Method for detecting semeglutide in food

Abstract

The invention discloses a method for detecting semeglutide in food, which comprises the following steps: S1, adding a to-be-detected food sample into methanol or a methanol aqueous solution, mixing, and sequentially carrying out homogenization, ultrasonic extraction and filtration to obtain an extracting solution; s2, carrying out liquid chromatography and tandem mass spectrometry analysis on the extracting solution; wherein the elution mode of the liquid chromatography is gradient elution. According to the method, the semeglutide in the to-be-detected food sample can be effectively extracted by adopting homogeneous extraction and ultrasonic extraction of the methanol aqueous solution, then the extracting solution is analyzed by adopting the liquid chromatography tandem mass spectrometer, and the used detection method has the advantages of being high in separation capacity, simple in operation process, wide in applicability and short in analysis time. By utilizing the method disclosed by the invention, qualitative and quantitative analysis of the semeglutide in the food can be quickly and accurately carried out, the liquid phase analysis accuracy is high, the repeatability is good, trace detection of the semeglutide in the food can be realized, and the detection limit is less than or equal to 1.5 mu g / kg.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting semaglutide in food. Background Art

[0002] Semaglutide is a drug developed and produced by Danish pharmaceutical company Novo Nordisk. It is used to treat adult patients with type 2 diabetes and obesity. The drug can activate GLP-1 (glucagon-like peptide-1) receptors, promote insulin secretion in a glucose concentration-dependent manner, inhibit glucagon secretion, delay gastric emptying to increase satiety, and inhibit appetite by inhibiting the action of the hypothalamic feeding center, thereby achieving the effect of lowering blood sugar and reducing weight. However, the use of semaglutide often causes side effects on the gastrointestinal tract, such as bloating, abdominal pain, constipation and even vomiting. In addition, GLP-1 increases the risk of gallbladder or biliary diseases by inhibiting cholecystokinin secretion and delaying gallbladder emptying.

[0003] Semaglutide has the effect of weight loss. Recently, Novo Nordisk's semaglutide tablets were approved for marketing. This is the first oral GLP-1 (glucagon-like peptide-1) receptor agonist approved for marketing in China. At present, semaglutide has not been approved for weight loss in China, nor has it been approved for use as a food raw material or as a food additive. Since this type of drug can also reduce weight without exercise, and due to the hype on social media, there are health foods on the market that use this as a publicity hotspot. There may be unauthorized production and sale of semaglutide oral dosage forms, so it is necessary to identify related foods. However, since semaglutide is defined as a drug both at home and abroad, no standard and literature have been found so far to record the detection method of semaglutide content in food. There are existing literature on semaglutide detection, mainly molecular peptide structure detection, plasma matrix detection, pure target and impurity analysis and research, and no detection method for semaglutide in food has been reported, and there is still a large gap in related detection technology. Summary of the invention

[0004] In order to overcome the problems existing in the prior art, the object of the present invention is to provide a method for detecting semaglutide in food.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a method for detecting semaglutide in food, comprising the following steps:

[0007] S1. Add the food sample to be tested into methanol or methanol-water solution, mix, homogenize, ultrasonically extract and filter in sequence to obtain an extract;

[0008] S2. The extract is subjected to liquid chromatography and tandem mass spectrometry analysis; wherein the elution mode of the liquid chromatography is gradient elution, with aqueous formic acid as mobile phase A and methanol formic acid as mobile phase B.

[0009] Preferably, the homogenization is performed in a homogenizer, wherein the homogenization speed is 8000-18000 rpm and the homogenization time is 0.5-3 min.

[0010] More preferably, the method further comprises the following steps: cleaning the blade of the homogenizer with a methanol-water solution after homogenization, and combining the cleaning liquid with the homogenization treatment liquid.

[0011] Preferably, the mass volume ratio of the food sample to be tested to methanol or methanol aqueous solution is (0.5-2) g:10 mL.

[0012] Preferably, the food sample to be tested is added to a methanol aqueous solution having a volume concentration of 5% to 75% and mixed.

[0013] Preferably, the temperature of the ultrasonic extraction is 40-60°C.

[0014] Preferably, the ultrasonic extraction time is 25-40 min.

[0015] Preferably, for oil-containing samples, n-hexane is added to the ultrasonic extraction, vortexed and allowed to stand until stratification, and the upper n-hexane solution is discarded.

[0016] Preferably, the filtration is performed using a 0.22 μm filter membrane.

[0017] Preferably, the method further comprises the following steps: using an HLB solid phase extraction column to perform solid phase extraction on the ultrasonic extraction treatment liquid, and then filtering.

[0018] Considering that different types of food have different matrices, those with simpler matrices can be directly tested on the machine, but the matrix effect of samples with complex matrices will affect the accuracy of the results of liquid chromatography tandem mass spectrometry, so the samples need to be purified. The present invention adopts HLB solid phase extraction column (SPE) purification. Through selective adsorption and elution, SPE can effectively improve the recovery rate of target analytes, reduce losses during sample pretreatment, reduce sample matrix interference, and improve detection sensitivity.

[0019] More preferably, the solid phase extraction comprises the following steps: activating a solid phase extraction column with methanol and a methanol aqueous solution respectively, transferring the extract to an HLB solid phase extraction column, performing solid phase extraction on the ultrasonic extraction treatment liquid with the HLB solid phase extraction column, eluting with a methanol solution, collecting the eluate, vortex mixing the solid phase extraction liquid and the eluate, and then filtering.

[0020] Preferably, the procedure of the gradient elution is:

[0021] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80±2 20±2 0.50 80±2 20±2 1.50 5±2 95±2 3.00 5±2 95±2 3.01 80±2 20±2 4.00 80±2 20±2 .

[0022] Preferably, the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid methanol solution.

[0023] Preferably, the analysis conditions of the liquid chromatography include:

[0024] Chromatographic column: a column filled with trifunctional n-octadecyl bonded silica gel;

[0025] Flow rate: 0.35±0.05mL / min; injection volume: 5.00±0.5μL; column temperature: 40±5℃.

[0026] Preferably, the mass spectrometry analysis conditions include:

[0027] Ionization mode: electrospray ion source; positive and negative ion simultaneous detection mode; multiple reaction monitoring;

[0028] Ion source parameters: air curtain gas flow rate: 25-35L / min; atomization gas flow rate: 45-55L / min; auxiliary heating gas flow rate: 45-55L / min:

[0029] Ion source spray voltage: 5000±100V in positive ion mode;

[0030] Auxiliary heating gas temperature: 500℃.

[0031] Preferably, the method further comprises the following steps: subjecting the matrix standard working solution of semaglutide to liquid chromatography and tandem mass spectrometry analysis to obtain a standard curve with peak area as the ordinate and concentration as the abscissa; and testing the food sample to be tested according to the standard curve to obtain the content of semaglutide in the food sample to be tested.

[0032] Preferably, the method further comprises the following steps: subjecting the matrix standard working solution of semaglutide to liquid chromatography and mass spectrometry, and the retention time of semaglutide in the food sample to be tested is within ±2.5% of the reference retention time of semaglutide in the matrix standard working solution; and the relative abundance of the component qualifier ions in the sample is compared with the relative abundance of the corresponding qualifier ions in the matrix standard working solution with a similar concentration. Under the conditions that the relative ion abundance is greater than 50% and the allowable relative error is ±20%, the relative ion abundance is greater than 20% and less than 50%, and the allowable relative error is ±25%, the relative ion abundance is greater than 10% and less than 20%, and the allowable relative error is ±30%, and the relative ion abundance is less than or equal to 10%, and the allowable relative error is ±50%, it can be determined that the corresponding analyte exists in the food sample to be tested.

[0033] Preferably, the food types of the food samples to be tested are selected from oils and fats and their products, condiments, meat products, dairy products, beverages, convenience foods, biscuits, canned foods, potatoes and puffed foods, candy products, alcoholic beverages, roasted foods and nut products, egg products, sugar, starch and starch products, cakes, and soy products.

[0034] The beneficial effects of the present invention are:

[0035] The present invention establishes a method for detecting semaglutide in food, and adopts methanol aqueous solution homogenization extraction and ultrasonic extraction to effectively extract semaglutide in the food sample to be tested, and the wall of the solid food embedded drug sample is broken to improve the extraction efficiency, and then the extract is analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS), and the detection method used has the advantages of strong separation ability, simple operation process, wide applicability and short analysis time. The method of the present invention can quickly and accurately perform qualitative and quantitative analysis of semaglutide in food, and the liquid phase analysis has high accuracy and good repeatability, and can realize trace detection of semaglutide in food, and the detection limit is ≤1.5μg / kg, which fills the gap in semaglutide detection technology in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of fitting curves for different extraction solvents;

[0037] Figure 2 This is the Q1MS scan of the semaglutide standard stock solution;

[0038] Figure 3 This is the Q3MS scan of the semaglutide standard stock solution;

[0039] Figure 4 is a multiple reaction monitoring mass chromatogram of a test sample containing semaglutide;

[0040] Figure 5 Schematic diagram of the fitting curve of semaglutide standard stock solution. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below through specific examples. The raw materials used in the following examples, unless otherwise specified, can be obtained from conventional commercial sources or prepared and separated by simple synthesis; the processes used, unless otherwise specified, are conventional processes in the art.

[0042] Instruments and reagents

[0043] Reagents and consumables: analytical grade formic acid (China Resources Chemical, 500 mL), chromatographic grade methanol (CNW), chromatographic grade n-hexane (CNW), mass spectrometry grade formic acid (Fisher 50 mL), HLB solid phase extraction column (CNW, 60 mg, 3 mL), and standard semaglutide were purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.

[0044] Instruments and equipment: AB 4500 ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry (SCIEX, USA), ultrasonic cleaner (Tianjin Aotesense Instrument Co., Ltd.), ME203E electronic analytical balance (Mettler, Switzerland), high-speed refrigerated centrifuge (Sichuan Shuke), solid phase extraction device (Anpu), T18 homogenizer (IKA).

[0045] 10% methanol solution: Measure 10 mL of methanol and 90 mL of water and mix well.

[0046] 1% formic acid methanol solution: Pipette 1 mL of formic acid, dilute to 100 mL with methanol, and mix well.

[0047] Preparation of standard stock solution: Accurately weigh an appropriate amount of semaglutide standard substance, dissolve and dilute with 1% formic acid methanol solution to form a quasi-stock solution of 1 μg / mL.

[0048] Preparation of standard working solution: Pipette the standard stock solution separately and dilute it with methanol solution to a lismaglutide concentration of 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL.

[0049] Solid beverage test matrix: compound probiotic powder with added semaglutide (content 2.8 mg / g).

[0050] Oral solution test matrix: oligofructose oral solution with semaglutide added (content 0.7 mg / mL).

[0051] Tablet candy test matrix: plant fruit and vegetable tablet candy encapsulating semaglutide (content 12.5 mg / g).

[0052] Optimization of extraction conditions

[0053] The matrix of this study is food. When semaglutide is used as an oral drug, the drug needs to be embedded. Therefore, the drug wall needs to be broken during pretreatment. Therefore, the homogenization method is considered to homogenize the sample at 12000r / min for 1 minute to make the drug easy to extract. According to the chemical properties of semaglutide, methanol and methanol-water mixtures of different proportions (volume ratios are 5:95, 10:90, 25:75, 50:50, 75:25, and 90:10, respectively) were tested. The extraction efficiency of semaglutide under ultrasonic extraction for 30 minutes was tested, and 3 parallels were set for each sample. The results are shown in Figure 1 , indicating that the extraction effect of semaglutide is best when the extraction solvent is 10% methanol water. Therefore, after comprehensive consideration, 10% methanol water is used as the extraction solvent. In addition, in order to select the optimal extraction time and extraction efficiency, the extraction efficiency of 10, 20, 30, 40, 50, and 60 min ultrasonic extraction time was compared with 10% methanol water solution as the extraction solvent at 50°C, 200W, and 40kHz. Within 0-25min, the content of the target gradually increased with the increase of ultrasonic time. After 25-40min, the content of the target remained basically stable. In addition, the ultrasonic energy at 50°C can further dissolve the target and improve the extraction efficiency. In summary, 10% methanol water solution was selected as the extraction solvent and ultrasonic extraction at 50°C for 25min was used as the extraction method of this study.

[0054] Optimization of purification conditions

[0055] Considering that different types of food have different matrices, samples with simpler matrices (such as lighter-colored oral liquids, beverages, and alcoholic beverages) can be directly tested on the machine, but the matrix effect of samples with complex matrices will affect the accuracy of the results of liquid chromatography-tandem mass spectrometry, so the samples need to be purified. An attempt was made to purify the sample using an SPE column. According to the molecular structure of the target, this study selected HLB and C18 solid phase extraction columns, respectively, and the purification effects were compared experimentally. A standard mixed solution of semaglutide with a mass concentration of 100 ng / mL was prepared and passed through the purification column. The purification results are shown in Table 1. It can be seen from the results that the recovery rate of the HLB solid phase extraction column is higher than that of the C18 column, so the HLB solid phase extraction column was selected as the purification column.

[0056] Table 1 Recovery rate of semaglutide by HLB column and C18 column

[0057]

[0058] Optimization of mass spectrometry conditions

[0059] The standard stock solution of semaglutide (1 μg / mL) was subjected to full mass spectrometry scanning detection by needle pump flow injection continuous injection. The mass spectrometry conditions were: curtain gas flow rate: 30 L / min; nebulizer gas flow rate (GS1): 50 L / min; auxiliary heating gas flow rate (GS2): 50 L / min; collision gas (CAD): medium intensity (medium); auxiliary heating gas temperature: 500 ° C; spray voltage: 5000 V (ESI+); scanning mode: multiple reaction monitoring mode, and the primary mass spectrum of the tested compound was obtained to find the parent ion of the compound. Then a secondary mass spectrometry scan was performed to obtain the corresponding daughter ions. The characteristic ion pairs were found, and the collision energy and declustering voltage were optimized respectively, and finally the best quality spectrum parameters of semaglutide were obtained. When the Q1MS scan of semaglutide was performed, it was found that [M+H]+ (m / z 956.4) had the highest abundance, so [M+H] was selected. + is the parent ion of semaglutide, Q1MS scan shows Figure 2 When performing product ion scanning, it was found that there were multiple fragment ions with good responses. After optimizing the MRM voltage parameters, the corresponding strongest m / z 710.3, m / z 595.3, and m / z 496.1 were selected as product ions. Figure 3 .

[0060] Example 1

[0061] This embodiment provides a method for detecting semaglutide in food, and the detection method is specifically as follows:

[0062] 1.1. Extraction of simple matrices (such as light-colored oral liquids, beverages, and alcoholic beverages)

[0063] Weigh 1g (accurate to 0.001g) of the oral liquid test matrix into a glass beaker, add 10% methanol aqueous solution to 10mL, homogenize in a homogenizer at 12000r / min for 1min, clean the homogenizer blade with 5mL 10% methanol aqueous solution, combine the extracts, and perform ultrasonic extraction at 50℃ for 25min (for oil-containing samples, add 5mL of n-hexane, vortex and oscillate for 2min, then stand until stratification, and discard the upper n-hexane solution). Take the lower layer solution and filter it through a 0.22μm filter membrane for determination by liquid chromatography-mass spectrometry.

[0064] 1.2. Extraction and purification of all matrices (including simple matrices)

[0065] (1) Extraction

[0066] Weigh 1g of solid beverage test matrix and 1g of compressed candy test matrix (accurate to 0.001g) in a glass beaker, add 10mL of 10% methanol aqueous solution, homogenize at 12000r / min for 1min in a homogenizer, clean the homogenizer blade with 5mL of 10% methanol aqueous solution, combine the extracts, and perform ultrasonic extraction at 50℃ for 25min (for oil-containing samples, add 5mL of n-hexane, vortex and oscillate for 2min, then stand until stratification, and discard the upper n-hexane solution). Transfer all to a glass centrifuge tube, centrifuge at 3000r / min for 5min, take all the supernatant and filter with filter paper, add 15mL of water to the filtrate, and wait for purification.

[0067] (2) Purification

[0068] Transfer all the liquid to be purified to the HLB solid phase extraction column (before using the solid phase extraction column, activate it with 3mL methanol and 3mL 5% methanol water in sequence). Pass the liquid to be purified through the HLB solid phase extraction column, rinse with 3mL water in sequence, drain under negative pressure for 3min, elute with 5mL methanol solution, and collect the eluate. (The entire solid phase extraction process should not exceed 1mL / min). Vortex for 1min, filter through a 0.22μm filter membrane, and provide for liquid chromatography-mass spectrometry.

[0069] 2. Liquid chromatography-tandem mass spectrometry conditions

[0070] (1) Liquid chromatography conditions

[0071] T3 chromatographic column (1.8 μm, 2.1×100 mm); injection volume 5 μL; flow rate: 0.35 mL / min; column temperature 40° C.; gradient elution: eluent A is 0.1% formic acid in water, eluent B is 0.1% formic acid in methanol, and the elution program is shown in Table 2.

[0072] Table 2 Mobile phase gradient elution program

[0073]

[0074]

[0075] (2) Mass spectrometry conditions

[0076] Curtain gas flow rate: 30 L / min; Nebulizer gas flow rate (GS1): 50 L / min; Auxiliary heating gas flow rate (GS2): 50 L / min; Collision gas (CAD): medium intensity (medium); Auxiliary heating gas temperature: 500 °C; Spray voltage: 5000 V (ESI+); Scan mode: multiple reaction monitoring mode. The mass spectrometry detection parameters of the target compounds are shown in Table 3, and the total ion current is shown in Figure 4 .

[0077] Table 3 Mass spectrometry detection parameters of target compounds

[0078]

[0079] 3. Qualitative testing

[0080] According to the above LC-MS / MS conditions, the solid beverage test matrix, oral liquid test matrix, compressed candy test matrix and standard working solution are prepared. If the mass chromatographic peak retention time of the test matrix is ​​consistent with that of the standard, and all selected ions should appear, then the ion ratio of the qualitative selected ion pair (mass spectrometry ion ratio refers to the ratio between the relative abundance and relative quantity of ions) is used for positive confirmation. In qualitative analysis, if the deviation of the relative abundance ratio (k) does not exceed the range specified in Table 4, it can be determined that the corresponding analyte exists in the sample.

[0081] Table 4 Relative abundance deviation

[0082] Relative abundance ratio k＞50％ 50％≥k＞20％ 20％≥k＞10％ k≤10％ Permissible relative deviation ±20％ ±25％ ±30％ ±50％

[0083] 4. Linear range, detection limit and quantification limit

[0084] Draw a standard curve with the concentration of the mixed standard series working solution as the horizontal axis and the peak area as the vertical axis. Figure 5 Schematic diagram of the fitting curve of the semaglutide standard stock solution. The linear regression equation of semaglutide is Y=340.93185X-24.21887, and the correlation coefficient r is 0.99940. In the range of 1 to 200 ng / mL, the method has a good linear relationship.

[0085] Taking the concentration when the quantitative ion signal-to-noise ratio is 3 as the detection limit, and the concentration when the signal-to-noise ratio is 10 as the quantification limit, the test results of the solid beverage test matrix, oral liquid test matrix, and compressed candy test matrix are shown in Table 5.

[0086] Table 5 Detection limit and quantification limit

[0087]

[0088]

[0089] As can be seen from Table 5, the detection limit of semaglutide is less than or equal to 1.5 μg / kg, and the quantification limit is less than or equal to 5 μg / kg, and both the detection limit and the quantification limit are low.

[0090] 5. Method accuracy and precision

[0091] The recovery and precision experiments were conducted using oral liquid negative samples. The negative samples were spiked with semaglutide at 5 μg / kg, 10 μg / kg, and 50 μg / kg, and the three concentration levels were measured six times in parallel at each spike level according to the optimized pretreatment detection method. The results are shown in Table 6.

[0092] Table 6 Recovery rate experimental results

[0093]

[0094] It can be seen from Table 6 that the precision is between 2.7% and 3.9%, and the recovery rate is between 85.4% and 91.4%. This method has good accuracy and precision.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for detecting semaglutide in food, characterized in that: The following steps are involved: S1. Add the food sample to be tested into methanol or methanol-water solution, mix, homogenize, ultrasonically extract and filter in sequence to obtain an extract; S2. The extract is subjected to liquid chromatography and tandem mass spectrometry analysis; wherein the elution mode of the liquid chromatography is gradient elution, with aqueous formic acid as mobile phase A and methanol formic acid as mobile phase B.

2. The method for detecting semaglutide in food according to claim 1, characterized in that: The homogenization is carried out in a homogenizer, wherein the homogenization speed is 8000-18000 rpm and the homogenization time is 0.5-3 min.

3. The method for detecting semaglutide in food according to claim 1, characterized in that: The mass volume ratio of the food sample to be tested to methanol or methanol aqueous solution is (0.5-2) g:10 mL.

4. The method for detecting semaglutide in food according to claim 1, characterized in that: The temperature of the ultrasonic extraction is 40-60°C.

5. The method for detecting semaglutide in food according to claim 1, characterized in that: The method also includes the following steps: using an HLB solid phase extraction column to perform solid phase extraction on the ultrasonic extraction treatment liquid, and then filtering.

6. The method for detecting semaglutide in food according to claim 1, characterized in that: The procedure of the gradient elution is: 。 7. The method for detecting semaglutide in food according to claim 1, characterized in that: The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid methanol solution.

8. The method for detecting semaglutide in food according to claim 1, characterized in that: The mass spectrometry analysis conditions include: Ionization mode: electrospray ion source; positive and negative ion simultaneous detection mode; multiple reaction monitoring; Ion source parameters: air curtain gas flow rate: 25-35L / min; atomizing gas flow rate: 45-55L / min; auxiliary heating gas flow rate: 45-55L / min: Ion source spray voltage: 5000±100V in positive ion mode; Auxiliary heating gas temperature: 500℃.

9. The method for detecting semaglutide in food according to claim 1, characterized in that: The method also includes the following steps: subjecting the matrix standard working solution of semaglutide to liquid chromatography and tandem mass spectrometry analysis to obtain a standard curve with peak area as the ordinate and concentration as the abscissa; and testing the food sample to be tested according to the standard curve to obtain the content of semaglutide in the food sample to be tested.

10. The method for detecting semaglutide in food according to claim 1, characterized in that: The food types of the food samples to be tested are selected from oils and fats and their products, condiments, meat products, dairy products, beverages, convenience foods, biscuits, canned foods, potatoes and puffed foods, candy products, alcoholic beverages, roasted foods and nut products, egg products, sugar, starch and starch products, cakes, and bean products.

Images