A method for distinguishing natural and artificial royal jelly acid based on liquid chromatography

Abstract

The application relates to the field of food physical and chemical detection technology, and discloses a natural and artificial royal jelly acid distinguishing method based on liquid chromatography, which comprises the following steps: processing target royal jelly samples to obtain a test sample solution; injecting the test sample solution into a high-performance liquid chromatograph, adopting a binary mobile phase step elution program for separation, and obtaining ultraviolet chromatographic data under ultraviolet detection at a wavelength of 210 nm; determining reference retention times of four preset characteristic substances according to an artificial 10-HDA positive control sample and calculating characteristic peak areas; establishing a background peak area benchmark value of a natural royal jelly negative control sample, multiplying the background peak area benchmark value by 3 to obtain corresponding positive determination threshold values; comparing the characteristic peak areas with the positive determination threshold values, combining a preset detection state of the characteristic substance D, and making a distinguishing conclusion. The application combines preset characteristic substances and background test logic, and solves the problem of physical and chemical distinction caused by consistent main component structures.

Description

Technical Field

[0001] This invention relates to the field of food physicochemical testing technology, specifically a method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography. Background Technology

[0002] Royal jelly is a natural substance, and its quality is usually evaluated based on the content of 10-hydroxy-2-decenoic acid (10-HDA), also known as royal jelly acid. In actual production and distribution, there is a phenomenon of artificially adding synthetic 10-HDA to increase the detected levels of royal jelly acid in low-quality products.

[0003] Existing physicochemical detection techniques mainly focus on the quantitative analysis of the main component content of 10-HDA. Since the chemical molecular structure of artificially synthesized 10-HDA is identical to that of natural royal jelly, conventional analytical methods can only reflect the total amount of 10-HDA in the sample, making it difficult to determine whether the component originates naturally or synthetically. This results in a deficiency in the detection system when dealing with royal jelly samples adulterated with artificially synthesized components.

[0004] Furthermore, the royal jelly matrix system contains components such as proteins, lipids, and colloids. These matrix components are difficult to remove during conventional sample pretreatment and interfere with the signal recognition of trace components after entering the chromatographic analysis system. Simultaneously, conventional liquid chromatography elution procedures lack specific separation mechanisms for synthetic byproducts, making it difficult to overcome overlapping matrix signal interference within the corresponding chromatographic range. This makes it difficult for existing technologies to extract characteristic data to establish criteria for distinguishing between natural and synthetic royal jelly acids. Summary of the Invention

[0005] The technical problem solved by this invention is that existing conventional physicochemical analysis techniques are difficult to distinguish between natural royal jelly and counterfeit products adulterated with synthetic 10-hydroxy-2-decenoic acid (10-HDA). Because synthetic 10-HDA and the 10-HDA in natural royal jelly have the same main chemical structure, existing detection methods are difficult to differentiate between royal jelly samples adulterated with synthetic 10-HDA.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography, comprising the following steps: The target royal jelly sample was processed to obtain a test solution; The test solution was injected into a high performance liquid chromatograph, and separation was performed using a binary mobile phase stepwise elution program. Ultraviolet detection was performed at a wavelength of 210 nm to obtain ultraviolet chromatographic data. Based on the positive control chromatographic data obtained by detecting artificial 10-HDA positive control samples, the reference retention times of preset characteristic A, preset characteristic B, preset characteristic C and preset characteristic D are determined, and the characteristic peak area of ​​the target royal jelly sample is calculated at the corresponding reference retention time in the ultraviolet chromatographic data. Based on the negative control chromatographic data obtained from the detection of the negative control sample of natural royal jelly, a baseline peak area value for the negative control sample of natural royal jelly is established. The baseline peak area values ​​of the preset feature A, the preset feature B and the preset feature C are multiplied by 3 respectively to obtain the corresponding positive judgment threshold. The characteristic peak area of ​​the target royal jelly sample is compared with the corresponding positive determination threshold, and an identification conclusion is made on the target royal jelly sample in combination with the detection status of the preset characteristic substance D.

[0007] Furthermore, the identification conclusion of the target royal jelly sample includes: When the peak area of ​​any of the preset feature A, preset feature B and preset feature C is greater than the corresponding positive determination threshold, or when the preset feature D is detected, a positive identification conclusion of artificial 10-HDA is output. When the peak areas of the preset feature A, the preset feature B, and the preset feature C are all not greater than the corresponding positive determination threshold, and the preset feature D is not detected, a negative identification conclusion is output indicating that no artificial 10-HDA feature signal is detected within the detection range of this method.

[0008] In a preferred embodiment of the present invention, the step of processing the target royal jelly sample to obtain the test solution includes: The target royal jelly sample was subjected to acid dispersion, acetonitrile extraction, and protein precipitation to obtain a mixture that formed a precipitate. The test solution is obtained by ultrasonic treatment, solid-liquid separation and filtration of the mixture that forms the precipitate.

[0009] Furthermore, the acid dispersion, acetonitrile extraction, and protein precipitation treatment of the target royal jelly sample includes: Royal jelly matrix was dispersed using a dilute phosphoric acid aqueous solution; Acetonitrile is added to form a mixed extraction system of acetonitrile and water; Add the pre-set Carrez I reagent and the pre-set Carrez II reagent to cause the protein and colloidal components to form flocculation and precipitation.

[0010] Furthermore, the step of obtaining the test solution by ultrasonic treatment, solid-liquid separation, and filtration of the mixture that forms the precipitate includes: The mixture that forms the precipitate is first subjected to ultrasonic degassing, then acetonitrile is added to make up the volume, and then ultrasonic extraction is performed to obtain the extract. The extract is centrifuged, and the supernatant is filtered through a microporous membrane to remove suspended particles, thus obtaining the test solution.

[0011] In another preferred embodiment of the present invention, the step of injecting the test solution into a high-performance liquid chromatograph and separating it using a binary mobile phase stepwise elution program includes: The test solution is injected into a high-performance liquid chromatograph equipped with a preset SMH column, wherein the SMH column has a specification of 150 mm × 4.6 mm and a packing particle size of 5 μm, the injection volume is 10 μL, and the column temperature is 40 °C. Mobile phase A and mobile phase B are used as binary mobile phases. Mobile phase A is a solution of acetonitrile and water mixed in a volume ratio of 36:64 and containing 0.4% phosphoric acid. Mobile phase B is methanol.

[0012] Furthermore, the binary mobile phase stepwise elution process includes: The mobile phase A was 100% used from 0 to 12.00 min at a flow rate of 1.2 mL / min. The mobile phase B, consisting of 100% of the mobile phase described above, was used at a flow rate of 1.5 mL / min from 12.01 to 15.00 min. Restore 100% of the mobile phase A within 15.01–27.00 min, maintaining a flow rate of 1.5 mL / min.

[0013] Furthermore, the calculation of the characteristic peak area of ​​the target royal jelly sample includes: Identify the corresponding target characteristic peaks in the ultraviolet chromatographic data; Determine the peak start point, peak end point, and integration baseline; Integrate the region between the integral baseline and the target feature peak contour to calculate the feature peak area; If no chromatographic peak meeting the identification criteria is formed at the corresponding reference retention time, the corresponding characteristic substance is recorded as not detected, and the corresponding peak area is recorded as 0.

[0014] Furthermore, a baseline value for the background peak area of ​​the negative control sample of natural royal jelly was established, including: A natural royal jelly sample without added artificial 10-HDA was used as the negative control sample. The preset feature A, preset feature B, and preset feature C each use the upper limit of the peak area in the multiple negative control samples as the corresponding background peak area reference value.

[0015] This invention provides a method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography. It has the following beneficial effects: 1. This invention uses pre-defined characteristic components A, B, C, and D from the production process of artificially synthesized 10-HDA as identification indicators. Combining liquid chromatography separation technology with the background testing mechanism of natural royal jelly negative control samples, and establishing a data discrimination logic based on a set positive judgment threshold and the detection status of characteristic component D, this invention solves the problem of physicochemical differentiation caused by the identical main component structure of artificially synthesized 10-HDA and natural royal jelly acid. It provides an analytical method that can objectively assess whether artificially synthesized components are added to a target royal jelly sample.

[0016] 2. This invention uses dilute phosphoric acid aqueous solution for matrix dispersion and combines it with acetonitrile to form a mixed extraction system. On this basis, a pre-set Carrez reagent is added to cause the protein and colloidal components in royal jelly to form flocculation and precipitation. Combined with ultrasonic treatment and centrifugal filtration, the target characteristic is extracted while eliminating suspended solids and matrix interference components. This reduces the risk of blockage of the subsequent liquid chromatography separation column and interference with the ultraviolet detection signal by the complex sample matrix, thereby improving the preparation quality and detection reproducibility of the test solution.

[0017] 3. This invention establishes a binary mobile phase stepwise elution program containing acetonitrile phosphate aqueous solution and methanol. By stepwise switching of the mobile phase ratio and flow rate within different time periods, the retention time differences of the 10-HDA main component, artificial 10-HDA characteristic substances, and natural royal jelly matrix components are created within the detection range. After data acquisition, the impurities retained in the chromatographic column are washed with methanol, and the column is equilibrated by restoring the initial ratio. This ensures the reliability of chromatographic data when the system continuously processes multiple batches of target royal jelly samples. Attached Figure Description

[0018] Figure 1 This is a system architecture diagram of the present invention; Figure 2 This is a flowchart of the method of the present invention; Figure 3 This is a flowchart of the sample preprocessing steps of the present invention; Figure 4 This is a flowchart of the chromatographic separation and elution process of the present invention; Figure 5 This is a flowchart of the data processing and integration steps of the present invention; Figure 6 This is a flowchart of the data comparison and judgment steps of the present invention; Figure 7 This is a preliminary characteristic peak chromatogram of an artificial 10-HDA sample under the C18 column conditions of this invention; Figure 8 The chromatogram of the negative control sample of natural royal jelly under the C18 chromatographic column conditions of this invention is shown. Figure 9 This is a chromatogram of a royal jelly sample spiked with artificial 10-HDA under the C18 chromatographic column conditions of the present invention; Figure 10 This is a superimposed chromatogram of the 0.5% artificial 10-HDA spiked sample and natural royal jelly samples from different floral sources according to the present invention; Figure 11 This is a superimposed chromatogram of a 0.5% artificial 10-HDA-spikeped royal jelly sample and a royal jelly residue sample from the present invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] See attached document Figure 1 This invention provides a liquid chromatography-based system for distinguishing between natural and artificial royal jelly acids. It includes a sample pretreatment module, a chromatographic separation module, and a data determination module.

[0021] The sample pretreatment module is used to receive the target royal jelly sample, perform acid dispersion, acetonitrile extraction, protein precipitation, solid-liquid separation and filtration on the target royal jelly sample to obtain the test solution, and transfer the test solution to the chromatographic separation module.

[0022] The chromatographic separation module, connected to the sample pretreatment module, receives the test solution and performs liquid chromatography separation according to a preset binary mobile phase stepwise elution program. This ensures that the main component of 10-hydroxy-2-decenoic acid, the synthetic 10-hydroxy-2-decenoic acid characteristic compound, and the natural royal jelly matrix components exhibit retention differences. Synthetic 10-hydroxy-2-decenoic acid is abbreviated as artificial 10-HDA. The components separated by the chromatographic column enter a UV detector, which records the change in absorbance over retention time, generating UV chromatographic data, which is then transmitted to the data interpretation module.

[0023] The data determination module, connected to the chromatographic separation module, receives ultraviolet chromatographic data. Based on the positive control chromatographic data obtained from the detection of an artificial 10-HDA positive control sample, it determines the reference retention times of preset characteristic compounds A, B, C, and D, and performs peak finding, baseline determination, and peak area integration at the corresponding reference retention times. The data determination module also uses negative control chromatographic data obtained from the detection of a natural royal jelly negative control sample to call the background peak area benchmark value of the natural royal jelly negative control sample, compares the characteristic peak area of ​​the target royal jelly sample with the corresponding positive determination threshold, and outputs an identification conclusion based on the comparison result.

[0024] See attached document Figure 2 This invention provides a method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography, comprising the following steps: S100 is used to perform acid dispersion, acetonitrile extraction and protein precipitation on the target royal jelly sample, and then obtain the test solution after ultrasonic treatment, solid-liquid separation and filtration.

[0025] The royal jelly matrix was dispersed in a dilute phosphoric acid aqueous solution, and then acetonitrile was added to form a mixed extraction system of acetonitrile and water. Pre-set Carrez I and Carrez II reagents were added to cause the protein and colloidal components to flocculate and precipitate. The sample solution was obtained after ultrasonic treatment, centrifugation, and microporous membrane filtration.

[0026] S200 injects the test solution into the high-performance liquid chromatograph, uses a preset chromatographic column and binary mobile phase stepwise elution program for separation, and performs ultraviolet detection at a wavelength of 210 nm to obtain ultraviolet chromatographic data.

[0027] Mobile phase A was an aqueous solution of acetonitrile containing phosphoric acid, and mobile phase B was methanol. The liquid chromatography system switched the mobile phase composition and flow rate according to a set time program to create retention differences among the 10-HDA main component, artificial 10-HDA characteristic compounds, and natural royal jelly matrix components. A UV detector continuously recorded the change in absorbance over retention time, generating UV chromatographic data for subsequent peak finding and integration.

[0028] S300, based on the positive control chromatographic data obtained by detecting artificial 10-HDA positive control samples, determines the reference retention times of preset characteristic A, preset characteristic B, preset characteristic C and preset characteristic D, identifies the target characteristic peaks at the corresponding reference retention times and calculates the peak areas.

[0029] Artificial 10-HDA samples capable of exhibiting preset characteristic substances A, B, C, and D were used as positive control samples and detected under the same chromatographic conditions as the target royal jelly sample. The data determination module used the measured retention time of the positive control sample as the peak-finding benchmark to identify the corresponding characteristic peaks in the UV chromatographic data of the target royal jelly sample. After identifying the target characteristic peaks, the onset point, termination point, and integration baseline were determined, and the characteristic peak area was calculated. If no chromatographic peak meeting the identification criteria was formed at the corresponding reference retention time, the characteristic substance was recorded as undetectable, and the peak area was recorded as 0.

[0030] S400, based on the negative control chromatographic data obtained by detecting the negative control sample of natural royal jelly, establishes the baseline peak area of ​​the negative control sample of natural royal jelly, multiplies the baseline peak area of ​​the preset characteristic A, preset characteristic B and preset characteristic C by 3 respectively to obtain the corresponding positive judgment threshold; combined with the detection status of the preset characteristic D, makes an identification conclusion on the target royal jelly sample.

[0031] Natural royal jelly samples with a clear source and no added artificial 10-HDA were used as negative control samples. Preset characteristic A, preset characteristic B, and preset characteristic C were each used as background peak area benchmarks based on the upper limit of the peak area in multiple negative control samples. Preset characteristic D did not form a chromatographic peak meeting the identification criteria in the negative control samples; its detection status was used for determination. A positive result was determined when the peak area of ​​any of the preset characteristic A, preset characteristic B, or preset characteristic C in the target royal jelly sample exceeded the corresponding positive determination threshold; a positive result was also determined when preset characteristic D was detected. If the peak areas of the preset feature A, preset feature B, and preset feature C are all not greater than the corresponding positive judgment threshold, and the preset feature D is not detected, it is determined that no artificial 10-HDA feature signal is detected within the detection range of this method.

[0032] This method is applicable to the identification of cases where at least one of the following pre-defined features A, B, C, and D associated with artificial 10-HDA is retained in royal jelly samples.

[0033] See attached document Figure 3 In the identification method provided by the present invention, step S100 includes the following steps: S110: Obtain the target royal jelly sample and disperse it in a dilute phosphoric acid aqueous solution. Royal jelly contains proteins, carbohydrates, lipids, and colloidal components. After adding the dilute phosphoric acid aqueous solution, the royal jelly matrix is ​​dispersed in the liquid phase system, establishing a processing basis for acetonitrile extraction and Carrez reagent precipitation of proteins.

[0034] S120, acetonitrile is added to the dispersion to extract the artificial 10-HDA characteristic. After being added to the dispersion, acetonitrile forms a mixed solvent system with water. Through mixing, the artificial 10-HDA characteristic in the sample enters the liquid phase. This step uses mixed solvent extraction, and no independent organic phase is formed in the system.

[0035] S130, pre-set Carrez I reagent and pre-set Carrez II reagent are added sequentially to the extract to cause proteins and colloidal interference components to form flocculation and precipitation. After adding the pre-set Carrez I reagent, the mixture is stirred, and then the pre-set Carrez II reagent is added and mixed again. Preferably, the added volumes of the pre-set Carrez I reagent and pre-set Carrez II reagent can be adjusted according to the mass of the royal jelly sample; at this ratio, the removal effect of proteins and colloidal components is optimal. The two reagents work together to cause proteins and colloidal components dispersed in the liquid phase to form precipitation, reducing the impact of suspended matter on centrifugation, filtration, and chromatographic analysis.

[0036] S140 involves ultrasonic degassing and extraction of the precipitated mixture, followed by volume adjustment with acetonitrile. Ultrasonic degassing is performed first to reduce air bubbles in the mixture. After degassing, acetonitrile is added to bring the volume to a final volume, and then ultrasonic extraction is performed to introduce the artificial 10-HDA characteristic into the mixed solvent system.

[0037] S150: The extract is centrifuged and filtered through a microporous membrane to obtain the test solution. Centrifugation separates the flocculated precipitate from the liquid phase. After centrifugation, the supernatant is collected and suspended particles are removed by passing it through a microporous membrane to obtain the test solution for liquid chromatography analysis.

[0038] See attached document Figure 4 In the identification method provided by the present invention, step S200 includes the following steps: S210 configures the separation and detection conditions for the high-performance liquid chromatography (HPLC) system. A preset column is used, specifically a 150mm × 4.6mm SMH column with a 5μm particle size, or another column with equivalent separation performance. These dimensions and particle size provide optimal resolution under this elution program. Preferably, a combination of 10μL injection volume, 40℃ column temperature, and 210nm UV detection wavelength ensures sufficient heat exchange of the analyte within the column, avoids excessive column pressure, and achieves optimal signal response sensitivity and signal-to-noise ratio in the detector.

[0039] The pre-defined chromatographic column is used to separate the 10-HDA main component, artificial 10-HDA characteristic compounds, and natural royal jelly matrix components. Under specified chromatographic conditions, the 10-HDA main component elutes first, followed by pre-defined characteristic compounds A, B, C, and D, which elute within subsequent retention time ranges.

[0040] S220, configure a binary mobile phase. Mobile phase A is a solution of acetonitrile and water at a volume ratio of 36:64 and containing 0.4% phosphoric acid; mobile phase B is methanol. Preferably, mobile phase A, using the above-mentioned 36:64 volume ratio and a specific phosphoric acid concentration of 0.4%, can effectively inhibit the dissociation of royal jelly acids, maximize the improvement of the peak shape of the target chromatographic peak, and avoid tailing. Mobile phase A is used for the separation of the target characteristic peak, and mobile phase B is used for eluting the matrix components remaining in the preset chromatographic column.

[0041] S230, perform binary mobile phase stepwise elution and flow rate switching. Use 100% mobile phase A at a flow rate of 1.2 mL / min from 0 to 12.00 min; use 100% mobile phase B at a flow rate of 1.5 mL / min from 12.01 to 15.00 min; and switch back to 100% mobile phase A at a flow rate of 1.5 mL / min from 15.01 to 27.00 min.

[0042] Preferably, by employing the aforementioned specific stepwise elution time program and flow rate parameter switching, baseline separation of the four target characteristic peaks can be achieved within the shortest analysis cycle. Furthermore, high-speed elution with methanol can effectively prevent the degradation of strongly retained matrix interferences and mitigate column performance degradation. The time interval from 0 to 12.00 min is for target characteristic peak separation and data acquisition. 12.01 to 15.00 min is used to elute the pre-set matrix components retained within the column. 15.01 to 27.00 min is used to restore mobile phase A and complete the pre-set column rebalancing.

[0043] S240 acquires ultraviolet absorbance signals and generates ultraviolet chromatographic data. Components separated by a preset chromatographic column sequentially enter the ultraviolet detector. The ultraviolet detector continuously records the change in absorbance with retention time at a wavelength of 210 nm, generating ultraviolet chromatographic data including the retention time and response intensity of each chromatographic peak, and transmits the ultraviolet chromatographic data to the data judgment module.

[0044] See attached document Figure 5 In the identification method provided by the present invention, step S300 includes the following steps: S310, establish reference retention times for preset characteristic substances A, B, C, and D. Select an artificial 10-HDA sample capable of exhibiting the preset characteristic substances A, B, C, and D as a positive control sample. Perform detection under the chromatographic conditions specified in step S200 to obtain the corresponding positive control chromatographic data. Preferably, based on the positive control chromatographic data, determine the reference retention times for preset characteristic substances A, B, C, and D to be 3.1 min, 6.1 min, 7.1 min, and 10.4 min, respectively. At these retention times, the peak interference of the characteristic substances is minimized, and the response is most stable.

[0045] Artificial 10-HDA samples from different sources do not present entirely the same combination of characteristic analytes. Positive control samples are used to determine the reference retention times of the four characteristic analytes. The same positive control sample is used within the same testing batch to establish a peak-finding benchmark.

[0046] S320: Identify the target characteristic peak at the reference retention time and determine the integration baseline. The data determination module scans the UV chromatographic signal centered on the reference retention times of preset characteristic substances A, B, C, and D, identifying chromatographic peaks with a start point, peak apex, and peak fall point. After the target characteristic peak is identified, the line connecting the start point and the peak fall point is used as the integration baseline. The integration baseline and the target characteristic peak profile together define the integration region.

[0047] If no chromatographic peak meeting the identification criteria is formed at the corresponding reference retention time, the characteristic substance is recorded as undetectable, and the peak area is recorded as 0. The same peak-finding conditions and integration rules are used for the target royal jelly sample, the artificial 10-HDA positive control sample, and the natural royal jelly negative control sample.

[0048] S330, calculate the peak areas of preset feature A, preset feature B, preset feature C, and preset feature D. The data determination module integrates the region between the integration baseline and the target feature peak contour to obtain the peak area of ​​each target feature peak, and associates and saves the peak area with the corresponding feature name, reference retention time, and detection status as the data input for step S400.

[0049] See attached document Figure 6 In the identification method provided by the present invention, step S400 includes the following steps: S410, establish the baseline peak area of ​​the natural royal jelly negative control sample. Multiple natural royal jelly samples with clearly identified sources and without added artificial 10-HDA are selected as negative control samples. These samples are processed and detected according to steps S100 to S300 to obtain the corresponding negative control chromatographic data. Based on this negative control chromatographic data, the peak areas of each negative control sample at the preset reference retention times for characteristic A, preset characteristic B, and preset characteristic C are obtained.

[0050] The upper limits of the peak areas of pre-defined characteristic A, pre-defined characteristic B, and pre-defined characteristic C in the negative control sample were used as the baseline values ​​for the background peak areas of the corresponding characteristic. The negative control sample did not form a chromatographic peak meeting the identification criteria at the reference retention time of pre-defined characteristic D; therefore, the detection status of pre-defined characteristic D was used for determination. When establishing the baseline values ​​for the background peak areas, the negative control sample and the target royal jelly sample used the same pretreatment steps, chromatographic conditions, peak-finding conditions, and integration rules.

[0051] S420, Establish positive determination thresholds. Multiply the baseline peak area values ​​of preset feature A, preset feature B, and preset feature C by 3 to obtain the positive determination thresholds for each feature. Preferably, in the determination model of this method, using a specific multiple relationship of multiplying the baseline value by 3 as the positive determination threshold can maximize the inclusion of background fluctuations between different batches of natural royal jelly samples, effectively preventing false positives caused by background noise, thereby ensuring the accuracy of the final identification conclusion. Preset feature D is not calculated using background multiples. When the target royal jelly sample forms a chromatographic peak that meets the identification conditions at the reference retention time of preset feature D, it is recorded as the detection of preset feature D.

[0052] S430, Perform data comparison and output identification conclusion. The data judgment module compares the peak areas of preset feature A, preset feature B and preset feature C in the target royal jelly sample with the corresponding positive judgment thresholds, and reads the detection status of preset feature D.

[0053] A positive identification conclusion for adulteration with artificial 10-HDA will be output if the target royal jelly sample meets any of the following conditions: The peak area of ​​the preset feature A is greater than the preset positive determination threshold of feature A; the peak area of ​​the preset feature B is greater than the preset positive determination threshold of feature B; the peak area of ​​the preset feature C is greater than the preset positive determination threshold of feature C; and the preset feature D is detected.

[0054] If the peak areas of the preset feature A, preset feature B, and preset feature C are all not greater than the corresponding positive judgment threshold, and the preset feature D is not detected, the output is a negative identification conclusion that no artificial 10-HDA feature signal is detected within the detection range of this method.

[0055] Specific application examples: Example 1: Establishment of liquid chromatography conditions, investigation of natural background and verification of artificial 10-HDA spike.

[0056] In this embodiment, the separation effect of C18 column and preferred SMH column was first investigated. Then, the determined liquid chromatography conditions were used to detect natural royal jelly samples from different flower sources and samples spiked with 0.5% artificial 10-HDA to establish the baseline peak area and positive judgment threshold.

[0057] C18 column condition screening. Preparation of artificial 10-HDA test solution: Weigh 0.05 g of artificial 10-HDA sample, dissolve in 25% acetonitrile solution and dilute to 20 mL. Pass an appropriate amount of solution through a 0.22 μm microporous membrane and collect the filtrate as the artificial 10-HDA test solution.

[0058] C18 chromatographic analysis of artificial 10-HDA samples was performed by injecting the artificial 10-HDA test solution into a high-performance liquid chromatograph (HPLC). A C18 column (150 mm × 4.6 mm, 5 μm particle size) was used; the injection volume was 10 μL; the flow rate was 1.2 mL / min; the column temperature was 40 °C; the UV detection wavelength was 210 nm; the mobile phase was a solution of methanol and water at a volume ratio of 55:45 containing 0.16% phosphoric acid; and the acquisition time was 25 min.

[0059] After detection, three chromatographic peaks appeared at 6.3 min, 11.1 min, and 16.7 min, respectively. To distinguish the chromatographic peaks observed at this stage from the characteristic substances used for final determination under liquid chromatography column conditions, the three chromatographic peaks were designated as preliminary characteristic peak A′, preliminary characteristic peak B′, and preliminary characteristic peak C′, respectively. Preliminary characteristic peaks A′, B′, and C′ are only used to record the screening results of the C18 chromatographic column and are not used as determination peaks in the identification method of this invention.

[0060] See attached document Figure 7 The horizontal axis represents retention time in minutes, and the vertical axis represents UV response intensity in mV. The preliminary characteristic peaks A′, B′, and C′ are located around 6.3 min, 11.1 min, and 16.7 min, respectively.

[0061] C18 chromatographic analysis of natural royal jelly samples: 0.5g of a well-identified natural royal jelly sample was weighed and extracted with 25% acetonitrile solution. Carrez reagent was added to precipitate proteins. After centrifugation, the supernatant was filtered through a 0.22μm microporous membrane and analyzed under the chromatographic conditions described in Part 1.2 of this embodiment.

[0062] The natural royal jelly sample showed multiple matrix response peaks during the detection period, with some of the matrix response peaks located near the preliminary characteristic peaks A′, B′, and C′.

[0063] See attached document Figure 8 The horizontal axis represents retention time in minutes, and the vertical axis represents UV response intensity in mV. Multiple matrix response peaks from natural royal jelly are present in the initial segment of the chromatogram, and the time intervals of the preliminary characteristic peaks are influenced by the matrix response.

[0064] C18 chromatographic analysis of artificial 10-HDA-spiked royal jelly samples: Weigh 0.5g of natural royal jelly sample, add artificial 10-HDA, prepare the test solution according to the sample processing method of this embodiment, and then perform detection according to the chromatographic conditions of this embodiment.

[0065] In artificially spiked royal jelly samples with 10-HDA, the initial characteristic peaks overlap with the peaks of the natural royal jelly matrix. The onset and ending points of some chromatographic peaks cannot be determined according to a uniform rule. Therefore, these chromatographic conditions are not used for subsequent peak area determination.

[0066] See attached document Figure 9 The horizontal axis represents retention time in minutes, and the vertical axis represents UV response intensity in mV. The positions marked A′, B′, and C′ in the figure correspond to three preliminary characteristic peaks. The response peaks formed after spiking were not completely baseline separated from the response peaks of the natural royal jelly matrix.

[0067] Establishment of separation conditions for liquid chromatography column. Preparation of artificial 10-HDA samples and standards: Ten portions of artificial 10-HDA samples from different sources and one portion of artificial 10-HDA standard were prepared into test solutions with a concentration of 0.35 mg / mL. Each test solution was filtered through a 0.22 μm microporous membrane and then injected into the high-performance liquid chromatograph.

[0068] The liquid chromatography conditions were as follows: a preset column was used, with a column size of 150 mm × 4.6 mm and a packing particle size of 5 μm. SMH columns or equivalent columns were preferred. The injection volume was 10 μL. The column temperature was 40 °C. The UV detection wavelength was 210 nm.

[0069] Mobile phase A was a solution of acetonitrile and water at a volume ratio of 36:64, containing 0.4% phosphoric acid. Mobile phase B was methanol. From 0 to 12.00 min, 100% mobile phase A was used at a flow rate of 1.2 mL / min; from 12.01 to 15.00 min, 100% mobile phase B was used at a flow rate of 1.5 mL / min; from 15.01 to 27.00 min, 100% mobile phase A was restored, and the flow rate was maintained at 1.5 mL / min.

[0070] 0–12.00 min is used for the separation of artificial 10-HDA features and data acquisition; 12.01–15.00 min is used for eluting the matrix components retained in the preset column; 15.01–27.00 min is used for restoring mobile phase A and completing the preset column rebalancing.

[0071] For the identification of artificial 10-HDA characteristic components, under the above chromatographic conditions, the main 10-HDA component eluted first. Six chromatographic peaks (A, 1, B, C, D, and 2) were recorded within the subsequent retention time range, with reference retention times of 3.1 min, 4.9 min, 6.1 min, 7.1 min, 10.4 min, and 19.7 min, respectively.

[0072] Based on the detection results of artificial 10-HDA samples and natural royal jelly samples, four preset characteristic substances A, B, C, and D were selected as identification indicators. The reference retention times for the four preset characteristic substances were 3.1 min, 6.1 min, 7.1 min, and 10.4 min, respectively. Peak 1 showed a background response in natural royal jelly, while peak 2 was located outside the target data acquisition range; therefore, peaks 1 and 2 were not included in the determination of the identification conclusion.

[0073] Natural royal jelly samples and artificial 10-HDA samples were collected. Nineteen samples of royal jelly derived from buckwheat flowers, ten samples from locust flowers, ten samples from linden flowers, ten samples from vitex flowers, ten samples from camellia flowers, seven samples from fennel flowers, and two samples from rapeseed flowers. Each sample weighed 200g.

[0074] Ten additional artificial 10-HDA samples from different manufacturers were collected, each weighing 100g. These artificial 10-HDA samples were used to investigate the characteristic peak composition of different production sources, as shown in Table 1.

[0075] Table 1. Source and Quantity of Floral Elements in Natural Royal Jelly Samples Preparation of the test solution of natural royal jelly. Weigh 0.5g of natural royal jelly sample, with a weighing deviation controlled within ±0.02g, and place it in a 20mL volumetric flask. Add 10mL of 0.35% phosphoric acid solution, then add 5mL of acetonitrile, and vortex mix for 1min.

[0076] Add 1.5 mL of the pre-prepared Carrez I reagent and shake well, then add 1.5 mL of the pre-prepared Carrez II reagent and shake well. Place the volumetric flask in an ultrasonic cleaner and sonicate for 1 min to remove air bubbles from the mixture. Add acetonitrile to a final volume of 20 mL and sonicate for 15 min.

[0077] Take 10 mL of the extract and place it in a centrifuge tube. Centrifuge at 4000 rpm for 5 min. Take 1 mL of the supernatant and filter it through a 0.22 μm microporous membrane. Collect the filtrate as the test solution for natural royal jelly.

[0078] Preparation of 0.5% artificial 10-HDA spiked sample: Weigh 0.5g of natural royal jelly negative control sample, with the weighing deviation controlled within ±0.02g, and place it in a 20mL volumetric flask.

[0079] A 10 mg / mL artificial 10-HDA solution was prepared using acetonitrile. 250 μL of this solution was added to a volumetric flask, representing 2.5 mg of artificial 10-HDA, which is 0.5% of the 0.5 g royal jelly sample. After vortexing for 1 min, 10 mL of 0.35% phosphoric acid solution was added. Acetonitrile extraction, Carrez reagent precipitation, sonication, centrifugation, and filtration were then performed according to Part 4 of this embodiment to obtain the test solution of the 0.5% artificial 10-HDA spiked sample.

[0080] For each batch of samples tested, a negative control sample of natural royal jelly and a sample spiked with 0.5% artificial 10-HDA were prepared simultaneously to check the background response and characteristic peak response within the test batch.

[0081] For the selection of positive control samples, based on the test results of 10 artificial 10-HDA samples, 3 artificial 10-HDA samples that can simultaneously present the preset feature A, preset feature B, preset feature C and preset feature D were selected as positive control samples.

[0082] Positive control samples were prepared as test solutions at a concentration of 0.35 mg / mL and analyzed in each batch of tests to obtain corresponding positive control chromatographic data. Based on this positive control chromatographic data, the measured retention times of the four pre-defined characteristic compounds in the positive control sample were used as the peak-finding benchmark for the same batch of samples.

[0083] Liquid chromatography detection and peak area integration were performed on the natural royal jelly test solution, the 0.5% artificial 10-HDA spiked sample test solution, and the positive control sample under the SMH liquid chromatography conditions of this embodiment, and the corresponding negative control chromatographic data and target ultraviolet chromatographic data were obtained respectively.

[0084] Peak searching is performed at the reference retention times of preset feature A, preset feature B, preset feature C, and preset feature D. After identifying chromatographic peaks with a start point, peak apex, and peak fall point, the line connecting the start point and peak fall point is used as the integration baseline, and the peak area is calculated. If no chromatographic peak meeting the peak shape conditions is formed, it is recorded as not detected, and the peak area is recorded as 0.

[0085] Chromatographic results of natural royal jelly samples from different floral sources were obtained by overlaying samples from rapeseed, locust, vitex, linden, buckwheat, fennel, and camellia flowers onto samples spiked with 0.5% artificial 10-HDA.

[0086] See attached document Figure 10 In the figure, black represents negative samples spiked with 0.5% artificial 10-HDA; dark gray represents rapeseed pulp; blue represents locust pulp; red represents vitex pulp; green represents linden pulp; dark blue represents buckwheat pulp; yellow-green represents fennel pulp; and light gray represents camellia pulp.

[0087] Figure 10 The horizontal axis represents retention time in minutes, and the vertical axis represents UV response intensity in μV. Natural royal jelly samples from different floral sources exhibit varying degrees of background response at the reference retention times of preset characteristic compounds A, B, and C, but no characteristic peak meeting the identification criteria was formed at the reference retention time of preset characteristic compound D. Samples spiked with 0.5% artificial 10-HDA showed a characteristic response of artificial 10-HDA within the target retention time range.

[0088] The baseline peak area, positive threshold, and spiked validation results were determined. Peak area integration was performed on 10 artificial 10-HDA samples, 1 artificial 10-HDA standard, a natural royal jelly negative control sample, natural royal jelly samples from different flower sources, and a 0.5% artificial 10-HDA spiked sample. The results are shown in Tables 2-1 and 2-2.

[0089] Table 2-1. Peak Area of ​​Artificial 10-HDA Characteristic Components Table 2-2. Peak Area of ​​Artificial 10-HDA Characteristic Components Note: Tables 2-1 and 2-2 contain the same set of test data. Each row corresponds to the manual 10-HDA number and sample name. "—" indicates that there is no corresponding test data for this item; " / " indicates that no identifiable chromatographic peak was detected at the corresponding retention time.

[0090] Based on the integrated results of natural royal jelly listed in Tables 2-1 and 2-2, and using negative control chromatographic data, the baseline peak area of ​​the preset characteristic A was 7275, the baseline peak area of ​​the preset characteristic B was 2517, and the baseline peak area of ​​the preset characteristic C was 979. The response of the natural royal jelly sample at the preset reference retention time of characteristic D was lower than the display limit used in the original detection record, and no chromatographic peak meeting the identification criteria was formed.

[0091] Multiply the baseline values ​​of the background peak areas of the preset feature A, preset feature B, and preset feature C by 3 respectively to obtain the following positive judgment thresholds: The preset positive threshold for feature A is 21825; The preset positive threshold for feature B is 7551; The preset positive threshold for feature C is 2937.

[0092] The preset characteristic D is determined by its detection status. When a chromatographic peak that meets the identification criteria is formed at the preset characteristic D reference retention time, it is recorded as the preset characteristic D being detected.

[0093] In the 0.5% artificial 10-HDA spiked sample, the peak areas of the pre-defined characteristic A, pre-defined characteristic B, pre-defined characteristic C, and pre-defined characteristic D were 107688, 6576, 20834, and 4646, respectively. The peak area of ​​pre-defined characteristic A was greater than 21825, the peak area of ​​pre-defined characteristic C was greater than 2937, and pre-defined characteristic D formed a chromatographic peak that met the identification criteria. Therefore, the 0.5% artificial 10-HDA spiked sample was determined to be positive for artificial 10-HDA.

[0094] If the peak area of ​​the preset feature B does not exceed 7551, the positive result of the sample will not be changed. This method uses four preset features for joint determination. If the peak area of ​​any one of the preset features A, B, and C is greater than the corresponding positive determination threshold, and the preset feature D is detected, all of these will enter the positive determination branch.

[0095] The lowest detection levels of the 10 artificial 10-HDA samples recorded in Tables 2-1 and 2-2 range from 0.04% to 0.5%. Artificial 10-HDA samples from different sources have different combinations of characteristic components, and at least one of the four pre-defined characteristic components forms a response for identification.

[0096] Example 2: Application of royal jelly residue sample detection.

[0097] This embodiment uses the sample pretreatment conditions, SMH liquid chromatography conditions, and judgment rules established in Example 1 to detect royal jelly residue samples.

[0098] Pretreatment of royal jelly residue samples. Weigh 0.5g of royal jelly residue sample 1, royal jelly residue sample 2 and royal jelly residue sample 3 respectively, with the weighing deviation controlled within ±0.02g, and place them in 20mL volumetric flasks.

[0099] Add 10 mL of 0.35% phosphoric acid solution to each volumetric flask, then add 5 mL of acetonitrile, and vortex for 1 min. Add 1.5 mL of the pre-prepared Carrez I reagent and shake well, then add 1.5 mL of the pre-prepared Carrez II reagent and shake well.

[0100] Place the volumetric flask in an ultrasonic cleaner and sonicate for 1 min to remove air bubbles from the mixture. Add acetonitrile to bring the volume to 20 mL, and then sonicate for another 15 min. Take 10 mL of the extract and centrifuge at 4000 rpm for 5 min. Take 1 mL of the supernatant and filter it through a 0.22 μm microporous membrane. Collect the filtrate as the test solution for royal jelly residue.

[0101] Simultaneously prepare royal jelly samples spiked with 0.5% artificial 10-HDA to determine the position of the target characteristic peak.

[0102] Liquid chromatography detection. The royal jelly residue test solution was detected using the SMH liquid chromatography conditions of Example 1, generating ultraviolet chromatographic data.

[0103] The chromatographic responses of preset feature A, preset feature B, preset feature C and preset feature D were examined at approximately 3.1 min, 6.1 min, 7.1 min and 10.4 min, respectively, and were integrated according to the same baseline rules as in Example 1.

[0104] See attached document Figure 11 In the figure, purple represents negative samples spiked with 0.5% artificial 10-HDA; green represents pulp residue 1; red represents pulp residue 2; and blue represents pulp residue 3.

[0105] Figure 11 The horizontal axis represents retention time in minutes (min), and the vertical axis represents UV response intensity in μV. The target retention time positions in the figure correspond to preset characteristic A, preset characteristic B, preset characteristic C, and preset characteristic D, respectively. The chromatographic responses of the three royal jelly residue samples within the target retention time intervals were processed according to the same integration rules as those for natural royal jelly samples.

[0106] The determination of royal jelly residue samples involves calculating the peak areas of preset characteristic A, preset characteristic B, and preset characteristic C in the royal jelly residue samples, and checking the detection status of preset characteristic D.

[0107] A royal jelly residue sample will be considered positive for artificial 10-HDA if any of the following conditions are met: The peak area of ​​the preset characteristic A is greater than 21825; The peak area of ​​the preset feature B is greater than 7551; The peak area of ​​the preset characteristic C is greater than 2937; Preset feature D was detected.

[0108] If the peak areas of the preset feature A, preset feature B, and preset feature C do not exceed the corresponding positive judgment threshold, and the preset feature D is not detected, it is determined that no artificial 10-HDA feature signal is detected within the detection range of this method.

[0109] Figure 11 This is used to demonstrate that royal jelly residue samples can be separated, identified, integrated, and determined under the same chromatographic conditions as royal jelly samples.

Claims

1. A method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography, characterized in that, Includes the following steps: The target royal jelly sample was processed to obtain a test solution; The test solution was injected into a high performance liquid chromatograph, and separation was performed using a binary mobile phase stepwise elution program. Ultraviolet detection was performed at a wavelength of 210 nm to obtain ultraviolet chromatographic data. Based on the positive control chromatographic data obtained by detecting artificial 10-HDA positive control samples, the reference retention times of preset characteristic A, preset characteristic B, preset characteristic C and preset characteristic D are determined, and the characteristic peak area of ​​the target royal jelly sample is calculated at the corresponding reference retention time in the ultraviolet chromatographic data. Based on the negative control chromatographic data obtained from the detection of the negative control sample of natural royal jelly, a baseline peak area value for the negative control sample of natural royal jelly is established. The baseline peak area values ​​of the preset feature A, the preset feature B and the preset feature C are multiplied by 3 respectively to obtain the corresponding positive judgment threshold. The characteristic peak area of ​​the target royal jelly sample is compared with the corresponding positive determination threshold, and an identification conclusion is made on the target royal jelly sample in combination with the detection status of the preset characteristic substance D.

2. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 1, characterized in that, The reference retention time for the preset feature A is 3.1 min, the reference retention time for the preset feature B is 6.1 min, the reference retention time for the preset feature C is 7.1 min, and the reference retention time for the preset feature D is 10.4 min.

3. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 1, characterized in that, In the step of comparing the characteristic peak area of ​​the target royal jelly sample with the corresponding positive determination threshold, and combining this with the detection status of the preset characteristic substance D to make an identification conclusion on the target royal jelly sample, the step of making an identification conclusion on the target royal jelly sample includes: When the peak area of ​​any of the preset feature A, preset feature B and preset feature C is greater than the corresponding positive determination threshold, or when the preset feature D is detected, a positive identification conclusion of artificial 10-HDA is output. When the peak areas of the preset feature A, the preset feature B, and the preset feature C are all not greater than the corresponding positive determination threshold, and the preset feature D is not detected, a negative identification conclusion is output indicating that no artificial 10-HDA feature signal is detected within the detection range of this method.

4. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 1, characterized in that, The step of processing the target royal jelly sample to obtain the test solution includes: The target royal jelly sample was subjected to acid dispersion, acetonitrile extraction, and protein precipitation to obtain a mixture that formed a precipitate. The test solution is obtained by ultrasonic treatment, solid-liquid separation and filtration of the mixture that forms the precipitate.

5. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 4, characterized in that, In the step of performing acid dispersion, acetonitrile extraction, and protein precipitation treatment on the target royal jelly sample to obtain a mixture forming a precipitate, the acid dispersion, acetonitrile extraction, and protein precipitation treatment on the target royal jelly sample includes: Royal jelly matrix was dispersed using a dilute phosphoric acid aqueous solution; Acetonitrile is added to form a mixed extraction system of acetonitrile and water; Add the pre-set Carrez I reagent and the pre-set Carrez II reagent to cause the protein and colloidal components to form flocculation and precipitation.

6. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 4, characterized in that, The step of obtaining the test solution after ultrasonic treatment, solid-liquid separation, and filtration of the mixture that forms the precipitate includes: The mixture that forms the precipitate is first subjected to ultrasonic degassing, then acetonitrile is added to make up the volume, and then ultrasonic extraction is performed to obtain the extract. The extract is centrifuged, and the supernatant is filtered through a microporous membrane to remove suspended particles, thus obtaining the test solution.

7. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 1, characterized in that, In the step of injecting the test solution into a high-performance liquid chromatograph, separating it using a binary mobile phase stepwise elution program, and performing ultraviolet detection at a wavelength of 210 nm to obtain ultraviolet chromatographic data, the step of injecting the test solution into the high-performance liquid chromatogram and separating it using a binary mobile phase stepwise elution program includes: The test solution is injected into a high-performance liquid chromatograph equipped with a preset SMH column, wherein the SMH column has a specification of 150 mm × 4.6 mm and a packing particle size of 5 μm, the injection volume is 10 μL, and the column temperature is 40 °C. Mobile phase A and mobile phase B are used as binary mobile phases. Mobile phase A is a solution of acetonitrile and water mixed in a volume ratio of 36:64 and containing 0.4% phosphoric acid. Mobile phase B is methanol.

8. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 7, characterized in that, In the step of injecting the test solution into a high-performance liquid chromatograph, separating it using a binary mobile phase stepwise elution program, and performing ultraviolet detection at a wavelength of 210 nm to obtain ultraviolet chromatographic data, the binary mobile phase stepwise elution program includes: The mobile phase A was 100% used from 0 to 12.00 min at a flow rate of 1.2 mL / min. The mobile phase B, consisting of 100% of the mobile phase described above, was used at a flow rate of 1.5 mL / min from 12.01 to 15.00 min. Restore 100% of the mobile phase A within 15.01–27.00 min, maintaining a flow rate of 1.5 mL / min.

9. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 1, characterized in that, In the step of determining the reference retention times of preset characteristic A, preset characteristic B, preset characteristic C, and preset characteristic D based on the positive control chromatographic data obtained from the detection of artificial 10-HDA positive control samples, and calculating the characteristic peak area of ​​the target royal jelly sample at the corresponding reference retention times in the ultraviolet chromatographic data, the calculation of the characteristic peak area of ​​the target royal jelly sample includes: Identify the corresponding target characteristic peaks in the ultraviolet chromatographic data; Determine the peak start point, peak end point, and integration baseline; Integrate the region between the integral baseline and the target feature peak contour to calculate the feature peak area; If no chromatographic peak meeting the identification criteria is formed at the corresponding reference retention time, the corresponding characteristic substance is recorded as not detected, and the corresponding peak area is recorded as 0.

10. The method for distinguishing between natural and artificial royal jelly acids based on liquid chromatography according to claim 1, characterized in that, The step of establishing a baseline peak area value for a natural royal jelly negative control sample based on negative control chromatographic data obtained from detecting a natural royal jelly negative control sample includes: A natural royal jelly sample without added artificial 10-HDA was used as the negative control sample. The preset feature A, preset feature B, and preset feature C each use the upper limit of the peak area in the multiple negative control samples as the corresponding background peak area reference value.

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