CN108490087A - A kind of high performance liquid chromatography measuring content of taurine based on Composition distribution - Google Patents
A kind of high performance liquid chromatography measuring content of taurine based on Composition distribution Download PDFInfo
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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
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- 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
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention belongs to Pharmaceutical Analysis technical fields, and in particular to a kind of efficient liquid-phase chromatography method measuring content of taurine in taurine particles or taurine eye drops based on differential refraction detector.The high performance liquid chromatography of the present invention that content of taurine is measured based on differential refraction detector, high performance liquid chromatography is combined with differential refraction detector, the high performance liquid chromatography Composition distribution method for directly measuring content of taurine is established, with NH2Column is splitter; it is detected with differential refraction display; entire detection process; without using carrier gas; without carrying out derivation process to taurine, and without intermediate reaction process, the range of linearity is wide; can it is easy, fast and accurately directly measure content of taurine, this method can realize the measurement of content of taurine in taurine particles and taurine eye drops.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical fields, and in particular to one kind measuring taurine based on differential refraction detector
The efficient liquid-phase chromatography method of content of taurine in particle or taurine eye drops.
Background technology
Taurine (Taurine), also known as beta-amino ethanesulfonic acid, molecular formula C2H7NO3S, molecular weight 125.15 are a kind of
The non-protein amino acid of sulfur-bearing, earliest by being separated in cow-bezoar.Taurine molecule is small, does not have phenyl class group, chemically
Matter is stablized, and insoluble in organic solvents such as ether, exists in vivo with free state, is not involved in the biosynthesis of vivo protein, but
It is but closely related with the metabolism of cystine, cysteine, is the important nutrient of people and animal.It is deposited in a free form
It is in the almost all of internal organs of mammal, content is especially abundant in central nervous system.Taurine has important
Physiological action has and adjusts nervous system excitability, antipyretic, analgesia, bring down a fever, enhance the effects that resistance of human body, can promote big
Brain cell development improves immunity, relieves fatigue, adjusts nerve conduction；In addition, taurine also has important pharmacological action,
With anti-inflammatory, it is antipyretic, analgesia, it is antiviral the effect of.Taurine particles are a kind of to can be used for alleviating the common of common cold initial stage fever
Drug, taurine eye drops can be used for cataract conditions caused by taurine metabolism disorder and treat.Effectively to control taurine
A kind of taurine preparations quality such as particle or taurine eye drops, it is necessary to accurate, convenient and effective content of taurine of exploitation
Assay method.
《Chinese Pharmacopoeia》The taurine bulk pharmaceutical chemicals content assaying method that version in 2015 is recorded uses potentiometric titration；It is limited
In the limitation of taurine ultraviolet absorptivity,《Chinese Pharmacopoeia》In taurine particles and taurine eye drops that version in 2015 is recorded
Content of taurine assay method, be all made of in efficient liquid phase after chromatographic column in reaction tube, with o-phthalaldehyde to ox sulphur
Sour derivatization, using the ultraviolet feature of phenyl in derivative, with the content of UV detector detection method indirect determination taurine,
This method not only needs to be equipped with reaction tube in high performance liquid chromatography, higher to equipment requirement, at the same operate it is comparatively laborious, to anti-
It answers process, reaction conversion ratio to be difficult to intuitive judgement, influences the accuracy of content of taurine.National drug standards new drug turns
In the compound taurine eye drops quality standard that positive standard the 42nd is recorded, using the dilute copolymer of benzene second and sulfo group (Ca- coordinations)
In conjunction with gel be filler, water-acetonitrile (3：2) it is mobile phase, meso inositol is that the high performance liquid chromatography-of internal standard compound shows
Poor refraction detector method, this method are to be separately added into meso in solution in sample and reference substance on the basis of external standard method
Inositol internal standard compound obtains the content of taurine in sample by the variation relative value of the peak area or peak height that compare internal standard compound.
But this method relative external standerel method operation sequence is more troublesome, it is desirable that internal standard compound and sample will precises, usually use when analysis
It is unstable in system, the case where carrier gas sampling volume is difficult to grasp during operating condition variation error is big or gas-chromatography, pass through
The error that internal standard compound eliminates system is introduced, meets examination requirements to reach its accuracy.
In recent years, the document report content assaying method of different taurines, after mainly having acetylacetone,2,4-pentanedione chromogenic reaction
Spectrophotometry, HPLC ELSD detection method, mass spectrography etc..Potentiometric titration is commonly available to without dry
The measurement of substance or the less bulk pharmaceutical chemicals sample of impurity is disturbed, acetylacetone,2,4-pentanedione chromogenic reaction light splitting afterwards photometry also needs to develop the color
Reaction, cumbersome, to reaction process, reaction conversion ratio has been difficult to intuitive judgement, influences the accuracy of taurine measurement；
High performance liquid chromatography-evaporative light-scattering detector principle is first to be atomized column eluent to form aerosol, then in the drift of heating
It moves in pipe and is detected evaporation of the solvent, last remaining fixedness particles of solute in scattering measuring pond, need high pressure
Nitrogen or air can all influence to survey as carrier gas, inconvenient for use, carrier gas stability, atomization process, the thermal stability of sample to be tested
The accuracy of amount；Mass spectrography high specificity, accuracy are high, but somewhat expensive, and instrumentation is complicated, be usually used in material purity,
Structure or unknown sample it is qualitative or quantitative, but it is used for the routine check of conventionally known sample, instrument use cost and inspection
It tests that time cost is relatively high, is unfavorable for the raising of the saving and checkability of routine inspection room inspection cost.
Differential refraction detector is a kind of common detector, and measuring principle is by continuously detecting reference cell and inspection
The specific refractivity for surveying solution in pond carrys out determination sample concentration, commonly uses the measurement without UV absorption or weak uv-absorbing substance, makes
With simple and convenient, it is not necessarily to carrier gas, it is not necessary that taurine derivatization treatment, the range of linearity is wide, can directly measure taurine.This hair
Bright to be combined high performance liquid chromatography with differential refraction detector, the high performance liquid chromatography for establishing directly measurement content of taurine shows
Difference detector method, cost is relatively low, quick, accurate, high sensitivity, it is ensured that taurine raw material is wanted and the quality of preparation.
Therefore, a kind of accurate, convenient and effective content of taurine assay method of exploitation, for the drug containing taurine
Content detection and quality control all have important meaning.
Invention content
For this purpose, technical problem to be solved by the present invention lies in provide it is a kind of without it is derivative, be not necessarily to carrier gas, it is easy to operate,
High sensitivity, the high performance liquid chromatography differential pulse polarograpll method that can directly measure content of taurine.
In order to solve the above technical problems, a kind of height measuring content of taurine based on Composition distribution of the present invention
Effect liquid phase chromatogram method, including by taurine reference substance and taurine sample respectively to flow phased soln, be made reference substance solution and
The step of test solution, and reference substance solution and test solution are injected into high performance liquid chromatograph respectively, with NH2Column is
Splitter is that the mobile phase is eluted with water-acetonitrile, is detected with differential refraction detector, ox is calculated with external standard method
The content of sulfonic acid.
The high performance liquid chromatography that content of taurine is measured based on Composition distribution, controls the differential refraction
The detector reference cell temperature of detector is 35.0-50.0 DEG C, and control detection cell temperature is 35.0-50.0 DEG C.
The high performance liquid chromatography that content of taurine is measured based on Composition distribution, controls the reference cell temperature
Degree is identical as the detection cell temperature.
The high performance liquid chromatography detection condition includes：
Chromatographic column：NH2Column, 250mm × 4.6mm, 5 μm；
Mobile phase：Volume ratio is 40-45：Water-acetonitrile solution of 60-55；
Flow velocity：0.7-1.0ml/min；
Column temperature：30.0-40.0℃；
Sample size：20-30μL.
A concentration of 0.3-0.8mg/mL of the taurine reference substance solution.
A concentration of 0.3-0.8mg/mL of the test solution.
The taurine sample includes taurine particles or taurine eye drops.
The invention also discloses it is described based on Composition distribution measure content of taurine high performance liquid chromatography containing
Application in taurine medicine assay and field of quality control.
Include according to the side the invention also discloses content of taurine method for measuring in a kind of taurine particles
Method carries out the step of content of taurine detection.
Include according to the side the invention also discloses content of taurine assay method in a kind of taurine eye drops
Method carries out the step of content of taurine detection.
Differential refraction detector is a kind of common detector, and measuring principle is by continuously detecting reference cell and inspection
The specific refractivity for surveying solution in pond carrys out determination sample concentration, commonly uses the measurement without UV absorption or weak uv-absorbing substance.This
The invention high performance liquid chromatography that content of taurine is measured based on differential refraction detector, by high performance liquid chromatography with show
Poor refraction detector combines, and the high performance liquid chromatography Composition distribution method for directly measuring content of taurine is established, with NH2Column
It for splitter, is detected with differential refraction display, entire detection process, without using carrier gas, without being carried out to taurine
Derivation process, and without intermediate reaction process, the range of linearity is wide, can it is easy, fast and accurately directly measure content of taurine,
This method can realize the measurement of content of taurine in taurine particles and taurine eye drops；This method also has detector ginseng
The advantages that number setting is simple, and mobile phase is easy to prepare, high sensitivity, testing cost is relatively low, can fast and accurately detect ox
The content of taurine in sulfonic acid preparation, it is ensured that taurine raw material is wanted and the quality control of preparation.
The present invention utilizes the-NH of taurine2,-OH, with NH2- NH on column2Polar bond interattraction, with water-acetonitrile
System is mobile phase, is continuously increased the content of acetonitrile in mobile phase, and retention time of the taurine on nh 2 column gradually increases,
Separation efficiency gradually increases；In mobile phase water-acetonitrile, volume ratio 40：60, under conditions of flow velocity is 0.8ml/min, taurine
In NH2Retention time on column reaches 6min or so, and theoretical tray reaches 10000, good separation.The second in mobile phase
The increase of nitrile content is conducive to the separation efficiency for improving taurine, but since taurine does not dissolve in organic solvent, in mobile phase
Excessive ethane nitrile content limits the dissolving of taurine, therefore it is water-acetonitrile, volume ratio 40 to optimize mobile phase：60.The present invention
Research initial trial is shown, with water-acetonitrile, volume ratio (40：60) it is mobile phase, taurine retains extremely weak on a cl 8 column.
The present invention select alanine and taurine as system suitability solution investigate method specificity, alanine and
Taurine separating degree is good, and mark-on reclaims test result shows in taurine particles and taurine eye drops, Taurine pair
It is met the requirements according to the rate of recovery of product.
Description of the drawings
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and ties
Attached drawing is closed, the present invention is described in further detail.
Fig. 1 to Fig. 3 is the method for the present invention system suitability HPLC chromatogram, wherein A is system suitability spectrogram (figure
1), B is taurine reference substance chromatogram (Fig. 2), and C is blank solution chromatogram (Fig. 3)；
Fig. 4 to Fig. 5 is the HPLC spectrograms of taurine particles of the present invention and taurine eye drops, wherein A is ox sulphur
Granulates (Fig. 4), B are taurine eye drops (Fig. 5).
Specific implementation mode
The following embodiments of the present invention are related to equipment and include：Agilent 1260Infinity high performance liquid chromatographs,
1260 differential refraction detectors of Agilent；Mei Teletuo benefit XP205 type electronic balances.
Taurine reference substance (lot number：111616-201605, National Institute for Food and Drugs Control, content：
100.0%)；
Alanine reference substance (lot number：140680-200803, National Institute for Food and Drugs Control, content：99.9%)；
Taurine particles (A factories, lot number 160601,0.4g/ bags of specification；B factories, lot number 20160522,0.4g/ bags of specification；C
Factory, lot number 151108,0.4g/ bags of specification)；
Taurine eye drops (A factories, lot number 16011501, specification 0.4g：8mL；B factories, lot number 161204, specification 0.5g：
10mL；C factories, lot number 161001, specification 0.5g：10mL)；
Acetonitrile is chromatographically pure, and experimental water is ultra-pure water, remaining reagent is that analysis is pure.
Embodiment 1
The method that the present embodiment measures content of taurine based on differential refraction detector method, includes the following steps：
(1) precision weighs taurine reference substance about 10mg, sets in 25mL measuring bottles, and volume ratio 40 is added：60 water-acetonitrile
Solution dissolved dilution shakes up to scale, and taurine reference substance solution is made；
Precision weighs alanine reference substance 10mg, sets in 25mL measuring bottles, and volume ratio 40 is added：60 water-acetonitrile solution is molten
Solution is diluted to scale, shakes up to get alanine reference substance solution；
(2) precision weighs taurine particles fine powder in right amount (being equivalent to taurine 100mg), sets in 50mL measuring bottles, and body is added
Product ratio 40：60 water-acetonitrile solution dissolved dilution to scale, shakes up, filtration；Precision measures subsequent filtrate 5mL, sets 25mL measuring bottles
In, volume ratio 40 is added：60 water-acetonitrile solution dissolved dilution to scale is to get taurine particles test solution；
Precision measures taurine eye drops 1mL (being equivalent to taurine 50mg), sets in 25mL measuring bottles, and volume ratio 40 is added：
60 water-acetonitrile solution dissolved dilution to scale, shakes up, filtration；Precision measures subsequent filtrate 5mL, sets in 25mL measuring bottles, is added
Volume ratio 40：60 water-acetonitrile solution dissolved dilution to scale is to get taurine eye drops test solution；
(3) high performance liquid chromatography is shone, with NH2Column is packed column, is detected with differential refraction detector；
The testing conditions include：
High performance liquid chromatograph：Agilent 1260Infinity；
Differential refraction detector：Agilent 1260；
Chromatographic column：Waters NH2Column, 250mm × 4.6mm, 5 μm；
Mobile phase：Volume ratio 40：60 water-acetonitrile solution；
Flow velocity：0.8mL/min；
Column temperature：35.0℃；
Controlling the detector parameters includes：
Reference cell temperature：35.0℃；
Detection cell temperature：35.0℃；
Sample size：20μL；
(4) taurine reference substance solution is drawn respectively, alanine reference substance solution injects high performance liquid chromatograph, progress
System suitability measures, while with volume ratio 40：60 water-acetonitrile mobile phase is measured as blank solution, detects chromatogram
As shown in Figure 1.
As it can be seen that taurine retention time 6.217min, alanine retention time 6.873min, alanine and taurine point
From degree 2.66, number of theoretical plate is calculated as 11614 with taurine.
The investigation of 2 linear relationship of embodiment
Using mobile phase as solvent, take respectively concentration 1mg/mL, 0.8mg/mL, 0.6mg/mL, 0.4mg/mL, 0.2mg/mL,
0.1mg/mL and 0.05mg/mL taurine reference substance solutions.
According to chromatographiccondition in embodiment 1, with peak area to taurine reference substance concentration (mg/mL) linear regression,
Obtain regression equation Y=195768.56X+1085.93, r=0.99858 (n=7).The result shows that in sample introduction concentration 0.05mg/
Linear relationship is good in the range of mL-1mg/mL.
3 Precision Experiment of embodiment
1mg/mL, 0.4mg/mL and 0.05mg/mL taurine reference substance solution are taken respectively, according to chromatography in embodiment 1 point
Analysis condition, each to repeat sample introduction 5 times, the RSD for acquiring peak area is respectively 0.31%, 0.37%, 0.36%.
4 stability experiment of embodiment
Take 0.4mg/mL taurine reference substance solutions, according to chromatographiccondition in embodiment 1,0h, 1h, 2h, 4h,
6h, 20h, sample introduction being repeated for 24 hours, measuring peak area, it is 0.28% to calculate peak area RSD, and interior solution is basicly stable for 24 hours.
5 mark-on reclaims of embodiment are tested
Mark-on blank solution：The taurine particles test sample of 2mg/mL is prepared according to step in embodiment (2) method respectively
Solution and eye drops test solution, filtration, precision measure subsequent filtrate 4mL, set in 20mL measuring bottles, mobile phase is added to be diluted to quarter
Degree to get.
Mark-on solution：The taurine particles test solution of 2mg/mL is prepared according to step in embodiment (2) method respectively
With eye drops test solution, filtration is accurate respectively to measure subsequent filtrate 4mL, sets in 20mL measuring bottles；It is separately added into 4mL, 5mL,
A concentration of 1.6mg/mL's of 6mL shines product solution to taurine, adds volume ratio 40：60 water-acetonitrile mobile phase is diluted to scale,
It is respectively 0.8 that mark-on ratio is made respectively：1、1：1、1.2：1 taurine particles mark-on solution and taurine eye drops mark-on
Solution.
Taurine mark-on blank solution and different proportion taurine mark-on solution are taken respectively, according to chromatography in embodiment 1 point
Analysis condition calculates taurine reference substance and measures concentration, and is actually added into concentrations control, acquires the rate of recovery, be recorded in the following table 1,2.
1 taurine particles recovery test result (n=9) of table
2 taurine eye drops recovery test result (n=9) of table
The average recovery rate 100.27% of taurine particles, RSD are 1.26% (n=9)；Taurine eye drops are averaged
The rate of recovery is 100.62%；RSD is 1.58% (n=9)；RSD is respectively less than 2%.The detection limit of embodiment 6
It according to chromatographiccondition in embodiment 1, is gradually diluted with 0.4mg/mL taurine reference substance solutions, takes 20 μ L
It injects in high performance liquid chromatograph, records chromatogram, be 3/1 for condition with signal-to-noise ratio, measure detection limit, must detect and limit concentration
0.5μg/mL。
The assay of 7 taurine sample of embodiment
The content of taurine in three batch taurine particles and three batch taurine eye drops is detected respectively, is pressed
Taurine particles test solution is prepared respectively according to the method for step (2) in embodiment 1 and taurine eye drops test sample is molten
Liquid, and peak area is measured according to chromatographiccondition in embodiment 1, content of taurine is calculated based on external standard method.It is provided in Fig. 2
The HPLC spectrograms of the taurine particles and taurine eye drops of detection.In taurine particles spectrogram, taurine retention time
Occurs a peak at 6.273min, 5.601min, two peak separating degrees are 3.03；In taurine eye drops spectrogram, do not find except molten
Apparent peak outside agent.
It is computed, the content of taurine is respectively in taurine particles：98.33%, 103.42%, 100.54%；Ox sulphur
The content of sour Taurine Eye Drops is respectively：93.67%, 96.65%, 99.08%.
As it can be seen that the method for the invention can effectively realize content of taurine in taurine particles and taurine eye drops
It measures, meanwhile, without carrying out additional derivatization treatment, detection method is simple and practicable, and sensitivity is higher, can be widely applied to
The measurement of content of taurine and quality control.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.
For those of ordinary skill in the art, other various forms of changes can also be made on the basis of the above description
Change or changes.There is no necessity and possibility to exhaust all the enbodiments.And obvious change extended from this
Change or changes still within the protection scope of the invention.
Claims (10)
1. a kind of high performance liquid chromatography measuring content of taurine based on Composition distribution, which is characterized in that including by ox sulphur
Respectively to flow phased soln reference substance solution is made and the step of test solution in sour reference substance and taurine sample, and respectively
Reference substance solution and test solution are injected into high performance liquid chromatograph, with NH2Column is splitter, with water-acetonitrile for the stream
Dynamic phase is eluted, and is detected with differential refraction detector, and the content of taurine is calculated with external standard method.
2. the high performance liquid chromatography according to claim 1 for measuring content of taurine based on Composition distribution, feature
It is：The detector reference cell temperature for controlling the differential refraction detector is 35.0-50.0 DEG C, and control detection cell temperature is
35.0-50.0℃。
3. the high performance liquid chromatography according to claim 2 for measuring content of taurine based on Composition distribution, feature
It is, it is identical as the detection cell temperature controls the reference cell temperature.
4. the high performance liquid chromatography of content of taurine is measured based on Composition distribution according to claim 1-3 any one of them
Method, which is characterized in that the high performance liquid chromatography detection condition includes：
Chromatographic column：NH2Column, 250mm × 4.6mm, 5 μm；
Mobile phase：Volume ratio is 40-45：Water-acetonitrile solution of 60-55；
Flow velocity：0.7-1.0ml/min；
Column temperature：30.0-40.0℃；
Sample size：20-30μL.
5. the high performance liquid chromatography of content of taurine is measured based on Composition distribution according to claim 1-4 any one of them
Method, which is characterized in that a concentration of 0.3-0.8mg/mL of the taurine reference substance solution.
6. the high performance liquid chromatography according to claim 5 for measuring content of taurine based on Composition distribution, feature
It is, a concentration of 0.3-0.8mg/mL of the test solution.
7. the high performance liquid chromatography of content of taurine is measured based on Composition distribution according to claim 1-6 any one of them
Method, which is characterized in that the taurine sample includes taurine particles or taurine eye drops.
8. claim 1-7 any one of them based on Composition distribution measure content of taurine high performance liquid chromatography containing
Application in taurine medicine assay and field of quality control.
9. content of taurine method for measuring in a kind of taurine particles, which is characterized in that including any according to claim 1-7
The step of method described in carries out content of taurine detection.
10. content of taurine assay method in a kind of taurine eye drops, which is characterized in that including appointing according to claim 1-7
The step of method described in one carries out content of taurine detection.
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Cited By (2)
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| CN113045457A (en) * | 2019-12-27 | 2021-06-29 | 湖北远大生命科学与技术有限责任公司 | Equipment for purifying taurine and system for producing taurine |
| CN113049540A (en) * | 2019-12-27 | 2021-06-29 | 湖北远大生命科学与技术有限责任公司 | Method for determining content of taurine in solution and method for purifying taurine |
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| CN103454358A (en) * | 2013-09-13 | 2013-12-18 | 厦门大学 | Method for detecting taurine |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113045457A (en) * | 2019-12-27 | 2021-06-29 | 湖北远大生命科学与技术有限责任公司 | Equipment for purifying taurine and system for producing taurine |
| CN113049540A (en) * | 2019-12-27 | 2021-06-29 | 湖北远大生命科学与技术有限责任公司 | Method for determining content of taurine in solution and method for purifying taurine |
| CN113049540B (en) * | 2019-12-27 | 2023-05-30 | 湖北远大生命科学与技术有限责任公司 | Method for determining taurine content in solution and method for purifying taurine |
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Application publication date: 20180904 |