A method for simultaneously detecting simediglutide and SNAC in slimming traditional Chinese medicines or health foods
By employing a multi-reaction monitoring-information-dependent acquisition-enhanced daughter ion scanning mode of ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry, simultaneous detection of smegglutinin and SNAC was achieved, solving the problem of insufficient qualitative accuracy in existing technologies and improving the reliability and efficiency of detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 岳磊
- Filing Date
- 2026-05-22
- Publication Date
- 2026-07-24
AI Technical Summary
In the existing technology, the detection methods for semaglutide have insufficient qualitative accuracy, especially in the complex matrix of traditional Chinese medicine or health food, which is prone to false positive results, and there is a lack of methods for simultaneous detection of semaglutide and its characteristic excipient SNAC.
Using an ultra-high performance liquid chromatography-triple quadrupole linear ion trap combined mass spectrometer, in multiple reaction monitoring-information-dependent acquisition-enhanced daughter ion scanning mode, semaglutide and SNAC were simultaneously detected through triple judgment logic (MRM retention time, MRM ion pair abundance ratio, and EPI secondary fragment full scan spectrum) to form a complete chain of evidence.
It improves the qualitative reliability of detection, overcomes interference from complex matrices, significantly reduces the false positive rate, provides a more robust basis for determining illegal additive behavior, and saves detection time and costs.
Smart Images
Figure CN122449019A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to food and drug safety testing technology, specifically to a method for simultaneously detecting smegglutinin and SNAC in traditional Chinese medicine for weight loss or health food. Background Technology
[0002] Smegglutide is a GLP-1 receptor agonist initially used to treat type 2 diabetes. Due to its significant weight-loss effect, it has been widely used in the weight-loss field in recent years. Oral formulations of smegglutide are currently available. Unlike traditional injectable formulations, oral smegglutide faces challenges from gastric acid and enzyme degradation, as well as low intestinal mucosal permeability. Therefore, its oral formulations must include the absorption-enhancing excipient SNAC. SNAC protects smegglutide from pepsin degradation by creating a relatively high pH environment in the stomach, while simultaneously promoting its transcellular absorption; it is an indispensable characteristic excipient in oral smegglutide formulations.
[0003] With the surge in market demand for semaglutide and the gradual decline in the price of raw materials, unscrupulous individuals may illegally add semaglutide to traditional Chinese medicines and health foods that claim to have weight-loss effects in order to make huge profits, seriously endangering the health and safety of consumers.
[0004] Currently, most methods for detecting semaglutide employ liquid chromatography-tandem mass spectrometry (LC-MS / MS), but these methods have the following shortcomings: First, most methods only use multiple reaction monitoring (MRM) mode for qualitative and quantitative analysis. MRM mode only monitors preset precursor-daughter ion pairs. When dealing with the complex matrices of traditional Chinese medicine or health foods, interfering components in the sample may produce signals with retention times and ion pairs similar to the target analyte, easily leading to false positives and insufficient qualitative reliability. Second, there is currently no method for simultaneously detecting semaglutide and its characteristic excipient SNAC. SNAC serves as an identifying marker for oral semaglutide preparations; if semaglutide and SNAC could be detected simultaneously in the same sample, it would provide a more robust basis for determining whether illegal additives have occurred. Summary of the Invention
[0005] The purpose of this invention is to provide a method for simultaneously detecting smegglutinin and SNAC in traditional Chinese medicine for weight loss or health food, so as to solve the problem of insufficient qualitative accuracy in existing detection methods.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for simultaneously detecting smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food, comprising the following steps:
[0007] S1. Take the test sample, extract it with methanol and purify it to prepare the test sample solution;
[0008] S2. The test solution was detected using an ultra-high performance liquid chromatography-triple quadrupole linear ion trap combined mass spectrometer in multiple reaction monitoring-information-dependent acquisition-enhanced daughter ion scanning mode to obtain a multiple reaction monitoring chromatogram.
[0009] S3. When the signal of at least one of the monitoring ion pair channels of smegglutinin and SNAC in the multi-reaction monitoring chromatogram exceeds a preset threshold, the enhanced fragment ion scan is automatically triggered to obtain a full scan spectrum of secondary fragment ions.
[0010] S4. Based on the multiple reaction monitoring chromatogram, obtain the chromatographic retention time and multiple reaction monitoring ion pair abundance ratio data, and compare them with the enhanced daughter ion scanning secondary fragment ion full scan chromatogram, and compare them with the standards of smegglutinin and SNAC, respectively, to determine whether the test sample contains smegglutinin and SNAC.
[0011] Furthermore, the dosage form of the test sample is a capsule, tablet, oral liquid, or tea.
[0012] Furthermore, the step of preparing the test solution includes: taking the test sample, extracting it with methanol using ultrasound, cooling it, making up to volume with methanol, filtering it through a microporous membrane, and taking the filtrate as the test solution.
[0013] Furthermore, the conditions for the ultra-high performance liquid chromatography are as follows:
[0014] The chromatographic column is a C18 column;
[0015] The mobile phase system consisted of an acetonitrile solution containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid. The gradient elution program was as follows: 0–3 min, the acetonitrile solution containing 0.1% formic acid was increased from 20% to 90%; 3–4 min, it was maintained at 90%; 4–4.1 min, it was decreased from 90% back to 20%; 4.1–5 min, it was maintained at 20%.
[0016] The flow rate was 0.3 mL / min;
[0017] The column temperature is 30℃.
[0018] Furthermore, the conditions for the mass spectrometer are: using an electrospray ionization source and positive ion scanning mode.
[0019] Furthermore, in the multiple reaction monitoring mode, the monitoring ion pairs for smegglutinin are 1029.3→1238.5 Da and 1029.3→1303.1 Da; and the monitoring ion pairs for SNAC are 280.2→262.3 Da and 280.2→121.0 Da.
[0020] Furthermore, the determination of whether the test sample contains semaglutide and SNAC specifically involves determining semaglutide and SNAC separately. The result of the determination of the target analyte is positive when the following three conditions are met simultaneously:
[0021] S4.1 The retention time of the chromatographic peak of the analyte in the test solution is consistent with the retention time of the standard of the target analyte;
[0022] S4.2 The abundance ratio of two-to-many reaction monitoring ions of the analyte in the test solution shall conform to the specified ratio of the abundance ratio of the ion pair of the target analyte standard.
[0023] S4.3 The enhanced fragment ion scanning full scan spectrum of the analyte in the test solution is matched with the spectrum of the target analyte in the self-built standard spectral library.
[0024] Furthermore, if the results for both semaglutide and SNAC are positive in one of the test samples, then the test sample is determined to contain both semaglutide and SNAC.
[0025] Compared with existing technologies, the present invention provides a method for simultaneously detecting smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food. This method employs a triple-judgment logic: MRM retention time as the first qualitative criterion, MRM ion-pair abundance ratio as the second qualitative criterion, and EPI secondary fragment full-scan spectrum as the third confirmatory criterion. Building upon traditional MRM dual confirmation, it adds a pure secondary fragment spectrum provided by a linear ion trap as a molecular fingerprint for confirmation, overcoming the false-positive problem caused by complex matrix interference and significantly improving qualitative reliability.
[0026] This invention proposes for the first time a detection strategy for simultaneous detection of semaglutide and its characteristic excipient SNAC in oral formulations. If semaglutide and SNAC are detected simultaneously from the same sample, a more complete chain of evidence can be formed, providing strong technical support for determining whether there is illegal addition of oral dosage form semaglutide, and making up for the shortcomings of existing methods that only detect a single target.
[0027] This invention employs the MRM-IDA-EPI composite scanning mode, which can simultaneously complete high-sensitivity quantitative screening and high-quality secondary fragment full-scan confirmation with a single injection, eliminating the need for repeated injection analysis, significantly saving detection time and costs, and making it ideal for rapid screening and confirmation of large batches of samples. Attached Figure Description
[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0029] Figure 1 A flowchart illustrating the method steps provided in this embodiment of the invention;
[0030] Figure 2 MRM chromatogram and EPI secondary fragment full scan chromatogram of smegglutinin standard provided in the embodiments of the present invention;
[0031] Figure 3 The MRM chromatogram and EPI secondary fragment full scan chromatogram of the SNAC standard provided in the embodiments of the present invention are shown. Detailed Implementation
[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.
[0033] As attached Figure 1 To be continued Figure 3 As shown:
[0034] Example 1: Detection of Mixed Standard Solutions and Establishment of a Standard Spectral Library
[0035] Step 1: Preparation of reference stock solutions. Accurately weigh approximately 10 mg (accurate to 0.01 mg) each of smegglutide sodium salt reference standard and SNAC sodium salt reference standard, place them in separate 50 mL volumetric flasks, dissolve and dilute to the mark with methanol, shake well, and prepare single reference stock solutions with a concentration of approximately 200 μg / mL for each.
[0036] Step 2: Preparation of the mixed standard working solution. Accurately measure appropriate amounts of the stock solutions of semaglutide and SNAC, and dilute them stepwise with methanol to obtain a mixed standard working solution with a semaglutide concentration of approximately 200 μg / mL and an SNAC concentration of approximately 2 μg / mL.
[0037] Step 3: Instrument Conditions. Ultra-high performance liquid chromatography (UHPLC) conditions: Column: CORTECS UPLC C18 column (2.1 mm × 50 mm, 1.6 μm); Mobile phase A: acetonitrile solution containing 0.1% formic acid; Mobile phase B: aqueous solution containing 0.1% formic acid; Gradient elution program: 0–3 min, mobile phase A linearly increases from 20% to 90%; 3–4 min, mobile phase A remains at 90%; 4–4.1 min, mobile phase A linearly decreases from 90% to 20%; 4.1–5 min, mobile phase A remains at 20% for system equilibration; Flow rate: 0.3 mL / min; Column temperature: 30℃; Injection volume: 2 μL.
[0038] Mass spectrometry conditions: 4000 QTrap linear ion trap mass spectrometer, equipped with an electrospray ionization source; positive ion scan mode; curtain gas CUR 35 psi; ionization voltage IS 5500 V; ion source temperature 550 °C; nebulizer gas GAS1 50 psi; auxiliary gas GAS2 50 psi; collision gas CAD High; operating mode: multiple reaction monitoring - information-dependent acquisition - enhanced daughter ion scan mode.
[0039] MRM ion pairs and parameters are shown in Table 1:
[0040] Table 1. Mass spectrometry parameters of smegglutinin and SNAC
[0041]
[0042] IDA parameter settings: The trigger condition is to automatically trigger EPI scanning when the signal intensity of any MRM ion pair channel exceeds 5000 cps; the EPI scanning range is 100~1500Da, the collision energy is 40eV; the collision energy diffusion is ±15eV; the scan rate is 20000Da / s; and dynamic background subtraction is enabled.
[0043] Step 4: Detection and Results. Take the mixed standard working solution and analyze it under the conditions described above. In the MRM chromatogram, the semaglutide quantitative ion pair shows a symmetrical and sharp chromatographic peak at a retention time of approximately 2.61 min, and the SNAC quantitative ion pair shows a peak at a retention time of approximately 2.76 min. When the signals of both channels exceed the trigger thresholds, the instrument automatically performs an EPI scan, obtaining full-scan chromatograms of secondary fragment ions for semaglutide and SNAC, respectively. See the MRM chromatogram and EPI chromatogram of the semaglutide standard for reference. Figure 2 For the MRM chromatogram and EPI chromatogram of the SNAC standard, please refer to [link / reference needed]. Figure 3 .
[0044] Step 5: Establishment of Standard Spectral Library. After removing background noise from the obtained EPI spectra, they are entered into the analysis software to establish standard EPI spectral libraries for semaglutide and SNAC. The spectral library records the characteristic fragment ion distribution of each target analyte in detail, serving as a comparison benchmark for spectral matching and retrieval during subsequent actual sample detection.
[0045] Example 2: Simulated Detection and Triple Judgment Process of Positive Samples
[0046] Step 1: Preparation of Simulated Positive Samples. The contents of a commercially available weight-loss capsule, confirmed negative by the method in Example 1, were used as a blank matrix. Approximately 0.5 g of this blank matrix was accurately weighed and placed in a 50 mL volumetric flask. An appropriate amount of the mixed standard working solution prepared in Example 1 was added to the flask, ensuring that the addition level of smegglutinin was approximately 20 mg / kg and the addition level of SNAC was approximately 2000 mg / kg, simulating a positive sample containing both target analytes. Approximately 40 mL of methanol was added, and the sample was extracted by sonication for 20 minutes. The extract was then removed, cooled to room temperature, and diluted to the mark with methanol, and shaken well. An appropriate amount of the extract was filtered through a 0.22 μm microporous membrane, and the filtrate was used as the simulated positive test solution.
[0047] Step 2: Instrument Detection. The simulated positive test solution was detected under the ultra-high performance liquid chromatography and mass spectrometry conditions described in Example 1. The injection volume was 2 μL, and data was acquired using MRM-IDA-EPI mode to obtain the MRM chromatogram and the triggered EPI chromatogram.
[0048] Step 3: Triple determination of smegglutinin.
[0049] First step: In the MRM chromatogram, the extracted quantitative ion pair 1029.3→1238.5 showed a distinct chromatographic peak at a retention time of approximately 2.61 min. Under the same batch testing conditions, the retention time of the smegglutide standard was also 2.61 min, with a deviation of less than 2.5%, meeting the retention time consistency requirement.
[0050] The second step involves extracting the chromatographic peak areas of the quantitative ion pair 1029.3→1238.5 and the qualitative ion pair 1029.3→1303.1, respectively, calculating the peak area ratio of the two ion pairs, and comparing it with the ion pair abundance ratio measured by the standard. The relative deviation is 4%, which is within the allowable range of ±20%, and the abundance ratio meets the requirements.
[0051] The third step: During MRM detection, if the signal intensity at this retention time exceeds the 5000 cps trigger threshold, the instrument automatically performs an EPI scan to obtain a full scan spectrum of the secondary fragments of the target analyte in the sample. This spectrum is then compared with the standard spectrum of semaglutide in the standard spectral library established in Example 1. The matching score is 95.2%, higher than the 80% pass threshold, and all major characteristic fragment ions (m / z 1238.5, m / z 1303.1, etc.) appear completely, indicating a successful spectral match.
[0052] Conclusion: If all three conditions are met simultaneously, the simulated positive sample is determined to contain semaglutide.
[0053] Step 4: Triple determination for SNAC.
[0054] First step: In the MRM chromatogram, the chromatogram of the quantitative ion pair 280.2→262.3 was extracted, and a chromatographic peak appeared at a retention time of approximately 2.76 min. The retention time deviation from the standard was less than 2.5%, which met the consistency requirements.
[0055] Second: The peak area ratio of the quantitative ion pair 280.2→262.3 to the qualitative ion pair 280.2→121.0, compared with the abundance ratio of the standard ion pair, has a relative deviation within ±20%, which meets the requirements.
[0056] The third step: When the MRM signal exceeds the threshold, the instrument automatically triggers an EPI scan to obtain a full scan spectrum of the secondary fragments of the target analyte in the sample. After comparison with the SNAC standard spectral library, the matching score is 97.6%, and the characteristic fragment ions (m / z 262.3, m / z 121.0, etc.) are complete, indicating successful spectral matching.
[0057] Conclusion: If all three conditions are met simultaneously, the simulated positive sample is determined to contain SNAC.
[0058] Step 5: Comprehensive Judgment. Since the independent determination results of semaglutide and SNAC in the same test sample are both positive, it is determined that the simulated positive test sample contains both semaglutide and SNAC.
[0059] This embodiment fully demonstrates the entire process of the method, from sample pretreatment to instrument analysis, then to the triple determination of each target analyte, and finally to the comprehensive determination, verifying the detection capability of the method for samples containing two target analytes.
[0060] Example 3: Application of Screening for Actual Weight Loss Products
[0061] Step 1: Sample Selection and Preparation. One batch each of a commercially available brand of slimming capsules and slimming tea were selected as test samples. For the capsule sample, approximately 0.5g of the contents were taken, and for the tea sample, approximately 0.5g of the contents were ground and weighed. Both samples were extracted and purified according to the sample pretreatment method in Example 2 to prepare the test solution.
[0062] Step 2: Detection. Using the instrument conditions described in Example 1, detect both batches of test solutions in MRM-IDA-EPI mode.
[0063] Step 3: Result Determination. In the MRM chromatograms of both batches of samples, no obvious chromatographic peaks were detected within the target retention time windows for smegglutinin (2.61 min) and SNAC (2.76 min). The MRM ion pair channel signals were all below the trigger threshold, and EPI scanning was not triggered. Comparison with the standard revealed that none of the three determination criteria were met.
[0064] Step 4: Conclusion. It was determined that neither batch of commercially available weight-loss products contained smegglutinin or SNAC. This method was successfully applied to the screening and detection of actual samples.
[0065] Methodological Validation
[0066] To demonstrate the scientific validity, accuracy, and reliability of this method, a systematic methodological verification was conducted, as detailed below:
[0067] Linearity assessment: A series of standard solutions with varying concentration gradients were prepared by precisely measuring the mixed standard working solution and serially diluting it with methanol. The linear concentration range of semaglutide was 0.02–10 μg / mL, and that of SNAC was 2–1000 μg / mL. Linear regression was performed with the target analyte concentration on the x-axis and the peak area of the quantitative ion pair on the y-axis. The results showed that semaglutide and SNAC exhibited good linearity within their respective linear ranges, with correlation coefficients (R²) greater than 0.995.
[0068] Limits of detection (LOD) and limits of quantitation (LOQ): The LOD is defined as the concentration corresponding to a signal-to-noise ratio (S / N) of 3, and the LOD is defined as the concentration corresponding to an S / N of 10. In this method, the LOD of smegglutinin is 50 μg / mL (equivalent to 2.5 g / kg in the sample), and the LOD is 150 μg / mL (equivalent to 7.5 g / kg in the sample); the LOD of SNAC is 0.1 μg / mL (equivalent to 5 mg / kg in the sample), and the LOD is 0.3 μg / mL (equivalent to 15 mg / kg in the sample). These indicators demonstrate that this method has the sensitivity to meet practical detection requirements.
[0069] Precision: The same mixed standard working solution was precisely pipetted and injected six times consecutively under the above chromatographic and mass spectrometric conditions. The relative standard deviations (RSDs) of the peak areas of smegglutinin and SNAC were calculated. The results showed that the RSDs of the peak areas of both were less than 3.5%, indicating good instrument precision.
[0070] Recovery: Known negative samples (commercially available weight-loss capsules containing no semaglutide or SNAC, as tested and confirmed to be free of these two compounds) were used as blank matrices. Low, medium, and high concentrations of mixed standard solutions of semaglutide and SNAC were added. Six replicates of the spiked test solution were prepared for each spiking level and injected for analysis. The recoveries and RSDs were calculated. Results showed that the average recoveries of semaglutide ranged from 90.5% to 105.2%, with RSDs ranging from 2.8% to 5.6%; the average recoveries of SNAC ranged from 92.3% to 103.8%, with RSDs ranging from 3.1% to 4.9%. Both met the accuracy and precision requirements of the analytical method.
[0071] The above methodological validation data fully demonstrate that the method established in this invention has a wide linear range, high sensitivity, good precision and accuracy, and can meet the needs of simultaneous qualitative detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine and health food.
[0072] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. A method for simultaneous detection of smegglutinin and SNAC in traditional Chinese medicine or health food for weight loss, characterized in that, Includes the following steps: S1. Take the test sample, extract it with methanol and purify it to prepare the test sample solution; S2. The test solution was detected using an ultra-high performance liquid chromatography-triple quadrupole linear ion trap combined mass spectrometer in multiple reaction monitoring-information-dependent acquisition-enhanced daughter ion scanning mode to obtain a multiple reaction monitoring chromatogram. S3. When the signal of at least one of the monitoring ion pair channels of smegglutinin and SNAC in the multi-reaction monitoring chromatogram exceeds a preset threshold, the enhanced fragment ion scan is automatically triggered to obtain a full scan spectrum of secondary fragment ions. S4. Based on the multiple reaction monitoring chromatogram, obtain the chromatographic retention time and multiple reaction monitoring ion pair abundance ratio data, and compare them with the enhanced daughter ion scanning secondary fragment ion full scan chromatogram, and compare them with the standards of smegglutinin and SNAC, respectively, to determine whether the test sample contains smegglutinin and SNAC.
2. The method for simultaneous detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food according to claim 1, characterized in that, The dosage form of the test sample is capsule, tablet, oral liquid or tea.
3. The method for simultaneous detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food according to claim 1, characterized in that, The steps for preparing the test solution include: taking the test sample, extracting it with methanol using ultrasound, cooling it, making up to volume with methanol, filtering it through a microporous membrane, and taking the filtrate as the test solution.
4. The method for simultaneous detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food according to claim 1, characterized in that, The conditions for the ultra-high performance liquid chromatography are as follows: The chromatographic column is a C18 column; The mobile phase system consisted of an acetonitrile solution containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid. The gradient elution program was as follows: 0–3 min, the acetonitrile solution containing 0.1% formic acid was increased from 20% to 90%; 3–4 min, it was maintained at 90%; 4–4.1 min, it was decreased from 90% back to 20%; 4.1–5 min, it was maintained at 20%. The flow rate was 0.3 mL / min; The column temperature is 30℃.
5. The method for simultaneous detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food according to claim 1, characterized in that, The conditions for the mass spectrometer are: using an electrospray ionization source and positive ion scanning mode.
6. The method for simultaneous detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food according to claim 5, characterized in that, In the multiple reaction monitoring mode, the monitoring ion pairs for smegglutinin are 1029.3→1238.5 Da and 1029.3→1303.1 Da; the monitoring ion pairs for SNAC are 280.2→262.3 Da and 280.2→121.0 Da.
7. The method for simultaneous detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food according to claim 1, characterized in that, The determination of whether the test sample contains semaglutide and SNAC specifically involves determining semaglutide and SNAC separately. A positive result for the target analyte is achieved when all three of the following conditions are met: S4.1 The retention time of the chromatographic peak of the analyte in the test solution is consistent with the retention time of the standard of the target analyte; S4.2 The abundance ratio of two-to-many reaction monitoring ions of the analyte in the test solution shall conform to the specified ratio of the abundance ratio of the ion pair of the target analyte standard. S4.3 The enhanced fragment ion scanning full scan spectrum of the analyte in the test solution is matched with the spectrum of the target analyte in the self-built standard spectral library.
8. The method for simultaneous detection of smegglutinin and SNAC in weight-loss traditional Chinese medicine or health food according to claim 7, characterized in that, If the results for both semaglutide and SNAC are positive in one of the test samples, then the test sample is determined to contain both semaglutide and SNAC.