CN104865324A - Method for detecting content of 5,6-dimethyl benzimidazole - Google Patents
Method for detecting content of 5,6-dimethyl benzimidazole Download PDFInfo
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- CN104865324A CN104865324A CN201510295550.5A CN201510295550A CN104865324A CN 104865324 A CN104865324 A CN 104865324A CN 201510295550 A CN201510295550 A CN 201510295550A CN 104865324 A CN104865324 A CN 104865324A
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Abstract
The invention relates to a method for detecting the content of 5,6-dimethyl benzimidazole. The method is characterized in that a high-performance liquid chromatography is adopted for detection. As the high-performance liquid chromatography is adopted for detecting the content of 5,6-dimethyl benzimidazole, no strongly-corrosive and strongly-irritant solvent used in a titration method exists, and the safety and reliability are achieved; the content of 5,6-dimethyl benzimidazole in a sample is quantified according to an external standard method, so that the method provided by the invention is accurate, simple, and high in repeatability and sensitivity; a relatively good linear relationship is formed when the 5,6-dimethyl benzimidazole is between 0.1-2.0 mg/ml, and the linear correlation coefficient is 0.9996.
Description
Technical field
This method of the present invention belongs to chemical analysis field, particularly relates to a kind of method of Syrups by HPLC 5,6-dimethylbenzimidazole content.
Background technology
5,6-dimethylbenzimidazole is as Cobastab
12the exogenous precursors thing of synthesis, joins in fermentation medium, is directly attached to VB by the biosynthetic process of microorganism
12go in molecular structure, and the structure of himself not much changes, but VB
12output but improve a lot because adding 5,6-dimethylbenzimidazole.Therefore the height of 5,6-dimethylbenzimidazole content directly affects VB
12output.
5,6-dimethylbenzimidazole structure is as follows:
In prior art, 5, what the assay of 6-dimethylbenzimidazole adopted is perchloric acid titration method, the reagent such as glacial acetic acid, perchloric acid is used in testing process, there is severe corrosive, strong and stimulating, human body is had and burns, therefore develop one can safety, Accurate Determining 5,6 dimethylbenzimidazole the method for content be have problem to be solved always.
Summary of the invention
The object of the invention is to the defect overcoming above-mentioned prior art, provide a kind of safe and reliable, and method is simple and easy in operation, result is accurate, 5,6-reproducible dimethylbenzimidazole detection method of content.
Technical scheme taked for achieving the above object is:
A kind of 5,6 dimethylbenzimidazole detection method of content, is characterized in that adopting high performance liquid chromatography to detect.
The detailed process that described high performance liquid chromatography carries out detecting is: adopt mobile phase to be solvent preparation standard product solution and need testing solution, after the sample size sample introduction of 10-20ul, calculates content according to external standard method;
Wherein high-efficient liquid phase chromatogram condition is:
Chromatographic column: C8 or C18 chromatographic column,
Detecting device: DAD detecting device,
Mobile phase: methyl alcohol: disodium phosphate soln (10g/L) (pH2.5-3.5)=(25.0:75.0), the ratio of methyl alcohol is 20.0-30.0%;
Flow velocity: 0.60-1.20ml/min;
Determined wavelength: 265-276.
Described standard solution and need testing solution concentration are 0.1mg/ml-2.0mg/ml.
The present invention adopts high performance liquid chromatography to carry out the content detection of 5,6-dimethylbenzimidazole, without the severe corrosive that titrimetry uses, and strong and stimulating solvent, safe and reliable.And by external standard method, 5,6-dimethylbenzimidazole content in sample are carried out quantitatively, method is accurate, simply, reproducible, highly sensitive, when 5, when 6 dimethylbenzimidazoles are between 0.1mg/ml-2.0mg/ml, all have good linear relationship, its linearly dependent coefficient is 0.9996.
Accompanying drawing explanation
Fig. 1 is that the HPLC of 5,6-dimethylbenzimidazole reference substance detects collection of illustrative plates;
Fig. 2 is that the HPLC of 5,6-dimethylbenzimidazole detects collection of illustrative plates;
Fig. 3 is linear relationship chart and the regression equation of 5,6-dimethylbenzimidazole.
Embodiment
In order to the technical scheme making those skilled in the art better understand invention, below in conjunction with specific implementation method, technical scheme of the present invention is described in further detail.
Embodiment 1
5,6-dimethylbenzimidazole analyzes the preparation of sample:
The preparation of standard solution: take 5,6-dimethylbenzimidazole standard items 50mg, accurately weighed, put in 100ml volumetric flask, add mobile phase and dissolve and be settled to scale, shake up.Get the organic filter filtration of solution through 0.45um with 1ml syringe, obtain standard solution.
The preparation of need testing solution: take 5,6-dimethylbenzimidazole sample 50mg, accurately weighed, be placed in 100ml volumetric flask, add mobile phase appropriate, be settled to scale after dissolving, shake up.Get the organic filter filtration of solution through 0.45um with 1ml syringe, obtain need testing solution.
Standard solution and each obtained 5 parts of need testing solution.
Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, Waters C18 chromatographic column (250*4.6,5um); Mobile phase: methyl alcohol: disodium phosphate soln (10g/L) (pH3.0)=(25.0:75.0); Flow velocity: 0.80ml/min; Determined wavelength: 270nm; Sample size 10ul, detecting device: UV-detector; Column oven 35 DEG C.
By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, sample introduction gained peak area is 1500428, RSD to standard solution 5 samples is respectively 0.15%; 5 sample gained peak area mean values of need testing solution are 1454257, RSD is 0.09%.
According to formula:
The content of 5,6-dimethylbenzimidazole: a=A
samplem
mark/ A
markm
sample* 100%.
Adopt external standard method standard items and test sample on average to be substituted into the content of test sample 5,6-dimethylbenzimidazole is 99.55%.
The methodological study of detection method described in the embodiment of the present invention 1:
(1) Precision Experiment
Take same 5,6-dimethylbenzimidazole samples, carry out 6 revision tests, peak area table 1:
The area of table 15,6-dimethylbenzimidazole revision test
| Number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 1455237 | 1453970 | 1454068 | 1454853 | 1453371 | 1452912 |
Peak area RSD is 0.06%, and result shows, it is high that this method detects 5,6-dimethylbenzimidazole content precision, favorable reproducibility.
(2) linear relationship is investigated
According to the described method of invention, be the storing solution of 10mg/ml with the volumetric flask compound concentration of 50ml, add the standard solution that mobile phase is diluted to 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml respectively, according to this method, carry out data regression to sample concentration (X) and peak area (Y), equation of linear regression is Y=2.84143 × 10
6+ 24645, as shown in Figure 3, R=0.9996, result shows that 5,6-dimethylbenzimidazole is in 0.1mg/ml-2.0mg/ml concentration range to linear relationship, and peak area and concentration are good linear relationship.
(3) application of sample recovery experiment
5, the 6-dimethylbenzimidazoles accurately taking known content are appropriate, then precision adds a certain amount of 5 respectively, 6-dimethylbenzimidazole sample 80%, 100%, 120%, make 5,6-dimethylbenzimidazole concentration in need testing solution in the high, medium and low region of 5,6-dimethylbenzimidazole typical curve each 3 parts, according to the operation under item of the present invention, measure, calculate the recovery, result average recovery rate is 99.58% in accordance with the law, RSD is 0.58%, shows that the method average recovery is good.
To sum up show detection method of content good stability of the present invention, precision is high, is applicable to the assay of 5,6-dimethylbenzimidazole.
Embodiment 2
Sample and standard items preparation are with embodiment 1.
Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, Waters C8 chromatographic column (150*4.6,3.5um); Mobile phase: methyl alcohol: disodium phosphate soln (10g/L) (pH3.0)=(20.0:80.0); Flow velocity: 0.60ml/min; Determined wavelength: 260nm; Sample size 10ul, detecting device: UV-detector; Column oven 30 DEG C.
By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, sample introduction gained peak area is 1558746, RSD to standard solution 5 samples is respectively 0.25%; 5 sample gained peak area mean values of need testing solution are 1498754, RSD is 0.31%.
According to formula:
The content of 5,6-dimethylbenzimidazole: a=A
samplem
mark/ A
markm
sample* 100%.
Adopt external standard method standard items and test sample on average to be substituted into the content of test sample 5,6-dimethylbenzimidazole is 99.52%.
Embodiment 3
Sample and standard items preparation are with embodiment 1.
Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, Waters C8 chromatographic column (150*4.6,3.5um); Mobile phase: methyl alcohol: disodium phosphate soln (10g/L) (pH3.5)=(30.0:70.0); Flow velocity: 1.2ml/min; Determined wavelength: 276nm; Sample size 10ul, detecting device: UV-detector; Column oven 40 DEG C.
By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, standard solution 5 samples respectively sample introduction gained peak area 1432451 are that RSD is 0.30%; 5 sample gained peak area mean values of need testing solution are 1455312, RSD is 0.12%.
According to formula:
The content of 5,6-dimethylbenzimidazole: a=A
samplem
mark/ A
markm
sample* 100%.
Adopt external standard method standard items and test sample on average to be substituted into the content of test sample 5,6-dimethylbenzimidazole is 99.68%.
Embodiment 4
Sample and standard items preparation are with embodiment 1.
Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, Waters C8 chromatographic column (150*4.6,3.5um); Mobile phase: methyl alcohol: disodium phosphate soln (10g/L) (pH2.5)=(25.0:75.0); Flow velocity: 1.0ml/min; Determined wavelength: 270nm; Sample size 20ul, detecting device: UV-detector; Column oven 35 DEG C.
By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, standard solution 5 samples respectively sample introduction gained peak area 1504624 are that RSD is 0.22%; 5 sample gained peak area mean values of need testing solution are 1496221, RSD is 0.28%.
According to formula:
The content of 5,6-dimethylbenzimidazole: a=A
samplem
mark/ A
markm
sample* 100%.
Adopt external standard method standard items and test sample on average to be substituted into the content of test sample 5,6-dimethylbenzimidazole is 99.70%.
Above-mentioned detailed description of carrying out with reference to the detection method of content of embodiment to this one 5,6-dimethylbenzimidazole, is illustrative instead of determinate, can lists several embodiments according to institute's limited range.Therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.
Claims (3)
1. one kind 5,6 dimethylbenzimidazole detection method of content, is characterized in that adopting high performance liquid chromatography to detect.
2. according to according to claim 15,6 dimethylbenzimidazole detection method of content, it is characterized in that the detailed process that described high performance liquid chromatography carries out detecting is: adopt mobile phase to be solvent preparation standard product solution and need testing solution, after the sample size sample introduction of 10-20ul, calculate content according to external standard method;
Wherein high-efficient liquid phase chromatogram condition is:
Chromatographic column: C8 or C18 chromatographic column,
Detecting device: DAD detecting device,
Mobile phase: methyl alcohol: disodium phosphate soln (10g/L) (pH2.5-3.5)=(25.0:75.0), the ratio of methyl alcohol is 20.0-30.0%;
Flow velocity: 0.60-1.20ml/min;
Determined wavelength: 265-276.
3., according to according to claim 15,6 dimethylbenzimidazole detection method of content, it is characterized in that described standard solution and need testing solution concentration are 0.1mg/ml-2.0mg/ml.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628844A (en) * | 2015-12-31 | 2016-06-01 | 中国农业科学院兰州畜牧与兽药研究所 | Method determining residue benzimidazole medicine in animal tissue |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB766749A (en) * | 1954-01-11 | 1957-01-23 | Aschaffenburger Zellstoffwerke | Benzimidazole cobalamines and process for their preparation and separation |
| CN1054799A (en) * | 1990-01-31 | 1991-09-25 | 罗纳·布朗克医药公司 | The relevant polypeptide of biosynthesizing cobalami and/or cobamide, dna sequence dna, its preparation method and the application of these polypeptide of encoding |
-
2015
- 2015-06-02 CN CN201510295550.5A patent/CN104865324A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB766749A (en) * | 1954-01-11 | 1957-01-23 | Aschaffenburger Zellstoffwerke | Benzimidazole cobalamines and process for their preparation and separation |
| CN1054799A (en) * | 1990-01-31 | 1991-09-25 | 罗纳·布朗克医药公司 | The relevant polypeptide of biosynthesizing cobalami and/or cobamide, dna sequence dna, its preparation method and the application of these polypeptide of encoding |
Non-Patent Citations (4)
| Title |
|---|
| C. PAKIN等: "α-Ribazole, a fluorescent marker for the liquid chromatographic determination of vitamin B12 in foodstuffs", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| MARIJANA M. AČANSKI等: "CORRELATION BETWEEN RETENTION CONSTANTS OBTAINED IN REVERSED-PHASE LIQUID CHROMATOGRAPHY AND PARTITION COEFFICIENTS OF SOME BENZIMIDAZOLE DERIVATIVES", 《ACTA PERIODICA TECHNOLOGICA》 * |
| S.M. PETROVIC等: "Reversed Phase Liquid Chromatography of Some Benzimidazole Derivatives", 《CHROMATOGRAPHIA》 * |
| 殷莉莉等: "高效液相色谱法测定腺苷钴胺片中的羟钴胺素含量", 《中南药学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628844A (en) * | 2015-12-31 | 2016-06-01 | 中国农业科学院兰州畜牧与兽药研究所 | Method determining residue benzimidazole medicine in animal tissue |
| CN105628844B (en) * | 2015-12-31 | 2017-04-19 | 中国农业科学院兰州畜牧与兽药研究所 | Method determining residue benzimidazole medicine in animal tissue |
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Application publication date: 20150826 |