response and separation of PACs. The optimized results were achieved by using the gradient eluted mobile phase containing 0.1% (V/V) formic acid. The gradient program of HPLC was modified according to the content of acetonitrile in the mobile phase, and all studied PACs were eluted within 10 min under the described chromatographic conditions (the supplementary Fig. S2). 3.2 Optimization of pretreament efficiency However, the determination of target components in tea samples is seriously deteriorated by the interference of tea polyphenols (Cladière, M. et al., 2018; Jiao, W. T. et al., 2016). Effective and selective extraction of PACs is essential for the analysis of tea beverages. Herein, several pretreatment parameters, including the amount of activated carbon, the pretreatment system, and pretreatment time were optimized. There are a lot of tea polyphenols and other additives in the tea beverages, and direct analysis of these samples will seriously deteriorate the detection sensitivity and cause great pollution to the ion source of the mass spectrometer. Activated carbon can absorb tea polyphenols and organic additives, and the amount of activated carbon has a significant effect on the removal efficiency of these matrices Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 in beverage samples. Fig. 2 compares the UV spectra of tea beverages treated with different amounts of activated carbon (see Section 2.3). As can be seen, the untreated tea beverage shows much UV absorption in the wavelength range of 230-360 nm, with a maximum wavelength of 270 nm. When treated with only 0.01g activated carbon, there is a distinctive drop in the UV absorption. The UV absorption decreases with increasing the amount of the activated carbon treated. There is almost no UV absorption in the tea beverage when treated with 0.04 g activated carbon. Thus, the dosage of the activated carbon for the treatment of tea beverages was selected as 0.04 g in the following experiments. Pretreatment time also significantly affects the removal efficiency of the matrix in the tea beverages. Improvement in the removal efficiency of the matrix can improve the ionization efficiency and the MS signal of PACs. Fig. 3 shows the sum MS signal of PACs in the same tea beverage with different stirring times. As shown in the figure, the sum of MS area of PACs increases with extending the stirring time, and reaches an equilibrium when it is over 2 h. In consideration of the overall experimental efficiency, the stirring time was selected as 2 h in the following experiments. Besides the matrix, activated carbon can also absorb the target PACs in the samples. Thus, it is essential to develop a pretreatment method for selective absorption of tea polyphenols rather than PACs. To our interest, PACs exist in two forms in aqueous solutions, the neutral molecule and the anion, where the anionic form can not be effectively absorbed by the non-polar activated carbon. Reaction with Na CO contributes to the transformation of the neutral PACs into the anionic ones. 2 3 The acidity of PACs is stronger than that of carbonic acid. Fig. 4 compares the sum MS signal of PACs in 0.5 mL tea beverage treated with a series of Na CO solutions with different concentrations. 2 3 As can be seen, the sum MS signal rises with increasing the concentration of Na CO from 0 to 0.2 2 3 mM, indicating that the addition of Na CO facilitates activated carbon’s selective absorption of tea 2 3 polyphenols. Then, the sum MS signal decreases with continuously increasing the concentration of Na CO , because it inhibited ionization efficiency. Thus, the concentration of Na CO in the 2 3 2 3 pretreatment process was selected as 0.2 mM in the following experiments. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 3.3 Determination of PCAs in beverages Under the optimized conditions described above, a series of mixed standard solutions of the seven PACs were analyzed by LC-MS/MS, and the corresponding calibration graphs were constructed by plotting peak area vs concentration (Table 2). Good linearity was achieved in the range studied for each organic acid with the correlation coefficient (R ) between 0.9907 and 0.9999 (Table 2). The limit of detection (LOD, S/N=3) ranged from 1 to 50 ng/mL, and the limit of quantification (LOQ, S/N=10) were measured to be in the range of 1-50 ng/mL. Intraday reproducibility (RSD) was determined to be among 1.51% and 3.01% for every PAC at 100 ng/mL, and the interday reproducibility (RSD) was 2.37% and3.54% . Then, the developed method was applied for the determination of the PACs in 8 beverage samples (Table 3), and the results had a good reproducibility with RSD (N=3) ranging from 0.9 % to 7.6% (Table S2 in the supplementary information). The content of each PAC ranged from 10.5 to 3387.3 ng/mL, and the corresponding standard recovery ranged from 72.2 % to 110.0 % (Table S3 in the supplementary information), indicating the validity of the results. Analysis of Table 3 indicated that tartaric acid (10.5 - 32.8 ng/mL), α-ketoglutaric acid (34.9 - 92.4 ng/mL), cis-aconitic acid (210.5 - 359.6 ng/mL) and fumaric acid (42.5 - 130.6 ng/mL) show similar content in both tea beverages and non-tea beverages. Interestingly, the concentration of malic acid (1140.6 - 3387.3 ng/mL), malonic acid (215.0 - 270.4 ng/mL), and succinic acid (92.9 - 275.9 ng/mL) in tea beverages was found to be significantly more than that (88.7 - 573.7 ng/mL, 11.8 - 16.7 ng/mL, and 15.3 - 22.5 ng/mL) in non- tea beverages, respectively. The difference in the concentration might be due to the characteristic metabolic pathways in tea. Finally, the content of PACs was compared among the qualified and the spoiled ones (Table S4). From the determined results, the content of PACs in the beverages increased greatly during the spoilage period. Take jasmine tea (Fig. 5) as an example, the content of tartaric acid increased from 15.9 ng/mL in the qualified one, to 42.3 ng/mL in the 4 days’ open one at room temperature (about 15 C), and to 105.2 ng/mL in the spoiled one. The content of succinic acid increased 75 times in during the spoilage period. The increasement was 2, 5, 7, 14 and 15 times for cis-aconitic acid, malonic acid, Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 malic acid, fumaric acid and α-ketoglutaric acid, respectively. Among them, the increasement is particularly obvious for succinic acid and α-ketoglutaric acid. Similar results were obtained for other beverages (Table S4 in the supplementary information). Also, analytical methods were developed to determine PACs in various food substrates, and the results indicated that the amount of PACs varied in different substrates or during fermentation and storage period. A liquid chromatographic method was developed for the investigation of tartaric, malic, ascorbic and citric acids in fruit juices, and their content was found to in the range of 8.42-228 mg/mL (Scherer, R. et al., 2012). Moreover, a simple and rapid method was developed for the determination of 5 PACs (gluconic acid, tartaric acid, malic acid, citric acid, and succinic acid) in honey by liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Suto, M. et al., 2020). The corresponding limit of detection was ranged from 0.005 to 0.70 mg/kg. Six targeted organic acids (tartaric, malic, shikimic, lactic, citric, and succinic) were determined in Chardonnay and Merlot wines by high-performance liquid chromatography method coupled with diode array detector (Ivanova-Petropulos, V. et al., 2020). During fermentation and storage, the content of malic acid decreases and the content of lactic acid increases in wine. In general, the sum of all determined organic acids was higher in white wines (mean 6.18 g/L) compared with red wines (mean 5.62 g/L). It has been reported that the concentration levels of fumaric acid in apple juice could be important indicators of microbial spoilage of juices such as fumaric acid produced by moulds (Tricard, C. et al., 1986 ). The TCA cycle is a metabolic pathway utilized by aerobic organisms to generate cellular energy and intermediates for biosynthetic pathways (Eniafe, J. and Jiang, S., 2021). Thereby, the increase of polycarboxylic acids’ content in tea beverages, such as succinic acid and α-ketoglutaric acid, is likely to be produced in the tricarboxylic acid cycle of microorganisms during the deterioration of beverages. Thus, the acidification in the spoiled beverages is partly attributed to the formed PACs, besides acetic acid. Therefore, the above PACs can be selected as a criteria to differentiate the qualified tea beverages from the spoiled ones. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 4. Conclusion In this work, we have developed a proper and simple pretreatment method to effectively remove the complex matrix in the tea beverages, and to establish an LC-MS method for the simultaneous determination of the PACs in these samples. According to the validation study, the sensitivity, linearity, repeatability and recovery of the method are satisfactory. The method has been applied for the determination of PACs in 8 commercially available beverage samples among the qualified and the spoiled ones, and the results showed that the content of PACs in the beverages increased greatly during the spoilage period. And the contents of PACs were compared between the commercial tea beverages and non-tea beverages, the results showed that some PACs content varied greatly among different beverages. The described method can be used in the routine analysis for the determination of PACs in tea beverages, and may be easily extended to other matrices and PACs. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Credit authorship contribution statement Yuting Kang: Investigation, Writing-original draft, Writing - review & editing. Chenghua Li: Formal analysis. Huiru Li: Formal analysis. Jing Li: Formal analysis. Kezhi Jiang: Initiantion, Supervision, Writing - review & editing, Project administration, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Captions of Tables and Figures: Table 1 Retention times (RT) and SRM transitions for the studied PACs Table 2: Linear ranges (μg/mL), linear equations and correlation coefficients (R ) for the target polybasic carboxylic acid Table 3 The determined content of 7 PACs in tea beverages Fig. 1 MS/MS of [M-H] of α-ketoglutaric acid (A) and malic acid (B) Fig. 2 UV image of a certain tea beverage treated with different dosages of activated carbon (A, treated by 0.01g activated carbon; B, treated by 0.02g activated carbon; C, treated by 0.03g activated carbon; D, treated by 0.04g activated carbon) Fig. 3 The effect of the stirring time on the response of PCAs in LC-MS. Fig. 4 The effect of the Na CO concentration (mM) on the response of PACs in LC-MS. 2 3 Fig. 5 Comparison of the content of PACs in the qualified, 4 day’s open and the spoiled beverage. (A) Tartaric acid. (B) Aconitic acid. (C) α-Ketoglutaric acid, Malonic acid and Fumaric acid. (D) Succinic acid and Malic acid. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Table 1 Retention times (RT) and SRM transitions for the studied PACs Compound Molecular RT Transition Fragmentor Collision formula (min) (m/z) voltage (V) energy (V) Tartaric acid C H O 4.099 149→88 380 10 4 6 6 α-Ketoglutaric acid C H O 4.844 145→101 380 10 5 6 5 Malic acid C H O 4.696 133→115 380 10 4 6 5 Malonic acid C H O 4.851 103→59 380 10 3 4 4 Cis-aconitic acid C H O 6.232 173→85 380 14 6 6 6 Succinic acid C H O 7.743 117→73 380 10 4 6 4 Fumaric acid C H O 7.036 115→71 380 10 4 4 4 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Table 2: Linear ranges (μg/mL), linear equations and correlation coefficients (R ) for the target polybasic carboxylic acid Intra-day Inter-day Range Compound Equation R RSD RSD (ng/mL) (100ng/mL) (100ng/mL) Tartaric Acid y = 11.5x + 4.28 0.9907 1~200 1.51% 2.69% Malonic Acid y = 8.04x + 9.11 0.9971 5~500 2.00% 3.36% α-Ketoglutaric acid y = 13.34x - 190.97 0.9999 50~5000 2.87% 2.99% Succinic Acid y = 99.64x - 271 0.9976 5~500 2.58% 2.37% Malic Acid y = 66.93x - 558.95 0.9991 10~1000 1.62% 3.42% Fumaric Acid y = 2.80x - 89.64 0.9999 50~5000 1.57% 3.72% Cis-aconitic Acid y = 25.313x - 163.45 0.9994 50~5000 3.01% 3.54% Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Table 3 The determined content of 7 PACs in tea beverages Concentration (ng/mL) beverage α- Cis- Tartaric Malic Malonic Succinic Fumaric Ketoglutaric aconitic acid acid acid acid acid acid acid Jasmine tea 15.9 69.7 1848.9 215.0 236.0 115.9 89.9 Jasmine grapefruit tea 32.8 34.9 1140.6 243.4 224.6 92.9 70.2 Jasmine honey tea 16.2 71.4 2399.1 270.4 259.5 191.0 99.2 oolong tea 20.8 92.4 2815.8 226.5 237.2 253.6 130.6 peach oolong 32.8 48.9 2334.4 259.1 359.6 236.1 82.7 cold brew tea 25.9 44.1 3387.3 261.3 269.0 259.9 54.2 peach juice 10.5 65.2 573.7 16.7 210.5 22.5 50.2 Fanta Orange Juice 21.8 43.3 88.7 11.8 285.4 15.3 42.5 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 1 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 2 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 3 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 4 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac067/6827100 by DeepDyve user on 16 November 2022 Figure 5 Accepted Manuscript

Journal

Food Quality and SafetyOxford University Press

Published: Nov 14, 2022

Keywords: Tea beverage; deterioration; polycarboxylic acid; simultaneous determination; liquid chromatography–mass spectrometry

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