Determination of Diacetyl in Beer by a Precolumn Derivatization-HPLC

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Determination of Diacetyl in Beer by a Pre-Column Derivatization-HPLC-UV Method Using 4-(2,3-dimethyl-6quinoxalinyl)-1,2-benzenediamine as a Derivatizing Reagent Ji-Yu Wang, Xin-Jie Wang, Xian Hui, Shui-Hong Hua, Heng Li, and Wen-Yun Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00990 • Publication Date (Web): 11 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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Journal of Agricultural and Food Chemistry

Determination of Diacetyl in Beer by a Pre-Column Derivatization-HPLC-UV Method Using 4-(2,3-dimethyl-6-quinoxalinyl)-1,2-benzenediamine as a Derivatizing Reagent

Ji-Yu Wang, Xin-Jie Wang, Xian Hui, Shui-Hong Hua, Heng Li*, and Wen-Yun Gao*

National Engineering Research Center for Miniaturized Detection Systems and College of Life Sciences, Northwest University, 229 North Taibai Road, Xi’an, Shaanxi 710069, China

*To whom correspondence should be addressed (Heng Li: Fax, +86 29 88303572; Tel, +86 29 88303446 ext. 834; E-mail: [email protected]. Wen-Yun Gao: Fax, +86 29 88303572; Tel, +86 29 88303446 ext. 832; E-mail: [email protected])

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ACS Paragon Plus Environment


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ABSTRACT: Diacetyl is an important flavoring compound in many foods, especially in beer.

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In the present study, we developed and validated a new pre-column derivatization HPLC-UV

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method for the determination of diacetyl using 4-(2,3-dimethyl-6-quinoxalinyl)-1,2-

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benzenediamine as a novel derivatizing reagent. After derivatization with the reagent at a pH

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value 4.0 at ambient temperature for 10 min, diacetyl was analyzed on an ODS column and

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detected at 254 nm. The results show that the correlation coefficient of the method is 0.9991

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in the range of 0.10 to 100.0 µM diacetyl, and the limit of detection is 0.02 µM. The method

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was further evaluated in the analysis of beer samples with the recoveries ranging from 94.4%

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to 102.6% and RSDs from 1.36% to 3.33%. The concentrations of diacetyl in 8 beer samples

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were determined in the range of 0.19 to 0.42 µM. The method established in this study may be

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well suitable for the determination of diacetyl in beer.

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KEYWORDS: Diacetyl, determination, 4-(2,3-dimethyl-6-quinoxalinyl)-1,2-benzenediamine,

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HPLC, pre-column derivatization

14 15 16 17 18 19 20 21 22 2


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INTRODUCTION

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2,3-Butanedione, also known as diacetyl or biacetyl, is a natural byproduct of fermentation

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that is responsible for the aroma of many food products and beverages. It is also widely used

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as a food additive for improving the flavor of popcorn, candy, chocolate, and roasted foods.1,2

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However, the compound has some undesirable impacts on health safety and the flavor of wine

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and beer. Research has shown that diacetyl may be harmful when inhaled over a long duration

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and may cause various toxic responses, for example, lung disease, Alzheimer’s disease,

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mutagenesis, and carcinogenesis.3,4 The odor threshold of diacetyl for wine can be up to 58.14

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µM depending on the type of wine, 5 and this value is extremely low for beer (approximately

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1.16 µM).6 If the concentration of diacetyl is higher than the sensory threshold, wine and beer

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will smell and taste like spoiled food. Therefore, it is necessary to establish efficient and

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practical methods to determine the concentration of this compound not only for health safety

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but also for controlling the quality of various fermented foods such as wine and beer.

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As a consequence of this concern, many methods have been developed for measuring

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diacetyl concentrations.7,8 Among these methods, the pre-column derivatization HPLC is

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currently the most popular way to quantify this compound in different samples, not only in

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various food products such as wine,9-15 beer,9,16,17 coffee,18-19 soy sauce,13,19 honey,20,21

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vinegar,22 baby food,23 soft drink,24 fructose agave syrups,25 and other foodstuffs,26 but also in

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body liquids such as urine27-31 and plasma.32 The most frequently employed diacetyl

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derivatization reagent is o-phenylenediamine (1, Figure 1) because it is readily available and

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has a low price. It forms a quinoxaline derivative with diacetyl, which can be easily separated

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and determined by HPLC equipped with various detectors such as UV, fluorescence, and 3



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MS.9-13,16,18-20,22-24,33 The drawback of 1 is that the derivatization reaction needs to be heated at

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approximately 40°C or higher for at least 10 min or longer to ensure its completion. If the

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reaction is carried out at room temperature, then it has to be kept in the dark for at least a few

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hours or even overnight.20,23,33 Both high reaction temperatures and long reaction times could

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lower the accuracy of the determination, especially for wine and beer because of the

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nonenzymatic oxidation of acetolactate to diacetyl.37,38 Other derivatization reagents used for

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HPLC determination of diacetyl are analogues or derivatives of 1; however, these compounds

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should also react with diacetyl under heating conditions, for example

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5,6-diamino-2,4-hydroxypyrimidine (2, Figure 1, 60-80°C, 30 min),14,29 3,4-diaminopyridine

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(3, 90°C, 2 hr),15 4-nitro-1,2-diaminobenzene (4, 45°C, 20 min),17 5,6-diamino-1-methyluracil

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(5, 60% acetic acid, reflux for 24 hr),21 6-hydroxy-2,4,5-triaminopyrimidine (6, 60°C, 45

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min),27,30 4-methoxy-1,2-diaminobenzene (7, 40°C, 4 hr or reflux in ethanol for 40 min),25,26,31

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2,3-diaminonaphthalene (8, rt, overnight),32 4,5-dimethoxy-1,2-diaminobenzene (9, 60°C, 4

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hr),28,34 1,2-diamino-4,5-methylenedioxybenzene (10, 60°C, 40 min),35 and rhodamine

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B-hydrozine (11, 37°C, 3 hr).36 Therefore, it is necessary to develop novel reagents that can

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quickly react with diacetyl under ambient conditions.

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3,3΄-Diaminobenzidine (12) has been widely used in various scientific fields and is

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currently utilized as a monomer to prepare high-temperature resistant synthetic resins and

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fibers. However, in the analytical field, it has been employed only as a chromogenic reagent

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to determine selenium.39,40 In this research, we found that the mono-quinoxaline derivative of

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12, 4-(2,3-dimethyl-6-quinoxalinyl)-1,2-benzenediamine (13) (Figure 2) can react with

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diacetyl quickly at room temperature and can be used to determine diacetyl concentrations in 4


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beer with high sensitivity and accuracy. An additional advantage of the derivatization is that it

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is almost independent of the acidity of the reaction mixture. Here, we will discuss the

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experimental details.

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MATERIALS AND METHODS

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Chemicals and Reagents. Compounds 4, 12 and diacetyl are of analytical-grade and

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were purchased from Fluka (Shanghai). HPLC-grade methanol was purchased from

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Sigma-Aldrich (Beijing). Millipore water was obtained using a Milli-Q water purification

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system. Compound 13 was synthesized in this lab from 12. All other chemicals and solvents

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are of analytical-grade and were obtained from commercial sources. The stock solution of 100

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mM diacetyl was prepared in Millipore water; 5 mM reagent 13 was prepared in 0.1 M HCl;

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and 1.3 mM (200 mg/L) reagent 4 was prepared in methanol. The stock solutions were stored

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in at 4°C before use.

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Instrumentation. An Agilent 1200 HPLC system (Agilent Technologies, Shanghai)

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equipped with a DAD detector and a Shim-pack VP-ODS column (250 × 4.6 mm, 4.6 µm,

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Shimadzu, Japan) were used for the separation and the analysis. 1H-NMR spectra were

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collected on a Varian Inova-600 MHz NMR spectrometer. HRESI-MS was performed with a

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Thermo Fisher LTQ XL system.

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Preparation of 13. One hundred and eight milligrams (0.3 mmol) of 12 was added to

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a round bottom flask containing 10 mL of water. The mixture was stirred at room temperature

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(rt) for approximately 20 min before 35 mg (0.4 mmol) diacetyl was added to the mixture.

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After further stirred for half an hour, the mixture was basified with 1 M NaOH and extracted

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with CHCl3 (15 mL × 3). The CHCl3 layers were pooled and dried over anhydrous MgSO4 for 5



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approximately 2 hr. The solid was then filtered out, and the organic solvent was evaporated

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under reduced pressure. The dark red powder that was obtained was further isolated on a

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silica gel column using CH3OH-CHCl3-diethylamine = 6-1-0.1 (V/V/V) as a developing

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solvent. Two compounds were acquired, and their structures were elucidated by HRESI-MS

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and 1H-NMR. The results showed that one product was the desired compound 13 and the

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other was fully diacetylated compound 2,2΄,3,3΄-tetramethyl-6,6΄-biquinoxaline (14).

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4-(2,3-Dimethyl-6-quinoxalinyl)-1,2-benzenediamine (13). Obtained as dark red

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1 powder (45.2 mg, yield 57.1%). H-NMR (D2O, 600 MHz): 7.51 (1H, brs), 7.41 (1H, brs),

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7.15 (2H, brs), 7.00 (2H, m), 2.38 (3H, s, Me), 2.31 (3H, s, Me). HRESI-MS (positive mode)

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m/z: calcd for C16H18N4, [M+H]+ 265.1453, found 265.1461.

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2,2΄,3,3΄-Tetramethyl-6,6΄-biquinoxaline (14). Obtained as dark red powder (18.4

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mg, yield 24.6%). 1H-NMR (D2O, 600 MHz): 7.58 (2H, brs), 7.33 (2H, d, J = 6), 7.15 (2H, d,

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J = 6), 2.41(6H, s, Me × 2), 2.36 (6H, s, Me × 2). HRESI-MS (positive mode) m/z: calcd for

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C20H19N4, [M+H]+ 315.1623, found 315.1617.

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Derivatization Procedure and Identification of the Derivative. Ten microliters of a

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diacetyl solution (0.2 mM) was added to 50 µL of a solution of 13 (0.2 mM, containing 40%

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methanol), and the resulting solution was mixed well using a vortex mixer. The total solution

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was then kept at rt for 10 min, and 10 µL was used for HPLC analysis. All the samples were

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filtered through a 0.22-µm filter membrane before they were injected into the HPLC system.

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The derivative was then characterized by LC-MS.

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Chromatographic Method. The mobile phase comprised methanol and water. The gradient used was as follows: 0 min, 60% MeOH; 10 min, 100% MeOH; and 15-17 min, 60% 6


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MeOH. Both of the mobile-phase solvents were filtered with a 0.22-µm membrane before use.

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The analysis was carried out at ambient temperature with an injection volume of 10 µL, a

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flow rate of 0.7 mL/min, and UV detection at 254 nm. Each sample was injected in triplicate.

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The retention times of compounds 13 and 14 were 7.55 and 13.03 min, respectively, and the

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separation could be completed within 15 min.

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Stability Test of Compound 14. Fifty microliters of a diacetyl solution (0.2 mM) was

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added to 50 µL of a solution of 13 (1 mM, containing 40% methanol), and the resulting

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solution was mixed well with vortex mixer. The total solution was then kept on the bench top

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at rt for 4 days. Aliquots of 10 µL were used for HPLC analysis at different time points, and

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the peak areas of 13 and the product 14 were recorded.

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Analysis of Diacetyl in Beer Samples. Eight beers were purchased at local

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supermarkets in Xi’an and stored at 4°C before determination. Then, 10 mL of each beer

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sample was degassed with magnetic stirring at rt and 0.9 mL was transferred to a 2-mL

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Eppendorf vial. Each vial was supplemented with 90 µL of a solution of 13 (0.5 mM), and 10

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µL of Millipore water and homogenized with a vortex mixer. After been kept at rt for 10 min,

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the resulting solutions were filtered through a 0.22-µm membrane and analyzed using the

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HPLC system. For spiking experiments, the addition of 10 µL of Millipore water was

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replaced by addition of 10 µL of a diacetyl standard solution. Each sample was measured in

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triplicate.

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RESULTS AND DISCUSSION

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Preparation of Compound 13 and Identification of its Derivatives. The preparation of quinoxalines has been reported to require a high temperature, a strong acidic medium, and 7



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a long time.41 However, a recent paper showed that vicinal diamines could react with

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1,2-dicarbonyl compounds in water at rt.42 Thus, we carried out the reaction between 12 and

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diacetyl using the reaction conditions with a 12 to diacetyl ratio approximately 1 to 1 (Figure

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2). Because 12 possesses two sets of the reactive vicinal diamine moiety, we deduced that two

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compounds, i.e., 13 and 14, could have formed. HPLC and LC-MS analyses of the reaction

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mixture confirmed that these two compounds were produced (Figure 3). After isolation on a

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silica gel column 13 and 14 were obtained and their structures were elucidated by interpreting

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the 1H-NMR and HRESI-MS spectra. Then 13 was utilized to derivatize diacetyl and the

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HPLC analysis of the reaction mixture showed that a new compound formed whose HPLC

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behavior and HRESI-MS datum were identical with that of 14.

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Optimization of Derivatization Conditions. To determine the optimum

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derivatization conditions, the effects of four factors, including the concentration ratio of

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compound 13 to diacetyl, the pH of the reaction mixture, the reaction time, and the reaction

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temperature, on the resulting peak areas of the reaction products were investigated, and the

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results are shown in Figure 4.

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Different concentrations of 13 (0.1-1.0 mM) were reacted with 0.1 mM diacetyl at rt

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for 10 min (pH 4.0). The results (Figure 4A) indicate when the ratio reaches 4 to 1, the

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maximum production of 14 could be detected in the reaction mixture. Therefore, the ratio of

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13 to diacetyl was set to 5 to 1 in the following experiments to ensure the sensitivity of the

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determination. In the test of the optimum acidity, we found (Figure 4B) that the pH values

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ranging from 1-10 did not affect the formation of 14. Since the pH value of the reaction

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mixture resulted from 90 µL of a solution of 13 (0.5 mM) and 0.9 mL beer is approximately 8


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4.0, we selected this acidity for the derivatization procedure. From the data shown in Figure

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4C, we were able to determine that rt would be the best choice for the derivatization of

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diacetyl with 13. Figure 4D depicted the influence of the reaction time on the production of

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14. The results revealed that the reaction went to its end within 5 min. To ensure the

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completeness of the derivatization and the accuracy of the analysis, we selected 10 min as the

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best time duration.

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Based on the above assays, we decided the optimum conditions of the derivatization

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reaction between the reagent 13 and diacetyl were as follows: ratio of 13 to diacetyl: 5 to 1;

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reaction acidity: pH 4.0; reaction temperature: rt; and reaction time: 10 min. Due to the

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relatively poor water solubility of the product 14, we added methanol to the reaction mixture

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to avoid its precipitation. We found that 20% methanol in the derivatization solution was

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enough to keep the compound soluble.

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Stability of Compound 14. The stability test results showed that there were no

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obvious changes in the peak area of 14 after it has been kept on the bench top at room

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temperature for 4 days, indicating their stability under testing conditions.

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Validation of the Method. Totally ten samples at diacetyl concentrations between 0.1

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and 200 µM were derivatized with 13 separately and measured and the linear calibration

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curve was fitted. We then evaluated the sensitivity of the procedure by determining the limit

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of detection (LOD) at a signal-to-noise ratio of 3 and the limit of quantification (LOQ) at a

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signal-to-noise ratio of 10.43 Subsequently, we assessed the reproducibility of the method by

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determining the relative standard deviation (RSD) with a diacetyl concentration of 10 µM. As

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the mean value of six measurements, RSD of method reached 2.83%. The linear calibration 9



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ranges, regression equations, regression coefficient (R2), LOD, LOQ, and RSD of the new

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method were calculated and are listed in Table 1.

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Application of Compound 13 to Beer Analysis. Based on the above validation, we

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evaluated the accuracy of the developed method by determining the concentrations of diacetyl

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in different beer samples using the standard addition method. Three different concentrations

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of standard diacetyl were added to the beer samples. Three replicates were used for each

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concentration, and each sample was injected in six replicates. The recovery and RSD values

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were calculated and are listed in Table 2. The representative chromatograms of the blank of

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compound 13, the standard diacetyl derivatized with 13, the beer sample without 13

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derivatization, and 13 derivatized diacetyl in the beer sample before and after spiking are

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shown in Figure 5. The data exhibit that the recoveries of diacetyl are between 94.4% and

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102.6% and the RSDs are between 1.36% and 3.33%, relating to the different beer samples.

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Therefore, the new method established in this study is well suitable for the quantification of

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diacetyl in beer samples.

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Comparison of Compounds 4 and 13. Up to now, quite a few derivatizing reagents

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have been employed to measure diacetyl in various samples by HPLC methods (Figure 1).

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Among all the HPLC procedures using UV detection, the method utilizing 4 as a derivatizing

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reagent displays the lowest LOD and LOQ (Tables 1).8,17 To compare 4 and 13, we validated

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the published method in the derivatization of standard diacetyl and in the determination of

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beer samples and the results were listed in Tables 1 and 2, respectively. Combining these

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validation data and the optimized derivatization conditions for each reagent, we would

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conclude that: 10


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i)

Compound 13 is more reactive than 4 because 13 could derivatize diacetyl under

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milder conditions (rt, 5min at pH from 1-10) than 4 (45°C, 20 min at pH from 1-3).

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This could be owing to the structural difference of the two compounds. Nitro

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group possesses strong electron withdrawing effect and its existence can largely

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reduce the nucleophilicity of the amino groups in compound 4; whereas the

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quinoxalinyl moiety of 13 shows mainly its conjugation effect which can enhance

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to some extent the reactivity of its amino groups.

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ii) Although there is no significant difference in the recoveries and RSD of the two

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methods (Table 2), compound 13 is more suitable than 4 for quantification of

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diacetyl because the slope of the calibration curve of 13 is about three times

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bigger than that of 4; whereas 4 is a better option in the qualification of diacetyl

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because it shows lower LOD than 13 (Table 1). The larger slope of the method

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using 13 should result from the bigger conjugation system of this compound, but

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the reason that the method using 4 shows a lower LOD remains ambiguous.

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In summary, we set up in this study a novel derivatization procedure for the measurement

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of diacetyl in beer by HPLC employing 13 as a new derivatizing reagent. The advantage of

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the method is that the derivatization reaction could be carried out quickly under mild

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conditions (rt, 5 min) and in a wide pH range (1-10). Moreover, the process also exhibits

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effective chromatographic separation, satisfactory linearity, and excellent repeatability. The

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mild conditions for diacetyl derivatization can efficiently prevent the formation of extra

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diacetyl under heating condition owing to nonenzymatic oxidation of the precursor of diacetyl

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in the samples and thus ensure the accuracy of the determination. These conditions effectively 11



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simplify the operation and expedite the measurement as well.

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Funding

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This work was supported by the National Science Foundation of China (Grants 21172179,

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21402152), the Program for Changjiang Scholars and Innovative Research Team in

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University (No. IRT_15R55), and the Scientific Research Project of Education Department of

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Shaanxi Provincial Government (No. 15JK1710).

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Acknowledgments

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The authors gratefully acknowledge Dr. Xinfeng Zhao and Dr. Chaoni Xiao of the College of

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Life Sciences, Northwest University for their kind help with the LC-MS and 1H-NMR

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analyses.

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Notes

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The authors declare no competing financial interest.

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diacetyl in urine using 4-methoxy-o-phenylenediamine as derivatizing reagent. Anal. 16


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assay of methylglyoxal. Anal. Chim. Acta 1992, 263,137–142.

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363

364

365

366

367

368

369

370

371

372 18


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373

Figure captions

374

Figure 1. Chemical structures of the reagents used for the pre-column derivatization HPLC

375

determination of diacetyl.

376

Figure 2. Reaction of diacetyl with compound 12 (1 : 1) in acidic water and with 13 (1 : 5) in

377

acidic aqueous MeOH at room temperature.

378

Figure 3. HPLC and LC-MS profiles of the reaction of compound 12 and diacetyl. Line 1,

379

compound 12; line 2, compound 12 to diacetyl ratio is 1 : 1. Reaction conditions: pH 3.0, rt

380

for 10 min.

381

Figure 4. Optimization of derivatization conditions. Effects of the (A) concentration ratio of

382

compound 13 to diacetyl, (B) derivatization pH, (C) derivatization temperature, and (D)

383

derivatization time on the peak areas of the product 14. Reaction conditions: (A) 0.1 mM

384

diacetyl, pH 4.0, rt, 10 min; (B) 0.1 mM diacetyl, 0.5 mM 13, rt, 10 min; (C) 0.1 mM diacetyl,

385

0.5 mM 13, pH 4.0, 10 min; (D) 0.1 mM diacetyl, 0.5 mM 13, pH 4.0, rt.

386

Figure 5. HPLC chromatograms obtained from (A) the blank of compound 13 (line 1) and

387

compound 13 derivatized standard diacetyl (line 2), (B) beer sample without derivatization,

388

(C) beer sample blank, and (D) beer sample spiked with diacetyl standard (2.0 µM). HPLC

389

conditions: column, Shim-pack VP-ODS column (250 × 4.6 mm inner diameter, Shimadzu,

390

Japan); UV detection, λ = 254 nm; gradient, 0 min, 60% MeOH; 10 min, 100% MeOH;

391

15-17min, 60% MeOH; flow rate, 0.7 mL/min; temperature, ambient.

392 393 394 19



395

Tables Table 1. Linear Calibration Ranges, Regression Equations, and Detection Limits (LOD and LOQ) of the Methods Using Compounds 4 or 13 as Derivatizing Reagents.

a

parameters

4d

4e

13

calibration range (µM)

0.058-116

0.075-120

0.10-100

regression equation, ya

330507x +337

112348x +319

411610x + 481

R2

0.9992

0.999

0.9992

RSD (%) (n = 6)

1.76

2.06

2.83

LOD (µM)b

0.009

0.01

0.02

LOQ (µM)c

0.03

0.05

0.10

x, concentration of diacetyl (µM); y, peak area of the products. bS/N = 3, per 10 µL injection

volume. cS/N = 10, per 10 µL injection volume. ddata extracted from [17]. edata obtained in this study under the optimized conditions for reagent 4.17

20


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Table 2. The Recoveries of Diacetyl from Different Beer Samples by the Methods Using Compounds 4 or 13 as Derivatization Reagents samples

lager Ae

lager Be

lager Ce

lager De

lager Ee

lager Fe

initiala

spiked

totalb

foundc

recovery

RSDd

(µM)

(µM)

(µM)

(µM)

(%)

(%)

0.20

0.383

0.193

96.5

3.29

0.50

0.663

0.473

94.6

3.17

2.00

2.154

1.964

98.2

2.32

0.20

0.569

0.199

99.5

3.24

0.50

0.872

0.502

100.4

2.33

2.00

2.302

1.932

96.6

1.98

0.50

0.782

0.472

94.4

3.02

2.00

2.352

2.042

102.1

2.65

5.00

5.335

5.025

100.5

1.67

0.50

0.762

0.512

102.4

2.98

2.00

2.286

2.036

101.8

1.36

5.00

5.225

4.975

99.5

2.03

0.50

0.897

0.477

95.4

3.02

2.00

2.414

1.994

99.7

2.89

5.00

5.235

4.815

96.3

2.56

0.50

0.812

0.512

102.4

3.33

2.00

2.302

2.002

100.1

2.79

0.19

0.37

0.31

0.25

0.42

0.30

21



lager Ge

lager He

lager Af

lager Bf

a

0.26

0.22

0.21

0.37

Page 22 of 28

5.00

5.190

4.890

97.8

1.99

0.50

0.773

0.513

102.6

3.10

2.00

2.270

2.010

100.5

1.98

5.00

5.345

5.085

101.7

2.22

0.50

0.696

0.476

95.2

3.01

2.00

2.132

1.912

95.6

2.95

5.00

5.260

5.040

100.8

1.89

0.20

0.407

0.197

98.5

2.19

0.50

0.685

0.475

95.0

2.86

2.00

2.114

1.904

95.2

3.11

0.20

0.575

0.205

102.5

3.14

0.50

0.849

0.479

95.8

1.66

2.00

2.342

1.972

98.6

2.18

Mean value of diacetyl content in beer before spiking (n=6). bMean value of diacetyl content

in beer after spiking (n=6). cThe total measured diacetyl content minus the initial measured diacetyl content. dMean value of six determinations. eCompound 13 as a derivatizing reagent. f

Compound 4 as a derivatizing reagent.

22


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Figure graphics

23



24


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TOC graphic

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Determination of Diacetyl in Beer by a Precolumn Derivatization-HPLC

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