Method for simultaneously detecting multiple risky substances in radix scrophulariae through liquid chromatography-mass spectrometry

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting multiple risky substances in radix scrophulariae through liquid chromatography-mass spectrometry. According to the analysis method, the contents of various risky substances in the black ginseng are simultaneously determined by utilizing a triple quadrupole-liquid chromatograph-mass spectrometer, the detection limit (LOD) of the determination method is 0.5-2.5 mu g / kg, the limit of quantitation (LOQ) is 0.9-6.8 mu g / kg, and the limit covers 1 / 10-1 / 5 of the national standard limit; the linear range is 2-500 [mu] g / kg (Rgt; 0.999), and the requirements of trace and constant analysis are met. The within-day precision RSD < lt >; the daily precision is 3.5%, and the daily precision is RSD < lt >; 4.2% (n = 6); the standard recovery rate is 85%-112%, and the standard recovery rate meets the ISO 17025 certification requirement. The method has the advantages of rapidness, accuracy, high sensitivity, high efficiency, strong specificity and simplicity in operation.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for simultaneously detecting multiple risk substances in black ginseng by liquid chromatography-mass spectrometry. Background Art

[0002] As a processed product of ginseng, black ginseng is rich in nutritional value and has multiple active ingredients beneficial to the human body, such as active ingredients like ginsenosides, flavonoids, and trace elements. They have benefits such as enhancing immunity, anti-stress, and anti-tumor effects. Therefore, black ginseng has always held an important research position in the international market and scientific field, and also has good research value in the field of healthcare and health preservation. The formation process of black ginseng involves complex processing techniques (such as steaming, drying, fermentation, etc.), and some risk substances may be introduced or generated during these processes. To ensure the safety of black ginseng, it is necessary to detect and control potential risk substances during the processing. During the processing of black ginseng, Maillard reaction, lipid oxidation, and microbial metabolism may produce harmful substances such as acrylamide (AA), heterocyclic amines (HAs), and advanced glycation end products (AGEs), and the detection needs to take into account complex matrix interference.

[0003] Acrylamide mainly exists in fried and baked foods and is generated by the Maillard reaction of starchy ingredients at high temperatures. Long-term intake may cause neurotoxicity and has attracted attention due to its potential carcinogenicity (classified as a Group 2A carcinogen by IARC) and genotoxicity. Its detection usually relies on gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS / MS). The former requires derivatization to improve sensitivity, while the latter is suitable for complex matrices; enzyme-linked immunosorbent assay (ELISA) can also be used for rapid screening, but it depends on the development of specific antibodies.

[0004] Heterocyclic amines (HAs) are commonly found in meat cooked at high temperatures and are generated by the reaction of creatine, amino acids, and sugars. Some types such as PhIP and MeIQx are classified as Group 2A or 2B carcinogens and are closely related to colorectal cancer, breast cancer, etc. Their strong carcinogenicity and genotoxicity are mainly achieved by forming DNA adducts after metabolic activation. During detection, high-performance liquid chromatography-fluorescence detection (HPLC-FLD) combined with solid-phase extraction (SPE) can enrich the target substances, and liquid chromatography-mass spectrometry (LC-MS / MS) has become the mainstream method for simultaneously detecting multiple HAs due to its high selectivity. In addition, pretreatment techniques such as accelerated solvent extraction (ASE) can effectively handle complex sample matrices.

[0005] Advanced glycation end products (AGEs) are widely present in high-sugar diets or foods cooked at high temperatures. They are generated through the non-enzymatic reaction of sugars with proteins and are closely related to diabetic complications, atherosclerosis, and neurodegenerative diseases. Their harm stems from the oxidative stress and chronic inflammation triggered by activating the RAGE receptor. Detection methods include fluorescence spectroscopy (utilizing the autofluorescence characteristics of AGEs), immunological methods (such as ELISA), and liquid chromatography-mass spectrometry (LC-MS). The latter can accurately determine specific AGEs molecules such as carboxymethyllysine (CML).

[0006] Dicarbonyl compounds (such as methylglyoxal MGO and glyoxal GO), as key precursors for AGEs formation, not only accelerate protein cross-linking and oxidative damage but also have direct cytotoxicity, affecting mitochondrial function and promoting metabolic disorders. Their detection is often achieved through derivatization combined with HPLC or gas chromatography (GC). For example, after derivatizing with o-phenylenediamine (OPD) to generate fluorescent products for analysis; enzymatic detection uses the glyoxalase system for indirect quantification. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this section, the abstract, and the title. However, such simplifications or omissions shall not be used to limit the scope of the present invention.

[0008] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0009] Therefore, the present invention provides a method that is fast, accurate, highly sensitive, efficient, specific, and easy to operate. It not only fills the gap in the systematic detection of risk substances in black ginseng but also provides data support for optimizing processing technology and safety evaluation, having important practical value for promoting the standardization and internationalization of the black ginseng industry.

[0010] The technical solutions adopted by the present invention are as follows: A method for simultaneously detecting multiple risk substances in black ginseng by liquid chromatography-mass spectrometry, comprising the following steps: (1) Sample pretreatment: Using a sodium hydroxide-methanol mixed solution as a solvent, add the test sample into it, mix evenly to obtain a test sample solution. Subsequently, homogenize the test sample solution and then activate and elute it. After concentrating the eluate to dryness with nitrogen, add 1.0 mL of an acetic acid buffer-acetonitrile mixed solution, vortex and mix well, filter through a membrane, and then inject the sample. (2) Set the parameter conditions for the liquid chromatography and mass spectrometry of the triple quadrupole-liquid chromatography-mass spectrometry instrument. (3) Inject the prepared test solution into an injection vial, directly inject the sample through the instrument, collect data, and then perform data analysis. After the analysis, compare the peak area of N-nitrosodiclofenac measured in the test solution with the standard curve, and calculate the content of N-nitrosodiclofenac in the test sample.

[0011] 2. Further, in the step (1): the sodium hydroxide-methanol mixed solution is 9.8 mL of 40 g / L; 3. Further, the parameter conditions of the liquid chromatography are as follows: The chromatographic column is a C18 chromatographic column (Phenomenex SynergiPolar-R) filled with octadecylsilane-bonded silica gel, with a specification of 2×150 mm, 4 μm; mobile phase A is an aqueous solution of formic acid and ammonium acetate, and mobile phase B is methanol; the flow rate is 0.3 mL / min; the column temperature is 35°C; the injection volume is 2 μL; the gradient program is as follows: At 0.01 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; At 2.00 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; At 3.00 min, the volume percentage of mobile phase A is 30%, and the volume percentage of mobile phase B is 70%; At 3.01 min, the volume percentage of mobile phase A is 20%, and the volume percentage of mobile phase B is 80%; At 4.20 min, the volume percentage of mobile phase A is 20%, and the volume percentage of mobile phase B is 80%; At 4.80 min, the volume percentage of mobile phase A is 30%, and the volume percentage of mobile phase B is 70%; At 6.50 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; 4. Further, the parameter conditions of the mass spectrometry are as follows: The MRM positive ion mode is adopted; the ion source is an ESI source, the nebulizer pressure is 40 psi; the capillary voltage is 4000 V; the dryer temperature is 350°C; the heater flow rate is 10 L / min; other mass spectrometry parameters are shown in the following table.

[0012] Table 1 Mass spectrometry MRM parameters Compound Ion pair (m / z) Declustering voltage (eV) Collision energy (eV) GS 251.2 / 233.1251.2 / 173.0 9090 1015 3-DGal 235.2 / 199.1235.2 / 145.1 100100 2025 GO 131.1 / 104.1131.1 / 77.0 140140 2030 MGO 145.1 / 118.1145.1 / 77.0 130130 2030 2,3-BD 159.2 / 131.0159.2 / 118.1 150150 2530 MeIQ 213.0 / 197.0213.0 / 198.1 3737 4736 MeIQx 214.0 / 199.0214.0 / 173.1 6161 3730 PhIP 225.0 / 183.0225.0 / 210.0 108108 4737 CML 205.0 / 84.0205.0 / 130.0 3838 1610 CEL 219.0 / 84.0219.0 / 130.1 2424 2012 4,8-DIMeIQx 228.0 / 160.0228.0 / 121.0 8181 3943 The advantages and positive effects achieved by the present invention are: 1. This research breaks through the limitations of traditional detection and processing technologies, and for the first time realizes the full-chain innovation of "analysis of generation mechanism - highly sensitive detection - precise process control" of risk substances in black ginseng, providing theoretical support and technical paradigms for the safety evaluation and industrial upgrading of medicine and food homologous products, and promoting the transformation of traditional Chinese medicinal materials to standardized and intelligent manufacturing.

[0013] 2. The present invention selects a Phenomenex Polar-R C18 column (2.1×150 mm, 4μm), and its polar embedded groups effectively separate compounds with similar polarities (such as acrylamide and 3-DGal, resolution R > 1.8). Methanol / acetonitrile - 0.1% ammonium formate gradient elution (0~8 min: 5%~95% organic phase) takes into account both hydrophobicity and ionization efficiency; by optimizing chromatographic conditions (such as using a Phenomenex Polar-R C18 chromatographic column) and mass spectrometry parameters (multiple reaction monitoring mode), the separation efficiency of the target substances is significantly improved.

[0014] 3. The detection limit (LOD) of the present invention is 0.5~2.5 μg / kg, and the quantification limit (LOQ) is 0.9~6.8 μg / kg, covering 1 / 10~1 / 5 of the national standard limit; the linear range is 2~500 μg / kg (R² > 0.999), meeting the requirements of trace to macro analysis. The within-day precision RSD < 3.5%, and the between-day precision RSD < 4.2% (n = 6); the standard recovery rate is 85%~112%, meeting the requirements of ISO 17025 certification.

[0015] 4. The present invention develops a method for simultaneously detecting multiple risk substances in black ginseng by liquid chromatography - mass spectrometry. Description of the Drawings

[0016] Figure 1 It is the chromatogram of the mixed standard of the present invention. Detailed Embodiments

[0017] The following further illustrates the present invention in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0018] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For the parts not specifically annotated in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the filing date of this invention application for implementation. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0019] The parameter conditions of the liquid chromatography and mass spectrometry used in the embodiments are as follows: The chromatographic column was a C18 chromatographic column (Phenomenex SynergiPolar-R) packed with octadecylsilyl-bonded silica gel, with a specification of 2×150 mm, 4 μm; mobile phase A was an aqueous solution of ammonium formate and acetic acid, and mobile phase B was methanol; the flow rate was 0.3 mL / min; the column temperature was 35°C; the injection volume was 2 μL; the gradient program was as follows: At 0.01 min, the volume percentage of mobile phase A was 70%, and the volume percentage of mobile phase B was 30%; At 2.00 min, the volume percentage of mobile phase A was 70%, and the volume percentage of mobile phase B was 30%; At 3.00 min, the volume percentage of mobile phase A was 30%, and the volume percentage of mobile phase B was 70%; At 3.01 min, the volume percentage of mobile phase A was 20%, and the volume percentage of mobile phase B was 80%; At 4.20 min, the volume percentage of mobile phase A was 20%, and the volume percentage of mobile phase B was 80%; At 4.80 min, the volume percentage of mobile phase A was 30%, and the volume percentage of mobile phase B was 70%; At 6.50 min, the volume percentage of mobile phase A was 70%, and the volume percentage of mobile phase B was 30%; The parameter conditions of the mass spectrometry were as follows: The MRM positive ion mode was adopted; the ion source was an ESI source, the nebulizer pressure was 40 psi; the capillary voltage was 4000 V; the dryer temperature was 350°C; the heater flow rate was 10 L / min; other mass spectrometry parameters are shown in the following table.

[0020] Table 1 Mass spectrometry MRM parameters Compound Ion pair (m / z) Declustering voltage (eV) Collision energy (eV) GS 251.2 / 233.1251.2 / 173.0 9090 1015 3-DGal 235.2 / 199.1235.2 / 145.1 100100 2025 GO 131.1 / 104.1131.1 / 77.0 140140 2030 MGO 145.1 / 118.1145.1 / 77.0 130130 2030 2,3-BD 159.2 / 131.0159.2 / 118.1 150150 2530 MeIQ 213.0 / 197.0213.0 / 198.1 3737 4736 MeIQx 214.0 / 199.0214.0 / 173.1 6161 3730 PhIP 225.0 / 183.0225.0 / 210.0 108108 4737 CML 205.0 / 84.0205.0 / 130.0 3838 1610 CEL 219.0 / 84.0219.0 / 130.1 2424 2012 4,8-DIMeIQx 228.0 / 160.0228.0 / 121.0 8181 3943 Sample pretreatment: Weigh 2 g of the sample (accurate to 0.01 g) into a 50 mL centrifuge tube, then add 9.8 mL of a 40 g / L sodium hydroxide - methanol mixed solution, and homogenize for 1 min. The homogenizer blade head is washed twice with 5.0 mL of the 40 g / L sodium hydroxide - methanol mixed solution each time, and the washing solutions are combined into the sample extraction centrifuge tube. The sample is centrifuged at 10000 r / min for 10 min. The solid-phase extraction column is pre-activated successively with 2 mL of methanol and 3 mL of a 4 g / L sodium hydroxide solution. Measure 10 mL of the extract and add it to the solid-phase extraction column. After discarding the effluent, wash it successively with 3 mL of the 4 g / L sodium hydroxide - methanol mixed solution and 2 mL of n-hexane. After each washing, the washing solution in the column body needs to be drained completely. Finally, elute with 1.5 mL of an ethanol - dichloromethane solution, and the elution flow rate is less than 1 mL / min. The eluate is concentrated to nearly dry under nitrogen at 35°C in a water bath, then add 1.0 mL of an acetic acid buffer - acetonitrile mixed solution, vortex and mix well, filter through a microporous filter membrane into an injection vial, and wait for LC-MS / MS analysis and determination. Example

[0021] Linear range analysis: The steps are as follows: Prepare a series of standard solutions with different concentrations including low, medium, and high levels, use the detection method to measure each standard solution, and record the corresponding response values. Then, plot the measured response values against the concentrations to obtain the standard curve. Through linear regression analysis, calculate the linear correlation coefficient (R2) of the standard curve. The closer the R2 value is to 1, the better the linear relationship. According to the linear correlation coefficient and linear equation of the standard curve, determine the linear range. The linear range is usually expressed as the interval from the lowest concentration to the highest concentration. Within this interval, the relationship between the response value and the concentration is linear. When evaluating the linear range, it should be noted that the concentration range of the standard solution should cover the expected detection range, and each standard solution at each concentration should be measured at least three times to ensure the reliability and repeatability of the data. In addition, when plotting the standard curve, appropriate software should be used for linear regression analysis to ensure the accuracy of the linear correlation coefficient. Finally, confirm the accuracy of the linear range through additional verification experiments. For example, different batches of standard solutions can be used for verification to ensure the stability and reliability of the linear range.

[0022] Table 2 Analytical characteristic quantities of the LC-MS / MS detection method for compounds Compound Linear equation Linear range (ug / L) <![CDATA[Coefficient of correlation r 2 > GS Y = 598.34X + 894.21 2-500 0.9995 3-DGal Y = 267.29X + 457.57 2-500 0.9995 GO Y = 621.58X + 312.35 2-500 0.9996 MGO Y = 1367.54X + 336.76 2-500 0.9998 2,3-BD Y = 831.84X + 741.63 2-500 0.9996 MeIQ Y = 717.26X + 846.22 2-500 0.9997 MeIQx Y = 459.36X + 945.53 2-500 0.9995 PhIP Y = 1249.68X + 245.17 2-500 0.9998 CML Y = 298.65X + 624.39 2.5-500 0.9993 CEL Y = 434.58X + 159.67 2.5-500 0.9996 4,8-DIMeIQx Y = 527.61X + 185.69 2-500 0.9997 The linear ranges of the compounds in the table are mostly between 2 - 500 μg / L, indicating that the detection method has a good linear relationship within a relatively wide concentration range. The correlation coefficients of the compounds in the table are all above 0.9993, indicating that these compounds have extremely high linear correlations within their respective linear ranges. These high correlation coefficients indicate that the detection method has high accuracy and reliability within the linear ranges of these compounds. Example

[0023] Analysis of detection limit and quantification limit: The steps are as follows: Prepare standard solutions with low concentrations of 0.5 ug / L, 1 ug / L, 2 ug / L, 5 ug / L, and 10 ug / L respectively, and perform LC-MS / MS detection in ascending order. When the signal-to-noise ratio S / N reaches 5, the concentration of the target substance detected by the instrument is the detection limit; when the signal-to-noise ratio S / N reaches 10, the concentration of the target substance detected by the instrument is the quantification limit. Example

[0024] Precision analysis: The steps are as follows: When evaluating the precision of the method, a standard substance of 100 pg / kg is used as the research object, and it is measured in parallel three times and continuously for three days. The intra-day precision is represented by the average of the relative standard deviations of the response values of the target substances measured every day, while the inter-day precision is represented by the total relative standard deviation of the measurement results for three days.

[0025] Table 3 Instrument detection limits, quantification limits and precision of compounds Compound Limit of detection LOD (ug / L) Limit of quantification LOQ (ug / L) Intra-day precision (%) Inter-day precision (%) GS 2.5 3.6 0.98 1.36 3-DGal 0.5 4.1 1.13 1.68 GO 1.0 3.5 1.26 0.96 MGO 0.5 1.2 0.57 2.62 2,3-BD 0.7 1.9 1.87 2.17 MeIQ 0.6 0.9 2.03 2.63 MeIQx 0.5 1.2 0.27 3.24 PhIP 0.6 1.8 2.17 1.63 CML 1.3 2.5 1.56 0.82 CEL 1.3 2.5 3.67 0.75 4,8-DIMeIQx 0.9 1.3 2.59 1.28 The LOD (0.5 - 2.5 μg / L) and LOQ (0.9 - 6.8 μg / L) of all compounds are within the reasonable range of trace detection, indicating that the method is suitable for the trace analysis of target substances in complex matrices. For example, the LOD of 3-DGal is 0.5 μg / L, which is consistent with the detection sensitivity of sugar derivatives in the literature; the LOQ of CML and CEL is 2.5 μg / L, which is in line with the common limits for the detection of advanced glycation end products. The intra-day precision (0.27% - 3.67%) and inter-day precision (0.75% - 3.24%) are both far lower than the conventional threshold of 15%, indicating that the method has good repeatability. For example, the intra-day precision of MeIQ is 0.27%, indicating excellent instrument stability; while the intra-day precision of CEL is relatively high (3.67%), which may be related to its strong polarity and susceptibility to fluctuations in the pH of the mobile phase, and further improvement is required by adding internal standards (such as isotope-labeled substances).

[0026] As can be seen from the above embodiments, the analysis method of the present invention is applicable to detecting the contents of various risk substances during the formation of black ginseng, overcoming problems such as low impurity limit, complex sample matrix being prone to interference, and similar impurity structure to the main component of the test sample being prone to false positives. The method has good precision, high accuracy, low detection limit, saves detection time, and the detection method is efficient and accurate.

[0027] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that: without departing from the spirit and scope of the present invention and the appended claims, various substitutions, changes, and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A method for simultaneously detecting multiple risk substances in black ginseng by liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: (1) Sample pretreatment: Use sodium hydroxide-methanol mixed solution as solvent, add the test sample into it, mix well to obtain the test sample solution, then homogenize the test sample solution and activate and elute it. After the eluent is concentrated to dryness with nitrogen, add 1.0 mL of acetic acid buffer-acetonitrile mixed solution, vortex mix, filter through a filter membrane, and inject; (2) Setting the parameters of liquid chromatography and mass spectrometry of triple quadrupole liquid chromatography-mass spectrometry instrument; (3) injecting the prepared test solution into the injection bottle, directly injecting the sample through the instrument, collecting data, and then performing data analysis; (4) After the analysis is completed, the peak area of ​​N-nitroso-diclofenac measured in the test solution is compared with the standard curve, and the content of N-nitroso-diclofenac in the test sample is calculated.

2. The method for simultaneously detecting multiple risk substances in black ginseng by liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step (1): the sodium hydroxide-methanol mixed solution is 9.8mL 40g / L.

3. The method for simultaneously detecting multiple risk substances in black ginseng by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The parameters of the liquid chromatography are as follows: The chromatographic column was a C18 column (Phenomenex Synergi Polar-R) filled with octadecylsilane bonded silica gel, with specifications of 2×150 mm, 4 μm; mobile phase A was ammonium formic acid acetate aqueous solution, and mobile phase B was methanol; the flow rate was 0.3 mL / min; The column temperature was 35°C; the injection volume was 2 μL; the gradient program was as follows: 0.01min, the volume percentage of mobile phase A was 70%, and the volume percentage of mobile phase B was 30%; 2.00 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; 3.00 min, the volume percentage of mobile phase A is 30%, and the volume percentage of mobile phase B is 70%; 3.01 min, the volume percentage of mobile phase A is 20%, and the volume percentage of mobile phase B is 80%; 4.20 min, the volume percentage of mobile phase A is 20%, and the volume percentage of mobile phase B is 80%; 4.80 min, the volume percentage of mobile phase A is 30%, and the volume percentage of mobile phase B is 70%; 6.50 min, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%.

4. The method for simultaneously detecting multiple risk substances in black ginseng by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass spectrometry parameters are as follows: The MRM positive ion mode was used; the ion source was ESI source, the nebulizer pressure was 40psi; the capillary voltage was 4000V; the dryer temperature was 350°C; the heater flow rate was 10L / min; other mass spectrometry parameters are detailed in the table below.

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