Liquid chromatography-tandem mass spectrometry detection method for dibenzoylthiamine in food

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry detection method for dibenzoylthiamine in food, which comprises the following steps: extracting a sample by using an extracting solution, performing acidolysis, performing composite enzymolysis, performing centrifugal purification and diluting, detecting by using a liquid chromatography-tandem mass spectrometer, and quantifying by using an external standard method. The qualitative and quantitative method for the content of dibenzoylthiamine in food with different matrixes is researched and formulated for the first time, the method is easy and convenient to operate and high in qualitative and quantitative capacity, and the method can serve as a reliable method for detecting the content of dibenzoylthiamine in food. The detection technology blank of the additive products is filled up, and technical guarantee is provided for entry food supervision.

Description

Technical Field

[0001] The invention relates to a liquid chromatography tandem mass spectrometry detection method for dibenzoylthiamine in food, belonging to the technical field of instrument detection. Background Art

[0002] Dibenzoyl Thiamine, English name Dibenzoyl Thiamine, molecular formula C 26 H 26 N4O4S, molecular weight 490.574, CAS number: 299-88-7, is a derivative of thiamine, a thiol thiamine composed of thiamine and benzoic acid. It will not be decomposed by neuraminidase (thiamine degrading enzyme). It can be quickly absorbed in the body and metabolized into thiamine (vitamin B1) and benzoic acid. Since dibenzoylthiamine is not allowed to be used in food in my country, there is currently no detection method and limit requirement for dibenzoylthiamine.

[0003] It is of great significance to conduct timely follow-up investigation and risk assessment on food additives in imported food, timely discover or eliminate potential risks, prepare for timely risk monitoring, and study and establish detection methods for dibenzoylthiamine. Starting from food safety testing, in view of the lack of detection methods for dibenzoylthiamine in food and the situation that it is outside the scope of food safety supervision, "quality improvement, standard first", establish detection technology for dibenzoylthiamine in food, fill the detection technology gap of such additive products, provide technical guarantee for imported food supervision, and effectively promote the strengthening of technical trade measures, increase bargaining chips in international trade negotiations, and enhance the ability to predict and respond to risks caused by changes in trade policies. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a liquid chromatography tandem mass spectrometry detection method for dibenzoylthiamine in food.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] After extraction with extracting solution, acid hydrolysis, compound enzymatic hydrolysis, centrifugal purification and dilution, the samples were detected by liquid chromatography-tandem mass spectrometry and quantified by external standard method.

[0007] There is no relevant detection method reported for dibenzoylthiamine at present. The present invention adopts the method of acid hydrolysis and composite enzymatic hydrolysis for the first time to extract dibenzoylthiamine added in food.

[0008] The purpose of subjecting the sample to composite enzymatic hydrolysis is to degrade the bound state of dibenzoylthiamine as well as proteins, fats, etc. in the food, and to release dibenzoylthiamine to the maximum extent.

[0009] Among the imported foods currently, the types of food that use dibenzoylthiamine as a nutritional enhancer are mainly instant noodles, instant rice, puffed foods, etc.

[0010] The specific steps are:

[0011] (1) Sample pretreatment

[0012] Weigh 1 g (accurate to 0.01 g) of the mixed sample into a 50 mL centrifuge tube, add 20 mL of 0.01 mol / L hydrochloric acid solution, tighten the lid and mix well, and keep it in an autoclave at 121°C for 30 min; after the acid hydrolysis is completed, cool to room temperature, add 5 mL of enzyme mixture to the sample, immediately cover the lid and vortex to mix well, and enzymolyze in a 37°C water bath oscillator for 30 min; then add 50 mmol / L ammonium formate solution, make up to 50 mL, vortex to mix well; centrifuge, take the supernatant and filter it through a 0.22 μm microporous filter membrane (dilute with mobile phase if necessary) for liquid chromatography-tandem mass spectrometry determination.

[0013] Note: The operation should be carried out in a light-proof environment.

[0014] (2) Liquid chromatography tandem mass spectrometry

[0015] 1. Chromatographic conditions

[0016] a) Chromatographic column: HSS T3 column, 1.8 μm, 100 mm × 2.1 mm (inner diameter), or equivalent;

[0017] b) Mobile phase: A is 0.1% formic acid in water, B is 0.1% formic acid in methanol, elution gradient is shown in the table below;

[0018] Time (min) Mobile phase A (%) Mobile phase B (%) 0 99 1 3 99 1 5 5 95 8 50 50 10 99 1 12 99 1

[0019] c) Flow rate: 0.3 mL / min;

[0020] d) Column temperature: 30°C;

[0021] e) Injection volume: 2 μL.

[0022] 2. Mass spectrometry conditions

[0023] a) Ionization mode: electrospray positive ion mode.

[0024] b) Detection method: multiple reaction monitoring (MRM).

[0025] c) Atomizing gas pressure: 45 psi.

[0026] d) Ion spray voltage: 3500V.

[0027] e) Drying gas temperature: 300℃.

[0028] f) Drying gas flow rate: 9L / min.

[0029] g) The qualitative ion pairs, quantitative ions, fragmentation voltages and collision energies are shown in the table below.

[0030]

[0031] *Quantitative ion

[0032] (3) Qualitative determination

[0033] The sample and the mixed standard working solution are measured under the above conditions. If the retention time of the mass chromatographic peak in the sample is consistent with that of a component in the mixed standard working solution (within the range of variation of ±2.5%); the relative abundance of the qualitative ion pair in the sample is consistent with that of the mixed standard working solution with equivalent concentration, and the relative abundance deviation does not exceed the range specified in the table below, it can be determined that the component exists in the sample.

[0034] Relative ion abundance (%) ＞50 ＞20～50 ＞10～20 ≤10 Maximum allowable deviation (%) ±20 ±25 ±30 ±50

[0035] (4) Quantitative determination

[0036] 1. Preparation of standard curve

[0037] The matrix standard working solution is measured according to the instrument reference conditions to obtain the chromatographic peak area of ​​the corresponding standard solution. The standard curve is drawn with the concentration of the matrix standard working solution as the horizontal axis and the peak area of ​​the chromatographic peak as the vertical axis.

[0038] 2. Determination of sample solution

[0039] The sample solution is measured according to the instrument reference conditions to obtain the corresponding chromatographic peak area of ​​the sample solution. The concentration of the components in the test solution is obtained according to the standard curve; if the response value of the sample solution exceeds the linear range of the standard curve, it should be diluted for analysis.

[0040] Preferably, the enzyme mixture used in the sample pretreatment is prepared by weighing 200 mg of amylase and 200 mg of papain, dissolving them in 200 mL of 50 mmol / L ammonium formate buffer, and adjusting the pH to 4.0-4.5 with formic acid.

[0041] Beneficial effects:

[0042] The present invention studies and develops, for the first time, a qualitative and quantitative method for the content of dibenzoylthiamine in different matrix foods. The method is simple to operate and has strong qualitative and quantitative capabilities. It can be used as a reliable method for detecting the content of dibenzoylthiamine in food. It fills the gap in the detection technology of this type of additive products and provides technical support for the supervision of imported food. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0044] Figure 1 This is a diagram of the molecular structure of dibenzoylthiamine.

[0045] Figure 2 It is the extracted ion chromatogram and total ion chromatogram of dibenzoylthiamine standard solution.

[0046] Figure 3 This is the standard working curve of dibenzoylthiamine. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Example 1 Establishment of a method for detecting dibenzoylthiamine

[0049] This example establishes a method for detecting dibenzoylthiamine in food

[0050] 1. Principle of dibenzoylthiamine determination:

[0051] After extraction with extracting solution, acid hydrolysis, compound enzymatic hydrolysis, centrifugal purification and dilution, the sample was detected by liquid chromatography-tandem mass spectrometry and quantified by external standard method.

[0052] Dibenzoylthiamine is different from other additives. The present invention adopts the methods of acid hydrolysis and enzymolysis to extract dibenzoylthiamine from food to the maximum extent.

[0053] 2 Reagents and Materials

[0054] Unless otherwise specified, all reagents used were analytically pure. Water was first-grade water in accordance with GB / T 6682.

[0055] 2.1 Reagents

[0056] 2.1.1 Methanol: mass spectrometry grade.

[0057] 2.1.2 Formic acid: mass spectrometry grade.

[0058] 2.1.3 Ammonium formate: mass spectrometry grade.

[0059] 2.1.4 Ammonia water: content 26%.

[0060] 2.1.5 Glacial acetic acid.

[0061] 2.1.6 Concentrated hydrochloric acid.

[0062] 2.1.7 Amylase: activity unit 0.5-3.0U / mg.

[0063] 2.1.8 Papain: activity unit ≥ 3U / mg.

[0064] 2.1.9 Ammonia water (1+5): Measure 100 mL of ammonia water and slowly pour it into 500 mL of water and mix well.

[0065] 2.1.10 Hydrochloric acid (0.01 mol / L): Take 9 mL of concentrated hydrochloric acid and dissolve it in 1000 mL of water. Take 50 mL of the solution, dilute it with water and make up to 500 mL.

[0066] 2.1.11 Formic acid aqueous solution (0.1%): Take 1 mL of formic acid and dilute it to 1 000 mL with water. Filter it with a 45 μm microporous filter membrane and set aside.

[0067] 2.1.12 Formic acid methanol solution (0.1%): Take 1 mL of formic acid and dilute it to 1000 mL with methanol.

[0068] 2.1.13 50mmol / L ammonium formate buffer solution: Dissolve 3.15g of ammonium formate in water and dilute to 1000mL.

[0069] 2.1.14 Mixed enzyme solution: Weigh 200 mg of amylase and 200 mg of papain, dissolve them in 200 mL of 50 mmol / L ammonium formate buffer, and adjust the pH to 4.0-4.5 with formic acid.

[0070] 2.2 Standards

[0071] Dibenzoylthiamine, purity ≥98%.

[0072] 2.3 Preparation of standard solution

[0073] 2.3.1 Standard stock solution (1.0 mg / mL)

[0074] Weigh 0.1 g (accurate to 0.0001 g) of dibenzoylthiamine standard, dissolve it in 0.01 mol / L hydrochloric acid and dilute to 100 mL in a brown volumetric flask.

[0075] 2.3.2 Preparation of blank matrix solution

[0076] Weigh 1 g of blank sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add 20 mL of 0.01 mol / L hydrochloric acid solution, tighten the lid and mix, and keep it in an autoclave at 121°C for 30 min; after acid hydrolysis, cool to room temperature, add 5 mL of enzyme mixture to the sample, immediately cover the lid and vortex mix, and enzymolyze in a 37°C water bath oscillator for 30 min; then add 50 mmol / L ammonium formate solution, make up to 50 mL, vortex mix; centrifuge, take the supernatant and filter it through a 0.22 μm microporous filter membrane for liquid chromatography-tandem mass spectrometry determination.

[0077] 2.3.3 Matrix standard working solution

[0078] Accurately pipette an appropriate amount of dibenzoylthiamine standard stock solution and dilute it with blank matrix to make matrix standard working solutions with contents of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, and 1.0 μg / mL, respectively.

[0079] Note: The operation should be carried out in a light-proof environment.

[0080] 3. Instruments and equipment

[0081] 3.1 High performance liquid chromatography-tandem mass spectrometer: equipped with an electrospray ion source.

[0082] 3.2 Analytical balance: sensitivity is 0.0001g.

[0083] 3.3 pH meter: accuracy is 0.01.

[0084] 3.4 Vortex mixer.

[0085] 3.5 Ultrasonic oscillator.

[0086] 4 Analysis steps

[0087] 4.1 Sample extraction

[0088] Weigh 1 g (accurate to 0.01 g) of the mixed sample into a 50 mL centrifuge tube, add 20 mL of 0.01 mol / L hydrochloric acid solution, tighten the lid and mix well, and keep it in an autoclave at 121°C for 30 min; after the acid hydrolysis is completed, cool to room temperature, add 5 mL of enzyme mixture to the sample, immediately cover the lid and vortex to mix well, and enzymolyze in a 37°C water bath oscillator for 30 min; then add 50 mmol / L ammonium formate solution, make up to 50 mL, vortex to mix well; centrifuge, take the supernatant and filter it through a 0.22 μm microporous filter membrane (dilute with mobile phase if necessary) for liquid chromatography-tandem mass spectrometry determination.

[0089] Note: The operation should be carried out in a light-proof environment.

[0090] 4.2 Instrument testing conditions

[0091] 4.2.1 Chromatographic conditions

[0092] a) Chromatographic column: HSS T3 column, 1.8 μm, 100 mm × 2.1 mm (inner diameter), or equivalent;

[0093] b) Mobile phase: A is 0.1% formic acid in water, B is 0.1% formic acid in methanol, elution gradient is shown in Table 1;

[0094] c) Flow rate: 0.3 mL / min;

[0095] d) Column temperature: 30°C;

[0096] e) Injection volume: 2 μL.

[0097] Table 1 Elution gradient

[0098] Time (min) Mobile phase A (%) Mobile phase B (%) 0 99 1 3 99 1 5 5 95 8 50 50 10 99 1 12 99 1

[0099] 4.2.2 Mass spectrometry conditions

[0100] a) Ionization mode: electrospray positive ion mode.

[0101] b) Detection method: multiple reaction monitoring (MRM).

[0102] c) Atomizing gas pressure: 45 psi.

[0103] d) Ion spray voltage: 3500V.

[0104] e) Drying gas temperature: 300℃.

[0105] f) Drying gas flow rate: 9L / min.

[0106] g) The qualitative ion pairs, quantitative ions, fragmentation voltages and collision energies are shown in Table 2.

[0107] Table 2 Qualitative ion pairs, quantitative ions, fragmentor voltages and collision energies of water-soluble vitamins

[0108]

[0109] *Quantitative ion

[0110] 4.3 Qualitative determination

[0111] The sample and the mixed standard working solution are measured under the above conditions. If the retention time of the mass chromatographic peak in the sample is consistent with that of a component in the mixed standard working solution (within the range of variation of ±2.5%); the relative abundance of the qualitative ion pair in the sample is consistent with that of the mixed standard working solution with equivalent concentration, and the relative abundance deviation does not exceed the range specified in Table 3, then it can be determined that the component exists in the sample.

[0112] Table 3 Maximum allowable deviation of relative ion abundance for qualitative confirmation

[0113] Relative ion abundance (%) ＞50 ＞20～50 ＞10～20 ≤10 Maximum allowable deviation (%) ±20 ±25 ±30 ±50

[0114] 4.4 Quantitative determination

[0115] 4.4.1 Preparation of standard curve

[0116] The matrix standard working solution is measured according to the instrument reference conditions to obtain the chromatographic peak area of ​​the corresponding standard solution. The standard curve is drawn with the concentration of the matrix standard working solution as the horizontal axis and the peak area of ​​the chromatographic peak as the vertical axis.

[0117] 4.4.2 Determination of sample solution

[0118] The sample solution is measured according to the instrument reference conditions to obtain the corresponding chromatographic peak area of ​​the sample solution. The concentration of the components in the test solution is obtained according to the standard curve; if the response value of the sample solution exceeds the linear range of the standard curve, it should be diluted for analysis.

[0119] 5 Result calculation

[0120] The result is calculated according to formula (1):

[0121]

[0122] Where:

[0123] X—the content of the component to be tested in the sample, in micrograms per hundred grams (μg / 100g);

[0124] c—the concentration of a component in the sample solution obtained from the standard curve, in micrograms per milliliter (μg / mL);

[0125] V—the fixed volume of the sample solution, in milliliters (mL);

[0126] m—the mass of the sample weighed, in grams (g);

[0127] The calculation result is expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, and the result is rounded to three significant figures.

[0128] 6. Precision

[0129] The absolute difference between two independent determination results obtained under repeated conditions shall not exceed 10% of the arithmetic mean.

[0130] Example 2 Solvent blank experiment and sample addition experiment

[0131] The method provided in Example 1 was subjected to a solvent blank experiment and a sample addition experiment.

[0132] 2.1 Solvent Blank

[0133] Without adding a sample, the sample was treated in the same way as the sample.

[0134] 2.2 Sample addition experiment

[0135] Weigh 1.0g (accurate to 0.01g) of the sample to be tested, and perform the recovery test according to the three content levels of quantitative limit 0.1mg / 100g, concentration of concern 1.0mg / 100g and concentration of concern 2 3.0mg / 100g, and treat it in the same way as the sample. Quantitatively calculate the recovery rate according to the negative sample matrix standard curve, and the recovery rate is between 92.5% and 100%, and the relative standard deviation is between 0.97% and 3.49%.

[0136] The specific data are as follows:

[0137]

[0138] 7. Limit of detection and limit of quantification

[0139]

[0140] When the sample weight is 1g, the detection limit of the method is 0.03mg / 100g, and the quantification limit is 0.1mg / 100g. From the data in the above table, it can be seen that at the detection limit level, the signal-to-noise ratio of the analyte is greater than 3; at the quantification limit level, the signal-to-noise ratio of the analyte is greater than 10.

[0141] Example 3 Specific application of the detection method

[0142] Using the method described in Example 1, multiple batches of samples were randomly selected from samples of instant noodles, instant rice, potato chips, etc., imported and exported commodities in the applicant's business, as well as instant foods, puffed foods, condiments, etc. sold in the market. During the large-scale testing process, dibenzoylthiamine was tested. No dibenzoylthiamine was detected in the commodities sold in the domestic market. More than 500 batches of imported commodities were tested, and nearly 100 batches of samples were found to contain dibenzoylthiamine. The content of the positive samples ranged from 0.15 to 2.0 mg / 100 g, and the detection rate was nearly 20%.

[0143] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting dibenzoylthiamine in food by liquid chromatography tandem mass spectrometry, characterized in that: After extraction with extractant, acid hydrolysis, compound enzymatic hydrolysis, centrifugal purification and dilution, the samples were detected by liquid chromatography-tandem mass spectrometry and quantified by external standard method.

2. The method for detecting dibenzoylthiamine in food by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The specific steps are: (I) Sample pretreatment and preparation of dibenzoylthiamine standard solution 1. Sample pretreatment Weigh 1 g (accurate to 0.01 g) of the mixed sample into a 50 mL centrifuge tube, add 20 mL of 0.01 mol / L hydrochloric acid solution, tighten the lid and mix well, and keep it in an autoclave at 121°C for 30 min; after the acid hydrolysis is completed, cool to room temperature, add 5 mL of enzyme mixture to the sample, immediately cover the lid and vortex to mix well, and enzymolyze in a 37°C water bath oscillator for 30 min; then add 50 mmol / L ammonium formate solution, make up to 50 mL, vortex to mix well; centrifuge, take the supernatant and filter it through a 0.22 μm microporous filter membrane (dilute with mobile phase if necessary) for liquid chromatography-tandem mass spectrometry determination; 2. Preparation of standard solution 2.1 Standard stock solution (1.0 mg / mL) Weigh 0.1 g (accurate to 0.000 1 g) of dibenzoylthiamine standard, dissolve it in 0.01 mol / L hydrochloric acid and dilute to 100 mL in a brown volumetric flask; 2.2 Preparation of blank matrix solution Weigh 1 g of blank sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add 20 mL of 0.01 mol / L hydrochloric acid solution, tighten the lid and mix, and keep it in an autoclave at 121°C for 30 min; after the acid hydrolysis is completed, cool to room temperature, add 5 mL of enzyme mixture to the sample, immediately cover the lid and vortex mix, and enzymolyze in a 37°C water bath oscillator for 30 min; then add 50 mmol / L ammonium formate solution, make up to 50 mL, vortex mix; centrifuge, take the supernatant and filter it through a 0.22 μm microporous filter membrane for liquid chromatography-tandem mass spectrometry determination; 2.3 Matrix standard working solution Accurately pipette an appropriate amount of dibenzoylthiamine standard stock solution and dilute it with blank matrix to standard working solutions with contents of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, and 1.0 μg / mL, respectively; (II) Liquid chromatography tandem mass spectrometry 1. Chromatographic conditions a) Chromatographic column: HSS T3 column, 1.8 μm, 100 mm × 2.1 mm (inner diameter); b) Mobile phase: A is 0.1% formic acid in water, B is 0.1% formic acid in methanol, and the elution gradient is as follows: c) Flow rate: 0.3 mL / min; d) Column temperature: 30°C; e) Injection volume: 2 μL; 2. Mass spectrometry conditions a) Ionization mode: electrospray positive ion mode; b) Detection method: multiple reaction monitoring (MRM); c) Atomizing gas pressure: 45psi; d) Ion spray voltage: 3500 V; e) Drying gas temperature: 300°C; f) Drying gas flow rate: 9L / min; g) Qualitative ion pairs, quantitative ions, fragmentor voltages and collision energies are as follows: *Quantitative ion (III) Qualitative determination The sample and the mixed standard working solution are measured under the above conditions. If the retention time of the mass chromatographic peak in the sample is consistent with that of a certain component in the mixed standard working solution (within the variation range of ±2.5%); the relative abundance of the qualitative ion pair in the sample is consistent with that of the mixed standard working solution with equivalent concentration, and the relative abundance deviation does not exceed the range specified in Table 3, it can be determined that the component exists in the sample; The maximum allowable deviation of relative ion abundance in qualitative confirmation (IV) Quantitative determination 1. Preparation of standard curve The matrix standard working solution is measured according to the instrument reference conditions to obtain the chromatographic peak area of ​​the corresponding standard solution; the standard curve is drawn with the concentration of the matrix standard working solution as the abscissa and the peak area of ​​the chromatographic peak as the ordinate; 2. Determination of sample solution The sample solution is measured according to the instrument reference conditions to obtain the corresponding chromatographic peak area of ​​the sample solution; the concentration of the component in the test solution is obtained according to the standard curve; If the response value of the sample solution exceeds the linear range of the standard curve, it should be diluted before analysis.

3. The method for detecting dibenzoylthiamine in food by liquid chromatography tandem mass spectrometry according to claim 2, characterized in that: The enzyme mixture was prepared by weighing 200 mg of amylase and 200 mg of papain, dissolving them in 200 mL of 50 mmol / L ammonium formate solution, and adjusting the pH to 4.0-4.5 with formic acid.

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