CN105699517A - High performance liquid chromatography detection method for content of altrenogest - Google Patents
High performance liquid chromatography detection method for content of altrenogest Download PDFInfo
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- CN105699517A CN105699517A CN201610060735.2A CN201610060735A CN105699517A CN 105699517 A CN105699517 A CN 105699517A CN 201610060735 A CN201610060735 A CN 201610060735A CN 105699517 A CN105699517 A CN 105699517A
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- liquid chromatography
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- acetonitrile
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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
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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
- G01N30/60—Construction of the column
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
Abstract
The invention provides a high performance liquid chromatography detection method for the content of altrenogest. The high performance liquid chromatography detection method adopts the following chromatographic conditions to detect: a chromatographic column adopts a reversed phase chromatographic column which takes octadecyl silane bonded silica gel as fillers, and is an Agilent C18 column; and a mobile phase adopts an acetonitrile-0.01% phosphoric acid solution, wherein the volume percent of acetonitrile is 60%-80%, the flow speed is 0.8ml/min-1.2ml/min and the column temperature is 20-40 DEG C. The high performance liquid chromatography detection method has the advantages of simplicity, convenience, high efficiency, high sensitivity, good separation effect and high precision and accuracy.
Description
Technical field
The invention belongs to efficient liquid phase chromatographic analysis detection technique field, particularly to the high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content。
Background technology
RU-2267, has another name called altrenogest, English name: Azithromycin, and No. CAS is 850-52-2, and molecular formula is C21H26O2, molecular weight 310.43, white crystalline powder, it is soluble in the organic solvents such as methanol, acetonitrile, ethanol, is insoluble in water, structural formula is shown in Fig. 1。
RU-2267 plays an important role in the cultivation of animal。
Present livestock breed aquatics scale has been universal phenomenon。In the large-scale cultivation of pig, owing to the oestrus of sow is different, the workload of raiser can be greatly increased；The premature labor of sow can be substantially reduced nest number pigs born amount and piglet survival ratio, adds loss to raiser and increases cultivation input simultaneously。
RU-2267 can make substantial amounts of sow reach oestrus simultaneously, sow can be made to concentrate childbirth, wean, deliver for sale。Decrease transmission of pathogen, it is simple to scale manages。RU-2267 can also prevention of preterm birth and safely and effectively, it is possible to avoid the loss owing to premature labor brings。
Therefore, in the large-scale cultivation helped, usually adopt the injection of RU-2267 that cultivation pig is injected。The injection of RU-2267 is that the adjuvant that RU-2267 is equipped with necessity is prepared from, in order to control the content of RU-2267 in the injection of RU-2267, it is necessary to the injection of RU-2267 is detected accurately。But prior art not yet exists the accurate detecting method being applicable to the injection that this kind of effective ingredient is single RU-2267。
Summary of the invention
The invention is for solving the problems of the prior art, it is provided that high performance liquid chromatography (HPLC) detection method of a kind of RU-2267 content, has simplicity, the advantage that efficient, highly sensitive, good separating effect, preci-sion and accuracy are high。
The high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content that the invention provides, adopts following chromatographic condition to detect: chromatographic column adopts octadecylsilane chemically bonded silica to be the reversed phase chromatographic column of implant, AgilentC18 post；Mobile phase adopts acetonitrile-0.01% phosphoric acid solution, and wherein, the volume fraction of acetonitrile is 60%-80% (that is, the volume of acetonitrile accounts for the 60%-80% of the cumulative volume of acetonitrile and 0.01% phosphoric acid solution), flow velocity 0.8-1.2ml/min；Column temperature is 20-40 DEG C。
Wherein, chromatographic column preferably adopts AgilentC18 post (4.6*250mm, 5 μm) and AgilentC18 post (4.6*150mm, 5 μm) test, more preferably AgilentC18 post (4.6*250mm, 5 μm), the chromatographic column being suitable for makes sample have better separating effect and shorter retention time。
Wherein, mobile phase replace conventional water to mix use with acetonitrile with 0.01% phosphoric acid solution, control the volume content of acetonitrile simultaneously, the problem that can improve the peak shape hangover that the injection of RU-2267 causes when chromatograph detects well, and obtain in shorter retention time and go out peak effect preferably, be conducive to the raising of accuracy of detection。The preferred volume of acetonitrile is 70%。
Wherein, flow velocity and the control of column temperature, choosing in conjunction with above-mentioned mobile phase, it is possible to make the injection of RU-2267 better separate and form good peak shape, is conducive to improving accuracy of detection。Preferable flow rate is 1.0ml/min；Preferred column temperature is 30 DEG C。
Described method preferably adopts Shimadzu LC-20AT high performance liquid chromatograph, adopts UV-detector, and detection wavelength is 236nm, and sample size is advisable with 20 μ l。
The method of the invention specifically comprises the steps:
S1: configure the standard solution of known RU-2267 content concn with acetonitrile, and adopt chromatographic condition as above to carry out the detection under same chromatographic condition, the standard solution of calculating variable concentrations peak area under same retention time, makes and obtains peak area y standard curve equation y=f (x) about concentration of standard solution x；
S2: testing sample is obtained RU-2267 sample solution to be measured after necessity processes, adopt the liquid chromatographic detection condition (including chromatographic condition, retention time, sample size etc.) with step S1, obtain the RU-2267 sample solution to be measured peak area under same retention time, and according to standard curve equation y=f (x) that step S1 obtains, calculate the concentration obtaining RU-2267 sample solution to be measured；
S3: obtain the RU-2267 content data of testing sample according to the concentration of RU-2267 sample solution to be measured。
Wherein, in described step S1, the concentration of standard solution x of standard curve equation y=f (x) is in 50-1000 μ g/ml scope, and namely the present invention can obtain testing result accurately and reliably in bigger detection range。Further, described standard curve equation y=f (x) is linear equation with one unknown, coefficient R2>=0.99, it is possible to obtain the linear equation that degree of fitting is significantly high。
Wherein, in described step S2, the necessary process of testing sample can be included testing sample is carried out a certain proportion of dilution, and/or be filtered sample waiting and operate。
The invention uses UV-detector that the content of RU-2267 is measured by HPLC, simultaneously auxiliary。Through precision test, linear relationship test and application of sample recovery test etc., method having been verified, result shows that the method is accurately and reliably, and detection range is big, highly sensitive and accuracy height。
Accompanying drawing explanation
Fig. 1 is the structural formula of RU-2267；
Fig. 2 is the spectral scan figure of RU-2267；
The HPLC that Fig. 3 is RU-2267 standard solution in specific embodiment detects collection of illustrative plates；
Fig. 4 is the linear relationship chart of RU-2267 and standard curve equation in specific embodiment。
Detailed description of the invention
Below by conjunction with accompanying drawing, the invention is further described, but does not limit protection scope of the present invention。
Embodiment
(1) chromatographic condition
Adopt Shimadzu LC-20AT high performance liquid chromatograph associating UV-detector, detect wavelength 236nm。With octadecylsilane chemically bonded silica chromatographic column for chromatographic column, selecting AgilentC18 post (4.6*250mm, 5 μm), mobile phase is acetonitrile-0.01% phosphoric acid solution, and wherein the volume fraction of acetonitrile is 70%；Flow velocity is 1.0ml/min；Column temperature is 30 DEG C。
(2) acquisition of standard curve equation
Precision weighing RU-2267 reference substance 100mg, it is placed in the brown volumetric flask of 50ml, dissolve with acetonitrile, it is settled to scale as RU-2267 standard solution storing solution, then RU-2267 standard solution storing solution stepwise dilution being made RU-2267 concentration is 50, 100, 200, 500, 800, the RU-2267 standard solution of 1000 μ g/ml, the RU-2267 standard solution of variable concentrations is injected separately into the chromatographic column of the HPLC device of the entrance duty that step (1) is arranged, sample size 20 μ l, calculate the peak area under same retention time respectively, with concentration of standard solution x for abscissa, with peak area y for vertical coordinate, make standard curve equation y=59863.9636x-28630.6827, R2=0.9999, result shows that RU-2267 is within the scope of 50-1000 μ g/ml, and peak area becomes relation above with concentration。Acetonitrile can match with mobile phase, detection wavelength (detection wavelength is relatively low) well as solvent。
(3) content of RU-2267 in testing sample (RU-2267 injection):
Precision takes testing sample, dissolves with acetonitrile, is diluted (dilution gfactor is the inverse of dilution ratio) according to suitable dilution ratio, makes debita spissitudo, shake up and obtain RU-2267 sample solution to be measured, filters with 0.45 μm of organic filter。The chromatographic column of the HPLC device of the entrance duty that implantation step (1) is arranged, sample size 20 μ l, calculate and the peak area under step (2) same retention time, bring the standard curve equation of step (2) gained into, calculate the concentration of RU-2267 sample solution to be measured, calculate the content of the RU-2267 of testing sample again through the sampling amount of testing sample and dilution ratio。Computational methods are as follows:
(4) precision is investigated
To same testing sample (RU-2267 injection), continuous 6 sample introductions, peak area is table 1 such as:
Table 1
| Number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 30040787 | 30038685 | 30041634 | 30042589 | 30045958 | 30039737 |
The peak area relative standard deviation (RSD) of 6 sample introductions is 0.01%, it is shown that HPLC method of the present invention detection RU-2267 content precision is high, and favorable reproducibility。
(5) application of sample recovery test
The RU-2267 injection liquid samples accurately weighing known content is appropriate, respectively precision adds a certain amount of RU-2267 injection liquid samples 80%, 100%, 120% as test sample again, make the RU-2267 concentration in need testing solution respectively in the basic, normal, high region of RU-2267 standard curve, by the operation under item of the present invention, measuring in accordance with the law, calculate the response rate, result average recovery rate is 99.77%, RSD is 0.09%, it was shown that this average recovery of the present invention is good。
Claims (7)
1. a high-efficiency liquid chromatography method for detecting for RU-2267 content, adopts following chromatographic condition to detect: chromatographic column adopts octadecylsilane chemically bonded silica to be the reversed phase chromatographic column of implant, AgilentC18 post；Mobile phase adopts acetonitrile-0.01% phosphoric acid solution, and wherein, the volume fraction of acetonitrile is 60%-80%, flow velocity 0.8-1.2ml/min；Column temperature is 20-40 DEG C。
2. the high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content according to claim 1, it is characterized in that, chromatographic column adopts AgilentC18 post (4.6*250mm, 5 μm) and AgilentC18 post (4.6*150mm, 5 μm) test, preferred AgilentC18 post (4.6*250mm, 5 μm)。
3. the high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content according to claim 1, it is characterised in that in mobile phase, the volume of acetonitrile is 70%；Flow velocity is 1.0ml/min；Column temperature is 30 DEG C。
4. the high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content according to claim 1, it is characterised in that comprise the steps:
S1: configure the standard solution of known RU-2267 content concn with acetonitrile, and adopt chromatographic condition as claimed in claim 1 to carry out the detection under same chromatographic condition, the standard solution of calculating variable concentrations peak area under same retention time, makes and obtains peak area y standard curve equation y=f (x) about concentration of standard solution x；
S2: testing sample is obtained RU-2267 sample solution to be measured after necessity processes, adopt the liquid chromatographic detection condition with step S1, obtain the RU-2267 sample solution to be measured peak area under same retention time, and according to standard curve equation y=f (x) that step S1 obtains, calculate the concentration obtaining RU-2267 sample solution to be measured；
S3: obtain the RU-2267 content data of testing sample according to the concentration of RU-2267 sample solution to be measured。
5. the high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content according to claim 4, it is characterised in that in described step S1, the concentration of standard solution x of standard curve equation y=f (x) is in 50-1000 μ g/ml scope。
6. the high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content according to claim 4, it is characterised in that in described step S1, standard curve equation y=f (x) is linear equation with one unknown, coefficient R2≥0.99。
7. the high-efficiency liquid chromatography method for detecting of a kind of RU-2267 content according to claim 4, it is characterised in that in described step S2, includes testing sample is carried out a certain proportion of dilution to necessary process of testing sample, and/or sample is filtered。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110441411A (en) * | 2019-07-04 | 2019-11-12 | 天津市中升挑战生物科技有限公司 | A kind of high-efficiency liquid chromatography method for detecting of Altrenogest preparation |
-
2016
- 2016-01-28 CN CN201610060735.2A patent/CN105699517A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| M.MACHNIK ET AL.: "Pharmacokinetics of altrenogest in horses", 《J.VET.PHARMACOL.THERAP.》 * |
| MARTINA REJTHAROVA ET AL.: "Development and validation of an LC-MS/MS method for the determination of six gestagens in kidney fats", 《FOOD ADDITIVES&CONTAMINANTS》 * |
| MIKAEL PEDERSEN ET AL.: "Confirmatory analysis of steroids in muscle using liquid chromatography-tandem mass spectrometry", 《FOOD ADDITIVES AND CONTAMINANTS》 * |
| WWW.APEXBIO.COM: "《Altrenogest》", 13 January 2016 * |
| 康海宁 等: "超高效液相色谱-串联质谱法对河豚鱼、鳗鱼及烤鳗中烯丙孕素与氯地孕酮残留的同时测定", 《分析测试学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110441411A (en) * | 2019-07-04 | 2019-11-12 | 天津市中升挑战生物科技有限公司 | A kind of high-efficiency liquid chromatography method for detecting of Altrenogest preparation |
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