CN108107138A - The method that HPLC-CAD measures glucose in Dextrose and Sodium Chloride Inj. - Google Patents
The method that HPLC-CAD measures glucose in Dextrose and Sodium Chloride Inj. Download PDFInfo
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- CN108107138A CN108107138A CN201711365572.XA CN201711365572A CN108107138A CN 108107138 A CN108107138 A CN 108107138A CN 201711365572 A CN201711365572 A CN 201711365572A CN 108107138 A CN108107138 A CN 108107138A
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- sodium chloride
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- G—PHYSICS
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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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Abstract
The invention discloses a kind of methods that HPLC CAD measure glucose in Dextrose and Sodium Chloride Inj..Belong to field of chemical detection, it is desirable to provide a kind of to measure detection method accurate, easy to operate, this method comprises the following steps successively：1) preparation of test solution：Dextrose and Sodium Chloride Inj. 1ml methanol constant volumes is taken to shake up to 1000ml, filter to get；2) preparation of reference substance solution：Precision weighs glucose control product 15g, is placed in 250mL measuring bottles, and methanol is added to dissolve and is diluted to scale, shake up to get；3) analyzed using high performance liquid chromatography.The alternative traditional detection method of the present invention is used for the content assaying method of glucose in Dextrose and Sodium Chloride Inj..
Description
Technical field
The present invention relates to field of chemical detection, especially a kind of HPLC-CAD measures grape in Dextrose and Sodium Chloride Inj.
The method of sugar.
Background technology
2010 editions methods for describing glucose assays in glucose injection of Chinese Pharmacopoeia, 3.6.1 section records use
Optical Rotation measures the content of the glucose in parenteral solution, and this method controls requirement high environment temperature, during measure by environment because
Element is affected, and 3.6.2 sections record measures the content of the sodium chloride in parenteral solution using silver nitrate titration method, and this method is to behaviour
Make personnel specialty skill set requirements height, there are certain deviations for different tester's test results.It is not recorded in existing national standard
The assay method of glucose in HPLC-CAD methods measurement Dextrose and Sodium Chloride Inj., even if the measurement side using traditional HPLC
Method, linear rate is in entire measurement range and disobeys stringent linear distribution standard.
EFI fog detector (CAD) is detector general at present, and when being combined with HPLC, HPLC eluents are through in atomizer
The effect of nitrogen and be atomized, wherein larger drop flows out under the action of impinger through sewer pipe, smaller solute (analysis
Object) drop is dried at room temperature for, form particles of solute.Meanwhile it shunts to be formed second strand of nitrogen for the nitrogen of carrier gas and flows through
It crosses corona-type device (platinum wire electrode containing high pressure) and forms positively charged nitrogen particle, through touching when reversely meeting with particles of solute
Hitting makes particles of solute become positively charged.Its structure independent of analyte, it is not required that ionize analyte, as long as the substance category
It can be detected in half volatile or non-volatile compounds, and sensitivity can reach pg ranks, reappearance is good, is purple
The strong supplement of the common detector such as (UV), mass spectrum (MS), differential refraction (RI) and evaporative light-scattering (ELSD) outside.
The content of the invention
Against the above deficiency, the present invention is intended to provide a kind of measure HPLC-CAD measure glucose accurate, easy to operate
The method of glucose in sodium chloride injection.
In order to solve the above technical problem, the present invention provides technical solution be such：A kind of HPLC-CAD measures Portugal
The method of glucose, comprises the following steps successively in grape sugar sodium chloride injection：
1) preparation of test solution：Dextrose and Sodium Chloride Inj. 1ml methanol constant volumes is taken to be shaken up, mistake to 1000ml
Filter to get；
2) preparation of reference substance solution：Precision weighs glucose control product 15g, is placed in 250mL measuring bottles, methanol is added to dissolve
And be diluted to scale, shake up to get；
3) analyzed using high performance liquid chromatography, parameter setting is as follows：
Chromatographic column：Thermo Mixed-Mode Hilic, 2.1 × 150mm, 1.3 μm；
Flow velocity：0.3mL/min；
Column temperature：40℃；
Sample size：10μL；
Mobile phase：50mmol/L ammonium acetates-acetonitrile.
Further, the model of the high performance liquid chromatograph：Thermo-U3000 high performance liquid chromatographs, it is described
Thermo-U3000 high performance liquid chromatographs attach Corona Ultra EFI fog detectors.
Further, the volume ratio of 50mmol/L ammonium acetates-acetonitrile is 15~80 in the mobile phase：20~85.
Further, the mobile phase is added in using the method for gradient elution, wherein specific parameter is as follows：
It is 10 minutes to elute total time；
0-2 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 15 in mobile phase：85；
4-5 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 80 in mobile phase：20；
6-10 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 15 in mobile phase：85.
Further, the preparation of the test solution needed 0.45 μm of filter membrane.
Beneficial effects of the present invention are as follows：
The concentration of glucose in Dextrose and Sodium Chloride Inj. can be measured using the above program, avoid national standard test
Personnel operate and such environmental effects, while HPLC combination EFI fog detectors (CAD), it is ensured that in entire concentration range
Meet the rule of linear distribution, avoid the inaccuracy that traditional HPLC test methods under extremely low concentration are brought.
The operation principle of Corona Ultra EFI fog detector (CAD) technology is that analyte is changed into particles of solute,
The size of particle increases with the content of analyte；Particles of solute bumps against with positively charged nitrogen particle, and charge is therewith
It is transferred on particle (particles of solute is bigger, charged more)；Their electric charge transfer to collector, is passed through Gao Ling by particles of solute
The electrostatic detection of sensitivity measures the carried charge of particles of solute, and resulting signal code is directly proportional to the content of solute；Inspection
Survey result is related with analyte particles, and signal code is directly proportional to the quality of analyte in sample.
Above-mentioned test method is eluted by the way of gradient elution, can realize ingredient to be measured in a wider scope
Efficiently separate, meanwhile, accurate measurement during extremely low concentration can be realized using Corona Ultra EFI fog detectors, is overcome
The unstability that the manual operation of conventional test methodologies is brought.
Specific implementation method
Claims are described further with reference to specific embodiment, but do not form any limit to the present invention
System, it is any to still fall within invention which is intended to be protected making limited number of time to the present invention and change obtained technical solution.
Embodiment 1
1 instrument and reagent
Thermo-U3000 high performance liquid chromatographs (power ＆ light company of the U.S., subsidiary Corona Ultra electron sprays detection
Device), chromatographic column is Thermo Mixed-Mode Hilic (2.1 × 150mm, 1.3 μm).
Ammonium acetate (analyzes pure, Shanghai Aladdin biochemical technology limited company)；
Glucose control product (lot number：E1407051, aladdin, content >=99.5%)；
250ml Dextrose and Sodium Chloride Inj. (lot numbers：E131102K2, Hunan Cologne Pharmaceutical Co., Ltd)；250ml Portugals
Grape sugar sodium chloride injection (lot number：131228804, Shuanghe Pharmaceutical Ind Co., Ltd., Anhui)；250ml glucose sodium chlorides are noted
Penetrate liquid (lot number：H13121609, Guangxi Yu Yuan pharmaceutcal corporation, Ltds)；It is provided by hospital；
Acetonitrile (chromatographically pure, Sigma-aldrich companies of the U.S.)；
Extraction methanol is ultrapure methanol.
2. method
The preparation of 2.1 test solutions
Dextrose and Sodium Chloride Inj. 1ml methanol constant volumes is taken to shake up to 1000ml, are filtered, filtrate crosses 0.45 μm of filter membrane,
Obtain test solution.
The preparation of 2.2 reference substance solutions
Reference《Chinese Pharmacopoeia》It is prepared under middle Dextrose and Sodium Chloride Inj. specification (7) item of version two in 2015, takes grape
Sugar about 15g, it is accurately weighed, be placed in 250mL measuring bottles, methanol added to dissolve and is diluted to scale, shake up to get.
2.3 linear preparations
Precision pipettes reference substance stock solution 1ml and is placed in 10ml measuring bottles, adds ultrapure methanol dilution and is settled to scale, shakes
It is even, then precision pipettes each 1ml and is respectively placed in 100ml, 50ml, 25ml, 10ml, 5ml measuring bottle, adds ultrapure methanol dilution and constant volume
To scale, shake up to get.
Linear solvent shown in table 1 is made：
Table 1
2.4 chromatographic condition
Flow velocity：0.3mL/min, 40 DEG C of column temperature, 10 μ L of sample size, mobile phase are A (50mmol/L ammonium acetates)：B (acetonitrile).
The mobile phase is added in using the method for gradient elution, wherein specific parameter is as follows：
It is 10 minutes to elute total time；
0-2 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 15 in mobile phase：85；
4-5 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 80 in mobile phase：20；
6-10 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 15 in mobile phase：85.
3. result
3.1 specificities are tested
Appropriate ultrapure methanol is taken, by machine testing on chromatographic condition under " 2.4 " item, shows ClO in ultrapure methanol-To detecting nothing
Interference.
3.2 results and linear relationship
By chromatographic condition under " 2.4 " item, glucose is from impurity ClO in the ultrapure methanol of solvent-Deng interference.
Precision measures 10 μ l of test solution and reference substance solution, injects liquid chromatograph, peak area is recorded, with reference substance
Concentration is abscissa, using peak area as ordinate, draws standard curve, carries out linear regression.
Glucose is good with peak area linear relationship in the range of 0.5~1.0mg/ml, regression equation Y=125.8386X+
(1.2101 r=0.9993)；Chlorine, sodium are good with peak area linear relationship in the range of 0.09~1.80mg/ml, regression equation
Respectively Y=33.6836X+0.6429 (r=0.9999), Y=17.3087X+0.0395 (r=0.9999).
3.3 precision test
Accurate essence takes same reference substance solution, and continuous sample introduction 6 times records peak area, calculates the RSD of glucose, be
0.25%.The result shows that instrument precision is good.
3.4 repetitive test
6 parts of sample is taken, is prepared by " 2.1 " test sample method, measures peak area.Glucose in 6 parts of samples is calculated respectively
Average content is 12.51g/250ml, RSD 1.50%.The result shows that this method repeatability is good.
3.5 stability test
Take sample, 0,4,8,12,16,20, interior sample introduction 7 times for 24 hours, peak area is measured, when calculating different in sample respectively
Between the RSD of glucose content that puts be 0.93%, 0.85%, 1.04%.The result shows that test solution is in interior stabilization for 24 hours.
3.6 recovery test
6 parts of the sample of known content is taken, every part of about 1ml is accurately weighed.It is accurate respectively to add in reference substance storage under " 2.2 " item
Standby liquid 1ml, is prepared by " 2.1 " test sample method, is measured.It the results are shown in Table 2, glucose is flat in Dextrose and Sodium Chloride Inj.
The equal rate of recovery is respectively 100.4%, 99.8%, 100.1%, RSD be respectively 1.11%, 1.02%, 1.15%.
Following table is by taking the glucose rate of recovery as an example.
Table 2
4th, discuss
4.1 do not have UV absorption functional group due to glucose and sodium chloride, it is impossible to pass through conventional high performance liquid chromatography-purple
Outer detection method (HPLC-UV) is carried out at the same time detection.A kind of accurate, quick, easy high performance liquid chromatography-EFI is established herein
Fog detector method measures the content of glucose in Dextrose and Sodium Chloride Inj..It can be applied to its quality control and stability ground
Study carefully, also there is reference value to the measure of its similar compound.The common Dextrose and Sodium Chloride Inj. content of hospital can be used as
A kind of effective means of detection.
4.2, due to the use of 3 μm of 2.1 × 150mm chromatographic columns of Thermo Mixed-Mode Hilic-1, have mixed
Syntype stationary phase combines two kinds of reversed phase liquid chromatographies (RP) and methanolic hydrogen interaction (HILIC) attribute of parent, makes to measure chlorine simultaneously
It is possibly realized with two kinds of zwitterions of sodium.
4.3 EFI fog detectors (CAD) are a kind of novel universal detectors developed in recent years, in sensitivity, again
Existing property and linear aspect are respectively provided with excellent performance, and easy to operate.It is based on unique new testing principle, its detection technique
Independent of the molecular structure of compound, any non-volatile or half volatile compound can be applied to so that without showing difference
In the case of refraction detector and ion chromatography, the detection to glucose can be completed.
4.4《Chinese Pharmacopoeia》The content assaying method of two uses of version in 2015 is cumbersome to be taken, and is surveyed respectively with Optical Rotation
Determine glucose content, silver nitrate titration method measures chlorinity in sodium chloride.The present invention uses HPLC-CAD methods instead and is measured, sample
Machine is directly dissolved with ultrapure methanol, the pre-treatment time is greatly saved, while detects three component contents, ensure that result
Accuracy and durability.And the good separating effect of glucose, chlorine and sodium, the method strong antijamming capability.
These are only specific embodiments of the present invention, it is all made in the range of the spirit and principles in the present invention any repair
Change, equivalent substitution and improvement etc., should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of method that HPLC-CAD measures glucose in Dextrose and Sodium Chloride Inj., which is characterized in that under including successively
State step：
1) preparation of test solution：Dextrose and Sodium Chloride Inj. 1ml methanol constant volumes is taken to shake up, filter, i.e., to 1000ml
；
2) preparation of reference substance solution：Precision weighs glucose control product 15g, is placed in 250mL measuring bottles, add methanol dissolve and it is dilute
Release to scale, shake up to get；
3) analyzed using high performance liquid chromatography.
2. HPLC-CAD according to claim 1 measures the method for glucose in Dextrose and Sodium Chloride Inj., feature
It is, the detection parameters of the high performance liquid chromatography described in step 2 are as follows：
Chromatographic column：Thermo Mixed-Mode Hilic, 2.1 × 150mm, 1.3 μm；
Flow velocity：0.3mL/min；
Column temperature：40℃；
Sample size：10μL；
Mobile phase：50mmol/L ammonium acetates-acetonitrile.
3. HPLC-CAD according to claim 2 measures the method for glucose in Dextrose and Sodium Chloride Inj., feature
It is, the model of the high performance liquid chromatograph：Thermo-U3000 high performance liquid chromatographs, the Thermo-
U3000 high performance liquid chromatographs attach Corona Ultra EFI fog detectors.
4. HPLC-CAD according to claim 1 measures the method for glucose in Dextrose and Sodium Chloride Inj., feature
It is, the volume ratio of 50mmol/L ammonium acetates-acetonitrile is 15~80 in the mobile phase：20~85.
5. the method that the HPLC-CAD according to claim 1 or 4 is any measures glucose in Dextrose and Sodium Chloride Inj.,
It is characterized in that, the mobile phase is added in using the method for gradient elution, wherein specific parameter is as follows：
It is 10 minutes to elute total time；
0-2 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 15 in mobile phase：85；
4-5 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 80 in mobile phase：20；
6-10 minutes, the volume ratio of 50mmol/L ammonium acetates-acetonitrile was 15 in mobile phase：85.
6. HPLC-CAD according to claim 1 measures the method for glucose in Dextrose and Sodium Chloride Inj., feature
It is, the preparation of the test solution needed 0.45 μm of filter membrane.
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| WO2020020152A1 (en) * | 2018-07-24 | 2020-01-30 | 深圳市海普瑞药业集团股份有限公司 | Method for analyzing nitrous acid degradation product of dalteparin sodium and application thereof |
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| WO2020020152A1 (en) * | 2018-07-24 | 2020-01-30 | 深圳市海普瑞药业集团股份有限公司 | Method for analyzing nitrous acid degradation product of dalteparin sodium and application thereof |
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Application publication date: 20180601 |