阅读说明：本技术 一种晶体番茄红素中正己烷和丙酮溶剂残留量的检测方法 (Method for detecting residual quantity of n-hexane and acetone solvents in crystal lycopene ) 是由 连运河 张玉芬 程远欣 吴迪 夏卫东 王欢欢 于 2021-01-22 设计创作，主要内容包括：本发明公开了一种晶体番茄红素中正己烷和丙酮溶剂残留量的检测方法,包括如下步骤：S1、晶体番茄红素用超微粉碎机粉碎,混匀5min；S2、取混匀后样品0.3000±0.01 g到20 mL顶空瓶中,加入色谱级及以上级别的二氯甲烷涡旋溶解；S3、将步骤S2所得溶液加入色谱级及以上级别的N,N—二甲基甲酰胺,迅速加盖密封,加热,涡旋萃取,然后静置,直至分层完全,取上层待测；S4、将步骤S3溶液,放入气相色谱仪顶空进样器中,80℃平衡30min,吸取1mL上部气体,气相色谱-氢离子火焰检测器检测正己烷和丙酮溶剂残留量,本发明能够准确检测晶体番茄红素中正己烷和丙酮的残留量。(The invention discloses a method for detecting residual amounts of n-hexane and acetone solvents in crystal lycopene, which comprises the following steps: s1, crushing the crystal lycopene by using an ultrafine crusher, and uniformly mixing for 5 min; s2, taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, adding dichloromethane of chromatographic grade or above, and carrying out vortex dissolution; s3, adding the solution obtained in the step S2 into N, N-dimethylformamide with a chromatographic grade or above, quickly covering and sealing, heating, performing vortex extraction, then standing until layering is complete, and taking the upper layer to be tested; s4, placing the solution obtained in the step S3 into a gas chromatograph headspace sample injector, balancing for 30min at 80 ℃, absorbing 1mL of upper gas, and detecting the residual quantity of the n-hexane and acetone solvents by using a gas chromatograph-hydrogen ion flame detector.)

1. A method for detecting the residual quantity of n-hexane and acetone solvents in crystal lycopene is characterized by comprising the following steps: the method comprises the following steps:

s1, crushing the crystal lycopene by using an ultrafine crusher, and uniformly mixing for 5 min;

s2, taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, adding dichloromethane of chromatographic grade or above, and carrying out vortex dissolution;

s3, adding the solution obtained in the step S2 into N, N-dimethylformamide with a chromatographic grade or above, quickly covering and sealing, heating, performing vortex extraction, then standing until layering is complete, and taking the upper layer to be tested;

s4, placing the solution obtained in the step S3 into a gas chromatograph headspace sample injector, balancing for 30min at 80 ℃, sucking 1mL of upper gas, and detecting the residual quantity of the n-hexane and acetone solvents by a gas chromatograph-hydrogen ion flame detector.

2. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 1, characterized in that: and in the step S1, the crushing end point is that the passing rate of the 100-mesh sieve reaches more than 95%.

3. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 1, characterized in that: the addition amount of the dichloromethane in the step S2 is 0.2-1 mL.

4. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 1, characterized in that: and in the step S3, the adding amount of the N, N-dimethylformamide is 5-5.8 mL.

5. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 1, characterized in that: in the step S3, the heating temperature is 50-60 ℃, the heating time is 10min, and the vortex extraction is performed for 3-5 min.

6. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 1, characterized in that: and in the step S3, standing is carried out for 10-20 min at the temperature of 20-25 ℃.

7. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 1, characterized in that: the gas chromatography condition in the step S4 is a chromatographic column polyethylene glycol nitrobenzene modified capillary column, the column length is 60m, the column inner diameter is 0.25mm, the membrane thickness is 0.25 mu m, the carrier gas is nitrogen, the flow is 3.0mL/min, the injection port temperature is 220 ℃, the column temperature is programmed temperature rise, the detector temperature is 235 ℃, and the split-flow ratio is 25: 1.

8. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 7, characterized in that: the temperature programming condition is that the initial temperature is 60 ℃, the temperature is kept for 5min, the temperature is increased to 85 ℃ at 3.5 ℃/min, then the temperature is increased to 220 ℃ at 20 ℃/min, and the temperature is kept for 1 min.

9. The method for detecting the residual quantity of n-hexane and acetone solvents in crystalline lycopene according to claim 7, characterized in that: the quantitative method is an external standard curve method, 5-5.8 mL of N, N-dimethylformamide is taken as a matrix, and N-hexane and acetone standard substances are respectively added to prepare series of standard solutions of 0, 2, 5, 10, 20 and 50 mg/L.

Technical Field

The invention relates to a method for detecting residual amounts of n-hexane and acetone solvents in crystalline lycopene, and belongs to the technical field of detection.

Background

Lycopene (Lycopene) is a natural carotenoid, mainly found in ripe fruits such as tomato, watermelon, guava, rose hip, papaya and grapefruit. The molecular structure of the carotenoid is a straight-chain hydrocarbon and is also an oxygen-free carotenoid, the carotenoid consists of 11 conjugated C ═ C and two unconjugated C ═ C, and the carotenoid can efficiently quench singlet oxygen, eliminate peroxy radicals, regulate intercellular communication, enhance immunity, regulate cholesterol synthesis and the like. In addition, lycopene can also be used for preventing and treating atherosclerosis, cardiovascular diseases, cancer, etc. Lycopene has been recognized as a class a nutrient by the world food and agriculture organization/world health organization (FAO/WHO) and joint food additive Joint Experts Committee (JECFA), and has been widely used in the fields of medical materials, health products, food additives, cosmetics, and the like. In addition, lycopene also has advantages in improving animal health and improving animal product quality.

The crystal lycopene is obtained by crystallizing and purifying lycopene oleoresin, wherein the lycopene oleoresin is an oily substance obtained by extracting tomatoes and products thereof by using an organic solvent and removing the organic solvent in an extracting solution, the mass fraction of the lycopene is more than 6-20%, the lycopene cannot be directly eaten or used for medicines, and the lycopene can be used in health-care foods, medicines and cosmetics only by being purified. Researches on Qinqin and the like find that the antioxidant effect of the lycopene crystal is superior to that of lycopene oleoresin. The limit of n-hexane and ethyl acetate residues in lycopene is regulated to be 50mg/kg by national standards, and foreign customers also require that the acetone residue of high-end lycopene is below 200mg/kg, so that the establishment of the solvent residue detection method suitable for n-hexane and acetone in lycopene crystals is significant to the healthy development of the lycopene industry.

At present, the detection method of the residual solvent quantity in the tomato red comprises the following steps: the detection method of the n-hexane residue is water wetting detection, the detection method of the ethyl acetate residue is diethyl phthalate extraction detection, the simultaneous detection method of ethyl acetate and ethanol in lycopene oil resin is dimethyl sulfoxide and water extraction, no report is provided about the detection method of the acetone residue in lycopene, and the conventional detection method of the n-hexane residue is not suitable for crystal lycopene.

The crystal lycopene is a dark red needle-shaped crystal, the molecular structure of the crystal lycopene is a straight-chain hydrocarbon and is also a carotenoid without oxygen, the crystal lycopene is dissolved in chloroform, benzene and grease and insoluble in solvent residues such as polar solvents DMA, DMF, dimethyl sulfoxide and the like which are commonly used for detecting, if the solvent is used for detecting the conventional solvent residues, a sample is seriously caked at the bottom in the process, the dissolution of n-hexane and acetone solvents is seriously influenced, the residual quantity of the n-hexane and the acetone in the crystal lycopene can not be accurately detected, the bulk density of the crystal lycopene is small according to the detection of national standard GB5009.262, and the volume of 5g of the national standard sample in a 20mL headspace bottle is more than 10mL, and the detection cannot be carried out.

Therefore, the establishment of a method for reliably and accurately detecting the residual quantity of n-hexane and acetone in the crystal lycopene is an urgent problem to be solved.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for detecting the residual quantity of the solvent of n-hexane and acetone in crystal lycopene, and the residual quantity of the solvent of n-hexane and acetone in crystal lycopene can be reliably and accurately detected only by solving the problem that the solvent of n-hexane and acetone in crystal lycopene is difficult to fully extract.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for detecting residual quantity of n-hexane and acetone solvents in crystal lycopene comprises the following steps:

s1, crushing the crystal lycopene by using an ultrafine crusher, and uniformly mixing for 5 min;

s2, taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, adding dichloromethane of chromatographic grade or above, and carrying out vortex dissolution;

s3, adding the solution obtained in the step S2 into N, N-dimethylformamide with the chromatographic grade or above, quickly covering and sealing, heating, performing vortex extraction, then standing until the layering is complete, and taking the upper layer to be tested;

s4, placing the solution obtained in the step S3 into a gas chromatograph headspace sample injector, balancing for 30min at 80 ℃, sucking 1mL of upper gas, and detecting the residual quantity of the n-hexane and acetone solvents by a gas chromatograph-hydrogen ion flame detector.

The technical scheme of the invention is further improved as follows: and in the step S1, the crushing end point is that the passing rate of the 100-mesh sieve reaches more than 95%.

The technical scheme of the invention is further improved as follows: the addition amount of the dichloromethane in the step S2 is 0.2-1 mL.

The technical scheme of the invention is further improved as follows: and in the step S3, the adding amount of the N, N-dimethylformamide is 5-5.8 mL.

The technical scheme of the invention is further improved as follows: in the step S3, the heating temperature is 50-60 ℃, the heating time is 10min, and the vortex extraction is performed for 3-5 min.

The technical scheme of the invention is further improved as follows: and in the step S3, standing is carried out for 10-20 min at the temperature of 20-25 ℃.

The technical scheme of the invention is further improved as follows: the gas chromatography condition in the step S4 is a chromatographic column polyethylene glycol nitrobenzene modified capillary column, the column length is 60m, the column inner diameter is 0.25mm, the membrane thickness is 0.25 μm, the carrier gas is nitrogen, the flow rate is 3.0mL/min, the injection port temperature is 220 ℃, the column temperature is programmed temperature rise, the detector temperature is 235 ℃, and the split ratio is 25: 1.

The technical scheme of the invention is further improved as follows: the temperature programming condition is that the initial temperature is 60 ℃, the temperature is kept for 5min, the temperature is increased to 85 ℃ at 3.5 ℃/min, then the temperature is increased to 220 ℃ at 20 ℃/min, and the temperature is kept for 1 min.

The technical scheme of the invention is further improved as follows: the quantitative method is an external standard curve method, 5-5.8 mL of N, N-dimethylformamide is taken as a matrix, and N-hexane and acetone standard substances are respectively added to prepare series of standard solutions of 0, 2, 5, 10, 20 and 50 mg/L.

Due to the adoption of the technical scheme, the invention has the technical progress that:

the method can accurately detect the residual quantity of n-hexane and acetone in the crystal lycopene; the method is simple to operate, accurate and reliable in result and good in reproducibility; provides technical guarantee for the production and sale of the crystalline lycopene which controls the residual quantity of n-hexane and acetone solvents and the use of the crystalline lycopene by customers, and provides technical support for the follow-up research of the generation mechanism of acetone.

Detailed Description

The present invention will be described in further detail with reference to the following examples:

a method for detecting residual quantity of n-hexane and acetone solvents in crystal lycopene comprises the following steps:

s1, crushing the crystal lycopene by using an ultrafine grinder until the passing rate of a 100-mesh sieve reaches more than 95%, and uniformly mixing for 5 min;

s2, taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, adding 0.2-1 mL of dichloromethane with the chromatographic grade or above for vortex dissolution;

s3, adding 5-5.8 mL of N, N-dimethylformamide of a chromatographic grade or above into the solution obtained in the step S2, quickly covering and sealing, heating to 50-60 ℃, heating for 10min, performing vortex extraction for 3-5 min, then standing for 10-20 min at 20-25 ℃ until layering is complete, and taking the upper layer to be tested;

s4, placing the solution obtained in the step S3 into a gas chromatograph headspace sample injector, balancing for 30min at 80 ℃, sucking 1mL of upper gas, and detecting the residual quantity of the n-hexane and acetone solvents by a gas chromatograph-hydrogen ion flame detector.

The gas chromatography conditions are that a chromatographic column polyethylene glycol nitrobenzene modified capillary column has the length of 60m, the inner diameter of the column of 0.25mm, the thickness of the membrane of 0.25 mu m, the carrier gas is nitrogen, the flow rate is 3.0mL/min, the temperature of a sample inlet is 220 ℃, the temperature of the column is programmed temperature rise, the temperature of a detector is 235 ℃, and the split ratio is 25: 1. The programmed temperature rise condition is that the initial temperature is 60 ℃, the temperature is kept for 5min, the temperature is increased to 85 ℃ at 3.5 ℃/min, then the temperature is increased to 220 ℃ at 20 ℃/min, and the temperature is kept for 1 min.

The quantitative method is an external standard curve method, and 5-5.8 mL of N, N-dimethylformamide solvent is used as a matrix, and N-hexane and acetone standard substances are respectively added to prepare 0, 2, 5, 10, 20 and 50mg/L series of standard solutions.

Example 1

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, and adding 0.5mL of dichloromethane with the chromatographic grade and above for vortex dissolution; adding 5.5mL of N, N-Dimethylformamide (DMF) with a chromatographic grade or above, rapidly covering and sealing, heating at 55 deg.C for 10min, performing vortex extraction for 4min, standing at 22 deg.C for 10min, completely layering, and collecting the upper layer to be tested; placing into a headspace sample injector of a gas chromatograph, balancing for 30min at 80 ℃, sucking 1mL of upper gas, detecting the content of normal hexane and acetone in the crystal lycopene by using a gas chromatography-hydrogen ion flame detector, and detecting samples 1, 2 and 3, wherein the detection results are shown in Table 1; wherein, the gas chromatography condition is a chromatographic column polyethylene glycol nitrobenzene modified capillary column, the length of the column is 60m, the inner diameter of the column is 0.25mm, the thickness of the membrane is 0.25 μm, the carrier gas is nitrogen, the flow rate is 3.0mL/min, the temperature of a sample inlet is 220 ℃, the temperature of the column is programmed temperature, the temperature of a detector is 235 ℃, and the split ratio is 25: 1; the temperature programming condition is that the temperature is initially 60 ℃, is kept for 3min, is increased to 85 ℃ at 3.5 ℃/min, is increased to 220 ℃ at 20 ℃/min, and is kept for 1 min.

Table 1 table of test results of example 1

Example 2

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, and adding 0.6mL of dichloromethane with the chromatographic grade and above for vortex dissolution; adding 5.4mL of N, N-Dimethylformamide (DMF) with a chromatographic grade or above, rapidly covering and sealing, heating at 53 deg.C for 10min, performing vortex extraction for 5min, standing at 25 deg.C for 15min, completely layering, and collecting the upper layer to be tested; placing into a headspace sample injector of a gas chromatograph, balancing for 30min at 80 ℃, sucking 1mL of upper gas, detecting the content of normal hexane and acetone in the crystal lycopene by using a gas chromatography-hydrogen ion flame detector, and detecting samples 1, 2 and 3, wherein the detection results are shown in Table 2; wherein, the gas chromatography condition is a chromatographic column polyethylene glycol nitrobenzene modified capillary column, the length of the column is 60m, the inner diameter of the column is 0.25mm, the thickness of the membrane is 0.25 μm, the carrier gas is nitrogen, the flow rate is 3.0mL/min, the temperature of a sample inlet is 220 ℃, the temperature of the column is programmed temperature, the temperature of a detector is 235 ℃, and the split ratio is 25: 1; the temperature programming condition is that the temperature is initially 60 ℃, is kept for 3min, is increased to 85 ℃ at 3.5 ℃/min, is increased to 220 ℃ at 20 ℃/min, and is kept for 1 min.

Table 2 table of test results of example 2

Example 3

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, and adding 0.4mL of dichloromethane with the chromatographic grade and above for vortex dissolution; adding 5.6mL of N, N-Dimethylformamide (DMF) with a chromatographic grade or above, rapidly covering and sealing, heating at 60 ℃ for 10min, performing vortex extraction for 4min, standing at 20 ℃ for 20min, completely layering, and taking the upper layer to be tested; placing into a headspace sample injector of a gas chromatograph, balancing for 30min at 80 ℃, sucking 1mL of upper gas, detecting the content of normal hexane and acetone in the crystal lycopene by using a gas chromatography-hydrogen ion flame detector, and detecting samples 1, 2 and 3, wherein the detection results are shown in Table 3; wherein, the gas chromatography condition is a chromatographic column polyethylene glycol nitrobenzene modified capillary column, the length of the column is 60m, the inner diameter of the column is 0.25mm, the thickness of the membrane is 0.25 μm, the carrier gas is nitrogen, the flow rate is 3.0mL/min, the temperature of a sample inlet is 220 ℃, the temperature of the column is programmed temperature, the temperature of a detector is 235 ℃, and the split ratio is 25: 1; the temperature programming condition is that the temperature is initially 60 ℃, is kept for 3min, is increased to 85 ℃ at 3.5 ℃/min, is increased to 220 ℃ at 20 ℃/min, and is kept for 1 min.

Table 3 table of test results of example 3

Example 4

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; taking 0.3000 +/-0.01 g of the uniformly mixed sample into a 20mL headspace bottle, and adding 1.0mL of dichloromethane with the chromatographic grade and the grade above for vortex dissolution; adding 5.0mL of N, N-Dimethylformamide (DMF) with a chromatographic grade or above, rapidly covering and sealing, heating at 57 ℃ for 10min, performing vortex extraction for 3min, standing at 23 ℃ for 17min, completely layering, and taking the upper layer to be tested; placing into a headspace sample injector of a gas chromatograph, balancing for 30min at 80 ℃, sucking 1mL of upper gas, detecting the content of n-hexane and acetone in the crystal lycopene by using a gas chromatography-hydrogen ion flame detector, and detecting samples 1, 2 and 3, wherein the detection results are shown in Table 4; wherein, the gas chromatography condition is a chromatographic column polyethylene glycol nitrobenzene modified capillary column, the length of the column is 60m, the inner diameter of the column is 0.25mm, the thickness of the membrane is 0.25 μm, the carrier gas is nitrogen, the flow rate is 3.0mL/min, the temperature of a sample inlet is 220 ℃, the temperature of the column is programmed temperature, the temperature of a detector is 235 ℃, and the split ratio is 25: 1; the temperature programming condition is that the temperature is initially 60 ℃, is kept for 3min, is increased to 85 ℃ at 3.5 ℃/min, is increased to 220 ℃ at 20 ℃/min, and is kept for 1 min.

Table 4 table of test results of example 4

Example 5

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; taking 0.3000 +/-0.01 g of the uniformly mixed sample to a 20mL headspace bottle, and adding 0.2mL of dichloromethane with the chromatographic grade and above for vortex dissolution; adding 5.8mL of N, N-Dimethylformamide (DMF) with a chromatographic grade or above, rapidly covering and sealing, heating at 50 ℃ for 10min, performing vortex extraction for 5min, standing at 24 ℃ for 13min, completely layering, and taking the upper layer to be tested; placing into a headspace sample injector of a gas chromatograph, balancing for 30min at 80 ℃, sucking 1mL of upper gas, detecting the content of normal hexane and acetone in the crystal lycopene by using a gas chromatography-hydrogen ion flame detector, and detecting samples 1, 2 and 3, wherein the detection results are shown in Table 5; wherein, the gas chromatography condition is a chromatographic column polyethylene glycol nitrobenzene modified capillary column, the length of the column is 60m, the inner diameter of the column is 0.25mm, the thickness of the membrane is 0.25 μm, the carrier gas is nitrogen, the flow rate is 3.0mL/min, the temperature of a sample inlet is 220 ℃, the temperature of the column is programmed temperature, the temperature of a detector is 235 ℃, and the split ratio is 25: 1; the temperature programming condition is that the temperature is initially 60 ℃, is kept for 3min, is increased to 85 ℃ at 3.5 ℃/min, is increased to 220 ℃ at 20 ℃/min, and is kept for 1 min.

Table 5 table of test results of example 5

In summary, the following steps: in the parallel experiments of 3 samples in each embodiment of the invention, the contents of n-hexane and acetone measured by 5 parallel experiments of each sample are similar and have small difference, which shows that the invention can accurately detect the residual quantity of n-hexane and acetone in the crystal lycopene, and has accurate and reliable result and good reproducibility.

Comparative example 1 vortex dissolution without dichloromethane

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; the sample after mixing was taken at 0.3000. + -. 0.01g into a 20mL headspace bottle, and 6mL of DMF was added directly and sealed by snap-capping, otherwise as in example 1. The results are shown in Table 6.

Table 6 table of test results of comparative example 1

Comparative example 2 dissolution with larger amounts of dichloromethane without DMF

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; the sample was taken 0.3000. + -. 0.01g after mixing into a 20mL headspace bottle, 6mL of dichloromethane was added and sealed by snap-capping, otherwise as in example 1. The results are shown in Table 7.

Table 7 table of test results of comparative example 2

Comparative example 3 dissolution with DMA alone

Pulverizing crystalline lycopene with micronizer to 100 mesh, and mixing for 5min, wherein the passing rate of the screen is more than 95%; the mixed sample 0.3000. + -. 0.01g was taken into a 20mL headspace bottle, 6mL DMA was added and the bottle was quickly capped and sealed as in example 1. The results are shown in Table 8.

Table 8 table of test results of comparative example 3

Comparative example 4

Pulverizing crystal lycopene with pulverizer to 80 mesh sieve with a pass rate of more than 95%, and mixing for 5 min; the other steps were as in example 1. The results are shown in Table 9.

TABLE 9 TABLE of the test results of comparative example 4

The results of tables 6 to 9 were analyzed to show that: the n-hexane and acetone content detected in the comparative examples 1 and 3 is low, which indicates that the n-hexane and acetone are not completely dissolved out and the detection result is inaccurate; comparative examples 2 and 4 detected that the results of 5 parallel experiments of each sample were very different, indicating that the detection results were not accurate.