Method for detecting illegally added compounds in food

Abstract

The invention discloses a method for detecting illegally added compounds in food, which is characterized in that complex matrix interference such as saccharides, fatty acid and phospholipid is efficiently removed through a purification strategy of combining a solid-phase extraction column and an adsorbent, the anti-interference capability of the method is remarkably improved, and the method is ensured to have good accuracy and applicability in various food matrixes. In a separation and detection link, high-throughput analysis for synchronously detecting various illegally added compounds in a relatively short time is realized by optimizing chromatography and mass spectrometry conditions, and the screening efficiency is greatly improved. Based on a multi-reaction monitoring and positive and negative ion switching scanning technology, the method is excellent in sensitivity and selectivity, can cover hormone compounds, PDE5 inhibitor compounds, SSRI compounds and other compounds with remarkable structural difference, and greatly expands the screening range. And the method also has effective identification capability on nafil derivatives, structural analogues and other medicines which are not listed in supervision, and provides reliable technical support for systematic screening and supervision of kidney-tonifying and yang-strengthening illegal additives in food.

Description

Technical Field

[0001] This application relates to the field of detection technology for illegal additives in health products, and in particular to a method for detecting illegally added compounds in food. Background Technology

[0002] Currently, high-performance chromatography-mass spectrometry (HPLC-MS) has become the mainstream analytical method for detecting illegally added compounds in kidney-tonifying and aphrodisiac health products. However, this technology typically only detects a few specific compounds, making it difficult to cover the complex situation of the numerous and varied illegally added substances and failing to meet the growing market regulatory demands. Summary of the Invention

[0003] In view of this, this application provides a method for detecting illegally added compounds in food, in order to solve the problem that existing detection technologies have insufficient target coverage and are unable to cope with the diversification of illegal additives.

[0004] According to the first aspect of this application, a method for detecting illegally added compounds in food is provided, comprising: Set up a variety of preset illegal additive compounds and prepare standard working solutions of each preset illegal additive compound; Obtain food samples to be tested; Food samples are extracted using a pre-set extraction solvent to obtain an extract. The extract is first purified using a solid-phase extraction column, and then the purified liquid is further purified using a pre-defined adsorbent combination to obtain the purified test solution. Based on preset chromatographic separation conditions, the purified test solution is separated to obtain chromatographically separated sample components; Using a multi-reaction monitoring mode combined with a positive and negative ion switching scanning mode, mass spectrometry analysis was performed on standard working solutions of various preset illegally added compounds and sample components after chromatographic separation to obtain mass spectrometry identification parameters corresponding to various preset illegally added compounds and mass spectrometry analysis data corresponding to sample components. Based on the comparison results of mass spectrometry identification parameters and mass spectrometry analysis data, it can be determined whether there are illegally added compounds in food samples.

[0005] The aforementioned method for detecting illegally added compounds in food utilizes a purification strategy combining solid-phase extraction columns and adsorbents to efficiently remove interference from complex matrices such as sugars, fatty acids, and phospholipids, significantly enhancing the method's anti-interference capability and ensuring good accuracy and applicability across various food matrices. In the separation and detection stage, optimized chromatographic and mass spectrometric conditions enable high-throughput analysis of multiple illegally added compounds simultaneously within a short time, greatly improving screening efficiency. Based on multiple reaction monitoring and positive / negative ion switching scanning technology, the method exhibits excellent sensitivity and selectivity, covering compounds with significantly different structures, such as hormones, PDE5 inhibitors, and SSRIs, greatly expanding the screening scope. It also effectively identifies natriuretic derivatives, structural analogs, and other unregulated drugs, providing reliable technical support for the systematic screening and regulation of illegally added kidney-tonifying and aphrodisiac substances in food.

[0006] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0007] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a flowchart illustrating a method for detecting illegally added compounds in food according to one embodiment of this application; Figure 2 This is a total ion flow chromatogram of 138 pre-selected illegally added compounds in one embodiment of this application. Detailed Implementation

[0008] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0009] In recent years, with increasing life pressures, a growing number of people are experiencing decreased sexual function and fatigue, driving rapid growth in the market demand for kidney-tonifying, aphrodisiac, and anti-fatigue foods. However, some unscrupulous merchants, in pursuit of product efficacy and increased sales, illegally add chemically synthesized drugs or their structural derivatives to these foods; such practices are not uncommon. Long-term consumption of these products without the consumer's knowledge may harm their health, causing adverse reactions such as headaches, fainting, or even shock, posing a serious health risk, especially to individuals with underlying conditions such as cardiovascular disease or diabetes.

[0010] Currently, the main methods for detecting illegally added compounds in kidney-tonifying and aphrodisiac health products include high-performance liquid chromatography (HPLC), HPLC-tandem mass spectrometry (HPLC-MS / MS), and HPLC-high-resolution mass spectrometry (HPLC-MS / MS). Among these, HPLC has limitations such as low selectivity, insufficient sensitivity, and susceptibility to interference affecting qualitative and quantitative accuracy; while MS / MS offers high resolution, its quantitative capabilities are relatively weak; HPLC-MS / MS, due to its advantages of high selectivity, high sensitivity, and relatively simple sample preparation, has become the mainstream detection technology. However, although some studies have used HPLC-MS / MS to detect these illegally added compounds, most methods are only applicable to a few specific drugs, making it difficult to meet the market regulatory needs of the diverse range of illegally added substances.

[0011] The types of illegally added substances are numerous, and new chemical drugs are constantly emerging. Many similar drugs with the same or multiple effects are not yet included in the current regulatory scope. At the same time, counterfeiters are becoming increasingly sophisticated in their methods, often evading regulation by adding drugs with the same effects not covered by current testing standards, as well as derivatives or structural analogs of senna-like drugs. Therefore, given that the types of compounds covered by current testing standards are still not comprehensive enough, it is necessary to expand the screening scope of kidney-tonifying and aphrodisiac chemicals by combining drug efficacy characteristics and market monitoring feedback, and to establish a high-throughput analytical method capable of simultaneously detecting multiple compounds.

[0012] Based on this, this application provides a pickling method guided by scientific analysis, which can efficiently remove oxide scale while maximizing the protection of the substrate, reducing resource consumption, and mitigating environmental impact.

[0013] Please see Figure 1 As shown, Figure 1 A flowchart illustrating a method for detecting illegally added compounds in food provided in this application embodiment includes the following steps: S10: Set multiple preset illegal additive compounds and prepare standard working solutions of various preset illegal additive compounds.

[0014] In this step, multiple illegally added compounds are pre-defined as detection targets, and standard solutions are prepared for each target compound to serve as a basis for comparison in the subsequent qualitative and quantitative analysis of the target compounds in the samples.

[0015] Optionally, prior to the start of the experiment, a specific list of illegally added compounds requiring screening was determined based on regulations, market intelligence, and scientific literature. This list included 138 pre-selected illegally added compounds, covering a variety of substances such as hormones, anabolic steroids, adrenergic receptor antagonists, dopamines, SSRIs, alpha-receptor blockers, PDE5 inhibitors, and prostaglandins. It is understood that researchers may dynamically adjust the pre-selected list of illegally added compounds based on market dynamics and regulatory requirements; this application does not impose any restrictions on this.

[0016] In one embodiment of this application, a specific standard working solution preparation scheme is provided. In S10, that is, the preparation of standard working solutions with various pre-set illegally added compounds, the following steps S11-S13 are specifically included: S11: Obtain various pre-defined standards containing illegally added compounds.

[0017] In this step, high-purity, precisely known standards corresponding to all the pre-specified illegal compounds are procured from qualified and authoritative standard suppliers as legal chemical references.

[0018] S12: Mix each standard with methanol solution, and prepare standard stock solutions of various preset illegally added compounds based on the first preset concentration.

[0019] In this step, appropriate amounts of each compound standard are weighed into a volumetric flask, diluted with methanol solution, and brought to a first preset concentration to prepare standard stock solutions of each compound.

[0020] Optionally, the first preset concentration is 5000 ng / mL.

[0021] S13: Based on the second preset concentration, dilute each standard stock solution to obtain standard working solutions of various preset illegally added compounds.

[0022] In this step, appropriate amounts of each standard stock solution are taken into volumetric flasks, diluted with methanol solution, and brought to a second preset concentration to obtain standard working solutions of each preset illegally added compound.

[0023] Optionally, the second preset concentration is 200 ng / mL.

[0024] Through the above methods, standard working solutions covering a variety of pre-set illegally added compounds were systematically prepared, establishing a complete and reliable standard reference system. This provided accurate qualitative and quantitative benchmarks for subsequent instrumental analysis, effectively ensuring the accuracy and comparability of the test results.

[0025] In practical applications, appropriate amounts of 138 standards are accurately weighed, dissolved in methanol and diluted to 10 mL to prepare a single standard stock solution with a concentration of 5000 ng / mL. Using methanol as a solvent, the above single standard stock solution is uniformly diluted 25 times to obtain a mixed standard working solution with a concentration of 200 ng / mL.

[0026] S20: Obtain the food sample to be tested.

[0027] In this step, the food samples to be tested are representative samples drawn according to the prescribed procedures, which can truly reflect the quality and composition of the entire batch of products, thereby ensuring that the test results have reliable representativeness and accuracy.

[0028] Optionally, the food to be tested may be a health food product that tonifies the kidneys and enhances male virility.

[0029] S30: Extract food samples using a preset extraction solvent to obtain an extract.

[0030] In this step, a pre-selected extraction solvent that can efficiently dissolve the analyte in the sample is used to separate the analyte from the complex food matrix (such as pharmaceutical excipients, plant fibers, candy sugars, etc.) and convert it into a liquid form for subsequent analysis and detection.

[0031] In one embodiment of this application, a specific extraction solution preparation method is provided. In step S30, the food sample is extracted using a preset extraction solvent to obtain the extraction solution, specifically including the following steps S31-S33: S31: A food sample of a preset mass is vortex-mixed with a first preset volume of a preset extraction solvent to obtain a mixed solution including the food sample and the preset extraction solvent.

[0032] S32: Ultrasonic extraction of the mixed solution.

[0033] S33: Centrifuge the mixed solution after ultrasonic extraction to achieve solid-liquid separation (so that the mixed solution is separated into supernatant and solid residue), and collect the supernatant as the extract.

[0034] For steps S31-S33, after obtaining the food sample, it is pretreated as follows: a predetermined mass of the food sample is weighed and placed in a centrifuge tube, and a first predetermined volume of the predetermined extraction solvent is added. The sample and solvent are vortexed to obtain a mixed solution. The centrifuge tube is then placed in an ultrasonic extractor for ultrasonic extraction. The ultrasonic cavitation effect is used to forcefully and efficiently release the analyte from the complex matrix and dissolve it in the predetermined solvent. The ultrasonically extracted solution is centrifuged to completely separate the liquid containing the analyte from the solid residue. The upper clear liquid is collected as the extract, and the lower solid residue is discarded.

[0035] Optionally, the preset extraction solvent is a methanol solution. Methanol, as a moderately polar solvent, has good solubility for illegally added substances used in tonifying and aphrodisiac purposes (such as phenytoin, hormones, and other moderately polar compounds), enabling efficient extraction of the target analyte from complex food matrices. Simultaneously, as a commonly used mobile phase component in reversed-phase liquid chromatography, it can effectively reduce the solvent effect and improve chromatographic peak shape, thereby enhancing analytical sensitivity and stability. Further, the preset mass is 1 g; the first preset volume is 10 mL.

[0036] In one embodiment of this application, a specific extraction solution preparation method is provided. In step S32, namely, ultrasonic extraction of the mixed solution, the following steps S321-S325 are specifically included: S321: Determine whether the food sample contains oil. If yes, proceed to step S322; otherwise, proceed to step S325.

[0037] In this step, for oily matrix samples, a large amount of lipids will be co-extracted with the analyte, causing significant ion inhibition or enhancement effects in subsequent LC-MS / MS analysis, severely interfering with quantitative accuracy and leading to results deviating from the true value. Furthermore, lipids are non-volatile components; if they directly enter the liquid chromatography flow path and mass spectrometry ion source, they will irreversibly contaminate the chromatographic column, tubing, and precision components, causing instrument performance degradation, increased maintenance costs, and even permanent damage. Therefore, it is necessary to first determine whether the sample contains oil: if it is an oily matrix, lipid-soluble interfering substances must be removed before extraction; if it is not an oily matrix, the mixed solution can be directly subjected to ultrasonic extraction.

[0038] S322: Add a first preset volume of n-hexane solution to the mixed solution and vortex mix.

[0039] S323: Centrifuge the vortex-mixed solution to separate the solution into an oil-containing supernatant and a lower layer.

[0040] S324: Collect the lower layer liquid and perform ultrasonic extraction on the lower layer liquid based on a preset time.

[0041] For steps S322-S324, when the sample is an oil-based matrix, a first preset volume of n-hexane solution is added to a centrifuge tube containing the sample and methanol, and vortexed. Since n-hexane is a non-polar solvent and immiscible with methanol, the vortexing action ensures sufficient contact between it and the oil in the sample. Subsequently, high-speed centrifugation is performed, utilizing density differences to achieve liquid-layer separation: the low-density n-hexane carries the extracted oil to form the supernatant, while the high-density methanol becomes the lower layer. After discarding the oil-containing supernatant, the lower methanol solution is transferred to a new centrifuge tube for defatting, and then ultrasonically extracted from the lower layer for a preset time.

[0042] Optionally, the preset time is 10 minutes.

[0043] S325: Perform ultrasonic extraction on the mixed solution based on a preset time.

[0044] In this step, if it is determined that the sample does not contain oil, then the sample and methanol mixture is subjected to efficient ultrasonic extraction directly according to the preset time.

[0045] In practical applications, food sample dosage forms include tablets, capsules, powders, and oral liquids. For solid or semi-solid samples (such as protein powder, oyster powder, biscuits, candy, coffee, and corresponding dosage forms such as tablets and capsules), after sampling, mixing, and grinding, accurately weigh 1g (accurate to 0.001g) into a 50mL centrifuge tube, add 10mL of methanol solution, vortex to mix, and then sonicate for 10min, followed by centrifugation at 5000r / min for 5min. For liquid samples (such as beverages and alcoholic beverages), after shaking well, accurately measure 1mL into a 50mL centrifuge tube, add an appropriate amount of methanol solution, sonicate for 10min, and then centrifuge at 5000r / min for 5min. For oil-based samples (such as soft capsules), after mixing, accurately weigh 1g (accurate to 0.001g) and place it in a 50mL centrifuge tube. Add 10mL of methanol and vortex to mix. Then add 10mL of n-hexane and vortex for 1min. Centrifuge at 5000r / min for 3min and discard the n-hexane layer. Continue ultrasonic extraction for 15min and centrifuge at 5000r / min for 5min.

[0046] S40: The extract is initially purified using a solid-phase extraction column, and then the purified liquid is further purified using a pre-defined adsorbent combination to obtain the purified test solution.

[0047] In this step, the extracted solution has a complex matrix, making direct instrumental analysis impossible. Therefore, a solid-phase extraction column is first used to purify the extract, removing large molecules such as phospholipids, proteins, and some pigments, as well as highly polar interfering substances. Subsequently, a pre-set adsorbent combination is used to further purify the initially purified solution, removing residual small-molecule impurities, resulting in a purified analyte. The compounds in this analyte are enriched, and matrix interference is effectively eliminated, allowing direct analysis using liquid chromatography-mass spectrometry (LC-MS / MS).

[0048] By combining the two complementary purification mechanisms described above, a wider variety of matrix interferences with different properties can be effectively removed, thereby significantly enhancing the signal response and stability of the analyte, greatly improving detection sensitivity and quantitative accuracy, while effectively avoiding contamination of the chromatographic column and mass spectrometer, extending the instrument's lifespan, and ensuring data stability.

[0049] In one embodiment of this application, a specific extraction liquid purification scheme is provided. In S40, the extraction liquid is initially purified based on a solid-phase extraction column, and then the purified liquid is further purified based on a preset adsorbent combination to obtain the purified test liquid. Specifically, it includes the following steps S41-S42: S41: The extract is initially purified using a solid-phase extraction column, and the target fraction is collected.

[0050] S42: The target fraction is subjected to secondary purification based on a preset adsorbent combination to obtain a purified liquid. The purified liquid is then filtered to obtain the test liquid.

[0051] For steps S41-S42, the extract is loaded onto a solid-phase extraction column, allowing the target compound to pass through while its selective retention mechanism traps large molecules such as phospholipids, proteins, and some pigments, along with highly polar interfering substances, on the column, achieving preliminary purification and coarse screening of interfering substances. The resulting target fraction is then directly collected into a centrifuge tube containing a pre-set adsorbent combination, where adsorption removes residual small-molecule impurities, completing a secondary fine purification. Finally, suspended particles are removed by membrane filtration, yielding a clear, purified test solution.

[0052] The high-purity test solution obtained through the above-mentioned method and the tandem purification strategy can be directly and safely injected into the liquid chromatography-mass spectrometry instrument. This not only effectively ensures that the detection results have high sensitivity, accuracy and stability, but also significantly reduces the risk of instrument contamination and extends the service life of core components.

[0053] In one embodiment of this application, a specific initial purification treatment scheme is provided. In S41, the extract is initially purified based on a solid-phase extraction column, and the target fraction is collected. Specifically, this includes the following steps S411-S413: S411: Extract a second preset volume of extract and load it onto a solid-phase extraction column for purification.

[0054] S412: Discard the third preset volume of filtrate that initially flows out.

[0055] S413: Collect the fourth preset volume of eluent that flows out subsequently as the target fraction.

[0056] For steps S411-S413, a second preset volume of crude extract is loaded onto the top of the solid-phase extraction column and passed through the column under gravity or negative pressure. During this process, large molecular interferences such as phospholipids and proteins are retained by the packing material, while the target compound flows out with the solvent. Due to the existence of dead volume between the packing materials in the column, the initial effluent is the original solvent filled with voids, and its composition and concentration are unstable, failing to represent the true purification effect. Therefore, the first third preset volume of filtrate is discarded. Subsequently, the fourth preset volume of eluent is collected using centrifuge tubes containing a preset adsorbent combination, thus obtaining the high-quality target fraction after initial purification.

[0057] Optionally, the second preset volume is 7 mL, the third preset volume is 4 mL, and the fourth preset volume is 2 mL.

[0058] In one embodiment of this application, a specific secondary purification treatment scheme is provided. In S42, the target fraction is subjected to secondary purification treatment based on a preset adsorbent combination to obtain a purified liquid. The purified liquid is then filtered to obtain the test liquid. Specifically, the scheme includes the following steps S421-S422: S421: Place the target fraction in a centrifuge tube containing a preset adsorbent combination and vortex mix to obtain a mixture including the target fraction and the preset adsorbent combination.

[0059] S422: Allow the mixture in the centrifuge tube to stand, and collect the supernatant and pass it through a filter membrane with a preset pore size to obtain purified liquid.

[0060] For steps S421-S422, the target fraction flowing out of the solid-phase extraction column is directly received using a centrifuge tube containing a preset adsorbent combination. After sealing, the adsorbent particles are vortexed to ensure full contact with the liquid, effectively increasing their interaction area. During vigorous mixing, residual impurities are captured by the active surface of the adsorbent, forming a particle dispersion system after impurity adsorption, resulting in a turbid suspension. Subsequently, the centrifuge tube is allowed to stand, and the adsorbent particles settle to the bottom of the tube under gravity. The upper clarified liquid is then transferred and filtered through a microporous membrane to obtain a purified liquid without solid residue.

[0061] Optionally, the preset pore size is 0.22 μm. The preset adsorbent combination is PSA and C18. In practical applications, C18 columns are typically used for the detection of phenobarbital-like substances. Given that this embodiment also requires the simultaneous detection of multiple compounds such as hormones, anabolic steroids, and dopamines, T3 and C18 columns were selected to simultaneously determine 138 aphrodisiac compounds to compare their separation effects. Experimental results showed that both columns exhibited good overall separation performance, but the T3 column failed to effectively separate the isomers thio-idenafil and thio-homosidinenafil. Therefore, this embodiment ultimately selected the C18 column with superior separation capabilities. Furthermore, this embodiment compared the purification effects of four adsorbents—PSA, ALN, C18, and GCB—and two solid-phase extraction columns—PRIME HLB and HLB. When using GCB adsorbent, the recovery rate of 90% of the target compounds was less than 60%, indicating significant adsorption of the target substances. In comparison, C18 and PSA exhibited weaker adsorption of the target analyte, resulting in better extraction and recovery. Further comparison of HLB and PRIME HLB solid-phase extraction columns without activation, rinsing, or elution steps revealed that the PRIME HLB column produced a higher sample recovery rate and lower baseline noise. Therefore, this application ultimately selected a direct-flow purification method using PRIME HLB solid-phase extraction columns, combined with a combination of C18 and PSA adsorbents, to purify the sample. PSA effectively removes impurities such as sugars and fatty acids, while C18 removes nonpolar compounds and fats. The PRIME HLB column efficiently removes interfering substances such as proteins and phospholipids, thus achieving comprehensive purification of the extract.

[0062] By employing the purification strategy of combining PRIME HLB solid-phase extraction column with PSA and C18 adsorbents, the interference from complex matrices such as sugars, fatty acids, and phospholipids was effectively removed, significantly enhancing the method's anti-interference ability and ensuring its accuracy and applicability in various food matrices.

[0063] In practical applications, 7 mL of the supernatant is passed through an Oasis PRI ME HLB solid-phase extraction column. Its selective retention mechanism traps large molecules such as phospholipids, proteins, and some pigments, as well as strongly polar interfering substances, allowing the target compound to pass through with the solvent. The first 4 mL of filtrate is discarded, and the remaining 2 mL of eluent is collected in a 15 mL centrifuge tube containing 50 mg PSA and 50 mg C18. PSA adsorbs polar small molecules such as sugars, organic acids, and fatty acids, while C18 adsorbs non-polar impurities such as lipids and sterols. The mixture is then vortexed for 1 min, allowed to stand for 5 min, and the supernatant is filtered through a 0.22 μm filter membrane. The purified solution is then used for analysis by liquid chromatography-mass spectrometry (LC-MS).

[0064] S50: Based on preset chromatographic separation conditions, the purified test solution is separated to obtain the chromatographically separated sample components.

[0065] In this step, pre-set chromatographic separation conditions are used to separate different compounds in the purified mixed test solution over time, obtaining sample components arranged in a time sequence. This avoids signal interference caused by multiple compounds entering the mass spectrometer simultaneously, ensuring that the mass spectrometer can perform clear and accurate qualitative and quantitative analysis of each compound.

[0066] In one embodiment of this application, the preset liquid chromatography separation conditions are as follows: a C18 column is used, wherein the column specifications are 100×2.1mm and 2.5μm; the mobile phase is methanol and a 0.1% (v / v) formic acid aqueous solution; the column temperature is 30 degrees Celsius, the injection volume is 5μL, and the flow rate is 0.35mL / min.

[0067] Furthermore, to obtain good peak shape and ionization efficiency, this application compared four mobile phase systems: 0.1% (v / v) formic acid-water-methanol, 0.1% (v / v) formic acid-water-acetonitrile, 5 mmol / L ammonium acetate-methanol, and 5 mmol / L ammonium acetate-acetonitrile. The results showed that in the ammonium acetate system, clomiphene elution time was delayed and the peak shape was tailed, presumably because the lower pH of the mobile phase inhibited its ionization, causing it to exist more in molecular form, thus prolonging the retention time. In addition, acetonitrile had stronger elution ability than methanol; in the acetonitrile system, the peaks of each component were more concentrated, resulting in poor separation. Considering all factors, 0.1% (v / v) formic acid-water-methanol was ultimately selected as the mobile phase system. Subsequently, the separation effect was further improved by optimizing the gradient program. The study found that an excessively high initial methanol ratio led to insufficient early elution peak separation; while a too low ratio could improve separation, it prolonged peak width and analysis time. After system optimization, the gradient elution program was determined as follows: the initial ratio was 10% methanol, maintained for 1 min; the methanol ratio was linearly increased to 85% within 1–12 min, during which the effective elution and good separation of most target analytes could be achieved; subsequently, the organic phase ratio was further increased within 1 min to elute residual impurities. Table 1 shows the gradient elution program used in high-performance liquid chromatography (HPLC). Specifically, the initial methanol concentration was 10%; from 0 to 1 min, maintained at 10%; from 1 min to 12 min, 10% to 85%; from 12 min to 15 min, maintained at 85%; from 15 min to 15.1 min, 85% to 95%; from 15.1 min to 16 min, maintained at 95%; from 16 min to 16.1 min, 95% to 10%; and from 16.1 min to 18 min, maintained at 10%.

[0068] Table 1

[0069] S60: Employing a multi-reaction monitoring mode combined with a positive and negative ion switching scanning mode, it performs mass spectrometry analysis on standard working solutions of various preset illegally added compounds and sample components after chromatographic separation, obtaining mass spectrometry identification parameters corresponding to various preset illegally added compounds and mass spectrometry analysis data corresponding to sample components.

[0070] In this step, firstly, the standard working solutions are injected into an ultra-high performance liquid chromatography-tandem quadrupole mass spectrometer (UHPLC-MS / MS). Using multiple reaction monitoring (MRM) mode combined with positive and negative ion switching scanning, all target compounds are analyzed simultaneously in a single injection. Dedicated mass spectrometry identification parameters for each illegally added compound are established, including precursor ion, daughter ion, and optimized mass spectrometry voltage parameters (cluster removal voltage, inlet voltage, collision voltage, and outlet voltage). Subsequently, sample components are injected into the same system, and the precursor and daughter ions of each chromatographically separated component are sequentially verified using MRM mode to obtain qualitative and quantitative mass spectrometry analysis data.

[0071] The proprietary mass spectrometry identification parameter system (including precursor ion, daughter ion, and optimized voltage parameters) established through the above methods provides multiple verification dimensions for compound identification, significantly improving the accuracy and anti-interference capability of qualitative identification. The use of multiple reaction monitoring combined with positive and negative ion switching scanning technology significantly improves the sensitivity and selectivity of the method, effectively covering a variety of compounds with significant structural differences, such as hormones, PDE5 inhibitors, and SSRIs, greatly expanding the screening scope.

[0072] In one embodiment of this application, a specific geological spectrometry analysis scheme is provided. In S60, a multi-reaction monitoring mode combined with a positive and negative ion switching scanning mode is used to perform mass spectrometry analysis on standard working solutions of various preset illegally added compounds and sample components after chromatographic separation, to obtain mass spectrometry identification parameters corresponding to various preset illegally added compounds and mass spectrometry analysis data corresponding to sample components. Specifically, the scheme includes the following steps S61-S62: S61: Perform mass spectrometry analysis on standard working solutions of various preset illegally added compounds. Based on the multi-reaction monitoring mode combined with the positive and negative ion switching scanning mode, collect the mass spectrometry identification parameters of various preset illegally added compounds. Among them, the mass spectrometry identification parameters include the mass spectra of the parent ion and daughter ion, as well as the optimized mass spectrometry voltage parameters, including the declustering voltage, inlet voltage, collision voltage, and outlet voltage.

[0073] S62: Perform mass spectrometry analysis on sample components and acquire mass spectrometry analysis data of sample components based on multiple reaction monitoring mode.

[0074] For steps S61-S62, continuous injection via needle pump is used to perform a full mass spectrometry scan to obtain the primary mass spectrum of the compound and determine its parent ion; then, a secondary mass spectrometry scan is performed to obtain the corresponding daughter ions, thereby determining the characteristic ion pairs; by optimizing parameters such as the collision energy and declustering voltage of each ion pair, the optimal mass spectrometry identification parameters for various illegally added compounds are finally obtained.

[0075] Subsequently, the chromatographically separated sample components are injected into the mass spectrometer, and the mother ion-daughter ion pairs of various preset illegally added compounds are specifically acquired through the multiple reaction monitoring mode to obtain mass spectrometry analysis data containing only the response of the target analyte.

[0076] Optionally, the mass spectrometry conditions are as follows: curtain gas flow rate of 30 L / min, nebulizer gas flow rate of 50 L / min, auxiliary heating gas flow rate of 50 L / min, collision gas intensity of medium, auxiliary heating gas temperature of 500 °C, spray voltage of 5000 V (ESI+) or -4500 V (ESI-), and scanning mode of multiple reaction monitoring. Table 2 shows the mass spectrometry identification parameters for various preset illegally added compounds. The first ion is the quantitative ion.

[0077] Table 2

[0078] Optionally, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was performed on an AB 4500 ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry (ULMS) system. This mass spectrometer features simultaneous switching between positive and negative ion monitoring modes, enabling simultaneous acquisition of mass spectrometric signals of the target analyte in both positive and negative ionization modes during a single injection. It can simultaneously detect 138 illegally added compounds used in kidney-tonifying and aphrodisiac products in just 18 minutes, significantly improving analytical efficiency. For example... Figure 2The figure shows the total ion chromatograms of 138 pre-specified illegally added compounds. The vertical axis represents signal intensity in counts per second (0-3,500,000), and the horizontal axis represents time in minutes (0-18 min). The curves in the figure reflect the change in total ion intensity over time. Each chromatographic peak typically corresponds to the time point at which one or more compounds elute from the column and are detected. Among the 138 pre-specified illegally added compounds, letrozole, prostaglandins, tadalafil impurity 48, 3-deethyl-3-propylcarbadinafil, N-phenylpropenyltadalafil, and depiperazinylthiosildenafil exhibited ionization responses only in the electrospray negative ion mode (ESI-), or their response intensity in ESI- mode was significantly better than in the positive ion mode (ESI+). Therefore, these six drugs were detected using ESI- mode, while the remaining 132 compounds were analyzed using ESI+ mode.

[0079] In one embodiment of this application, a specific scheme for constructing geological spectrum identification parameters is provided. In S61, that is, mass spectrometry analysis is performed on standard working solutions of various illegally added compounds. Based on the multi-reaction monitoring mode combined with the positive and negative ion switching scanning mode, the mass spectrometry identification parameters of various illegally added compounds are collected. Specifically, it includes the following steps S611-S615: S611: Continuously inject standard working solutions of various pre-set illegally added compounds into the mass spectrometer.

[0080] S612: Perform a full first-order mass spectrometry scan on the standard working solution to obtain the first-order mass spectra of various pre-specified illegally added compounds, and determine the optimal parent ion of each pre-specified illegally added compound.

[0081] S613: Perform a secondary mass spectrometry scan on the parent ion to obtain the daughter ion and identify characteristic ion pairs.

[0082] S614: Optimize the mass spectrometry voltage parameters of various illegally added compounds based on characteristic ion pairs.

[0083] S615: Based on the parent ion, daughter ion, and mass spectrometry voltage parameters, various preset mass spectrometry identification parameters for illegally added compounds are constructed.

[0084] For steps S611-S615, the standard working solution is injected into the mass spectrometer for a first-stage full scan to identify the charged intact molecules formed after the ionization of each compound as the parent ion. After the parent ion undergoes collision-induced dissociation, its fragment ion spectrum is obtained through a second-stage mass spectrometry scan. From this fragment ion spectrum, 1-2 characteristic and stable daughter ions are selected to form a unique characteristic ion pair consisting of the parent ion and the daughter ion. Based on this ion pair, the mass spectrometry voltage parameters such as collision energy and declustering voltage are systematically optimized to obtain the parameter combination with the optimal signal intensity and stability. Finally, the parent ion, daughter ion, and optimal parameters are integrated to construct a dedicated identification parameter that can be directly used for mass spectrometry detection.

[0085] S70: Based on the comparison results of mass spectrometry identification parameters and mass spectrometry analysis data, determine whether there are illegally added compounds in food samples.

[0086] In this step, by comparing the mass spectrometry identification parameters of the standard with the mass spectrometry analysis data of the sample using both parent and daughter ion characteristics and performing quantitative calculations, the accurate detection of trace components can be achieved, thereby determining whether there is any illegal addition in the food sample and identifying the specific types and accurate amounts of the added compounds.

[0087] Optionally, this application uses food as the analytical matrix, which is diverse and exhibits significant differences. Therefore, protein solid beverages, soft capsules, candies, and oral liquids were selected as representative matrices. Standard curves were prepared using both solvent and blank matrices, and the matrix effect (ME) was calculated based on the slope: ME (%) = (slope of matrix standard curve / slope of solvent standard curve) 1) ×100. ME values ​​were defined as weak effect (0%-20%), moderate effect (20%-50%), and strong effect (greater than 50%), with negative values ​​indicating inhibition. Results showed that most compounds exhibited weak matrix effects across the four matrices, a few moderate effects, and no compounds with strong matrix effects were found. Although matrix-matched standard curves could be used to correct for matrix effects, it was difficult to obtain completely negative matrices in actual detection, and the 138 pre-specified compounds were illegal additives; their detection was considered valid. Therefore, this application ultimately used solvent-prepared standard curves for qualitative and quantitative analysis.

[0088] Furthermore, the limits of detection and limits of quantitation for sample components were defined as the concentrations corresponding to a signal-to-noise ratio (S / N) of 3 and 10, respectively, in quantitative ion chromatography. The specific results are shown in Table 3, which presents the regression equation, correlation coefficient R, limit of detection (μg / kg), limit of quantitation (μg / kg), and linear range (ng / mL) for each measured component.

[0089] Table 3

[0090] The above methods can effectively detect nafeta derivatives, structural analogs, and other unequal drugs, providing a reliable technical means to combat criminals who evade regulation by altering drug structures.

[0091] As can be seen, the purification strategy of combining solid-phase extraction columns and adsorbents in the above scheme effectively removes interference from complex matrices such as sugars, fatty acids, and phospholipids, significantly improving the method's anti-interference ability and ensuring good accuracy and applicability in various food matrices. In the separation and detection stage, by optimizing chromatographic and mass spectrometric conditions, high-throughput analysis of multiple illegally added compounds can be achieved simultaneously in a short time, greatly improving screening efficiency. Based on multiple reaction monitoring and positive / negative ion switching scanning technology, the method exhibits excellent sensitivity and selectivity, covering compounds with significant structural differences such as hormones, PDE5 inhibitors, and SSRIs, greatly expanding the screening scope. It also has effective identification capabilities for natriuretic derivatives, structural analogs, and other drugs not listed in the regulatory framework, providing reliable technical support for the systematic screening and regulation of illegally added kidney-tonifying and aphrodisiac substances in food.

[0092] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.

Claims

1. A method for detecting illegally added compounds in food, characterized in that, include: Set up a variety of preset illegal additive compounds and prepare standard working solutions of each preset illegal additive compound; Obtain food samples to be tested; The food sample is extracted using a preset extraction solvent to obtain an extract. The extract is first purified using a solid-phase extraction column, and then the purified liquid is secondly purified using a preset adsorbent combination to obtain the purified test solution. Based on preset chromatographic separation conditions, the purified test solution is separated to obtain chromatographically separated sample components; Using a multi-reaction monitoring mode combined with a positive and negative ion switching scanning mode, mass spectrometry analysis was performed on the standard working solutions of various preset illegally added compounds and the sample components after chromatographic separation to obtain the mass spectrometry identification parameters corresponding to various preset illegally added compounds and the mass spectrometry analysis data corresponding to the sample components. Based on the comparison results of the mass spectrometry identification parameters and the mass spectrometry analysis data, it is determined whether there are illegally added compounds in the food sample.

2. The method for detecting illegally added compounds in food according to claim 1, characterized in that, The steps for preparing standard working solutions of various pre-specified illegally added compounds specifically include: Obtain standard samples corresponding to various preset illegally added compounds; Mix each standard with methanol solution, and prepare standard stock solutions of various preset illegally added compounds based on a first preset concentration; Based on the second preset concentration, each standard stock solution is diluted to obtain the standard working solution containing various preset illegally added compounds.

3. The method for detecting illegally added compounds in food according to claim 1, characterized in that, The step of extracting the food sample using a preset extraction solvent to obtain an extract specifically includes: A food sample of a predetermined mass is vortex-mixed with a first predetermined volume of a predetermined extraction solvent to obtain a mixed solution comprising the food sample and the predetermined extraction solvent. The mixed solution was subjected to ultrasonic extraction; The mixed solution after ultrasonic extraction is centrifuged to achieve solid-liquid separation, and the supernatant is collected as the extract.

4. The method for detecting illegally added compounds in food according to claim 3, characterized in that, The step of ultrasonic extraction of the mixed solution specifically includes: Determine whether the food sample contains oil; If the food sample is the oil-containing sample, add a first preset volume of n-hexane solution to the mixed solution and perform vortex mixing; The vortex-mixed solution is centrifuged to separate the solution into an oil-containing supernatant and a lower layer. Collect the lower layer liquid and perform ultrasonic extraction on the lower layer liquid based on a preset time; If the food sample is a non-oil-containing sample, the mixed solution is subjected to ultrasonic extraction based on a preset time.

5. The method for detecting illegally added compounds in food according to claim 1, characterized in that, The steps of performing a primary purification treatment on the extract based on a solid-phase extraction column, and a secondary purification treatment on the purified liquid based on a preset adsorbent combination to obtain a purified test solution specifically include: The extract is initially purified using the solid-phase extraction column, and the target fraction is collected. The target fraction is subjected to secondary purification based on a preset adsorbent combination to obtain a purified liquid. The purified liquid is then filtered to obtain the test liquid.

6. The method for detecting illegally added compounds in food according to claim 5, characterized in that, The step of performing initial purification of the extract based on the solid-phase extraction column and collecting the target fraction specifically includes: The second preset volume of extract is loaded onto a solid-phase extraction column for purification; Discard the initial third preset volume of filtrate that flows out; Collect the fourth preset volume of eluent that flows out subsequently, as the target fraction.

7. The method for detecting illegally added compounds in food according to claim 5, characterized in that, The step of performing secondary purification treatment on the target fraction based on a preset adsorbent combination to obtain a purified liquid, and filtering the purified liquid to obtain the test liquid specifically includes: The target fraction is placed in a centrifuge tube containing a preset adsorbent combination and vortexed to obtain a mixture including the target fraction and the preset adsorbent combination. The mixture in the centrifuge tube is allowed to stand, and the supernatant is collected and passed through a filter membrane with a preset pore size to obtain the purified liquid.

8. The method for detecting illegally added compounds in food according to claim 1, characterized in that, The preset liquid chromatography separation conditions are as follows: A C18 column was used, with column dimensions of 100 × 2.1 mm and 2.5 μm. The mobile phase is: methanol and a 0.1% (v / v) aqueous solution of formic acid; The column temperature was 30 degrees Celsius, the injection volume was 5 μL, and the flow rate was 0.35 mL / min. The chromatographic gradient elution conditions were as follows: initial methanol concentration of 10%; 0 to 1 min, maintain 10%; 1 min to 12 min, 10% to 85%; 12 min to 15 min, maintain 85%; 15 min to 15.1 min, 85% to 95%; 15.1 min to 16 min, maintain 95%; 16 min to 16.1 min, 95% to 10%; 16.1 min to 18 min, maintain 10%.

9. The method for detecting illegally added compounds in food according to claim 1, characterized in that, The step of employing a multi-reaction monitoring mode combined with a positive and negative ion switching scanning mode to perform mass spectrometry analysis on the standard working solutions of various preset illegally added compounds and the sample components after chromatographic separation, and obtaining the mass spectrometry identification parameters corresponding to various preset illegally added compounds and the mass spectrometry analysis data corresponding to the sample components, specifically includes: Mass spectrometry analysis was performed on standard working solutions of various pre-specified illegally added compounds. Based on multiple reaction monitoring mode combined with positive and negative ion switching scanning mode, mass spectrometry identification parameters of various pre-specified illegally added compounds were collected. The mass spectrometry identification parameters include the mass spectra of the parent ion and daughter ion, as well as the optimized mass spectrometry voltage parameters, including the declustering voltage, inlet voltage, collision voltage, and outlet voltage. Mass spectrometry analysis of sample components was performed, and mass spectrometry data of sample components were acquired based on multiple reaction monitoring mode.

10. The method for detecting illegally added compounds in food according to claim 9, characterized in that, The steps of performing mass spectrometry analysis on standard working solutions of various pre-defined illegally added compounds, based on multiple reaction monitoring mode combined with positive and negative ion switching scanning mode, and acquiring mass spectrometry identification parameters of various pre-defined illegally added compounds, specifically include: Standard working solutions of various pre-pre-selected illegally added compounds were continuously injected into the mass spectrometer; A full-scan primary mass spectrometry scan was performed on the standard working solution to obtain primary mass spectra of various pre-specified illegally added compounds, and the optimal precursor ion of each illegally added compound was determined. The parent ion was subjected to a secondary mass spectrometry scan to obtain the daughter ions and identify characteristic ion pairs; Based on characteristic ion pairs, the mass spectrometry voltage parameters of various preset illegally added compounds are optimized; Based on the parent ion, daughter ion, and mass spectrometry voltage parameters, various preset mass spectrometry identification parameters for illegally added compounds are constructed.

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