CN107782829A - One kind utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products（Tris）Method - Google Patents
One kind utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products（Tris）Method Download PDFInfo
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- CN107782829A CN107782829A CN201711047617.9A CN201711047617A CN107782829A CN 107782829 A CN107782829 A CN 107782829A CN 201711047617 A CN201711047617 A CN 201711047617A CN 107782829 A CN107782829 A CN 107782829A
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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
- G01N30/96—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 using ion-exchange
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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 one kind to utilize trishydroxymethylaminomethane in sulfate by ion chromatography biological products（Tris）Method.This method is using method derived from fluid infusion after pulsed amperometry, post, with CS16 cation-exchange chromatography posts, mobile phase is pyrovinic acid solution, fluid infusion is alkaline solution after post, Tris concentration peak area standard curve is drawn using external standard method, the regression equation of standard curve is drawn, Tris content is obtained by the regression equation calculation.This method Tris minimum detectability is 3.14E 5 mmol/L, minimum to be quantitatively limited to the mmol/L of 1.05E 4, and the rate of recovery is 93.8% 100.1%, and reappearance scope is 0.10% 0.52% (RSD, n=5).This method is without derivative and complex sample pre-treatment, the acid and alkaline group not carried in by biological products on protein is disturbed, and also zwitterion is not disturbed in by sample, and detection sensitivity is high, analyze speed is fast, can be applied to Tris content detection and quality assessment in relevant industries biological products.
Description
Technical field
The present invention relates to one kind to utilize trishydroxymethylaminomethane in sulfate by ion chromatography biological products（Tris）Side
Method, belong to analysis detection field.
Background technology
Trishydroxymethylaminomethane（English abbreviation Tris）Also known as tromethamine；Trometamol；2- amino -2- (hydroxyl first
Base) -1,3- propane diols.Tris is a kind of weak base, the biological buffer system being commonly used in biological products production process.Tris can draw
Hypoglycemia, low blood pressure, Nausea and vomiting are played, can also suppress to breathe, or even stop breathing.When Excess free enthalpy or renal insufficiency
Alkalemia can be caused.
The method for mainly using acid base titration on Tris quantitative detection at present, quantitative limit is higher and is vulnerable to various acid
The interference of base groups.And because containing substantial amounts of carboxyl, amino and zwitterion, the Tris in product can not lead in biological products
The method for crossing titration carries out quantitative detection.Therefore a kind of Tris established in fast and accurately biological products detection method, it is
Urgent problem to be solved at present.
Content of the chromatography of ions pulsed amperometry commonly used to small-molecule substances such as carbohydrate of the detection rich in hydroxyl.
The content of the invention
In order to solve the above problems, the invention provides one kind to utilize trihydroxy methyl in sulfate by ion chromatography biological products
Aminomethane（Tris）Method.The method of the present invention is using method derived from fluid infusion after pulsed amperometry, post, is used
CS16 cation-exchange chromatography posts, mobile phase are pyrovinic acid solution, and fluid infusion is alkaline solution after post, is drawn using external standard method
Tris concentration-peak area standard curve, draws the regression equation of standard curve, obtains Tris's by the regression equation calculation
Content.
The detection Tris of present invention method, is to use ion chromatograph, and test sample is treated in method detection derived from fluid infusion after post
Product；Wherein chromatographic column is CS16 cation-exchange chromatography posts；Column temperature is 29-31 DEG C；Fluid infusion is potassium hydroxide or hydroxide after post
Sodium solution；Rehydration solution concentration is 200-500 mmol/L after post；Fluid infusion flow velocity is 0.29-0.31 ml/min after post；Mobile phase
For 40mmol/L pyrovinic acid solution；Flow rate of mobile phase is 0.95-1.05mL/min；Detector is pulsed amperometry；
Using Au as electrode, Ag-AgCl electrodes are reference electrode, waveform Carbohydrates.
In one embodiment, the chromatography of ions uses the ion colors of Thermo Fisher company ICS 5000
Spectrometer.
In one embodiment, the chromatographic column pacifies Ionpac CS16 cation exchange columns to wear, including analytical column
(250mm × 5mm) and guard column (50mm × 5mm).Column temperature is 30 DEG C.
In one embodiment, the sampling volume of testing sample is 25 μ L in methods described.
In one embodiment, methods described is using fluid infusion after 500 mmol/L sodium hydroxide solutions progress post.
In one embodiment, mobile phase is 40 mmol/L pyrovinic acid solution；Flow rate of mobile phase is 1.0 mL/
min。
In one embodiment, methods described, the Tris standard liquids of configuration various concentrations are included, using ion color
Spectrum detection, obtains the peak area under different Tris standard liquids, draws the standard curve between peak area and Tris concentration, obtains
Go out the regression equation of standard curve；The peak area that testing sample detects to obtain is substituted into the regression equation of standard curve, you can obtain
Know the Tris concentration in testing sample.
Beneficial effects of the present invention
(1) the inventive method detection Tris minimum detectability is 3.14E-5 mmol/L, minimum to be quantitatively limited to 1.05E-4
Mmol/L, rate of recovery 93.8%-100.1%, reappearance scope are 0.10%-0.52% (RSD, n=5)；
(2) the inventive method detection limit and quantitative limit compared with conventional acid-base titration method is lower, and not by being carried on protein
The interference of acid and alkaline group, also zwitterion is not disturbed in by sample；
(3) the inventive method is that can detect the content of Tris in different biological products without derivative and complex sample pre-treatment；
(4) the inventive method has preferably selectivity, does not have other chromatographic peaks to disturb in Tris retention times region.
Brief description of the drawings
Fig. 1：Tris chromatography of ions figure under the conditions of embodiment 1.
Fig. 2：Tris chromatography of ions figure under the conditions of embodiment 2.
Fig. 3：Tris chromatography of ions figure under the conditions of embodiment 3.
Embodiment
Embodiment given below, the invention will be further described.
Embodiment 1：
Test sample sample is the freeze dried human zymoplasm of Shanghai Laishi Blood Product Co., Ltd's production, by regulation plus injection
Water redissolves, the sample detection after 500 times of dilutions.It is the ion colors of Thermo Fisher company ICS 5000 to test the instrument used
Spectrometer, chromatographic condition are：Wear peace Ionpac CS16 cation exchange columns, including analytical column (250mm × 5mm) and guard column
(50mm × 5mm), column temperature is 30 DEG C；Using method derived from fluid infusion after post, fluid infusion is that 500mmol/L sodium hydroxides are molten after post
Liquid；Mobile phase is pyrovinic acid solution, flow rate of mobile phase 1.0mL/min；Detector is pulsed amperometry, using Au as electricity
Pole, Ag-AgCl electrodes are reference electrode, waveform Carbohydrates；Tris chromatography of ions figure is shown in Fig. 1.
By the standard liquid of various concentrations, carry out testing and analyzing under the chromatographic condition of the present embodiment using Tris concentration as
Abscissa, peak area is that ordinate draws standard working curve, and is calculated by returning, and it is Y=1755.3 to obtain standard curve
X+1.838, coefficient correlation is between 0.9998.According to test result and baseline noise, Tris theoretical detection limit is extrapolated
For 3.33E-5 mmol/L, theory is quantitatively limited to 1.05E-4 mmol/L.According to result is calculated, preparing Tris concentration is respectively
1.0E-4 mmol/L and 3.33E-5 mmol/L solution, detected under the conditions of the present embodiment to confirm test limit and determine
Amount limit.Test limit measured value is 2.99E-5 mmol/L, and quantitative limit measured value is 1.05E-4 mmol/L.Meet theoretical test limit
Result is calculated with quantitative limit.Parallel 5 measure of test sample, the relative standard deviation between parallel sample is in 0.10%-0.52%
Between.Recovery testu is carried out to test sample, the rate of recovery is between 93.8%-100.1%.
Embodiment 2
Test sample sample is 1% glycine solution, the sample detection after 500 times dilute.It is Thermo to test the instrument used
The ion chromatographs of Fisher companies ICS 5000, chromatographic condition are：Wear peace Ionpac CS16 cation exchange columns, including analysis
Post (250mm × 5mm) and guard column (50mm × 5mm), column temperature is 30 DEG C；Using method derived from fluid infusion after post, fluid infusion after post
For 500mmol/L sodium hydroxide solutions；Mobile phase is pyrovinic acid solution, flow rate of mobile phase 1.0mL/min；Detector is arteries and veins
Ampere detector is rushed, using Au as electrode, Ag-AgCl electrodes are reference electrode, waveform Carbohydrates；Tris ion colors
Spectrogram is shown in Fig. 2.
Because pulsed amperometry can detect the micromolecular compound of hydroxyl and amino, it can be seen from the figure that exists
Interference without glycine on Tris retention time position.
Embodiment 3
Test sample sample is the lyophilized human blood coagulation FVIII of Shanghai Laishi Blood Product Co., Ltd's production（AHF）, press
Regulation plus water for injection redissolve, the sample detection after 500 times of dilutions.It is Thermo Fisher company ICS to test the instrument used
5000 ion chromatographs, chromatographic condition are：Wear peace Ionpac CS16 cation exchange columns, including analytical column (250mm × 5mm)
With guard column (50mm × 5mm), column temperature be 30 DEG C；Using method derived from fluid infusion after post, fluid infusion is 500mmol/L hydrogen after post
Sodium hydroxide solution；Mobile phase is pyrovinic acid solution, flow rate of mobile phase 1.0mL/min；Detector is pulsed amperometry,
Using Au as electrode, Ag-AgCl electrodes are reference electrode, waveform Carbohydrates；Tris chromatography of ions figure is shown in Fig. 3.
The lyophilized human blood coagulation FVIII of Shanghai Laishi Blood Product Co., Ltd's production（AHF）Production work
Tris is not added with skill.It can be seen from the figure that interference without other materials on Tris retention time position.
Outside above-described embodiment, the research of detection method durability of the present invention has also been carried out.
Column temperature excursion is ± 3.3%, distinguishes sample introduction in 29 DEG C, 30 DEG C and 31 DEG C of column oven, as a result all conforms to
Ask.
Flow rate of mobile phase excursion is ± 5%, is 0.95 ml/min, 1.00 ml/min and 1.05 in flow rate of mobile phase
Sample introduction is distinguished during ml/min, is as a result all met the requirements.
Fluid infusion change in flow scope is ± 3.3%, is 0.29 ml/min, 0.30 ml/min and 0.31 in fluid infusion flow velocity
Sample introduction is distinguished during ml/min, is as a result all met the requirements.
Described above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvement and modification can also be made, these are improved and modification also should
It is considered as the scope of the present invention.
Claims (7)
1. one kind utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products（Tris）Method, its feature exists
In：The assay method comprises the following steps：
（1）The preparation of test sample and reference substance：Biological freeze-dried products add water for injection dissolution filter or Biological water solution system
Product directly filter, and take filtrate as need testing solution；Tris is weighed, water for injection dissolving is added, difference is obtained after gradient dilution
The Tris solution of concentration is as reference substance solution；
（2）The chromatography of ions：Using pulsed amperometry, method derived from fluid infusion after post；Chromatographic column is handed over for CS16 cations
Change chromatographic column；Mobile phase is pyrovinic acid solution；Fluid infusion is alkaline solution after post；Using Au as electrode, Ag-AgCl electrodes are reference
Electrode；Waveform is Carbohydrates；Column temperature is 30 DEG C；
（3）Calculate Tris contents in test sample：Quantitative analysis uses external standard method, and the reference substance of the various concentration prepared is molten
Liquid liquid sample introduction, testing result, and Tris concentration-peak area standard curve is drawn out, draw the regression equation of standard curve；
With entering need testing solution under reference substance solution identical sampling condition, obtained by above-mentioned regression equation calculation in test sample
Tris content.
2. one kind according to claim 1 utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products
（Tris）Method, it is characterised in that：The step（1）In test sample be biological aqueous liquid preparations or biological freeze-dried products
Water for injection redissolves the one or more of liquid.
3. one kind according to claim 1 utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products
（Tris）Method, it is characterised in that：The step（1）In test sample be freeze dried human zymoplasm, lyophilized human blood coagulation
FVIII, intravenous injection human gamma globulin (HGG), intramuscular injection human gamma globulin (HGG), hypodermic injection human gamma globulin (HGG), Lyophilized Human coagulate
The one or more of hemase original compound, cryodesiccant human fibrinogen and human serum albumin.
4. one kind according to claim 1 utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products
（Tris）Method, it is characterised in that：The step（2）In chromatographic column be CS16 cation-exchange chromatography posts.
5. one kind according to claim 1 utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products
（Tris）Method, it is characterised in that：The step（2）In pyrovinic acid solution concentration be 40 mmol/L, flow velocity 1
mL/min。
6. one kind according to claim 1 utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products
（Tris）Method, it is characterised in that：The alkaline solution of fluid infusion is one kind or several of sodium hydroxide or potassium hydroxide solution after post
Kind, concentration is 500 mmol/L, and flow velocity is 0.3 mL/min.
7. one kind according to claim 1 utilizes trishydroxymethylaminomethane in sulfate by ion chromatography biological products
（Tris）Method, it is characterised in that：After post after fluid infusion, the pH value of solution is between 12-14.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112946123A (en) * | 2021-02-01 | 2021-06-11 | 中山未名海济生物医药有限公司 | Method for detecting tromethamine content |
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| EP1530721B1 (en) * | 2002-08-22 | 2008-12-24 | Applera Corporation | Method for characterizing biomolecules utilizing a result driven strategy |
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Cited By (1)
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| CN112946123A (en) * | 2021-02-01 | 2021-06-11 | 中山未名海济生物医药有限公司 | Method for detecting tromethamine content |
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Application publication date: 20180309 |