CN106226256A - A kind of surfactant quantitative detecting method - Google Patents

A kind of surfactant quantitative detecting method Download PDF

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Publication number
CN106226256A
CN106226256A CN201610841839.7A CN201610841839A CN106226256A CN 106226256 A CN106226256 A CN 106226256A CN 201610841839 A CN201610841839 A CN 201610841839A CN 106226256 A CN106226256 A CN 106226256A
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surfactant
light absorption
protein
absorption value
tween80
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田志刚
程永凤
郑晓东
魏海明
刘玉玲
孙汭
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University of Science and Technology of China USTC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning

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  • Spectroscopy & Molecular Physics (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

The open a kind of surfactant quantitative detecting method of the present invention, a kind of detects the method for surfactant in protein solution.The method includes being optionally added into protein precipitant protein precipitation, separates albumen and surfactant；Then by removing the surfactant solution of removing protein after drying, redissolve；Its light absorption value is detected with surfactant solution after being mixed by Coomassie brilliant blue dye liquor again；Finally calculate the amount of surfactant.The features such as it is simple that the present invention has operating procedure, and detection is quickly, highly sensitive, high flux, are particularly well-suited to the quantitative of surfactant in pharmaceutical grade protein.

Description

A kind of surfactant quantitative detecting method
Technical field
A kind of method that the present invention relates to Coomassie brilliant blue dye liquor detection by quantitative surfactant.
Background technology
In the preparation of pharmaceutical preparation, surfactant, because having hydrophilic and two kinds of groups of lipotropy, can significantly reduce Surface tension and be widely used, be the important adjuvant of acceptable emulsions, suspensoid, liposome etc..Such as, produced at Chinese medicine Journey, often adds polysorbate as the extraction of active substance in solubilizing agent and cosolvent, beneficially Chinese medicine.In bio-pharmaceuticals Cheng Zhong, particularly pharmaceutical grade protein often add poloxamer (poloxamer), polysorbate (polysorbate, Etc. Tween) its biological activity is affected for preventing protein aggregation.Additionally, in the virus removal technique of bio-pharmaceuticals, surface activity Agent such as polysorbate80 or triton x-100 (Triton X-100) are used to remove enveloped virus.
Recent studies have shown that surfactant-based pharmaceutic adjuvant such as Tween80 etc. may result in certain untoward reaction (Wu Yi etc., " pharmacology of pharmaceutic adjuvant Tween80, pharmacokinetics and analysis Advances in Methods ", Chinese Pharmaceutical Affairs, 2008,22 (8): 717-720；Marlene Isaksson,et al.Contant allery to Tween 80in an inhalation suspension.Contact Dermatitis,2001,44:313).Such as: Tween80 can produce retting conditions by inducing cell Effect, causes activity substance release, acute allergy occurs, and causes hepatotoxicity, peripheral nervous toxicity etc. no Good reaction.Accordingly, it would be desirable to the activating agent in medicine preparation is carried out quantitatively, to determine that it is in safety range.
The common method of the surfactant measured at present is that (Lv Peng etc. " live by surface for spectrophotography, liquid chromatography etc. Property agent detection method summary " daily chemical industry 2015,45 (11): 643-647).Its principle of spectrophotography is surface activity Agent (such as Tween80, sodium lauryl sulphate (SDS) etc.) can be anti-with organic dyestuff (such as ammonium cobalt rhodanate, methylene blue) The blue complex that should generate, this complex is soluble in organic solvent (such as dichloromethane, chloroform), and at 620nm Have obtained the maximum absorption, and in range of doses, its absorption value be dose-dependence (Chinese Pharmacopoeia 3 2015 editions, in State's standard GB/T/T 7494-1987).Although the method is simple, but needs the albumen in sample is carried out precipitate and separate, and And the complex of surfactant with organic dyestuff can not be extracted from aqueous solution by organic solvent completely, thus operation Error is bigger.
Another conventional method is high performance liquid chromatography, and with efficient liquid phase-evaporative light-scattering detector.This Class methods and results is more accurate, but the requirement to instrument and equipment and personnel is the highest.(M Hu,et al.High performance liquid chromatographic determination of polysorbate 80in pharmaceutical suspensions.J Chromatogr A,2003,984(2):233-236)。
Therefore, strongly need to provide a kind of and measured the side containing the surfactant in protein example by colorimetric analysis Method, wherein can by a kind of rapidly and simply in the way of eliminate the interference of protein.
Summary of the invention
A kind of method relating to Coomassie brilliant blue dye liquor detection by quantitative surfactant in the present invention, will coomassie bright After blue dye liquor and standard substance or sample mix, detect the light absorption value of its 630nm.Surfactant concentration and OD value with standard substance Make linear regression analysis, obtain regression equation.Utilize regression equation, calculate the content of surfactant in sample.
The principle of the method is that Coomassie brilliant blue dye liquor can react aobvious blue, at 630nm with surfactant There is obtained the maximum absorption, and its light absorption value is the most linear with the concentration of surfactant.
If containing protein in sample, because it can react with Coomassie brilliant blue, thus disturbing the detection of this method, therefore Need to use acetone precipitation such as to remove disturbance of protein.The solution removing isolating protein is removed protein precipitation institute by seasoning again Organic solvent.Therefore, the invention is particularly applicable to the quantitative of surfactant in protein example.
In particular it relates to the following:
1. the method detecting surfactant, said method comprising the steps of:
1) Coomassie brilliant blue dye liquor is mixed standing with surfactant solution；
2) detection sample light absorption value；
3) concentration of standard substance surfactant and itself and Coomassie brilliant blue dye liquor reacted standard substance extinction are utilized Value sets up linear regression equation；
4) utilize step 3) in the linear regression equation set up, according to step 2) in the sample light absorption value that obtains calculate table The amount of face activating agent.
2., according to the method described in 1, wherein said surfactant includes anion surfactant, such as dodecyl sulfur Acid sodium (SDS)；Cationic surfactant, such as benzalkonium chloride (DDBAC)；Nonionic surfactant, such as polysorbate (Tween)；High molecular surfactant, such as Polyethylene Glycol (PEG)；And the mixing of above-mentioned various surfactant.
3., according to the method described in 1, the detection wavelength of wherein said light absorption value is 600-650nm.
4., according to the method described in 1, the detection wavelength of wherein said light absorption value is 620-640nm.
5., according to the method described in 1, described linear regression equation is by calculating the light absorption value at 620-640nm and 440- The ratio of the light absorption value at 460nm, then carry out linear regression analysis with the concentration of surfactant, set up regression equation.
6., according to the method described in 1, described method is in step 1) the most optionally comprise the following steps before:
Add protein precipitant protein precipitation, albumen is separated with surfactant；And the surfactant that will separate Solution is dried, redissolves.
7., according to the method described in 6, wherein said protein precipitant agent includes acetone, methanol, ethanol or its combination in any.
A kind of method relating to quantitative surfactant of Coomassie brilliant blue dye liquor of the present invention, it comprises the following steps: The preparation of standard substance, the preparation of sample, mensuration light absorption value and gauging surface active agent content.
The preparation of 1 standard substance
Weigh surfactant, be configured to 4-6 concentration with water by certain Concentraton gradient.
The preparation of 2 samples
To nonprotein sample, can directly detect.If containing protein in sample, then with protein precipitant acetone As a example by remove by the following method.
Pre-cold acetone and sample at least more than 1h.Being added in 0.6ml sample by 1.4ml acetone and shake up, ice bath is overnight； 12000g, centrifugal 15min, collect supernatant.The acetone adding 1ml pre-cooling shakes up, ice bath 15min, centrifugal 12000g, 10min, receives Collection supernatant.Repeat to drill and make 1 time.After all of supernatant is dried removal acetone, adds 0.6ml water and redissolve.If surface activity Agent concentration is higher, can carry out the dilution of certain multiple.
3 measure light absorption value
Take standard substance and sample 0.1ml in 96 orifice plates, each add the mixing of 0.1ml Coomassie brilliant blue dye liquor, colour developing 10min, measures OD value at 630nm.
4 gauging surface active agent content
Surfactant concentration and OD value with standard substance make linear regression analysis, obtain regression equation.Utilize recurrence side Journey, calculates the content of surfactant in sample.
The method of the present invention has the advantages that
(1) present invention passes through colorimetric determination surface-active contents, easy and simple to handle, quick.
(2) present invention goes the interference of removing protein by the sedimentation method, is particularly well-suited to surfactant in pharmaceutical grade protein Quantitatively.
(3) present invention can detect with microwell plate, it is adaptable to the detection of large sample amount.
Accompanying drawing explanation
Fig. 1, Tween80 and Coomassie brilliant blue dye liquor reactive absorption spectrum change.
Fig. 2, the range of linearity of coomassie bright method detection Tween80.
Fig. 3, the ruggedness of coomassie bright method detection Tween80.
The standard curve (light absorption value method) of Fig. 4, Tween80.
The standard curve (ratio method) of Fig. 5, Tween80.
The standard curve of Fig. 6, SDS.
The standard curve of Fig. 7, DDBAC.
The standard curve of Fig. 8, PEG4000.
Detailed description of the invention
Following example will assist in those of ordinary skill in the art and are further appreciated by the present invention, but the most in any form Limit the present invention.Experimental technique in embodiment, if no special instructions, all uses this area routine techniques.Institute without specified otherwise Some chemical reagent are analytical pure, are commercial conventional reagent without all reagent of specified otherwise, can buy or by specification Preparation.
In the examples below, Coomassie brilliant blue dye liquor is purchased from Thermo company, Tween 80, BSA (bovine serum albumin In vain), Sigma-Aldrich company it is purchased from.DDBAC, SDS, PEG4000 are purchased from Shanghai Sheng Gong company.
Embodiment 1. variable concentrations Tween80 change of absorption spectrum in Coomassie brilliant blue dye liquor
1) prepared by Tween80 standard substance: the Tween80 solution of preparation 100,500,1000,5000 μ g/ml.
2) spectral scan: respectively take the Tween80 solution 0.5ml of variable concentrations, adds 0.5ml Coomassie brilliant blue dye liquor, mixed Even placement 10min, carries out continuous spectrum scanning at 350-700nm.
3) result is shown in Fig. 1, and the light absorption value at 600nm-650nm rises along with the increase of Tween80 concentration, both promptings Being proportionate, its peak value is 620-640nm.
The range of linearity of embodiment 2.Tween80 detection.
1) preparation of Tween80 standard substance: preparation 0,5,10,20,40,80,160,320 μ g/ml7 kind variable concentrations Tween80 solution.
2) OD value is measured: in the Tween80 solution of variable concentrations, add Coomassie brilliant blue dye liquor by 1: 1 (volume ratio), Each 0.2ml that draws is in 96 orifice plates, and mixing, develop the color 10min, measures OD value at 630nm.
3) result is shown in Fig. 2, by linear regression analysis, when Tween80 concentration range is at 0-80 μ g/ml, its correlation coefficient R2>0.998；When concentration range is at 0-160 μ g/ml and 0-320 μ g/ml, its coefficient R2It is respectively less than 0.998.This explanation is worked as When Coomassie brilliant blue concentration in solution is fixed, along with the increase of Tween80, the complex of Coomassie brilliant blue formation in combination The most gradually increasing, the OD value at 630nm the most gradually strengthens, and Tween80 concentration is linear with OD value.If but During Tween80 substantially excess, then both deflect away from linear relationship, also it is accomplished by improving the concentration of Coomassie brilliant blue.Therefore, exist Need in actually detected to estimate Tween80 concentration range, to determine optimal Coomassie brilliant blue usage amount；Or by high concentration Tween80 carries out the dilution of certain multiple.
The ruggedness of embodiment 3.Tween80 detection.
1) preparation of Tween80 standard substance: preparation 0,5,10,20,40,80,160,320 μ g/ml7 kind variable concentrations Tween80 solution.
2) OD value is measured: in the Tween80 solution of variable concentrations, press 1:1 (volume ratio) add Coomassie brilliant blue dye liquor, Each 0.2ml that draws is in 96 orifice plates, and mixing, develop the color 10min, 1.5h, 3h, 24h, measures OD value at 630nm.
3) after result is shown in Fig. 3, Tween80 and Coomassie brilliant blue dye liquor colour developing 24h, its OD value almost without change, this explanation Tween80 is sufficiently stable with the chromogenic reaction of Coomassie brilliant blue dye liquor.
The standard curve of embodiment 4 Tween80
1) prepared by standard substance: weigh Tween80, is configured to 0,20,40,60,80,100 g/ml6 concentration of μ with water, each Parallel 2 pipes of concentration.
2) measuring OD value: take the standard substance 0.1m of each concentration in 96 orifice plates, each to add 0.1ml Coomassie brilliant blue dye liquor mixed Even, the multiple hole of 2, each sample, develop the color 10min, measures OD value at 630nm and 450nm.
3) linear regression analysis: concentration and OD value with Tween80 make linear regression analysis, obtain regression equation.
4) result is shown in Fig. 4, and in the range of 0-100 μ g/ml, Tween80 concentration is the most linear with the OD value of 630nm, Coefficient R2=0.995.
5) if doing rectilinear regression with the ratio of the OD value of 630nm and 450nm with Tween80 concentration, its coefficient R2 =0.999.By these computational methods, its linear correlation is more preferably.See Fig. 5
The detection by quantitative of Tween80 in the protein sample of embodiment 5. variable concentrations
1) prepare protein-contg Tween80 solution: preparation containing 0.01,0.1,1, the Tween80 solution of 10mg/ml BSA, Tween80 content is 100 μ g/ml, 4 DEG C of more than pre-cooling 1h.
2) albumen precipitation: being added in 0.6ml sample by the acetone of 1.4ml pre-cooling and shake up, ice bath is overnight.12000g, centrifugal 15min, collects supernatant.The acetone adding 1ml pre-cooling shakes up, ice bath 15min, centrifugal 12000g, 10min, collects supernatant.Repeat Drill and make 1 time.After all of supernatant is dried removal acetone, adds 0.6ml water and redissolve.
3) prepared by standard substance: with embodiment 4.
4) OD value is measured: with embodiment 4.
5) linear regression analysis: with embodiment 4.Utilize regression equation, calculate the content of Tween80.
6) the results are shown in Table 1: after acetone precipitation removes removing protein, Tween80 can by accurate quantitative analysis, its precision < 6%, accurate Exactness is between-5.6%--1.7%.
Table 1 is the detection by quantitative of Tween80 in the albumen of variable concentrations
The detection by quantitative of embodiment 7 high concentration Tween80.
1) sample preparation: containing the Tween80 solution of 200 μ g/ml BSA, Tween80 concentration is 20,50,100,500, 1000μg/ml。
2) albumen precipitation: with embodiment 4, Tween80 concentration is after the sample of 500,1000 μ g/ml finally redissolves, respectively Dilute 5 and 10 times.
3) prepared by standard substance: with embodiment 4
4) OD value is measured: with embodiment 4
5) linear regression analysis: with embodiment 4.Utilize regression equation, calculate the content of Tween80.
6) the results are shown in Table 2: by dilution and albumen precipitation, the Tween80 of high concentration also can be by accurate quantitative analysis, its precision < 6.5%, accuracy is between-4.7%-5.0%.
The detection by quantitative of table 2 high concentration Tween80
The standard curve of embodiment 8 SDS.
1) prepared by standard substance: SDS water is configured to 4 concentration of 50,100,150,200 μ g/ml, each concentration parallel 2 Pipe.
2) colour developing: take in the standard substance 0.1m ELISA Plate of each concentration, each 0.1ml Coomassie brilliant blue dye liquor that adds mixes, often The multiple hole of 2, individual sample, develop the color 10min, measures OD value at 630nm and 450nm.
3) linear regression analysis: make linear regression analysis with the concentration of SDS and the ratio of 630nm/450nm, returned Equation.
4) result is shown in Fig. 6, and in the range of 50-200 μ g/ml, SDS concentration is the most linear with ratio, coefficient R2> 0.99。
The detection of SDS in embodiment 9 protein sample
1) preparing protein-contg SDS solution: the preparation SDS solution containing 0.1mg/ml BSA, SDS content is 100 μ g/ml, 4 DEG C more than pre-cooling 1h.
2) albumen precipitation: with embodiment 5.
3) prepared by standard substance: with embodiment 8.
4) OD value is measured: with embodiment 8.
5) linear regression analysis: with embodiment 8.Utilize regression equation, calculate the content of SDS.
6) the results are shown in Table 3: after acetone precipitation removes removing protein, SDS can be by accurate quantitative analysis, and its precision is 2.9%, accurately Degree is for-12.8%.
The standard curve of embodiment 10 DDBAC.
1) prepared by standard substance: DDBAC water is configured to 0,40,80,120,160,200 g/ml6 concentration of μ, Mei Genong Spend parallel 2 pipes.
2) colour developing: take in the standard substance 0.1m ELISA Plate of each concentration, each 0.1ml Coomassie brilliant blue dye liquor that adds mixes, often The multiple hole of 2, individual sample, develop the color 10min, measures OD value at 630nm and 450nm.
3) linear regression analysis: make linear regression analysis with the concentration of DDBAC and the ratio of 630nm/450nm, returned Return equation.
4) result is shown in Fig. 7, and in the range of 0-200 μ g/ml, SDS concentration is the most linear with ratio, coefficient R2> 0.99。
The detection of DDBAC in embodiment 11 protein sample
1) protein-contg SDS solution is prepared: the preparation DDBAC solution containing 0.1mg/ml BSA, DDBAC content is 100 μ g/ Ml, 4 DEG C of more than pre-cooling 1h.
2) albumen precipitation: with embodiment 5.
3) prepared by standard substance: with embodiment 10.
4) OD value is measured: with embodiment 10.
5) linear regression analysis: with embodiment 10.Utilize regression equation, calculate the content of DDBAC.
6) the results are shown in Table 3: after acetone precipitation removes removing protein, DDBAC can be by accurate quantitative analysis, and its precision is 7.2%, accurate Exactness is 8.6%.
The standard curve of embodiment 12 PEG4000.
1) prepared by standard substance: PEG4000 water is configured to 0,20,40,60,80,100mg/ml6 concentration, Mei Genong Spend parallel 2 pipes.
2) colour developing: take in the standard substance 0.1m ELISA Plate of each concentration, each 0.1ml Coomassie brilliant blue dye liquor that adds mixes, often The multiple hole of 2, individual sample, develop the color 10min, measures OD value at 630nm and 450nm.
3) linear regression analysis: make linear regression analysis with the concentration of PEG4000 and the ratio of 630nm/450nm, obtain Regression equation.
4) result is shown in Fig. 8, and in the range of 0-100mg/ml, PEG4000 concentration is the most linear with ratio, correlation coefficient R2>0.99。
The detection of PEG4000 in embodiment 13 protein sample
1) protein-contg PEG4000 solution is prepared: the preparation PEG4000 solution containing 0.1mg/ml BSA, PEG4000 content For 50mg/ml, 4 DEG C of more than pre-cooling 1h.
2) albumen precipitation: with embodiment 5.
3) prepared by standard substance: with embodiment 11.
4) OD value is measured: with embodiment 11.
5) linear regression analysis: with embodiment 11.Utilize regression equation, calculate the content of PEG4000.
6) the results are shown in Table 3: after acetone precipitation removes removing protein, PEG4000 can be by accurate quantitative analysis, and its precision is 0.7%, Accuracy is 4.4%.
The detection by quantitative of table 3 different surfaces activating agent

Claims (7)

1. the method detecting surfactant, said method comprising the steps of:
1) Coomassie brilliant blue dye liquor is mixed standing with surfactant solution；
2) detection sample light absorption value；
3) concentration and its standard substance light absorption value reacted with Coomassie brilliant blue dye liquor that utilize standard substance surfactant are built Vertical linear regression equation；
4) utilize step 3) in the linear regression equation set up, according to step 2) in the sample light absorption value that obtains calculate surface and live The amount of property agent.
Method the most according to claim 1, wherein said surfactant includes anion surfactant, such as dodecane Base sodium sulfate (SDS)；Cationic surfactant, such as benzalkonium chloride (DDBAC)；Nonionic surfactant, such as polysorbate Ester (Tween)；High molecular surfactant, such as Polyethylene Glycol (PEG)；And the mixing of above-mentioned various surfactant.
Method the most according to claim 1, the detection wavelength of wherein said light absorption value is 600-650nm.
Method the most according to claim 1, the detection wavelength of wherein said light absorption value is 620-640nm.
Method the most according to claim 1, described linear regression equation by calculate 620-640nm place light absorption value and The ratio of the light absorption value at 440-460nm, then carry out linear regression analysis with the concentration of surfactant, set up regression equation.
Method the most according to claim 1, described method is in step 1) the most optionally comprise the following steps before:
Add protein precipitant protein precipitation, albumen is separated with surfactant；And the surfactant solution that will separate After drying, redissolve.
Method the most according to claim 6, wherein said protein precipitant agent includes acetone, methanol, ethanol or it is any Combination.
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CN109254086A (en) * 2017-06-20 2019-01-22 海南先声药业有限公司 A kind of HPLC detection method of lauryl sodium sulfate in cefaclor dry suspensoid sample

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