CN112067707A - Method for detecting vitamin E in multi-vitamin tablets - Google Patents

Method for detecting vitamin E in multi-vitamin tablets Download PDF

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CN112067707A
CN112067707A CN202010273583.0A CN202010273583A CN112067707A CN 112067707 A CN112067707 A CN 112067707A CN 202010273583 A CN202010273583 A CN 202010273583A CN 112067707 A CN112067707 A CN 112067707A
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vitamin
mixed sample
sample
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pretreatment
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许巧红
郑敏敏
林锋华
赵艳霞
卢玲玲
裘蒋剑
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Zhejiang Nutrasis Biotech Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/047—Standards external

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Abstract

The invention aims to provide a detection method capable of quickly and effectively extracting vitamin E in multi-dimensional tablets in pretreatment, which adopts enzymolysis for pretreatment and comprises the following steps: step 1, adding water to wet a multi-dimensional sheet sample, adding a predetermined amount of protease, and shaking up to obtain a mixed sample I; step 2, placing the mixed sample I in a water bath with a preset temperature for ultrasonic preset time, and then adding a preset amount of ethanol to obtain a mixed sample II; step 3, continuing ultrasonic treatment on the mixed sample II at a preset temperature for preset time, cooling to room temperature, and diluting with ethanol to obtain a mixed sample III; and 4, filtering the mixed sample III, detecting by adopting HPLC, and calculating based on a reference substance detected by adopting HPLC to obtain the content of the vitamin E in the sample. The method adopts protease for enzymolysis pretreatment, so that the loss of vitamin E in the pretreatment process can be reduced, and the detection result is more accurate.

Description

Method for detecting vitamin E in multi-vitamin tablets
Technical Field
The invention belongs to the field of health care products, relates to a detection method of vitamin, and particularly relates to a detection method of vitamin E in a multi-vitamin tablet.
Background
The multivitamin tablet is a health product tablet containing multiple vitamins, and is usually composed of multiple vitamins and auxiliary materials with different components. Accurate detection of different kinds of vitamins in a multi-dimensional tablet is necessary to ensure the product quality.
Some multi-vitamin tablets contain vitamin E, the vitamin E is unstable in property and easy to oxidize, common solid raw materials in the industry are subjected to oxidation resistance by adopting an embedding technology, and an embedding layer needs to be broken first during detection, so that the detection is difficult. In the prior art, the detection of vitamin E mostly adopts Gas Chromatography (GC) or High Performance Liquid Chromatography (HPLC), and certain pretreatment is needed before the determination so as to separate the vitamin E from a sample to be detected. For example, the national standard (GB5009.82-2016) specifies a method for detecting vitamin E contained in food, which comprises the steps of saponifying and then extracting, concentrating the obtained organic phase, filtering and measuring by HPLC. The method has more steps and complicated operation, has higher requirements on the operation experience and skill of a person due to longer process, and the vitamin E is easy to generate content change due to oxidation and other reasons during the process, thereby influencing the accuracy of the final determination result. Moreover, for health care products with complex components such as multi-dimensional tablets, the pretreatment method of saponification extraction is difficult to effectively extract vitamin E, so that the final detection result has larger error.
Disclosure of Invention
In order to solve the problems, the invention provides a detection method capable of quickly and effectively extracting vitamin E in multi-dimensional tablets in pretreatment, which is characterized in that a protein embedding layer is destroyed by constant temperature enzymolysis, and then the pretreatment is carried out by directly extracting with an organic solvent, and the detection method comprises the following steps: step 1, adding water to wet a multi-dimensional sheet sample, adding a predetermined amount of protease, and shaking up to obtain a mixed sample I; step 2, placing the mixed sample I in a water bath with a preset temperature for ultrasonic preset time, and then adding a preset amount of ethanol to obtain a mixed sample II; step 3, continuing ultrasonic treatment on the mixed sample II at a preset temperature for preset time, cooling to room temperature, and diluting with ethanol to obtain a mixed sample III; and 4, filtering the mixed sample III, detecting by adopting HPLC, and calculating based on a reference substance detected by adopting HPLC to obtain the content of the vitamin E in the sample.
The method for detecting vitamin E in the multi-dimensional tablets provided by the invention can also have the technical characteristics that the preset temperature in the step 2 and the step 3 is 60-65 ℃, and the preset time in the step 2 and the step 3 is 20 min.
The method for detecting vitamin E in the multi-dimensional tablet provided by the invention can also have the technical characteristics that the volume mass ratio of the protease added in the step 1 to the sample is 1: 1-5: 1.
The method for detecting vitamin E in the multi-dimensional tablets provided by the invention can also have the technical characteristics that the enzymatic activity of the protease used in the step 1 is more than or equal to 3 AU-A/g.
Action and Effect of the invention
According to the method for detecting the vitamin E in the multi-dimensional tablets, the protease is adopted for enzymolysis pretreatment, and the enzymolysis process is carried out under the ultrasonic condition of 60-65 ℃, so that the loss of the vitamin E in the pretreatment process can be reduced, the requirement on experience of operators is low, a transfer extraction step is avoided, samples are basically free of loss, and the detection result is more accurate. In addition, the pretreatment process is simple to operate and short in time consumption, and the rapid and accurate detection of the vitamin E in the multi-dimensional tablets can be realized.
Detailed Description
The following describes specific embodiments of the present invention.
In the following examples and comparative examples, the vitamin E reference substance was obtained from the institute of medical sciences and has a purity of 98%; the protease manufacturer is novozymes; other reagents were obtained by general commercial procurement, and the conditions and operation were not specified, referring to the conventional techniques. In addition, the multi-dimensional tablets used in the examples and comparative examples were self-made by the applicant and comprised, as main components: calcium carbonate, manganese sulfate, ferrous fumarate, zinc sulfate, sodium selenite, copper sulfate, retinyl acetate, vitamin D3, thiamine nitrate, riboflavin, pyridoxine hydrochloride, cyanocobalamine, nicotinamide, folic acid, D-biotin, L-ascorbic acid, sodium L-ascorbate, calcium D-pantothenate, dl-alpha-tocopherol acetate, dl-alpha-tocopherol (vitamin E), microcrystalline cellulose, sodium starch octenyl succinate, maltodextrin, silicon dioxide, acacia gum, croscarmellose sodium, magnesium stearate, white granulated sugar, hydroxypropyl methyl cellulose, low-substituted hydroxypropyl cellulose, edible corn starch, tricalcium phosphate, citric acid, sodium citrate, caprylic acid, capric glyceride and a coating premix.
< example >
The detection method of vitamin E in the multi-dimensional tablets comprises the following steps:
step 1, putting 0.3g of a multidimensional sheet sample into a 50ml brown volumetric flask, adding 3ml of water for wetting, adding 0.8g of protease (novozymes Alcalase 3.0T), and shaking up to obtain a mixed sample I;
step 2, placing the mixed sample I in a water bath at the temperature of 60-65 ℃ for ultrasonic treatment for 20min, and then adding 40ml of ethanol to obtain a mixed sample II;
step 3, continuing to perform ultrasonic treatment on the mixed sample II in a water bath at the temperature of 60-65 ℃ for 20min, cooling to room temperature, diluting to a scale with ethanol, and fully shaking up to obtain a mixed sample III;
and 4, filtering the mixed sample III by using a 0.45-micron filter membrane, detecting by using HPLC, and calculating based on a reference substance detected by using HPLC to obtain the content of the vitamin E in the sample.
In this example, the HPLC conditions for step 4 were as follows:
high performance liquid chromatograph: agilent, type 1260;
a chromatographic column: ZORBAX SB-C18;
mobile phase: methanol: water 98: 2
Flow rate: 1.0ml/min
Sample introduction amount: 100 μ L
Detection wavelength: 264nm
Column temperature: 30 deg.C
In this embodiment, an external standard method is used for content calculation, and the specific operations are as follows: and (4) taking a vitamin E control product, diluting the vitamin E control product by using the HPLC mobile phase to a sample equivalent concentration, and detecting by adopting the HPLC condition. And (3) filtering the mixed sample III, calculating the peak area obtained by HPLC detection by using an external standard method formula to obtain the concentration of the vitamin E in the mixed sample III, and then converting to obtain the content of the vitamin E in the multi-dimensional tablet sample.
< comparative example 1>
The method for detecting vitamin E in the multi-dimensional tablets of comparative example 1 comprises the following steps:
step 1, taking 0.3g of a multi-dimensional sheet sample, placing the multi-dimensional sheet sample in a 50ml brown volumetric flask, adding 3ml of water for wetting, adding 1.0ml of protease (novozymes Alcalase 3.0T or the same enzyme with the enzyme activity more than or equal to 3AU-A/g) for shaking uniformly to obtain a mimutexed sample I;
step 2, placing the mixed sample I in a water bath at the temperature of 60-65 ℃ for heating for 20min, and then adding 40ml of ethanol to obtain a mixed sample II;
step 3, continuing to heat the mixed sample II in a water bath at the temperature of 60-65 ℃ for 20min, cooling to room temperature, diluting to a scale with ethanol, and fully shaking up to obtain a mixed sample III;
and 4, filtering the mixed sample III by using a 0.45-micron filter membrane, detecting by using HPLC, and calculating based on a reference substance detected by using HPLC to obtain the content of the vitamin E in the sample.
The HPLC conditions used in this comparative example were the same as those in the examples.
As described above, the present comparative example differs from the examples mainly in that only the mixed sample is heated without being subjected to ultrasonication in step 2 and step 3.
< comparative example 2>
The method for detecting vitamin E in the multi-dimensional tablets of comparative example 2 comprises the following steps:
step 1, taking 0.3g of a multi-dimensional sheet sample, placing the multi-dimensional sheet sample in a 50ml brown volumetric flask, adding 3ml of water for wetting, adding 1.0ml of protease (novozymes Alcalase 3.0T or the same enzyme with the enzyme activity more than or equal to 3AU-A/g) for shaking uniformly to obtain a mimutexed sample I;
step 2, placing the mixed sample I in a room-temperature water bath for ultrasonic treatment for 20min, and then adding 40ml of ethanol to obtain a mixed sample II;
step 3, continuing to perform ultrasonic treatment on the mixed sample II in a room-temperature water bath for 20min, diluting the mixed sample II to a scale with ethanol, and fully shaking up to obtain a mixed sample III;
and 4, filtering the mixed sample III by using a 0.45-micron filter membrane, detecting by using HPLC, and calculating based on a reference substance detected by using HPLC to obtain the content of the vitamin E in the sample.
The HPLC conditions used in this comparative example were the same as those in the examples.
As described above, the present comparative example differs from the examples mainly in that the temperature condition when only the mixed sample is subjected to ultrasonication in step 2 and step 3 is room temperature.
< comparative example 3>
The method for detecting vitamin E in the multi-dimensional tablets of comparative example 3 comprises the following steps:
step 1, taking 0.3g of a multi-dimensional sheet sample, placing the multi-dimensional sheet sample in a 50ml brown volumetric flask, adding 3ml of water for wetting, adding 1.0ml of protease (novozymes Alcalase 3.0T or the same enzyme with the enzyme activity more than or equal to 3AU-A/g) for shaking uniformly to obtain a mimutexed sample I;
step 2, standing the mixed sample I at room temperature for 20min, and then adding 40ml of ethanol to obtain a mixed sample II;
step 3, shaking the mixed sample II uniformly, continuing standing at room temperature for 20min, diluting with ethanol to a scale, and fully shaking uniformly to obtain a mixed sample III;
and 4, filtering the mixed sample III by using a 0.45-micron filter membrane, detecting by using HPLC, and calculating based on a reference substance detected by using HPLC to obtain the content of the vitamin E in the sample.
The HPLC conditions used in this comparative example were the same as those in the examples.
As described above, the comparative example differs from the examples mainly in that the mixed sample was not subjected to sonication nor heated in step 2 and step 3.
< comparative example 4>
The method for detecting vitamin E in the multi-dimensional tablets of comparative example 4 comprises the following steps:
step 1, putting 0.3g of a multidimensional sheet sample into a 50ml brown volumetric flask, adding 3ml of water for wetting, and shaking up to obtain a mixed sample I;
step 2, placing the mixed sample I in a water bath at the temperature of 60-65 ℃ for heating and ultrasonic treatment for 20min, and then adding 40ml of ethanol to obtain a mixed sample II;
step 3, continuing heating and ultrasonic treating the mixed sample II in a water bath at the temperature of 60-65 ℃ for 20min, cooling to room temperature, diluting to a scale with ethanol, and fully shaking up to obtain a mixed sample III;
and 4, filtering the mixed sample III by using a 0.45-micron filter membrane, detecting by using HPLC, and calculating based on a reference substance detected by using HPLC to obtain the content of the vitamin E in the sample.
The HPLC conditions used in this comparative example were the same as those in the examples.
As described above, the comparative example differs from the examples mainly in that no protease was added to the sample, that is, no enzymatic hydrolysis was performed.
< comparative example 5>
The vitamin E in the multi-dimensional tablets of the comparative example 5 is detected by adopting a national standard method, and the method specifically comprises the following steps:
saponification: weighing a proper amount of homogenized multidimensional slice solid sample into a 150mL flat-bottomed flask, adding about 20mL warm water, mixing uniformly, adding 1.0g ascorbic acid and 0.1g BHT, mixing uniformly, adding 30mL absolute ethyl alcohol, adding 10 mL-20 mL potassium hydroxide solution, shaking while adding, shaking, mixing uniformly, oscillating soap in a constant-temperature water bath at 80 ℃ for 30min, and immediately cooling to room temperature by cold water after saponification.
And (3) extraction: transferring the saponified solution into 250mL separating funnel with 30mL water, adding 50mL petroleum ether-ether mixture, shaking for 5min, transferring the lower layer solution into another 250mL separating funnel, adding 50mL mixed ether solution, extracting again, and combining ether layers.
Washing: the ether layer was washed with about 100mL of water and repeated about 3 times until the ether layer was washed neutral (the pH of the lower solution was checked using pH paper) and the lower aqueous phase was removed.
Concentration: filtering the washed ether layer into a 250mL rotary evaporation bottle or a nitrogen concentration tube through anhydrous sodium sulfate (about 3g), flushing a separating funnel and the anhydrous sodium sulfate for 2 times by using about 15mL petroleum ether, merging the mixture into the evaporation bottle, connecting the evaporation bottle onto a rotary evaporator or a gas concentrator, carrying out reduced pressure distillation or gas flow concentration in a water bath at 40 ℃ or carrying out gas flow concentration, taking down the evaporation bottle when about 2mL of ether liquid in the bottle is left, and immediately blowing the evaporation bottle to be nearly dry by using nitrogen. The residue in the evaporation flask was dissolved with methanol in portions and transferred to a 10mL volumetric flask to volume. The solution was passed through a 0.22 μm organic filter and subjected to HPLC.
The HPLC conditions and the calculation method of the content used in this comparative example are the same as those in the examples.
< comparative example 6>
The method for detecting vitamin E in the multi-dimensional tablets of comparative example 6 comprises the following steps:
step 1, putting 0.3g of a multidimensional slice sample into a 50ml brown volumetric flask, adding 3ml of water for wetting, adding 0.8g of protease (Tengheng 20 ten thousand enzyme active protease; the enzyme activity is less than 3AU-A/g) for shaking uniformly to obtain a mimutexed sample I;
step 2, placing the mixed sample I in a water bath at the temperature of 60-65 ℃ for ultrasonic treatment for 20min, and then adding 40ml of ethanol to obtain a mixed sample II;
step 3, continuing to perform ultrasonic treatment on the mixed sample II in a water bath at the temperature of 60-65 ℃ for 20min, cooling to room temperature, diluting to a scale with ethanol, and fully shaking up to obtain a mixed sample III;
and 4, filtering the mixed sample III by using a 0.45-micron filter membrane, detecting by using HPLC, and calculating based on a reference substance detected by using HPLC to obtain the content of the vitamin E in the sample.
The HPLC conditions used in this comparative example were the same as those in the examples.
Effects and effects of the embodiments
The multidimensional slices of the same batch were tested by the methods of the above examples and comparative examples, each method was performed in 6 replicates, and the test results are shown in table 1 below. In table 1, the data of the 6 parallel detection results are separated by a semicolon.
Figure BDA0002443993530000061
As shown in Table 1, the average value of the test results of the examples was 26mg/g for the same lot of multi-dimensional sheet samples.
Compared with the examples, the detection results of the comparative examples 1 to 3 are about 20mg/g and lower than the detection results of the examples. After protease is added, the ultrasonic treatment is not carried out in the comparative example 1, the heating treatment is not carried out in the comparative example 2, and the heating treatment and the ultrasonic treatment are not carried out in the comparative example 3.
Compared with the examples and the comparative examples 1 to 3, the comparative example 4 is not added with protease, which means that no enzymolysis treatment exists, and the detection result of the comparative example 4 is about 14mg/g, which is far lower than that of the examples and is obviously lower than that of the comparative examples 1 to 3, which shows that although the enzymolysis treatment in the comparative examples 1 to 3 is insufficient, the insufficient enzymolysis treatment still has a certain effect of reducing the loss in the pretreatment process. In addition, novozymes Alcalase 3.0T is adopted for enzymolysis in the embodiment, but further emutexperiments show that the same kind of enzyme with the enzyme activity being more than or equal to 3AU-A/g can achieve the same detection effect, the detection result is basically the same as the embodiment, and the description is omitted.
The detection method of the comparative example 5 adopts the saponification pretreatment method of the national standard in the prior art, the detection result is about 23mg/g, which is lower than that of the embodiment, and the effect of the pretreatment method of the embodiment on loss reduction is better than that of the saponification pretreatment method in the prior art.
The test method of comparative emutexample 6 was the same as in emutexample emutexcept that an active enzyme having an enzyme activity of < 3AU-A/g was used, and the result was lower than that of emutexample. This result indicates that the recovery of the assay is lower when treated with protease of lower enzyme activity, probably because the magnitude of the enzyme activity correlates with the degree of reduction of pretreatment loss. Correspondingly, the novozymes Alcalase 3.0T or similar enzymes with the enzyme activity more than or equal to 3AU-A/g are adopted for enzymolysis treatment, so that the loss in pretreatment can be reduced, and the final detection result is more consistent with the actual content of the vitamin E in the multi-dimensional tablets.
In summary, in the method for detecting vitamin E in a multi-dimensional tablet of the embodiment, since the protease with the enzyme activity of not less than 3AU-a/g is used for the pretreatment of the enzymolysis, and the enzymolysis process is performed under the ultrasonic condition of 60-65 ℃, the loss of the vitamin E in the pretreatment process can be reduced, so that the detection result is more accurate. In addition, the pretreatment operation of the embodiment mainly comprises enzymolysis, dilution and filtration, and compared with saponification, extraction, concentration and the like in the prior art, the method is simpler in operation, shorter in time consumption, lower in experience requirement on operators, and capable of realizing rapid and accurate detection of vitamin E in the multi-dimensional tablet.

Claims (4)

1. A method for detecting vitamin E in multi-vitamin tablets is characterized in that enzymolysis is adopted for pretreatment, and the method comprises the following steps:
step 1, adding water to wet a sample of the multidimensional sheet, adding a predetermined amount of protease, and shaking up to obtain a mixed sample I;
step 2, placing the mixed sample I in a water bath with a preset temperature for ultrasonic preset time, and then adding a preset amount of ethanol to obtain a mixed sample II;
step 3, continuing ultrasonic treatment on the mixed sample II at the preset temperature for preset time, cooling to room temperature, and diluting with ethanol to obtain a mixed sample III;
and 4, filtering the mixed sample III, detecting by adopting HPLC, and calculating based on a reference substance detected by adopting HPLC to obtain the content of the vitamin E in the sample.
2. The method for detecting vitamin E in a multi-dimensional tablet according to claim 1, wherein:
wherein the preset temperature in the step 2 and the step 3 is 60-65 ℃,
the predetermined time in step 2 and step 3 is 20 min.
3. The method for detecting vitamin E in a multi-dimensional tablet according to claim 1, wherein:
wherein the volume-mass ratio of the protease added in the step 1 to the sample is 1: 1-5: 1.
4. The method for detecting vitamin E in a multi-dimensional tablet according to claim 1, wherein:
wherein the enzymatic activity of the protease used in the step 1 is more than or equal to 3 AU-A/g.
CN202010273583.0A 2020-04-09 2020-04-09 Method for detecting vitamin E in multi-vitamin tablets Pending CN112067707A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102253130A (en) * 2010-12-24 2011-11-23 天津天狮生物发展有限公司 Method for measuring vitamins A, D and E in compound vitamin
CN105510454A (en) * 2015-11-24 2016-04-20 威海百合生物技术股份有限公司 A method of rapidly measuring vitamin E in a healthcare product

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102253130A (en) * 2010-12-24 2011-11-23 天津天狮生物发展有限公司 Method for measuring vitamins A, D and E in compound vitamin
CN105510454A (en) * 2015-11-24 2016-04-20 威海百合生物技术股份有限公司 A method of rapidly measuring vitamin E in a healthcare product

Non-Patent Citations (1)

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Title
叶燕: "几种保健食品中特定功效成分的质量标准初探", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 *

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Application publication date: 20201211