CN103913521B - Novel method for detecting hydrogen peroxide residues in heparin sodium - Google Patents
Novel method for detecting hydrogen peroxide residues in heparin sodium Download PDFInfo
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- CN103913521B CN103913521B CN201310684303.5A CN201310684303A CN103913521B CN 103913521 B CN103913521 B CN 103913521B CN 201310684303 A CN201310684303 A CN 201310684303A CN 103913521 B CN103913521 B CN 103913521B
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- hydrogen peroxide
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- heparin sodium
- triphenylphosphine
- acetonitrile
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Abstract
The invention discloses a novel method for detecting hydrogen peroxide residues in heparin sodium raw materials, and aims at increasing sensitivity of detection limit, accuracy of results and eliminating interference of external environments. The novel detection method developed by the invention is characterized in that interference of heparin sodium to hydrogen peroxide detection is eliminated by passing a heparin sodium sample solution through an anion exchange column; and the influences of environmental temperature and light to the content of hydrogen peroxide in an experimental process are prevented by subjecting hydrogen peroxide to a derivatization reaction. Hydrogen peroxide reacts with excess triphenylphosphine solution to produce stable triphenylphosphine oxide; then triphenylphosphine oxide is injected in a high performance liquid chromatograph; triphenylphosphine oxide is separated by a chromatographic column and detected by an ultraviolet detector; and the content of hydrogen peroxide is determined quantitatively and accurately by an external standard method. The method is good in separation degree, high in accuracy, reasonable in process and simple in operation.
Description
One, technical field
The present invention relates to the method for residual peroxide content detection in a kind of liquaemin.
Two, background technology
Liquaemin bulk drug is the effective constituent of heparin preparations and the initiation material of low molecular weight heparin preparations, and needs to add hydrogen peroxide and carry out oxidative decoloration in the process of producing, and inevitably produces residual peroxide.Therefore measure the residual of hydrogen peroxide in liquaemin to be necessary.The existing mensuration to hydrogen peroxide in water mainly chemical titration, measures with liquor potassic permanganate titration.Chemical titration detectability is higher, and amount of samples is large, about 10g; And error is higher, complex operation, titration end-point is not easily held.And titrimetry terminal trace residue is not easily grasped, detect and be limited to 100PPM, the assay method of residual hydrogen dioxide in " National Standard of the People's Republic of China " food, have employed titrimetry.One is also had to be that high-efficient liquid phase technique directly measures, but it is more weak for uv absorption hydrogen peroxide, also not easily measure concerning trace residue, measure a method for content of hydrogen peroxide in solution, the method that patent No. CN02158703.5 adopts, the method is UV-detector direct-detection, but hydrogen peroxide easily decomposes, temperature and light is according to all having impact to it, and cause testing result inaccurate, detection limit is higher.Adopt derivatization method, hydrogen peroxide and triphenylphosphine reaction generate triphenylphosphine oxide, and triphenylphosphine oxide uv absorption is good, and avoids the resolution problem of hydrogen peroxide.The people such as Stefan with hydrogen peroxide oxidation triphenyl phosphorus (TPP) oxide (TPPO) that is triphenyl phosphorus for principle, measure hydrogen peroxide by the change between liquid chromatogram measuring TPP and TPPO, and measure the hydrogen peroxide in washing agent.This method is also adopt derivatization method, sample solution eliminates the impact that liquaemin detects hydrogen peroxide after crossing anion-exchange column after, then adopts the method for triphenylphosphine derivatization, effectively determines remaining of hydrogen peroxide.
Key of the present invention is: liquaemin is a kind of potpourri of different molecular weight mucopolysaccharide sulfuric acid ester, produces huge interference to the analysis of hydrogen peroxide.Use existing detection method, be embodied in the detection of interference triphenylphosphine oxide, the peak of liquaemin potpourri and triphenylphosphine oxide do not reach good separating effect (see accompanying drawing 1).We have employed the pre-treating method of anion-exchange column, eliminated liquaemin to a great extent to the impact detected, reached good baseline separation (see accompanying drawing 2).Therefore Detection results of the present invention is more excellent compared with existing detection method.
Three, summary of the invention
The object of the present invention is to provide a kind of detectability low, disturb little, simple to operate, sensitivity is good, and accuracy is high, and error is little, particularly for the detection method of residual peroxide in liquaemin.Reducing end on liquaemin sugar chain can and hydroperoxidation.Object of the present invention passes through realization like this: after liquaemin sample purified water is dissolved, after crossing anion-exchange column with syringe pipette samples solution, eliminate the impact of heparin, then add triphenylphosphine solution derivatization, the laggard high performance liquid chromatography of lucifuge reaction 30min detects.
Shown in assay method following steps of the present invention:
(1) demarcation of Hydrogen peroxide standard product
Hydrogen peroxide standard commercially available reagent is demarcated according to standard GB/T 9984-89.
(2) preparation of sample solution
Taking heparin sodium raw materials medicine 1g is dissolved in 10ml water, and constant volume, shakes up for subsequent use.
(3) preparation of derivatization reagent
Take triphenylphosphine 52.46mg, dissolve with acetonitrile, be settled to 1000mL, concentration is 0.0002mol/L, matching while using.
(4) chromatographic condition
Instrument: waters e2695 high performance liquid chromatograph, detecting device is waters2489 UV-detector
Chromatographic column: C18 chromatographic column, 4.6mm × 250mm, 5 μm;
Mobile phase: be acetonitrile by volume by acetonitrile and water: water=70:30 mixes, degassed.
Flow velocity: 1.0mL/min;
Determined wavelength: 225nm;
Sample size: 10 μ L.
(5) derivative reaction
After pipette samples solution and standard working solution cross anion-exchange column respectively with syringe, eliminate the impact of heparin.Pipette the Hydrogen peroxide standard working fluid after post and each 1mL of sample solution more respectively in 10mL brown bottle, added 2mL triphenylphosphine acetonitrile solution, jolting, be placed in dark at room temperature reaction 30min.
After derivative reaction terminates, start stratographic analysis immediately, draw 10 μ L sample solutions and inject high performance liquid chromatograph.Under above-mentioned chromatographic condition, measure its peak area, utilize the concentration of hydrogen peroxide in external standard method calculation sample solution.
four, accompanying drawing explanation
Fig. 1 is the spectrogram of sample without anion-exchange column process; Fig. 2 is the spectrogram of sample through anion-exchange column process.
Claims (1)
1. measure a detection method for residual peroxide in liquaemin bulk drug, shown in assay method following steps of the present invention:
(1) demarcation of Hydrogen peroxide standard product
Hydrogen peroxide standard commercially available reagent is demarcated according to standard GB/T 9984-89;
(2) preparation of sample solution
Taking heparin sodium raw materials medicine 1g is dissolved in 10ml water, and constant volume, shakes up for subsequent use;
(3) preparation of derivatization reagent
Take triphenylphosphine 52.46mg, dissolve with acetonitrile, be settled to 1000mL, concentration is 0.0002mol/L, matching while using;
(4) chromatographic condition
Instrument: waters e2695 high performance liquid chromatograph, detecting device is waters 2489 UV-detector;
Chromatographic column: C18 chromatographic column, 4.6mm × 250mm, 5 μm;
Mobile phase: be acetonitrile by volume by acetonitrile and water: water=70: 30 mix, degassed;
Flow velocity: 1.0mL/min;
Determined wavelength: 225nm;
Sample size: 10 μ L;
(5) derivative reaction
After pipette samples solution and standard working solution cross anion-exchange column respectively with syringe, eliminate the impact of heparin; Pipette the Hydrogen peroxide standard working fluid after post and each 1mL of sample solution more respectively in 10mL brown bottle, added 2mL triphenylphosphine acetonitrile solution, jolting, be placed in dark at room temperature reaction 30min;
After derivative reaction terminates, start stratographic analysis immediately, draw 10 μ L sample solutions and inject high performance liquid chromatograph; Under above-mentioned chromatographic condition, measure its peak area, utilize the concentration of hydrogen peroxide in external standard method calculation sample solution.
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| CN201310684303.5A CN103913521B (en) | 2013-12-05 | 2013-12-05 | Novel method for detecting hydrogen peroxide residues in heparin sodium |
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| CN201310684303.5A CN103913521B (en) | 2013-12-05 | 2013-12-05 | Novel method for detecting hydrogen peroxide residues in heparin sodium |
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| CN103913521B true CN103913521B (en) | 2015-07-08 |
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| CN106908524B (en) * | 2015-12-23 | 2021-05-04 | 重庆华邦胜凯制药有限公司 | Separation and determination method of calcipotriol intermediate L and potential genotoxic impurities thereof |
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Address after: 264006 Yantai economic and Technological Development Zone, Changbai Mountain Road, No. 7, No. Applicant after: YANTAI DONGCHENG PHARMACEUTICAL GROUP CO., LTD. Address before: 264006 Yantai economic and Technological Development Zone, Changbai Mountain Road, No. 7, No. Applicant before: Yinan Dongyuan Bioengineering Co., Ltd. |
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