CN108226318A - A kind of analyzing detecting method of N- fluorenylmethyloxycarbonyls-l-Alanine - Google Patents
A kind of analyzing detecting method of N- fluorenylmethyloxycarbonyls-l-Alanine Download PDFInfo
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- CN108226318A CN108226318A CN201611192923.7A CN201611192923A CN108226318A CN 108226318 A CN108226318 A CN 108226318A CN 201611192923 A CN201611192923 A CN 201611192923A CN 108226318 A CN108226318 A CN 108226318A
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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Abstract
The present invention relates to a kind of analyzing detecting methods of N fluorenylmethyloxycarbonyls L alanine, for the quality control of N fluorenylmethyloxycarbonyl L alanine, it is the chromatographic column (C18 using octadecylsilane chemically bonded silica as filler, 4.6 × 250mm, 5 μm), using trifluoroacetic acid, acetonitrile and water mixed solution system as mobile phase, using Detection wavelength as 220nm, high-efficient liquid phase chromatogram technique analysis detection is carried out.The analyzing detecting method of the present invention can effectively separate N fluorenylmethyloxycarbonyl L alanine and its impurity, and this method has the advantages of linear relationship is good, easy to operate, repeatability and durability are good, as a result reliable and stable.
Description
Technical field
The present invention relates to a kind of HPLC analytical method, especially a kind of N- fluorenylmethyloxycarbonyls-l-Alanine
Analyzing detecting method.
Background technology
Thymalfasin is chemical synthesis the polypeptide being made of 28 amino acid, is mainly used for treating chronic hepatitis B, hepatitis,
Also the treatment diseases such as tumour and immune deficiency can be combined with other drugs.
N- fluorenylmethyloxycarbonyls-l-Alanine is to synthesize the important starting material of thymalfasin, and structural formula is as follows：
Up to the present, N- fluorenylmethyloxycarbonyls-L- is not recorded in USP, EP, BP, JP and Chinese Pharmacopoeia and document
The analyzing detecting method of alanine, but the analysis of N- fluorenylmethyloxycarbonyls-l-Alanine is detected to thymalfasin reaction controlling and receipts
Rate raising has important role, while also directly affect the quality of finished product, is effectively analyzed so establishing a kind of stabilization
Detection method carries out quality control to N- fluorenylmethyloxycarbonyls-l-Alanine to be very important.
Invention content
The purpose of the present invention is to provide a kind of efficient liquid phase chromatographic analysis detection sides of N- fluorenylmethyloxycarbonyls-l-Alanine
Method, for the quality control of N- fluorenylmethyloxycarbonyls-l-Alanine.
In order to achieve the object of the present invention, inventor finally obtains following technical solution by a large number of experiments：
A kind of analyzing detecting method of N- fluorenylmethyloxycarbonyls-l-Alanine is using octadecylsilane chemically bonded silica to fill out
The chromatographic column (C18,4.6 × 250mm, 5 μm) of material, with water-trifluoroacetic acid (1000:1) it is mobile phase A, with acetonitrile-trifluoroacetic acid
(1000:1) it is Mobile phase B, gradient elution using Detection wavelength as 220nm, carries out high-efficient liquid phase chromatogram technique analysis detection.
The gradient elution setting is as follows：
Further, it is preferable to gradient elution setting it is as follows：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 20 | 0 | 100 |
| 25 | 0 | 100 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
Analyzing detecting method of the present invention can be realized by following steps：
A, it takes N- fluorenylmethyloxycarbonyls-l-Alanine sample appropriate, is dissolved with acetonitrile, be configured to every 1ml and risen containing thymalfasin
The sample solution of beginning 0.1~0.6mg of material；
B, setting flow rate of mobile phase is 0.9~1.1ml/min, and Detection wavelength 220nm, column temperature is 20~30 DEG C；
C, the 10 μ l of sample solution of A is taken to inject liquid chromatograph, complete the analysis inspection of N- fluorenylmethyloxycarbonyls-l-Alanine
It surveys；
High performance liquid chromatograph：Waters e2695 liquid chromatographic systems；
Chromatographic column：Yi Lite, SinoChromODS-BP C18；4.6 × 250mm, 5 μm；
Mobile phase：With water-trifluoroacetic acid (1000:1) it is mobile phase A, with acetonitrile-trifluoroacetic acid (1000:1) it is mobile phase
B, gradient elution setting are as follows：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 20 | 0 | 100 |
| 25 | 0 | 100 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
Detection wavelength：220nm；
Column temperature：25℃；
Flow velocity：1.0ml/min.
Analyzing detecting method of the present invention, can be effectively by N- fluorenylmethyloxycarbonyls-l-Alanine and its impurity point
It opens, and this method linear relationship is good, easy to operate, repeatability and durability are good, as a result reliable and stable, so as to be used for N-
The quality control of fluorenylmethyloxycarbonyl-l-Alanine, the quality for final finished provide effective guarantee.
Description of the drawings
The N- fluorenylmethyloxycarbonyls of Fig. 1 embodiments 1-l-Alanine HPLC collection of illustrative plates.
The N- fluorenylmethyloxycarbonyls of Fig. 2 embodiments 2-l-Alanine HPLC collection of illustrative plates.
The N- fluorenylmethyloxycarbonyls of Fig. 3 embodiments 3-l-Alanine HPLC collection of illustrative plates.
The N- fluorenylmethyloxycarbonyls of Fig. 4 embodiments 6-l-Alanine linear work curve.
Specific embodiment
The following is specific embodiments of the present invention, and technical scheme of the present invention is done and is further described, but of the invention
Protection domain be not limited to these embodiments.It is every to be included in this hair without departing substantially from the change of present inventive concept or equivalent substitute
Within bright protection domain.
Embodiment 1
Instrument and condition：Waters e2695 liquid chromatographic systems, 2489 ultraviolet/visible light detectors of Waters, chromatography
Column：Yi Lite, SinoChrom ODS-BP C18；4.6 × 250mm, 5 μm；Detection wavelength：220nm；Column temperature is 25 DEG C；Flow velocity
1.0ml/min；Mobile phase：With water-trifluoroacetic acid (1000:1) it is mobile phase A, with acetonitrile-trifluoroacetic acid (1000:1) it is flowing
Phase B, gradient elution setting are as follows：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 20 | 0 | 100 |
| 25 | 0 | 100 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
Experimental procedure：N- fluorenylmethyloxycarbonyls-l-Alanine with acetonitrile is dissolved and quantifies dilution, fluorenes containing N- in every 1ml is made
The solution of methoxycarbonyl group-l-Alanine 0.5mg, as test solution；Precision measures 10 μ l of test solution injection liquid phase colors
Spectrometer carries out efficient liquid phase chromatographic analysis by above-mentioned condition, records chromatogram, as a result see attached drawing 1.
Attached drawing 1 shows that under the chromatographic condition N- fluorenylmethyloxycarbonyls-l-Alanine and its impurity peaks can be kept completely separate,
And the retention time of N- fluorenylmethyloxycarbonyls-l-Alanine is in 8.368min or so.
Embodiment 2
Instrument and condition：Waters e2695 liquid chromatographic systems, 2489 ultraviolet/visible light detectors of Waters, chromatography
Column：Yi Lite, SinoChrom ODS-BP C18；4.6 × 250mm, 5 μm；Detection wavelength：220nm；Column temperature is 20 DEG C；Flow velocity
0.9ml/min；Mobile phase：With water-trifluoroacetic acid (1000:1) it is mobile phase A, with acetonitrile-trifluoroacetic acid (1000:1) it is flowing
Phase B, gradient elution setting are as follows：
Experimental procedure：N- fluorenylmethyloxycarbonyls-l-Alanine with acetonitrile is dissolved and quantifies dilution, fluorenes containing N- in every 1ml is made
The solution of methoxycarbonyl group-l-Alanine 0.5mg, as test solution；Precision measures 10 μ l of test solution injection liquid phase colors
Spectrometer carries out efficient liquid phase chromatographic analysis by above-mentioned condition, records chromatogram, as a result see attached drawing 2.
Attached drawing 2 shows that under the chromatographic condition N- fluorenylmethyloxycarbonyls-l-Alanine and its impurity peaks can be kept completely separate,
And the retention time of N- fluorenylmethyloxycarbonyls-l-Alanine is in 9.078min or so.
Embodiment 3
Instrument and condition：Waters e2695 liquid chromatographic systems, 2489 ultraviolet/visible light detectors of Waters, chromatography
Column：Yi Lite, SinoChrom ODS-BP C18；4.6 × 250mm, 5 μm；Detection wavelength：220nm；Column temperature is 30 DEG C；Flow velocity
1.1ml/min；Mobile phase：With water-trifluoroacetic acid (1000:1) it is mobile phase A, with acetonitrile-trifluoroacetic acid (1000:1) it is flowing
Phase B, gradient elution setting are as follows：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 40 | 60 |
| 20 | 0 | 100 |
| 25 | 0 | 100 |
| 30 | 40 | 60 |
| 35 | 40 | 60 |
Experimental procedure：N- fluorenylmethyloxycarbonyls-l-Alanine with acetonitrile is dissolved and quantifies dilution, fluorenes containing N- in every 1ml is made
The solution of methoxycarbonyl group-l-Alanine 0.5mg, as test solution；Precision measures 10 μ l of test solution injection liquid phase colors
Spectrometer carries out efficient liquid phase chromatographic analysis by above-mentioned condition, records chromatogram, as a result see attached drawing 3.
Attached drawing 3 shows that under the chromatographic condition N- fluorenylmethyloxycarbonyls-l-Alanine and its impurity peaks can be kept completely separate,
And the retention time of N- fluorenylmethyloxycarbonyls-l-Alanine is in 7.755min or so.
Embodiment 4
Repeated experiment
Instrument and condition：With embodiment 1.
Experimental procedure：Take this product appropriate, it is accurately weighed, it acetonitrile is added to dissolve and is diluted is made in every 1ml containing the molten of 0.5mg
As test solution, 6 parts of test solutions are prepared with method for liquid.Take test solution, continuous sample introduction six times, by area normalization
Change method calculates N- fluorenylmethyloxycarbonyls-l-Alanine content, and calculates its relative standard deviation, and experimental result is shown in Table 1.
1 N- fluorenylmethyloxycarbonyls of table-l-Alanine repeated experiment result
As shown in Table 1, the content of N- fluorenylmethyloxycarbonyls-l-Alanine does not have notable difference in each test solution, relatively
Standard deviation is 0.008%, it is seen that the repeatability of this analyzing detecting method is good.
Embodiment 5
Durability is tested
Instrument and condition：Waters e2695 liquid chromatographic systems, 2489 ultraviolet/visible light detectors of Waters；Detection
Wavelength：220nm；Mobile phase：With water-trifluoroacetic acid (1000:1) it is mobile phase A, with acetonitrile-trifluoroacetic acid (1000:1) it is stream
Dynamic phase B, gradient elution setting are as follows：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 20 | 0 | 100 |
| 25 | 0 | 100 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
Experimental procedure：Take this product appropriate, it is accurately weighed, it acetonitrile is added to dissolve and is diluted is made in every 1ml containing the molten of 0.5mg
Liquid, as test solution.Respectively by changing column temperature, flow velocity and chromatographic column, record N- fluorenylmethyloxycarbonyls-l-Alanine is related
The situation of change of substance and content, experimental result are shown in Table 2.
2 N- fluorenylmethyloxycarbonyls of table-l-Alanine durability experimental result
As shown in Table 2, after changing column temperature, flow velocity and chromatographic column, N- fluorenylmethyloxycarbonyls-related substance of l-Alanine and content
Measurement result there is no notable difference, it is seen that the good tolerance of analyzing detecting method of the present invention.
Embodiment 6
Linearity and range is tested
Instrument and condition：With embodiment 1.
Experimental procedure：Take this product appropriate, it is accurately weighed, acetonitrile is added to dissolve and dilute be made it is molten containing about 1mg in every 1ml
Liquid, as linear storing solution.Precision measures linear storing solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, puts respectively in 10ml measuring bottles,
Dilution in acetonitrile is added to shake up, measure in accordance with the law, record chromatogram to scale.With the concentration C (mg/ml) of test sample for abscissa, with N-
Fluorenylmethyloxycarbonyl-l-Alanine peak area carries out linear regression for ordinate, obtains equation of linear regression y=22265440.81x+
887005.60, as a result see attached drawing 4.
By attached drawing 4 it is found that in figure Trendline correlation coefficient r=0.995, it is seen that under the chromatographic condition, N- fluorenes methoxies
Carbonyl-l-Alanine linear relationship in the concentration range of 0.1004~0.6024mg/ml is good.
Claims (4)
1. a kind of analyzing detecting method of N- fluorenylmethyloxycarbonyls-l-Alanine, analysis detection is carried out using high performance liquid chromatography,
Characterized by the following steps：
A, it takes N- fluorenylmethyloxycarbonyls-l-Alanine sample appropriate, is dissolved with acetonitrile, be configured to every 1ml starting materials containing thymalfasin
Expect the sample solution of 0.1~0.6mg；
B, setting flow rate of mobile phase is 0.9~1.1ml/min, and Detection wavelength 220nm, column temperature is 20~30 DEG C；
C, the 10 μ l of sample solution of A is taken to inject liquid chromatograph, complete the analysis detection of N- fluorenylmethyloxycarbonyls-l-Alanine；
Wherein, chromatographic column：C18,4.6 × 250mm, 5 μm；
Mobile phase：With water-trifluoroacetic acid (1000:1) it is mobile phase A, with acetonitrile-trifluoroacetic acid (1000:1) it is Mobile phase B, ladder
Degree elution setting is as follows：
2. analyzing detecting method as described in claim 1, it is characterised in that：A concentration of 0.5mg/ml of the sample solution.
3. analyzing detecting method as described in claim 1, it is characterised in that：The flow rate of mobile phase be 1.0ml/min, column
Temperature is 25 DEG C.
4. analyzing detecting method as described in claim 1, it is characterised in that：Eluent gradient elution setting is as follows：
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| CN201611192923.7A CN108226318A (en) | 2016-12-21 | 2016-12-21 | A kind of analyzing detecting method of N- fluorenylmethyloxycarbonyls-l-Alanine |
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| CN201611192923.7A CN108226318A (en) | 2016-12-21 | 2016-12-21 | A kind of analyzing detecting method of N- fluorenylmethyloxycarbonyls-l-Alanine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111505160A (en) * | 2020-05-11 | 2020-08-07 | 成都市科隆化学品有限公司 | Fmoc-protected amino acid purity and related substance analysis method |
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| CN101709079A (en) * | 2009-12-22 | 2010-05-19 | 江苏诺泰制药技术有限公司 | Synthesis method of romurtide |
| WO2010099746A1 (en) * | 2009-03-05 | 2010-09-10 | 连云港恒邦医药科技有限公司 | Glucagon-like peptide-2 analogues, preparation method and use thereof |
| CN102229649A (en) * | 2011-05-05 | 2011-11-02 | 中国人民解放军第四军医大学 | Preparation method of body protection polypeptide (BPC 157 peptide) |
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2016
- 2016-12-21 CN CN201611192923.7A patent/CN108226318A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU1139301A (en) * | 1999-10-18 | 2001-04-30 | Solvay Pharmaceuticals B.V. | Peptides having corticotropin releasing factor (crf) antagonist activity |
| WO2007041775A1 (en) * | 2005-10-10 | 2007-04-19 | The University Of Queensland | Cysteine protease inhibitors incorporating azide groups |
| WO2010099746A1 (en) * | 2009-03-05 | 2010-09-10 | 连云港恒邦医药科技有限公司 | Glucagon-like peptide-2 analogues, preparation method and use thereof |
| CN101709079A (en) * | 2009-12-22 | 2010-05-19 | 江苏诺泰制药技术有限公司 | Synthesis method of romurtide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111505160A (en) * | 2020-05-11 | 2020-08-07 | 成都市科隆化学品有限公司 | Fmoc-protected amino acid purity and related substance analysis method |
| CN111505160B (en) * | 2020-05-11 | 2021-04-06 | 成都市科隆化学品有限公司 | Fmoc-protected amino acid purity and related substance analysis method |
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Application publication date: 20180629 |