Method for detecting illegally added saccharide substances in sea cucumbers and their products by ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The present invention discloses a method for detecting illegally added saccharide substances in sea cucumbers and their products by ultra-high performance liquid chromatography-tandem mass spectrometry. The samples are extracted with ethanol-water (8 + 2, v + v) solution, rapidly purified by an improved QuEChERS method, and detected by atmospheric pressure chemical ionization-multiple reaction monitoring mode. In the range of 10-750 mg / L, the linear relationships of the 5 target substances are good, the calibration curves are quadratic curves, and their correlation coefficients are all greater than 0.999. This method is used for pretreatment and determination of sea cucumber products such as dried sea cucumbers and ready-to-eat sea cucumbers. The detection limits of the method are 40 and 80 mg / kg, and the quantification limits are 100 and 200 mg / kg. In the recovery experiment, the recovery ranges of the 5 target substances are 83.1% - 103.2%, and the RSDs are all less than 8.7%. The method has simple pretreatment, high extraction efficiency, low detection limit, can effectively avoid the interference of impurities, and realizes rapid, accurate qualitative and quantitative detection.

Description

Technical Field

[0001] The present invention relates to the field of food detection, and particularly to a method for detecting illegally added saccharide substances in sea cucumbers and their products by ultra-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] As one of the "Eight Treasures of the Sea" in China, sea cucumbers have always been a delicacy on the table. In recent years, the quality of sea cucumbers and their products has received high social attention. Some illegal vendors add saccharide substances during the process of making dried sea cucumbers for purposes such as plasticity and weight gain, seriously affecting the quality and nutritional value of dried sea cucumber products. Exogenous sugars mainly refer to saccharide components artificially added during the processing of sea cucumbers, including fructose, glucose, sucrose, maltose, oligosaccharides, etc. Currently, the analysis and research on exogenous saccharides in sea cucumbers and their products are mostly limited to the field of reducing sugars, which cannot fully reflect the total sugar content in the products (non-reducing sugars cannot be measured), nor can it reflect which exogenous sugars are specifically added. In relevant standards, the limit of water-soluble total sugar in sea cucumbers is set at ≤3.0 g / 100 g, and the detection method is the method for determining reducing saccharide substances. Currently, sea cucumber processing and production enterprises in the market intentionally avoid standard indicators and add non-reducing sugars during the processing of dried sea cucumbers to obtain sugar-adulterated dried sea cucumbers.

[0003] Currently, there are few studies on detecting saccharide substances in sea cucumber matrices. The pretreatment methods mainly focus on derivatization methods and protein precipitation methods, and the detection methods focus on colorimetry, liquid chromatography, and ion chromatography. The above methods have defects such as only being able to measure total sugar and requiring cumbersome derivatization operations. Due to the advantages of high performance liquid chromatography-tandem mass spectrometry such as strong selectivity, high sensitivity, and large detection throughput, in this study, ultra-high performance liquid chromatography-tandem mass spectrometry was used, the ion source was optimized, and finally the APCI source was selected for detection. Summary of the Invention

[0004] In the face of public opinion, the existing methods mainly detect reducing sugars and do not include sucrose with the highest risk. In this application, a high proportion of organic phase high-temperature ultrasonic extraction is used to extract the target substance, the QuEChERS method is optimized to select a new type of QuEChERS purification material, and an amino chromatographic column and an APCI ion source are used to achieve precise separation of the target substance and enhancement of the detection signal. It is extremely urgent to establish a rapid and high-throughput sea cucumber sugar adulteration detection technology suitable for current market supervision. This study is of great significance for maintaining and regulating the sea cucumber market and protecting the rights and interests of consumers.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for detecting illegally added saccharide substances in sea cucumbers and their products by ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0007] (1) Sample pretreatment:

[0008] Extraction: Take the sample, homogenize, pulverize, accurately weigh the homogenized sample into a stoppered centrifuge tube, add ethanol-aqueous solution, vortex, shake in a water bath, centrifuge, and wait for purification;

[0009] Purification: Take the above extraction solution, inject it into a purification tube containing purification filler, vortex and mix evenly, filter with a centrifugal filter membrane for detection;

[0010] (2) Preparation of standard solution:

[0011] Accurately weigh 5 kinds of carbohydrate standard substances into a 10 mL volumetric flask respectively, dissolve with water and make up to the mark, prepare a standard stock solution with a mass concentration of 5.00 mg / mL, and store it in the dark at 4 °C; Transfer 2.00 mL of each standard substance stock solution to a 10 mL volumetric flask respectively, dilute with methanol to prepare a mixed standard intermediate solution with a concentration of 1 mg / mL; Transfer an appropriate amount of the mixed standard intermediate solution, and dilute it step by step with the mobile phase to prepare a mixed standard working solution with a concentration of 10 - 750 μg / mL;

[0012] (3) Ultra-high performance liquid chromatography - mass spectrometry detection.

[0013] Furthermore, the extraction conditions are: Take an appropriate amount of the sample, homogenize and pulverize, accurately weigh 1.0 g of the homogenized sample into a 50 mL stoppered centrifuge tube, add 20 mL of ethanol-aqueous solution with a volume ratio of 8:2, vortex and mix evenly for 30 s, shake in a water bath at 50 °C for 150 min, centrifuge at 8000 r / min for 5 min, and wait for purification;

[0014] Purification conditions: Take 1.5 mL of the above extraction solution and inject it into a purification tube containing purification filler. The purification filler is C18: 100 mg; EMR: 100 mg, vortex and mix evenly for 1 min, centrifuge at 10000 r / min for 3 min; Take the supernatant and filter it through a 0.22 μm filter membrane for UPLC-MS / MS detection.

[0015] Furthermore, the liquid chromatography conditions are: Chromatographic column: Acchrom NH2 column (5 μm, 2.1 * 100 mm); Column temperature: 40 °C; Mobile phase A: aqueous solution; Mobile phase B: acetonitrile; Gradient elution program: 0 - 2 min, 90% B - 90% B; 2.0 - 5.0 min, 90% - 80% B; 5.0 - 9 min, 80% - 60% B; 9.0 - 9.2 min, 60% - 45% B; 9.2 - 10 min, 45% B; 10 - 10.5 min, 45% - 90% B; 10.5 - 14 min, 90% B; Injection volume: 2 μL.

[0016] Furthermore, the mass spectrometry conditions were as follows: atmospheric pressure chemical ionization ion source, negative ion mode; scanning mode: multiple reaction monitoring mode; nebulizer gas flow rate: 3 L / min; interface temperature: 350 °C; desolvation gas temperature: 600 °C; DL temperature: 200 °C; heating block temperature: 200 °C; dryer gas flow rate: 6 L / min. The monitored ion pairs, declustering voltage (DP) and collision energy parameters of the 5 target substances are shown in Table 1.

[0017] Table 1 Retention times, parent ions, product ions and collision energies of 5 saccharide substances

[0018]

[0019] Note: * represents the quantitative ion

[0020] Beneficial effects

[0021] This application established an improved QuEChERS purification liquid chromatography-tandem mass spectrometry method for the determination of illegally added saccharide substances in sea cucumbers and their products. The chromatographic conditions, extraction solvents, extraction conditions and purification methods were optimized. The application of liquid chromatography-tandem mass spectrometry with an APCI source for sugar determination greatly reduced the detection limits of the target substances. The application of the improved QuEChERS purification effectively improved the purification efficiency, reduced the interference of impurities, and achieved rapid, accurate qualitative and quantitative detection. More importantly, it filled the loopholes in the relevant standards, provided an effective detection method for cracking down on illegally added sugared sea cucumbers, and safeguarded the legitimate rights and interests of consumers. Description of the drawings

[0022] Figure 1 is the total ion current chromatogram (50 μg / mL) of 5 saccharide substances;

[0023] Figure 2 is the comparison of the separation effects of three chromatographic columns on the target substances (A: Waters BEH AMIDE; B: Waters NH2; C: Acchrom NH2);

[0024] Figure 3 is the content of positive samples measured by ethanol extracts with different contents;

[0025] Figure 4 is a) the content of positive samples extracted at different extraction temperatures; b) the content of positive samples extracted at different extraction times;

[0026] Figure 5 is the matrix effect K values of 5 saccharide substances before and after matrix effect elimination;

[0027] Figure 6 is the MRM chromatogram of 5 saccharide substances in dried sea cucumbers (concentration: 50 μg / mL; (a)-(e): fructose, glucose, sucrose, maltose, lactose). Detailed implementation mode

[0028] Example 1

[0029] 1 Experimental part

[0030] 1.1 Instruments, reagents and materials

[0031] Shimadzu LCMS-8050 triple quadrupole liquid chromatography-mass spectrometry (Shimadzu Corporation, Japan) with APCI ion source; MilliQ deionized water generator (Millipore Corporation, USA), IKA MS3 vortex mixer; 0.22 μm organic phase needle filter (ANPEL Corporation, China).

[0032] Acetonitrile (Merck, USA); formic acid (Fisher Scientific, USA); ethanol and anhydrous magnesium sulfate (Shanghai National Pharmaceutical Group). C18 purification filler, PSA purification filler, graphitized carbon black (GCB), acidic alumina purification filler, neutral alumina purification filler, basic alumina purification filler, Florisil purification filler were all purchased from Tianjin Agela Technologies Company; enhanced lipid removal purification agent (QuEChERS dSPE EMR-Lipid) (Agilent Technologies, USA).

[0033] Glucose (CAS NO.50-99-7), fructose (CAS NO.7660-25-5), sucrose (CAS NO.57-50-1), maltose (CAS NO.69-79-4), lactose (CAS NO.63-42-3) were all from Dr. Ehrenstorfer GmbH, Germany, with a purity greater than 97.0%;

[0034] 1.2 Preparation of standard solutions

[0035] Accurately weigh 5 kinds of carbohydrate standard substances into 10 mL volumetric flasks respectively, dissolve them with water and make up to the mark to prepare standard stock solutions with a mass concentration of 5.00 mg / mL, and store them in the dark at 4°C; respectively transfer 2.00 mL of each standard substance stock solution into 10 mL volumetric flasks, dilute with methanol to prepare a mixed standard intermediate solution with a concentration of 1 mg / mL. Transfer an appropriate amount of the mixed standard intermediate solution and dilute it step by step with the mobile phase to prepare a mixed standard working solution with a concentration of 10 - 750 μg / mL, and prepare it immediately before use.

[0036] 1.3 Sample pretreatment

[0037] 1.3.1 Extraction

[0038] Appropriately take samples of sea cucumbers and sea cucumber products, homogenize them. For dried sea cucumbers, they need to be pulverized with a high-speed pulverizer and stored at 4°C. Accurately weigh 1.0 g (accurate to 0.01 g) of the homogenized test sample into a 50 mL stoppered centrifuge tube, add 20 mL of ethanol-water (8 + 2, v+v) solution, vortex and mix evenly for 30 s, then shake in a water bath at 50°C for 150 min, centrifuge at 8000 r / min for 5 min, and wait for purification.

[0039] 1.3.2 Purification

[0040] Take 1.5 mL of the above extraction solution and inject it into a purification tube containing purification packing (C18: 100 mg; EMR: 100 mg), vortex and mix evenly for 1 min, centrifuge at 10000 r / min for 3 min. Take the supernatant and filter it through a 0.22 μm filter membrane for UPLC-MS / MS detection.

[0041] 1.4 Instrument analysis conditions

[0042] 1.4.1 Liquid chromatography conditions

[0043] Chromatographic column: Acchrom NH2 column (5 μm, 2.1 * 100 mm); Column temperature: 40°C; Mobile phase A: aqueous solution; Mobile phase B: acetonitrile. Gradient elution program: 0 - 2 min, 90% B - 90% B; 2.0 - 5.0 min, 90% - 80% B; 5.0 - 9 min, 80% - 60% B; 9.0 - 9.2 min, 60% - 45% B; 9.2 - 10 min, 45% B; 10 - 10.5 min, 45% - 90% B; 10.5 - 14 min, 90% B. Injection volume: 2 μL.

[0044] 1.4.2 Mass spectrometry conditions

[0045] Atmospheric pressure chemical ionization ion source (APCI), negative ion mode; Scanning mode: multiple reaction monitoring (MRM); Nebulizing gas flow rate: 3 L / min; Interface temperature: 350°C; Desolvation gas temperature: 602°C; DL temperature: 200°C; Heating block temperature: 200°C; Dryer flow rate: 6 L / min. The monitored ion pairs, declustering voltage (DP) and collision energy parameters of 5 target substances are shown in Table 1.

[0046] Table 1 Retention time, parent ion, daughter ion and collision energy of 5 saccharide substances

[0047]

[0048]

[0049] Note: * is the quantitative ion

[0050] 2 Results and discussion

[0051] 2.1 Optimization of Mass Spectrometry Conditions

[0052] 2.1.1 Selection of Ion Source

[0053] The chromatographic conditions such as the chromatographic column and mobile phase were determined through experiments. When trying to use the ESI ion source, it was found that the instrument response signal was poor and the detection limit was high. Since the APCI source has less dependence on the mobile phase and volatile salts, an attempt was made to replace the APCI ion source to improve the ionization efficiency and increase the response of chromatographic peaks. The effect was significant. Taking the chromatographic peak of fructose as an example, the signal-to-noise ratio increased by 12 times. The total ion current chromatogram of APCI is shown in Figure 1 。

[0054] 2.1.2 Determination of Mass Spectrometry Parameters

[0055] To improve the sensitivity, the APCI source requires sufficient flow rate and solvent ions to generate the best ionization signal. Through optimization experiments, 0.35 mL / min was finally selected as the mobile phase, and the dryer flow rate was increased from 5 L / min to 6 L / min to improve the desolvation efficiency.

[0056] A standard solution with a concentration of 50.0 μg / mL was prepared and injected into the mass spectrometer by means of flow injection. In the negative ion scanning mode, [M+H]- quasi-molecular ions were formed. Using [M+H]- as the parent ion, the collision energy (Collision energys, CE), Q1 Prerod deviation, and Q3 Prerod deviation were optimized, and the obtained parent ion was subjected to a second-stage mass spectrometry scan to obtain fragment ion information. Ion pairs with strong relative abundance and little interference were selected; the best mass spectrometry conditions were determined, as shown in Table 1.

[0057] 2.2 Optimization of Chromatographic Conditions

[0058] Five kinds of carbohydrate compounds are all monosaccharides or disaccharides, and their molecular structures are extremely similar. Among them, fructose and glucose are isomers, and sucrose, maltose, and lactose are isomers; in addition, the ion pairs generated by the mass spectrometry of the five substances are basically the same. Therefore, it is necessary to achieve chromatographic separation of the retention times of the compounds, and it is crucial to optimize the chromatographic conditions. Three chromatographic columns were tried in the experiment: Waters BEH AMIDE (1.7 μm, 2.1*100 mm), Waters NH2 column (3 μm, 4.6*100 mm), and Acchrom NH2 column (5 μm, 2.1*100 mm). The chromatographic conditions were optimized, and gradient elution was performed on the target substances. The separation effect is shown in Figure 2 。It can be clearly seen that the chromatographic peak shape of the Acchrom NH2 column is good and the resolution of the chromatographic peaks is better. Therefore, this chromatographic column was finally selected.

[0059] The separation effect and chromatographic peak shape of the target when acetonitrile-water and acetonitrile-0.1% ammonia water were used as the mobile phase were investigated. The experiment showed that when 0.1% ammonia water was used as the mobile phase, the target peak response was slightly improved, but the separation degree of sucrose, maltose and lactose decreased, and the effect of baseline separation could not be achieved. When acetonitrile-water was used as the mobile phase, the peak shape, separation degree and response could meet the requirements. Finally, acetonitrile-water (v+v) solution was selected as the mobile phase.

[0060] 2.3 Selection of pretreatment conditions

[0061] 2.3.1 Selection of extraction agent

[0062] Ethanol + water (v + v) can effectively extract exogenous sugars. According to the polarity and solubility of sugar substances and the principle that like dissolves like, the higher the ethanol content in the extraction solvent, the less impurities such as polysaccharides, proteins, and oligopeptides extracted from sea cucumbers, but the solubility rate of exogenous oligosaccharides in sea cucumbers is reduced. Experiments were designed to investigate the ethanol concentration of the extract.

[0063] A known positive sample with high exogenous sugar content (sucrose content accounts for 40.3% of the weight of dry sea cucumber) is used as a sample to measure the extraction effect. The ethanol content of the extract is set at 10 levels from 10% to 100%, namely 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. The figure below is a line chart of the extraction effect of ethanol solutions with different proportions. Under the premise of meeting the extraction efficiency, a high ethanol concentration of 80% ethanol aqueous solution is selected.

[0064] 2.3.2 Selection of extraction time and temperature

[0065] The extraction time and extraction temperature were two key factors to be investigated, and the content of positive samples was used as the evaluation index. Samples were prepared according to the above sample pretreatment conditions. 1) The extraction time was fixed at 120 min, and the extraction temperature (℃) was set to 30, 40, 50, 60, 70 and 80, respectively, with 6 levels; 2) The extraction temperature was fixed at 50℃, and the extraction time (min) was set to 30, 60, 90, 120, 150, 180, with 6 levels. Figure 4 (a) As the temperature rises, the matrix effect of impurities such as polysaccharides, collagen, and amino acids in sea cucumbers increases, resulting in a decrease in the measured content; secondly, the high temperature causes oxidation, reduction, and dehydration of exogenous sugars, leading to the decomposition of the target, so 50°C alcohol is selected as the optimal extraction temperature. Figure 4 (b) Prolonging the extraction time will improve the extraction efficiency. When the extraction time is 120 minutes, the extraction rate is the highest. If the extraction time is further extended, the extraction rate will decrease. The reason for the decrease may be that the heating time is long, and some exogenous sugar substances undergo dehydration and other reactions. Therefore, 120 minutes is selected as the optimal extraction time.

[0066] 2.3.3 Selection of purification conditions

[0067] Compared with plant-derived foods and soil, dried sea cucumbers contain high levels of protein (collagen), fat, a small amount of polysaccharides, and a complex matrix. When optimizing the purification conditions, not only should the removal rates of protein, fat, and polysaccharide impurities in the complex matrix be increased as much as possible, but also the extraction efficiency of the target substance should be considered.

[0068] Since the extraction solvent used in this experiment is an ethanol aqueous solution (8 + 2), which is different from the traditional acetonitrile extractant, the commonly used dispersive purifying agents were investigated: C18, PSA, graphitized carbon black (GCB), Florisil, enhanced lipid removal purifying agent (QuEChERS dSPE EMR-Lipid), acidic alumina, neutral alumina, and basic alumina. First, it was investigated whether the eight purifying agents would adsorb the target substance. 2 mL of the extractant was added to a purification tube containing 200 mg of a single purifying agent. It was found that two purifying agents, PSA purifying agent and neutral alumina, caused a decrease in the detection value of the positive sample, adsorbed the target substance, and had a low recovery rate. Further, it was investigated whether the purifying agents had a purification effect. Using the matrix effect reduction rate as the evaluation index, the standard solution was diluted with the purified blank matrix extract and the unpurified blank matrix extract respectively. The matrix effect reduction rate (A), B1 is the chromatographic peak signal peak area of the standard solution diluted with the purified blank matrix extract; B0 is the chromatographic peak signal peak area of the standard solution diluted with the unpurified blank matrix extract. It was found that three purifying agents, acidic alumina, basic alumina, and Florisil, did not significantly reduce the matrix effect. Therefore, GCB, C18, and EMR-Lipid were finally selected as the final purifying agents for optimization. In the experiment of investigating the purification effect, the purification effects were ranked as follows: C18, EMR-Lipid, GCB. C18 adsorbs non-polar interfering substances such as fat and lipids; EMR-Lipid has a strong selective adsorption for C5 and higher carbon chain compounds and can remove interfering lipids without adsorbing the target substance; GCB removes non-polar interfering substances but will adsorb planar structure molecules.

[0069] Using blank dried sea cucumbers as the matrix, the effects of 4 combinations of purification agents C18+GCB+EMR-Lipid, C18+GCB, C18+EMR-Lipid, EMR-Lipid+GCB (①100mg C18+100mg EMR-Lipid+30mg GCB, ②100mg C18+30mg GCB, ③100mg C18+100mg EMR-Lipid, ④100mg EMR-Lipid+30mg GCB) were investigated with the matrix effect as the index. The reduction rates of the matrix effect were found to be 8.1%, 5.6%, 8.5%, and 4.7% respectively. Therefore, 100mg C18+100mg EMR-Lipid was selected as the final purification combination.

[0070] 2.4 Matrix effect

[0071] The slope ratio K value of the matrix standard curve and the reagent standard curve of the target compound was used as the evaluation criterion for each compound. A ratio greater than 1 indicates matrix enhancement, and a ratio less than 1 indicates matrix suppression. The closer the K value is to 1, the smaller the matrix effect. After purification, the K values measured in this experiment were: 0.852 - 0.935, all showing weak matrix effects (0.8 < K < 1.2), see Figure 5 , and the matrix effect decreased significantly after purification, meeting the requirements of normal detection.

[0072] 2.5 Linear range and detection limit of the method

[0073] The experiment used the external standard method with a pure standard working curve for quantification. The experiment showed that according to the chromatographic peak response value law caused by the ionization of the APCI source and the target carbohydrate substances, a quadratic fitting standard curve was selected. For charged particles in the range of 10 - 750 mg / L, the linear equation correlation coefficients of the 5 carbohydrate substances were all greater than 0.999. The sample was pretreated and measured according to this method. The spiked concentration corresponding to the signal-to-noise ratio (S / N) ≥ 3 of the chromatographic peak was taken as the detection limit (LOD) of the method, and the spiked concentration corresponding to S / N ≥ 10 was taken as the lower limit of quantification (LOQ) of the method. The results are shown in Table 2. The LOD of the method was 40 and 80 mg / kg, and the LOQ was 100 and 200 mg / kg, with high sensitivity.

[0074] 2.6 Recovery and precision of the method

[0075] Carbohydrate standard solutions were added to the matrix of sea cucumbers and sea cucumber products. Mixed standard solutions at 3 different concentration levels were added respectively, and each level was measured 6 times repeatedly. The external standard method was used for quantification, and the recovery and precision (expressed as relative standard deviation RSD) were calculated. Figure 6It is the MRM spectrogram of 5 target substances in the sea cucumber matrix. The results show that the recovery rates of the 5 target substances by this method range from 83.1% to 103.2%, and the RSDs are all less than 8.7% (see Table 3). The data indicate that this method has good accuracy and precision and can meet the requirements of trace analysis of target substances.

[0076] Table 2 Linear ranges, regression equations, correlation coefficients, detection limits and lower limits of quantification of 5 saccharide substances

[0077]

[0078] 2.7 Determination of actual samples

[0079] A total of 54 batches of dried sea cucumber samples and 25 batches of ready-to-eat sea cucumber products in circulation in local supermarkets and markets were selected and detected according to this method. Exogenous saccharide substances were detected in 13 batches of dried sea cucumbers and 5 batches of ready-to-eat sea cucumber products. Among them, 1 batch of glucose, 16 batches of sucrose, and 3 batches of maltose were detected. Referring to the provisions of the national standard GB31602-2015 in China, the water-soluble total sugar index: ≤3.0 g / 100 g. Among them, 7 batches of samples did not meet this index, and all were caused by the addition of a large amount of sucrose. Special attention should be paid.

[0080] Table 3 Recovery rates and relative standard deviations of 10 QNs in sea cucumbers and sea cucumber products (n = 6)

[0081]

[0082] The specific recovery rate table is as follows:

[0083]

[0084]

[0085]

[0086]

Claims

1. A method for detecting illegally added saccharide substances in sea cucumbers and their products by ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that, It includes the following steps: (1) Sample pretreatment: Extraction: Take the sample, homogenize, pulverize, accurately weigh the homogenized sample into a stoppered centrifuge tube, add ethanol-aqueous solution, vortex, shake in a water bath, centrifuge, and wait for purification; Purification: Take the above extraction solution and inject it into a purification tube containing purification packing. The purification packing is C18: 100 mg, EMR: 100 mg. Vortex and mix evenly, centrifuge, filter with a membrane, and use for detection; (2) Preparation of standard solutions: Accurately weigh 5 kinds of carbohydrate standard substances into a 10 mL volumetric flask respectively, dissolve with water and make up to the mark, prepare a standard stock solution with a mass concentration of 5.00 mg / mL, and store it in the dark at 4°C. Respectively transfer 2.00 mL of each standard substance stock solution into a 10 mL volumetric flask, dilute with methanol, and prepare a mixed standard intermediate solution with a concentration of 1 mg / mL. Transfer an appropriate amount of the mixed standard intermediate solution and dilute it step by step with the mobile phase to prepare a mixed standard working solution with a concentration of 10 - 750 μg / mL. The 5 kinds of carbohydrates are: fructose, glucose, sucrose, maltose, lactose; (3) Ultra-high performance liquid chromatography-mass spectrometry detection; Liquid chromatography conditions are: Chromatographic column: Acchrom NH2 column, size 5 μm, 2.1 * 100 mm; Column temperature: 40°C; Mobile phase A: aqueous solution; Mobile phase B: acetonitrile; Gradient elution program: 0 - 2 min, 90% B - 90% B; 2.0 - 5.0 min, 90% - 80% B; 5.0 - 9 min, 80% - 60% B; 9.0 - 9.2 min, 60% - 45% B; 9.2 - 10 min, 45% B; 10 - 10.5 min, 45% - 90% B; 10.5 - 14 min, 90% B; Injection volume: 2 μL; Mass spectrometry conditions are: Atmospheric pressure chemical ionization ion source, negative ion mode; Scanning mode: multiple reaction monitoring; Nebulizing gas flow rate: 3 L / min; Interface temperature: 350°C; Desolvation gas temperature: 602°C; DL temperature: 200°C; Heating block temperature: 200°C; Dryer flow rate: 6 L / min.

2. The method for detecting illegally added saccharide substances in sea cucumbers and their products by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein, Extraction conditions: Take an appropriate amount of the sample, homogenize and pulverize, accurately weigh 1.0 g of the homogenized sample into a 50 mL stoppered centrifuge tube, add 20 mL of an ethanol-aqueous solution with a volume ratio of 8:2, vortex and mix evenly for 30 s, shake in a water bath at 50°C for 150 min, centrifuge at 8000 r / min for 5 min, and wait for purification; Purification conditions: Take 1.5 mL of the above extraction solution and inject it into a purification tube containing purification packing, vortex and mix evenly for 1 min, centrifuge at 10000 r / min for 3 min; Take the supernatant and filter it through a 0.22 μm membrane for UPLC-MS / MS detection.

Images