Method suitable for purification, enrichment and determination of citrinin in food

Abstract

The invention discloses a method suitable for purification, enrichment and determination of citrinin in food, and belongs to the technical field of chemical analysis. The method suitable for purifying and enriching citrinin in food comprises the following steps: preparing a sample to be detected into an extracting solution to be purified; the extracting solution passes through a solid-phase extraction column with MAX serving as an upper-layer filler and GCB serving as a lower-layer filler, elution is conducted, and a citrinin-enriched solution to be detected is obtained. And further determining the content of citrinin in the to-be-detected liquid to obtain the content of citrinin in the food. The method provided by the invention is suitable for purification and enrichment of citrinin in various foods, can effectively control the influence of co-extraction impurities, and can obtain purification and recovery capacities comparable to those of an immunoaffinity column. The citrinin content of the enriched solution is further measured, and the citrinin content in various foods can be obtained. The detection method provided by the invention is simple and convenient to operate, low in cost, high in sensitivity and good in accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a method suitable for purifying, enriching and determining citrinin in food. Background Art

[0002] Currently, the main detection methods for citrinin include immunoaffinity column method, C18 column method and QuEChERS method.

[0003] Immunoaffinity Column Method: Principle: Utilizes citrinin monoclonal antibodies to specifically adsorb the target, achieving highly selective purification through an antigen-antibody reaction. Procedure: Sample extraction → PBS dilution → immunoaffinity column loading → PBST wash → methanol elution → HPLC detection. Advantages: High specificity and excellent purification (83.7% to 93.2% recovery).

[0004] C18 Extraction Column: Principle: Adsorbs the target compound based on hydrophobic interactions and then elutes it with an organic solvent. Application: Commonly used in the cleanup step of QuEChERS pretreatment.

[0005] QuEChERS method: Principle: Dispersive solid-phase extraction (d-SPE), using PSA, C18, and other fillers to adsorb impurities. Advantages: Simple operation and low cost.

[0006] The immunoaffinity column method has the following problems and defects: (1) High cost and harsh storage conditions: The unit price of the immunoaffinity column is about 180 yuan (4500 yuan / 25 pieces), and it needs to be strictly refrigerated at 2-8°C. Freezing is prohibited, otherwise the antibody will be inactivated. The reason is that this technology relies on the biospecific recognition of monoclonal antibodies, the cost of antibody production and purification is high, and the stability of biological materials is poor and easily affected by temperature fluctuations; (2) The operation is cumbersome and the flow rate control is sensitive: the temperature must be strictly controlled to return to room temperature (22-25°C) before use, and the loading, washing, and elution flow rates must be controlled at 1-2 drops / second. Too fast will lead to insufficient binding of the target (recovery rate reduction> 10%). The reason is that the kinetics of antigen-antibody reaction is affected by temperature and contact time, and a too fast flow rate reduces the effective binding time; (3) Limited column capacity: The column capacity of the immunoaffinity column is essentially the saturation threshold of the antibody-antigen binding site, which is limited by the number of antibodies, carrier space and operating conditions. The reasons include the limited number of immobilized antibodies, attenuation of antibody activity, and carrier space limitations; (4) The immunoaffinity column cannot tolerate a high proportion of organic solvents. Increasing the proportion of organic solvents will significantly increase the risk of protein denaturation, destroy the antigen binding site, and cause damage to the carrier structure.

[0007] C18 extraction columns have the following problems and drawbacks: The C18 packing has poor adsorption selectivity and a low removal rate for polar impurities (such as organic acids and pigments). Its mechanism of action relies on hydrophobic interactions, leading to the co-elution of citrinin (log P ≈ 2.5) and polar impurities (such as citric acid and red yeast rice pigments). This makes the C18 packing only suitable for simple matrices such as rice and wheat, but ineffective for red yeast rice and its products, which are heavily contaminated with citrinin. Furthermore, due to the pore size (typically 8 to 10 nm) and carbon loading of the C18 packing, citrinin is protonated in an acidic environment (pH < 4), increasing its hydrophobicity and making elution difficult, resulting in low recovery rates.

[0008] The QuEChERS method suffers from the following issues and drawbacks: Adsorbents such as PSA (ethylenediamine-N-propylsilane) and NH2 (amino) can bind to the carboxyl groups of citrinin, resulting in inadequate elution and recoveries of less than 80%. C18 adsorbents and solid-phase extraction techniques also suffer from poor purification effectiveness. Furthermore, the salting-out and layered purification step of QuEChERS can cause the highly water-soluble citrinin to partition between the aqueous and organic phases, reducing recovery. Summary of the Invention

[0009] The present invention aims to provide a method suitable for the purification, enrichment, and determination of citrinin in food. By precisely controlling the functional complementarity and spatial arrangement of fillers, the method significantly suppresses the interference of co-extracted impurities in complex food matrices (such as pigments and lipids) on the analysis of citrinin by liquid chromatography and LC-MS / MS.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] One of the technical solutions of the present invention is to provide a method for purifying and enriching citrinin in food, comprising the following steps:

[0012] The sample to be tested is prepared into an extract to be purified; the extract is eluted through a solid phase extraction column with MAX (mixed anion exchange reverse phase adsorbent) as the upper filler and GCB (graphitized carbon black) as the lower filler to obtain a test solution enriched with citrinin.

[0013] The schematic diagram of the solid phase extraction column in the present invention is shown in Figure 1 .

[0014] The functional division and action mechanism of the filler in the present invention are as follows:

[0015] MAX filler: It specifically captures negatively charged citrinin through an anion exchange mechanism (carboxyl group ionization at pH>4). At the same time, this filler has good tolerance to neutral, high-proportion organic solvents (100% acetonitrile), and can retain citrinin while eluting and eliminating interference from neutral lipids and non-polar pigments.

[0016] GCB filler: With the π-π stacking structure on the surface of graphitized carbon black, it produces irreversible adsorption of planar conjugated pigment molecules such as chlorophyll, carotenoids and monascus pigments, eliminating pigment background signals.

[0017] The synergistic effect of the spatial order of the fillers in the present invention is:

[0018] Hierarchical design (MAX upper layer, GCB lower layer): During sample loading, MAX preferentially adsorbs citrinin and retains some fat-soluble pigments. During the subsequent elution phase, pigments escaping from MAX are recaptured by the GCB lower layer, ultimately resulting in a clear and transparent eluate (absorbance <0.1 AU, 400-600 nm), avoiding chromatographic interference caused by pigment co-elution. Finally, acidic acetonitrile is used to selectively elute citrinin, while impurities are retained on the extraction column.

[0019] Preferably, the upper filler layer and the lower filler layer are separated by a sieve plate.

[0020] Preferably, the particle size of the MAX is 40 to 60 μm.

[0021] Preferably, the particle size of the GCB is 120-400 mesh.

[0022] Preferably, when the volume of the solid phase extraction column tube used in the solid phase extraction column is 6 mL, the amount of MAX used is ≥200 mg, and the amount of GCB used is 50-250 mg.

[0023] Experiments show that when the solid phase extraction column volume is 6mL: when the MAX dosage is <200mg, the recovery rate of citrinin is proportional to its dosage. When the MAX is ≥200mg, the adsorption of citrinin tends to be saturated. Increasing the amount will not increase the recovery rate of the target compound (Δ<3%), but will prolong the column time (flow rate reduction>40%). When GCB is in the range of 50-250mg, the recovery rate of citrinin remains>90%; however, excessive GCB (>300mg) has a high specific surface area (1200m 2 / g) caused a strong hydrophobic effect, resulting in partial irreversible adsorption of citrinin (the recovery rate decayed to <70%).

[0024] Preferably, the elution solution is a mixture of formic acid, water and acetonitrile in a volume ratio of 1:1:18.

[0025] Preferably, a rinsing step is further included before the elution, and the eluent used for rinsing is acetonitrile.

[0026] The second technical solution of the present invention is to provide a method for detecting citrinin in food, comprising the following steps:

[0027] The content of citrinin in the test solution enriched with citrinin obtained by the above-mentioned method for purifying and enriching citrinin in food is determined by liquid chromatography or liquid chromatography-mass spectrometry, and the content of citrinin in the food is calculated.

[0028] The beneficial technical effects of the present invention are as follows:

[0029] The method provided by the present invention is suitable for purifying and enriching citrinin in various foods, effectively controlling the impact of co-extracted impurities and achieving purification and recovery capabilities comparable to those of immunoaffinity columns. Further assaying the enriched solution for citrinin content can determine the citrinin content in various foods. The provided detection method is simple to operate, low-cost, highly sensitive, and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the solid phase extraction column in the present invention.

[0031] Figure 2 The schematic diagram of the method selectivity verification of Example 1 of the present invention using a double-layer composite filler extraction column to purify and enrich citrinin is shown in FIG. Figure 2 .

[0032] Figure 3 The liquid chromatograms of rice, corn, wheat, barley, jam, fermented bean curd, ham, soy sauce, chili powder, and red yeast rice in the selectivity verification of the method in Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0034] It should be pointed out that the matters not described in detail in the present invention are conventional operating means in this field and are not the focus of the present invention.

[0035] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intervening value in the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] (1) Preparation of reagents and materials:

[0040] Acetonitrile and formic acid were of LCMS grade, PBS (pH = 7.2-7.4) salt packing, GCB (graphitized carbon black, 120-400 mesh) and MAX (mixed anion exchange reversed phase adsorbent, 40-60 μm) fillers, empty solid phase extraction column tubes (6 mL) and matching frits (10 μm), and experimental water was grade 1 water as specified in GB / T 6682.

[0041] 0.5% formic acid-water solution (v / v): Take 5.0 mL of formic acid and dilute it to 1000 mL with pure water; 0.5% formic acid-acetonitrile solution (v / v): Take 5.0 mL of formic acid and dilute it to 1000 mL with acetonitrile;

[0042] Citrinin (C 13 H 14 O5, CAS No.: 518-75-2), purity> 99%, or certified standard. 13 C 13 -Citrin internal standard solution ( 13 C 13 H 14 O5, 10 μg / mL), or certified reference material.

[0043] Citrinin stock solution (100 μg / mL): Weigh 10.0 mg (accurate to 0.1 mg) of citrinin into a small beaker and dissolve in acetonitrile. Transfer to a 100 mL volumetric flask and dilute to the mark with acetonitrile. Mix thoroughly to obtain a 100 μg / mL standard stock solution. Transfer the solution to a brown reagent bottle and store at -18°C in the dark. The expiration date is 12 months.

[0044] Citrinin working solution (1.0 μg / mL): Accurately pipette 1.0 mL of the stock solution into a 100 mL volumetric flask, dilute to the mark with acetonitrile, and mix thoroughly to obtain a 1.0 μg / mL standard working solution. Transfer the solution to a brown reagent bottle and store at -18°C in the dark. The expiration date is 1 month.

[0045] 13 C 13 Citrinin internal standard working solution (1.0 μg / mL): Accurately pipette 1.0 mL of the isotope internal standard stock solution (10.0 μg / mL) into a 10 mL volumetric flask, dilute to the mark with acetonitrile, and mix thoroughly to obtain a 1.0 μg / mL internal standard working solution. Transfer the solution to a brown reagent bottle and store at -18°C in the dark. The expiration date is 6 months.

[0046] Preparation of standard curve: A series of standard solutions with concentrations of citrinin of 0.0, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, and 100 ng / mL were prepared using acetonitrile-water solution (20+80, v / v). 13 C 13 -The internal standard concentration of citrinin is 10 ng / mL and is prepared immediately before use.

[0047] (2) Sample preparation:

[0048] Dry samples such as grains, chili peppers, and red yeast rice (red yeast health capsules should be removed from the capsule shell, no less than 200g) should be ground evenly in a solid grinder and passed through a 60-mesh sieve. Grind the edible portion of fermented meat products such as ham. Semi-solid samples such as fermented bean curd and jam should be homogenized in a homogenizer. Liquid samples such as soy sauce, rice vinegar, red yeast rice wine, yellow rice wine, rice wine, and fruit and vegetable juices should be properly degassed and mixed evenly. Each sample for testing should be no less than 100g and stored in a sealed, refrigerated sample bottle.

[0049] (3) Sample extraction:

[0050] Grains and chili powder: Accurately weigh 5.0 g into a 50 mL centrifuge tube. Add 50 μL of isotope internal standard solution (1.0 μg / mL), mix thoroughly, and let stand for 30 minutes. Subsequently, vortex and extract with 25.0 mL of 80% acetonitrile in water (v / v) for 25 minutes. Centrifuge at 8500 rpm for 3 minutes and collect 5.0 mL of the supernatant (equivalent to 1.0 g of sample) for purification.

[0051] Ham, fermented bean curd, and jam: Accurately weigh 5.0 g into a 50 mL centrifuge tube, add 250 μL of isotope internal standard solution (1.0 μg / mL), mix thoroughly, and let stand for 30 minutes. Subsequently, vortex and extract with 25.0 mL of 80% acetonitrile aqueous solution (v / v) for 25 minutes. Centrifuge at 8500 rpm for 3 minutes, and collect 1.0 mL of supernatant (equivalent to 0.2 g sample) for purification.

[0052] Soy sauce: Accurately weigh 5.0 g of soy sauce into a 50 mL centrifuge tube, add 50 μL of isotope internal standard solution (1.0 μg / mL), mix thoroughly, and let stand for 30 minutes. Subsequently, vortex and extract with 25.0 mL of 80% acetonitrile in water (v / v) for 25 minutes. Centrifuge at 8500 rpm for 3 minutes, and collect 5.0 mL of the supernatant (equivalent to 1.0 g of sample) for purification.

[0053] Red Yeast Rice: Accurately weigh 1.0 g of red yeast rice into a 50 mL centrifuge tube, add 250 μL of isotope internal standard solution (1.0 μg / mL), mix thoroughly, and let stand for 30 minutes. Subsequently, vortex and extract with 25.0 mL of 80% acetonitrile (v / v) for 25 minutes. Centrifuge at 8500 rpm for 3 minutes, and collect 1.0 mL of the supernatant (equivalent to 0.04 g of sample) for purification.

[0054] (4) Sample purification:

[0055] Use PBS (pH = 7.2-7.4) to dilute the above sample extract and control the proportion of organic phase to less than 20% before passing through the column. The double-layer composite filler extraction column (the upper layer contains 221 mg of MAX and the lower layer contains 250 mg of GCB) is activated with 6 mL of acetonitrile and 6 mL of pure water in sequence before use. It is necessary to control the speed of the sample solution passing through the column to no more than 1 drop / s to ensure that the sample solution is in full contact with the filler. After the sample solution has passed through the column, it is rinsed with 6 mL of pure water and 6 mL of acetonitrile in sequence, and the eluent is discarded. Use 6 mL of 5% formic acid-5% water-90% acetonitrile for elution, collect the eluate, blow dry with nitrogen at 40°C, add 1.0 mL of 20% acetonitrile-water to reconstitute, vortex for 30 seconds, pass through a 0.22 μm microporous filter membrane, collect in a sample injection bottle, and wait for testing.

[0056] (5) Instrument detection parameters:

[0057] Chromatographic conditions: A C18 column with 1.7 μm particle size, 100 mm length, and 2.1 mm inner diameter, or equivalent, was used. Mobile phase A consisted of 0.5% formic acid in water, and mobile phase B consisted of 0.5% formic acid in acetonitrile. The flow rate was 0.3 mL / min, the column temperature was 40°C, the injection volume was 1 μL, and the gradient elution program was as shown in Table 1.

[0058] Table 1 Mobile phase gradient elution program

[0059] Time / min Mobile phase A / % Mobile phase B / % 0.0 50.0 50.0 2.5 50.0 50.0 2.6 2.0 98.0 4.5 2.0 98.0 4.6 50.0 50.0 7.0 50.0 50.0

[0060] UV detector parameters: wavelength 335nm.

[0061] Mass spectrometry conditions: ionization mode (ESI +), multiple reaction monitoring mode (parameters are detailed in Table 2), capillary voltage 5.5 kV, ion source temperature 550 °C, curtain gas 30 psi, desolvation gas flow 50 psi, declustering voltage 80 V.

[0062] Table 2 Multiple reaction monitoring parameters of citrinin and its isotope internal standard

[0063]

[0064] (6) Methodological validation and quality control:

[0065] Rice, corn, wheat, barley, jam, fermented bean curd, ham, soy sauce, chili powder, and red yeast rice were selected as representative matrices, and the above matrices were screened and verified with no background value or low background value as the test matrix.

[0066] Method sensitivity test: In the above-mentioned matrices, a series of concentrations such as 0.05μg / kg, 0.1μg / kg, 0.2μg / kg, 0.5μg / kg, 1.0μg / kg, 2.0μg / kg, 5.0μg / kg, and 10.0μg / kg were set for spiked test, with 3 times the signal-to-noise ratio (S / N) and 10 times the signal-to-noise ratio (S / N) as the method detection limit (LOD) and quantification limit (LOQ), respectively; Spiked accuracy (recovery rate) and precision (RSD) test: Spiked test was performed at three levels of LOQ, 10*LOQ, and 50*LOQ in six parallels in most matrices, except for red yeast rice (50μg / kg, 500μg / kg, and 1000μg / kg).

[0067] Evaluation of linear range and matrix effect: The instrument linear range was tested at a concentration series of 0.0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL. A matrix-matching curve was established in the blank or low-background matrix (including at least five concentration points, namely 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL). The slope of the matrix-matching curve was compared with the slope of the standard solution curve. A ratio between 0.8 and 1.2 indicated that the matrix effect was acceptable and not significant. A matrix effect <0.8 indicated a significant inhibitory effect. Conversely, a matrix effect >1.2 indicated a matrix enhancement effect. Whether it is an inhibition or enhancement effect, it indicates that the matrix components have an impact on the analysis of target compounds. The matrix effect can also be used as one of the evaluation indicators of the pretreatment purification effect.

[0068] (7) Screening of composite filler types:

[0069] Considering the inherent signal fluctuations of mass spectrometry and the inability to use isotopic internal standards for optimization, optimization was performed using ultra-high performance liquid chromatography (UPLC) with a highly stable UV detector. Initially, a preliminary experiment was conducted with commonly used adsorbents (all fillers except GCB had particle sizes between 40 and 60 μm), including C18, PSA (ethylenediamine-N-propyl), HLB (polystyrene divinylbenzene), NH2 amino, Phenyl phenyl, Thiol metal ion, GCB, MAX, MCX (mixed cation exchange reversed-phase), WAX (mixed weak anion exchange reversed-phase), and WCX (mixed weak cation exchange reversed-phase). A 200 mg / species solution was filled to the center of an empty 6 mL solid-phase extraction column. The sample was then loaded with 0.1 mL of a 1.0 μg / mL citrinin solution mixed with 5 mL of PBS. The column load liquid, acetonitrile eluent, and 5% formic acid-5% water-90% acetonitrile eluent were intercepted and analyzed respectively. Among them, except the column load liquid, all were nitrogen-purged and concentrated before analysis. The results are shown in Table 3.

[0070] Table 3 Recovery results of citrinin in adsorbent sample solution, eluent and eluent

[0071]

[0072]

[0073] Analysis of the data in Table 3 reveals that commonly used adsorbents such as PSA and C18 exhibit weak adsorption of citrinin. PSA primarily adsorbs negatively charged compounds through the anion exchange capacity of its amino functional group and hydrogen bonding. Citrinin is ionic in PBS, but its interaction with PSA is weak, resulting in its complete elution during the elution step. Similar results were observed for the NH2 amino adsorbent and PSA. C18 relies on hydrophobic interactions (van der Waals forces) to adsorb non-polar or weakly polar compounds. Citrinin contains multiple polar groups (carboxyl groups and lactone rings), making its ionic state highly hydrophilic. Therefore, C18 has a weak adsorption effect on it, resulting in significant loss during sample loading.

[0074] Citrinin is in an ionic state in neutral or weakly alkaline PBS and cannot undergo ion exchange with the negatively charged MCX / WCX filler to generate strong electrostatic forces. Its adsorption relies solely on reversed-phase hydrophobic interaction (HLB backbone), which is weak and is eluted during the elution process. This is why the results of MCX / WCX are similar to those of HLB.

[0075] Thiol metal ion adsorbent has a certain selectivity for citrinin, and there is no obvious loss during the loading and elution processes. However, its adsorption effect on citrinin is too strong, resulting in a low final recovery rate. The benzene ring structure of citrinin can undergo multiple interactions with the phenyl adsorbent, including π-π conjugation, hydrophobic interaction, and van der Waals force, resulting in high-intensity adsorption, which makes citrinin unable to be eluted.

[0076] Although the mechanism of action of GCB and Phenyl phenyl adsorbent with citrinin is similar, mainly relying on π-π stacking and molecular planar matching, acidic acetonitrile can weaken and destroy the π-π stacking of GCB and citrinin, thereby completely releasing it; while the -N + It forms a strong electrostatic attraction with the -COO- of citrinin, and acidic acetonitrile will also destroy this electrostatic force. In summary, considering the different adsorption mechanisms of GCB and MAX, these two adsorbents were selected for subsequent optimization.

[0077] (8) Functional form of composite fillers:

[0078] Two common pretreatment methods for adsorbents, dispersive solid-phase extraction and solid-phase extraction, were investigated. Dispersive solid-phase extraction (n=3): 0.2 mL of a 1.0 μg / mL citrinin solution (80% acetonitrile in water, simulating the sample extract) was added to 200 mg of adsorbent and vortexed for 3 minutes for adsorption. The solution was then centrifuged, the entire supernatant was transferred, concentrated, and analyzed. 6.0 mL of acetonitrile was then added for a rinse step. The solution was centrifuged, the supernatant was concentrated, and analyzed. Finally, 6.0 mL of 5% formic acid-5% water-90% acetonitrile was added for desorption. The solution was centrifuged, the supernatant was concentrated, and analyzed. The recovery of citrinin under each step was evaluated. Simultaneously, 2.0 mL of the actual red yeast rice sample extract was treated, and 1.0 mL of the treated solution was concentrated by nitrogen purging to evaluate the purification effect.

[0079] Solid-phase extraction (n=3): 0.1 mL of a 1.0 μg / mL citrinin solution plus 5 mL of PBS was passed through an extraction column containing 200 mg of adsorbent (GCB, MAX). The column load, acetonitrile eluate, and acidic acetonitrile eluate were collected, concentrated, and analyzed to evaluate the recovery of citrinin. Simultaneously, 1.0 mL of red yeast rice extract was used for pretreatment, and the purification effect was evaluated by reconstitution of the residue. The recovery results for different fillers are shown in Table 4.

[0080] Table 4 Recovery results of citrinin in sample solution, eluent and eluent after adsorbent treatment

[0081]

[0082] From the results in Table 4, it can be seen that when GCB, MAX, or GCB+MAX are used in the form of dispersed solid phase extraction, the adsorption effect of citrinin is not ideal, whether in the initial adsorption stage or the acetonitrile elution stage. The target compound is largely desorbed, resulting in a very unsatisfactory recovery rate in the final acidic acetonitrile elution stage. The above phenomenon may be explained by the following reasons: (1) In the dispersed solid phase extraction mode, the environment is usually high in organic phase, and the pH cannot be controlled by PBS, causing some citrinin to be in ionic state; (2) The adsorbent and the target compound are in disordered contact, lacking an ordered adsorption path; (3) Dispersed solid phase extraction is stirred by vortex, and turbulence dominates the dispersion. While increasing the contact between the target compound and the adsorbent, it also inevitably promotes the desorption of citrinin; (4) The vortex in the acetonitrile dispersion elution step forms a disordered fluid environment, intensifying molecular collisions and triggering the desorption of citrinin.

[0083] Compared to dispersed solid-phase extraction (SPE), SPE particles are densely packed in the column bed, forming ordered pore channels. The target substance must penetrate multiple layers of packing, resulting in a long adsorption path. This allows ample time for citrinin to form a stable π-π conjugation with the π-electron cloud of GCB and fully interact with MAX for ion exchange. Therefore, SPE was chosen as the pretreatment method. In SPE, single adsorbents are inferior to composite adsorbents in terms of citrinin recovery, and the color of the purified liquid is darker than that of the composite adsorbent extraction column. Therefore, a composite adsorbent combination of GCB and MAX was selected, and the dosage of the two adsorbents needs to be further optimized. As for how the composite adsorbents were layered, no significant differences were found in the experimental results. However, we observed that when the GCB packing was in the lower layer, the overall resistance to the sample liquid passing through was less, and the flow rate was faster under the same conditions. Therefore, a composite adsorbent design with MAX as the upper layer and GCB as the lower layer was ultimately selected.

[0084] (9) Single-factor experiment:

[0085] Single-factor tests were conducted on GCB and MAX, respectively, with recovery as the evaluation indicator. The experimental conditions were as follows: adsorbent weight 50, 100, 200, 300, 400, and 500 mg, and the loading solution was prepared by adding 0.1 mL of 1.0 μg / mL citrinin solution + 5 mL of PBS. The sample solution was passed through the above-mentioned adsorbent extraction column. The recovery results are shown in Table 5.

[0086] Table 5 Adsorbent single factor test results

[0087] Number of trials n = 3 MAX recovery rate (%) GCB recovery rate (%) 50mg 56.1±8.4 40.5±5.8 100mg 70.4±5.6 76.2±3.5 200mg 84.5±4.9 81.1±3.7 300mg 85.0±2.5 84.3±4.4 400mg 84.3±2.7 75.6±5.1 500mg 83.5±5.0 72.2±3.4

[0088] As shown in Table 5, when using MAX adsorbent, the recovery rate of citrinin is proportional to the dosage, reaching its highest value at a dosage of 200-300 mg. Excessive use has no significant effect on the recovery rate. In contrast, the relationship between the amount of GCB used and the recovery rate first increases and then decreases, reaching its highest point at a dosage of around 300 mg. Excessive use can lead to irreversible adsorption of citrinin, significantly reducing the recovery rate.

[0089] (10) Response surface-central composite design experiment:

[0090] A central composite design matrix (see Table 6) was designed based on the dosage of GCB and MAX and a dummy variable (X). GCB and MAX were centered at 200 mg, with 100 mg and 300 mg representing the low level (-1) and high level (+1), respectively. The ±alpha level (±1.68) was 31.8 mg and 368.2 mg, respectively. This part of the experiment used spiked red yeast rice extract as the test subject: 1.0 mL of supernatant (equivalent to 0.04 g of red yeast rice) was mixed with 0.1 mL of a 1.0 μg / mL citrinin solution and 5 mL of PBS, and the sample was then loaded. UPLC-UV detection was used for detection. The response values ​​were the citrinin signal intensity (to evaluate method recovery) and the total UV signal intensity excluding citrinin from 0-6 minutes (to evaluate the purification performance of the extraction column; poorer performance results in stronger UV signals from co-extracts). The results are shown in Table 7.

[0091] Table 6 Response surface-central composite design matrix

[0092]

[0093]

[0094] Table 7 Summary of response surface-variance analysis results

[0095]

[0096] First, from the perspective of model terms, the signal intensity of citrinin and the total signal intensity of the UV spectrum excluding citrinin from 0-6 min were both significant (P<0.0001), indicating that the selected regression model (quadratic polynomial model) was effective as a whole and suitable for analysis under the current conditions. The vector terms of the two response values ​​were not significant (P: 0.1167-0.1384), indicating that the model did not have systematic underfitting, did not omit important high-order terms or unknown interference factors, and the random error of the experimental data was controllable. By analyzing the P values ​​of the factor terms, it can be found that the linear term (X A 、X B ), the quadratic term (X A 2) have significant main effects and nonlinear relationships on the two response values. The interaction term (X A ×X B ) For the total signal intensity of impurities, X B 2 There are significant effects on the signal intensity of citrinin. All the factors related to the virtual variables are not significant, indicating that the design matrix system has good operation stability and there is no uncontrolled variation. 2 、Adjusted R 2 and Predicted R 2 Evaluate the goodness of fit of the model. 2 The values ​​were 0.9705-1.000 (all greater than 0.9), indicating that the model had a high goodness of fit. 2 It is a more robust goodness-of-fit indicator, suitable for multiple regression, and the |R 2 -AdjustedR 2 ∣<0.2, indicating that the model has no redundant variables; PredictedR 2 The model's predictive ability for new samples can be evaluated by cross-validation calculation, the |Predicted R of the two response values 2 -Adjusted R 2 ∣<0.2, indicating that the model's predictive ability is reliable. The model's discriminability index (AdeqPrecision) is mainly used to measure the model's ability to distinguish changes in response values. In the current model, AdeqPrecision is 17.0379-576.0167, which is greater than 4.0, indicating that the model has sufficient discriminability and ensures that the model can clearly capture the effect.

[0097] After completing the above analysis, the Numerical Optimization function was used, with the GCB and MAX dosages set to minimize, the dummy variable set to In range, the citrinin signal intensity set to maximize, and the 0-6 min impurity signal set to minimize. Ultimately, the optimal GCB dosage was 251 mg, and the optimal MAX dosage was 221 mg. Based on the center group and the designed sample size (n = 20), the residual degrees of freedom (df) = 10, and the t-distribution critical value (t0.025,10) was 2.228 (95% confidence level). The optimal solution predicted by the citrinin signal model is 478668, the residual sum of squares is 8.479E+07, the residual mean square is 8.479E+06, and the residual standard deviation is 2911. The solution predicted by the impurity signal model is 7.39E+06, the residual sum of squares is 3.817E+09, the residual mean square is 3.817E+08, and the residual standard deviation is 19541. The confidence interval is calculated according to the following formula:

[0098] Var 预测值 = residual mean square × (1 + h)

[0099]

[0100] Among them, the leverage value h = 1 / n = 1 / 20 = 0.05, and the conservative estimate is h = 0.1.

[0101] Var 拮青霉素信号强度预测值 = residual mean square × (1 + h)

[0102] =8.479×10 6 ×(1+0.1)=9.3269×10 6

[0103]

[0104] Var 杂质信号强度预测值 = residual mean square × (1 + h)

[0105] =3.817×10 8 ×(1+0.1)=4.1987×10 8

[0106]

[0107] The calculation results showed that the response intensity of citrinin was 479067±5229, and the total signal intensity of impurities was 7380515±38621, both of which were within the confidence interval, indicating that the prediction results of the above model were consistent with the actual results.

[0108] (11) Method selectivity verification:

[0109] To comprehensively evaluate the selectivity of the established method and the purification efficiency of the extraction column, the present invention selected 10 foods potentially contaminated with citrinin (rice, corn, wheat, barley, jam, fermented bean curd, ham, soy sauce, chili powder, and red yeast rice) as representative matrices. Blank background and spiked tests were performed according to the above-mentioned optimization process, focusing on the presence of interfering peaks near the target retention time of citrinin (~1.90 min). The purification effect of the double-layer composite filler extraction column was evaluated using an ultraviolet optical detector. The schematic diagram of the purification and enrichment of citrinin using the double-layer composite filler extraction column is shown in FIG. Figure 2 .result( Figure 3) clearly showed that after cleanup using this extraction column, no significant interference peaks were observed within the retention time window of citrinin in all 10 sample solutions, demonstrating that the current pretreatment conditions can effectively separate citrinin from matrix impurities and that the method has good selectivity. It is particularly noteworthy that although the matrices of chili powder (containing strongly polar interferences) and red yeast rice (containing weakly polar co-extracted impurities) are extremely complex, resulting in a low relative height of the chromatographic peak of citrinin after cleanup, its absolute peak height (~0.020 AU) and accurate retention time (~1.90 min) are clearly discernible and unaffected by matrix interference. This fully demonstrates that this cleanup method has reliable anti-interference capabilities even for the most complex matrices, and can be used to determine citrinin in most foods even with UV detectors with weaker anti-interference capabilities.

[0110] (12) Linear range, matrix effect and sensitivity verification

[0111] Standard solutions of concentrations of 0.0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 50.0 ng / mL, 100.0 ng / mL, 200.0 ng / mL, and 500.0 ng / mL were prepared using the initial mobile phase and analyzed by liquid chromatography-mass spectrometry. Under current instrument sensitivity conditions, concentrations of 200.0 ng / mL and above may cause mass spectrometry signal saturation and prevent linearity. Therefore, the highest linear point is generally set at 100.0 ng / mL. Using the isotope internal standard method for quantification, the linear equation is y = 0.101x, and the linear correlation coefficient is above 0.999, indicating that the method has good linearity in the range of 0.05 to 100.0 ng / mL.

[0112] To assess matrix effects, matrix-matched standard curves were prepared using extracts from 10 different matrices at concentrations of 5.0, 10.0, 20.0, 50.0, and 100.0 ng / mL. The influence of the matrix on quantitative accuracy was examined by comparing the slope of each matrix-matched curve with the slope of the solvent standard curve (i.e., the standard curve prepared using the initial mobile phase). The results are shown in Table 8.

[0113] Table 8 Matrix effects of typical matrices

[0114] matrix Matrix effect (%) rice 101.4 corn 98.5 wheat 96.6 barley 87.6 jam 88.1 fermented bean curd 92.1 Ham 93.8 soy sauce 99.1 chili powder 111.0 Red Yeast Rice 93.2

[0115] Table 8 shows that the matrix effects of the sample solutions after purification by the composite filler solid phase extraction column are all within the acceptable range of 80% to 120%, confirming that this pretreatment method can effectively eliminate matrix interference and ensure the reliability of the quantitative results.

[0116] Subsequently, gradient spike experiments were performed in the above matrix within the concentration range of 0.05 μg / kg to 10.0 μg / kg. The sensitivity of the method was calculated using a 3-fold and 10-fold signal-to-noise ratio, respectively. The results are shown in Table 9.

[0117] Table 9 Detection limits and quantification limits of typical matrix methods

[0118] matrix Detection limit (μg / kg) Limit of quantification (μg / kg) rice 0.15 0.5 corn 0.3 1.0 wheat 0.3 1.0 barley 0.3 1.0 jam 0.15 0.5 fermented bean curd 2.0 5.0 Ham 2.0 5.0 soy sauce 2.0 5.0 chili powder 5.0 10.0 Red Yeast Rice 5.0 10.0

[0119] The results in Table 9 are compared with two methods in the current national food safety standard GB5009.222-2016 (immunoaffinity column method: the detection limit and quantification limit for rice, corn, and chili powder samples are 8 μg / kg and 25 μg / kg, respectively; the detection limit and quantification limit for red yeast rice and its products are 25 μg / kg and 80 μg / kg, respectively; C18 extraction column method: the method detection limit is 3 μg / kg and the quantification limit is 10 μg / kg). The results show that the detection method provided by the present invention has a significant advantage in sensitivity. In addition to the improved sensitivity of the instrument itself, the double-layer composite filler extraction column has a purification effect comparable to that of the immunoaffinity column, and significantly reduces the negative impact of matrix interference on sensitivity, which is also one of the main reasons.

[0120] (13) Method accuracy, precision verification and column capacity test:

[0121] The accuracy and precision verification method is shown in step (6), and the results are shown in Table 10.

[0122] Table 10 Accuracy and precision results of typical matrix methods

[0123]

[0124]

[0125] The accuracy and precision of the method for detecting citrinin in 10 matrices were evaluated using three-level spiked experiments. Results showed that across cereals (rice, corn, wheat, and barley), processed foods (jam, fermented bean curd, ham, and soy sauce), and complex matrices (chili powder and red yeast rice), average recoveries across all spiked levels ranged from 90.4% to 99.1%, with relative standard deviations (RSDs) ranging from 1.4% to 8.9%. No target compound was detected in unspiked background samples, except for fermented bean curd (10.9 to 11.8 μg / kg) and red yeast rice (78.8 to 82.3 μg / kg). The recovery rate of the low concentration spike point (0.5-10 μg / kg) reached 90.5%-97.6%, and the recovery rate of the medium and high concentration point (25-1000 μg / kg) was stable at 90.4%-98.4%, and the RSD of all matrices was ≤8.9%, confirming that the method has excellent accuracy and reproducibility in different matrix types and concentration ranges. Subsequently, quality control samples (n=6) were used to analyze the accuracy of the method, specifically including MRM-CTORK0-20 red yeast rice powder citrinin (Purebon Biological Co., Ltd., labeled value: 10476 ± 2148 μg / kg) and QC-red yeast rice wine (Zhejiang Provincial Center for Disease Control and Prevention, labeled range: 38.4-42.0 μg / kg). The measurement results showed that the red yeast rice: 9964 ± 543 μg / kg, and the red yeast rice wine: 40.5 ± 1.2 μg / kg, once again proving that the detection method provided by the present invention is reliable in accuracy.

[0126] Comparisons were made with two pretreatment methods specified in GB 5009.222-2016 (i.e., immunoaffinity column and C18 column methods). Spike tests were conducted at 5 times the LOQ, and analysis was performed based on operator performance, processing time, absolute recovery, and matrix compatibility. The results are shown in Table 11. The specifications and performance of both the IAC and C18 columns must comply with the requirements of GB 5009.222-2016. The results indicate that while the IAC method offers high absolute recoveries (75.4%-98.0%) and is suitable for complex food matrices, it requires a longer processing time. This is due to the low tolerance of the IAC column for organic solvents, requiring significant dilution of the organic extract prior to sample loading. This increases the sample loading time and can lead to the precipitation of impurities (e.g., chili powder and red yeast rice). Failure to filter through glass fiber filter paper can lead to clogging of the IAC column, increasing analytical costs and significantly prolonging the processing time. The C18 column method is suitable for simple sample matrices, but even so, its recovery rate fluctuates significantly (RSD: 7.5%-10.9%). This may be related to the column flow rate and the general adsorption properties of citrinin and C18. If the flow rate is too fast, the recovery rate results will fluctuate significantly between parallel samples. However, the extraction column designed by the present invention has satisfactory applicability, and there are no significant differences between different personnel and different batches of fillers, indicating its reliability. At the same time, due to its good organic solvent tolerance (>50%) and low sample volume, the time required is the shortest among several methods, and there is no loss of recovery due to complex matrices (compared to the recovery rate of the IAC method in red yeast rice and chili powder).

[0127] Table 11 Test results of different extraction column methods / different operators

[0128]

[0129] On the other hand, the maximum column capacity of the citrinin immunoaffinity column is specified in GB 5009.222-2016 as exceeding 20 ng. The present invention collects five commercial citrinin immunoaffinity columns and tests their maximum column capacity. The test is based on the method for verifying the capacity of the immunoaffinity column in the determination of aflatoxins group B and group G in food in GB 5009.22-2016 National Food Safety Standard. The method is as follows: PBS solution (pH = 7.2-7.4) is used to prepare the citrinin solution, and the column loading amount is 100 ng, 500 ng, 1000 ng, 2000 ng, 3000 ng, 4000 ng and 5000 ng respectively. After loading, rinsing and eluting, the eluate is collected, dried with nitrogen, and the volume is adjusted to 1 mL with the initial mobile phase. The citrinin content is separated and determined by liquid chromatography, and the maximum concentration with a test recovery rate of ≥80% is the available column capacity. The test results are shown in Table 12.

[0130] Table 12 Column capacity test results

[0131]

[0132]

[0133] The results in Table 12 indicate that the maximum column capacity for IAC-1, IAC-4, and IAC-5 is approximately 1000 ng, while that for IAC-2 and IAC-3 is 2000 ng. This is primarily due to the technical aspects of the immunoaffinity column (antibody-carrier coupling efficiency, with directional coupling > random coupling) and the production level (antibody coupling amount). Currently, the column capacity of citrinin immunoaffinity columns typically does not exceed 2000 ng. However, the double-layer composite filler extraction column designed in this invention has a maximum capacity of 5000 ng, thereby reducing the possibility of repeated measurements or low results due to sample concentrations exceeding the column capacity.

[0134] (14) Applicability test of the method on actual samples:

[0135] A total of 252 samples were collected, including rice (n=34), wheat (n=42), barley (n=15), oats (n=20), corn (n=44), fruit and vegetable juices, jams and products (n=24), fermented meat products (n=13), red yeast rice (n=12), red yeast rice (n=16), chili peppers (n=32), soy sauce (n=20), rice vinegar (n=16), red yeast rice wine, yellow rice wine and rice wine (n=36). They were purified and measured based on the optimized method described above, and the results are summarized in Table 13.

[0136] Table 13 Actual sample measurement results

[0137]

[0138]

[0139] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for purifying and enriching citrinin in food, characterized in that: The following steps are involved: The sample to be tested is prepared into an extract to be purified; the extract is passed through a solid phase extraction column with MAX as the upper filler and GCB as the lower filler, and eluted to obtain a test solution enriched with citrinin.

2. The method for purifying and enriching citrinin in food according to claim 1, characterized in that: The upper layer of filler and the lower layer of filler are separated by a sieve plate.

3. The method for purifying and enriching citrinin in food according to claim 1, characterized in that: The particle size of the MAX is 40 to 60 μm.

4. The method for purifying and enriching citrinin in food according to claim 1, characterized in that: The particle size of the GCB is 120-400 meshes.

5. The method for purifying and enriching citrinin in food according to claim 1, characterized in that: When the volume of the solid phase extraction column tube used in the solid phase extraction column is 6 mL, the dosage of MAX is ≥200 mg, and the dosage of GCB is 50-250 mg.

6. The method for purifying and enriching citrinin in food according to claim 1, characterized in that: The elution solution is a mixture of formic acid, water and acetonitrile in a volume ratio of 1:1:

18.

7. The method for purifying and enriching citrinin in food according to claim 1, characterized in that: The method further comprises a rinsing step before the elution, and the rinsing liquid used is acetonitrile.

8. A method for detecting citrinin in food, characterized in that: The following steps are involved: The content of citrinin in the test solution enriched with citrinin obtained by the method for purifying and enriching citrinin in food according to any one of claims 1 to 7 is determined by liquid chromatography or liquid chromatography-mass spectrometry to calculate the content of citrinin in the food.

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