CN106324164A - Method for determining content of betaine in Chinese wolfberry formula granules - Google Patents
Method for determining content of betaine in Chinese wolfberry formula granules Download PDFInfo
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- CN106324164A CN106324164A CN201510370464.6A CN201510370464A CN106324164A CN 106324164 A CN106324164 A CN 106324164A CN 201510370464 A CN201510370464 A CN 201510370464A CN 106324164 A CN106324164 A CN 106324164A
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Abstract
The invention provides a method for determining the content of betaine in Chinese wolfberry formula granules. The method adopts high performance liquid chromatography for determination, an optimum chromatographic condition and a reasonable test solution treatment method are determined by tests, research of methodology is carried out, the content of betaine of the Chinese wolfberry formula granules produced by different manufacturers is determined, and the reasonable limit is defined. The method has the advantages of high specificity, high precision and good reproducibility, and can effectively control the quality of the Chinese wolfberry formula granules.
Description
Technical field: biological medicine
Background technology:
Chinese medicinal granule is the class new product that Chinese medicine slice decoction carries out form improvement, and medlar dispensing granule is one therein.Fructus Lycii is the prepared slices of Chinese crude drugs that Chinese medicine decoction is conventional, and therefore medlar dispensing granule is a kind of conventional Chinese medicinal granule naturally.There is no the content assaying method of medlar dispensing granule at present, be unprofitable to clinical practice and the management of medlar dispensing granule.For ensureing safety and the effectiveness of the clinical practice of medlar dispensing granule, formulate controlled, feasible content assaying method imperative.
With glycine betaine as reference substance under Chinese Pharmacopoeia one Fructus Lycii medical material item of version in 2010, using thin layer chromatography dual-wavelength scanning to measure its content, the method operation complexity, repeatability is bad, and poor accuracy seldom uses.Also having document to report, use HPLC method to measure the beet alkali content in Fructus Lycii medical material, but use ion exchange resin method to the process of need testing solution more, operate miscellaneous, error is relatively big, and repeatability is bad.In Fructus Lycii medical material, the content assaying method of glycine betaine is little to setting up beet alkali content method for measuring reference value in medlar dispensing granule at present.
Summary of the invention:
The present invention uses HPLC method to measure the content of glycine betaine in medlar dispensing granule, first chromatographic condition is studied with system suitability, different chromatographic columns and flowing phase, the chromatographic condition finally determined and system suitability: with amino bonded silica gel as filler are investigated；With acetonitrile-water (85:15) for flowing phase；Detection wavelength is 200nm.Number of theoretical plate is calculated by glycine betaine peak and is not less than 3000.
After determining chromatographic condition and system suitability, the high spot reviews processing method of need testing solution.Physicochemical property due to glycine betaine, under above-mentioned chromatographic condition, other composition in medlar dispensing granule is bigger to its determination influences, therefore the key that remove impurity is need testing solution processing method is purified, the present invention has investigated the method for multiple purification remove impurity, finally determining the method using alkali alumina column chromatography, concrete experimental study is as follows:
Take test sample appropriate, finely ground, accurately weighed, put in tool plug conical flask, add methanol 50ml, supersound process 30min, let cool, filter, and with a small amount of methanol washing container and residue, washing liquid is incorporated in filtrate, reclaim methanol to small size, add alkali alumina 2g, mix sample, join the alkali alumina post (8g installed, with 1, 2-dichloroethanes wet method dress post) on, it is 1 component with 50ml, press table 1 sequentially eluting successively, collect each component respectively, it is evaporated, residue is dissolved in water and is transferred in 25ml measuring bottle, it is diluted with water to scale, shake up, filter, take subsequent filtrate, obtain.Measure by the chromatographic condition determined, calculate the amount containing glycine betaine in every component.The results are shown in Table 1.
The measurement result of the different eluting solvent of table 1
| Numbering | Eluting solvent | The amount (mg) of glycine betaine |
| 1 | 1,2-dichloroethanes | 0 |
| 2 | 1,2-dichloroethanes-methanol (95:5) | 0 |
| 3 | 1,2-dichloroethanes-methanol (90:10) | 0 |
| 4 | 1,2-dichloroethanes-methanol (85:15) | 0 |
| 5 | 1,2-dichloroethanes-methanol (80:20) | 0.0386 |
| 6 | 1,2-dichloroethanes-methanol (70:30) | 0.3153 |
| 7 | 1,2-dichloroethanes-methanol (60:40) | 0.4132 |
| 8 | 1,2-dichloroethanes-methanol (50:50) | 0.5163 |
| 9 | 1,2-dichloroethanes-methanol (40:60) | 0.0871 |
| 10 | 1,2-dichloroethanes-methanol (30:70) | 0.1195 |
| 11 | 1,2-dichloroethanes-methanol (20:80) | —— |
| 12 | 1,2-dichloroethanes-methanol (10:90) | —— |
| 13 | Methanol | —— |
Result shows: glycine betaine is 1, during 2-dichloroethanes-methanol (80:20), start to elute, 1, during 2-dichloroethanes-methanol (30:70), start that impurity peaks occurs, interference measurement, during 1,2-dichloroethanes-methanol (70:30) to (50:50), eluting is most, therefore eluting solvent selects 1,2-dichloroethanes-methanol (50:50)；Through verification experimental verification, with 1,2-dichloroethanes-methanol (50:50) is to extract the extraction effect of solvent and methanol quite, and easy and simple to handle, therefore extracts solvent and also select 1,2-dichloroethanes-methanol (50:50).In need testing solution processing method, the investigation content of the test of each parameter is as follows:
(1) selection of extraction time: take test sample appropriate, finely ground, weigh about 400mg, accurately weighed, totally 4 parts, put in tool plug conical flask, add 1, mixed liquor (50:50) 50ml of 2-dichloroethanes-methanol, supersound process 10min respectively, 20min, 30min, 40min, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, filtrate passes through alkali alumina post (internal diameter 1.5cm, 10g, dry column-packing), with 150ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, obtain.By determining that chromatographic condition measures, calculate the amount containing glycine betaine in the granule being often equivalent to 1g decoction pieces.Result shows: the amount of the glycine betaine that ultrasonic 30min extracts is compared with during 40min, and its content is basically identical, therefore extraction time selects 30min.The results are shown in Table 2.
The measurement result of table 2 different extraction time
| Extraction time (min) | 10 | 20 | 30 | 40 |
| Content (mg/g) | 1.8056 | 1.9108 | 2.0804 | 2.0892 |
(2) selection of solvent amount is extracted: take test sample appropriate, finely ground, weigh about 400mg, accurately weighed, totally 3 parts, put in tool plug conical flask, it is separately added into 1, mixed liquor (50:50) 25ml of 2-dichloroethanes-methanol, 50ml, 100ml, supersound process 30min, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, filtrate passes through alkali alumina post (internal diameter 1.5cm, 10g, dry column-packing), with 150ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, obtain.By determining that chromatographic condition measures, calculate the amount containing glycine betaine in the granule being often equivalent to 1g decoction pieces.Result shows: the amount of the glycine betaine that 50ml mixed liquor extracts is compared with during 100ml, and its content is basically identical, therefore the amount extracting solvent selects 50ml.The results are shown in Table 3.
The different measurement result extracting solvent consumption of table 3
| Solvent consumption (ml) | 25 | 50 | 100 |
| Content (mg/g) | 1.9344 | 2.0824 | 2.0792 |
(3) selection of alkali alumina consumption: take test sample appropriate, finely ground, weigh about 400mg, accurately weighed, totally 4 parts, put in tool plug conical flask, add 1, mixed liquor (50:50) 50ml of 2-dichloroethanes-methanol, supersound process 30min, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, filtrate is respectively by alkali alumina post (internal diameter 1.5cm, 4g, 6g, 8g, 10g, dry column-packing), with 150ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, obtain.By determining that chromatographic condition measures, calculate the amount containing glycine betaine in the granule being often equivalent to 1g decoction pieces.Result shows: the amount of glycine betaine is affected little by the consumption of alkali alumina, but when consumption is 4g, its impurity is the most compared with when 6g, 8g and 10g, therefore alkali alumina consumption selects 6 ~ 10g.The results are shown in Table 4.
The measurement result of the different alkali alumina consumption of table 4
| Alkali alumina consumption (g) | 4 | 6 | 8 | 10 |
| Content (mg/g) | 2.1404 | 2.1224 | 2.1088 | 2.0932 |
(4) selection of elution amount: take test sample appropriate, finely ground, weigh about 400mg, accurately weighed, totally 4 parts, put in tool plug conical flask, add 1, mixed liquor (50:50) 50ml of 2-dichloroethanes-methanol, supersound process 30min, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, filtrate passes through alkali alumina post (internal diameter 1.5cm, 8g, dry column-packing), respectively with 50ml, 100ml, 150ml, 200ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, obtain.By determining that chromatographic condition measures, calculate the amount containing glycine betaine in the granule being often equivalent to 1g decoction pieces.Result shows: when elution amount is 100ml the amount of glycine betaine with 150ml and 200ml time compare, its content is basically identical, thus select elution amount be 100ml, the results are shown in Table 5.
The measurement result of the different elution amount of table 5
| Elution amount (ml) | 50 | 100 | 150 | 200 |
| Content (mg/g) | 1.9692 | 2.0980 | 2.1024 | 2.0912 |
Finally determine need testing solution processing method: take this product under content uniformity item, finely ground, take appropriate (being equivalent to 1.0g decoction pieces), accurately weighed, put in tool plug conical flask, add 1, mixed liquor (50:50) 50ml of 2-dichloroethanes-methanol, supersound process 30 minutes, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, filtrate passes through alkali alumina post (internal diameter 1.5cm, 8g, dry column-packing), with 100ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, obtain.
Methodological study:
(1) linear relationship test: precision weighs glycine betaine reference substance 10.64mg, put in 25ml measuring bottle, add flowing phased soln and be diluted to scale, shake up (concentrated solution), respectively precision measure 0,1.0,2.0,3.0,4.0,5.0ml put in 10ml measuring bottle, with the phase dilution that flows to scale, shake up, take above-mentioned solution 20 μ l respectively and inject chromatograph of liquid, measure by above-mentioned chromatographic condition.The results are shown in Table 6.
Table 6 linear relationship measurement result
(2) precision test: under line taking sexual relationship item, concentration is the solution of 127.68 μ g/ml, and continuous acupuncture 6 times, peak area value is shown in Table 7.Result meets the requirements.
Table 7 Precision test result
| Measure number of times | 1 | 2 | 3 | 4 | 5 | 6 | Meansigma methods | RSD(%) |
| Peak area value | 144376 | 142488 | 142923 | 146093 | 144366 | 145497 | 144291 | 0.97 |
(3) stability test: placed 24 hours by need testing solution, measured in 0,2,4,6,8,24 hours, and before placing, peak area value is 138140, and after placement, peak area value is 138409, illustrated that measuring solution measures stable within 24 hours.The results are shown in Table 8.
Table 8 stability test result
| Minute (h) | 0 | 2 | 4 | 6 | 8 | 24 |
| Peak area value | 138140 | 137050 | 138268 | 139057 | 137572 | 138409 |
(4) repeatability test: take test sample appropriate, finely ground, weigh about 300mg, accurately weighed, 6 parts, it is measured by the method for assay, calculates the amount containing glycine betaine in medlar dispensing granule.The results are shown in Table 9.Result meets the requirements.
Table 9 reproducible test results
| Measure number of times | 1 | 2 | 3 | 4 | 5 | 6 | Meansigma methods | RSD(%) |
| Content (%) | 0.4022 | 0.3988 | 0.4093 | 0.4002 | 0.4076 | 0.3998 | 0.4030 | 1.09 |
(5) recovery test: take test sample appropriate, finely ground, weigh about 150mg, accurately weighed, 6 parts, put in tool plug conical flask, precision adds reference substance solution (concentration is 0.1222mg/ml) respectively, add 1, the mixed liquor (50:50) of 2-dichloroethanes-methanol is to 50ml, supersound process 30 minutes, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, filtrate passes through alkali alumina post (internal diameter 1.5cm, 8g, dry column-packing), with 100ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, as measuring response rate solution.Taking above-mentioned solution 20 μ l respectively and inject chromatograph of liquid, measure by above-mentioned chromatographic condition, the solution of 127.68 μ g/ml under linear relationship item is comparison, calculates the response rate.The results are shown in Table 10.
Table 10 determination of recovery rates result
Meansigma methods is 99.87%(n=6), RSD=1.77%, meet the requirements.
The assay of sample:
By above-mentioned chromatographic condition, the medlar dispensing granule of each manufacturer production is measured, calculates the amount being often equivalent to the granule of 1g decoction pieces containing glycine betaine, and with pharmacopeia decoction pieces comparision contents, calculate the rate of transform.The results are shown in Table 11.
Table 11 assay result
States Pharmacopoeia specifications every g decoction pieces (C Han glycine betaine5H11NO2) 3.0mg must not be less than, the content of the average glycine betaine of granule that above-mentioned 9 batch samples are often equivalent to 1 gram of decoction pieces is 1.5220mg.Define the granule (C Han glycine betaine being often equivalent to 1 gram of decoction pieces5H11NO2) must not to compare the rate of transform with pharmacopeia medical material content limit be 40% less than 1.20mg().
Embodiment:
(1) preparation of reference substance solution: precision weighs glycine betaine reference substance 10.64mg, puts in 25ml measuring bottle, adds flowing phased soln be diluted to scale, shakes up, obtain concentrated solution, and precision measures 3.0ml, puts in 10ml measuring bottle, adds flowing phase dilution to scale, shakes up, to obtain final product.
(2) preparation of need testing solution: take this product, finely ground, take appropriate (being equivalent to the granule of 1.0g decoction pieces), accurately weighed, put in tool plug conical flask, add 1, mixed liquor (50:50) 50ml of 2-dichloroethanes-methanol, supersound process 30 minutes, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, filtrate passes through alkali alumina post (internal diameter 1.5cm, 8g, dry column-packing), with 100ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, obtain.
(3) chromatographic condition:
Chromatographic column: Kromasil NH2 (4.6 × 250mm, 5 μ)
Flowing phase: acetonitrile-water (85:15)
Flow velocity: 1.0ml/min
Detection wavelength: 200nm
Column temperature: 30 DEG C
Sample introduction: 20ul
(4) measure: calculating is often equivalent to the granule amount containing glycine betaine of 1g decoction pieces, and calculates the rate of transform.The results are shown in Table 12.
Table 12
Measurement result
Claims (6)
1. beet alkali content method for measuring in a medlar dispensing granule, it is characterised in that: this method specificity is strong, and precision is high, favorable reproducibility.
Beet alkali content method for measuring in a kind of medlar dispensing granule the most according to claim 1, it is characterised in that: use high effective liquid chromatography for measuring, its chromatographic condition and system suitability: with amino bonded silica gel as filler；With acetonitrile-water (85:15) for flowing phase；Detection wavelength is 200nm；Number of theoretical plate is calculated by glycine betaine peak should be not less than 3000.
Beet alkali content method for measuring in a kind of medlar dispensing granule the most according to claim 1, it is characterised in that: take glycine betaine reference substance appropriate, accurately weighed, add flowing and make every 1ml solution containing 10～50 μ g mutually, obtain reference substance solution.
Beet alkali content method for measuring in a kind of medlar dispensing granule the most according to claim 1, it is characterized in that: medlar dispensing granule is finely ground, take appropriate (being equivalent to 1.0～5.0g decoction pieces), accurately weighed, put in tool plug conical flask, add 1, mixed liquor (50:50) 50ml of 2-dichloroethanes-methanol, supersound process 30 minutes, let cool, filter, and with a small amount of mixed liquor washing container and medicinal residues, washing liquid is incorporated in filtrate, by alkali alumina post (internal diameter 1.0～2.0cm, 6～10g, dry column-packing), with 100ml mixed liquor eluting, collect effluent and eluent, it is evaporated, residue adds flowing phased soln and is transferred in 10ml measuring bottle, add flowing phase dilution to scale, shake up, filter, take subsequent filtrate, obtain need testing solution.
Beet alkali content method for measuring in a kind of medlar dispensing granule the most according to claim 1, it is characterised in that: its linear relationship result of the test shows that the concentration of glycine betaine and its peak area are good linear relationship；Precision test RSD is 0.97%；Repeatability test RSD is 1.09%；Recovery test average recovery rate is 99.87%, and RSD is 1.77%；It is stable that need testing solution measured in 24 hours.
Beet alkali content method for measuring in a kind of medlar dispensing granule the most according to claim 1, it is characterized in that: by the mensuration of beet alkali content in 9 batch medlar dispensing granule samples that different manufacturers is produced, the granule average content being often equivalent to 1g decoction pieces is 1.5220mg, defines and is often equivalent to the granule (C Han glycine betaine in 1 gram of decoction pieces5H11NO2) 1.20mg must not be less than.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108645949A (en) * | 2018-07-06 | 2018-10-12 | 河北华北制药华恒药业有限公司 | A kind of method of beet alkali content in detection zymotic fluid |
| CN112229939A (en) * | 2020-11-27 | 2021-01-15 | 中国科学院兰州化学物理研究所 | Method for analyzing and detecting octadecyl dimethyl betaine in crude oil by liquid chromatography |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103630618A (en) * | 2013-04-02 | 2014-03-12 | 汕头市创美中药饮片有限公司 | Method for determining betaine content in wolfberry |
| CN103910739A (en) * | 2014-03-26 | 2014-07-09 | 山东师范大学 | Bisbenzylisoquinoline glycine betaines as well as preparation method and application thereof in preparing anti-tumor medicines |
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- 2015-06-30 CN CN201510370464.6A patent/CN106324164A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103630618A (en) * | 2013-04-02 | 2014-03-12 | 汕头市创美中药饮片有限公司 | Method for determining betaine content in wolfberry |
| CN103910739A (en) * | 2014-03-26 | 2014-07-09 | 山东师范大学 | Bisbenzylisoquinoline glycine betaines as well as preparation method and application thereof in preparing anti-tumor medicines |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108645949A (en) * | 2018-07-06 | 2018-10-12 | 河北华北制药华恒药业有限公司 | A kind of method of beet alkali content in detection zymotic fluid |
| CN112229939A (en) * | 2020-11-27 | 2021-01-15 | 中国科学院兰州化学物理研究所 | Method for analyzing and detecting octadecyl dimethyl betaine in crude oil by liquid chromatography |
| CN112229939B (en) * | 2020-11-27 | 2022-02-01 | 中国科学院兰州化学物理研究所 | Method for analyzing and detecting octadecyl dimethyl betaine in crude oil by liquid chromatography |
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Application publication date: 20170111 |