CN107561172B

CN107561172B - Method for simultaneously detecting content of multiple vitamins in nutrient soft capsule

Info

Publication number: CN107561172B
Application number: CN201710565812.4A
Authority: CN
Inventors: 洪继兵; 赵小伟; 欧阳康乐
Original assignee: Ferguson Wuhan Biotechnology Co ltd
Current assignee: Ferguson Wuhan Biotechnology Co ltd
Priority date: 2017-07-12
Filing date: 2017-07-12
Publication date: 2020-04-17
Anticipated expiration: 2037-07-12
Also published as: CN107561172A

Abstract

The invention discloses a method for simultaneously detecting the content of multiple vitamins in a nutrient soft capsule, which comprises the step of extracting a sample to be detected and then detecting the sample by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph contains an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 250-280 nm, the chromatographic column of the high performance liquid chromatograph is a C18 chromatographic column, the mobile phase is a mixed solution of a buffer solution and acetonitrile, uniform elution is adopted, and the column temperature is 25-35 ℃. Compared with the prior art, the invention can complete the content determination of six vitamins at one time, and has the advantages of high accuracy of detection result, safer and simpler operation process and more accurate and stable result.

Description

Method for simultaneously detecting content of multiple vitamins in nutrient soft capsule

Technical Field

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for simultaneously detecting the content of multiple vitamins in a nutrient soft capsule.

Background

With the development of society and the increase of nutritional requirements, more and more products containing compound vitamins appear in the market, and the excessive supplement of the vitamins is also unfavorable for the body, so that the vitamin content in the products needs to be monitored, and a method for rapidly determining the vitamin content is needed, and the high performance liquid chromatography is a feasible method for determining the vitamin content. However, the method can only detect one or two components, which causes large reagent consumption, long detection period, high detection cost, and poor accuracy and sensitivity of the existing detection method.

CN 1582756A discloses a nutrient soft capsule for pregnant women, which consists of a plurality of vitamins and minerals such as vitamin C, nicotinamide, vitamin E, pantothenic acid, vitamin B6, vitamin B1, vitamin B2, β -carotene, folic acid, vitamin D2, vitamin B12, anhydrous calcium hydrogen phosphate, heavy magnesium oxide and the like, wherein at present, the vitamin components in the soft capsule can only be detected for a plurality of times, and no literature report for simultaneously detecting a plurality of components exists.

Disclosure of Invention

The invention aims to provide a method for simultaneously detecting the content of multiple vitamins in a nutrient soft capsule aiming at the defects in the prior art, and simultaneously improve the accuracy and the sensitivity of detection.

The technical scheme adopted by the invention is as follows:

a method for simultaneously detecting the content of multiple vitamins in a nutrient soft capsule is disclosed, wherein the vitamins comprise folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6 and vitamin C, the method comprises the step of extracting a sample to be detected and then detecting the sample by using a high performance liquid chromatograph, the high performance liquid chromatograph comprises an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 250-280 nm, the chromatographic column of the high performance liquid chromatograph is a C18 chromatographic column, a mobile phase is a mixed solution of a buffer solution and acetonitrile, uniform elution is adopted, and the column temperature is 25-35 ℃.

Preferably, the method for extracting the sample to be detected is as follows: placing 1g of soft capsule content in a 50ml volumetric flask, adding mobile phase, performing ultrasonic treatment at 45 deg.C for 30min, cooling to room temperature, fixing volume to scale with mobile phase, shaking, centrifuging 10ml, filtering supernatant with 0.45 μm filter membrane, discarding primary filtrate, and collecting subsequent filtrate as sample solution.

Preferably, the detection wavelength is 260 nm.

Preferably, the volume ratio of the buffer solution to the acetonitrile in the mobile phase is 80-95: 20-5.

Further preferably, the volume ratio of the buffer to the acetonitrile in the mobile phase is 90: 10.

Preferably, the buffer solution contains 1-5 mM of ion pair reagent and 30-100 mM of buffer salt, the pH value is adjusted to 2-4 by phosphoric acid, and the ion pair reagent is selected from tetrabutylammonium hydroxide, tetrabutylammonium bromide, dodecyl trimethyl ammonium chloride, sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate and sodium decane sulfonate, and preferably sodium heptane sulfonate; the buffer salt is selected from sodium salt or potassium salt of phosphoric acid or acetic acid.

Further preferably, the buffer contains 2mM ion pair reagent and 50mM buffer salt, and the pH is adjusted to 3 with phosphoric acid.

Preferably, the column temperature is 30 ℃.

Preferably, the flow rate of the uniform elution is 0.5-2 mL/min.

The invention has the beneficial effects that:

1) the invention adopts the mobile phase as the extracting solution, and the weak acid ensures that the solution environment is in proper acidity, so that the folic acid, the nicotinamide, the vitamin B1, the vitamin B2, the vitamin B6 and the vitamin C are more stable in the extracting solution, and the accurate detection result is facilitated; in addition, when the high performance liquid chromatography is carried out, the ion pair is used as a mobile phase, so that the vitamins can be stabilized, the retention time and the peak shape of the vitamins in a chromatographic column can be adjusted, and the effective separation and accurate detection of folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6 and vitamin C are realized. Particularly, when an ion pair solution containing 50mmol/L potassium dihydrogen phosphate solution and 2mmol/L sodium heptanesulfonate is selected, the separation effect and the detection accuracy can be optimized.

2) The detection pretreatment of the invention adopts a filter membrane with the diameter of 0.45 mu m or less to filter the detection liquid, the filtering effect is good, the column is effectively protected from being influenced by impurities, and the service life of the chromatographic column is prolonged.

3) The detection method can finish detection of six vitamins by one-time pretreatment, and has simple treatment process and high detection result accuracy.

4) The invention has the advantages of short detection time, safer and simpler operation process, small using reagent amount and accurate and stable result.

Drawings

FIG. 1 is a liquid chromatogram obtained after extraction with a mobile phase in example 1.

FIG. 2 is a liquid chromatogram obtained after extraction with water in example 1.

FIG. 3 is a liquid chromatogram obtained after extraction with 50% ethanol in example 1.

FIG. 4 is a liquid chromatogram obtained at a detection wavelength of 290 nm.

FIG. 5 is a liquid chromatogram obtained with fluidity A of a buffered saline solution containing 50mM disodium hydrogen phosphate, with phosphoric acid adjusted to pH 3.

FIG. 6 is a liquid chromatogram obtained with fluidity A being a buffered salt solution containing 2mM sodium heptanesulfonate, with phosphoric acid adjusting the pH to 3.

FIG. 7 is a liquid chromatogram obtained with fluidity A of a buffered salt solution containing 2mM sodium heptanesulfonate and 50mM disodium hydrogen phosphate, with phosphoric acid adjusted to pH 5.

FIG. 8 is a high performance liquid chromatogram obtained with a flow ratio of 90: 20.

FIG. 9 is a high performance liquid chromatogram obtained at a column temperature of 20 ℃.

FIG. 10 is a high performance liquid chromatogram obtained at a column temperature of 40 ℃.

FIG. 11 is a high performance liquid chromatogram of a standard solution.

Detailed Description

The present invention will be described in detail with reference to examples.

EXAMPLE 1 selection of sample solution preparation methods

1. Taking 1g of capsule content, placing in a 50ml volumetric flask, adding appropriate amount of mobile phase, performing ultrasonic treatment at 45 deg.C for 30min, cooling to room temperature, diluting to scale with the mobile phase, shaking, centrifuging 10ml, collecting supernatant, filtering with 0.45 μm filter membrane, discarding the primary filtrate, and collecting the subsequent filtrate as sample solution.

2. Taking 1g of capsule content, placing in a 50ml volumetric flask, adding appropriate amount of water, performing ultrasonic treatment at 45 deg.C for 30min, cooling to room temperature, diluting with water to scale, shaking, centrifuging 10ml, filtering supernatant with 0.45 μm filter membrane, discarding primary filtrate, and taking subsequent filtrate as sample solution.

3. Taking 1g of capsule content, placing in a 50ml volumetric flask, adding a proper amount of 50% ethanol, performing ultrasonic treatment at 45 ℃ for 30min, cooling to room temperature, diluting to scale with 50% ethanol, shaking up, taking 10ml for centrifugation, taking supernatant, filtering with a 0.45 μm filter membrane, discarding primary filtrate, and taking subsequent filtrate as sample solution.

The instrument comprises the following steps: waters model 2695/2998 HPLC series (Waters, including quaternary pump, autosampler, column oven, PDA detector, Empower3 chromatography workstation)

The solution is detected by high performance liquid chromatography, and the detection conditions are as follows:

the stationary phase of the chromatographic column is octadecyl bonded silica gel, and the particle size of the silica gel is 3-10 μm; the height of the column is 250mm, and the inner diameter is 4.6 mm;

the sample injection amount is 10 mu L;

the volume ratio of the mobile phase is 90:10 buffer solution and acetonitrile; the buffer contained 2mM sodium heptanesulfonate and 50mM disodium hydrogen phosphate, the balance being water, the pH being adjusted to 3.0 with phosphoric acid; the elution mode is uniform;

the flow rate is 1.0 mL/min;

the column temperature is 30 ℃;

the detection wavelength is 260 nm;

as a result: as shown in fig. 1-3; after extraction with the mobile phase, compared with extraction with water and ethanol, the method can obviously reduce impurities, has good separation effect on the main peak and the impurities, and is preferably used for extraction with the mobile phase.

EXAMPLE 2 selection of detection wavelength

Preparation of a sample solution: taking 1g of capsule content, placing in a 50ml volumetric flask, adding appropriate amount of mobile phase, performing ultrasonic treatment at 45 deg.C for 30min, cooling to room temperature, diluting to scale with the mobile phase, shaking, centrifuging 10ml, collecting supernatant, filtering with 0.45 μm filter membrane, discarding the primary filtrate, and collecting the subsequent filtrate as sample solution.

Detection wavelength: 260nm and 290nm

Other conditions were the same as in example 1.

As a result: as can be seen from FIGS. 1 and 4, vitamin B1(17.652min) and vitamin B1(18.539min) can not be detected effectively at a wavelength of 290nm, and 3.304min more of the detection of the interfering vitamin C is carried out; when the wavelength is 260nm, vitamin C (2.830min), nicotinamide (4.262min), vitamin B6(6.962min), folic acid (14.438min), vitamin B2(17.652min) and vitamin B1(18.539min) can be effectively detected, and other interference measurement is avoided, so 260nm is the most suitable detection wavelength.

EXAMPLE 3 selection of Mobile phase

(1) A is a buffered saline solution containing 50mM disodium hydrogen phosphate, pH adjusted to 3 with phosphoric acid; b is acetonitrile

(2) A is a buffered salt solution containing 2mM sodium heptanesulfonate, the pH is adjusted to 3 with phosphoric acid; b is acetonitrile

(3) A is a buffered salt solution containing 2mM sodium heptanesulfonate and 50mM disodium hydrogen phosphate, pH adjusted to 5.0 with phosphoric acid; b is acetonitrile

(4) A is a buffered salt solution containing 2mM sodium heptanesulfonate and 50mM disodium hydrogen phosphate, pH adjusted to 3.0 with phosphoric acid; b is acetonitrile

The volume ratio of the mobile phase is 90: 10;

other conditions were the same as in example 1.

As a result: as can be seen from FIGS. 1 and 5-7, the spectral peaks in FIG. 5 are not completely separated and the tailing is serious; in FIG. 6, although the separation of chromatographic peaks is changed, the peak pattern is not ideal enough, and the retention time is also prolonged; the number of the mixed peaks of the sample in FIG. 7 is increased, the separation effect of chromatographic peaks at 2.8min is poor, and the separation is incomplete; the peak pattern and degree of separation are best in figure 1.

Example 4 mobile phase ratio selection

Changing the volume ratio of the mobile phase: 90: 20; 90:10

Other conditions were the same as in example 1.

As a result: as shown in fig. 8 and 1, in fig. 8, peaks at 2.8min, 4.2min and 11.0min were trailing and the peak-off time was faster than that in fig. 1, but the peak areas at 6min and 14min were smaller than that in fig. 1, and the retention time of each peak was changed mainly due to the large amount of acetonitrile, and the mobile phase ratio was 90:10, which is better in fig. 1 than that in fig. 8.

EXAMPLE 5 selection of column temperature

Column temperature of the chromatographic column: 20 deg.C, 30 deg.C, 40 deg.C

Other conditions were the same as in example 1.

As a result: as shown in fig. 1, 9 and 10, when the column temperature is 20 ℃, the peak-out time is prolonged to a certain extent, the peak is trailing at 2.8min and 4.2min, and the peak is cracked at 19.9min, so that the separation effect is poor; the column temperature of 40 ℃ leads the peak-out time to a certain extent, but the temperature is increased, the number of peaks is increased (about 3min and 5 min), the separation is poor, the measurement is influenced, and the phenomenon is obviously improved when the column temperature is 30 ℃, so that the optimal selection is 30 ℃.

Example 6 determination of the contents of folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6, and vitamin C in the nutrient soft capsule.

The instrument adopts a Waters2695/2998 high performance liquid chromatograph;

METTLER XP205 electronic balance;

the nutrient soft capsules for pregnant women are prepared by the method of CN 1582756A example 1;

the folic acid, the nicotinamide, the vitamin B1, the vitamin B2, the vitamin B6 and the vitamin C standard are all purchased from China food and drug testing research institute;

other reagents were analytical grade.

Preparation of standard solution

Mixing stock solution: respectively and accurately weighing 30mg of a vitamin B1 standard substance and 40mg of a vitamin B6 standard substance, dissolving the vitamin B1 standard substance and the vitamin B6 standard substance in 2ml of purified water in a 10ml measuring flask, then diluting the solution to a scale with methanol, and shaking up the solution.

Vitamin B2 stock solution: accurately weighing 20mg of vitamin B2 standard substance in a 10ml volumetric flask, dissolving with dilute acetic acid solution (6ml glacial acetic acid diluted with water to 100ml), diluting with methanol to scale, and shaking.

Folic acid stock solution: accurately weighing 10mg folic acid standard substance in a 100ml volumetric flask, adding 2ml0.1mol/L sodium hydroxide solution for dissolving, diluting to scale with methanol, and shaking up.

Standard solution: respectively weighing 100mg of vitamin C standard substance and 20mg of nicotinamide in a 100ml measuring flask, adding 2ml of purified water for dissolving, respectively accurately transferring 1ml of mixed stock solution, 1ml of vitamin B2 stock solution and 10ml of folic acid stock solution in the measuring flask, diluting to scale with a mobile phase, and shaking up.

The standard solution contains folic acid 0.01mg/ml, nicotinamide 0.2mg/ml, vitamin B1 0.03mg/ml, vitamin B2 0.02mg/ml, vitamin B6 0.04mg/ml and vitamin C1.0 mg/ml.

Second, preparation of test solution

Taking 1g of sample content, placing in a 50ml volumetric flask, adding mobile phase, performing ultrasonic treatment at 45 ℃ for 30min, cooling to room temperature, diluting to scale with the mobile phase, shaking uniformly, taking 10ml for centrifugation, taking supernatant, filtering with a 0.45 μm filter membrane, discarding primary filtrate, and taking subsequent filtrate as sample solution.

Thirdly, measuring the contents of folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6 and vitamin C in the test solution

Detecting the standard solution and the sample solution by high performance liquid chromatography under the following detection conditions:

the sample injection amount is 10 mu L;

the mobile phase is formed by mixing the following components in a volume ratio of 90:10 buffer solution and acetonitrile; buffer containing 2mM sodium heptane sulfonate and 50mM disodium hydrogen phosphate, pH adjusted to 3.0 with phosphoric acid; the flow mode is uniform flow;

the flow rate is 1.0 mL/min;

the column temperature is 30 ℃;

the detection wavelength is 260 nm;

the contents of folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6 and vitamin C are calculated according to an external standard method.

Recording the peak area concentration of each vitamin under the corresponding concentration of the standard substance and the peak area of the corresponding position of the test substance and the standard substance, and calculating a correction factor of the reference substance according to the following formula:

calculating the formula:

in the formula:

wi is the content of vitamin in the test sample, mg/100 g;

ai is the peak area of the test solution;

mt is the standard sample size, mg;

si is the dilution multiple of the test solution;

at is the peak area of the standard solution;

mi is sample size of the test sample, g;

st is the dilution factor of the control solution.

According to the above calculation formula: the test sample contains folic acid 49.3mg/100g, nicotinamide 1118.5mg/100g, vitamin B1 122.3mg/100g, vitamin B2 126.8mg/100g, vitamin B6 198.9mg/100g, and vitamin C6016.3 mg/100 g.

Example 7 Linear test

Firstly, preparing a series of folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6 and vitamin C standard solutions with different concentrations;

precisely measuring appropriate amounts of folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6 and vitamin C, and preparing into a series of solutions with different concentrations.

Secondly, detecting a series of folic acid, nicotinamide, vitamin B1, vitamin B2, vitamin B6 and vitamin C standard solutions with different concentrations by using a high performance liquid chromatography method, wherein the detection conditions are the same as the chromatographic conditions in example 6;

and after the examination is finished, recording the peak area of the standard substance under each concentration, taking the concentration as a self variable, and taking the corresponding peak area as a dependent variable to obtain a linear regression equation.

The results obtained are shown in tables 1 to 6 below.

TABLE 1 folate Linearity relationship

TABLE 2 Linear relationship of nicotinamide

TABLE 3 vitamin B1 Linear relationship

TABLE 4 vitamin B2 Linear relationship

TABLE 5 vitamin B6 Linear relationship

TABLE 6 vitamin C Linear relationship

The linear equation of the vitamins is as follows:

the folic acid has a good linear relation with the peak area within the concentration range of 0.0040-0.0162 mg/ml; the linear equation is that y is 9,228,585.2948x-1,565.9115, R²＝0.9998，R＝0.9998；

The nicotinamide has a good linear relation with the peak area within the concentration range of 0.0811-0.3244 mg/ml; the linear equation is that y is 18,872,719.5603x-10,963.3636, R²＝0.9999，R＝0.9999；

The vitamin B1 has a good linear relation with the peak area within the concentration range of 0.0123-0.0493 mg/ml; the linear equation is that y is 21,881,044.8134x-2,716.1803, R²＝0.9998，R＝0.9999；

The vitamin B2 has a good linear relation with the peak area within the concentration range of 0.0082-0.0328 mg/ml; linear equation is y 30,779,341.4634x +1,502.3000, R²＝0.9999，R＝0.9999；

The vitamin B6 has a good linear relation with the peak area within the concentration range of 0.0165-0.0661 mg/ml; the linear equation is that y is 2,122,967.8476x-641.9721, R²＝0.9999，R＝0.9999；

The vitamin C has a good linear relation with the peak area within the concentration range of 0.2728-1.0912 mg/ml; linear equation is y 16,931,678.8856x +35,501.0000, R²＝0.9999，R＝0.9999。

Example 8 recovery test

Taking a commercially available sample, measuring according to the national standard method (measuring folic acid in GB 5009.211-2014 food safety national standard food, measuring nicotinic acid and nicotinamide in GB 5009.89-2016 food safety national standard food, measuring vitamin B1 in GB 5009.84-2016 food safety national standard food, measuring vitamin B2 in GB 5009.85-2016 food safety national standard food, measuring vitamin B6 in GB 5009.154-2016 food safety national standard food, measuring folic acid in GB 5009.211-2014 food safety national standard food, measuring vitamin C in GB 5413.18-2010 food safety national standard infant food and milk product, measuring ascorbic acid in GB 5009.86-2016 food safety national standard food), adding a certain amount of standard solution, measuring according to the method, and obtaining the result shown in the following table 7, Table 8, table 9.

TABLE 7 determination of vitamin content in sample 1

TABLE 8 sample 2 vitamin assay

TABLE 9 determination of vitamin content in sample 3

As can be seen from tables 7, 8 and 9, the recovery rates of the samples are all between 90% and 105%, and the RSD% is less than 2%, indicating that the method has good accuracy.

Example 9 precision test

Precisely absorbing 10 mu L of test solution, and repeatedly injecting sample for 6 times according to the chromatographic conditions of the example 1, wherein the RSD% of each vitamin peak area is less than 2%, which indicates that the method has better precision. The results are shown in Table 10.

TABLE 10 precision test measurement of test solutions

EXAMPLE 10 repeatability test

6 parts of the same batch of test sample are taken, the test sample solution is prepared according to the preparation method of the test sample solution in the embodiment 1, the results of sample injection measurement are shown in a table 11, and the RSD% of each vitamin peak area is less than 2.0%, which shows that the method has good repeatability.

TABLE 11 test article repeatability test determination

Example 11 stability test

The same sample solution was injected at 0, 2, 4, 8, and 12 hours after the preparation according to the chromatographic conditions of example 1, and the RSD% of the peak area measured in the standing time point was less than 2.0%, and the results are shown in table 12; as can be seen from the results, the test solution had excellent stability.

TABLE 12 test article stability test determination

Claims

1. A method for simultaneously detecting the content of multiple vitamins in a nutrient soft capsule is characterized in that: the method comprises the steps of extracting a sample to be detected, detecting the sample by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph contains an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 260nm, a chromatographic column of the high performance liquid chromatograph is a C18 chromatographic column, a mobile phase is a mixed solution of buffer solution and acetonitrile, the volume ratio of the buffer solution to the acetonitrile in the mobile phase is 90:10, uniform elution is adopted, the column temperature is 30 ℃,

the extraction method of the sample to be detected comprises the following steps: placing 1g of soft capsule content in 50ml volumetric flask, adding mobile phase, performing ultrasonic treatment at 45 deg.C for 30min, cooling to room temperature, fixing volume to scale with mobile phase, shaking, centrifuging 10ml, filtering supernatant with 0.45 μm filter membrane, discarding primary filtrate, taking subsequent filtrate as sample solution,

the buffer solution contains 2mM ion pair reagent and 50mM buffer salt, the pH value is adjusted to 3 by phosphoric acid, and the ion pair reagent is sodium heptanesulfonate; the buffer salt is selected from sodium salt or potassium salt of phosphoric acid or acetic acid.

2. The method for simultaneously detecting the content of multiple vitamins in a nutrient soft capsule as claimed in claim 1, wherein the method comprises the following steps: the flow rate of the uniform elution is 0.5-2 mL/min.