CN115453033A - Method for detecting content of vitamin D in vitamin D drops - Google Patents
Method for detecting content of vitamin D in vitamin D drops Download PDFInfo
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- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 title claims abstract description 87
- 229930003316 Vitamin D Natural products 0.000 title claims abstract description 86
- 235000019166 vitamin D Nutrition 0.000 title claims abstract description 86
- 239000011710 vitamin D Substances 0.000 title claims abstract description 86
- 150000003710 vitamin D derivatives Chemical class 0.000 title claims abstract description 86
- 229940046008 vitamin d Drugs 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 38
- 239000000243 solution Substances 0.000 claims description 48
- 239000013558 reference substance Substances 0.000 claims description 32
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 30
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 claims description 22
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 239000000741 silica gel Substances 0.000 claims description 13
- 229910002027 silica gel Inorganic materials 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 238000012937 correction Methods 0.000 claims description 12
- 230000014759 maintenance of location Effects 0.000 claims description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims description 9
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 claims description 7
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 5
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- 238000001035 drying Methods 0.000 claims description 3
- 238000010812 external standard method Methods 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 6
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- 238000012360 testing method Methods 0.000 description 22
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 description 20
- 239000012085 test solution Substances 0.000 description 14
- 238000005303 weighing Methods 0.000 description 14
- 239000012088 reference solution Substances 0.000 description 10
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- 238000000746 purification Methods 0.000 description 7
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
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- DMBIRDATGKQYEH-UHFFFAOYSA-N hexane pentan-1-ol Chemical compound CCCCCC.CCCCCO DMBIRDATGKQYEH-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention provides a method for detecting the content of vitamin D in a vitamin D drop, which solves the technical problems of poor specificity and low production efficiency of the existing detection method. The invention can be widely applied to the technical field of drug analysis.
Description
Technical Field
The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the content of vitamin D in vitamin D drops.
Background
Vitamin D drops is a medicine for preventing and treating rickets in children. A large number of evidence-based medical researches and basic researches find that the vitamin D drops have certain curative effects on the treatment of cancer, hypertension, diabetes, tuberculosis and immunologic dysfunction. At present, the detection method of the content of vitamin D drops collected in United states pharmacopoeia, british pharmacopoeia, european Union pharmacopoeia and Chinese pharmacopoeia is quite complicated and has low detection efficiency. Although patent CN201410387198 discloses a method for detecting the content of vitamin D in vitamin D drops, the vitamin D drop sample is directly injected without being purified and collected after being dissolved, and the specificity is poor, thereby affecting the service life of a chromatographic column.
Disclosure of Invention
The invention aims to solve the defects of the background technology and provides the method for detecting the content of the vitamin D in the vitamin D drop, which has the advantages of good stability and repeatability, high analysis efficiency, low production cost and high production efficiency.
Therefore, the invention provides a method for detecting the content of vitamin D in vitamin D drops, which comprises the following specific steps: (1) Performing HPLC detection on a methanol-acetonitrile solution of a vitamin D reference substance with a known concentration, recording the peak area of the vitamin D reference substance with each concentration, and calculating a reference substance correction factor according to the following formula:
control correction factor = concentration/peak area;
(2) Dissolving vitamin D drops to be detected in n-hexane, purifying and collecting HPLC (high performance liquid chromatography), drying the purified and collected effluent liquid of vitamin D retention time by using nitrogen, dissolving the effluent liquid by using a methanol-acetonitrile solution, performing HPLC detection on the obtained solution again, and recording the peak area of the vitamin D;
(3) And (3) according to the calculation of an external standard method, multiplying the peak area of the vitamin D obtained in the step (2) by the correction factor of the reference substance obtained in the step (1) to obtain the concentration of the vitamin D.
Preferably, in the step (1) and step (2) HPLC detection, both the chromatographic columns are reversed-phase chromatographic columns, and the stationary phase of the reversed-phase chromatographic column is a C18 column;
and (3) in the step (2) of purifying and collecting HPLC detection, the chromatographic column is a normal phase chromatographic column, and the stationary phase of the normal phase chromatographic column is a silica gel column.
Preferably, the particle size of the silica gel column is 3-10 μm, the column height is 100-250 mm, the inner diameter is 4.6mm, and the sample injection amount is 100-500 μ L; the grain diameter of the C18 column is 3-10 mu m, the column height is 100-250 mm, the inner diameter is 4.6mm, and the sample injection amount is 10-20 mu L.
Preferably, the first mobile phase corresponding to the normal phase chromatographic column is a mixture of two mobile phases with a volume ratio of 997:3, mixed solution consisting of n-hexane and n-pentanol;
the volume ratio of the second mobile phase corresponding to the reversed phase chromatographic column is 95-99: 5-1 of acetonitrile and methanol;
preferably, the second mobile phase is a mixture of 97:3 acetonitrile and methanol.
Preferably, the first mobile phase and the second mobile phase flow at constant speed, the flow rate is 0.8-1.5 mL/min, the column temperature is 25-35 ℃, the detection wavelength is 254nm, and the retention time is 20-30 min.
Preferably, the flow rate of the first mobile phase is 1.5mL/min, the flow rate of the second mobile phase is 1.0mL/min, the column temperature is 30 ℃ and the retention time is 25min.
Preferably, in the step (1) and the step (2), the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solution is 3.
Preferably, the vitamin D is vitamin D 3 。
The invention has the beneficial effects that:
according to the method for detecting the content of the vitamin D in the vitamin D drop, provided by the invention, positive and negative two-phase chromatographic columns are innovatively used, namely, the positive phase chromatographic column with a silica gel column as a stationary phase and the reverse phase chromatographic column with a C18 column as a stationary phase are used for purifying and collecting the vitamin D to be detected and then detecting the vitamin D, so that interfering substances appearing in the detection can be effectively removed, and the detection precision is improved. And the two chromatographic columns are combined with corresponding mobile phases, and the flow rate, the detection wavelength, the chromatographic columns and other analysis conditions are limited, so that the method for detecting the content of the vitamin D in the vitamin D drop with good stability and repeatability, high analysis efficiency and good separation degree is finally obtained, and the method can be used for qualitatively and quantitatively detecting the vitamin D sensitively and accurately 3 Objective, comprehensive and accurateThe quality of the vitamin D drops is evaluated, and the method has important significance for controlling the quality of the vitamin D drops and ensuring the clinical curative effect.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.
FIG. 1 is a chromatogram of a purification control in example 1 of the present invention;
FIG. 2 is a chromatogram of test sample 1 in example 1 of the present invention;
FIG. 3 is a chromatogram of a blank solution of example 1 of the present invention;
FIG. 4 is vitamin D of control 1 in example 1 of the present invention 3 A content determination chromatogram;
FIG. 5 shows vitamin D content in test sample 1 in example 1 of the present invention 3 A content determination chromatogram;
FIG. 6 is vitamin D of example 2 of the present invention 3 And (5) a linear spectrum of a reference substance.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application clearer, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
Example 1 vitamin D in vitamin D drops 3 Determination of content
The instrument adopts an Agilent1260 high performance liquid chromatograph; METTLER XP205 electronic balance.
The material is vitamin D drop produced in the soft capsule workshop of the company.
Vitamin D 3 The reference substance is purchased from China pharmaceutical and biological product institute, and has a batch number of 100061-201809;
the reagents are all chromatographically pure.
1. Preparing a purified reference substance solution
Precisely weighing 28mg of vitamin D 3 Adding n-hexane for dissolving, fixing the volume, shaking up, precisely measuring a volumetric flask of 1ml to 100ml, adding n-hexane for fixing the volume, and shaking up to obtain the purified reference solution.
2. Preparing test solution
Precisely weighing 2g (containing 55.55 μ g vitamin D) 3 ) And (3) placing the content of the vitamin D drop into a 20mL brown volumetric flask, adding n-hexane for dissolving, fixing the volume, and shaking up to obtain the purified test solution.
Setting two purified test solution to be respectively a test article 1 and a test article 2; wherein the sample weighing amount of the sample 1 is 2.0007g; sample 2 was weighed to 1.9997g.
3. Preparation of vitamin D 3 Control solution
Precisely weighing 14mg of vitamin D 3 And (3) putting a reference substance into a 100mL brown volumetric flask, adding acetonitrile-methanol (the same below) with the volume ratio of 97 to dissolve, shaking up, precisely measuring 5mL to 50mL volumetric flask, diluting the volumetric flask with acetonitrile-methanol solution to a scale, shaking up, precisely measuring 2mL to 50mL volumetric flask, diluting the volumetric flask with acetonitrile-methanol solution to a scale, and shaking up to obtain the vitamin D 3 And (4) a reference substance solution.
A total of two vitamin D 3 Reference substance solutions, namely reference substance 1 and reference substance 2; wherein, the reference 1 contains vitamin D 3 The concentration of (A) is 0.5533ug/mL; control 2 vitamin D 3 The concentration of (2) is 0.5570ug/mL.
4. Preparing a blank solution
Acetonitrile-methanol at a volume ratio of 97.
5. Vitamin D in test solution 3 Determination of the content
HPLC detection is carried out on the purification reference substance solution, a purification collection interval is determined, and the chromatogram of the purification reference substance solution is shown in figure 1.
And (3) carrying out purification collection HPLC detection on the test sample 1 and the test sample 2, wherein the purification collection HPLC detection conditions are as follows:
the stationary phase of the normal phase chromatographic column is silica gel column with particle size of 3-10 μm, column height of 250mm, inner diameter of 4.6mm, and sample injection amount of 500 μ L.
The first mobile phase is a mixed solution consisting of n-hexane and n-pentanol in a volume ratio of 997:3, and the flow mode is uniform; the flow rate is 1.5mL/min, the column temperature is 30 ℃, the detection wavelength is 54nm, and the retention time is 17min; a VWD detector.
Separating the test sample 1 and test sample 2 solutions by normal phase chromatographic column, and collecting eluate of vitamin D retention time in the separated product, wherein chromatogram of test sample 1 is shown in FIG. 2. And after drying by nitrogen, dissolving the nitrogen by using a methanol-acetonitrile solution to obtain content test sample solutions, namely a content test sample 1 and a content test sample 2.
Respectively carrying out HPLC detection on the blank solution, the reference substance 1, the reference substance 2, the content test article 1 and the content test article 2, wherein the detection conditions are as follows:
the stationary phase of the reversed phase chromatographic column is C18, the particle size of silica gel is 3-10 μm, the column height is 250mm, the inner diameter is 4.6mm, and the sample injection amount is 20 μ L.
The second mobile phase is a mixed solution consisting of acetonitrile and methanol in a volume ratio of 97:3, and the flow mode is constant; the flow rate is 1.0mL/min, the column temperature is 30 ℃, the detection wavelength is 54nm, and the retention time is 22min; a VWD detector. The HPLC chromatograms of the blank solution, the reference 1 and the test sample 1 are shown in fig. 3, fig. 4 and fig. 5, respectively.
Control correction factor = concentration/peak area;
control correction factor f1=0.5533/127.1/0.025=0.1741 from control 1;
control correction factor f2=0.5570/129.0/0.025= -0.1727 from control 2;
wherein, 1 unit of vitamin D 3 ＝0.025ug。
Averaging F1 and F2 to obtain a reference substance correction factor F =0.1734;
calculating vitamin D in the sample 1 and the sample 2 according to an external standard method 3 The content of (a).
For supply ofSample 1: vitamin D in test article 1 3 The peak area of (a) is 120.9, and the product is multiplied by a reference substance correction factor F =0.1734 and multiplied by a dilution factor, and the product is divided by the sample weight to obtain the vitamin D in the test sample 1 3 Content of 1048 units/g.
For test article 2: vitamin D in sample 2 3 The peak area of (1) is multiplied by a reference substance correction factor F =0.1734, multiplied by a dilution factor, and divided by the sample amount to obtain the content of vitamin D3 in the test sample 2 of 1050 units/g. Specific data and results are shown in table 1.
TABLE 1 vitamin D 3 Assay data
Example 2 Linear test
1. Preparing vitamin D with different concentrations 3 Control solution
Precisely measuring vitamin D 3 0.8mL, 0.9mL, 1.0mL, 1.1mL and 1.2mL of reference substance solution (28 mu g/mL) are respectively placed in a 50mL brown measuring flask, acetonitrile-methanol is added for dilution to scale and shaking is carried out uniformly, thus obtaining the vitamin D 3 The control solutions were 0.46. Mu.g/mL, 0.51. Mu.g/mL, 0.57. Mu.g/mL, 0.63. Mu.g/mL, and 0.68. Mu.g/mL, respectively.
2. Mixing vitamin D 3 HPLC detection is carried out on the reference solution, and the detection conditions are as follows:
the stationary phase of the reversed phase chromatographic column is C18 column, the particle size of the C18 column is 3-10 μm, the column height is 250mm, the inner diameter is 4.6mm, and the sample injection amount is 20 μ L.
The second mobile phase is a mixed solution consisting of acetonitrile and methanol with the volume ratio of 97: 3; the flow rate is 1.0mL/min, the column temperature is 30 ℃, the detection wavelength is 254nm, and the retention time is 22min; a VWD detector.
Recording vitamin D at each concentration after HPLC detection 3 The peak area of the reference substance was used as the independent variable and the corresponding peak area as the dependent variable to obtain a linear regression equation, and the specific data are shown in table 2.
TABLE 2
| Concentration (μ g/mL) | 0.46 | 0.51 | 0.57 | 0.63 | 0.68 |
| Peak area | 112.5 | 125.9 | 139.8 | 153.3 | 167.6 |
The experimental results are as follows: vitamin D 3 Has good linear relation with the peak area in the concentration range of 0.46-0.68 mu g/mL. Linear regression equation: y =245.382X-0.048, r =0.998, and the corresponding linear curve of this equation is shown in fig. 6.
Example 3 specificity test
1. Preparation of blank solution
Taking mobile phase acetonitrile-methanol with the volume ratio of 97:3 as a blank solution.
2. Preparation of blank adjuvant solution
Precisely weighing refined peanut oil 2.0g, placing in 20mL volumetric flask, adding appropriate amount of n-hexane, ultrasonic treating for 1min to dissolve completely, adding n-hexane to desired volume, mixing, precisely weighing500uL, separating with liquid chromatographic column using silica gel as filler and n-hexane-n-pentanol solution at 997:3 volume ratio as mobile phase at flow rate of 1.5mL/min, column temperature of 30 deg.C and detection wavelength of 254nm, recording HPLC chromatogram, and collecting vitamin D-containing extract 3 The effluent liquid is dried by nitrogen, 2.5mL of acetonitrile-methanol solution with the volume ratio of 97:3 is added precisely, and the mixture is treated by ultrasonic wave to be dissolved, thus obtaining blank auxiliary material solution.
3. Preparation of control solutions
Collecting vitamin D 3 The reference substance is about 14mg, accurately weighed, placed in a 100mL volumetric flask, added with an appropriate amount of acetonitrile-methanol solution with a volume ratio of 97, ultrasonically treated for 1min to completely dissolve, subjected to constant volume to scale with acetonitrile-methanol solution with a volume ratio of 97.
Precisely measuring 20uL of each solution, taking a C18 column (octadecyl bonded silica gel) as a filler, taking acetonitrile-methanol as a mobile phase, carrying out HPLC detection at a flow rate of 1.0mL/min and a column temperature of 30 ℃ and a detection wavelength of 254nm, and recording an HPLC chromatogram.
The experimental conclusion is that: comparing the blank solution HPLC chromatogram, the blank adjuvant solution HPLC chromatogram and the reference solution HPLC chromatogram to show that the blank solution and the blank adjuvant solution are used for detecting vitamin D 3 The contents are all free of interference.
Example 4 recovery test
1. Preparation of control solutions
Collecting vitamin D 3 Precisely weighing the reference substance to 100mL volumetric flask, adding a proper amount of acetonitrile-methanol (the same below) with a volume ratio of 97, performing ultrasonic treatment for 1min to completely dissolve the reference substance, performing constant volume to scale with the acetonitrile-methanol, shaking up, precisely weighing 5mL to 50mL volumetric flask, diluting to scale with the acetonitrile-methanol, shaking up, precisely weighing 2mL to 50mL volumetric flask, diluting to scale with the acetonitrile-methanol, and shaking up to obtain the reference substance solution.
2. Preparation of reference stock solution (1)
Collecting vitamin D 3 Precisely weighing 14mg of reference substance, placing in a 100mL volumetric flask, adding a proper amount of n-hexane, performing ultrasonic treatment for 1min to completely dissolve, metering volume to scale with n-hexane, shaking up, precisely weighing 10mL, placing in a 50mL volumetric flask, diluting to scale with n-hexane, and shaking up to obtain reference substance stock solution (1).
3. Preparation of test solution
Weighing 2.0g of refined peanut oil, weighing 9 parts, dividing into 3 groups, respectively placing into 20mL volumetric flasks, adding 1.8mL of reference stock solution (1) into group 1, adding 2.0mL of reference stock solution (1) into group 2, adding 2.2mL of reference stock solution (1) into group 3, dissolving and diluting to scale with n-hexane, precisely weighing 500ul, and collecting vitamin D by chromatography system for purification 3 The following method was followed to purify the sample group 1 solution of 90% concentration, the sample group 2 solution of 100% concentration and the sample group 3 solution of 110% concentration.
The control solution, the sample group 1 solution, the sample group 2 solution and the sample group 3 solution were measured by precision measurement at 20uL each, and the recovery rate of the sample solution was calculated using a C18 column (octadecyl-bonded silica gel) as a filler, acetonitrile-methanol as a mobile phase at a flow rate of 1.0mL/min and a column temperature of 30 ℃ and a detection wavelength of 254nm, and the results are shown in table 3.
TABLE 3 recovery results
Example 5 solution stability
Storing the test solution at-20 + -5 deg.C for 24 hr and 48 hr, storing the reference solution at-20 + -5 deg.C for 1 day, 3 days, 5 days and 7 days, comparing the stored reference solution with the test solution, and comparing the measured result with the initial result.
1. Preparation of control solutions
Collecting vitamin D 3 Precisely weighing reference substance about 14mg, placing in 100mL volumetric flask, adding appropriate amount of acetonitrile-methanol, ultrasonic treating for 1min to dissolve completely, and dissolving with ethyl acetateAnd (3) fixing the volume of the nitrile-methanol to a scale, shaking up, precisely measuring a 5 mL-50 mL volumetric flask, diluting the volumetric flask to the scale by using acetonitrile-methanol, shaking up, precisely measuring a 2 mL-50 mL volumetric flask, diluting the volumetric flask to the scale by using acetonitrile-methanol, and shaking up to obtain a reference solution. Preparing 2 parts by the same method to obtain reference solution 1# and reference solution 2#, the concentrations are shown in Table 5.
2. Preparation of test solution
2g of the content of a finished vitamin D drop product is precisely weighed and placed in a 20mL volumetric flask, n-hexane is used for dissolving and diluting the content to a scale, the content is uniformly mixed, 500 mu L of the content is precisely measured, a liquid chromatographic column with silica gel as a filling agent is used, n-hexane-n-amyl alcohol with the volume ratio of 997 is used as a mobile phase for separation, the flow rate is 1.5mL/min, the column temperature is 30 ℃, the detection wavelength is 254nm, and the vitamin D drop product is purified and collected, wherein the detection wavelength is 254nm 3 The effluent liquid is dried by nitrogen, 2.5mL of acetonitrile-methanol is precisely added, and ultrasonic treatment is carried out to dissolve the effluent liquid, so as to obtain a test solution. Preparing 2 parts by the same method to obtain sample solution 1# and sample solution 2#.
Precisely measuring 20ul of the reference solution 1#, the reference solution 2#, the test solution 1# and the test solution 2# for HPLC determination, using a C18 column (octadecyl bonded silica gel) as a filling agent, acetonitrile-methanol as a mobile phase, at a flow rate of 1.0mL/min, a column temperature of 30 ℃, and a detection wavelength of 254nm. The measurement data of the test solution 1# and the test solution 2# are shown in table 4, and the measurement data of the control solution 1# and the control solution 2# are shown in table 5.
TABLE 4 measurement data of test solutions
TABLE 5 control solution measurement data
And (4) experimental conclusion: as can be seen from the data in Table 4, the concentration of the sample solution after storing at-20 + -5 deg.C for 24h and 48h respectively is not more than + -2.0% than 0 h. As can be seen from the data in Table 5, the concentration values of the control solutions after storage at-20. + -. 5 ℃ for 1 day, 3 days, 5 days and 7 days, respectively, were not more than. + -. 2.0% than that of 0 h.
In conclusion, according to the method for detecting the content of the vitamin D in the vitamin D drop, provided by the invention, positive and negative two-phase chromatographic columns are innovatively used, namely, the positive phase chromatographic column with the fixed phase being a silica gel column and the negative phase chromatographic column with the fixed phase being a C18 column are used for purifying and collecting the vitamin D to be detected and then detecting the vitamin D, so that interfering substances in the detection can be effectively removed, and the detection precision is improved. And the two chromatographic columns are combined with corresponding mobile phases, the flow rate, the detection wavelength, the chromatographic columns and other analysis conditions are limited, and multiple experiments verify that the method for detecting the content of the vitamin D in the vitamin D drop with good stability and repeatability, high analysis efficiency and good separation degree is finally obtained, and the method can be used for qualitatively and quantitatively detecting the vitamin D sensitively and accurately 3 Therefore, the quality of the vitamin D drops is objectively, comprehensively and accurately evaluated, and the method has important significance for controlling the quality of the vitamin D drops and ensuring the clinical curative effect.
The above description is only a preferred embodiment of the present application and should not be taken as limiting the present application, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
1. A method for detecting the content of vitamin D in vitamin D drops is characterized by comprising the following steps:
(1) Carrying out HPLC detection on a methanol-acetonitrile solution of a vitamin D reference substance with a known concentration, recording the peak area of the vitamin D reference substance with each concentration, and calculating a reference substance correction factor according to the following formula:
control correction factor = concentration/peak area;
(2) Dissolving vitamin D drops to be detected in n-hexane, purifying and collecting HPLC (high performance liquid chromatography) detection, drying the purified and collected vitamin D retention time effluent by using nitrogen, dissolving the effluent by using a methanol-acetonitrile solution, performing HPLC detection on the obtained solution again, and recording the peak area of the vitamin D;
(3) And (3) according to the calculation of an external standard method, multiplying the peak area of the vitamin D obtained in the step (2) by the correction factor of the reference substance obtained in the step (1) to obtain the concentration of the vitamin D.
2. The method for detecting the content of vitamin D in vitamin D drops according to claim 1, wherein in the HPLC detection in the step (1) and the step (2), a chromatographic column is a reversed-phase chromatographic column, and the stationary phase of the reversed-phase chromatographic column is a C18 column;
and (3) in the step (2) of purifying and collecting HPLC detection, the chromatographic column is a normal phase chromatographic column, and the stationary phase of the normal phase chromatographic column is a silica gel column.
3. The method for detecting the content of vitamin D in vitamin D drops as claimed in claim 2, wherein the silica gel column has a particle size of 3 to 10 μm, a column height of 100 to 250mm, an inner diameter of 4.6mm, and a sample introduction amount of 100 to 500 μ L; the grain diameter of the C18 column is 3-10 mu m, the column height is 100-250 mm, the inner diameter is 4.6mm, and the sample injection amount is 10-20 mu L.
4. The method of claim 2, wherein the first mobile phase of the normal phase chromatography column is 997:3, mixed solution consisting of n-hexane and n-pentanol;
the volume ratio of the second mobile phase corresponding to the reversed phase chromatographic column is 95-99: 5-1 of acetonitrile and methanol.
5. The method of detecting vitamin D levels in vitamin D drops of claim 4, wherein the second mobile phase is 97:3 acetonitrile and methanol.
6. The method for detecting the content of vitamin D in the vitamin D drop according to claim 4, wherein the first mobile phase and the second mobile phase both flow at a constant speed, the flow rate is 0.8-1.5 mL/min, the column temperature is 25-35 ℃, the detection wavelength is 254nm, and the retention time is 20-30 min.
7. The method of detecting the content of vitamin D in vitamin D drops according to claim 6, wherein the flow rate of the first mobile phase is 1.5mL/min, the flow rate of the second mobile phase is 1.0mL/min, the column temperature is 30 ℃, and the retention time is 25min.
8. The method of detecting the content of vitamin D in vitamin D drops according to claim 1, wherein in the step (1) and the step (2), the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solution is 3.
9. The method of claim 1, wherein the vitamin D is vitamin D 3 。
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