Rapid determination method for cyanide content in sorghum

Abstract

The rapid determination method for the cyanide content in the sorghum is efficient, accurate, simple and convenient in detection, adopts a full-automatic flow injection analyzer to rapidly determine the cyanide content in the sorghum, and comprises the following steps: S10, a pretreatment step: adding an inorganic acid solution into a crushed sorghum sample to be detected to hydrolyze and extract the cyanide in the sorghum, the method comprises the following steps: uniformly mixing the raw materials, performing ultrasonic treatment at a first temperature for a first time, cooling to room temperature, adding an alkali solution, and uniformly mixing, the first temperature is higher than the room temperature, so as to obtain a supernatant which is an acid hydrolysis extracting solution, and the supernatant is used as a basic stock solution for preparing a sample introduction solution of a full-automatic flow injection analyzer. The pretreatment ensures the extraction effect of the cyanide in the sorghum, a distillation device does not need to be arranged in the pretreatment process, the determination is simple, the accuracy is higher, the consumed time is short, and the efficiency is high.

Description

Technical Field

[0001] This application relates to the technical field of detecting the cyanide content in sorghum, and particularly to a rapid determination method for the cyanide content in sorghum. Background Art

[0002] Cyanide is one of the main endogenous risk components in Maotai-flavor Baijiu and is also a key precursor for the formation of ethyl carbamate (EC). Sorghum is the main source of cyanide in Maotai-flavor Baijiu. Cyanide mainly exists in sorghum in the form of cyanogenic glycosides (mainly dhurrin), and is released in the form of hydrocyanic acid after degradation. Sorghum raw materials themselves contain a certain amount of cyanide. Selecting sorghum varieties with less cyanide can directly reduce the content of these precursor substances during solid-state fermentation, thus avoiding the risk brought by high EC content in the finished Baijiu. Therefore, establishing a detection method for the cyanide content in brewing raw materials can assist in the optimization of sorghum varieties, which is of great significance for improving the quality of Baijiu brewing.

[0003] Currently, the main methods for determining cyanide in food are spectrophotometry, gas chromatography, gas chromatography-mass spectrometry, ion chromatography, and flow injection-spectrophotometry in national standards. The above methods are mainly applicable to the detection of cyanide in liquid samples such as formulated liquor, drinking water, and beverages. There are fewer detection methods for the cyanide content in solid samples, mainly spectrophotometry and gas chromatography, but these methods have defects such as complex sample pretreatment operations, low automation, and low analysis efficiency, and are not suitable for batch sample detection. In recent years, more and more laboratories have used continuous flow injection spectrophotometry to determine cyanide. This method can automatically complete the derivation of cyanide in the sample solution and the rapid determination of absorbance values. However, the injection volume of this method is relatively large, so the preparation of the cyanide extract in solid samples is a difficult point. A large number of studies have used distillation-automatic flow injection analyzers to achieve the determination of cyanide in solid samples. This method uses distillation for sample pretreatment extraction, but the distillation device is relatively large, and volatile hydrocyanic acid is easily lost in the distillation stage of solid samples, affecting the results.

[0004] Therefore, there is an urgent need for a rapid determination method for the cyanide content in sorghum that is accurate and efficient. Summary of the Invention

[0005] This application provides a rapid determination method for the cyanide content in sorghum, which uses an automatic flow injection analyzer to rapidly determine the cyanide content in sorghum, including:

[0006] S10: Pretreatment step. Add an inorganic acid solution to the crushed sorghum sample to be tested to hydrolyze and extract cyanide in the sorghum. After mixing evenly, perform ultrasonic treatment at a first temperature for a first duration. After cooling to room temperature, add an alkali solution and mix evenly. The first temperature is higher than normal temperature. The obtained supernatant is the acid hydrolysis extract, and the supernatant is used as the basic stock solution for preparing the injection solution of the fully automatic flow injection analyzer.

[0007] In some optional embodiments of the first aspect of the present application, it further includes:

[0008] S20: Analysis and detection step. Filter and dilute the supernatant to obtain the injection solution of the fully automatic flow injection analyzer.

[0009] Use the fully automatic flow injection analyzer to detect the injection solution to obtain the cyanide detection value in the injection solution, and calculate the content of cyanide in the crushed sorghum sample based on the cyanide detection value in the injection solution, the mass of the crushed sorghum sample to be tested, and the dilution factor of cyanide in the supernatant.

[0010] In some optional embodiments of the first aspect of the present application, the inorganic acid solution is any one of H2SO4 solution, HCl solution, H3PO4 solution, and HF solution.

[0011] In some optional embodiments of the first aspect of the present application, the inorganic acid solution is HCl solution;

[0012] The concentration of the HCl solution is 3 mol / L to 5 mol / L.

[0013] In some optional embodiments of the first aspect of the present application, the concentration of the HCl solution is 4 mol / L.

[0014] In some optional embodiments of the first aspect of the present application, the first duration is 30 min to 150 min.

[0015] In some optional embodiments of the first aspect of the present application, the first duration is 30 min to 60 min.

[0016] In some optional embodiments of the first aspect of the present application, the first duration is 30 min.

[0017] In some optional embodiments of the first aspect of the present application, the alkali solution is NaOH solution, and the concentration of the NaOH solution is 15 g / L to 25 g / L.

[0018] In some optional embodiments of the first aspect of the present application, the concentration of the NaOH solution is 20 g / L.

[0019] In some optional embodiments of the first aspect of the present application, the value range of the first temperature is 60 °C to 80 °C.

[0020] In some alternative embodiments of the first aspect of the present application, the first temperature ranges from 65 °C to 75 °C.

[0021] In some alternative embodiments of the first aspect of the present application, the first temperature is 70 °C.

[0022] In some alternative embodiments of the first aspect of the present application, an aqueous microporous membrane is used to filter the supernatant.

[0023] In some alternative embodiments of the first aspect of the present application, the specification of the aqueous microporous membrane is a 0.22 μm aqueous microporous membrane.

[0024] Beneficial effects:

[0025] The rapid determination method for the content of cyanide in sorghum provided by the present application proposes to first hydrolyze and extract cyanide from sorghum with an acid solution, and adding an alkali solution after ultrasonic treatment can enable the alkali solution to also undergo a neutralization reaction with the residual acid during the acid hydrolysis process, so that the alkali solution can better play the role of fixing hydrocyanic acid. The pretreatment of the present application ensures the extraction effect (such as the extraction amount) of cyanide in sorghum. There is no need to set up a distillation device during the pretreatment process, avoiding the loss of cyanide caused by the large size of the distillation device. The pretreatment method of the present application is simple, has higher accuracy, shorter time consumption, and higher efficiency. Description of the drawings

[0026] Figure 1 It is a schematic diagram of the results of the extraction effect of different acid hydrolysis solutions on cyanide in sorghum;

[0027] Figure 2 It is a schematic diagram of the results of the extraction effect of different concentrations of HCl solutions on cyanide in sorghum;

[0028] Figure 3 It is a schematic diagram of the results of the extraction effect of different hydrolysis times on cyanide in sorghum;

[0029] Figure 4 It is a schematic diagram of the results of the extraction effect of different concentrations of NaOH solutions on cyanide in sorghum;

[0030] Figure 5 It is a schematic diagram of the results of the extraction effect of different hydrolysis temperatures on cyanide in sorghum. Specific embodiments

[0031] The following further details the present application in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0032] The present application provides a rapid determination method for the content of cyanide in sorghum, which uses a fully automatic flow injection analyzer to rapidly determine the content of cyanide in sorghum, including:

[0033] S10: Pretreatment step. Add an inorganic acid solution to the crushed sorghum sample to be tested for hydrolysis and extraction of cyanide in the sorghum. After mixing evenly, perform ultrasonic treatment at a first temperature for a first duration. After cooling to room temperature, add an alkali solution and mix evenly. The first temperature is higher than normal temperature. The obtained supernatant is the acid hydrolysis extraction solution, and the supernatant is used as the basic stock solution for preparing the injection solution of the fully automatic flow injection analyzer.

[0034] The purpose of cooling to room temperature is to avoid excessive pressure in the closed reactor due to too high temperature, resulting in easy overflow of hydrocyanic acid generated by the acid hydrolysis reaction during the process of opening the lid and adding the alkali solution for fixation.

[0035] In some optional embodiments of the first aspect of the present application, it further includes:

[0036] S20: Analysis and detection step. Filter and dilute the supernatant to obtain the injection solution of the fully automatic flow injection analyzer.

[0037] Use the fully automatic flow injection analyzer to detect the injection solution to obtain the cyanide detection value in the injection solution, and calculate the content of cyanide in the crushed sorghum sample based on the cyanide detection value in the injection solution, the mass of the crushed sorghum sample to be tested, and the dilution multiple of cyanide in the supernatant.

[0038] In some optional embodiments of the first aspect of the present application, the inorganic acid solution is any one of H2SO4 solution, HCl solution, H3PO4 solution, and HF solution.

[0039] In some optional embodiments of the first aspect of the present application, the inorganic acid solution is HCl solution;

[0040] The concentration of the HCl solution is 3 mol / L to 5 mol / L.

[0041] In some optional embodiments of the first aspect of the present application, the concentration of the HCl solution is 4 mol / L.

[0042] In some optional embodiments of the first aspect of the present application, the first duration is 30 min to 150 min.

[0043] In some optional embodiments of the first aspect of the present application, the first duration is 30 min to 60 min.

[0044] In some optional embodiments of the first aspect of the present application, the first duration is 30 min.

[0045] In some optional embodiments of the first aspect of the present application, the alkali solution is NaOH solution, and the concentration of the NaOH solution is 15 g / L to 25 g / L.

[0046] In some optional embodiments of the first aspect of the present application, the concentration of the NaOH solution is 20 g / L.

[0047] In some alternative embodiments of the first aspect of the present application, the first temperature ranges from 60 °C to 80 °C.

[0048] In some alternative embodiments of the first aspect of the present application, the first temperature ranges from 65 °C to 75 °C.

[0049] In some alternative embodiments of the first aspect of the present application, the first temperature is 70 °C.

[0050] In some alternative embodiments of the first aspect of the present application, an aqueous microporous membrane is used to filter the supernatant.

[0051] In some alternative embodiments of the first aspect of the present application, the specification of the aqueous microporous membrane is a 0.22 μm aqueous microporous membrane.

[0052] Beneficial effects:

[0053] The rapid determination method for the content of cyanide in sorghum provided by the present application proposes to first hydrolyze and extract cyanide from sorghum with an acid solution. Adding an alkali solution after ultrasonic treatment can enable the alkali solution to also undergo a neutralization reaction with the residual acid during the acid hydrolysis process, so that the alkali solution can better play the role of fixing hydrocyanic acid. The pretreatment of the present application ensures the extraction effect (such as the extraction amount) of cyanide in sorghum, eliminates the need to set up a distillation device during the pretreatment process, and avoids the loss of cyanide caused by the large size of the distillation device. Subsequently, the automatic flow injection analyzer automatically completes the determination of the derivative and absorbance value during the test, and the cyanide content is obtained through calculation. The pretreatment method of the present application is simple, has higher accuracy, short time consumption, and high efficiency.

Specific embodiments

[0055] I. Materials and reagents

[0056] Chloramine T: Analytical pure (purity ≥ 95%), purchased from Shanghai Macklin Biochemical Co., Ltd.

[0057] Tartaric acid: Analytical pure (purity ≥ 95%), purchased from Shanghai Macklin Biochemical Co., Ltd.

[0058] Isonicotinic acid: Analytical pure (purity ≥ 95%), purchased from Shanghai Macklin Biochemical Co., Ltd.

[0059] Potassium dihydrogen phosphate: Analytical pure (purity ≥ 95%), purchased from Shanghai Macklin Biochemical Co., Ltd.

[0060] 1,3-Dimethylbarbituric acid: Analytical pure (purity ≥ 95%), purchased from Shanghai Macklin Biochemical Co., Ltd.

[0061] Sodium hydroxide: Analytical pure (purity ≥ 95%), purchased from Sinopharm Chemical Reagent Co., Ltd.

[0062] Hydrochloric acid: guaranteed reagent (purity ≥ 99.8%), purchased from Chongqing Chuandong Chemical Co., Ltd.

[0063] Cyanide standard compound in water: mass concentration of 50 mg / L, purchased from Beijing Tanmo Quality Inspection Technology Co., Ltd.

[0064] II. Equipment

[0065] Crushing: Controlled-type test tube grinder (IKA Werke GmbH & Co. KG, Germany).

[0066] Ultrasonic: Ultrasonic cleaner (Shanghai Chenhui Instrument Co., Ltd.).

[0067] Centrifugation: 5804 centrifuge (Eppendorf AG, Germany).

[0068] L8 fully automatic rapid cyanide analyzer for liquor: Beijing Jitian Instrument Co., Ltd.

[0069] ME204T electronic balance: Shanghai Mettler Toledo Co., Ltd.

[0070] EQ7000 ultrapure water instrument: Merck Millipore China Co., Ltd.

[0071] III. Optimization of pretreatment method

[0072] 3.1 Preparation of standard solution

[0073] Accurately pipette 2.00 mL of cyanide standard solution (50.00 mg / L), and make up the volume to 100 mL with sodium hydroxide solution (1.00 g / L) to prepare a cyanide standard working solution with a mass concentration of 1.0 mg / L.

[0074] Accurately pipette 1.00 mL of cyanide standard working solution (1.0 mg / L), and make up the volume to 10 mL with sodium hydroxide solution (1.00 g / L) to prepare a cyanide standard working solution with a mass concentration of 0.10 mg / L.

[0075] 3.2 Preparation of standard solution

[0076] Preparation of mobile phase solution

[0077] (1) Carrier solution and absorption solution: Weigh 1.0 g of sodium hydroxide, make up the volume to 1 L with water, mix well, and degas for later use.

[0078] (2) Distillation reagent: Add 800 mL of deionized water to a 1000 mL beaker, add 13.21 g of tartaric acid and stir to dissolve, add 3.5 g of sodium hydroxide, make up the volume to 1 L with water, mix well, degas, and store in a refrigerator at 4 °C for later use.

[0079] (3) Buffer solution: Weigh 97.0 g of potassium dihydrogen phosphate, make up the volume to 1 L with water, mix well, degas and reserve for use.

[0080] (4) Chloramine T solution: Weigh 2 g of chloramine T and dissolve it in water, make up the volume to 500 mL with water, mix well, degas and store in the dark for use (prepare fresh before use).

[0081] (5) Iso-nicotinic acid-barbituric acid color reagent: Weigh 12.0 g of sodium hydroxide, 16.8 g of 3-dimethylbarbituric acid, and 13.6 g of iso-nicotinic acid in sequence, dissolve them in water, make up the volume to 1 L with water, mix well, degas and store in the dark for use.

[0082] 3.3 Principle of Determining Cyanide in Water by Flow Injection Analysis

[0083] Principle of iso-nicotinic acid-barbituric acid method in the embodiments of this application: In a medium with pH < 4, simple cyanide and part of complex cyanide are distilled out in the form of hydrogen cyanide through on-line distillation, separated from other interfering substances. The gaseous hydrogen cyanide passes through a gas permeable membrane and is absorbed by sodium hydroxide solution. Under weakly acidic conditions, cyanide ions react with the active chlorine of chloramine T to form cyanogen chloride. Cyanogen chloride reacts with iso-nicotinic acid, undergoes hydrolysis to form glutaconaldehyde, and glutaconaldehyde condenses with two barbituric acid molecules to form a blue-violet dye, and colorimetric determination is carried out at 600 nm.

[0084] Analysis steps of the flow injection analyzer: (1) Power on and preheat: After powering on, perform instrument self-check. After the self-check is completed, install the pump tubes, put the capillaries connected to each pump tube into ultrapure water for pre-experiment cleaning for 300 s, and at the same time wait for the heating component to heat up. (2) Run and detect: After the instrument heats up, run the workstation, edit the injection program, put the capillaries of all pump tubes into the corresponding mobile phase solution. Before sample determination, 3 - 4 blank samples need to be made to balance the instrument. After confirming that there are no bubbles in the flow cell, start sample detection. (3) Clean and shut down: After sample detection is completed, put all capillaries into ultrapure water for post-experiment cleaning for 300 s to ensure that there is no residual reagent and sample contaminating or corroding the instrument. Finally, pull out all capillaries, place them in the air for instrument evacuation for 300 s. After the liquid in the pump tubes is drained, loosen the pump tubes, and close the software and the instrument.

[0085] 3.4 Specific Pretreatment Steps

[0086] S10: Pretreatment step. Add 5 mL of inorganic acid solution to 0.5 g of the crushed sorghum sample to be tested to hydrolyze and extract the cyanide in the sorghum. Tighten the bottle cap and mix the sample solution evenly. After mixing, perform ultrasonic treatment at 70 °C for 30 min and then cool it with running water. After cooling to room temperature, add 45 mL of 20 g / L NaOH solution and mix evenly for fixation. The obtained supernatant is the acid hydrolysis extract, and the supernatant is used as the basic stock solution for preparing the injection solution of the fully automatic flow injection analyzer. Take 5 mL of the supernatant, filter it through a 0.22 μm aqueous microporous membrane into the injection tube, add 5 mL of ultrapure water, dilute and mix evenly, and then directly detect it on the machine.

[0087] 3.5 Analytical detection step

[0088] S20: Analytical detection step. Filter and dilute the supernatant to obtain the injection solution of the fully automatic flow injection analyzer. Use the fully automatic flow injection analyzer to detect the injection solution to obtain the cyanide detection value in the injection solution, and calculate the content of cyanide in the crushed sorghum sample based on the cyanide detection value in the injection solution, the mass of the crushed sorghum sample to be tested, and the dilution factor of cyanide in the sorghum.

[0089] Specifically:

[0090] S21: Take 10 mL of the cyanide standard working solution (100 μg / L) into the injection tube, and use the fully automatic rapid cyanide detector for liquor to automatically dilute it online by 1 time, 2 times, 5 times, 10 times, and 20 times respectively to prepare a series of standard working solutions with mass concentrations of 100 μg / L, 50 μg / L, 20 μg / L, 10 μg / L, and 5 μg / L. The cyanide in the standard working solution and the sample solution automatically completes the derivation and absorbance value determination in the closed system of the fully automatic flow injection analyzer. Take the cyanide mass concentration as the abscissa and the absorbance value peak area of the cyanide derivative as the ordinate to draw the standard curve. The cyanide detection value in the injection solution is obtained by substituting the absorbance value peak area detected by the instrument into the standard curve regression equation. The cyanide detection value in the injection solution is the cyanide mass concentration in the injection solution.

[0091] S22: According to the following formula (1), calculate the content of cyanide in the crushed sorghum sample based on the cyanide detection value in the injection solution, the mass of the crushed sorghum sample to be tested, and the dilution factor of cyanide in the sorghum:

[0092]

[0093] In the formula:

[0094] W - Content of cyanide in the solid sample, μg / kg;

[0095] C - Cyanide detection value in the acid hydrolysis extract, μg / L;

[0096] M - mass of the solid sample taken, g;

[0097] N - dilution factor of cyanide in sorghum, N is 100 in a specific example.

[0098] The calculation process of the dilution factor N of cyanide in sorghum is as follows: the sum of the volume V1 of the inorganic acid solution and the volume V2 of the NaOH solution added in the pretreatment step is multiplied by the dilution factor for filtering and diluting into the sample injection solution. For example, in an embodiment of the present application, 5 mL of the inorganic acid solution plus 45 mL of the NaOH solution means that the cyanide in sorghum is diluted 50 times. Then, 5 mL of the supernatant is taken before sample injection, and 5 mL of ultrapure water is added to the 5 mL of the supernatant for dilution, that is, it is further diluted 2 times. Therefore, N = (5 + 45) * 2 = 100.

[0099] 3.6 Further optimization of the specific conditions in the pretreatment step

[0100] 3.6.1 Selection of acidic solution

[0101] In this part, the final cyanide content is used to measure whether the hydrolysis is complete.

[0102] In the experiment, 4 kinds of acids, namely H2SO4 (2 mol / L), HCl (4 mol / L), H3PO4 (2 mol / L) and HF (4 mol / L), were respectively used to extract cyanide in sorghum to investigate the influence of different acid solutions on the extraction effect of cyanide in sorghum. The results are as Figure 1 shown. The specific experimental steps are as follows: Accurately weigh 0.5 g of the ground sorghum sample into a 50 mL brown stoppered reagent bottle, add 5 mL of the above different acid solutions respectively, tighten the bottle stopper and mix the sample solution evenly. After ultrasonic treatment at 70 °C for 30 min, cool it with running water. Quickly and accurately add 45 mL of 20 g / L NaOH solution for fixation. Take 5 mL of the supernatant, filter it through a 0.22 μm aqueous microporous membrane into the sample injection tube, add 5 mL of ultrapure water for dilution and mixing, and then directly detect it on the machine. Calculate the content of cyanide in sorghum according to the analysis and detection steps in part 3.5.

[0103] As Figure 1 can be seen, different acid solutions have little influence on the detection results of cyanide in sorghum, and the cyanide content ranges from 202.04 to 242.35 μg / kg. Among them, the extraction effects of H2SO4 solution and HCl solution on cyanide are relatively good, and the difference in the cyanide content extracted by the two is not significant. It was found during the sample pretreatment process that the injection volume of the flow injection analyzer is large, and the extraction solution prepared with HCl solution is easier to filter than the other three acid solutions, which can save a large amount of sample preparation time. Generally speaking, HCl solution is a better acid hydrolysis solution.

[0104] 3.6.2 Selection of hydrochloric acid concentration

[0105] The experiments were carried out to hydrolyze and extract cyanide from sorghum using HCl solutions with concentrations of 0.5, 1, 2, 4, 6, and 8 mol / L respectively, and to investigate the effects of HCl solutions with different concentrations on the extraction efficiency of cyanide. The results are as follows. Figure 2 The specific experimental steps are as follows: Accurately weigh 0.5 g of pulverized sorghum samples into a 50 mL brown stoppered reagent bottle, add 5 mL of the above-mentioned HCl solutions with different concentrations respectively, tighten the bottle stopper and mix the sample solution evenly, ultrasonicate at 70 °C for 30 min and then cool with running water. Quickly and accurately add 45 mL of 20 g / L NaOH solution for fixation. Take 5 mL of the supernatant, filter it through a 0.22 μm aqueous microporous membrane into the injection tube, add 5 mL of ultrapure water for dilution and mixing, and then directly detect it on the machine. Calculate the content of cyanide in sorghum according to the analysis and detection steps in Section 3.5.

[0106] As Figure 2 can be seen, with the increase of the concentration of HCl hydrolysis solution, the content of cyanide in the sorghum extract shows a trend of first increasing and then decreasing. Among them, the content of cyanide extracted by 4 mol / L HCl solution is the highest, reaching 238.63 μg / kg. The concentration of HCl solution has a great influence on the extraction efficiency of cyanide in sorghum. Among them, too low a concentration of HCl solution leads to incomplete hydrolysis of sorghum, poor extraction efficiency of cyanide, and low content of cyanide. While too high a concentration of HCl solution has higher volatility and is more likely to volatilize during the fixation stage of adding NaOH solution, which may carry away the generated hydrocyanic acid and result in a decrease in the content of cyanide. Therefore, 3 mol / L - 5 mol / L HCl solution is more suitable, and 4 mol / L HCl solution is preferably used for the extraction of cyanide in sorghum.

[0107] 3.6.3 Hydrolysis time

[0108] The experiments investigated the extraction of cyanide in sorghum when the acid hydrolysis times were 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min respectively. The results are as follows. Figure 3 The specific experimental steps are as follows: Accurately weigh 0.5 g of pulverized sorghum samples into a 50 mL brown stoppered reagent bottle, add 5 mL of 4 mol / L HCl solution, tighten the bottle stopper and mix the sample solution evenly, ultrasonicate at 70 °C for 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min respectively, and then cool with running water. Quickly and accurately add 45 mL of 20 g / L NaOH solution to the samples with different hydrolysis times for fixation. Take 5 mL of the supernatant, filter it through a 0.22 μm aqueous microporous membrane into the injection tube, add 5 mL of ultrapure water for dilution and mixing, and then directly detect it on the machine. Calculate the content of cyanide in sorghum according to the analysis and detection steps in Section 3.5.

[0109] AsFigure 3 It can be seen that the cyanide content in the sorghum extract shows an increasing trend in the first 30 minutes before hydrolysis (i.e., the ultrasonic treatment duration), remains stable from 30 minutes to 150 minutes, with the content range of 235.85 - 245.3 μg / kg, and significantly decreases at 180 minutes. If the hydrolysis time is too short, the hydrolysis of linamarin in sorghum is incomplete, resulting in poor extraction effect of hydrocyanic acid. While if the hydrolysis time is too long, more of the extracted hydrocyanic acid volatilizes and is lost, leading to a low detection result of the cyanide content. In summary, the hydrolysis time of 30 minutes to 60 minutes is better, especially when the hydrolysis time is 30 minutes, both the extraction content and extraction efficiency of hydrocyanic acid are the best.

[0110] 3.6.4 Concentration of NaOH solution

[0111] During the hydrolysis process of sorghum, the extracted hydrocyanic acid is prone to volatilization. NaOH solution can be used to fix it for subsequent on-machine detection. In the experiment, NaOH solutions with concentrations of 5, 10, 20, 30, 40, and 50 g / L were respectively selected to fix the hydrocyanic acid in the extract, and the effects of NaOH solutions with different concentrations on the cyanide content were investigated to optimize the best NaOH concentration. The specific experimental steps are as follows: Accurately weigh 0.5 g of the ground sorghum sample into a 50 mL brown stoppered reagent bottle, add 5 mL of 4 mol / L HCl solution, tighten the bottle stopper and mix the sample solution evenly. After ultrasonic treatment at 70 °C for 30 minutes, cool it with running water. Quickly and accurately add 45 mL of the above-mentioned NaOH solutions with different concentrations for fixation. Take 5 mL of the supernatant, filter it through a 0.22 μm aqueous microporous membrane into the injection tube, add 5 mL of ultrapure water for dilution and mixing, and then directly conduct on-machine detection. Calculate the cyanide content in sorghum according to the analysis and detection steps in Section 3.5.

[0112] It can be Figure 4 seen that with the increase of the NaOH solution concentration, the cyanide content shows a trend of increasing first and then decreasing. When the NaOH solution concentration is 20 g / L, the cyanide content is the largest, reaching 304.75 μg / kg. The residual acid solution in the acid hydrolysis process will undergo a neutralization reaction with the NaOH solution, and only after neutralizing the excess acid solution can the alkaline solution better play the role of fixing hydrocyanic acid. Therefore, when the NaOH solution concentration is relatively low, it may lead to poor fixation effect of hydrocyanic acid and a low cyanide detection value. While when the NaOH solution concentration is relatively high, the strongly alkaline cyanide extract is difficult to distill during on-machine detection, resulting in a low cyanide detection result. Generally speaking, the NaOH solution concentration is 15 g / L - 25 g / L, and a NaOH solution concentration of 20 g / L is preferred.

[0113] 3.6.5 Hydrolysis temperature

[0114] The experiment investigated the extraction of cyanide in sorghum when the hydrolysis temperatures were 30 °C, 50 °C, 70 °C, 90 °C, and 100 °C respectively. The results are asFigure 5 As shown below. The specific test steps are as follows: Accurately weigh 0.5 g of the ground sorghum sample into a 50 mL brown stoppered reagent bottle, add 5 mL of 4 mol / L HCl solution, tighten the bottle stopper and mix the sample solution evenly. After ultrasonic treatment for 30 min at 30 °C, 50 °C, 70 °C, 90 °C and 100 °C respectively, cool it with running water. Quickly and accurately add 45 mL of 20 g / L NaOH solution to the samples treated under different hydrolysis temperature conditions for fixation. Take 5 mL of the supernatant, filter it through a 0.22 μm aqueous microporous membrane into the injection tube, add 5 mL of ultrapure water for dilution and mixing, and then directly detect it on the machine. Calculate the content of cyanide in sorghum according to the analysis and detection steps in Section 3.5.

[0115] As Figure 5 can be seen, with the increase of hydrolysis temperature, the cyanide content in the sorghum extract shows a trend of first increasing and then decreasing, reaching the highest at 70 °C, which is 246.83 μg / kg. The cyanide content in the sorghum extract under different temperature conditions is significantly different, indicating that the hydrolysis temperature has a great influence on the extraction effect of cyanide. When the hydrolysis temperature is too low, the hydrolysis of cyanogenic glycoside in sorghum is incomplete, resulting in poor extraction effect of hydrocyanic acid. When the hydrolysis temperature is too high, the extracted hydrocyanic acid is easily decomposed and volatilized by heat, resulting in a decrease in cyanide content. When the first temperature range is 60 °C to 80 °C, the cyanide content in the sorghum extract is approximately greater than 220 μg / kg, proving that cyanide can be well extracted from the sorghum extract within this temperature range.

[0116] In some examples, the first temperature range is 65 °C to 75 °C. Specifically, in some examples, when the first temperature is 70 °C, the cyanide content in the sorghum extract reaches the maximum.

[0117] IV. Methodological verification

[0118] In some embodiments, a specific rapid determination method for the cyanide content in sorghum:

[0119] Pretreatment step method: Accurately weigh 0.5 g of the ground sorghum sample into a 50 mL brown stoppered reagent bottle, add 5 mL of HCl (4 mol / L) solution, tighten the bottle stopper and mix the sample solution evenly. After ultrasonic treatment at 70 °C for 30 min, cool it with running water. Quickly and accurately add 45 mL of 20 g / L NaOH solution for fixation. Take 5 mL of the supernatant, filter it through a 0.22 μm aqueous microporous membrane into the injection tube, add 5 mL of ultrapure water for dilution and mixing, and then directly detect it on the machine.

[0120] 4.1 Method linear range, detection limit and quantification limit

[0121] Prepare a series of standard working solutions of cyanide with concentrations of 100 μg / L, 50 μg / L, 20 μg / L, 10 μg / L, and 5 μg / L respectively, and perform flow injection analysis and detection according to the analysis and detection steps in 3.5 to obtain the standard curve spectrum of cyanide. Plot the standard curve with the cyanide mass concentration (X) as the abscissa and the corresponding absorbance peak area (Y) as the ordinate. The regression equation of the standard curve of cyanide is Y = 0.4574X - 0.1008, R 2 = 0.9996, indicating good linearity of cyanide in the range of 5 - 100 μg / L. Gradually dilute the acid hydrolysis extract prepared by this method (including two steps of acid hydrolysis and alkali fixation) and then inject it for analysis. The limit of detection (LOD) of this method is 8.32 μg / kg, and the limit of quantitation (LOQ) is 27.34 μg / kg.

[0122] 4.2 Precision Test

[0123] For the precision test, sorghum samples from three different origins were selected. Prepare the injection liquid samples according to the pretreatment method described in the fourth part. The extraction solutions of each variety of sorghum were prepared 6 times repeatedly, and the cyanide content of sorghum in each of the 6 detections of each variety of sorghum was calculated according to the calculation method in part 3.5.

[0124] Calculate the relative standard deviation (RSD) based on the detection results. The results are shown in Table 1. It can be seen from Table 1 that the RSD values of the precision tests for the three varieties of sorghum are all < 10%, meeting the requirements of GB 5009.36 - 2023 "National Food Safety Standard - Determination of Cyanide in Foods", indicating that this method can better meet the determination of cyanide content in sorghum.

[0125] Table 1 Results of Precision and Repeatability Tests

[0126]

[0127] 4.3 Stability Test

[0128] For the stability test, sorghum samples from three different origins were selected for further pretreatment. Inject and analyze them at 0 h, 0.5 h, 1.0 h, 2.0 h, and 3.0 h after pretreatment respectively. Calculate the RSD value based on the detection results. The results are shown in Table 2. It can be seen from Table 2 that there are no significant differences in the cyanide content of the detected sorghum samples within 1 - 3 h, and the RSD of the stability test results is 0.70% - 2.04%, indicating that this method has good stability.

[0129] Table 2 Results of Stability Test

[0130]

[0131] In Table 2, a is the result of the significance analysis of differences. The same a indicates that there is no significant difference between the measurement results.

[0132] 4.4 Spike recovery test

[0133] For the spike recovery experiment, a sorghum sample was selected. 0.5 g of the ground sorghum sample was accurately weighed into a stoppered test tube, and cyanide standard solutions with low (100 μg / kg), medium (200 μg / kg), and high concentrations (400 μg / kg) were added respectively. After pretreatment and instrumental analysis, the detection results and spike recoveries were calculated. As shown in Table 3, the spike recoveries of this method were all in the range of 81.93% - 108.27%, meeting the requirements of methodological investigation and showing good accuracy.

[0134] Table 3 Results of spike recovery test

[0135]

[0136]

[0137] 4.5 Comparison of detection results with the national standard detection method

[0138] In this experiment, the first method of GB 5009.36—2023 National Food Safety Standard - Determination of Cyanide in Foods and this method were used to determine the cyanide content in sorghum from three different producing areas. The specific experimental steps of the first method of GB 5009.36—2023 National Food Safety Standard - Determination of Cyanide in Foods are as follows:

[0139] 4.5.1 Reagent preparation

[0140] (1) Phosphate buffer solution (0.5 mol / L, pH = 7.0): Weigh 3.40 g of anhydrous potassium dihydrogen phosphate and 3.55 g of anhydrous disodium hydrogen phosphate, dissolve them in water and make up to 100 mL.

[0141] (2) Acetic acid solution (1 + 6): Mix acetic acid and water according to a volume ratio of 1:6.

[0142] (3) Iso-nicotinic acid - pyrazolone solution: Weigh 1.5 g of iso-nicotinic acid and dissolve it in 24 mL of sodium hydroxide solution (20 g / L), add water to make up to 100 mL. Separately, weigh 0.25 g of pyrazolone and dissolve it in 20 mL of absolute ethanol. Combine the above two solutions and shake well. Prepare freshly before use.

[0143] (4) Chloramine T solution (20 g / L): Weigh 2 g of chloramine T, dissolve it in water and dilute to 100 mL. Prepare freshly before use.

[0144] 4.5.2 Preparation of Sample Solution and Standard Solution

[0145] (1) Preparation of sample solution: Accurately weigh 10 g of sorghum into a 250 mL distillation flask, add 150 mL of ultrapure water, stopper and seal, then carry out magnetic stirring for 2 h. Add 20 mL of zinc acetate solution and 2.0 g of tartaric acid, quickly connect the distillation device, insert the lower end of the condenser into the liquid surface of a 100 mL volumetric flask containing 10 mL of sodium hydroxide solution (20 g / L), start distillation. When about 100 mL of distillate is collected, make up the volume to the mark with water. Accurately pipette 1.00 mL of the distillate into a 10 mL colorimetric tube, add 2 g / L sodium hydroxide solution to 5 mL, shake well and use as the test sample solution.

[0146] (2) Preparation of standard solution: Accurately pipette 0 mL, 0.40 mL, 0.80 mL, 1.20 mL, 1.60 mL and 2.00 mL of cyanide standard working solution (1.0 mg / L) into 10 mL colorimetric tubes respectively, i.e., the masses of cyanide (calculated as CN - ) are 0 μg, 0.40 μg, 0.80 μg, 1.20 μg, 1.60 μg, 2.00 μg respectively. Finally, add 2 g / L sodium hydroxide solution to 5 mL.

[0147] 4.5.3 Determination of Sample

[0148] Add 2 drops of phenolphthalein indicator solution to the above-mentioned sample solution and standard solution respectively, add acetic acid solution to adjust until the red color fades, then use 20 g / L sodium hydroxide solution to adjust to near red color, then add 2 mL of phosphate buffer solution, then add 0.2 mL of chloramine T solution, shake well and let stand for 3 min, then add 2 mL of isonicotinic acid-pyrazolone solution, make up the volume to the mark with water, stopper and shake well to mix evenly, and place in a 37 °C constant temperature water bath for 40 min. Use a 1 cm colorimetric cell, adjust the zero point with 2 g / L sodium hydroxide solution as the blank solution, measure the absorbance of each solution at a wavelength of 638 nm. Plot a standard curve with the mass of cyanide ion as the abscissa and the absorbance of the ion as the ordinate. The content of cyanide (calculated as CN

[0149]

[0150] In the formula:

[0151] X - content of cyanide in the sample (calculated as CN - ), mg / kg;

[0152] A - mass of cyanide determined in the test sample solution (calculated as CN - ), μg;

[0153] V1 - total volume of the sample distillate, mL;

[0154] m - mass of the sample, g;

[0155] V2 - volume of the distillate for determination, mL;

[0156] 1000 - unit conversion factor.

[0157] As can be seen from Table 4, the detection deviations of the cyanide contents in the three varieties of sorghum determined by the national standard method and this method are all < 10%, and the detection results are accurate and reliable. Moreover, the pretreatment of the national standard method includes three key steps such as magnetic stirring (2 h), distillation, and water bath heating (40 min), which takes a long time, while this method only requires ultrasonic hydrolysis (30 min - 60 min, the shortest is 30 min), which takes a short time. Generally speaking, this method can achieve the accurate and rapid determination of cyanide in sorghum.

[0158] Table 4 Detection results of cyanide content in sorghum

[0159]

[0160]

[0161] Generally speaking, this application provides a rapid determination method for the cyanide content in sorghum that is efficient, accurate, and simple to detect. In the embodiments of this application, only ultrasonic hydrolysis with an acidic solution is used to extract hydrocyanic acid without distillation, and then an automatic flow injection analyzer is used to detect the cyanide in the extract, thereby realizing the rapid determination of cyanide in sorghum. This extraction method is simpler, takes less time, and has higher accuracy compared with the distillation extraction method.

[0162] The above specific embodiments do not constitute a limitation on the protection scope of this application. Those skilled in the art should understand that various modifications, combinations, sub - combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application shall be included within the protection scope of this application.

Claims

1. A method for rapidly determining the cyanide content in sorghum, characterized in that: Rapid determination of cyanide content in sorghum using a fully automated flow injection analyzer, including: S10: Pretreatment step, adding inorganic acid solution to the sorghum crushed sample to be tested to hydrolyze and extract cyanide in the sorghum, mixing and ultrasonically treating at a first temperature for a first time, cooling to room temperature and adding alkaline solution to mix, the first temperature is higher than room temperature, and the obtained supernatant is an acid hydrolysis extract, and the supernatant is used as a basic stock solution for preparing the injection solution of the fully automatic flow injection analyzer.

2. The method for rapid determination of cyanide content in sorghum according to claim 1, characterized in that: Also includes: S20: analysis and detection step, filtering and diluting the supernatant to obtain an injection solution for a fully automatic flow injection analyzer, The fully automatic flow injection analyzer is used to detect the sample liquid to obtain the cyanide detection value in the sample liquid, and the cyanide content in the sorghum crushed sample is calculated based on the cyanide detection value in the sample liquid, the mass of the sorghum crushed sample to be tested and the dilution multiple of the cyanide in the sorghum.

3. The method for rapid determination of cyanide content in sorghum according to claim 1, characterized in that: The inorganic acid solution is any one of a H2SO4 solution, a HCl solution, a H3PO4 solution and a HF solution.

4. The method for rapid determination of cyanide content in sorghum according to claim 1, characterized in that: The inorganic acid solution is HCl solution; The concentration of the HCl solution is 3 mol / L to 5 mol / L; Preferably, the concentration of the HCl solution is 4 mol / L.

5. The method for rapid determination of cyanide content in sorghum according to claim 1, characterized in that: The first duration is 30 minutes to 150 minutes; Preferably, the first duration is 30 minutes to 60 minutes; Preferably, the first duration is 30 minutes.

6. The method for rapid determination of cyanide content in sorghum according to claim 1, characterized in that: The alkaline solution is a NaOH solution, and the concentration of the NaOH solution is 15 g / L to 25 g / L; Preferably, the concentration of the NaOH solution is 20 g / L.

7. The method for rapid determination of cyanide content in sorghum according to claim 1, characterized in that: The first temperature ranges from 60°C to 80°C; Preferably, the first temperature ranges from 65°C to 75°C; Preferably, the first temperature is 70°C.

8. The method for rapid determination of cyanide content in sorghum according to claim 2, characterized in that: Filtering the supernatant using an aqueous microporous filter membrane; Preferably, the specification of the aqueous phase microporous filter membrane is 0.22 μm aqueous phase microporous filter membrane.

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