CN106018336A - Method for near infrared spectral analysis technology-based monitoring of human albumin acetic acid buffer deposition process - Google Patents
Method for near infrared spectral analysis technology-based monitoring of human albumin acetic acid buffer deposition process Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 127
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 53
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 53
- 238000005516 engineering process Methods 0.000 title claims abstract description 13
- 238000010183 spectrum analysis Methods 0.000 title claims abstract description 11
- 238000012544 monitoring process Methods 0.000 title claims abstract description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 title claims abstract 19
- 239000000872 buffer Substances 0.000 title abstract description 7
- 238000005137 deposition process Methods 0.000 title abstract 5
- 239000008351 acetate buffer Substances 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 33
- 238000001228 spectrum Methods 0.000 claims description 30
- 238000001556 precipitation Methods 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- 238000002329 infrared spectrum Methods 0.000 claims description 11
- 238000004088 simulation Methods 0.000 claims description 9
- 238000005259 measurement Methods 0.000 claims description 7
- 238000007796 conventional method Methods 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 2
- 229960000583 acetic acid Drugs 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 238000013494 PH determination Methods 0.000 abstract 1
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- 238000007427 paired t-test Methods 0.000 description 5
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- 238000003556 assay Methods 0.000 description 4
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- 108010088751 Albumins Proteins 0.000 description 1
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- 238000004497 NIR spectroscopy Methods 0.000 description 1
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- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
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- 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
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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- 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
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- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
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- 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
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N2021/3595—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using FTIR
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Abstract
The invention discloses a method for near infrared spectral analysis technology-based monitoring of a human albumin acetic acid buffer deposition process. The method comprises building a model for determining HA content in a human albumin acetic acid buffer deposition process through a near infrared spectral analysis technology, building a model for pH determination and carrying out process determination on HA content and pH in a human albumin acetic acid buffer deposition process through the models. The method can realize process determination of HA content and pH in a human albumin acetic acid buffer deposition process and can timely adjust an acetic acid buffer adding rate and accurately and timely determine a reaction terminal point according to HA content change.
Description
Technical field
The present invention relates to a kind of method based on near-infrared spectral analysis technology monitoring human albumin's acetate buffer solution precipitation process.
Background technology
Human albumin (HA) is the protein that in human plasma, content is the highest, accounts for the 40%-60% of plasma protein.HA is facing
Bed is used for treating various disease, including hypovolaemia, hypoalbuminemia, suffer a shock, burn, perform the operation lose blood, wound, slow
Property hepatopathy, nutritional support, critical patient recovery etc..The isolated from human plasma mostly due to HA, raw material sources are deficient,
The situation that supply falls short of demand often occurs in market.
The production of HA at present uses cold ethanol method to separate from human plasma substantially, due to put in the industrialized production of HA
Blood plasma enormous amount (typically with ton measure), the volume of retort is the biggest, huge volume to concentration of alcohol, pH regulator and
The mensuration of HA content brings a lot of technical difficult point, and endpoint pH determines the bar needing to be less than 10% at ethanol content
Measure at room temperature condition with pH meter under part.Thus be monitored in the mode of actual industrial production process many employings off-line and judge
Reaction end, greatly reduces production efficiency.And offline mode lags behind production process, seriously constrain carrying of HA yield
The high lifting with product quality.
Near-infrared spectral analysis technology (NIRS) through development in recent years, be widely used at present petrochemical industry, pharmacy and
The fields such as Nicotiana tabacum L., and achieve good economic benefit.But it is white at monitoring human blood to yet there are no near-infrared spectral analysis technology both at home and abroad
Albumen acetic buffer precipitation process or the report in terms of setting up correlation model.
Summary of the invention
For above-mentioned prior art, it is an object of the invention to provide a kind of based on near-infrared spectral analysis technology monitoring human albumin
The method of acetate buffer solution precipitation process, the method for the present invention can effectively realize in human albumin's acetate buffer solution precipitation process
The process of HA content and pH value measures, in order to immediately adjust according to the changes of contents of HA add acetate buffer solution speed and can standard
Really judge reaction end (pH value reaches between 4.6-4.7, is i.e. reaction end during albumin isoelectric point, IP) timely, improve production
Efficiency.
For achieving the above object, the present invention uses following technical proposals:
Method based on near-infrared spectral analysis technology monitoring human albumin's acetate buffer solution precipitation process provided by the present invention,
Comprise the steps:
(1) model mensuration for HA content is set up:
A simulation different batches human albumin's acetate buffer solution precipitation process also samples, and gathers the original near infrared spectrum of sample,
And use conventional method that HA content in sample is detected, obtain off-line index components chemical measurements；
The sample of step A is divided into calibration set and checking collection by B, and sample spectra carries out pretreatment, the most different variablees
The system of selection impact on modeling result, selects to participate in the variable of modeling, sets up the PLSR model mensuration for HA content；
C uses the predictive ability of the sample checking model of checking collection；
(2) mensuration of model pH value is set up:
A simulation different batches human albumin's acetate buffer solution precipitation process also samples, and gathers the original near infrared spectrum of sample,
And use conventional method that ethanol content in sample is detected, and be the sample of 10% according to measurement result preparation ethanol content,
Measure the pH value of sample；
The sample of step A is divided into calibration set and checking collection by B, sample spectra carries out pretreatment, and carries out modeling interval
Investigate, set up the PLSR model mensuration for pH value；
C uses the predictive ability of the sample checking model of checking collection；
(3) HA of human albumin's acetate buffer solution precipitation process is contained by the model utilizing step (1) and step (2) to set up
Amount and pH carry out process mensuration.
Said method, in step (1), described simulation different batches human albumin's acetate buffer solution precipitation process also samples, tool
Body method is: the Adding Way of every a batch of acetate buffer solution is: every 4min adds an acetate buffer solution, front 9 times every time
Add 0.2mL, add 0.8mL latter 6 times every time, before adding acetate buffer solution, sample 1mL centrifugal treating every time；Simulation at least 8
Individual batch.
Said method, in step (1), gathers spectrum, near infrared spectrum with Antaris II Fourier Transform Near Infrared instrument
Acquisition condition be: spectral resolution is 8cm-1, scanning times is 32, and spectral region is 10000-4000cm-1。
Said method, in step (1), described PLSR model of setting up is during the mensuration of HA content, and introducing is reduced
The thought of concentration ranges, removes the sample spot that HA changes of contents is inconspicuous and less to model application effect, retains changes of contents
Model set up by the sample that trend is big, thus improves accuracy and the effectiveness of model.
Preferably, participating in front 11 sample points that sample is each batch of modeling, the content range of HA is 1.22-13.00g/L.
Said method, in step (1), it is smooth and equal that the method for described sample spectra pretreatment is preferably first derivative SG21 point
Value centralization.
Said method, in step (1), the described variable selecting participation modeling method particularly includes: with RMSECV+RMSEP
Be worth as the evaluation index of model, investigate correlation coefficient process (CC), successive projection method (SPA), without information variable method of elimination (UVE),
Compete adaptive weighted method (CARS), stability compete adaptive weighted method (SCARS), forward direction partial least square method (FiPLS),
Moving window partial least square method (MWPLS), reverse partial least square method (BiPLS) and distinct methods are combined with SPA
The method impact on modeling result, final preferably Variable Selection is: based on SPA associated with method, select to participate in modeling
Variable be 35.
Said method, in step (2), described simulation different batches human albumin's acetate buffer solution precipitation process also samples, tool
Body is: simulate 6 batch acetate buffer solution precipitation processes, obtains 74 pH value sample between 4.50-5.55.
Said method, in step (2), sample is divided into calibration set and checking collection division methods include at random (RS) method,
Concentration gradients method, KS method, SPXY method and Duplex method；Preferably, division methods is Duplex method, the calibration set of selection
49, sample, checking 25, sample of collection.
Said method, in step (2), the method for described sample spectra pretreatment includes: Orthogonal Signal Correction Analyze (OSC),
Order derivative SG15 point is smooth, second dervative SG15 point is smooth, OSC+ first derivative SG15 point is smooth, OSC+ second dervative
SG15 point is smooth, the smooth+OSC of first derivative SG15 point, the smooth+OSC of second dervative SG15 point.
Preferably, the method for described sample spectra pretreatment is the smooth+OSC of first derivative SG15 point.
Said method, in step (2), the spectrum range investigating out participation modeling is 4613-4000cm-1、6538-5404cm-1、
10000-7355cm-1。
It should be noted that NIRS is a kind of indirect determination technology, the linear relationship of one-level sample has the biggest for model result
Impact, sample used in the present invention, the difficulty that its level one data stably measures is big, and the foundation for corresponding model brings one
Fixed difficulty；And, protein structure is complicated, has absorption in whole near infrared spectrum district, thus results in the choosing of characteristic interval
Select more difficulty；Additionally, normal saline has strong absorption near infrared spectrum region, the spectrum letter of other materials can be covered
Breath, therefore, the extraction of effective information is also the key issue that model is set up.
Comprise in the original spectrogram of the near infrared spectrum gathered is not necessarily all useful information, in order to ensure sample spectra
Differentiating, the data of spectrum typically have hundreds of to thousands of data points, but the information that in spectrum district, each data point comprises differs,
Need spectrum is carried out pretreatment, and use compression algorithm spectroscopic data, select partial spectrum district to be used for setting up number in full spectrum
Learn model.And chemistry of based on material, the information of physical arrangement and the requirement to spectral instrument, the interval of spectrum is examined
It is necessary for examining, and is not elective.Founding mathematical models needs to be investigated by concrete test, constantly
Carry out choice of parameters, parameter optimization, stability and the most reasonable model of reliability could be obtained.
Based on this, the rating model for HA content that the present invention is set up and the model measured for pH value, due to sample
During the difficulty that the character of itself is brought by the foundation of corresponding near-infrared spectroscopy, and model foundation, pre-for spectrum
Processing method, the system of selection of spectral band, different Variable Selection, etc., it is required for technical staff according to actual sample
System carries out many-sided multi-angle and investigates, the category that the difficulty existing for this process selects far beyond those skilled in the art's routine.
Beneficial effects of the present invention:
The present invention utilizes near-infrared spectral analysis technology to establish a kind of human albumin acetate buffer solution precipitation process of monitoring first
Method.The process that can effectively realize HA content and pH value in human albumin's acetate buffer solution precipitation process measures, and can
Immediately adjust according to HA changes of contents during the course and add acetate buffer solution speed judgement reaction end accurately and timely, improve
Production efficiency.
Accompanying drawing explanation
Fig. 1 is the acetate buffer solution precipitation process original near infrared light spectrogram recorded during HA assay model is set up；
Fig. 2 is to participate in the variable that HA assay model is set up；
Fig. 3 is HA content prediction value and reference value comparison diagram；
Fig. 4 is the acetate buffer solution precipitation process original near infrared light spectrogram recorded during pH value quantitative determination model is set up；
Fig. 5 is to participate in the variable that pH value quantitative determination model is set up；
Fig. 6 is pH value predictive value and reference value comparison diagram.
Detailed description of the invention
The present invention is further illustrated in conjunction with the embodiments, it should explanation, the description below merely to explain the present invention,
Its content is not defined.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channel.
Embodiment 1: for the foundation of the rating model of HA content
(1) acetate buffer solution precipitation process: take supernatant (Shandong Taibang Biological Product Co., Ltd. after 100mL FIV filter pressing
There is provided) it is placed in round-bottomed flask, then round-bottomed flask is placed in the low-temp reaction instrument of temperature constant；Process samples 1mL before starting
Then starting to drip acetate buffer solution, the feed postition of acetate buffer solution is to add 0.2mL for first 9 times every time, adds 0.8 latter 6 times every time
mL；During every 4min add an acetate buffer solution and sample 1mL and do centrifugal treating, repeat altogether 8 batches.Closely
The collection of infrared spectrum: the transmission module of Antaris II Fourier Transform Near Infrared instrument carries out the collection of original near infrared spectrum.
Light path 4mm, spectral region 10000-4000cm-1, resolution is 8cm-1, scanning times 32；The most once carry on the back
The collection of scape, using air as reference.The mensuration of HA content: the assay method of HA content is BCG method, uses commercially available
Test kit is measured.
(2) introduce and reduce the thought of concentration ranges, will inconspicuous and less to the model application effect sample of HA changes of contents
Point removes, and model set up by the sample retaining changes of contents trend big, thus improves accuracy and the effectiveness of model.Preferably build
Apperance product are front 11 sample points of each batch, and the content range of HA is 1.22-13.00g/L.Table 1 for bulk sample product modeling and
Locally sample modeling result contrast, as can be seen from Table 1, accuracy and the effectiveness of locally sample modeling are significantly improved.
Spectrum uses multiple Variable Selection to select to participate in after the smooth process with average centralization of first derivative SG 21
The variable of modeling, table 2 be different Variable Selections, wherein based on be combined with SPA refer to CC-SPA, UVE-SPA,
35 altogether of the method choice such as CARS-SPA, SCARS-SPA, FiPLS-SPA, MWPLS-SPA, BiPLS-SPA
Variable combines.Using RMSECV+RMSEP value as the evaluation index of model, its value the least explanation model is the best.Result
Display BiPLS-SPA obtains minimum result, but model exists Expired Drugs, therefore based on 35 with SPA combination method selection
Individual variable modeling has obtained optimal model result, the R of modelc 2、Rp 2, RMSEC, RMSECV, RMSEP be respectively
0.977、0.978、0.5971g/L、0.7038g/L、0.5893g/L。
Table 1 bulk sample product and local sample modeling result compare
| Modeling sample | Rc 2 | Rp 2 | RMSECV(g/L) | RMSEP(g/L) |
| Bulk sample product | 0.733 | 0.740 | 2.5402 | 2.3755 |
| Locally sample | 0.890 | 0.838 | 1.6075 | 1.5692 |
Table 2 different Variable Selection modeling contrast
(3) investigate the predictive ability of model with the collection sample of testing of 3 batches, utilize paired t-test to carry out statistical analysis.By
Table 3 paired t-test statistical result understands, and average and the standard deviation of the result that NIRS method obtains with traditional BCG method connect very much
Closely, the P=0.374 obtained under the confidence limit of 95% > 0.05, illustrate to there is not significant difference between two kinds of method measurement results,
The alternative traditional method of NIRS method carries out the quick mensuration of acetate buffer solution precipitation process HA content.
Table 3 checking collection sample paired t-test statistical result
Embodiment 2: for the foundation of pH value rating model
(1) acetate buffer solution precipitation process: simulate 6 batches of acetate buffer solution precipitation processes, in addition to the 6th batch, other 5 batches of acid are sunk
Step keeps consistent with the process in embodiment 1 (1).6th batch is added ethanol 1ml after acid adding 13 times every time, mends altogether
Add 2 times, remaining condition with front 5 batches the most consistent.Every sub-sampling 1.2ml also does centrifugal treating.Original near infrared spectra collection bar
Part is identical with in embodiment 1 (1), gas chromatography determination sample ethanol content, is 10% (volume according to content preparation ethanol
Mark) sample, pH meter measure sample pH value.There are the pH value of 74 samples, for the foundation of quantitative model,
Table 4 is sample pH information table.
Table 4 sample pH information table
| Numbering | A collection of | Two batches | Three batches | Four batches | Five batches | Six batches |
| 3 | 5.49 | 5.55 | 5.43 | —— | —— | —— |
| 4 | 5.34 | 5.32 | 5.31 | —— | 5.43 | 5.38 |
| 5 | 5.24 | 5.30 | 5.21 | 5.28 | 5.34 | 5.25 |
| 6 | 5.15 | 5.23 | 5.14 | 5.17 | 5.25 | 5.18 |
| 7 | 5.09 | 5.11 | 5.07 | 5.06 | 5.17 | 5.11 |
| 8 | 5.04 | 5.08 | 5.00 | 5.03 | 5.09 | 5.06 |
| 9 | 4.99 | 5.02 | 4.96 | 4.98 | 5.05 | 5.01 |
| 10 | 4.94 | 4.89 | 4.84 | 4.85 | 4.92 | 4.86 |
| 11 | 4.80 | 4.80 | 4.75 | 4.76 | 4.79 | 4.77 |
| 12 | 4.74 | 4.70 | 4.67 | 4.65 | 4.71 | 4.68 |
| 13 | 4.65 | 4.62 | 4.62 | 4.63 | 4.64 | 4.60 |
| 14 | 4.60 | 4.61 | 4.55 | 4.57 | 4.58 | 4.60 |
| 15 | 4.55 | 4.56 | 4.50 | 4.52 | 4.54 | 4.55 |
(2) for obtaining the sample sets division result of optimum, random (RS) method, concentration gradients method, KS method, SPXY have been investigated
The sample sets division methods impacts on model such as method, Duplex method, table 5 is sample sets division information, and table 6 is different sample sets
Division methods modeling result, using RMSEP value as model-evaluation index, the best sample collection division methods of final selection be
Duplex, obtains 49 samples of calibration set and checking 25 samples of collection.
Table 5 sample sets division information table
The different sample sets division methods modeling result of table 6
| Method | Rc 2 | Rp 2 | RMSEC | RMSECV | RMSEP |
| RS | 0.828 | 0.749 | 0.1142 | 0.1478 | 0.1576 |
| Concentration gradients method | 0.830 | 0.794 | 0.1172 | 0.1736 | 0.1342 |
| KS | 0.825 | 0.712 | 0.1117 | 0.1400 | 0.1953 |
| SPXY | 0.782 | 0.762 | 0.1351 | 0.1584 | 0.1276 |
| Duplex | 0.855 | 0.814 | 0.1081 | 0.1597 | 0.1251 |
Full band range interior focusing spectrum preprocess method investigate, compared for OSC, first derivative SG15 point smooth, two
The smooth impact on modeling result of order derivative SG15 point, table 7 is the modeling result obtained after different pretreatments, with RMSEP
Value is as model-evaluation index, and the optimal preprocess method preferably gone out is the smooth+OSC of first derivative SG15 point.
Modeling result after the process of table 7 different pretreatments method
Interval for obtaining optimal modeling, the absorbance of sample spectra and background spectrum and spectrum is investigated, is removed raw
The background spectrum scope of reason saline is: 5400-5200cm-1And 7350-6950cm-1, the optimal spectrum range of absorbency of investigation is
0.63-2.88.Finally, selecting three spectrum ranges for modeling is 4613-4000cm-1、6538-5404cm-1、10000-7355
cm-1, model parameter is Rc 2=0.968, Rp 2=0.956, RMSEC=0.0512, RMSECV=0.0875, RMSEP=0.0594.
(3) concentrating 25 samples to verify the predictive ability of model with checking, table 8 is the statistics knot of paired t-test
Really, it is seen that the result average that the meansigma methods of pH meter measurement result obtains with NIRS is identical.Under the confidence limit of 95%,
P=0.847 > 0.05, illustrates that the result that the measurement result of pH meter and near-infrared model measure does not has significant difference it was confirmed NIRS
For acid sink process pH value measure effectiveness.
Table 8 paired t-test statistical result
Embodiment 1 and embodiment 2 show, the present invention set up the model for HA assay and for pH value measure model
Accurately, reliably, it is possible to the process effectively realizing HA content and pH value in human albumin's acetate buffer solution precipitation process measures.
Although the detailed description of the invention of the present invention is described by the above-mentioned accompanying drawing that combines, but not limit to scope
System, one of ordinary skill in the art should be understood that on the basis of technical scheme, and those skilled in the art need not pay
Go out various amendments or deformation that creative work can make still within protection scope of the present invention.
Claims (10)
1. a method based on near-infrared spectral analysis technology monitoring human albumin's acetate buffer solution precipitation process, its feature exists
In, comprise the steps:
(1) model mensuration for HA content is set up:
A, simulation different batches human albumin's acetate buffer solution precipitation process also sample, and gather the original near infrared spectrum of sample,
And use conventional method that HA content in sample is detected, obtain off-line index components chemical measurements；
B, the sample of step A is divided into calibration set and checking collection, sample spectra is carried out pretreatment, the most different changes
The amount system of selection impact on modeling result, selects to participate in the variable of modeling, sets up the PLSR model mensuration for HA content；
The predictive ability of model verified by C, the sample using checking to collect；
(2) mensuration of model pH value is set up:
A, simulation different batches human albumin's acetate buffer solution precipitation process also sample, and gather the original near infrared spectrum of sample,
Use conventional method that ethanol content in sample is detected, and be the sample of 10% according to measurement result preparation ethanol content, survey
The pH value of random sample product；
B, the sample of step A is divided into calibration set and checking collection, sample spectra is carried out pretreatment, and modeling interval is entered
Row is investigated, and sets up the PLSR model mensuration for pH value；
The predictive ability of model verified by C, the sample using checking to collect；
(3) HA of human albumin's acetate buffer solution precipitation process is contained by the model utilizing step (1) and step (2) to set up
Amount and pH carry out process mensuration.
2. the method for claim 1, it is characterised in that in step (1), described PLSR model of setting up is for HA
During the mensuration of content, introduce the thought reducing modeling district, remove HA changes of contents inconspicuous and to model application effect
Less sample spot, model set up by the sample retaining changes of contents trend big.
3. method as claimed in claim 2, it is characterised in that in step (1), described PLSR model of setting up is for HA
During the mensuration of content, participating in front 11 sample points that sample is each batch of modeling, the content range of HA is
1.22-13.00g/L。
4. the method for claim 1, it is characterised in that in step (1), the method for described sample spectra pretreatment is
First derivative SG21 point smooths and average centralization.
5. the method for claim 1, it is characterised in that in step (1), Variable Selection is: based on SPA
Associated with method, select participate in modeling variable be 35.
6. the method for claim 1, it is characterised in that in step (2), the described simulation white egg of different batches human blood
White vinegar acid buffer precipitation process also samples, particularly as follows: simulation 6 batch acetate buffer solution precipitation processes, obtains 74 pH value
Sample between 4.50-5.55.
7. the method for claim 1, it is characterised in that in step (2), the division methods of sample sets be randomized,
At least one in concentration gradients method, KS method, SPXY method and Duplex method, preferably Duplex method.
8. method as claimed in claim 7, it is characterised in that in step (2), the method for described sample spectra pretreatment is
OSC, first derivative SG15 point is smooth, second dervative SG15 point is smooth, OSC+ first derivative SG15 point is smooth, OSC+
Second dervative SG15 point is smooth, the smooth+OSC of first derivative SG15 point and second dervative SG15 point smooth in+OSC extremely
Few one.
9. method as claimed in claim 8, it is characterised in that in step (2), the method for described sample spectra pretreatment
For the smooth+OSC of first derivative SG15 point.
10. the method for claim 1, it is characterised in that in step (2), investigates out the spectrum range of participation modeling
For 4613-4000cm-1、6538-5404cm-1、10000-7355cm-1。
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