CN101620206B - A method for detecting ethyl carbamate in yellow rice wine - Google Patents

Abstract

The invention discloses a method for detecting ethyl carbamate in yellow wine, comprising the following steps: (1) pre-processing the yellow wine by using reduced pressure distillation so as to reduce the volume of the yellow wine to 1/5-1/10 of the initial volume; (2) mixing the pre-processed yellow wine and hydrochloric acid as well as 9-hydroxyl solutions by tons to carry out derivatizing reaction to obtain the derivatized yellow wine; and (3) carrying out component separation on the derivatized yellow wine by using an effective liquid chromatograph and calculating the content of the ethyl carbamate by using an external standard method. The method has simple operation and low detection cost, and has simpler needed apparatus, easier sample pre-processing, more convenient needed detection condition and shorter spent detection time when compared with an analysis method for solid phase extraction-gas chromatography/mass spectrometry (SPE-GC/MS).

Description

A method for detecting ethyl carbamate in yellow rice wine

technical field

The invention belongs to the technical field of food analysis and food safety, and in particular relates to a method for detecting the content of ethyl carbamate in rice wine.

technical background

Urethane (abbreviated as EC, also known as urethane) widely exists in various fermented foods, especially in drinking wine, and is a potential carcinogen. The International Agency for Research on Cancer of the World Health Organization has classified ethyl carbamate (EC) as a 2A carcinogen, which is as dangerous as acrylamide. The World Health Organization and the United Nations Food and Agriculture Organization (WHO/FAO) Food Additives Committee (JECFA) conducted a systematic assessment of ethyl carbamate in 2005, emphasizing that the intake of ethyl carbamate mainly comes from drinking alcohol, and it is recommended that it must Take effective measures to reduce the content of ethyl carbamate in drinking alcohol.

Due to the low concentration of ethyl carbamate in drinking wine and the complex components in drinking wine, the interference to the detection is too large, which makes the detection of ethyl carbamate in drinking wine very difficult. The traditional solid-phase extraction-gas chromatography/mass spectrometry (SPE-GC/MS) has been written into the standard detection method by the authoritative organizations of the American Association of Official Analytical Chemists (AOAC), the International Office of Grape and Wine (OIV) and the European Union, but The pretreatment of samples by this method is extremely complicated, time-consuming, and the detection cost is high, so it is not suitable for routine analysis in laboratories.

The urethane in rice wine has faced the threat of technical barriers to international trade, and will become a major obstacle restricting the further healthy and sustainable development of my country's rice wine industry, and it will also pose a serious threat to the lives of consumers. Therefore, it is necessary to establish a method for the detection of ethyl carbamate in yellow rice wine. Judging from the current research situation, most of the detection methods of ethyl carbamate in rice wine still use the traditional solid-phase extraction-gas chromatography/mass spectrometry (SPE-GC/MS), which is expensive, time-consuming, and unsuitable. routine analysis.

Contents of the invention

The invention provides a method for detecting ethyl carbamate in rice wine by using a fluorescence detector of a high-performance liquid chromatography, which has the characteristics of simple operation, good repeatability, high precision, low detection cost and the like.

A method for detecting ethyl carbamate in yellow rice wine, the steps are as follows:

(1) Utilize vacuum distillation to pretreat the yellow rice wine, so that the volume of the yellow rice wine is reduced to 1/5-1/10 of the initial volume;

The temperature of described vacuum distillation is 30-40 ℃ (preferably 35 ℃);

(2) mixing the pretreated yellow rice wine with hydrochloric acid and 9-hydroxyl anthene solution for derivatization reaction to obtain the derivatized yellow rice wine;

The derivatization reaction time is 30min, and the reaction is at room temperature;

The volume ratio of yellow rice wine, hydrochloric acid, 9-hydroxyl anthene solution is:

Yellow rice wine: hydrochloric acid: 9-hydroxyl anthene solution=(7～10): 1: 2, (preferably 10: 1: 2);

The concentration of the hydrochloric acid is 1～2.0mol/L (preferably 1.5mol/L);

Described 9-hydroxyxanthene solution is the n-propanol solution of 9-hydroxyxanthene, and the concentration of 9-hydroxyxanthene in the 9-hydroxyxanthene solution is 0.01-0.03mol/L (preferably 0.02mol/L);

The derivatization reaction between urethane and 9-hydroxyl anthene is as follows:

Urethane itself has no fluorescence characteristics, but the product tonyl urethane obtained after the derivatization reaction can emit 600nm fluorescence under the excitation of 233nm wavelength light, so it can be separated by high performance liquid chromatography (HPLC). quantified by a fluorescence detector (FLD).

The proper dosage ratio of yellow rice wine: hydrochloric acid: 9-hydroxyxanthene solution and the preferred concentration of hydrochloric acid can make the derivatization reaction more thorough and make the final detection result more accurate.

(3) Utilize high-performance liquid chromatography to carry out component separation to the yellow rice wine after derivatization, adopt external standard method to calculate the ethyl carbamate content;

The high-performance liquid chromatograph adopts a C18 reversed-phase column (column length 250×column diameter 4.6mm, packing material diameter 4 μ m parameter meaning to say), detection temperature 35 ℃, injection volume 20 μ L, urethane peak The time is 35-38min;

Methanol and water are used as the mobile phase for gradient elution, and the change of mobile phase with time during gradient elution is shown in the following table:

The ratio of methanol and water in the table is volume percent.

The present invention adopts specific separation conditions to realize the precise separation and content detection of ethyl carbamate. When the method of steps (1) to (3) is used to measure the content of ethyl carbamate, the standard curve obtained by the external standard method, The correlation coefficient R ² between the peak area and the concentration of ethyl carbamate = 0.9990; the precision RSD of the method obtained by performing multiple measurements on the same sample is less than 5%; the recovery rate of the method using the standard substance addition method is 98%-101% ; This shows that the inventive method has very high precision.

The invention adopts vacuum distillation to pretreat the rice wine sample to achieve the purpose of enriching the ethyl carbamate in the rice wine, and the treated sample can be used for the quantitative determination of the ethyl carbamate. The content of ethyl carbamate in rice wine was determined under specified HPLC conditions.

The method of the invention has simple operation and low detection cost. Compared with the traditional solid phase extraction-GC/MS analysis method, the required instruments and equipment are simple, the sample pretreatment is not complicated, the required detection conditions are simple and the detection time is short.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of blank control.

Fig. 2 is the high-performance liquid chromatogram of urethane standard substance.

Fig. 3 is the high performance liquid phase chromatogram of embodiment 1.

Fig. 4 is the high performance liquid phase chromatogram of embodiment 2.

Fig. 5 is the high performance liquid phase chromatogram of embodiment 3.

Fig. 6 is the high performance liquid phase chromatogram of embodiment 4.

Fig. 7 is the high performance liquid phase chromatogram of embodiment 5.

Fig. 8 is the high performance liquid chromatogram of embodiment 6.

Fig. 9 is the high performance liquid chromatogram of embodiment 7.

Fig. 10 is the high performance liquid phase chromatogram of embodiment 8.

Fig. 11 is the high performance liquid phase chromatogram of embodiment 9.

Fig. 12 is the high performance liquid phase chromatogram of embodiment 10.

Detailed ways

High performance liquid chromatography and fluorescence detector adopted: Waters2695 high performance liquid chromatography, Waters2475 fluorescence detector (U.S. Waters company)

Chromatographic conditions: C18 reverse phase column (250×4.6mm, 4μm), detection temperature 35°C, injection volume 20μL, excitation wavelength λ _ex =233nm, emission wavelength λ _em =600nm, mobile phase is methanol and water, gradient elution The procedure is as follows:

time/min Flow rate/mL·min ^-1 Methanol% water% 0 0.8 61 39 40 0.8 61 39 42 1 100 0 50 1 100 0

The relevant reagents used are as follows:

Urethane standard substance (EC, purchased from Sigma Company), 9-xanthydrol (purchased from Sigma Company), methanol (chromatographically pure), double distilled water, hydrochloric acid (analytical pure), n-propanol ( analytically pure).

Accurately weigh 3.6mg of urethane standard substance, dilute to 200mL with 30% methanol to make 18μg/mL standard stock solution, and accurately draw 2.5mL, 1.0mL, 0.5mL, 0.1mL standard stock solution respectively 30% methanol was fixed to 10mL to obtain standard urethane test solutions with concentrations of 4.5 μg/mL, 1.8 μg/mL, 0.9 μg/mL and 0.18 μg/mL.

Add 1mL of standard sample (urethane standard test solution), 0.1mL of hydrochloric acid solution and 0.2mL of 9-hydroxybenzene solution into the centrifuge tube, and place the centrifuge tube on a shaker for a few seconds to mix evenly, and place it for 30min about.

The concentration of described hydrochloric acid is 1.5mol/L; Described 9-hydroxyxanthene solution is the n-propanol solution of 9-hydroxyxanthene, and the concentration of 9-hydroxyxanthene in the 9-hydroxyxanthene solution is that concentration is 0.02mol/L .

After the solution after the derivatization reaction is filtered with a microfiltration membrane, it is injected into a high-performance liquid chromatograph, and the urethane standard test solution of different concentrations is injected for detection, and the corresponding peak area value Area (A) is recorded, and the data As in Table 1:

Table 1 method linear relationship inspection

Using the concentration C (the concentration of ethyl carbamate in the standard ethyl carbamate test solution) to perform linear regression on the area A, the linear regression equation for ethyl carbamate is A=17624C+377.3 (R ² =0.999). It can be seen from the linear correlation coefficient that the linear relationship between the concentration of ethyl carbamate and the peak area is good.

Carry out 6 consecutive repeated measurements on a standard substance, investigate the precision of retention time and peak area, the data are shown in Table 2:

Table 2 Method precision inspection

It can be seen from the above table that both the retention time and the peak area, the relative standard deviation RSD is less than 5%, the precision is high, and the ethyl carbamate in rice wine can be accurately determined.

Add 1.18 μg/mL and 2.36 μg/mL urethane standard substances to the yellow rice wine sample, measure the recovered amount of added urethane, and calculate the recovery rate. The results are shown in Table 3. The results show that the recovery rate of the method is between 98% and 101%, which can be used for the accurate determination of ethyl carbamate.

Table 3 Method spike recovery investigation

The mensuration of urethane content in yellow rice wine of embodiment 1～10

Take commercially available rice wine of 10 different grades, pretreat the rice wine through vacuum distillation respectively, mix the pretreated rice wine with hydrochloric acid and 9-hydroxyl tonne solution, and carry out derivatization reaction to obtain the derivatized rice wine. The reaction time was 30 min, and the reaction was carried out at room temperature.

According to the volume ratio, yellow rice wine: hydrochloric acid: 9-hydroxyxanthene solution=10: 1: 2; The concentration of hydrochloric acid is 1.5mol/L; The concentration of 9-hydroxyl anthene is 0.02mol/L.

The components of the derivatized rice wine were separated by high performance liquid chromatography, and the content of ethyl carbamate was calculated by using the external standard method under the obtained working area;

The high performance liquid chromatograph adopts C18 reverse phase column (250×4.6mm, 4μm), the detection temperature is 35°C, the injection volume is 20μL, and the peak elution time of urethane is 35-38min;

Using methanol and water as the mobile phase for gradient elution, gradient elution

Table 4 shows the detection results of the content of ethyl carbamate in yellow rice wine of each embodiment.

Table 4 The content of ethyl carbamate in yellow rice wine

The liquid chromatograms corresponding to each embodiment can be seen in Figure 3 to Figure 12, all of which show good separation effects.

Claims (2)

1. method that detects urethanes in the yellow rice wine is characterized in that:

(1) utilizes decompression distillation, yellow rice wine is carried out pre-service, make the yellow rice wine volume be reduced to 1/5～1/10 of initial volume;

(2) will be through pretreated yellow rice wine and hydrochloric acid and xanthydrol solution by 7～10: 1: 2 volume ratio be mixed, at room temperature derivative reaction 30min obtains the yellow rice wine behind the derivatization, described concentration of hydrochloric acid is that 1～2.0mol/L, xanthydrol solution are the n-propanol solution of xanthydrol, and dense the crossing of xanthydrol is 0.01～0.03mol/L;

(3) yellow rice wine after utilizing high performance liquid chromatograph to derivatization carries out component and separates, and utilizes fluorescence detector to detect and adopts external standard method to calculate the urethane ester content;

The excitation wavelength lambda of described fluorescence detector _Ex=233nm, emission wavelength lambda _Em=600nm, high performance liquid chromatograph adopts the C18 reversed-phase column, and specification is 250 * 4.6mm, 4 μ m, 35 ℃ of detected temperatures, sample size 20 μ L adopt the first alcohol and water to carry out gradient elution as moving phase, and moving phase is changed in time during gradient elution:

Time/min Flow velocity/mLmin ^-1 Methyl alcohol % Water % 0 0.8 61 39 40 0.8 61 39 42 1 100 0 50 1 100 0

The urethanes appearance time is 35-38min.

2. the method for claim 1, it is characterized in that: in the step (1), the pretreated temperature of yellow rice wine decompression distillation is 30～40 ℃.

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