CN107860847A - Vitamin B in one kind detection multivitamin preparation12Method about material - Google Patents
Vitamin B in one kind detection multivitamin preparation12Method about material Download PDFInfo
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- CN107860847A CN107860847A CN201711118637.0A CN201711118637A CN107860847A CN 107860847 A CN107860847 A CN 107860847A CN 201711118637 A CN201711118637 A CN 201711118637A CN 107860847 A CN107860847 A CN 107860847A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses one kind to detect vitamin B in multivitamin preparation12Method about material, methods described comprise the following steps, chromatographic condition：Using octadecylsilane chemically bonded silica as chromatogram column packing；Using disodium phosphate soln mobile phase A, methanol as Mobile phase B, gradient elution is carried out；Detection wavelength：300~400nm；Column temperature：20~50 DEG C；Flow velocity：0.5~2.0mL/min；Sample size：300~700 μ l；Run time：100~200 minutes.It is prepared by sample solution：Need testing solution is prepared using extraction, its concentration can be 0.5~100 μ g/ml, separately take need testing solution, contrast solution is used as after 50 times of dilution.Measure：Above-mentioned solution is injected into high performance liquid chromatograph, chromatogram is recorded and is analyzed.It is of the invention simple efficiently, specificity it is strong, easy to operate, can be rapidly and efficiently detect vitamin B in multivitamin preparation12Relevant material, effectively control the quality of multivitamin compound preparation.
Description
Technical field
The present invention relates to vitamin B in a kind of detection multivitamin preparation in Pharmaceutical Analysis field2Side about material
Method.
Background technology
Vitamin B12(VitaminB12) cobalamin or cyanogen cobalt element are also known as, it is a kind of polycyclic based compound of cobalt containing trivalent,
The pyrrole ring of 4 reduction, which connects together, is turned into 1 big ring of corrin (similar to porphyrin), is vitamin B12The core of molecule, contains
The compound of this ring is all referred to as corrinoid, and the uniquely vitamin containing metallic element.Vitamin B12For the pin of light red
Shape crystallizes, and soluble in water and ethanol is most stable under the mild acid conditions of pH value 4.5~5.0, strong acid (pH<2) or in alkaline solution divide
Solution, heat can have to be destroyed to a certain degree, but the high-temperature sterilization loss of short time is smaller, and it is easily destroyed to meet strong light or ultraviolet.From
Vitamin B in right boundary12All it is Microbe synthesis, high animals and plants can not manufacture vitamin B12.Vitamin B12Main life
Reason function is to participate in manufacture erythrocyte, prevents pernicious anaemia；Prevent cerebral nerve from being destroyed.
In view of vitamin B12Important function, containing vitamin B12Preparation quality it is most important, through consulting domestic and international medicine
Allusion quotation and document, vitamin B in CP2015, EP8.0 and BP201312Raw material standard only to its it is total it is miscellaneous controlled, it is uncharged
Vitamin B12Relevant material in preparation.And the compound capsule of multivitamin composition, complicated, there has been no a variety of at present
On vitamin B in vitamin preparation12Report about substance-measuring, the present invention is to vitamin B12Impurity carried out full side
The research of position, to vitamin B in multivitamin preparation12List it is miscellaneous and it is total it is miscellaneous controlled respectively, filled up a variety of dimension lifes
Vitamin B in the compound preparation of element composition12Blank about substance detecting method, product quality is more strictly controlled, ensured
Drug safety.
This method specificity, reappearance are good, fast and effectively separation determination can go out to tie up life in multivitamin preparation
Plain B12Relevant material, can effectively control the quality and curative effect of multivitamin preparation.
The content of the invention
It is an object of the invention to provide a kind of simple and easy to do, high sensitivity, favorable reproducibility multivitamin preparation in
Vitamin B12Detection method about material.Methods described includes：
Vitamin B will be contained12Multivitamin preparation by pre-treatment be configured to detect solution, with octadecylsilane key
Conjunction silica gel is chromatographic column, uses disodium phosphate soln mobile phase A, methanol as Mobile phase B, records chromatogram.
Preferably, take this product content appropriate, add 5~50ml of chloroform shakings to make to disperse, add water to extract 3 times, merge
Layer solution, then cleaned with appropriate chloroform, it is diluted with water in every milliliter of solution containing about vitamin B12For the molten of 10~100 μ g
Liquid, centrifugation, supernatant filtration, subsequent filtrate is taken as need testing solution.It is another to take need testing solution, it is molten as compareing after 50 times of dilution
Liquid.
Preferably, the extractant during prepared by the need testing solution is water；Diluent in prepared by reference substance solution is water.
Preferably, the mobile phase is disodium phosphate soln (mobile phase A), methanol (Mobile phase B).
Preferably, the column temperature is 20~50 DEG C.
Preferably, the flow velocity of the mobile phase is 0.5~2.0mL/min.
Preferably, the chromatographic column is octadecylsilane chemically bonded silica.
Preferably, the Detection wavelength is 300~400nm.
The present invention is characterized by us and efficiently quickly determines vitamin B in multivitamin preparation using HPLC methods12's
Relevant material.This method specificity is strong, favorable reproducibility, high sensitivity, and testing cost is low, can be widely used in Pharmaceutical Analysis
In field, contain vitamin B so as to better assure that12Multivitamin preparation quality and validity.
Brief description of the drawings
Fig. 1：Vitamin B in multivitamin preparation in embodiment 112Negative sample collection of illustrative plates about substance-measuring
Fig. 2：Vitamin B in multivitamin preparation in embodiment 112Contrast solution collection of illustrative plates about substance-measuring
Fig. 3：Vitamin B in multivitamin preparation in embodiment 112Test sample collection of illustrative plates about substance-measuring
Fig. 4：Vitamin B in multivitamin preparation in embodiment 212System suitability collection of illustrative plates about substance-measuring
Fig. 5：Vitamin B in multivitamin preparation in embodiment 312Relevant substance-measuring collection of illustrative plates
Fig. 6：Vitamin B in multivitamin preparation in embodiment 412Relevant substance-measuring collection of illustrative plates
Embodiment
Below in conjunction with the embodiment of the present invention, technical scheme is at large described, it is clear that described
Embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, this area
The every other embodiment that those of ordinary skill is obtained under the premise of creative work is not made, belongs to protection of the present invention
Scope.
Embodiment 1
1st, chromatographic condition
Instrument：High performance liquid chromatograph；
Chromatographic column：AQ-C18Post (model：Ultimate, 250 × 4.6mm, 5 μm)；
Mobile phase A：Disodium phosphate soln, Mobile phase B：Methanol；
Detection wavelength：361nm；
Column temperature：30℃；
Flow velocity：1.0ml/min；
Run time：About 160 minutes；
Sample size：500μl
According to the form below carries out gradient elution：
2nd, sample preparation
(1) preparation of mobile phase
Mobile phase A：Prepare 0.025mol/L disodium phosphate soln (being 3.5 with phosphorus acid for adjusting pH)；Mobile phase B：First
Alcohol.
(2) prepared by need testing solution
Lucifuge operates.This product content is taken (to be approximately equivalent to vitamin B in right amount1245 μ g), it is accurately weighed, put separatory funnel
In, add chloroform 15ml shakings to make to disperse, extracted 3 times, first time 15ml with water, later each 5ml, combining water layer solution,
Cleaned once with 10ml chloroforms again, water layer solution is put in 25ml measuring bottles, is diluted with water to scale, is shaken up, centrifugation (3000
Rev/min) 10 minutes, supernatant filtration, subsequent filtrate is taken as need testing solution.
(3) prepared by negative sample solution
Take negative sample appropriate, prepared with need testing solution method.
(4) prepared by contrast solution
Precision measures need testing solution 2ml, puts in 100ml measuring bottles, is diluted with water to scale, shake up, as contrast solution.
(5) experimental result
Precision measures each 500 μ l of need testing solution, negative sample, contrast solution, is injected separately into high performance liquid chromatograph, note
Chromatogram is recorded, collection of illustrative plates is shown in Figure of description 1,2,3.
Embodiment 2
Solvent：Water.
Need testing solution：Prepared with " need testing solution preparation " method in embodiment 1.
Negative sample solution：Prepared with " need testing solution preparation " method in embodiment 1.
Material solution：Take vitamin B12Appropriate raw material, accurately weighed, being dissolved in water and diluting is made in every 1ml containing about dimension
It is raw
Plain B121.8 μ g solution, shakes up, and filtration, produces.
Precision measures each 500 μ l of above-mentioned solution, is injected separately into liquid chromatograph, records chromatogram.Collection of illustrative plates is shown in that specification is attached
Fig. 4.
As a result show：Negative sample and solvent are to vitamin B12And its impurity peaks do not interfere with, tailing factor, number of theoretical plate
Symbol
Regulation is closed, separation is good, shows that system suitability result is good.
Embodiment 3
Instrument：High performance liquid chromatograph；
Chromatographic column：AQ-C18Post (model：Ultimate, 250 × 4.6mm, 5 μm)；
Mobile phase A：Disodium phosphate soln, Mobile phase B：Methanol；
Detection wavelength：355nm；
Column temperature：35℃；
Flow velocity：1.2ml/min；
Run time：About 160 minutes；
Sample size：500μl
According to the form below carries out gradient elution：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 85 | 15 |
| 30 | 85 | 15 |
| 31 | 84 | 16 |
| 100 | 84 | 16 |
| 101 | 40 | 60 |
| 110 | 85 | 15 |
| 160 | 85 | 15 |
Need testing solution preparation method is carried out according to the method for " need testing solution preparation " in embodiment 1, and measure collection of illustrative plates is shown in
Fig. 5 of bright book accompanying drawing.
Embodiment 4
Instrument：High performance liquid chromatograph；
Chromatographic column：AQ-C18Post (model：Ultimate, 250 × 4.6mm, 5 μm)；
Mobile phase A：Disodium phosphate soln, Mobile phase B：Methanol；
Detection wavelength：365nm；
Column temperature：25℃；
Flow velocity：0.8ml/min；
Run time：About 160 minutes；
Sample size：500μl
According to the form below carries out gradient elution：
Need testing solution preparation method is carried out according to the method for " need testing solution preparation " in embodiment 1, and measure collection of illustrative plates is shown in
Fig. 6 of bright book accompanying drawing.
Embodiment 5
Do not destroy：This product content is taken (to be approximately equivalent to vitamin B in right amount1245 μ g), it is accurately weighed, put in separatory funnel,
Chloroform 15ml shakings are added to make to disperse, then plus water shakes, and is extracted 3 times, first time 15ml with water, later each 5ml, merges
Water layer solution, cleaned once with 10ml chloroforms, water layer is put in 25ml measuring bottles, is diluted with water to scale, is shaken up, centrifugation
(3000 revs/min) 10 minutes, supernatant filtration, take subsequent filtrate, as need testing solution, produce.The same method of negative sample, auxiliary material
Prepare.
Table --- vitamin B12Sample specificity tests process
The preparation of raw material stock solution：Take vitamin B12Appropriate raw material, accurately weighed, being dissolved in water and diluting is made every 1ml
In containing about vitamin B1218 μ g solution, shakes up, produces.
Table --- vitamin B12Raw material specificity tests process
Precision measures above-mentioned test sample, raw material and corresponding negative sample solution and each 500 μ l of auxiliary material solution respectively, injection
Liquid chromatograph, record chromatogram.
Table --- sample specificity result of the test
| Content of the test | Vitamin B12(%) | Vitamin B12Main peak and forward and backward impurity separating degree | Maximum single miscellaneous (%) |
| Do not destroy | 100 | ---、--- | Do not detect |
| Acid destroys | 100.24 | ---、--- | Do not detect |
| Alkali destroys | 96.78 | ---、--- | Do not detect |
| Oxidative demage | 99.82 | ---、--- | Do not detect |
| Illumination destroys | 99.81 | Do not detect | |
| High temperature | 97.32 | ---、--- | Do not detect |
Table --- raw material specificity result of the test
| Content of the test | Vitamin B12(%) | Vitamin B12Main peak and forward and backward impurity separating degree | Maximum single miscellaneous (%) |
| Do not destroy | 100 | ---、--- | Do not detect |
| Acid destroys | 106.19 | ---、--- | Do not detect |
| Alkali destroys | 99.92 | ---、--- | Do not detect |
| Oxidative demage | 98.66 | ---、--- | Do not detect |
| Illumination destroys | 92.32 | ---、--- | 5.49 |
| High temperature | 103.06 | ---、--- | Do not detect |
Table --- peak purity result of the test
The above results show：This product test sample and vitamin B12Substantially impurity is not detected in raw material；Impurity under failure condition
Mainly produced through illumination degrading, other conditions are relatively stable, produced substantially without other impurity.
Under this chromatographic condition, auxiliary material and negative sample have many places appearance, but go out peak position (i.e. relatively in degradation impurity
Retention time is between 0.17~0.25) shown without peak, show auxiliary material and negative sample to vitamin B12Measure about material
It is noiseless；Vitamin B in test sample12Main peak can efficiently separate with caused each impurity after forced degradation, and peak purity accords with
Close and require, be not detected by foreign body, available for the Related substances separation of this product, show that the experiment of this method specificity is good.
Claims (6)
1. vitamin B in one kind detection multivitamin preparation12Method about material, it is characterised in that methods described includes
Following steps：
Chromatographic condition：With AQ-C18For chromatographic column；It is with 0.020~0.026mol/L disodium phosphate solns (phosphoric acid adjusts pH3.5)
Mobile phase A, using methanol as Mobile phase B, carry out gradient elution；Detection wavelength：300~400nm；Column temperature：20~50 DEG C；Flow velocity：
0.5~2.0mL/min；Sample size：300~700 μ l, run time 100~200 minutes.
The preparation of sample solution：Take this product content appropriate, make to disperse added with 5~50ml of solvent shakings, extractant extraction 3
It is secondary, combining water layer solution, then cleaned with appropriate organic solvent, it is diluted with water in every milliliter of solution containing about vitamin B12For 0.5
~100 μ g solution, centrifugation, supernatant filtration, subsequent filtrate is taken as need testing solution.Need testing solution separately is taken, is made after dilution
For contrast solution.Measure：Need testing solution and contrast solution are injected separately into liquid chromatograph, record chromatogram measure.
2. according to the method for claim 1, it is characterised in that the chromatographic column is using octadecylsilane chemically bonded silica to fill out
Material, its granularity are 3 μm, 5 μm, 7 μm or 10 μm；Its internal diameter is 2~5mm；Its length is 10~30cm.
3. according to the method for claim 1, it is characterised in that the disodium phosphate soln is mobile phase A, methanol is stream
Dynamic phase B；The disodium phosphate soln phosphorus acid for adjusting pH is 2.0~5.0.
4. the preparation of need testing solution according to claim 1, it is characterised in that the organic solvent is chloroform,
The extractant is water.
5. the preparation of need testing solution according to claim 1, it is characterised in that the rotating speed centrifuged in the experiment is every
2000~4000 turns of minute, time are 5~20 minutes.
6. the preparation of reference substance solution according to claim 1, it is characterised in that the contrast solution is the use of test sample
Water dilutes 50 times of gained.
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Cited By (4)
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| CN109239230A (en) * | 2018-10-30 | 2019-01-18 | 广州汉光药业股份有限公司 | The impurity analysis method of multivitamin preparation |
| CN111595961A (en) * | 2020-04-30 | 2020-08-28 | 南京海纳医药科技股份有限公司 | Detection method of vitamin B2 related substances |
| CN112540136A (en) * | 2020-11-18 | 2021-03-23 | 湖北远大天天明制药有限公司 | Method for separating and detecting components of naphthalene-sensitized vitamin eye drops |
| CN112684021A (en) * | 2020-12-01 | 2021-04-20 | 南京健友生化制药股份有限公司 | Vitamin B12Method for detecting related substances of injection |
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| CN112540136A (en) * | 2020-11-18 | 2021-03-23 | 湖北远大天天明制药有限公司 | Method for separating and detecting components of naphthalene-sensitized vitamin eye drops |
| CN112684021A (en) * | 2020-12-01 | 2021-04-20 | 南京健友生化制药股份有限公司 | Vitamin B12Method for detecting related substances of injection |
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Application publication date: 20180330 |