Method for rapidly and simultaneously detecting sulforaphane, sulforaphane and metabolites thereof in urine based on UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) method

Abstract

The invention discloses a method for rapidly and simultaneously detecting sulforaphane, sulforaphane and metabolites thereof in urine based on a UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) method, and belongs to the technical field of plant active substance detection. Based on solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry, the invention provides a method for simultaneously detecting sulforaphane, sulforaphane, cysteine-sulforaphane and N-acetylcysteine sulforaphane. A methodological test result shows that the detection method established by the invention is simple, rapid and efficient, shows excellent extraction efficiency and detection repeatability in a complex urine matrix, and successfully overcomes the technical difficulties that glucosinolate metabolites are easy to oxidize and large in polarity span. According to the method, high-credibility data support is provided for follow-up development of isothiocyanate compound bioavailability evaluation, metabolite kidney clearance dynamic modeling and dietary intervention clinical research, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant active substance detection, and particularly to a method for rapidly and simultaneously detecting sulforaphene, sulforaphane and their metabolites in urine based on UPLC-MS method. Background Art

[0002] Globally, people are increasingly aware of the impact of dietary factors on health. Increasing vegetable intake can effectively prevent diseases such as diabetes, cardiovascular diseases and cancers. Cruciferous vegetables are the largest and most widely consumed plants in the world. The beneficial effects of cruciferous vegetables on the human body include the ability to prevent oxidative stress, induce the production of detoxifying enzymes, stimulate the immune system, reduce the risk of cancer, inhibit cell malignancy and carcinogenic mutations, and reduce the proliferation of cancer cells, etc. This is mainly attributed to an important class of secondary metabolites in these plants, isothiocyanates. Cruciferous plants are mainly divided into two genera, Brassica (mainly including broccoli, cabbage and cauliflower, etc.) and Raphanus (mainly including radish and white radish). Sulforaphane (SFN), with the chemical name of 1-isothiocyanato-4-methylsulfinylbutane, molecular formula of C6H 11 S2NO and molecular weight of 177.29, is an isothiocyanate mainly present in the genus Brassica, and is most abundant in broccoli. It is one of the most effective natural inducers of cytoprotective enzymes (phase II enzymes) discovered so far and can inhibit the occurrence of various cancers. In addition, sulforaphane also plays an active role in preventing neurological diseases, antioxidation, antibacterial and inhibiting obesity. Sulforaphene (SFE), with the chemical name of 4-isothiocyanato-1-methylsulfinyl-1-butene, molecular formula of C6H9S2NO and molecular weight of 175.27, is an isothiocyanate mainly present in the genus Raphanus and also has the effects of anti-cancer, anti-inflammatory, antibacterial and preventing neurological diseases.

[0003] The metabolic pathway of SFN after ingestion has been elucidated, mainly through the mercapturic acid metabolic pathway for transformation. The chronological order of the generated products is glutathione-sulforaphane (SFN-GSH), cysteinylglycine-sulforaphane (SFN-CG), cysteine-sulforaphane (SFN-Cys) and N-acetylcysteine-sulforaphane (SFN-NAC). Currently, there are no coupled SFE metabolites on the market, and it is also unclear whether SFE also metabolizes through the mercapturic acid pathway. Some studies speculate that SFE may be metabolized through the mercapturic acid pathway by first being converted to SFN. Verifying whether the metabolic pathway of SFE is to be first converted to SFN and then further metabolized is a necessary process for understanding the metabolic pathway of SFE and an important step in clarifying the result basis of the biological function of SFE in the body.

[0004] In terms of chemical structure, SFE and SFN only differ by a double bond. Due to the structural similarity, it is difficult to separate the two by chromatography. Currently, the main method for determining SFN and its metabolites is high-performance liquid chromatography-mass spectrometry (HPLC-MS). There is little research on SFE, and there is no method for simultaneously detecting SFE, SFN, and their metabolites. Although someone has successfully separated SFE and SFN, it was only carried out in different plant tissues, and there is no method for separating SFE and SFN in human urine. The existing LC-MS methods for determining SFN and its metabolites have disadvantages such as cumbersome operation, poor sensitivity, narrow linear range, and long detection time in actual research. Since SFN and its metabolites are unstable in nature, the cumbersome operation and long detection time will cause partial decomposition of SFN and its metabolites, reducing the accuracy and reliability of the results.

[0005] Therefore, it is necessary to propose a high-performance liquid chromatography-mass spectrometry (HPLC-MS) technique that is simple, time-consuming, and highly accurate for simultaneously detecting SFE, SFN, and their metabolites in human urine. It can not only be applied to the pharmacokinetic analysis of SFN but also provide a new direction and new method for the metabolic research of SFE. Summary of the Invention

[0006] The object of the present invention is to provide a method for rapidly and simultaneously detecting sulforaphane, sulforaphane, and their metabolites in urine based on UPLC-MS to solve the problems existing in the above-mentioned prior art. The present invention provides a simple, rapid, and efficient method for detecting sulforaphane, sulforaphane, and their metabolites based on optimized ultra-high performance liquid chromatography-tandem mass spectrometry, showing outstanding methodological advantages in complex urine matrices and providing reliable technical support for the metabolism of isothiocyanate compounds and kidney clinical research.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for simultaneously detecting sulforaphane, sulforaphane, and sulforaphane metabolites in urine by LC-MS, comprising the following steps:

[0009] Collect urine samples from subjects;

[0010] Centrifuge the urine samples, collect the supernatant, perform solid-phase extraction on the supernatant, and collect the extracted samples;

[0011] Prepare standard solutions of sulforaphane, sulforaphane, and sulforaphane metabolites;

[0012] Perform ultra-high performance liquid chromatography-tandem mass spectrometry analysis on the extracted samples and the standard solutions respectively;

[0013] Among them, the sulforaphane metabolites are cysteine-sulforaphane and N-acetylcysteine sulforaphane.

[0014] Preferably, the solid-phase extraction includes the following steps:

[0015] Add 2 mL of methanol and 2 mL of ultrapure water to the extraction column at a flow rate of 2 mL / min, and add them 2 times in total;

[0016] Mix the supernatant with formic acid aqueous solution, load the sample, and the flow rate is 5 mL / min;

[0017] Wash the extraction column with formic acid aqueous solution and drain it;

[0018] Add methanol to the extraction column 3 times for elution at a flow rate of 2 mL / min. The total amount of methanol is 10 mL. Collect the eluate, concentrate it, and redissolve it with methanol to obtain the extracted sample.

[0019] Preferably, the extraction column is a Stara-X 33μm polymeric strong cation solid-phase extraction column;

[0020] The volume fraction of formic acid in the formic acid aqueous solution is 2%.

[0021] Preferably, the flow rate of the ultra-high performance liquid chromatography is 0.35 mL / min; the column temperature is 30 °C; the wavelength of the ultraviolet detector is 254 nm.

[0022] Preferably, the mobile phase A of the ultra-high performance liquid chromatography is formic acid ultrapure aqueous solution, and the volume fraction of formic acid is 0.1%;

[0023] The mobile phase B is formic acid acetonitrile solution, and the volume fraction of formic acid is 0.1%.

[0024] Preferably, the elution program of the ultra-high performance liquid chromatography is: 95% A at 0 min; gradually reduced to 65% A within 0 - 8 min; 5% A at 8 - 10 min.

[0025] Preferably, the ion source of the mass spectrometry is electrospray ionization; the scan mode is positive ion multiple reaction monitoring; the drying gas temperature is 350 °C; the drying gas flow rate is 5 L / min; the sheath gas temperature is 250 °C; the sheath gas flow rate is 11 L / min; the nebulizer pressure is 20 psi; the capillary voltage is 3500 V; the fragmentation voltage is 80 V; the collision energy is 5 eV.

[0026] Preferably, when using the mass spectrometry for quantitative detection of raphanin, the parent ion mass-to-charge ratio is 176, and the daughter ion mass-to-charge ratio is 112; when using the mass spectrometry for qualitative detection of raphanin, the parent ion mass-to-charge ratio is 176, and the daughter ion mass-to-charge ratio is 107.9;

[0027] When using the mass spectrometry for quantitative detection of sulforaphane, the parent ion mass-to-charge ratio is 178 and the daughter ion mass-to-charge ratio is 114; when using the mass spectrometry for qualitative detection of sulforaphane, the parent ion mass-to-charge ratio is 178 and the daughter ion mass-to-charge ratio is 72;

[0028] When using the mass spectrometry for quantitative detection of cysteine-sulforaphane, the parent ion mass-to-charge ratio is 299 and the daughter ion mass-to-charge ratio is 136; when using the mass spectrometry for qualitative detection of cysteine-sulforaphane, the parent ion mass-to-charge ratio is 299 and the daughter ion mass-to-charge ratio is 178;

[0029] When using the mass spectrometry for quantitative detection of N-acetylcysteine-sulforaphane, the parent ion mass-to-charge ratio is 341 and the daughter ion mass-to-charge ratio is 178; when using the mass spectrometry for qualitative detection of N-acetylcysteine-sulforaphane, the parent ion mass-to-charge ratio is 341 and the daughter ion mass-to-charge ratio is 136.

[0030] Preferably, the urine sample includes a blank control sample and an experimental sample;

[0031] Among them, the collection of the blank control sample includes: collecting the midstream urine of the subject in the morning on an empty stomach before the intervention experiment;

[0032] The collection of the experimental sample includes: the urine of the subject collected at different times after the intervention experiment, and boric acid is added.

[0033] The present invention also provides an application of the above method in monitoring the levels of raphanin, sulforaphane, cysteine-sulforaphane and N-acetylcysteine-sulforaphane in human urine for non-diagnostic or therapeutic purposes.

[0034] The present invention discloses the following technical effects:

[0035] The present invention provides a method for simple, rapid and efficient simultaneous detection of raphanin, sulforaphane and their metabolites based on solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry. The results of methodological tests show that the detection method established by the present invention exhibits excellent extraction efficiency (Recovery≥80%) and detection repeatability (RSD≤15%) in complex urine matrices, and successfully overcomes the technical difficulties of easy oxidation and large polarity span of glucosinolate metabolites. The present invention provides high-confidence data support for subsequent evaluation of the bioavailability of isothiocyanate compounds, kinetic modeling of renal clearance of metabolites, and clinical research on dietary intervention, and has broad application prospects.

[0036] Compared with the prior art, the present invention also has the following advantages:

[0037] (1) Detection substances

[0038] The analysis of glucosinolate (GSL) metabolites in the prior art mostly focuses on the detection of single-stage metabolites in specific biological matrices. For example, by systematically integrating and analyzing the natural content differences of sulforaphane (SFN) and sulforaphene (SFE) in fresh plant tissues (including seeds, leaves, and mature fruits), or by the dynamic changes in the urinary and blood concentrations of single isothiocyanates (ITCs) and their metabolites (such as SFN and SFN-GSH, SFN-Cys, and SFN-NAC) generated during the biotransformation process. However, so far, an analytical method capable of simultaneously detecting different glucosinolate metabolites in urine tissues (such as covering SFE, SFN, and their metabolites) has not been established. The present invention has successfully established an analytical method for simultaneously detecting SFE, SFN, and their metabolites (SFN-Cys and SFN-NAC).

[0039] (2) Extraction method

[0040] Based on the chemical instability characteristics of SFE, SFN, and their metabolites SFN-Cys and SFN-NAC in biological samples, this study established a standardized process for urine sample collection: adding boric acid crystals (H3BO3, purity ≥ 99.5%) in advance to the urine collection tube to form a weakly acidic environment through immediate acidification (pH 2.5 - 3.0), effectively inhibiting the hydrolysis reaction and oxidative degradation process of ITC derivatives in the collected urine. Its stabilization mechanism involves the protective effect of protonation on the active groups of ITCs and their metabolites. This pretreatment scheme prolongs the half-life of the target metabolites and significantly improves the accuracy of ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) quantitative analysis.

[0041] Aiming at the interference problem caused by the complex matrix of urine to trace analysis, the present invention innovatively introduces solid-phase extraction (SPE) pretreatment technology. Using a Strata-X adsorption column (30mg / 3mL) to specifically enrich SFE, SFN, and their main metabolites (SFN-Cys and SFN-NAC) through hydrophobic interaction. Compared with the interference of co-dissolved impurities caused by the simple centrifugation and precipitation method in the prior art, or the co-precipitation loss of the target substances caused by the methanol protein precipitation method, the method provided by the present invention reduces the matrix effect to less than 6%, and the absolute recovery rate of the target substances reaches 94.58% - 104.68%. Moreover, for the first time, the synchronous and efficient extraction of the SFN metabolite group in human urine is realized. The establishment of the technical system of the present invention provides a reliable methodological basis for the pharmacokinetic study of SFE and SFN compounds in vivo and the detection of exposure biomarkers.

[0042] (3) Chromatographic method

[0043] The present invention adopts an optimized ultra-high performance liquid chromatography method to achieve rapid and accurate separation of SFE, SFN and their metabolites (SFN-Cys and SFN-NAC). Based on the problems of too long chromatographic retention time and insufficient separation efficiency of SFE, SFN and their metabolites (SFN-Cys and SFN-NAC) in the existing chromatographic analysis methods, and combined with the characteristics of the unstable chemical structures of several metabolites and easy decomposition caused by long-term elution, the present invention makes a systematic improvement to the gradient elution program: by simplifying the gradient elution parameter combination and optimizing the flow rate to 0.35 mL / min (a 75% increase compared with the conventional 0.2 mL / min), the single detection cycle is shortened to 10 min. While significantly improving the analysis throughput, this strategy ensures that the characteristic peaks of SFE, SFN and their metabolites (SFN-Cys and SFN-NAC) are all concentrated in the stable peak elution interval of 4 - 6 min by precisely controlling the chromatographic retention behavior of the target substances, effectively avoiding the risk of compound degradation caused by retention time drift or elution delay. Experimental data show that the optimized method of the present invention still maintains the peak shape integrity and quantitative accuracy of each target substance while doubling the analysis speed, providing a reliable methodological solution for the rapid detection of unstable glucosinolate metabolites in biological samples.

[0044] (4) Mass spectrometry method

[0045] The present invention adopts an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) method for metabolite analysis, and its technical necessity is reflected in the following aspects: First, due to the biological limitations of the metabolism of GSL by intestinal flora, the concentration of endogenous ITCs and their metabolites in human urine is significantly reduced, and the traditional high performance liquid chromatography (HPLC) method is difficult to achieve accurate quantification due to insufficient detection sensitivity; Second, the retention times of SFN and SFE highly overlap in the conventional chromatographic system, while the mass spectrometry technology can achieve baseline separation and specific detection of the two through the differences in the accurate mass number and the mass-to-charge ratio of characteristic fragment ions; Third, the existing technology generally uses a single ion pair for metabolite qualitative and quantitative analysis, and this methodological design is vulnerable to matrix effect interference in complex biological matrices. The present invention first establishes a multi-ion pair monitoring strategy through systematic preliminary experiments, and sets qualitative ion pairs (for structural confirmation) and quantitative ion pairs (for concentration determination) for each target compound respectively, significantly improving the detection specificity and result credibility. In terms of instrument parameter optimization, a composite optimization mode combining automatic tuning and manual calibration is adopted. By systematically screening the signal response intensities under different fragmentation voltages and collision energy conditions, the optimal ionization parameter combination for each target substance is finally determined. The optimization strategy of the present invention effectively improves the sensitivity and quantitative accuracy of the detection system for ITCs (such as SFN and SFE) and their metabolites (SFN-Cys and SFN-NAC). Description of the drawings

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0047] Figure 1 Quantitative mass spectrometry diagrams of each standard; among them, A is the quantitative mass spectrometry diagram of SFE; B is the quantitative mass spectrometry diagram of SFN; C is the quantitative mass spectrometry diagram of SFN-Cys; D is the quantitative mass spectrometry diagram of SFN-NAC;

[0048] Figure 2 Quantitative mass spectrometry diagrams of each metabolite in the sample; among them, A is the quantitative mass spectrometry diagram of SFE in the sample; B is the quantitative mass spectrometry diagram of SFN in the sample; C is the quantitative mass spectrometry diagram of SFN-Cys in the sample; D is the quantitative mass spectrometry diagram of SFN-NAC in the sample;

[0049] Figure 3 Peak time comparison diagram of different standards;

[0050] Figure 4 Peak time comparison diagram of different metabolites in the sample;

[0051] Figure 5 Peak time comparison diagram of each metabolite standard and the peak time in the sample; among them, A is the peak time comparison diagram of SFN standard and the sample peak time; B is the peak time comparison diagram of SFN-Cys standard and the SFN-Cys peak time in the sample; C is the peak time comparison diagram of SFN-Cys standard and the sample peak time; D is the peak time comparison diagram of SFN-NAC standard and the sample peak time;

[0052] Figure 6 Standard curve diagrams of SFE, SFN, SFN-Cys and SFN-NAC. Detailed implementation manners

[0053] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0055] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0056] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0057] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0058] The key organic matter information related to this invention is as follows:

[0059] Sulforaphane (SFE), with the molecular formula C6H9NOS2, CAS number: 592-95-0.

[0060] Sulforaphane (SFN), with the molecular formula C6H 11 NOS2, CAS number: 4478-93-7.

[0061] Cysteine-sulforaphane (SFN-Cys), with the molecular formula C9H 18 N2O3S3, CAS number: 364083-21-6.

[0062] N-acetylcysteine sulforaphane (SFN-NAC), with the molecular formula C 11 H 20 N2O4S3, CAS number: 334829-66-2.

[0063] Example 1

[0064] 1. Materials and Reagents

[0065] 1.1 Collection of Experimental Materials

[0066] Blank urine: Subjects need to complete a 14-day washout period before sample collection. During this period, the intake of cruciferous vegetables (such as broccoli, cabbage, etc.) and their processed products is strictly prohibited to eliminate potential interference from dietary residues on the urine background value. Before the start of the intervention experiment (i.e., within 24 hours before the first intake of broccoli seed powder and radish seed powder), collect the midstream urine of the subjects in the morning on an empty stomach as the blank control sample.

[0067] Urine samples: Collect the urine of the subjects as experimental samples at different time periods after intervention (0-2, 2-4, 4-6, 6-8, 8-12, and 12-24 hours after consuming broccoli seed powder or radish seed powder).

[0068] All urine collection processes follow the ISO 15189 standard and are immediately sub-packed using sterile polypropylene containers (containing 0.1% sodium azide preservative); for the experimental group, boric acid granules are additionally added (to immediately acidify glucosinolate metabolites to prevent their decomposition) and stored in an ultra-low temperature freezer at -80°C until pre-analysis processing.

[0069] 1.2 Experimental reagents

[0070] Table 1 Experimental reagents used in the present invention

[0071]

[0072] 2. Instruments and equipment

[0073] Table 2 Instruments used in the present invention

[0074]

[0075] 3. Experimental methods

[0076] 3.1 Standard product gradient dilution

[0077] The standard product specifications of SFE, SFN-Cys, and SFN-NAC are all 5 mg. Add 1 mL of methanol to each to dissolve them completely; then transfer to a 10 mL volumetric flask and make up to the scale with methanol to prepare a stock solution of 500 ppm; the SFN standard product specification is 20 mg. Add 5 mL of methanol to dissolve it completely; then transfer to a 10 mL volumetric flask and make up to the scale with methanol to prepare a stock solution of 2000 ppm. Further dilute the stock solutions to obtain SFE, SFN, SFN-Cys, and SFN-NAC standard solutions with concentrations of 0.1 ppm, 0.2 ppm, 0.5 ppm, 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 5 ppm, and 10 ppm respectively. Filter the diluted standard solutions of each gradient through a 0.22 μm PTFE filter membrane and wait for further on-machine detection and standby.

[0078] 3.2 Sample preparation

[0079] After thawing the urine samples collected before and after the intervention of the subjects, centrifuge them at 4°C (13,000 rpm, 15 min), take 2 mL of the supernatant for standby. Use a solid-phase extraction SPE Strata-X column (33 μm polymeric strong cation) to extract the sample: First, add a total of 4 mL of methanol (2 mL each time) and 4 mL of ultrapure water (2 mL each time) to the column successively to activate the column, and keep the flow rate at 2 mL / min. Then dilute the above centrifuged supernatant to 10 mL of water / formic acid (98:2, v / v) solution and load it onto the extraction column, keeping the flow rate at 5 mL / min. After loading, continue to wash the column with water / formic acid (98:2, v / v) solution, and dry the system by suction for one hour to ensure that the column is completely dry. The target analytes are eluted with methanol. After drying, add a total of 10 mL of methanol to the column in 3 - 4 times, and keep the flow rate at 2 mL / min for sufficient elution. The obtained eluate is concentrated by a rotary evaporator (temperature set at 35°C), redissolve the concentrated product after rotary evaporation with 2 mL of methanol, and filter it through a 0.22 μm PTFE membrane for further on-machine detection standby.

[0080] 3.3 Chromatographic conditions

[0081] Agilent Zorbax reverse C 18 Chromatographic column (4.6 μm, 5×150 mm) Mobile phase: Phase A: Ultrapure water solution of 0.1% formic acid; Phase B: Acetonitrile solution of 0.1% formic acid. Injection volume: 2 μL; Flow rate: 0.35 mL / min; Column temperature: 30°C; Wavelength of ultraviolet detector: 254 nm. Gradient elution conditions: 0 - 8 min, Phase A: 95% - 65%, Phase B: 5% - 35%; 8 - 10 min (post-run), Phase A: 5%, Phase B: 95% (Table 3).

[0082] Table 3 Gradient elution conditions of high performance liquid chromatography

[0083]

[0084] 3.4 Mass spectrometry conditions:

[0085] Ion source: ESI source; Mode: MRM mode. Drying gas temperature: 350°C; Drying gas flow rate: 5 L / min; Sheath gas temperature: 250°C; Sheath gas flow rate: 11 L / min; Nebulizer pressure: 20 psi; Capillary voltage: 3500 V. The detection information of substances such as SFN is shown in Table 4:

[0086] Table 4 Mass spectrometry detection conditions of SFE, SFN and their metabolites

[0087]

[0088] Calibration of 3.5 Standard Solution

[0089] Inject the 10 final concentration solutions diluted from each standard in "3.1 Standard Product Gradient Dilution" into the instrument for detection in sequence according to the experimental chromatographic and mass spectrometric conditions. Use the concentration as the abscissa and the peak area as the ordinate, and select at least 6 points to plot the standard curve of each substance. Perform linear regression on the data of 6 or more concentrations obtained from the detection, fit to obtain the curve equation y = ax + b, and obtain the linear equation coefficients a and b.

[0090] Detection of Samples

[0091] Use a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument to perform on-machine detection of the samples prepared in "3.2 Sample Preparation", obtain the peak areas y of SFE, SFN, SFN-Cys, and SFN-NAC in the samples, substitute them into the standard curve y = ax + b of each substance, and calculate the concentrations x of SFE, SFN, SFN-Cys, and SFN-NAC in the samples.

[0092] 3.7 Matrix Effect

[0093] Matrix Effect is a key methodological interference factor in bioanalysis, specifically referring to the competitive inhibition or enhancement of the ionization process of target compounds by endogenous substances (such as phospholipids, urea, metabolites) or co-eluting substances in the sample, which then leads to the deviation of the mass spectrometry response signal from the true value. Especially in complex biological matrices such as urine and plasma, such effects may cause a quantitative deviation of up to ±30%. To systematically evaluate the anti-interference ability of the method established in the present invention, the following verification scheme is designed:

[0094] (1) Preparation of Blank Matrix

[0095] Collect the baseline urine of healthy subjects before ingesting radish seeds and broccoli seeds, and process it through the solid-phase extraction process in "3.2 Sample Preparation" to obtain a blank urine methanol complex solution without the target substance (matrix background response value ≤ LOQ).

[0096] (2) Construction of Standard Solutions

[0097] a) Pure Solvent Standard Curve: Dissolve the SFE, SFN, SFN-Cys, and SFN-NAC standard products in methanol to prepare gradient solutions of 5 ppm, 20 ppm, and 50 ppm;

[0098] b) Matrix-Matched Standard Curve: Mix the above solutions with the blank matrix complex solution according to a 10% addition ratio to finally obtain drug-containing simulated samples of 0.5 ppm, 2 ppm, and 5 ppm, simulating the coexistence state of the analyte and matrix in the actual sample.

[0099] (3) Matrix effect quantification

[0100] Analysis was carried out according to the parameters in "3.3 Chromatographic conditions" and "3.4 Mass spectrometric conditions". Each concentration level was determined in parallel 6 times, and the data were expressed as mean ± SD.

[0101] 3.8 Accuracy

[0102] Accuracy is one of the core parameters for the verification of mass spectrometry analysis methods, used to characterize the deviation degree between the measured value and the true concentration value, and reflects the comprehensive level of the systematic error of the method. Its calculation method is as follows:

[0103]

[0104] (1) Preparation of blank matrix: The same as "3.7 Matrix effect"

[0105] (2) Quantitative verification of accuracy: Analyze 6 batches repeatedly (intraday) and continuously for 3 days (interday), and analyze according to the chromatographic conditions in Section 3.3 and the mass spectrometric parameters in Section 3.4. The data were expressed as mean ± SD.

[0106] 3.9 Recovery rate and precision

[0107] Extraction recovery rate is the core index for evaluating the efficacy of sample pretreatment methods, which characterizes the ability of the target analyte to be selectively separated and quantitatively recovered from complex biological matrices (such as urine and blood). The present invention systematically evaluates the method performance through a standard addition recovery experiment, and the specific operation is as follows:

[0108] (1) Matrix selection and standard addition design

[0109] Fasting urine of healthy volunteers (blank matrix, verified by LC-MS / MS to have no residue of the target substance) was used as the research carrier, and standard solutions with concentrations of 0.5 ppm (low), 2 ppm (medium), and 5 ppm (high) prepared by "3.1 Standard product gradient dilution" were added respectively to simulate the occurrence state of the target substance in the actual sample.

[0110] (2) Experimental grouping and detection

[0111] a) Untreated group: Inject the spiked urine directly for analysis to determine the theoretical response value (reflecting the original signal intensity of the target substance);

[0112] b) Treated group: After completing the pretreatment according to the solid-phase extraction process in "3.2 Sample preparation", inject the sample for analysis to determine the actual response value.

[0113] (3) Calculate the extraction recovery rate at each concentration level through the following formula:

[0114]

[0115] (4) Precision verification

[0116] a) Intra-day precision: Six parallel samples were prepared from the same batch and analyzed within a single day. The relative standard deviation (RSD) was calculated.

[0117] b) Inter-day precision: The above experiment was repeated for three consecutive days, and the total RSD was calculated to evaluate the time stability of the method.

[0118] All data were presented in the form of mean ± standard deviation.

[0119] 3.10 Data analysis

[0120] All data were analyzed using Excel software and presented as mean and SEM unless otherwise specified. Origin software was used to generate charts.

[0121] 4. Experimental results

[0122] 4.1 Retention time

[0123] The retention time of SFE measured by mass spectrometry was 5.46 min, that of SFN was 5.50 min, that of SFN-Cys was 4.76 min, and that of SFN-NAC was 5.22 min. The specific chromatograms are shown in Figures 1 to 5 as follows.

[0124] Based on the optimized ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis method, the separation characteristics of glucosinolate metabolites were revealed through systematic chromatographic behavior studies ( Figures 1-5 ). The key findings are as follows:

[0125] (1) Pretreatment efficiency verification

[0126] By using a solid-phase extraction column (Stara-X 33μm 3mL) combined with a two-step elution strategy (2% methanol → 100% methanol), the efficient enrichment of SFE, SFN, and their metabolites in urine was successfully achieved. As shown in Figure 1 (standards) and Figure 2 (treated samples), the characteristic chromatographic peaks of each target showed a symmetric Gaussian distribution (symmetry factor 0.95 - 1.05), and no obvious tailing (tailing factor < 1.2) was observed. Notably, in the detection channel of the characteristic quantitative ion pair m / z 341 → 178 of SFN-NAC, the chromatographic peak purity index (≥0.99) was highly consistent with that of the standard (ΔRT < 0.03 min), confirming that the pretreatment process did not cause chemical degradation or artificial product formation of the target.

[0127] (2) Resolution of chromatographic retention behavior

[0128] As Figure 3 and Figure 4 shown, in the gradient elution system (0.1% formic acid in water - 0.1% formic acid in acetonitrile) of an Agilent Zorbax C 18 chromatographic column (4.6×150 mm, 5 μm), the retention times of various substances showed significant regularity: SFN-Cys (4.76 min) < SFN-NAC (5.22 min) < SFE (5.46 min) ≈ SFN (5.50 min). This retention difference is closely related to the polarity of the compounds - SFN-Cys has the strongest polarity due to the presence of a free mercapto group (-SH), while SFN-NAC has its polarity reduced through N-acetylation modification; SFE and SFN have similar isothiocyanate parent nuclear structures, resulting in similar retention characteristics (ΔRT = 0.04 min).

[0129] (3) Method specificity verification

[0130] By comparing the chromatographic behaviors of the standards and human urine samples ( Figure 5 ), it was found that the retention time deviations of the target substances were all less than ±0.05 min (RSD < 0.9%). After optimizing the gradient elution program (0 min 95% B; 0 - 8 min 5 - 35% B; 8 - 10 min 95% B), the baseline separation of each analyte from the matrix interference peaks was achieved (resolution Rs > 1.5).

[0131] 4.2 Methodological verification

[0132] 4.2.1 Standard curve linearity

[0133] According to the conditions in "3.3 Chromatographic conditions" and "3.4 Mass spectrometry conditions", standards of SFE, SFN, SFN-Cys, and SFN-NAC with concentrations in the range of 0.1 - 10 mg / L were successively determined from low to high concentration. A standard curve was plotted with concentration as the abscissa and peak area as the ordinate.

[0134] The results showed that in the liquid chromatography - mass spectrometry detection method, each substance had good linearity in the range of 0.1 mg / L - 10 mg / L, with R 2 > 0.995. The specific standard curves are shown in Table 5 and Figure 6 as follows.

[0135] Table 5 Standard curve equations of SFE, SFN, SFN-Cys, and SFN-NAC

[0136]

[0137] 4.2.2 Detection limit and quantification limit

[0138] Through systematic methodological verification, the present invention has clarified the detection sensitivity parameters of four glucosinolate metabolites. The experimental results show that there are significant differences in the limits of detection (LOD, signal-to-noise ratio S / N≥3) and limits of quantification (LOQ, S / N≥10) for each analyte: the LOD of SFE is 0.010 mg / L and the LOQ is 0.13 mg / L; the LOD of SFN is 0.017 mg / L and the LOQ is 0.12 mg / L; the detection sensitivity of its cysteine conjugate SFN-Cys is relatively high (LOD = 0.004 mg / L, LOQ = 0.06 mg / L), while the detection performance of the terminal metabolite SFN-NAC is the most prominent, with an LOD as low as 0.0003 mg / L and an LOQ of 0.028 mg / L. It is worth noting that the detection sensitivity of SFN-NAC is nearly 50 times higher than that of the prototype SFN, which is mainly due to the better ionization efficiency of its stable N-acetylcysteine modification structure in the electrospray ionization source (ESI). This result proves that by optimizing the chromatographic separation conditions and mass spectrometry parameters, combined with an improved solid-phase extraction pretreatment method, the highly sensitive detection of trace metabolites in complex biological matrices has been successfully achieved, providing a reliable technical guarantee for the accurate quantification of ultra-low concentration samples in subsequent pharmacokinetic studies.

[0139] 4.2.3 Matrix effect

[0140] Table 6 Matrix effect results of SFE, SFN, SFN-Cys and SFN-NAC (n = 6)

[0141]

[0142] To exclude the influence of biological matrix on the detection results, the system of the present invention systematically investigated the possible interference effects of urine on the mass spectrometry detection results. As shown in Table 6, at three concentration levels of low (0.5 ppm), medium (2 ppm), and high (5 ppm), the matrix effect values of each analyte (including the terminal metabolite SFN-NAC) were all stable between 85-115% (n = 6). Specifically, the matrix effect range of SFE was 97.28-103.25%, that of SFN was 95.87-102.43%, that of SFN-Cys was 90.70-101.33%, while the terminal metabolite SFN-NAC showed a slight matrix enhancement trend of 98.22-110.44%. All results met the acceptance criteria (80-120%) of the "Guidelines for the Validation of Quantitative Analysis Methods for Biological Samples" in the 2020 Edition of the Chinese Pharmacopoeia, indicating that through solid-phase extraction and methanol reconstitution strategies during the urine sample pretreatment process, this method effectively eliminated the ion suppression or enhancement phenomena caused by interfering substances such as urea. The research results confirmed that the established analytical method had good matrix tolerance and was applicable to the accurate quantification of isothiocyanates and their metabolites in complex biological matrices.

[0143] 4.2.4 Accuracy

[0144] To verify the accuracy of the detection method, standard drug solutions at three concentrations of low, medium, and high were taken respectively for accuracy verification, and the results are shown in Table 7.

[0145] Table 7 Matrix effect results of SFE, SFN, SFN-Cys, and SFN-NAC (n = 6, 3d)

[0146]

[0147]

[0148] Through comprehensive verification across multiple concentrations, multiple batches, and different time dimensions, the influences of instrument deviation and pretreatment error on the experimental results were excluded. Among them, the accuracy of SFE was 100.52%-101.54%, close to the theoretical value; the accuracy of SFN was 98.62%-100.98%, with controllable fluctuations; the accuracy of SFN-Cys was 94.58%-100.18%, slightly approaching the lower limit at the low concentration end but meeting the standards; the accuracy of SFN-NAC was 99.57%-104.68%, showing a slight matrix enhancement trend. All results met the requirements of the EMA (85%-115%), proving that this method was applicable to the accurate quantification of the target substances in actual biological samples and provided a reliable data basis for subsequent pharmacokinetic studies.

[0149] 4.2.5 Extraction recovery and precision

[0150] To investigate the effect of the pretreatment process on the metabolites to be detected, the extraction recovery was determined, and the intra-day precision and inter-day precision of the extraction recovery were calculated. The results are shown in Table 8.

[0151] Table 8 Recovery and precision results of SFE, SFN, SFN-Cys and SFN-NAC (n = 6, 3d)

[0152]

[0153] The results showed that the extraction recoveries of SFE at low, medium and high concentrations were 89.25% - 90.53%, the intra-day precision was 1.05% - 5.90%, and the inter-day precision was 1.03% - 4.74%; the extraction recoveries of SFN at low, medium and high concentrations were 92.27% - 94.52%, the intra-day precision was 0.66% - 10.99%, and the inter-day precision was 1.32% - 7.28%; the extraction recoveries of SFN-Cys at low, medium and high concentrations were 80.98% - 84.14%, the intra-day precision was 2.04% - 6.10%, and the inter-day precision was 3.03% - 5.20%; the extraction recoveries of SFN-NAC at low, medium and high concentrations were 84.96% - 88.27%, the intra-day precision was 1.11% - 5.03%, and the inter-day precision was 1.86% - 4.11%. All the recoveries were ≥80%, indicating that the pretreatment method could effectively retain the target analytes SFE, SFN, SFN-Cys and SFN-NAC. The solid-phase extraction process could effectively enrich the target compounds without significant degradation or irreversible adsorption; the precision of all metabolites within and between days was ≤15%, indicating good method reproducibility and detection stability.

[0154] The LC-MS / MS method established in the present invention exhibits excellent extraction efficiency (Recovery ≥ 80%) and detection repeatability (RSD ≤ 15%) in complex urine matrices, successfully overcomes the technical difficulties of easy oxidation and large polarity span of glucosinolate metabolites, and provides high-confidence data support for subsequent evaluation of the bioavailability of isothiocyanate compounds, kinetic modeling of metabolite renal clearance, and clinical studies on dietary intervention.

[0155] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for simultaneously detecting glucoraphanin, sulforaphane and sulforaphane metabolites in urine by LC-MS method, characterized in that Comprising the following steps: Collect urine samples from subjects; Centrifuge the urine samples, collect the supernatant, subject the supernatant to solid-phase extraction, and collect the post-extraction samples; Prepare standard solutions of sulforaphane, sulforaphane metabolite, and N-acetylcysteine sulforaphane; Subject the post-extraction samples and the standard solutions to ultra-high performance liquid chromatography-tandem mass spectrometry analysis respectively; Wherein, the sulforaphane metabolites are cysteine-sulforaphane and N-acetylcysteine sulforaphane.

2. The method according to claim 1, wherein The solid-phase extraction comprises the following steps: Add 2 mL of methanol and 2 mL of ultrapure water to the extraction column at a flow rate of 2 mL / min, and add a total of 2 times; Mix the supernatant with a formic acid aqueous solution, load the sample, and the flow rate is 5 mL / min; Wash the extraction column with a formic acid aqueous solution and drain it; Add methanol to the extraction column in 3 portions for elution, the flow rate is 2 mL / min, the total amount of methanol is 10 mL, collect the eluate, concentrate it, and re-dissolve it with methanol to obtain the post-extraction samples.

3. The method according to claim 2, wherein The extraction column is a Stara-X 33μm polymeric strong cation solid-phase extraction column; The volume fraction of formic acid in the formic acid aqueous solution is 2%; 4. The method according to claim 1, wherein The flow rate of the ultra-high performance liquid chromatography is 0.35 mL / min; the column temperature is 30°C; the wavelength of the ultraviolet detector is 254 nm.

5. The method according to claim 4, characterized in that The mobile phase A of the ultra-high performance liquid chromatography is a formic acid ultrapure water solution, and the volume fraction of formic acid is 0.1%; The mobile phase B is a formic acid acetonitrile solution, and the volume fraction of formic acid is 0.1%.

6. The method according to claim 5, characterized in that, The elution program of the ultra-high performance liquid chromatography is: 95% A at 0 min; gradually decreased to 65% A within 0 - 8 min; 5% A at 8 - 10 min.

7. The method according to claim 1, wherein The ion source of the mass spectrometry is electrospray ionization; the scanning mode is positive ion multiple reaction monitoring; the drying gas temperature is 350°C; the drying gas flow rate is 5 L / min; the sheath gas temperature is 250°C; the sheath gas flow rate is 11 L / min; the nebulizer pressure is 20 psi; the capillary voltage is 3500 V; the fragmentation voltage is 80 V; the collision energy is 5 eV.

8. The method according to claim 7, characterized in that, When using the mass spectrometry to quantitatively detect sulforaphane, the parent ion mass-to-charge ratio is 176, and the daughter ion mass-to-charge ratio is 112; when using the mass spectrometry to qualitatively detect sulforaphane, the parent ion mass-to-charge ratio is 176, and the daughter ion mass-to-charge ratio is 107.9; When using the mass spectrometry to quantitatively detect sulforaphane metabolite, the parent ion mass-to-charge ratio is 178, and the daughter ion mass-to-charge ratio is 114; when using the mass spectrometry to qualitatively detect sulforaphane metabolite, the parent ion mass-to-charge ratio is 178, and the daughter ion mass-to-charge ratio is 72; When using the mass spectrometry to quantitatively detect cysteine-sulforaphane, the parent ion mass-to-charge ratio is 299, and the daughter ion mass-to-charge ratio is 136; when using the mass spectrometry to qualitatively detect cysteine-sulforaphane, the parent ion mass-to-charge ratio is 299, and the daughter ion mass-to-charge ratio is 178; When using the mass spectrometry to quantitatively detect N-acetylcysteine sulforaphane, the parent ion mass-to-charge ratio is 341, and the daughter ion mass-to-charge ratio is 178; when using the mass spectrometry to qualitatively detect N-acetylcysteine sulforaphane, the parent ion mass-to-charge ratio is 341, and the daughter ion mass-to-charge ratio is 136.

9. The method according to claim 1, wherein The urine samples include blank control samples and experimental samples; Among them, the collection of the blank control sample includes: collecting the midstream urine of the subject on an empty stomach in the morning before the intervention experiment; The collection of the experimental sample includes: the urine of the subject collected at different times after the intervention experiment, and boric acid is added.

10. Use of the method according to any one of claims 1-9 for monitoring the levels of sulforaphane, sulforaphane, cysteine-sulforaphane and N-acetylcysteine sulforaphane in human urine for non-diagnostic or therapeutic purposes.

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