Preparation method of a coix seed aflatoxin B1 matrix standard substance and its determination method

Abstract

This invention provides a method for preparing and determining aflatoxin B1 matrix standard material from Job's tears. The method primarily employs a "reverse culture" technique to study the preparation technology of aflatoxin B1 matrix standard material from Job's tears. By investigating the culture conditions of Aspergillus flavus in Job's tears, and examining the effects of processes such as irradiation, drying, pulverization, and packaging on the uniformity and stability of the toxin, a preparation process for aflatoxin B1 matrix standard material from Job's tears is established. This technique is then applied to prepare aflatoxin B1 matrix standard material, providing a metrological standard for the detection, calibration, and quality control of aflatoxin in traditional Chinese medicine and agricultural products.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a method for preparing a coix seed aflatoxin B1 matrix standard and its value determination method. Background Technology

[0002] In recent years, traditional Chinese medicine (TCM) has gradually gained widespread recognition both domestically and internationally, especially for its unique efficacy demonstrated in the prevention and control of the COVID-19 pandemic. With the widespread application of traditional Chinese medicine, the issue of mycotoxin contamination in medicinal materials has attracted considerable attention. my country is a major producer of medicinal materials, and aflatoxin B1 (AFB1) is a mandatory testing indicator for various medicinal materials during import and export. However, currently, there is a lack of matrix standard materials for aflatoxin B1 in medicinal materials, which is insufficient to meet the needs of actual testing. Therefore, the development of relevant matrix standard materials is urgently needed.

[0003] Job's tears (Coix lacryma-jobi) is the dried, mature seed of the grass Coix lacryma-jobi L. var. ma-yuen (Roman.) Stapf. It is a high-quality food and medicine, cultivated throughout China with high yields and significant nutritional value. Aflatoxin is a fungal toxin of great concern both domestically and internationally. The 2020 edition of the Chinese Pharmacopoeia stipulates that the aflatoxin B1 content in 24 medicinal materials, including Job's tears, must not exceed 5 μg / kg. -1 Therefore, it is very necessary to develop a matrix standard for aflatoxin B1 in this type of medicinal material.

[0004] To address the shortcomings of existing technologies, the invention team conducted extensive experiments, employing a "reverse culture" technique to study the preparation of aflatoxin B1 matrix standard material from Job's tears. Through investigation of Aspergillus flavus culture conditions in Job's tears, and by examining the effects of processes such as irradiation, drying, pulverization, and packaging on toxin stability, a preparation process for the aflatoxin B1 matrix standard material from Job's tears was established. This technique was then applied to prepare the aflatoxin B1 matrix standard material, aiming to provide a metrological standard for the detection, calibration, and quality control of aflatoxin in traditional Chinese medicine. A method for preparing aflatoxin B1 matrix standard material from Job's tears was obtained, providing a metrological standard for the detection, calibration, and quality control of aflatoxin in the traditional Chinese medicine Job's tears. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing aflatoxin B1 matrix standard material from coix seed.

[0006] Another objective of this invention is to provide a method for determining the value of aflatoxin B1 matrix standard material from coix seed.

[0007] The method for preparing the aflatoxin B1 matrix standard material in coix seed according to the present invention includes the following steps:

[0008] (1) Preparation of Aspergillus flavus spore suspension;

[0009] (2) Inoculation of Coix seed medicinal material;

[0010] (3) Sterilization and drying of inoculated Coix seed medicinal materials;

[0011] (4) Crush, sieve and mix the inoculated Coix seed medicinal material;

[0012] (5) Aflatoxin B1 matrix standard material of coix seed is dispensed, sterilized and stored.

[0013] Preferred,

[0014] The preparation of the Aspergillus flavus spore suspension in step (1) is specifically as follows: Aspergillus flavus is inoculated into PDA medium and cultured for 3 days in a sterile operating table, then inoculated into PDA medium again and cultured in a light incubator for 5 days. The Aspergillus flavus spore suspension is collected, and the spore concentration is adjusted to 10⁵ CFU·mL using a hemocytometer. -1 A suspension of Aspergillus flavus spores was obtained.

[0015] Preferred,

[0016] The sterilization, inoculation, and culture of the Job's tears medicinal material in step (2) are as follows: Take the Job's tears medicinal material and sterilize it with microwave at a power of 650w for 90s. Then rinse it with sterile water in a clean bench to make the water activity greater than 0.8. Inoculate 5mL of spore suspension for every 500g of Job's tears. Shake to make the Aspergillus flavus solution evenly cover the surface of the Job's tears. Place it in an incubator at a temperature of 28℃ and a relative humidity of 90% for 5 days to obtain the inoculated Job's tears medicinal material and test its aflatoxin content.

[0017] Preferred,

[0018] The sterilization and drying of the cultured medicinal materials in step (3) are carried out by the following method: the cultured Coix seed medicinal materials are sterilized by radiation with a dose of 4kG of Cobalt-60; then dried at 50℃ for 3h to obtain the dried inoculated Coix seed medicinal materials.

[0019] Preferred,

[0020] The specific method for pulverizing, sieving, and mixing the inoculated coix seed material after drying in step (4) is as follows: Take the dried inoculated coix seed material, soak it in liquid nitrogen for 10 seconds, pulverize it with a blade pulverizer at 13000 r / min, and pass it through a No. 4 sieve to obtain toxin-producing coix seed powder. The concentration can be adjusted with blank coix seed powder. Place it in a V-type mixer and mix it at a speed of 240 r / min for 24 hours to obtain coix seed aflatoxin B1 matrix standard material.

[0021] Preferred,

[0022] The coix seed aflatoxin B1 matrix standard substance described in step (5) is dispensed, sterilized, and stored. The specific method is as follows: In a dry and clean environment, the coix seed aflatoxin B1 matrix standard substance is packed into a brown glass bottle with an inner cap, and vacuum-sealed with an outer aluminum foil bag. Each bottle contains 15g, which is sterilized by radiation with a dose of 4kG of cobalt-60 and stored in a low temperature environment at -20℃ in the dark.

[0023] A method for determining the value of aflatoxin B1 matrix standard material in coix seed, wherein the method is: detection is performed using an isotope internal standard combined with ultra-high performance liquid chromatography-tandem mass spectrometry.

[0024] The method of combining isotope internal standard with ultra-high performance liquid chromatography-tandem mass spectrometry is as follows:

[0025] Chromatographic and mass spectrometry conditions: Waters HSS T3 column, 100×2.1 mm, 1.8 μm; mobile phase A: 0.05-0.15% formic acid aqueous solution; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions:

[0026]

[0027] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0028]

[0029] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ;

[0030] Preparation of the test solution: Accurately weigh 3-5 g of coix seed powder, place it in an Erlenmeyer flask, add 1 g of sodium chloride, and accurately add 25 mL of 60-80% methanol solution. Stir at a frequency of 180 r·min.-1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1 Centrifuge for 5-10 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 14 μL of AFB to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0031] Determination method: Accurately pipette 3-10 μL of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0032] Preferred,

[0033] The method of combining isotope internal standard with ultra-high performance liquid chromatography-tandem mass spectrometry is as follows:

[0034] Chromatographic and mass spectrometry conditions: Waters HSS T3 column, 100×2.1 mm, 1.8 μm; mobile phase A: 0.08–0.12% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions:

[0035]

[0036] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0037]

[0038] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ;

[0039] Preparation of the test solution: Accurately weigh 4-5 g of coix seed powder, place it in an Erlenmeyer flask, add 1 g of sodium chloride, and accurately add 25 mL of 65-75% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1 Centrifuge for 5-8 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 14 μL of AFB to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0040] Determination method: Accurately pipette 3-8 μL of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0041] Further preferred,

[0042] Chromatographic and mass spectrometry conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 μm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions:

[0043]

[0044] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0045]

[0046] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. - 1;

[0047] Preparation of the test solution: Accurately weigh 5g of coix seed powder and place it in an Erlenmeyer flask. Add 1g of sodium chloride and accurately add 25mL of 70% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1 Centrifuge for 5 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0048] Determination method: Accurately pipette 5 μL each of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0049] Beneficial effects:

[0050] 1. This invention provides a method for preparing aflatoxin B1 matrix standard material from Coix seed and a method for determining the value of the prepared material. The method mainly employs a "reverse culture" technique to study the preparation technology of aflatoxin B1 matrix standard material from Coix seed. This preparation of aflatoxin B1 matrix standard material compensates for the deficiencies in the preparation process of AFB1 matrix standard material from traditional Chinese medicine, providing a metrological standard for the detection, calibration, and quality control of aflatoxin in traditional Chinese medicine and agricultural products.

[0051] 2. The preparation method of this invention has passed the examination of the sterilization method of Coix seed raw material, which includes high-pressure steam (121℃, 20min), ultraviolet sterilization (45min on the front and 45min on the back), and ozone (5g·h). -1 Microwave sterilization (650W 90s) was used, and the sterilization doses of gamma ray radiation (0, 2, 4, 6, 8, 10 kGy) were investigated. Finally, the sterilization effect was evaluated according to the method specified in the National Food Safety Standard—Mold and Yeast Count (GB 4789.15-2016). The results showed that microwave sterilization (650W 90s) had a better sterilization effect.

[0052] 3. The preparation method of this invention has been examined through the drying process of Coix seed. The key to ensuring the stability of subsequent standard substances lies in the changes in the Coix seed matrix and its moisture content. 10g of inoculated Coix seed material was sterilized and placed in an oven for 1h, 2h, 3h, 4h, 5h, and 6h, respectively, and the moisture content was measured using a rapid moisture analyzer. The results showed that the drying method that can control the moisture content of the standard substances below 8% is drying at 50℃ for 3 hours.

[0053] 4. The preparation method of this invention has passed the examination of raw material pulverization process. The dried medicinal materials were soaked in liquid nitrogen for 10 seconds and then pulverized using a blade pulverizer (13000 r / min). Using aflatoxin B1 content as an indicator, the uniformity of the powder after passing through a No. 2, No. 3, and No. 4 sieves was examined. The results showed that the RSD value of five parallel measurements of the pulverized coix seed after passing through a No. 2 sieve was 4.31%, the RSD value of five parallel measurements of the pulverized coix seed after passing through a No. 3 sieve was 3.81%, and the RSD value of five parallel measurements of the pulverized coix seed after passing through a No. 4 sieve was 3.79%. Only the particle size passing through the No. 4 sieve met the requirements. Therefore, the No. 4 sieve was selected as the particle size control scheme for the pulverized standard material.

[0054] 5. The preparation method of this invention passed the mixing process test. The powder was taken and placed in a V-type mixer (240 r·min). -1 The powder was mixed thoroughly, and aflatoxin B1 was used as the evaluation index to examine the uniformity of the powder after 6 h, 12 h, and 24 h of mixing. Six samples were taken using the quartering method, and the aflatoxin B1 content was determined, and the RSD value was calculated. The results showed that the RSD value of five parallel determinations after 6 h of mixing was 13.51%, the RSD value of five parallel determinations after 12 h of mixing was 6.04%, and the RSD value of five parallel determinations after 24 h of mixing was 2.34%. Only the RSD value measured after 24 h of mixing was the lowest, meeting the experimental requirements. Therefore, the mixing method of 24 h in a Knox mixer was selected, as the initial uniformity was good.

[0055] 6. The preparation method of this invention passed the test of the minimum sampling amount of the matrix standard material. The RSD value was 12.74% when the sample amount was 1g, and the measurement variability was large (greater than 3%) when the sample amount was 2g, which could not guarantee the accuracy of the measured sample. However, the RSD value of 2.26% when the sample amount was 5g met the detection requirements; therefore, the minimum sampling amount of 5g was selected.

[0056] 7. The preparation method of this invention passed the test of product radiation sterilization. Considering the infeasibility of dry heat sterilization and moist heat sterilization, this experiment adopted radiation sterilization and investigated the dose of gamma-ray radiation sterilization (0, 2, 4, 6, 8, 10 kGy). Finally, the sterilization effect was evaluated according to the national food safety standard—mold and yeast count (GB 4789.15-2016). When the dose of cobalt-60 radiation sterilization was 0 kGy, the colony count of yellow-green mold was 5 × 10⁻⁶. 4 cfu·g -1 When the cobalt-60 radiation sterilization dose was 2 kGy, the colony count of yellow-green mold was 34 cfu·g. -1 When the dose of cobalt-60 radiation sterilization exceeded 4 kGy, the yellow-green mold stopped growing. Considering the degradation rate of AFB1, one-way ANOVA showed no significant difference; therefore, a dose of 4 kGy for cobalt-60 radiation sterilization was selected.

[0057] 8. The stability and homogeneity of the prepared matrix standard material were investigated in this invention. Short-term and long-term stability studies showed that the standard material was stable within 12 months. The finished product of Coix seed was analyzed according to JJF1343-2012 "General Principles and Statistical Principles for Standard Material Value Determination". The AFB1 content was measured using the determination method, and the results were statistically analyzed using one-way ANOVA. The F-value was calculated by the ratio of between-group variance to within-group variance. The F-value was less than the critical value Fc. 0.01 (14,30) indicates that the standard substance has good homogeneity and meets the requirements of the standard substance technical specifications.

[0058] 9. This invention investigated the value determination of the prepared standard substances. To ensure the traceability and accuracy of the determination of the matrix standard substances, this study used an isotope internal standard combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) for detection, and employed a joint determination method involving multiple institutions. The use of UHPLC-MS / MS ensured the accuracy of the determination results. The test results show that the aflatoxin B1 matrix standard substance for Coix seed prepared in this experiment has good homogeneity and stability, meeting the requirements for the preparation of quality control samples in standard substances.

[0059] 10. This invention has investigated the detection method for aflatoxin B1 in Coix seed. Specificity results showed that AFB1 peak shape and separation were good, and analysis of the blank matrix spiked sample indicated that matrix components did not interfere with the detection. Linearity results showed good linearity (r > 0.999). The limit of detection (LOD) was set at a signal-to-noise ratio (S / N) ≥ 3, and the limit of quantitation (LOQ) was set at an S / N ≥ 10. The RSD of the measured recovery was 2.66%, less than 3.0%, which meets the requirements of the 2020 edition of the Chinese Pharmacopoeia. Repeatability results showed that the measured recovery... The RSD of the recovery rate was 2.66%, less than 3.0%, which meets the requirements of the 2020 edition of the Chinese Pharmacopoeia, indicating that the method has good repeatability. The stability test results showed that the test solution was stable within 24 hours. The precision test results showed that the RSD of the peak area of ​​AFB1 was 2.96%, less than 3.0%, indicating that the instrument precision was good. The recovery test results showed that the recovery rate of AFB1 was 92.74% to 109.87%, and the RSD value was 1.58% to 3.51%, indicating that the established method had good accuracy. Attached Figure Description

[0060] Figure 1 MRM diagram of aflatoxin and its internal standard;

[0061] Figure 2 Sterilization effect of coix seed samples treated with different sterilization methods (Note: CK is unsterilized coix seed, CY is coix seed after ozone sterilization, ZY is coix seed after ultraviolet sterilization, GY is coix seed after high pressure steam sterilization, and WB is coix seed after microwave sterilization).

[0062] Figure 3 Stereoscopic images of Job's tears samples after treatment with different sterilization methods (Note: CK is unsterilized Job's tears, CY is Job's tears after ozone sterilization, ZY is Job's tears after ultraviolet sterilization, GY is Job's tears after high-pressure steam sterilization, and 650W (90s) is Job's tears after microwave sterilization).

[0063] Figure 4 Figure showing the results of the investigation into the drying process;

[0064] Figure 5 The results of the investigation of sterilization conditions are shown in the figure. Detailed Implementation

[0065] Preparation method of Example 1

[0066] (1) Preparation of Aspergillus flavus spore suspension: In a sterile operating table, Aspergillus flavus was inoculated into PDA medium and cultured for 3 days, then inoculated into PDA medium again and cultured in a light incubator for 5 days. The Aspergillus flavus spore suspension was collected, and the spore concentration was adjusted to 10 using a hemocytometer. 5CFU·mL -1 A suspension of Aspergillus flavus spores was obtained.

[0067] (2) Inoculation of Coix Seed: Take Coix Seed and sterilize it with microwave at 650w for 90s. Then rinse it with sterile water in a clean bench to make the water activity greater than 0.8. Inoculate 5mL of spore suspension for every 500g of Coix Seed, shake it to make the Aspergillus flavus solution evenly cover the surface of Coix Seed, and place it in an incubator at 28℃ and 90% relative humidity for 5 days to obtain inoculated Coix Seed. Then test its aflatoxin content.

[0068] (3) Sterilization and drying of the cultured medicinal materials: The cultured Coix seed medicinal materials were sterilized by radiation with a dose of 4kG of cobalt-60; then dried at 50℃ for 3h to obtain the dried inoculated Coix seed medicinal materials.

[0069] (4) After the inoculated Coix seed material is dried, it is crushed, sieved and mixed. 5. Take the dried inoculated Coix seed material, soak it in liquid nitrogen for 10 seconds, crush it with a knife-type pulverizer at 13000r / min, and pass it through a No. 4 sieve to obtain toxin-producing Coix seed powder. The concentration can be adjusted with blank Coix seed powder. Place it in a V-type mixer and mix it at a speed of 240r / min for 24 hours to obtain Coix seed aflatoxin B1 matrix standard material.

[0070] (5) Dispensing, sterilizing, and storing the aflatoxin B1 matrix standard material from coix seed; 6. In a dry and clean environment, pack the aflatoxin B1 matrix standard material from coix seed into brown glass bottles with inner caps, vacuum seal them with an outer aluminum foil bag, each bottle containing 15g, sterilize with 4kG of cobalt-60 radiation, and store in a light-protected environment at -20℃.

[0071] Example 2: Value Setting Method

[0072] Chromatographic and mass spectrometry conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 μm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃, injection volume 5μL;

[0073] The gradient elution conditions are as follows:

[0074]

[0075] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0076]

[0077] Note:" 1"Indicates quantitative ions, " 2 "Indicates qualitative ions"

[0078] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ;

[0079] Preparation of the test solution: Accurately weigh 5.0 g of coix seed powder and place it in an Erlenmeyer flask. Add 1 g of sodium chloride and accurately add 25 mL of 70% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1 Centrifuge for 5 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0080] Determination method: Accurately pipette 5 μl of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0081] Example 3: Fixed Value Method

[0082] Chromatographic and mass spectrometric conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 μm; mobile phase A: 0.05% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃, injection volume 5μL;

[0083] The gradient elution conditions are as follows:

[0084]

[0085] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0086]

[0087] Note: "1" indicates quantitative ions, "2" indicates qualitative ions.

[0088] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ;

[0089] Preparation of the test solution: Accurately weigh 3.0 g of coix seed powder, place it in an Erlenmeyer flask, add 1 g of sodium chloride, and accurately add 25 mL of 60% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1 Centrifuge for 8 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0090] Determination method: Accurately pipette 3 μl of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0091] Example 4: Value Setting Method

[0092] Chromatographic and mass spectrometry conditions: Waters HSS T3 column, 100×2.1 mm, 1.8 μm; mobile phase A: 0.15% formic acid aqueous solution; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃, injection volume 5μL;

[0093] The gradient elution conditions are as follows:

[0094]

[0095] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0096]

[0097] Note:" 1 "Indicates quantitative ions, " 2 "Indicates qualitative ions"

[0098] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ;

[0099] Preparation of the test solution: Accurately weigh 3.0 g of coix seed powder, place it in an Erlenmeyer flask, add 1 g of sodium chloride, and accurately add 25 mL of 80% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1Centrifuge for 10 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0100] Determination method: Accurately pipette 10 μl of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0101] Example 5: Value Setting Method

[0102] Chromatographic and mass spectrometry conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 μm; mobile phase A: 0.08% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions:

[0103]

[0104] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0105]

[0106] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ;

[0107] Preparation of the test solution: Accurately weigh 5g of coix seed powder and place it in an Erlenmeyer flask. Add 1g of sodium chloride and accurately add 25mL of 65% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1 Centrifuge for 6 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure

[0108] [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0109] Determination method: Accurately pipette 4 μL each of the mixed reference solution and the test solution and inject them into an ultra-high performance liquid chromatography-tandem mass spectrometer for determination and calculation to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0110] Example 6: Fixed Value Method

[0111] Chromatographic and mass spectrometry conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 μm; mobile phase A: 0.12% formic acid aqueous solution; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions:

[0112]

[0113] Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode.

[0114]

[0115] Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ;

[0116] Preparation of the test solution: Accurately weigh 3g of coix seed powder and place it in an Erlenmeyer flask. Add 1g of sodium chloride and accurately add 25mL of 75% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 hour at a frequency of 5000 r·min -1 Centrifuge for 8 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0117] Determination method: Accurately pipette 8 μL each of the mixed reference solution and the test solution and inject them into an ultra-high performance liquid chromatography-tandem mass spectrometer for determination and calculation to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

[0118] To further verify the effectiveness of the present invention, the inventors conducted a series of verification experiments, excerpts of which are as follows:

[0119] Experimental Example

[0120] 1. Instruments and reagents

[0121] 1.1 Instruments

[0122] Ultra-high performance liquid chromatograph (Shimadzu Corporation, Japan, LC-30AD), mass spectrometer (AB4500, American company), vortex mixer (Tianjin Ruicheng Technology Co., Ltd., MS200), V-type mixer (Zhengzhou Great Wall Science & Industry Trade Co., Ltd., SHB-III), constant temperature and humidity incubator (Chengdu Alpha Technology Co., Ltd., SDS-3).

[0123] 1.2 Reagents

[0124] AFB1 standard (batch number 0291904), AFB1 internal standard solution (batch number 2A00I28), and aflatoxin immunoaffinity column (batch number 2J1J24) were purchased from Qingdao Pribang Biotechnology Co., Ltd., all with a purity greater than 99%; methanol (batch number 200343), acetonitrile (batch number 202888), and formic acid (batch number 166511) were purchased from Fisher Scientific, Inc., USA; and Aspergillus flavus strain (batch number 336678) was purchased from Chengdu Efa Biotechnology Co., Ltd.

[0125] 2 methods

[0126] 2.1 Establishment of a method for detecting aflatoxin B1 in Coix seed based on isotope internal standard

[0127] 2.1.1 Establishment of detection methods

[0128] 2.1.1.1 Chromatographic and Mass Spectrometry Conditions

[0129] The chromatographic column was a Waters HSS T3 (100 × 2.1 mm, 1.8 μm); the mobile phase was 0.1% formic acid-water (A) and acetonitrile (B), with gradient elution. The elution program is shown in Table 1. The flow rate was 0.4 mL / min. -1 Column temperature 40℃, injection volume 5μL.

[0130] Table 1 Gradient elution conditions for UPLC-MS / MS

[0131]

[0132] The mass spectrometry parameters of each compound are shown in Table 2. ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) were used to acquire the data.

[0133] Table 2 Mass spectrometry parameters of AFB1 and its internal standard

[0134]

[0135] Note:" 1 "Indicates quantitative ions, " 2 "Indicates a qualitative ion.

[0136] 2.1.1.2 Preparation of mixed reference solution

[0137] Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL, respectively. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 .

[0138] 2.1.1.3 Preparation of the test solution

[0139] Accurately weigh approximately 5.0 g of the test sample powder and place it in an Erlenmeyer flask. Add 1 g of sodium chloride and 25 mL of 70% methanol solution. Extract by shaking for 1 h (180 rpm). -1 Centrifuge for 5 minutes (5000 r·min). -1 Accurately measure 3 mL of the supernatant and place it in a 20 mL volumetric flask. Dilute to the mark with water, shake well, and filter through a 0.22 μm microporous membrane. Accurately measure 10 mL of the filtrate and pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min. Elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, and then elute with an appropriate amount of methanol. Collect the eluent, place it in a 2 mL volumetric flask, dilute to the mark with methanol, shake well, and filter through a 0.22 μm microporous membrane. Accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well.

[0140] 2.1.2 Validation of the detection method

[0141] 2.1.2.1 Specificity Examination

[0142] Under the conditions specified in section “2.1.1”, the MRM chromatograms of aflatoxin B1 and its internal standard are shown below. Figure 1 The results showed that AFB1 had good peak shape and separation, while the analysis of the blank matrix spiked sample showed that the matrix components did not interfere with the detection.

[0143] 2.1.2.2 Results of the examination of linear relationship

[0144] The standard solution prepared in "2.1.1.2" was diluted with blank matrix solution to a concentration gradient of 0.4, 0.8, 1.6, 2.4, and 3.2 ng / mL. The standard solution was then detected according to the conditions in "2.1.1.1". Linear regression was performed with concentration as the abscissa and peak area as the ordinate. The results showed a good linear relationship (r > 0.999). The limit of detection (LOD) was set at a signal-to-noise ratio (S / N) ≥ 3, and the limit of quantitation (LOQ) was set at an S / N ≥ 10, as shown in Table 3.

[0145] Table 3. Linearity, Limit of Detection, and Limit of Quantitation of AFB1

[0146]

[0147] 2.1.2.3 Repeatability Test

[0148] Accurately weigh approximately 5g of blank Coix seed sample and accurately add 0.2mL of mixed standard solution. Prepare six test solutions according to the method in section "2.1.1.3". Detect the AFB1 under the conditions in section "2.1.1.1" and calculate the RSD value of the recovery rate. The RSD of the measured recovery rate is 2.66%, which is less than 3.0%. The results are shown in Table 4 and meet the requirements of the 2020 edition of the Chinese Pharmacopoeia, indicating that the method has good repeatability.

[0149] Table 4 Results of Repeatability Testing

[0150]

[0151] 2.1.2.4 Stability Assessment

[0152] The same test solution was placed at room temperature for 0, 2, 4, 6, 12, and 24 hours, and then tested according to the conditions in section "2.1.1.1". The RSD value of the AFB1 content was calculated. The measured RSD value of AFB1 content was 2.11%, which is less than 3.0%. The results are shown in Table 5, which meets the experimental requirements and indicates that the test solution has good stability within 24 hours.

[0153] Table 5. Results of the stability test

[0154]

[0155] 2.1.2.5 Precision Examination

[0156] Take the reference solution from section "2.1.1.2" and inject it six times consecutively as described in section "2.1.1.1". Calculate the RSD value of the peak area for each toxin. The RSD value of the AFB1 peak area was found to be 2.96%, which is less than 3.0%. The results are shown in Table 6, indicating that the instrument has good precision.

[0157] Table 6. Precision test results

[0158]

[0159] 2.1.2.6 Recovery Test

[0160] Reference solutions were added at three levels: low (200 μL), medium (400 μL), and high (800 μL). Test solutions were prepared and measured according to the method described in section "2.1.1.3". The recoveries and relative standard deviations (RSDs) of AFB1 were calculated. The recoveries of AFB1 ranged from 92.74% to 109.87%, and the RSDs ranged from 1.58% to 3.51%, indicating that the established method was accurate. The results are shown in Table 7.

[0161] Table 7 Results of sample recovery rate

[0162]

[0163] 2.2 Research on the production process of aflatoxin B1 matrix standard material in coix seed

[0164] 2.2.1 Investigation on sterilization methods for Job's tears raw materials

[0165] Sterilization processes must remove or kill microorganisms, ensure the quality stability of the substrate material, and also consider sterilization costs. This experiment investigated high-pressure steam (121℃, 20 min), ultraviolet sterilization (45 min for the front, 45 min for the back), and ozone (5 g·h⁻¹). -1 Microwave (650W 90s) was used for radiation sterilization, and the doses of cobalt-60 radiation sterilization (0, 2, 4, 6, 8, 10 kGy) were investigated. Finally, the sterilization effect was evaluated according to the method specified in the National Food Safety Standard - Mold and Yeast Count (GB 4789.15-2016).

[0166] Results: Both cobalt-60 radiation sterilization (8 kGy) and microwave sterilization with different gradients (650 W, 90 s, 150 s; 910 W, 120 s; 1300 W, 90 s) achieved 100% sterilization efficiency (see Table 8). However, microwave sterilization (650 W, 150 s; 910 W, 120 s; 1300 W, 90 s) caused changes in the properties of the raw materials (see Table 8). Figure 2 , Figure 3 From the perspective of sterilization effect, both cobalt-60 radiation sterilization (8kGy) and microwave sterilization (650W, 90s) can be used for sterilizing Job's tears raw materials. However, cobalt-60 radiation sterilization is relatively expensive. Therefore, microwave sterilization (650W, 90s) was chosen as the sterilization method for the production of Job's tears aflatoxin B1 matrix standard material.

[0167] Table 8. Parameters and effects of different sterilization methods

[0168]

[0169]

[0170] 2.2.2 Activation of Aspergillus flavus and preparation of its spore suspension

[0171] This study cultured Aspergillus flavus by purchasing the necessary strains. In a sterile environment, the Aspergillus flavus was inoculated into PDA medium and cultured for 2-3 days, then aliquoted and stored. It was then re-inoculated into PDA medium and cultured in a light incubator for 5 days. The Aspergillus flavus spores were collected, and the spore concentration was adjusted to 10⁻⁶ using a hemocytometer. 5 CFU·mL -1 ,spare.

[0172] 2.2.3 Investigation of Aspergillus flavus culture conditions

[0173] Take 40g (3 portions) of Job's tears, sterilize them, and then rinse them with sterile water in a clean bench to make the water activity (aw) greater than 0.8. Inoculate each 500g of Job's tears with 5mL of spore suspension, shake to make the Aspergillus flavus solution evenly cover the surface, and incubate in an incubator at 28℃ and 90% relative humidity. Then, take 10g of the sample at 2, 4, 6 and 8 days to detect the content of aflatoxin.

[0174] Results: After culturing for 2 days, the toxin production of Aspergillus flavus on Coix seed was 1.34 μg·kg⁻¹. -1 The toxin production was 2.41 μg·kg after 4 days of cultivation. -1 After 6 days of cultivation, the toxin production was 10.25 μg·kg⁻¹. -1 After 8 days of cultivation, the toxin production was 103.11 μg·kg⁻¹. -1 This indicates that the aflatoxin production rate after 6 days of cultivation is too fast and unsuitable for large-scale production, while the production rate after 2 or 4 days of cultivation is too slow and inefficient. Therefore, Job's tears inoculated with Aspergillus flavus and cultured for 5 days were selected as the substrate for research and development.

[0175] 2.2.4 Investigation of raw material drying time

[0176] Take 10g of inoculated Coix seed material, sterilize it, place it in an oven, and dry it for 1h, 2h, 3h, 4h, 5h, and 6h. Then, use a rapid moisture analyzer to detect the moisture content.

[0177] Results: The drying process needs to consider changes in the coix seed matrix and its moisture content, which is crucial to ensuring the stability of subsequent standard substances. When the drying temperature is 50℃ and the drying time is 3 hours, the moisture content of the standard substances can be controlled below 8%, and the contents of the seven glycerides can be controlled within the range of 3.5-3.7%, i.e., within a range with no significant differences. (See...) Figure 4 Therefore, the drying method chosen is to dry at 50℃ for 3 hours.

[0178] 2.2.5 Investigation of raw material crushing process

[0179] After drying, the medicinal materials were soaked in liquid nitrogen for 10 seconds and then pulverized using a blade grinder (13000 r / min). The uniformity of the powder after passing through No. 2, No. 3 and No. 4 sieves was examined, with the aflatoxin B1 content as the indicator.

[0180] Results: The particle size and flowability after pulverization are key to ensuring the homogeneity of the standard material. Table 9 shows that the RSD value of five parallel determinations of contaminated coix seed after pulverization through a No. 2 sieve was 4.31%, the RSD value of five parallel determinations of contaminated coix seed after pulverization through a No. 3 sieve was 3.81%, and the RSD value of five parallel determinations of contaminated coix seed after pulverization through a No. 4 sieve was 3.79%. Only the particle size passing through the No. 4 sieve met the requirements. Therefore, the No. 4 sieve was selected as the particle size control method for the pulverized standard material.

[0181] Table 9 Investigation of particle size control conditions

[0182]

[0183] 2.2.6 Investigation of raw material powder mixing process and preliminary uniformity

[0184] Take the toxin-producing coix seed powder that has passed through a No. 4 sieve. Adjust its concentration using blank coix seed powder as needed, and place it in a V-type mixer (240 rpm). -1 Mix thoroughly, using aflatoxin B1 as the evaluation index, and examine the uniformity of the powder after 6h, 12h, and 24h of mixing. Take 6 samples using the quartering method, and test the aflatoxin B1 content according to the method under "2.1.1", and calculate the RSD value.

[0185] Results: As shown in Table 10, the RSD value of five parallel measurements after 6 hours of mixing was 13.51%, the RSD value of five parallel measurements after 12 hours of mixing was 6.04%, and the RSD value of five parallel measurements after 24 hours of mixing was 2.34%. Only the RSD value measured after 24 hours of mixing was the lowest, which meets the experimental requirements. Therefore, the mixing method selected was the 24-hour mixing mode in the Daoshi mixer, which resulted in good initial homogeneity.

[0186] Table 10 Results of the investigation on the mixing process

[0187]

[0188] 2.2.7 Dispensing of matrix standard materials

[0189] To ensure the stability of the samples, after the initial homogeneity was qualified, the powder was placed in brown glass bottles with inner caps in a dry and clean environment, and then vacuum-sealed in an outer aluminum foil bag. Each bottle contained 15g, for a total of 100 bottles. The bottles were stored at -20℃ in a light-proof environment.

[0190] Results: The minimum sample size is a key parameter for ensuring the homogeneity of the standard reference material. For this standard reference material, the RSD value of the candidate sample was 12.74% when the sample size was 1g. When the sample size was 2g, the measurement variability was significant, exceeding 3%, which could not guarantee the accuracy of the measured samples. However, the RSD value of 2.26% when the sample size was 5g met the detection requirements (see Table 11). Therefore, the minimum sample size of 5g was selected.

[0191] Table 11 Results of the investigation on minimum sampling size

[0192]

[0193] 2.2.8 Investigation of sterilization methods for matrix standard substances

[0194] Considering the impracticality of dry heat sterilization and moist heat sterilization, this experiment adopted radiation sterilization for the bottled products and investigated the dosage of gamma ray radiation sterilization (0, 2, 4, 6, 8, 10 kGy). Finally, the sterilization effect was evaluated according to the national food safety standard for mold and yeast count (GB 4789.15-2016).

[0195] Results: Table 12 shows that when the cobalt-60 radiation sterilization dose was 0 kGy, the colony count of yellow-green mold was 5 × 10⁻⁶. 4 cfu·g -1 When the cobalt-60 radiation sterilization dose was 2 kGy, the colony count of yellow-green mold was 34 cfu·g. -1 When the dose of cobalt-60 radiation sterilization exceeds 4 kGy, the yellow-green mold stops growing. The degradation rate of AFB1 should also be considered; see [reference needed]. Figure 5 One-way ANOVA showed no significant difference, therefore the recommended dose for cobalt-60 radiation sterilization was 4 kGy.

[0196] Table 12 Results of Cobalt-60 Radiation Sterilization

[0197]

[0198] 2.2.9 Inspection of the homogeneity of the matrix standard material

[0199] According to JJF1343-2012 "General Principles and Statistical Principles for Determining Standard Reference Values", this study randomly selected 15 samples from 100 bottles of the smallest packaging unit (15g / bottle) of Coix Seed, and took 3 subsamples from each bottle. The AFB1 content in the samples was determined according to the determination method in section "2.1". The results were statistically analyzed by one-way ANOVA.

[0200] The formula for uniformity assessment is as follows:

[0201] m units are randomly selected from the total number of standard reference units for measurement, and each unit is measured n times.

[0202]

[0203]

[0204]

[0205] γ1=m-1

[0206] (3-4)

[0207] γ2=Nm

[0208] (3-5)

[0209]

[0210]

[0211]

[0212] Note: γ1 is the overall mean; N is the total number of measurements; Q1 is the sum of squares of differences between groups; Q2 is the sum of squares of differences within groups; γ1 is the degrees of freedom between groups; γ2 is the degrees of freedom within groups. The variance between groups, The variance is the within-group variance.

[0213] Based on the degrees of freedom (γ1, γ2) and the given significance level (0.01), F(γ1, γ2) is obtained from the F critical value table. If F < Fα, then there is no significant difference between the data groups.

[0214] Results: The results of the homogeneity test are shown in Table 13. The homogeneity test was performed using one-way ANOVA. The F-value was calculated as the ratio of between-group variance to within-group variance. The F-value was less than the critical value Fc. 0.01 (14, 30) indicates that the standard substance has good homogeneity and meets the requirements of the standard substance technical specifications.

[0215] Table 13 Results of the uniformity test

[0216]

[0217]

[0218] 2.2.10 Short-term stability study of matrix reference materials

[0219] Short-term stability mainly examines the stability of substances during transportation. In this study, high-temperature conditions that may occur during transportation were simulated in an oven at 50°C, and the substances were stored at room temperature and in a 4°C refrigerator to investigate the effect of temperature on the candidate matrix reference materials. Twelve bottles of candidate reference materials were randomly selected and placed therein. Three samples were taken out on the 0th day, 2nd day, 7th day, and 14th day respectively, and the contents of AFB1 in these samples were measured. The measurement result at each time point is the average of the measurement results of the three samples at that time point.

[0220] Results: As can be seen from Table 14, based on the relative standard deviation of the linear model and judged by the t-test, the |β1| values under the conditions of -20°C, 4°C, and 50°C are all less than t (0.05,3) ·s(β1), which indicates that the reference material is stable within 14 days under the three investigated temperature conditions.

[0221] Table 14 Results of the investigation of short-term stability

[0222]

[0223]

[0224] 2.2.11 Investigation of the long-term stability of matrix reference materials

[0225] Stability is a basic property of reference materials, which is used to describe the property of reference materials changing over time, that is, to describe the time distribution characteristics of the characteristics of reference materials. In this study, 20 bottles were randomly selected from 100 prepared minimum packaging units (15 g / bottle) and stored at -20°C. At 5 time points of the 0th month, 1st month, 3rd month, 6th month, and 12th month, 3 bottles were randomly selected each time, and the contents of AFB1 in these samples were measured uniformly according to the method in "3". The measurement result at each time point is the average of the measurement results of the three samples at that time point.

[0226] Results: As can be seen from Table 15, based on the relative standard deviation of the linear model and judged by the t-test, │β1│ < t(0.05,3)·s(β1) is obtained, which indicates that the reference material is stable within 12 months.

[0227] Table 15 Results of the investigation of long-term stability

[0228]

[0229] 2.2.12 Certification of aflatoxin B1 matrix reference materials

[0230] To ensure the traceability and accuracy of the matrix standard reference material values, this study employed an isotope internal standard combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) for detection, and used a multi-institutional collaborative method for value determination. The Guizhou University of Traditional Chinese Medicine's Institute of Traditional Chinese and Ethnic Medicine Resources served as the organizing unit, screening laboratories qualified for aflatoxin B1 testing. Five laboratories were ultimately selected as collaborators for this value determination. The participating institutions in the AFB1 value determination of Coix seed are: 1. Guizhou Bolian Technology Testing Co., Ltd. (Qualification Certificate No.: 162412050352); 2. Anhui Microspection Testing Technology Co., Ltd. (Qualification Certificate No.: 211203141847); 3. Guangdong Hanchao Traditional Chinese Medicine Technology Co., Ltd. (Qualification Certificate No.: 202119135954); 4. Guizhou Zhongke Inspection and Testing Co., Ltd. (Qualification Certificate No.: 202418141527); 5. Guizhou Lvhuan Technology Testing Co., Ltd. (Qualification Certificate No.: 212400341669).

[0231] Six aliquots of sample were randomly distributed to each laboratory. Each sample was measured twice, and each laboratory provided at least six independent replicate measurements. The obtained data were analyzed using the Dixon test, Grubbs test, and Cochrane test to remove outliers, questionable values, and to determine the precision of the data between laboratories, respectively. The average of the processed data meeting the criteria was calculated as the final value.

[0232] Results: The values ​​of the reference materials were determined according to JJF 1343-2012 "General Principles and Statistical Principles for the Determination of Reference Materials," using a proven accurate method, and were determined collaboratively by multiple laboratories. The determination method adopted was the internationally accepted and highly accurate isotope dilution high-performance liquid chromatography-tandem mass spectrometry method, and was determined collaboratively by 5 laboratories. The results are shown in Table 16.

[0233] Table 16 Results of joint determination by multiple laboratories (μg·kg⁻¹) 1 )

[0234]

[0235] Result: Therefore, the average value of each group of data in Table 16 was recalculated to obtain the overall average value, which is the standard value, and the result is 11.4 μg·kg. -1 .

[0236] 2.2.13 Evaluation of uncertainty of matrix standard material

[0237] Uncertainty represents the error range of a standard substance, mainly including the uncertainty u caused by homogeneity. bb Uncertainty u arising from stability s and the uncertainty of a constant value u char.

[0238]

[0239] The formula for calculating the uncertainty caused by homogeneity is as follows:

[0240]

[0241] The formula for calculating the uncertainty introduced by stability is as follows:

[0242] u s =s(β1)×X (3-11)

[0243] Note: In the formula, s(β1) is the standard deviation of β1, and X is the time for stability testing.

[0244] Uncertainty of a constant value u char It consists of two parts. One part is the uncertainty calculated statistically from the values ​​measured by each laboratory under specified measurement conditions; this is the uncertainty of the determination process and belongs to Type A uncertainty. The other part is the uncertainty calculated by analyzing the factors affecting the measurement process; this is the measurement uncertainty and belongs to Type B uncertainty. The calculation formula is as follows:

[0245]

[0246] Result: The relative standard uncertainty caused by homogeneity, the relative standard uncertainty caused by stability, and the relative standard uncertainty caused by the determination process are combined according to the following formula to obtain the composite relative standard uncertainty of the standard substance.

[0247]

[0248] (1) Uncertainty introduced by homogeneity (u) bb )

[0249] The uncertainty introduced by homogeneity is given by Equation 3-10.

[0250]

[0251] From the data in Table 13, the uncertainty introduced by homogeneity is u. bb The relative uncertainty introduced by homogeneity is 0.63, and the relative uncertainty is u. bb (rel) = 0.055.

[0252] (2) Uncertainty introduced by stability (u) s1 u ss )

[0253] The uncertainty introduced by stability is obtained from Equation 3-11.

[0254] usl =s(β1) 长期 ×X=0.045×12=0.54

[0255] u ss =s(β1) 短期 ×X=0.055×12=0.66

[0256] From the data in Tables 14 and 15, the uncertainty introduced by long-term stability is 0.54, and the uncertainty introduced by short-term stability is 0.66. The relative standard uncertainty introduced by long-term stability is u. sl (rel) = 0.048, and the relative standard uncertainty introduced by short-term stability is u. ss (rel) = 0.059.

[0257] (3) Uncertainty introduced by the constant value (u) char )

[0258]

[0259] by u A =0.54, u B =0.34, u A (rel) = 0.047,

[0260] u B (rel) = 0.030, therefore u char =0.64.

[0261] (4) Combined standard uncertainty (u) CRM )

[0262] From formula 3-9, we get

[0263]

[0264] (5) Expanded uncertainty (U)

[0265] U = k × u CRM =2 × 0.65 = 1.29 μg·kg -1

[0266] k is the coverage factor, which is usually taken as k=2.

[0267] Therefore, the measured result of AFB1 in coix seed in this study was reported as (11.4±1.3) μg·kg. -1 k = 2.

[0268] 3 Discussion

[0269] The aflatoxin B1 matrix standard material developed in this experiment for Coix seed was prepared by inoculating Coix seed with Aspergillus flavus to ensure the homogeneity and stability of AFB1 in the matrix. The homogeneity and stability of AFB1 in the matrix were improved through investigation of different sterilization methods, pulverization processes, and mixing processes. This preparation process overcomes the deficiencies in the preparation process of matrix standard materials for AB1 in traditional Chinese medicine. Meanwhile, the determination method used was ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), ensuring the accuracy of the determination results. The test results show that the aflatoxin B1 matrix standard material for Coix seed developed in this experiment has good homogeneity and stability, meeting the requirements for the preparation of quality control samples in standard materials.

[0270] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a coix seed aflatoxin B1 matrix standard, characterized in that, Includes the following steps: (1) Preparation of Aspergillus flavus spore suspension: In a sterile operating table, Aspergillus flavus was inoculated into PDA medium and cultured for 3 days, then inoculated into PDA medium again and cultured in a light incubator for 5 days. The Aspergillus flavus spore suspension was collected, and the spore concentration was adjusted to 10 using a hemocytometer. 5 CFU·mL -1 A suspension of Aspergillus flavus spores was obtained; (2) Inoculation of Coix Seed: Take Coix Seed and sterilize it with microwave at 650w for 90s. Then rinse it with sterile water in a clean bench to make the water activity greater than 0.

8. Inoculate 5mL of spore suspension for every 500g of Coix Seed, shake it to make the Aspergillus flavus solution evenly cover the surface of Coix Seed, and place it in an incubator at 28℃ and 90% relative humidity for 5 days to obtain inoculated Coix Seed. Then test its aflatoxin content. (3) Sterilization and drying of inoculated Coix seed medicinal materials: The inoculated Coix seed medicinal materials were sterilized by radiation with a dose of 4 kG of cobalt-60; then dried at 50℃ for 3 h to obtain dried inoculated Coix seed medicinal materials; (4) Crushing, sieving and mixing of inoculated Coix seed material: Take the dried inoculated Coix seed material, soak it in liquid nitrogen for 10 seconds, crush it with a blade pulverizer at 13000 r / min, pass it through a No. 4 sieve to obtain toxin-producing Coix seed powder, adjust its concentration with blank Coix seed powder, place it in a V-type mixer, mix it at a speed of 240 r / min for 24 hours to obtain Coix seed aflatoxin B1 matrix standard material; (5) Dispensing, sterilization and storage of aflatoxin B1 matrix standard material from coix seed: In a dry and clean environment, the aflatoxin B1 matrix standard material from coix seed is dispensed into brown glass bottles with inner caps, and vacuum-sealed with an outer aluminum foil bag. Each bottle contains 15 g, which is sterilized by radiation with a dose of 4 kG of cobalt-60 and stored in a light-protected environment at -20℃.

2. A method for determining the value of the aflatoxin B1 matrix standard material prepared from coix seed according to claim 1, characterized in that, The determination method is as follows: detection is performed using an isotope internal standard combined with ultra-high performance liquid chromatography-tandem mass spectrometry. The method of combining isotope internal standard with ultra-high performance liquid chromatography-tandem mass spectrometry is as follows: Chromatographic and mass spectrometric conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 µm; mobile phase A: 0.05–0.15% formic acid aqueous solution; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions: ； Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode. ； Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ; Preparation of the test solution: Accurately weigh 3-5 g of coix seed powder, place it in an Erlenmeyer flask, add 1 g of sodium chloride, and accurately add 25 mL of 60-80% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 h at a frequency of 5000 r·min -1 Centrifuge for 5-10 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well to obtain the final product. Determination method: Accurately pipette 3-10 μL of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

3. The method for setting a value according to claim 2, characterized in that, The method of combining isotope internal standard with ultra-high performance liquid chromatography-tandem mass spectrometry is as follows: Chromatographic and mass spectrometric conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 µm; mobile phase A: 0.08–0.12% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions: ； Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode. ； Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ; Preparation of the test solution: Accurately weigh 4-5 g of coix seed powder, place it in an Erlenmeyer flask, add 1 g of sodium chloride, and accurately add 25 mL of 65-75% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 h at a frequency of 5000 r·min -1 Centrifuge for 5-8 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well to obtain the final product. Determination method: Accurately pipette 3-8 μL of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

4. The method for setting a value according to claim 3, characterized in that, The method of combining isotope internal standard with ultra-high performance liquid chromatography-tandem mass spectrometry is as follows: Chromatographic and mass spectrometric conditions: Waters HSS T3 column, 100 × 2.1 mm, 1.8 µm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; flow rate: 0.4 mL / min. -1 Column temperature 40℃; gradient elution conditions: ； Mass spectrometry parameters for each compound were acquired using an ESI ion source, positive ion mode scanning, and multiple reaction monitoring (MRM) mode. ； Preparation of mixed reference solution: Accurately measure an appropriate amount of aflatoxin B1 reference standard, dissolve it in methanol, and prepare a solution of 100 ng / kg. -1 AFB1 stock solution; accurately pipette 5, 10, 15, 20, 25, 30, 35, and 40 μL of AFB1 stock solution into 1 mL volumetric flasks, and add 4 μL of […] to each flask. 13 C 17 The AFB1 internal standard solution was diluted to the mark with methanol to prepare concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 ng / mL. -1 Standard solutions, each standard solution contains [ 13 C 17 The concentration of 1-AFB1 was 2 ng / mL. -1 ; Preparation of the test solution: Accurately weigh 5 g of coix seed powder and place it in an Erlenmeyer flask. Add 1 g of sodium chloride and accurately add 25 mL of 70% methanol solution. Stir at a frequency of 180 r·min. -1 Oscillating extraction for 1 h at a frequency of 5000 r·min -1 Centrifuge for 5 minutes, accurately measure 3 mL of the supernatant, place it in a 20 mL volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure 10 mL of the filtrate, pass it through an immunoaffinity column for total aflatoxin concentration at a flow rate of 3 mL / min, elute with 10 mL of water, discard the eluent, allow air to enter the column, expel the water from the column, elute again with an appropriate amount of methanol, collect the eluent, place it in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane; accurately measure [ 13 C 17 Add 4 μL of AFB1 to a high-volume vial, dilute to 1 mL with the filtrate, and shake well. Determination method: Accurately pipette 5 μL each of the mixed reference solution and the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometer, measure and calculate to obtain the value of the aflatoxin B1 matrix standard substance in coix seed.

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