A method for determining melatonin in legumes and grains

Abstract

This invention discloses a method for determining melatonin in legumes and cereals. First, the sample is pulverized, passed through a 50-80 mesh sieve, added to a centrifuge tube, methanol is added, and the mixture is shaken for 2-5 minutes. The mixture is then sonicated for 20-40 minutes, centrifuged at high speed for 3-8 minutes, and the supernatant is collected. This supernatant is transferred to a centrifuge tube containing a purifying agent, vortexed for 1-3 minutes, and centrifuged at high speed for 10-20 minutes. The supernatant is then collected again, water is added, and the mixture is vortexed and filtered into a sample vial. Finally, the sample is analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This method is a rapid, simple, highly sensitive, specific, and stable UHPLC-MS / MS method for determining melatonin in soybeans and cereals.

Description

Technical Field

[0001] This invention belongs to the field of food inspection and testing technology, and specifically relates to a method for determining melatonin in legumes and grains. Background Technology

[0002] Melatonin (MT) is an indole-like tryptamine (N-acetyl-5-methoxytryptamine) widely found in biological organisms, possessing functions such as regulating circadian rhythms, antioxidation, and neuroprotection. Melatonin is the most potent known endogenous free radical scavenger, playing a crucial role in sleep, promoting gonadal development, and regulating immunity, and exhibiting various regulatory functions in the human body. Currently, melatonin has been found in almost all organisms, including invertebrates, plants, unicellular algae, and even prokaryotes. It has also been found in tea and herbal infusions, baked goods, and fermented foods. Exogenous application of melatonin significantly improves seed germination rates, while excessively high concentrations inhibit seed germination. Melatonin participates in regulating seed and callus development, root development, and fruit ripening. Melatonin can enhance plant resistance to biotic and abiotic stresses.

[0003] Melatonin has the molecular formula C 13 N2H 16 O2 is a small indoleamine compound synthesized from the essential amino acid tryptophan. Its molecular structure contains an indole ring, a methoxy group, and an amide group. The indole ring structure imparts its high lipophilicity, while the methoxy and acetyl groups impart its hydrophobicity. Melatonin is a white or off-white crystalline powder, readily soluble in methanol, sensitive to light and air, and easily oxidized.

[0004] Currently, the main methods for determining melatonin include high-performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis (CE), gas chromatography-mass spectrometry (GC-MS), fluorescence method, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), photodetection method, electrophoresis method, and electrochemical method. Among these, HPLC-MS is a fast, accurate, and reliable method. Liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) has the characteristics of high sensitivity and good selectivity, which greatly improves the accuracy and precision of melatonin detection. It has been applied in vegetables, fruits, and grains. However, the pretreatment of extraction and purification in this method is complicated. After the sample is extracted, it needs to be adsorbed, eluted, purified, or concentrated and reconstituted using a solid phase extraction column. The operation steps are cumbersome and inefficient. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining melatonin in legumes and grains. Soybean and grain samples are extracted with methanol using ultrasound. The extract is purified by multi-walled carbon nanotubes and magnetic Fe3O4-humic acid. Detection is performed by ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) in multiple reaction monitoring mode, and quantification is performed using the external standard method.

[0006] A method for determining melatonin in legumes and grains, comprising the following steps:

[0007] (1) Crush the sample, pass it through a 50-80 mesh sieve, take 1-3g and add it to a centrifuge tube, add 15-25mL of methanol, shake and mix for 2-5min, place it in an ultrasonic bath for 20-40min, centrifuge at 8000-12000rpm for 3-8min, and take 1.5-2.5mL of supernatant.

[0008] (2) Transfer the supernatant to a centrifuge tube containing a purifying agent, vortex for 1-3 min, centrifuge at 8000-12000 rpm for 10-20 min, take 0.6-1.0 mL of supernatant, add 0.1-0.3 mL of water, vortex to mix, and filter the membrane into a sample bottle.

[0009] (3) The samples were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0010] The purifying agent consists of 40-80 mg of multi-walled carbon nanotubes and 40-80 mg of magnetic Fe3O4-humic acid. The multi-walled carbon nanotubes have an ID of 2 nm-7 nm, an OD of 11 nm, and a length of 10 μm.

[0011] The preparation method of the magnetic Fe3O4-humic acid is as follows: Weigh 4-6g of Fe3O4 powder into 100-200mL of distilled water, sonicate for 15-30min, add 20-40mL of NH3·H2O to control the pH of the solution system above 7, then add 0.5-1.5g of humic acid powder and 80-120mL of NaCl solution, place in a water bath at 80-100℃ and stir for 25-45min, cool to room temperature, wash with anhydrous ethanol until the supernatant is clear, separate with an external magnet, and dry in an oven at 40-80℃ to obtain Fe3O4-humic acid.

[0012] The filter membrane is a 0.22μm polyethersulfone filter membrane.

[0013] The mobile phase used for the ultra-high performance liquid chromatography-tandem mass spectrometry determination was 5 mmol ammonium formate, 0.1% formic acid in methanol, and 0.1% formic acid in water.

[0014] The mass spectrometer was acquired using multiple reaction monitoring (MRM), with an electrospray ionization source, capillary voltage of 3.90 kV, desolvation gas flow rate of 650 L / Hr, desolvation gas temperature of 350 °C, carrier gas N2, and collision gas Ar. The quantitative ion pair was 232.96 / 173.99, and the qualitative ion pair was 232.96 / 158.97.

[0015] The beneficial effects of this invention are as follows: The method of this invention utilizes multi-walled carbon nanotubes and magnetic Fe3O4-humic acid in the pretreatment process. Due to their unique structure, they possess numerous adsorption sites and a large surface area, resulting in strong adsorption capacity. Their adsorption performance is superior to commonly used adsorption materials such as C18 and PSA, effectively adsorbing interfering substances in complex mechanisms. The pretreatment is simple, requiring minimal equipment. After extraction and purification, the sample can be directly analyzed. The process is simple and efficient. Combined with the high sensitivity and selectivity of LC-MS / MS, it can stably perform qualitative and quantitative analysis of melatonin. This invention provides a rapid, simple, highly sensitive, specific, and stable ultra-high performance liquid chromatography-tandem mass spectrometry method for determining melatonin in soybeans and cereals. Attached Figure Description

[0016] Figure 1 Recovery rates of melatonin were added for different pretreatment methods.

[0017] Figure 2 Results of mobile phase determination and optimization for ultra-high performance liquid chromatography. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0019] Example 1

[0020] Accurately weigh 2g of uniformly pulverized sample (more than 90% passing through 60 mesh) (accurate to 0.01g) into a 50mL centrifuge tube, add 20mL of methanol, mix well and vortex at high speed for 3min, place in an ultrasonic bath for 30min, and protect from light during the process. Centrifuge at 10000rpm for 5min, take 2mL of supernatant, transfer to a 10mL centrifuge tube containing 60mg of multi-walled carbon nanotubes and 60mg of magnetic Fe3O4-humic acid, vortex for 1min, centrifuge at 10000rpm for 15min, take 0.8mL of supernatant, add 0.2mL of water, vortex to mix, filter through a 0.22μm polyethersulfone membrane into a sample vial, and determine by UPLC-MS / MS.

[0021] Chromatographic reference conditions:

[0022] a. Chromatographic column: BEH C18 (100mm×2.1mm, 1.7μm).

[0023] b. Column temperature: 30℃.

[0024] c. The types of mobile phases and gradient elution procedures for melatonin are shown in Table 1.

[0025] Table 1 Melatonin gradient elution procedure

[0026]

[0027] Mass spectrometry conditions:

[0028] a. Electrospray ionization source (ESI+).

[0029] b. Capillary voltage 3.90kV.

[0030] c. Solvent gas flow rate: 650 L / Hr.

[0031] d. Desolvent gas temperature 350℃.

[0032] e. Data collected using multiple response monitoring (MRM) methods.

[0033] Table 2 Reference Mass Spectrometry Conditions for Quantitative and Qualitative Ions in Melatonin Production

[0034]

[0035] The external standard method was used for quantitative determination, and the standard solution for quantification was prepared using 80% methanol aqueous solution.

[0036] Example 2 Method Optimization

[0037] Preprocessing method optimization:

[0038] (1) Selection of extraction solution: Melatonin is easily soluble in methanol. When using different solvents such as methanol, acetonitrile, and ethyl acetate for extraction, methanol extraction has the best effect.

[0039] (2) Optimization of extraction solvent dosage: 1g, 2g, and 4g of sample were weighed and added to 20ml of methanol, with extraction ratios of 1:20, 1:10, and 1:5, respectively. The ratio of sample to extraction solvent directly affects the extraction effect. The soybean sample is in powder form and has a certain volume. At an extraction ratio of 1:20, the solvent dosage is large, the sample weight is low, and the sample representativeness is poor. In the extraction ratio of 1:5, the volume ratio of sample powder to solution is close to 1:2, the mixture concentration is high, the liquid viscosity is high, and the shaking is uneven, which is not conducive to extraction. Therefore, the extraction ratio of 2g sample to 20ml of methanol has the best effect.

[0040] (3) Optimization of shaking time: Melatonin is sensitive to light and air, and is easily oxidized. Therefore, ensuring good extraction results and preventing oxidation are crucial during the extraction process. Rapid shaking extraction yields good results with a short time. A figure-eight shaker at 1000 rpm was used for rapid shaking extraction of the solid-liquid mixture. The shaking times were 1 min, 3 min, and 5 min. The 1 min shaking time was too short, resulting in insufficient wetting of a small amount of sample and poor shaking performance. The 3 min shaking time provided good results, ensuring thorough mixing of the sample and solvent. The 5 min shaking time did not significantly improve the extraction effect. Therefore, 3 min of shaking time was optimal.

[0041] (4) Optimization of extraction method: After oscillation for 3 minutes, four extraction modes were selected: overnight standing, overnight standing + 30 minutes of sonication, 30 minutes of sonication, and no treatment. The extraction results are as follows: Figure 1 As shown. From Figure 1 As can be seen, the overnight standing followed by 30-minute sonication resulted in the highest recovery rate, followed by overnight standing and 30-minute sonication. However, the recovery rates of the three methods were not significantly different, and all three methods showed higher recovery rates than the untreated method. Since the overnight standing period was relatively long and there was no significant difference among the three methods, the extraction method of oscillation followed by 30-minute sonication was the most suitable.

[0042] (5) Purification optimization: Soybeans contain a large amount of protein, fat, polysaccharide and other substances. After extraction, the purification of impurities is very important. In this experiment, multi-walled carbon nanotubes and magnetic Fe3O4-humic acid were selected as adsorbent materials. During the pretreatment process, due to the combined use of the two adsorbents, there are more adsorption sites and a huge surface area. The adsorption capacity is strong and the adsorption performance is better than that of commonly used adsorbent materials such as C18 and PSA. It can effectively adsorb interfering substances in complex mechanisms.

[0043] The use of adsorbents alone or in combination significantly affects the purification and adsorption effects. The dosage of multi-walled carbon nanotubes and magnetic Fe3O4-humic acid was selected based on the amount of supernatant. Purification was performed using supernatant concentrations of 10 mg / 1 ml, 30 mg / 1 ml, 60 mg / 1 ml, and 90 mg / 1 ml, respectively. Addition and recovery were conducted at a melatonin addition level of 40 ppb. The results are shown in Tables 3-4. The table shows that the purification effect was optimal at 30 mg / 1 ml supernatant. The combined use of multi-walled carbon nanotubes and magnetic Fe3O4-humic acid resulted in a stronger purification effect than using either alone.

[0044] Recovery rate % = (Measured value of sample after melatonin addition - Melatonin content of sample) / 40 ppb) × 100%

[0045] Table 3

[0046]

[0047] Note: * indicates p < 0.05 compared to composite group 1; # indicates p < 0.05 compared to composite group 2.

[0048] Table 4

[0049]

[0050]

[0051] Note: * indicates p < 0.05 compared to composite group 3; # indicates p < 0.05 compared to composite group 4.

[0052] (6) Optimization of ultra-high performance liquid chromatography-tandem mass spectrometry conditions

[0053] Mobile phase determination and optimization: Two mobile phase combinations were tested: methanol and 0.1% formic acid aqueous solution (combination 1), and 5 mmol ammonium formate, 0.1% formic acid, methanol, and 0.1% formic acid aqueous solution (combination 2). Figure 2 It can be seen that when the mobile phase is 5 mmol ammonium formate, 0.1% formic acid in methanol, and 0.1% formic acid in water, the melatonin response value is high and the baseline is stable. Therefore, this mobile phase system is used in this method. The column temperature is 30℃ and the flow rate is 0.200 mL / min.

[0054] Determination of mass spectrometry conditions: The mass spectrometry quantitative detection method for melatonin was selected using multiple reaction monitoring (MRM) mode, with an electrospray ionization source (ESI+), capillary voltage of 3.90 kV, desolvation gas flow rate of 650 L / Hr, desolvation gas temperature of 350 °C, carrier gas N2, collision gas Ar, quantitative ion pair of 232.96 / 173.99, and qualitative ion pair of 232.96 / 158.97.

[0055] Methodological evaluation:

[0056] Standard Curve: Accurately measure appropriate amounts of melatonin standard working solution and dilute with 80% methanol to prepare a series of standard working solutions with melatonin concentrations of 0.1, 0.5, 1, 2, 5, 10, 20, 50, 100, and 200 ng / mL. These solutions were then analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). Under the defined parameters, injections were performed from low to high concentrations, and a standard curve was plotted based on the average peak area ratio and the corresponding melatonin solution concentration (μg / L). The regression equation and correlation coefficient were calculated. The regression equation showed a good linear relationship between the melatonin standard solution concentrations and the target concentrations within the range of 1–200 μg / L. Specific linear equations and correlation coefficients are shown in Table 4.

[0057] Table 4. Linear Equation of Standard Curve

[0058] name Linear range μg / L Linear equations <![CDATA[Coefficient of correlation R 2 > melatonin 0.1～200 Y = 23635X + 5623 0.9991

[0059] Limits of Detection (LOD) and Limits of Quantification (LOD): External standard method was used for quantification in this experiment. Melatonin was prepared into standard working solutions containing 0.5–200 ng / mL, with nine concentration gradients. Working curves were plotted for the peak area y and concentration x (ng / mL) of the quantitative ion, and the results are shown in Table 4. It can be seen that the linear range of melatonin concentration exceeds three orders of magnitude. At a melatonin concentration of 0.1 μg / kg, the signal-to-noise ratio (SNR) is >3, and the LOD is 0.1 μg / kg; at a melatonin concentration of 0.5 μg / kg, the SNR is >10, and the LOD is 0.5 μg / kg.

[0060] Method precision: In the same laboratory, by the same operator using the same instruments and equipment, following the same test method, multiple determinations were performed on the same level of spiked samples within a short period of time. As shown in Table 5, the relative standard deviation of the reproducibility of this method is less than 5.0%, and the relative standard deviation of the reproducibility is less than 8.0%, indicating good repeatability and reproducibility.

[0061] Table 5. Repeatability and reproducibility results of different melatonin concentrations.

[0062] Spiked concentration (μg / kg) Repeatability RSD (%) Reproducibility RSD (%) 40 4.92％ 6.23％ 100 1.96％ - 400 2.18％ 2.98％

[0063] The melatonin content of soybeans, corn, rice, wheat, millet, and sorghum was determined using this method. The melatonin contents were 73.55 μg / kg for soybeans, 74.49 μg / kg for corn, 87.04 μg / kg for rice, 91.38 μg / kg for wheat, and 84.45 μg / kg for sorghum, respectively. This method is also applicable to grains such as soybeans, corn, and rice. Pretreatment can effectively extract melatonin and purify impurities, making the method effective and reliable.

[0064] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for determining melatonin in legumes and grains, characterized in that, Follow these steps: (1) Crush the sample, pass it through a 50-80 mesh sieve, take 1-3g and add it to a centrifuge tube, add 15-25 mL of methanol, shake and mix for 2-5 min, place it in an ultrasonic bath for 20-40 min, centrifuge at 8000-12000 rpm for 3-8 min, and take 1.5-2.5 mL of supernatant. (2) Transfer the supernatant to a centrifuge tube containing a purifying agent, vortex for 1-3 min, centrifuge at 8000-12000 rpm for 10-20 min, take 0.6-1.0 mL of supernatant, add 0.1-0.3 mL of water, vortex to mix, and filter the membrane into a sample bottle. The purifying agent consists of 40-80 mg of multi-walled carbon nanotubes and 40-80 mg of magnetic Fe3O4-humic acid. The preparation method of the magnetic Fe3O4-humic acid is as follows: Weigh 4-6 g of Fe3O4 powder into 100-200 mL of distilled water, sonicate for 15-30 min, add 20-40 mL of NH3·H2O to control the pH of the solution system above 7, then add 0.5-1.5 g of humic acid powder and 80-120 mL of NaCl solution, place in a water bath at 80-100℃ and stir for 25-45 min, cool to room temperature, wash with anhydrous ethanol until the supernatant is clear, separate with an external magnet, and dry in an oven at 40-80℃ to obtain Fe3O4-humic acid. (3) The samples were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry.

2. The method for determining melatonin in legumes and grains according to claim 1, characterized in that, The filter membrane is a 0.22μm polyethersulfone filter membrane.

3. The method for determining melatonin in legumes and grains according to claim 1, characterized in that, The mobile phase used for the ultra-high performance liquid chromatography-tandem mass spectrometry determination was 5 mmol ammonium formate, 0.1% formic acid in methanol, and 0.1% formic acid in water.

4. The method for determining melatonin in legumes and grains according to claim 1, characterized in that, The mass spectrometer was acquired using multiple reaction monitoring (MRM), with an electrospray ionization source, capillary voltage of 3.90 kV, desolvent gas flow rate of 650 L / Hr, desolvent gas temperature of 350 °C, carrier gas N2, collision gas Ar, quantitative ion pair of 232.96 / 173.99, and qualitative ion pair of 232.96 / 158.97.

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