Method for measuring content of taurine in functional beverage by HPLC (High Performance Liquid

Abstract

The invention discloses a method for determining the content of taurine in a functional beverage by using high performance liquid chromatography (HPLC), which comprises the following steps: by using 9-chloroformic acid fluorene methyl ester as a derivative, adding a sodium bicarbonate solution in a derivatization reaction process to promote the generation of a derivative, and then detecting by using the high performance liquid chromatography to determine the content of taurine in the functional beverage. And the content of taurine in different functional beverages can be quickly and accurately obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and specifically relates to a method for determining the taurine content in a functional beverage by utilizing high performance liquid chromatography (HPLC). Background Art

[0002] As an important amino acid, taurine plays a significant role in enhancing immunity and anti-fatigue. It can also promote intelligence and brain development, protect the heart and blood vessels, enhance immunity, promote carbohydrate and lipid metabolism, protect eyesight and promote visual development, maintain normal reproductive function, and resist oxidation and anti-aging. It is widely used in food, medicine and functional beverages. Therefore, accurate determination of taurine content in products is crucial for quality control and product development.

[0003] At present, the measurement methods of taurine include neutralization titration, spectrophotometry, liquid chromatography, etc. Among them, high performance liquid chromatography (HPLC) is favored due to its high sensitivity and accuracy. However, the existing HPLC method usually requires derivatization of samples when measuring taurine to increase the sensitivity and specificity of detection. However, the derivatization method is complicated to operate and may introduce errors, which cannot meet the needs of efficient and accurate detection. Summary of the invention

[0004] The object of the present invention is to provide a method for determining the taurine content in a functional beverage by utilizing HPLC.

[0005] To achieve the above object, the present invention adopts the following technical solution: A method for determining the content of taurine in a functional beverage by HPLC, comprising the following steps: 1) Preparation of standard solution: Accurately weigh taurine reference substance into a volumetric flask, dissolve it in water and dilute to the mark, shake well to obtain a standard stock solution with a concentration of 1.000 mg / mL; further dilute it into a series of standard solutions of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL before use; 2) Preparation of sample solution: Accurately measure the sample to be tested into a 50mL volumetric flask, dilute to the mark with water, and shake well to obtain a sample solution of appropriate concentration; 3) Preparation of derivatization agent: Accurately weigh an appropriate amount of 9-fluorenylmethyl chloroformate and dissolve it in acetonitrile to prepare a 1.0 mg / mL derivatization agent; 4) Derivatization reaction: Take 2 mL of the prepared series of standard solutions and sample solutions, put them in a 5 mL volumetric flask, add 1 mL of the derivatizing agent and mix well, then add acetonitrile to the mark and shake for 5 minutes; 5) High performance liquid chromatography detection: The derivatized series of standard solutions were filtered through a 0.45 μm microporous filter membrane and detected using a high performance liquid chromatograph. A standard curve was then drawn with the concentration of the series of standard solutions as the horizontal axis and the obtained chromatographic peak area as the vertical axis, and the taurine content in the sample to be tested was calculated.

[0006] Furthermore, in step 4), 0.5 mL of 2 wt % sodium bicarbonate solution may be added during the derivatization reaction.

[0007] Furthermore, the conditions of the high performance liquid chromatography used in step 5) are as follows: chromatographic column: Kromasil C18 chromatographic column (200 mm×4.6 mm, 5 μm); mobile phase: acetonitrile-0.05 mol / L potassium dihydrogen phosphate buffer (pH=7.5) isocratic elution, the volume ratio of the two is 28:72; flow rate: 1.0 mL·min -1 ; Column temperature: 40℃; Injection volume: 20μL; Detection wavelength: 265 nm.

[0008] Compared with the prior art, the present invention has the following excellent effects: (1) The present invention provides a method for determining taurine content that is simple to operate, highly accurate and highly sensitive. By optimizing the derivatization reagent and chromatographic conditions, the method can achieve efficient separation and accurate detection of taurine, thus meeting the industry's demand for accurate measurement of taurine content.

[0009] (2) The method of the present invention is suitable for the accurate measurement of taurine content in industries such as food, medicine, and functional beverages, and is of great significance for product quality control, new product development, and market supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The resulting standard curve was plotted for the Examples. DETAILED DESCRIPTION

[0011] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto. Example

[0012] 1. Reagents and Methods 1.1 Preparation of standard solution: Accurately weigh an appropriate amount of taurine reference substance into a 20 mL volumetric flask, dissolve it in water and dilute it to the mark, shake well to obtain a standard stock solution with a concentration of 1.000 mg / mL. Before use, further dilute it with water to a series of standard solutions of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL.

[0013] 1.2 Preparation of sample solution: Accurately measure an appropriate amount of the sample to be tested into a 50mL volumetric flask, add water to dilute to the scale, and shake well to obtain a sample solution of appropriate concentration.

[0014] 1.3 Preparation of derivatization agent: Accurately weigh an appropriate amount of 9-fluorenylmethyl chloroformate and dissolve it in acetonitrile to prepare a 1.0 mg / mL derivatization agent.

[0015] 1.4 Derivatization reaction Take 2 mL of the prepared series of standard solutions and sample solutions, put them in a 5 mL volumetric flask, add 1 mL of derivatizing agent and 0.5 mL of 2 wt% sodium bicarbonate solution, mix well, add acetonitrile to the scale, and shake for 5 minutes.

[0016] 1.5 High performance liquid chromatography detection The derivatized series of standard solutions were filtered through a 0.45 μm microporous filter membrane and detected by high performance liquid chromatography. A standard curve was then drawn with the concentration of the series of standard solutions as the horizontal axis and the obtained chromatographic peak area as the vertical axis, and the taurine content in the sample to be tested was calculated.

[0017] The HPLC conditions were as follows: chromatographic column: Kromasil C18 column (200 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-0.05 mol / L potassium dihydrogen phosphate buffer (pH = 7.5) isocratic elution, the elution ratio of the two was 28:72 (v / v); flow rate: 1.0 mL min -1 ; Column temperature: 40°C; Injection volume: 20 μL; Detection wavelength of diode array detector: 265 nm.

[0018] The calculation formula for taurine content is: C=A / A0×C0×V, where C is the taurine content in the sample to be tested (mg / mL), A is the peak area of ​​taurine in the sample solution, A0 is the peak area of ​​taurine in the standard solution, C0 is the taurine content in the standard solution (mg / mL), and V is the dilution multiple.

[0019] 2 Results and discussion 2.1 Selection of derivation conditions During the derivatization process, the effects of not adding salt solution, adding borate buffer solution, adding sodium bicarbonate solution and adding sodium carbonate solution on the determination results were investigated respectively. The results showed that if no salt solution was added, the peak area was small; when borate buffer solution was added, the retention time of the chromatographic peak was longer (greater than 15min); when sodium carbonate solution was added, the peak shape of the chromatographic peak was broadened and tailing was obvious; and when sodium bicarbonate solution was added, the peak shape of the chromatographic peak was sharper, the peak area was larger, and the retention time of the chromatographic peak was appropriate (8min). This may be because the addition of salt can react with the acid obtained by the derivatization reaction, promote the derivatization reaction, and the appropriate alkaline conditions can also make the obtained derivatives more stable; and compared with borate buffer solution and sodium carbonate solution, the product after the reaction of sodium bicarbonate solution with acid can overflow faster in gaseous state, thereby reducing the impact on the determination results.

[0020] 2.2 Selection of mobile phase During the experiment, methanol-sodium acetate buffer, acetonitrile-sodium acetate buffer, methanol-potassium dihydrogen phosphate buffer, and acetonitrile-potassium dihydrogen phosphate buffer were used as mobile phases, respectively. However, the use of other solution systems had problems such as failure to achieve good baseline separation, poor peak shape, or too long retention time of chromatographic peaks. Therefore, acetonitrile-potassium dihydrogen phosphate buffer was used as the mobile phase.

[0021] At the same time, the influence of different ratios of acetonitrile and potassium dihydrogen phosphate buffer on the determination results was further investigated, that is, acetonitrile and potassium dihydrogen phosphate buffer were used for isocratic elution at 30:70 (v / v), 28:72 (v / v), 25:75 (v / v), and 20:80 (v / v). The results showed that as the proportion of acetonitrile in the mobile phase decreased, although the peak shape of the chromatographic peak was improved, the peak area and peak height were increased, the retention time of the chromatographic peak was significantly prolonged. Therefore, when the ratio of acetonitrile to potassium dihydrogen phosphate buffer was 28:72 (v / v), the separation effect was better.

[0022] 2.3 Methodological considerations 2.3.1 Linear relationship investigation Figure 1 The standard curve is drawn. Figure 1 It can be seen that the regression equation obtained is y=4730.1X+137.17, and its linear correlation coefficient R 2 ≥0.999, indicating that there is a good linear relationship between the concentration and the corresponding peak area.

[0023] 2.3.2 Precision test Accurately pipette the prepared 0.4 mg / mL standard solution, perform the derivatization reaction according to item 1.4, and then perform HPLC determination by continuous injection 6 times according to the chromatographic conditions under item 1.5. Record the peak area and calculate the concentration of taurine. The results are shown in Table 1.

[0024] Table 1 Precision test results

[0025] From the results in Table 1, it can be seen that the method has good precision.

[0026] 2.3.3 Accuracy test Accurately weigh different amounts of taurine reference substance into a 20 mL volumetric flask, dissolve in water and dilute to the scale, shake well, perform derivatization reaction according to item 1.4, and perform HPLC determination according to the chromatographic conditions under item 1.5. Record the peak area and calculate the concentration of taurine. The results are shown in Table 2.

[0027] Table 2 Accuracy test results

[0028] The results in Table 2 show that the recovery rate of this method is in the range of 98.94% to 104.08%, indicating that this method has good reliability and accuracy.

[0029] 2.4 Determination of taurine content in samples Take 20 mL of each of two functional drinks of different brands and determine the taurine content therein according to the above method. The results are shown in Table 3.

[0030] Table 3 Results of taurine content determination in different samples

[0031] As can be seen from Table 3, this method can determine the different taurine contents in different samples.

[0032] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for determining the content of taurine in functional beverages by HPLC, characterized in that: The steps include: 1) Preparation of standard solution: Accurately weigh taurine reference substance into a volumetric flask, dissolve it in water and dilute to the mark, shake well to obtain a standard stock solution with a concentration of 1.000 mg / mL; further dilute it into a series of standard solutions of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL before use; 2) Preparation of sample solution: Accurately measure the sample to be tested into a 50mL volumetric flask, dilute to the mark with water, and shake well to obtain the sample solution; 3) Preparation of derivatization agent: Accurately weigh 9-fluorenylmethyl chloroformate and dissolve it in acetonitrile to prepare 1.0 mg / mL derivatization agent; 4) Derivatization reaction: Take 2 mL of the prepared series of standard solutions and sample solutions, put them in a 5 mL volumetric flask, add 1 mL of the derivatizing agent and mix well, then add acetonitrile to the mark and shake for 5 minutes; 5) High performance liquid chromatography detection: The derivatized series of standard solutions were filtered through a 0.45 μm microporous filter membrane and detected using a high performance liquid chromatograph. A standard curve was then drawn with the concentration of the series of standard solutions as the horizontal axis and the obtained chromatographic peak area as the vertical axis, and the taurine content in the sample to be tested was calculated.

2. The method for determining the taurine content in functional beverages by HPLC according to claim 1, characterized in that: Step 4) During the derivatization reaction, 0.5 mL of 2 wt % sodium bicarbonate solution was also added.

3. The method for determining the taurine content in functional beverages by HPLC according to claim 1, characterized in that: Step 5) The HPLC conditions used were: chromatographic column: Kromasil C18 column; mobile phase: acetonitrile-0.05 mol / L potassium dihydrogen phosphate buffer (pH=7.5) isocratic elution, the volume ratio of the two was 28:72; flow rate: 1.0 mL·min -1 ; Column temperature: 40℃; Injection volume: 20μL; Detection wavelength: 265 nm.

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