CN110441411A - A kind of high-efficiency liquid chromatography method for detecting of Altrenogest preparation - Google Patents
A kind of high-efficiency liquid chromatography method for detecting of Altrenogest preparation Download PDFInfo
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- CN110441411A CN110441411A CN201910597301.XA CN201910597301A CN110441411A CN 110441411 A CN110441411 A CN 110441411A CN 201910597301 A CN201910597301 A CN 201910597301A CN 110441411 A CN110441411 A CN 110441411A
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- 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
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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
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Abstract
The present invention provides a kind of high-efficiency liquid chromatography method for detecting of Altrenogest preparation, use following chromatographic condition to be detected: chromatographic column uses cyanoalkysilane bonded silica gel for filler, specially positive ZORBAX CN chromatographic column (4.6*250mm, 5 μm)；Mobile phase uses n-hexane-isopropanol, wherein the volume fraction of n-hexane is 90%-97% (i.e. the 90%-97% that the volume of n-hexane accounts for n-hexane and isopropanol total volume)；Flow velocity 0.8-1.2ml/min；Column temperature is 20-40 DEG C；Dilution: add and be settled to scale with n-hexane after 5ml isopropanol ultrasonic dissolution.This method has the advantages that easy, efficient, preci-sion and accuracy is high.
Description
Technical field
The invention belongs to efficient liquid phase chromatographic analysis detection technique field, in particular to a kind of Altrenogest preparation
High-efficiency liquid chromatography method for detecting.
Background technique
Altrenogest (structural formula is shown in Fig. 1) is a kind of 21 carbon steroid progestational hormone of triolefin of synthesis, belongs to 19- demethyl-
Stosterone is a kind of oral active progestational hormone.As all steroids, Altrenogest fat-soluble can be permeated by itself
Enter in target cell and then play a role in conjunction with special receptor, inhibits to promote sexual gland to be similar to the binding mode of Natural progesterone
The release of hormone.Altrenogest makes the ovarian follicle increased by inhibiting the concentration of endogenous promoting sexual gland hormone LH and FSH in blood
(20~25 millimeters of >) shrinks back, and then blocks heat and ovulation.In the rear half stage of medication, after all increase ovarian follicles are shunk back,
It followed by will appear the peak of FSH secretion, this will starting new round ovarian follicular growth and maturation.The phase can be followed by after the treatment
The regular increase of LH secretion, promotes the growth and maturation of ovarian follicle.This endocrine effect can make treated young dam
Enter heat after drug withdrawal, therefore Altrenogest can be used for domestic animal synchronization of Estrus.Estrus synchronization can make domestic animal delivery time
It concentrates, it is effective to improve the numerous of domestic animal convenient for concentrating feeding, wean, all-in and all-out, the same period being concentrated to enter next breeding cycle
Rate is grown, the propagation of cause of disease is reduced, carry out stringent epidemic prevention system and guarantees Biosecurity system, is conducive to scale management.
European EMEA and U.S. FDA approved Altrenogest solution are used for the synchronization of estrus of induction of mare and sow.By
The bulletin of agriculture rural area portion No. 16, No. 35 and No. 101 knows had more units to take in China's veterinary markets at present
The novel chiral synthon certificate of Altrenogest oral solution.
Altrenogest English entitled Altrenogest, No. CAS are as follows: 850-52-2, molecular formula C21H26O2, molecular weight
It is 310.43, white crystalline powder is soluble in the organic solvents such as methanol, acetonitrile, ethyl alcohol, is insoluble in water, in peanut oil, greatly
There is larger solubility in the vegetable oil such as soya-bean oil, in addition advantage of the vegetable oil with cheap suitable mass production, therefore Altrenogest
Oral solution often uses vegetable oil as solvent.Such as the Altrenogest oral preparations listed, the pregnant power of trade name is precious, and auxiliary material is soybean
Oil, content are planned to 0.4%；Altrenogest oral solution, the production of Ningbo the second hormone factory, lot number: 180706, content specification is
450mL:1800mg, solvent are peanut oil, and packing specification is 450mL/ bottles；Reversed-phase liquid chromatography method often uses water and certain proportion
Organic phase or salting liquid as mobile phase carry out test sample, but when formulation samples are finish, then need to pass through formulation samples
Complicated pretreatment process is such as repeatedly extracted, the elution of HLB solid phase extraction column with again with methanol after ether dissolution, is deoiled with removing
Agent.It not only needs also to expend a large amount of organic solvent, and treatment process excessively will cause sample damage by cumbersome step
Consumption, and then lead to measurement error, it is therefore desirable to develop a kind of simple and efficient accurate detection method.
Summary of the invention
In order to solve the problems in the prior art, the present invention provides a kind of inspections of the high performance liquid chromatography of Altrenogest preparation
Survey method.Using detection method of the invention, directly formulation samples dissolved dilution can be detected, without cumbersome preceding place
Step is managed, and detection method step of the invention is simple to operation, has the advantages that easy, efficient, preci-sion and accuracy is high.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of high-efficiency liquid chromatography method for detecting of Altrenogest preparation, comprising:
It is detected using following chromatographic condition:
The stationary phase of chromatographic column is cyanoalkysilane bonded silica gel；Mobile phase includes mobile phase A and Mobile phase B, the flowing
The polarity of phase A is less than the polarity of the Mobile phase B.
In above-mentioned detection method, the specification of the chromatographic column as a preferred implementation manner, are as follows: internal diameter 4.6mm,
Length is 150-250mm, and packing material size is 5 μm.Further, the chromatographic column is ZORBAX CN column (4.6*250mm, 5 μm)
With ZORBAX CN column (4.6*150mm, 5 μm)；Preferably ZORBAX CN column (4.6*250mm, 5 μm).Due in the prior art
Rarely have and Altrenogest is detected using normal phase column, therefore selects suitable chromatographic column and pre-treating method most important；For
This, the present inventor after having paid sufficient creative work, obtains cyanoalkysilane bonded silica gel by a large amount of further investigation
Normal phase column makes sample have better separating effect.
In above-mentioned detection method, the mobile phase A is n-hexane as a preferred implementation manner,；Mobile phase B is different
Propyl alcohol or ethyl alcohol, preferably isopropanol.
In some specific embodiments, the mobile phase A is n-hexane, Mobile phase B is isopropanol, and volume ratio is
90%:10%~97%:3%.
In some specific embodiments, the volume ratio of n-hexane and isopropanol is 95%:5%.By controlling n-hexane
Volume content, can not only improve well Altrenogest oral liquid formulations feed liquor phase front need before handle extraction step,
Preferable appearance effect can also be obtained in suitable retention time, be conducive to the raising of detection accuracy.
In some specific embodiments, the flow velocity of the efficient liquid phase is 0.8-1.2mL/min；Column temperature is 20-40 DEG C；
Detection wavelength is 230-240nm；Preferably, flow velocity 1.0ml/min；Column temperature is 30 DEG C；Detection wavelength is 235nm.Wherein, it flows
Speed with the control of column temperature, in conjunction with the selection of above-mentioned mobile phase, enable to the acquisition of Altrenogest preparation preferably to separate and formed
Good peak shape is conducive to improve detection accuracy.
In some specific embodiments, the solvent of the Altrenogest preparation is vegetable oil；Preferably, the vegetable oil
For one of soybean oil, peanut oil, castor oil, palm oil.
In above-mentioned detection method, as a preferred implementation manner, further include:
The preparation of standard solution: it is appropriate that precision weighs Altrenogest standard items, is dissolved with isopropanol, prescribed concentration is made
Solution, as standard solution；
The preparation of test solution: taking Altrenogest oral liquid formulations sample, with using n-hexane after appropriate isopropanol
It is settled to scale, as test solution；
Standard solution, test solution are detected respectively using the chromatographic condition.
Using said determination method, without being pre-processed to Altrenogest preparation, carried out by properly mixed solvent
Simple dilution can directly test it, not only easy to operate, and save cost, be easy to industrial applications.
In some specific embodiments, when detecting to standard solution, test solution, sampling volume is 10 μ l.
Technical effect of the invention is as follows:
The present invention assisted using HPLC, simultaneously using UV detector to the content of Altrenogest in Altrenogest preparation into
Row directly measures, and without pre-processing to Altrenogest preparation, carrying out simple dilution by properly mixed solvent can be straight
It connects and it is tested, it is not only easy to operate, and cost is saved, it is easy to industrial applications.In addition, by precision test, line
Method is verified in sexual intercourse test and sample-adding recovery test etc., the results showed that and measuring method of the invention is accurate and reliable,
Detection range is big, and high sensitivity and accuracy are high.
Detailed description of the invention
Fig. 1 is the structural formula of Altrenogest；
Fig. 2 is the HPLC test map of Altrenogest standard solution in embodiment 1；
Fig. 3 is the linear relationship chart and calibration curve equation of Altrenogest oral liquid formulations in embodiment 1.
Specific embodiment
Below by conjunction with attached drawing, invention is further explained, but the scope of protection of the present invention is not limited.
ZORBAX CN column, Altrenogest oral solution and chemical reagent etc. used in the embodiment of the present invention are commercially available quotient
Product.
Embodiment 1
(1) chromatographic condition screens
UV detector, Detection wavelength 235nm are combined using 1260 high performance liquid chromatograph of Agilent, chromatographic column uses cyanogen
Base silane bonded silica gel is the forward chromatographic column of filler, is selected ZORBAX CN column (4.6*250mm, 5 μm)；Mobile phase is just
Hexane-isopropanol system, inventor have found when screening n-hexane-Isopropanol Solvent: then main peak goes out n-hexane volume fraction height
Peak time evening, the test sample efficiency rate of exchange are low；The too low appearance of volume fraction is too fast, blank solvent peak (i.e. Altrenogest oral liquid formulations
Solvent in sample) and main peak also do not separate；Therefore, in optimal flow visualizing, the volume fraction of n-hexane is 95%；Stream
Fast 1.0ml/min；Column temperature is 30 DEG C；Altrenogest oral liquid formulations sample dilution: it is used after adding 5ml isopropanol ultrasonic dissolution
N-hexane is settled to scale；10 μ l of sample volume.
(2) acquisition of calibration curve equation
The preparation of standard solution: precision weighing Altrenogest standard 100mg is placed in 50ml brown volumetric flask, uses isopropyl
Alcohol dissolved dilution is simultaneously settled to scale as Altrenogest standard solution stock solution, then by Altrenogest standard solution stock solution
The Altrenogest standard solution that Altrenogest concentration is 400,450,500,550,600 μ g/ml is made in dilution step by step.
The acquisition of calibration curve equation: being respectively the Altrenogest standard of 400,450,500,550,600 μ g/ml by concentration
Solution is injected separately into the chromatographic column of the HPLC device into working condition of step (1) setting, 10 μ l of sample volume.Wherein, 500 μ
The HPLC test map of the Altrenogest standard solution of g/ml is shown in Fig. 2.The peak area under same retention time is calculated separately, with mark
Quasi- solution concentration x makes calibration curve equation y=8569.5x+93.206, R using peak area y as ordinate for abscissa2=
0.9998, see Fig. 3.Then the peak area y above-mentioned test obtained, substitutes into calibration curve equation, and calculated result shows that allyl is pregnant
Plain concentration within the scope of 401-601 μ g/ml, illustrate peak area and concentration at the relationship in the above calibration curve equation, further
Illustrate the pass that can be fitted the peak area that efficient liquid phase obtains and Altrenogest concentration well using detection method of the invention
System, the solvent for also further proving that isopropanol is prepared as standard solution can match with mobile phase, Detection wavelength well,
The selection of solvent provides foundation when also configuring for test solution.
(3) Altrenogest oral liquid formulations to be measured (manufacturer: rise challenge Biotechnology Co., Ltd, rule in Tianjin
Lattice: 0.4%, solvent: soybean oil) in Altrenogest content:
Precision weighs sample to be tested 2.87812g into 25ml brown volumetric flask, in this patent method dilute: first plus
Entering 5ml isopropanol makes to dissolve, then is settled to scale with n-hexane, shakes up, and it is molten to obtain Altrenogest oral liquid formulations sample to be measured
Liquid is filtered with 0.45 μm of organic filter.The chromatographic column of the HPLC device into working condition of injection step (1) setting, sample introduction
10 μ l are measured, are calculated and the peak area under step (2) same retention time, substitution step (2) resulting calibration curve equation y=
8569.5x+93.206 R2=0.9998, calculate the concentration of Altrenogest oral liquid formulations sample solution to be measured, then by
The density and dilution ratio of sample calculate the content of the Altrenogest oral liquid formulations of sample to be tested.
(4) precision is investigated
To same sample to be tested (Altrenogest oral solution), continuous 6 sample introductions, peak area such as table 1:
Table 1
| Number | 1 | 2 | 3 | 4 | 5 | 6 | Average value |
| Peak area | 4413.7 | 4420.5 | 4417.7 | 4425.3 | 4429.4 | 4439.5 | 4424.35 |
(n is measurement number,
Each measured value is respectively x1, x2..., xB,For the arithmetic mean of instantaneous value of measured value) it is 0.21%, the results showed that, the present invention
HPLC method detects Altrenogest oral liquid formulations sample size precision height, favorable reproducibility.
(5) it is loaded recovery test
The Altrenogest oral liquid formulations sample for accurately weighing known content is appropriate, then accurate a certain amount of alkene of addition respectively
Third pregnant plain oral solution 80%, 100%, 120% is used as test sample, makes the Altrenogest oral liquid formulations concentration in test solution
It is measured in accordance with the law in the basic, normal, high region of Altrenogest standard curve by being operated under item of the present invention respectively,As a result average recovery rate is 100.3%, RSD 0.67%, shows this
Sample recovery rate of the present invention is good.
It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to limit protection model of the invention
It encloses.In addition, it should also be understood that, after reading the technical contents of the present invention, those skilled in the art can make the present invention each
Kind change, modification and/or variation, all these equivalent forms equally fall within guarantor defined by the application the appended claims
Within the scope of shield.
Claims (10)
1. a kind of high-efficiency liquid chromatography method for detecting of Altrenogest preparation, which is characterized in that including using following chromatographic condition
It is detected:
The stationary phase of chromatographic column is cyanoalkysilane bonded silica gel；Mobile phase includes mobile phase A and Mobile phase B, the mobile phase A
Polarity is less than the polarity of the Mobile phase B.
2. high-efficiency liquid chromatography method for detecting according to claim 1, which is characterized in that the specification of the chromatographic column are as follows:
Internal diameter is 4.6mm, and length 150-250mm, packing material size is 5 μm.
3. high-efficiency liquid chromatography method for detecting according to claim 2, which is characterized in that the chromatographic column is ZORBAX
CN column (4.6*250mm, 5 μm) and ZORBAX CN column (4.6*150mm, 5 μm)；Preferably ZORBAX CN column (4.6*250mm, 5
μm)。
4. high-efficiency liquid chromatography method for detecting according to claim 1, which is characterized in that the mobile phase A is n-hexane；
Mobile phase B is isopropanol or ethyl alcohol, preferably isopropanol.
5. high-efficiency liquid chromatography method for detecting according to claim 4, which is characterized in that the mobile phase A be n-hexane,
Mobile phase B is isopropanol, and volume ratio is 90%:10%~97%:3%.
6. high-efficiency liquid chromatography method for detecting according to claim 5, which is characterized in that the n-hexane and isopropanol
Volume ratio is 95%:5%.
7. high-efficiency liquid chromatography method for detecting according to claim 1, which is characterized in that the flow velocity of the efficient liquid phase is
0.8-1.2mL/min；Column temperature is 20-40 DEG C；Detection wavelength is 230-240nm；Preferably, flow velocity 1.0ml/min；Column temperature is
30℃；Detection wavelength is 235nm.
8. high-efficiency liquid chromatography method for detecting according to claim 1, which is characterized in that the Altrenogest preparation it is molten
Agent is vegetable oil；Preferably, the vegetable oil is one of soybean oil, peanut oil, castor oil, palm oil.
9. high-efficiency liquid chromatography method for detecting according to claim 1, which is characterized in that further include:
The preparation of standard solution: it is appropriate that precision weighs Altrenogest standard items, is dissolved with isopropanol, the molten of prescribed concentration is made
Liquid, as standard solution；
The preparation of test solution: taking Altrenogest oral liquid formulations sample, with after appropriate isopropanol use n-hexane constant volume
To scale, as test solution；
Standard solution, test solution are detected respectively using the chromatographic condition.
10. high-efficiency liquid chromatography method for detecting according to claim 1, which is characterized in that the standard solution, for examination
When product solution is detected, sampling volume is 10 μ l.
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| CN105699517A (en) * | 2016-01-28 | 2016-06-22 | 天津市中升挑战生物科技有限公司 | High performance liquid chromatography detection method for content of altrenogest |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105699517A (en) * | 2016-01-28 | 2016-06-22 | 天津市中升挑战生物科技有限公司 | High performance liquid chromatography detection method for content of altrenogest |
Non-Patent Citations (2)
| Title |
|---|
| ARUNA V.KRISHNAN ET AL.: "Bisphenol-A: An Estrogenic Substance Is Released from Polycarbonate Flasks during Autoclaving", 《ENDOCRINOLOGY》 * |
| MATTHEW J.BECKMAN ET AL.: "Human 25-Hydroxyvitamin D3-24-Hydroxylase, a Multicatalytic Enzyme", 《BIOCHEMISTRY》 * |
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