A method for detecting 3-nitropropionic acid residues in sugar cane and sugar cane juice by gas chromatography-tandem mass spectrometry

Abstract

The application discloses a kind of 3-nitropropionic acid residues in sugar cane and sugar cane juice Gas Chromatography-Tandem Mass Spectrometry detection method.The application uses acetyl chloride-methanol method, acetyl chloride and methanol are reacted to obtain hydrochloric acid-methanol, which makes 3-nitropropionic acid in sugar cane and sugar cane juice carry out methyl esterification derivatization reaction, and 3-nitropropionic acid methyl ester is generated.After extraction with toluene and centrifugation, the upper liquid is added to the QuEChERS extraction bag, vortexed for 1 min, centrifuged at 10000 rpm for 5 min, and then the upper liquid is filtered through a 0.22 μm filter membrane.The sample is detected by Gas Chromatography-Tandem Mass Spectrometry in multiple reaction monitoring mode, and quantified by external standard method.The method has high extraction efficiency and sensitivity, good accuracy and repeatability, and can be used to determine the residue amount of 3-nitropropionic acid in sugar cane and sugar cane juice.

Description

Technical Field

[0001] This invention relates to the field of food safety testing, specifically to a gas chromatography-tandem mass spectrometry method for detecting 3-nitropropionic acid residues in sugarcane and sugarcane juice. Background Technology

[0002] 3-Nitropropionic acid (3-NPA) is a toxic metabolite produced by pathogens such as *Fusarium moniliforme* and *Aspergillus oryzae*, primarily originating from moldy sugarcane. 3-Nitropropionic acid is a neurotoxin that irreversibly inhibits mitochondrial succinate dehydrogenase, thereby hindering the normal tricarboxylic acid cycle in the human body. The resulting uncoupling of oxidative phosphorylation severely impairs the energy metabolism of aerobic neurons, damaging the central nervous system. 3-Nitropropionic acid is a potent neurotoxin that damages the central nervous system. Consuming spoiled sugarcane can cause vomiting, diarrhea, and even convulsions, coma, paroxysmal seizures, and even death, posing a risk to public health.

[0003] Sugarcane has a complex matrix with numerous interfering components, containing large amounts of sugar and pigments. Analyzing 3-nitropropionic acid residues within it places higher demands on sample extraction, purification, and detection. Current detection methods primarily utilize liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, LC can only provide qualitative and quantitative analysis based on retention time and peak area, lacking substantial definitive judgment criteria. LC-MS / MS, combining the superior separation capabilities of chromatography with the high sensitivity and specificity of mass spectrometry, is undoubtedly the ideal choice. Gas chromatography-tandem mass spectrometry (GC-MS / MS) offers significant advantages unmatched by other detection methods: high sensitivity, high selectivity, and high specificity. It can not only accurately quantify and confirm known substances but also efficiently identify and structurally analyze unknown compounds. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a gas chromatography-tandem mass spectrometry (GC-MS) method for detecting 3-nitropropionic acid residues in sugarcane and sugarcane juice. This method combines derivatization-QuEChERS purification-GC-MS, enabling the determination of 3-nitropropionic acid residues in sugarcane and sugarcane juice. This method has the advantages of simple and rapid pretreatment, strong resistance to matrix interference, accurate confirmation, and high sensitivity, providing a reliable analytical method for the rapid and accurate detection of 3-nitropropionic acid residues in sugarcane and sugarcane juice.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a gas chromatography-tandem mass spectrometry method for detecting 3-nitropropionic acid residues in sugarcane and sugarcane juice, the method comprising the following steps:

[0006] Step (1): Sample preparation: Peel and cut the sugarcane into pieces, grind it with a grinder and test it; if the sample is sugarcane juice, filter it directly to remove the residue and obtain the sugarcane juice sample.

[0007] Step (2): Derivatization, extraction and purification: Weigh 5.0 g of the sample from step (1) into a centrifuge tube, add 6 mL of 10% acetylchloromethanol and 8 mL of toluene solution; then fill the centrifuge tube with nitrogen and tighten the screw cap; after shaking and mixing, place in an 80℃ water bath for 2 h, take it out and shake it once every 20 min, take it out after water bath and cool it to room temperature, centrifuge at 10000 rpm for 5 min, transfer the upper liquid of the sample after reaction to a centrifuge tube, add QuEChERS extraction package for purification, vortex for 1 min, centrifuge at 10000 rpm for 5 min, take 1 mL of the upper liquid, filter it through a 0.22 μm filter membrane and then analyze it by GC-MS / MS;

[0008] Step (3): Preparation of matrix-matched standard curves: Take 20 μL, 40 μL, 80 μL, 160 μL and 320 μL of 1.0 μg / mL 3-nitropropionic acid solution into centrifuge tubes containing 5.0 g blank sample, perform the operation in step (2) and analyze by GC-MS / MS to obtain standard curve solutions of 4.0 μg / kg, 8.0 μg / kg, 16.0 μg / kg, 32.0 μg / kg and 64.0 μg / kg.

[0009] Step (4): Detection of the sample solution to be tested: The sample solution after extraction and purification in step (2) and the standard curve solution prepared in step (3) are injected into a gas chromatograph-tandem mass spectrometer respectively. The standard curve is plotted with the peak area of ​​the quantitative ion as the ordinate (Y) and its mass concentration as the abscissa (X, ng / mL). Based on the standard curve, the concentration of 3-nitropropionic acid in the sample solution to be tested is obtained, and then the content of 3-nitropropionic acid in sugarcane and sugarcane juice is obtained.

[0010] Furthermore, in the derivatization method of step (2), the acetyl chloride-methanol method is adopted. The hydrochloric acid-methanol obtained by reacting acetyl chloride with methanol causes 3-nitropropionic acid in sugarcane and sugarcane juice to undergo a methyl esterification derivatization reaction to generate methyl 3-nitropropionic acid. In the purification method of step (2), the liquid to be purified is purified by QuEChERS extraction package to remove the pigment and sugar content in the sugarcane and sugarcane juice sample, so as to avoid contaminating the gas chromatography-tandem mass spectrometer and interfering with the detection of 3-nitropropionic acid.

[0011] Furthermore, the QuEChERS extraction kit is for PSA / GCB / C. 18 (1.2g MgSO4, 400mg PSA, 400mg GCB, 400mg C 18 ).

[0012] Furthermore, the parameter conditions for gas chromatography-tandem mass spectrometry in step (4) are as follows:

[0013] Chromatographic conditions:

[0014] Column: Agilent RXI-5Sil MS capillary column, 30m x 0.25mm x 0.25μm; Column temperature program: 50℃, hold for 0min, increase to 150℃ at 10℃ / min, hold for 5min; Carrier gas: Helium, purity 99.999%; Carrier gas flow rate: 1.0mL / min; Injector temperature: 260℃; Injection volume: 1μL; Injection method: Split injection, split ratio: 10:1, purge flow rate 3.0mL / min, purge time 1min;

[0015] Mass spectrometry conditions:

[0016] Electron impact ionization source: 70 eV; ion source temperature: 230°C; transfer line temperature: 280°C; collision gas: nitrogen; scan type: multiple reaction monitoring mode (MRM); scan time: 5-10 min; the mass spectrometry parameters of qualitative and quantitative ion pairs and collision energies of methyl 3-nitropropionate, i.e., the derivative of 3-nitropropionic acid, are shown in Table 1. * in the table indicates quantitative ion pairs.

[0017] Table 1. GC-MS / MS analytical parameters of methyl 3-nitropropionate

[0018]

[0019] *Quantitative ion analysis.

[0020] The beneficial effects of this invention are as follows: This invention involves derivatization of 3-nitropropionic acid residues in sugarcane and sugarcane juice samples, purification using a QuEChERS extraction kit, and then sample injection for detection. The first step is derivatization and extraction: the sample is first derivatized with 10% acetylchloromethanol solution and then extracted with toluene; the second step is purification: the derivatized solution from the first step is purified using a QuEChERS extraction kit to remove pigments, sugars, and other impurities, and then filtered through a 0.22 μm organic phase filter membrane for GC-MS / MS detection. This purified test solution not only does not contaminate the detection instrument but also does not interfere with the detection of 3-nitropropionic acid residues. This invention is characterized by simple operation, good reproducibility, high extraction efficiency, and enhanced safety.

[0021] Secondly, the present invention provides a qualitative and quantitative analysis of 3-nitropropionic acid residues in sugarcane and sugarcane juice samples using gas chromatography-tandem mass spectrometry (GC-MS). First, a set of step-shaped standard curve solutions is prepared. Then, the standard curve solutions and the test solution are injected separately into the GC-MS instrument. If the retention time of the chromatographic peak in the test solution is consistent with that of the standard working solution, with an allowable deviation of less than ±2.5%, and the relative abundance of the mass spectrometric qualitative ion pair corresponding to this chromatographic peak is consistent with the relative abundance of the standard working solution at a comparable concentration, and the relative abundance deviation does not exceed the specified limit, then it can be determined that the test solution contains 3-nitropropionic acid. Attached Figure Description

[0022] Figure 1 The chemical structural formula of 3-nitropropionic acid in a specific embodiment of the present invention is C3H5NO4;

[0023] Figure 2 The extraction ion pair spectrum of methyl 3-nitropropionate at 4.0 μg / kg (spectrum of quantitative ion pair 102→55);

[0024] Figure 3 This is a standard working curve (sugarcane) in a specific embodiment of the present invention;

[0025] Figure 4 This is a secondary mass spectrum of 3-nitropropionic acid in a specific embodiment of the present invention. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] This invention provides a sugarcane and sugarcane juice containing 3-nitropropionic acid (e.g., ...). Figure 1 The method for detecting residual gas chromatography-tandem mass spectrometry (GC-MS / MS) includes the following steps.

[0028] Step (1): Sample preparation: Peel and cut the sugarcane into pieces, then grind it with a grinder for testing. If the sample is sugarcane juice, filter it directly to remove the residue and obtain the sugarcane juice sample.

[0029] Step (2): Derivatization, extraction, and purification: Weigh 5.0 g (accurate to 0.001 g) of the sample into a 50 mL centrifuge tube, add 6 mL of 10% acetylchloromethanol (volume fraction) and 8 mL of toluene solution. Then fill the centrifuge tube with nitrogen and tighten the screw cap. After shaking and mixing, place in an 80 °C water bath for 2 h, remove and shake once every 20 min, remove from the water bath and cool to room temperature, centrifuge at 10000 rpm for 5 min, transfer the supernatant of the sample after reaction to a 50 mL centrifuge tube, add a QuEChERS extraction kit for purification, vortex for 1 min, centrifuge at 10000 rpm for 5 min, take 1 mL of the supernatant, filter through a 0.22 μm filter membrane, and analyze by GC-MS / MS.

[0030] In step (2), the derivatization method uses the acetyl chloride-methanol method. The hydrochloric acid-methanol obtained by reacting acetyl chloride with methanol causes 3-nitropropionic acid in sugarcane and sugarcane juice to undergo a methyl esterification derivatization reaction to generate methyl 3-nitropropionate. In the purification method of step (2), the liquid to be purified is extracted using a QuEChERS extraction package (PSA / GCB / C). 18 (1.2g MgSO4, 400mg PSA, 400mgGCB, 400mg C 18 Purification removes pigments and sugars from sugarcane and sugarcane juice samples to avoid contaminating the gas chromatography-tandem mass spectrometry instrument and interfering with the detection of 3-nitropropionic acid.

[0031] Step (3): Preparation of matrix-matched standard curves: Take 20 μL, 40 μL, 80 μL, 160 μL, and 320 μL of 1.0 μg / mL 3-nitropropionic acid solution into 50 mL centrifuge tubes containing 5.0 g blank samples, add 6 mL of 10% acetylchloromethanol (volume fraction) and 8 mL of toluene solution, and then proceed as in step (2). Take 1 mL of the supernatant and filter it through a 0.22 μm organic phase filter membrane into a sample vial for gas chromatography-tandem mass spectrometry to prepare standard curve solutions of 4.0 μg / kg, 8.0 μg / kg, 16.0 μg / kg, 32.0 μg / kg, and 64.0 μg / kg. Figure 2 The image shows the extracted ion pair spectrum (quantitative ion pair 102→55) of 4.0 μg / kg methyl 3-nitropropionate.

[0032] Step (4): Detection of the sample solution to be tested: The purified sample solution extracted in step (2) and the standard curve solution prepared in step (3) are injected separately into a gas chromatograph-tandem mass spectrometer. A standard curve is plotted with the peak area of ​​the quantitative ion as the ordinate (Y) and its mass concentration as the abscissa (X, ng / mL). Based on the standard curve, the concentration of 3-nitropropionic acid in the sample solution to be tested is obtained, and thus the content of 3-nitropropionic acid in sugarcane and sugarcane juice is obtained. Figure 4 The image shown is a secondary mass spectrum of 3-nitropropionic acid.

[0033] The parameters for gas chromatography-tandem mass spectrometry are as follows:

[0034] Chromatographic conditions: Column: Agilent RXI-5Sil MS capillary column, 30m x 0.25mm x 0.25μm; Column temperature program: 50℃, hold for 0 min, increase to 150℃ at 10℃ / min, hold for 5 min; Carrier gas: Helium, purity 99.999%; Carrier gas flow rate: 1.0mL / min; Injector temperature: 260℃; Injection volume: 1μL; Injection method: Split injection, split ratio: 10:1, purge flow rate 3.0mL / min, purge time 1 min;

[0035] Mass spectrometry conditions: electron impact ionization source 70 eV, ion source temperature: 230°C; transfer line temperature: 280°C; collision gas: nitrogen; scan type: multiple reaction monitoring mode (MRM), scan time: 5-10 min; the mass spectrometry parameters of qualitative and quantitative ion pairs and collision energies of methyl 3-nitropropionate, i.e., the derivative of 3-nitropropionic acid, are shown in the table below, where * indicates quantitative ion pairs.

[0036] GC-MS / MS analytical parameters of methyl 3-nitropropionate

[0037] *Quantitative Ions

[0038] As attached Figure 3 As shown, this is the standard working curve in a specific embodiment of the present invention. 3-Nitropropionic acid exhibits good linearity in the range of 4.0–64.0 μg / kg, with a correlation coefficient R0. 2 The value was greater than 0.99, and the limit of quantitation for this method was 4.0 μg / kg. The results are shown in Table 2.

[0039] Table 2. Linear equation, correlation coefficient, and limit of quantitation for 3-nitropropionic acid.

[0040] matrix Linear equations <![CDATA[Coefficient of correlation R 2 > Limit of quantitation (μg / kg) sugar cane Y = 312.84X + 249.54 0.9976 4.0 Sugarcane juice Y = 292.96X + 159.62 0.9964 4.0

[0041] The limit of quantitation (LOQ) of this method was calculated using a signal-to-noise ratio (S / N) of 10. The LOQ for 3-nitropropionic acid was 4.0 μg / kg, demonstrating that the method of this invention has high sensitivity.

[0042] 3-Nitropropionic acid was added to blank samples at concentrations of 4.0, 8.0, and 32.0 μg / kg, and the determination was performed in six parallel trials. The recovery rate and relative standard deviation (RSD) are shown in Table 3. As shown in Table 3, the average recovery rate of the method of this invention is 80.6%–96.2%, and the relative standard deviation is no greater than 8.3%, indicating good recovery rate and precision. This method has high extraction efficiency, good purification effect, high sensitivity, and good repeatability, and can be used for the determination of 3-nitropropionic acid residues in sugarcane and sugarcane juice.

[0043] Table 3. Recovery, precision, and limit of quantitation of 3-nitropropionic acid (n=6)

[0044]

[0045]

[0046] This invention is not limited to the specific embodiments described above. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any simple changes or modifications made to the design structure and concept of this invention fall within the protection scope of this invention.

Claims

1. A method for detecting 3-nitropropionic acid residues in sugar cane and sugar cane juice by gas chromatography-tandem mass spectrometry, characterized by, The method comprises the following steps: Step (1): sample preparation: the sugarcane is peeled and cut into pieces, and then stirred and crushed with a grinder; if the sample is sugarcane juice, the residue is removed by filtration to obtain a sugarcane juice sample; Step (2): derivatization, extraction and purification: weigh 5.0 g of sample in step (1) into a centrifuge tube, add 6 mL of acetyl chloride methanol solution with a volume fraction of 10% and 8 mL of toluene solution; then fill the centrifuge tube with nitrogen, tighten the screw cap; after shaking and mixing, place in a water bath at 80°C for 2 h, take out and shake once every 20 min, take out after water bath, cool to room temperature, centrifuge at 10000 rpm for 5 min, transfer the upper liquid of the reacted sample to a centrifuge tube, add QuEChERS extraction kit for purification, QuEChERS extraction kit is PSA / GCB / C 18 , including 1.2 g of MgSO4, 400 mg of PSA, 400 mg of GCB, 400 mg of C 18 , vortex for 1 min, centrifuge at 10000 rpm for 5 min, take 1 mL of the upper liquid, pass through a 0.22 μm filter membrane, and then analyze on a GC-MS / MS; Step (3): preparation of a matrix-matched standard curve: 20 μL, 40 μL, 80 μL, 160 μL and 320 μL of 3-nitropropionic acid solution with a concentration of 1.0 μg / mL are taken in centrifuge tubes containing 5.0 g of a blank sample, and then the operation in step (2) is performed, and finally GC-MS / MS analysis is performed to prepare standard curve solutions of 4.0 μg / kg, 8.0 μg / kg, 16.0 μg / kg, 32.0 μg / kg and 64.0 μg / kg; Step (4): detection of the sample solution to be detected: the sample solution extracted and purified in step (2) and the standard curve solution prepared in step (3) are respectively injected into a gas chromatograph-tandem mass spectrometer, the peak area of a quantitative ion is taken as the vertical coordinate, and the mass concentration is taken as the horizontal coordinate to draw a standard curve; based on the standard curve, the concentration of 3-nitropropionic acid in the sample solution to be detected is obtained, and then the content of 3-nitropropionic acid in the sugarcane and the sugarcane juice is obtained.

2. A method for detecting 3-nitropropionic acid residues in sugar cane and sugar cane juice by gas chromatography-tandem mass spectrometry according to claim 1, characterized by: In the derivatization method of step (2), acetyl chloride-methanol is used to perform a methylation derivatization reaction on 3-nitropropionic acid in the sugarcane and the sugarcane juice to generate 3-nitropropionic acid methyl ester; in the purification method of step (2), the QuEChERS extraction kit is used to remove the pigment and sugar content in the sample in the sugarcane and the sugarcane juice to avoid pollution of the gas chromatograph-tandem mass spectrometer and interference with the detection of 3-nitropropionic acid.

3. A method for detecting 3-nitropropionic acid residues in sugar cane and sugar cane juice by gas chromatography-tandem mass spectrometry according to claim 1, characterized by: The parameter conditions of the gas chromatograph-tandem mass spectrometer in step (4) are as follows: Chromatographic conditions: Chromatographic column: Agilent RXI-5Sil MS capillary column 30 m x 0.25 mm x 0.25 μm; chromatographic column temperature program: 50 ℃ for 0 min, increased to 150 ℃ at a rate of 10 ℃ / min and maintained for 5 min; carrier gas: helium, purity 99.999%; carrier gas flow rate: 1.0 mL / min; injection port temperature: 260 ℃; injection volume: 1 μL; injection mode: split injection, split ratio: 10:1, purge flow 3.0 mL / min, purge time 1 min; Mass spectrometry conditions: Electron impact ion source 70 eV, ion source temperature: 230 C; transmission line temperature: 280 C; collision gas: nitrogen; scan type: multiple reaction monitoring mode MRM, scan time 5-10 min; the qualitative and quantitative ion pairs and the collision energy of 3-nitropropionic acid methyl ester, i.e., the derivatization product of 3-nitropropionic acid, are shown in Table 1, and the quantitative ion pairs are marked with * in the table; Table 1 GC-MS / MS analysis parameters of 3-nitropropionic acid methyl ester * Quantitative ion.

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