CN109387584A - One kind improving olanzapine in treatment schizophrenia based on aripipazole and causes Anomalous lipid metablism patient blood plasma metabonomic analysis methods - Google Patents
One kind improving olanzapine in treatment schizophrenia based on aripipazole and causes Anomalous lipid metablism patient blood plasma metabonomic analysis methods Download PDFInfo
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Abstract
The present invention provides a kind of based on aripipazole improvement olanzapine in treatment schizophrenia cause Anomalous lipid metablism patient blood plasma metabonomic analysis methods, Patients with Chronic Schizophrenia blood plasma including collection olanzapine in treatment merges the Patients with Chronic Schizophrenia plasma sample of aripipazole treatment with Olanzapine, through chromatography post separation after processing, and it is analyzed using Mass Spectrometer Method, instrument initial data is extracted and is aligned after carrying out LC/MS detection and analysis again, the data for obtaining noiseless interference, can be used for statisticalling analyze.Data are analyzed to obtain metabolism group profile, establish double cross verifying screening model, screening individually merges the lipid difference specific marker object of aripipazole treatment patient with Olanzapine and Olanzapine.
Description
Technical field
The present invention relates to technical field of analytical chemistry, and in particular to one kind improves olanzapine in treatment spirit based on aripipazole
Split disease causes Anomalous lipid metablism patient blood plasma metabonomic analysis methods.
Background technique
Two generation antipsychotic drugs treatment schizophrenia often occurs together Anomalous lipid metablism, occurs that fat even other are female
Side effect, such as lactation, Adam's apple become smaller, ED, thus reduce the compliance and quality of life of patient.Excessive body
Cardiovascular disease and the relevant pathogenicity rate of diabetes and the death rate can be can increase by increasing again, increased schizophrenia and suffered from Metabolic syndrome
The risk of sign.Olanzapine is most common two generations antipsychotic drug, and side effect is more so.Aripipazole is used as and controls
It treats one of schizoid mainstay, in clinic, use is merged with Olanzapine, can largely improve exclusive use
Anomalous lipid metablism caused by olanzapine in treatment schizophrenia not only mitigates the pain of patient, also increases the compliance of patient medication
Property.
Metabolism group is a kind of high sensitivity, and method for high-flux analysis can be used for studying low molecule under pathological and physiological condition
Measure the changing features of metabolin.Iipidomic is an important branch of metabolism group, it is intended to detailed analysis lipid species and its
Multiple action in biosystem.Ultra performance liquid chromatography-mass spectrum (LC/MS) joint technology is as wide in iipidomics analysis
One of the technology of general use, has the characteristics that high separation, high speed and high sensitivity.
This technology method and existing patent, application number 201310568472.2, application publication number CN103592389A are close
Seemingly, but not quite identical, technical method uses serum before rather than blood plasma is as detection sample: blood plasma negative ions total amount phase
Deng, keep electroneutral, ion concentration fluctuation biological metabolism is influenced it is significant, while blood plasma physicochemical property it is relative constant be inner ring
The primary performance of border stable state, the two show bigger to metabolism group Clinical significance of detecting using blood plasma；Schizophrenia suffers from crowd not
It is disconnected to increase, it is not retrieved similar to patent before this, therefore develop such technical method using new technology and novel sample, there is weight
Want meaning.
Technical solution
It is provided by the invention that olanzapine in treatment schizophrenia cause Anomalous lipid metablism patient blood plasma is improved based on aripipazole
Metabonomic analysis methods can synthetically embody Olanzapine and merge aripipazole treatment Patients with Chronic Schizophrenia metabolism production
The variation situation of object can be used for being used alone Olanzapine, merge aripipazole treatment Patients with Chronic Schizophrenia improvement metabolism
The early prediction of syndrome and prognosis provide advantageous technical support.
Based on this, the present invention provides a kind of based on aripipazole improvement olanzapine in treatment schizophrenia cause Anomalous lipid metablism
Patient's blood plasma metabonomic analysis methods, specifically include:
The first step, sample preparation；
Second step, ultra performance liquid chromatography-Mass Spectrometer Method analysis；
Third step, data analysis obtain otherness metabolin, are identified and verified.
The first step is further specially that collection is applied alone Olanzapine and shares the blood of Aripiprazole schizophreniac
Sample is starched, after processing for carrying out ultra performance liquid chromatography-Mass Spectrometer Method analysis, the processing method of the plasma sample are as follows:
By 100 μ l blood plasma and 900 μ l mixed with propylene glycol, it is ultrasonically treated 10 minutes after being vortexed more than 10 seconds, -20 DEG C of mixture to freeze 1 small
When, it is then centrifuged 10 minutes, collects upper layer (800 μ l), as sample under the action of the centrifugal force of 9500g.
Mobile phase A in ultra performance liquid chromatography in the second step-Mass Spectrometer Method analysis is mixed using acetonitrile and water
It closes solution and makees solvent, the two volume ratio is 95:5, is adopted in the mobile phase A containing 0.1% formic acid and 10mM ammonium formate, Mobile phase B
Solvent is made according to the mixed solution that volume ratio 9: 1 obtains with propylene glycol and acetonitrile, which contains 0.1% formic acid and 10mM first
Sour ammonium.
The third step is further specially that instrument initial data is extracted and be aligned after being tested and analyzed, and obtains nothing and makes an uproar
Sound interference, the data that can be used for statistical analysis.It is examined using orthogonal partial least squares discriminant analysis (0PLS-DA) and independent sample T
Test and data analyzed, wherein OPLS-DA according to OPLS VIP value excavate difference metabolin, according to independent sample T examine with
VIP value is excavated respectively, respectively excavates one group of difference metabolin, and 2 groups of difference lipid moleculars are obtained, and is compared by intersecting, is obtained
Shared difference lipid molecular.Again to shared lipid molecular and patient's Olanzapine, aripipazole drug concentration and biochemical indicator (ALT,
AST, GGT, TC, TG, LDL) correlation analysis is done, it is tested by the identical property of the pathology of analysed for relevance result and clinical symptoms
Card obtains metabolic disorder prediction lipid molecular.
Beneficial effect
In previous research, metabolin screening model is established frequently with cross validation method, feature is by the metabolism group of sample
Data are divided into two parts, and a portion is used to establish model, and another part is used to the predictive ability of evaluation model, according to pre-
The fine or not preference pattern parameter of survey ability, so that it is determined that final mask.But there are certain defects for this modeling method: differentiating mould
Type predictive ability is not independent sample.Because all samples can all be used to build up model in modeling process, the mistake
There are a degree of nonindependences for journey, disturb the differentiation of model prediction ability and the estimation of model predictive error.Lack
The modeling section of full independence leads to the possibility of over-fitting, and such model cannot reflect the letter of metabolism group data truly and effectively
Breath, so that deviation occurs in the screening to marker.
The present invention extracts the excellent characteristics of cross validation, using orthogonal partial least squares discriminant analysis (OPLS-DA) and solely
The shortcomings that vertical sample T inspection carries out intersection comparison, avoid cross validation method nonindependence to data analysis result, is applied
In the research of lipid-metabolism marker that aripipazole merges that olanzapine in treatment has larger improvement result to Anomalous lipid metablism, lead to
It crosses true with the correlation analysis of patient's Olanzapine, aripipazole drug concentration and biochemical indicator (ALT, AST, GGT, TC, TG, LDL)
Difference lipid-metabolism object is recognized and verifies, thus different to lipid metaboli comprehensively, synthetically investigating aripipazole merging olanzapine in treatment
While often having the lipid-metabolism correlation marker of larger improvement result, the reasonable screening of marker is realized.This method is to sharing
Aripipazole, which treats Patients with Chronic Schizophrenia, improves the early prediction and the advantageous technology branch of prognosis offer of metabolic syndrome
It holds.
Attached drawing
Fig. 1 improves olanzapine in treatment schizophrenia using aripipazole provided by the invention and causes Anomalous lipid metablism patient blood
Starch metabonomic analysis methods flow chart.
Specific embodiment
The present invention provides a kind of based on aripipazole improvement olanzapine in treatment schizophrenia cause Anomalous lipid metablism patient blood
Metabonomic analysis methods are starched, the Patients with Chronic Schizophrenia blood plasma including collecting olanzapine in treatment merges A Li with Olanzapine
The Patients with Chronic Schizophrenia plasma sample for sending azoles to be treated through chromatography post separation after processing, and is analyzed using Mass Spectrometer Method, then
Instrument initial data is extracted and is aligned after carrying out LC/MS detection and analysis, the number for obtaining noiseless interference, can be used for statisticalling analyze
According to.Data are analyzed to obtain metabolism group profile, establish double cross verifying screening model, Olanzapine and Austria are individually used in screening
The flat lipid difference specific marker object for merging aripipazole treatment patient of nitrogen.
This method specifically includes:
The first step, sample preparation；
Second step, ultra performance liquid chromatography-Mass Spectrometer Method analysis；
Third step, data analysis obtain otherness metabolin, are identified and verified.
The first step is further specially that collection is applied alone Olanzapine and shares the blood of Aripiprazole schizophreniac
Sample is starched, after processing for carrying out ultra performance liquid chromatography-Mass Spectrometer Method analysis, the processing method of the plasma sample are as follows:
By 100 μ l blood plasma and 900 μ l mixed with propylene glycol, it is ultrasonically treated 10 minutes after being vortexed more than 10 seconds, -20 DEG C of mixture to freeze 1 small
When, it is then centrifuged 10 minutes, collects upper layer (800 μ l), as sample under the action of the centrifugal force of 9500g.
Place blank sample and Quality control samples in every 5 samples, the blank sample be propylene glycol and water according to
The cooperation of volume ratio 9: 1 obtains, and Quality control samples are by merging all plasma samples according to being configured to after equal part and blank sample
Same volume obtains.
Mobile phase A in ultra performance liquid chromatography in the second step-Mass Spectrometer Method analysis is mixed using acetonitrile and water
It closes solution and makees solvent, the two volume ratio is 95: 5, contains 0.1% formic acid and 10mM ammonium formate, and Mobile phase B uses propylene glycol and second
Nitrile makees solvent according to the mixed solution that volume ratio 9: 1 obtains, and contains 0.1% formic acid and 10mM ammonium formate.
The third step is further specially that instrument initial data is extracted and be aligned after being tested and analyzed, and obtains nothing and makes an uproar
Sound interference, the data that can be used for statistical analysis.It is examined using orthogonal partial least squares discriminant analysis (OPLS-DA) and independent sample T
Test and data analyzed, wherein OPLS-DA according to OPLS VIP value excavate difference metabolin, according to independent sample T examine with
VIP value is excavated respectively, respectively excavates one group of difference metabolin, and 2 groups of difference lipid moleculars are obtained, and is compared by intersecting, is obtained
Shared difference lipid molecular.Again to shared lipid molecular and patient's Olanzapine, aripipazole drug concentration and biochemical indicator (ALT,
AST, GGT, TC, TG, LDL) correlation analysis is done, it is tested by the identical property of the pathology of analysed for relevance result and clinical symptoms
Card obtains metabolic disorder prediction lipid molecular.
The present invention will be described in detail below with reference to the drawings of preferred embodiments, whereby to the present invention how applied technology method
Technical problem is solved, and the realization process for reaching technical effect can fully understand and implement.
As shown in Figure 1, method provided by the invention mainly includes three steps: 1. sample preparations collection be applied alone Olanzapine and
The plasma sample of Aripiprazole schizophreniac is shared, is ready for ultra performance liquid chromatography-Mass Spectrometer Method after processing
Analysis；2. ultra performance liquid chromatography-Mass Spectrometer Method analysis: by serum sample through chromatography post separation, and being analyzed using Mass Spectrometer Method；
3. instrument initial data is extracted and is aligned in data analysis, the data for obtaining noiseless interference, can be used for statisticalling analyze, by orthogonal
Data are analyzed in partial least squares discriminant analysis (OPLS-DA) and independent sample T inspection, obtain the otherness between two groups
Metabolin, and combine existing document report to carry out Preliminary Identification and verifying these substances.
Processing method in step 1 are as follows: mix 100 μ l blood plasma with 900 μ l propan-2-ols.After being vortexed more than 10 seconds at ultrasound
Reason 10 minutes, -20 DEG C of mixture freeze 1 hour, and then 9500g is centrifuged 10 minutes.It collects upper layer (800 μ l) and is transferred to
Layer.Quality controls (QC) sample and represents the blood serum sample in analyzing by merging the aliquot of all blood serum samples.Every 5 samples
Blank sample (propan-2-ol/water, 9: 1v/v) and QC sample are placed in product.
Ultra performance liquid chromatography-Mass Spectrometer Method analysis instrument analysis platform is UPLC-QTOF/MS in step 2
(ACQUITY UPLC I-Class, Waters, Manchester, UK) carries out four times of time of flight mass light of electrospray ionisation
Spectrometer (Xevo G2-S Q-TOF, A Waters, Manchester, UK).[1.7 μm of chromatographic column of ACQUITY UPLC CSH；
50mm (length) × 2.1mm (i.d.)] for LC separation and chromatographic column remain 55 DEG C.Flow velocity is 0.4 ml/min sample injection
It is 2 μ l.Chromatographic separation condition are as follows: column temperature is that 40 DEG C of flow velocitys are 0.4 ml/min；Mobile phase A composition, 0.1% formic acid/10mM
Acetonitrile/water (95: 5v/v) in ammonium formate；Mobile phase B composition, B are 0.1% formic acid acid/10mM ammonium formate in propan-2-ol/acetonitrile
Solution in (9: 1v/v)；Gradient elution.Sample volume is 4 microlitres, and keeping autosampler is 4 DEG C.Initial linear gradient: 40%
B, 0-2 minutes 40%B to 43%B, 2-2.1 minutes: 43%B to 50%B, 2.1-12 minutes: 50%B to 54%B, 12-12.1
Minute: 54%B to 70%B, 12.1-18 minute: 70%B to 99%B, 18-18.1 minute: 99%B to 40%B, 18.1-20 points
Clock: 40%B.
Mass Spectrometry Conditions positive ion mode condition is with nitrogen as atomization, taper hole gas flight detection model.Positive and negative anodes capillary
Tube voltage is 3kV mode, and the taper voltage under both of which is 25V, and source is set as 120 DEG C, conical gas flow is 50 liters/
H, desolvation temperature are set as 450 DEG C, 850 ls/h of dissolve gas flow.Leucine enkephalin (Waters
Co., Manchester, UK) it is used as lock mass with reference to ion, m/z under 556.2771 holotype of m/z and negative mode
554.2615, locking is spraying with l/ minutes velocity calibration data of 5 μ.It is obtained using the continuous mode of angular impact energy scan twice
Take MSE data.Low energy model, scanning range 50-2000Da, sweep time 0.2s, collision energy are set as 6V.High-energy mould
Formula, scanning range 50-2000Da, sweep time 0.2s, angular impact energy 15-60 volt.
Step 3 collects plasma sample, through chromatography post separation after processing, and is analyzed using Mass Spectrometer Method, is carrying out detection point
Instrument initial data is extracted and is aligned after analysis, the data for obtaining noiseless interference, can be used for statisticalling analyze.Using orthogonal partially minimum
Two multiply discriminant analysis (OPLS-DA) and independent sample T inspection data are analyzed, wherein OPLS-DA according to OPLS VIP value
Difference metabolin is excavated, two groups of discrepant lipid moleculars are obtained, is compared by intersecting, shared difference lipid molecular is obtained.Again
To shared lipid molecular and patient's Olanzapine, aripipazole drug concentration and biochemical indicator (ALT, AST, GGT, TC, TG, LDL)
Correlation analysis is done, is verified by the identical property of the pathology of analysed for relevance result and clinical symptoms, obtains metabolic disorder prediction
Lipid molecular.
All above-mentioned this intellectual properties of primarily implementation, there is no this new products of implementation of setting limitation other forms
And/or new method.Those skilled in the art will utilize this important information, above content modification, to realize similar execution feelings
Condition.But all modifications or transformation belong to the right of reservation based on new product of the present invention.
The above described is only a preferred embodiment of the present invention, being not that the invention has other forms of limitations, appoint
What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc.
Imitate embodiment.But without departing from the technical solutions of the present invention, according to the technical essence of the invention to above embodiments institute
Any simple modification, equivalent variations and the remodeling made, still fall within the protection scope of technical solution of the present invention.
Claims (4)
1. one kind, which improves olanzapine in treatment schizophrenia based on aripipazole, causes Anomalous lipid metablism patient blood plasma metabolism group point
Analysis method characterized by comprising
The first step, sample preparation；
Second step, ultra performance liquid chromatography-Mass Spectrometer Method analysis；
Third step, data analysis obtain otherness metabolin, are identified and verified.
2. metabonomic analysis methods as described in claim 1, it is characterised in that: the first step is further specially to collect
Olanzapine is applied alone and shares the plasma sample of Aripiprazole schizophreniac, after processing for carrying out ultra high efficiency liquid phase color
Spectrum-Mass Spectrometer Method analysis, the processing method of the plasma sample are as follows: by 100 μ l blood plasma and 900 μ l mixed with propylene glycol, be vortexed super
It is ultrasonically treated after spending 10 seconds 10 minutes, -20 DEG C of mixture freeze 1 hour, are then centrifuged 10 under the action of the centrifugal force of 9500g
Minute, collect upper layer, as sample.
3. metabonomic analysis methods as claimed in claim 1 or 2, it is characterised in that: the ultra high efficiency liquid in the second step
Mobile phase A in the detection and analysis of phase chromatography-mass spectroscopy makees solvent using the mixed solution of acetonitrile and water, and the two volume ratio is 95: 5,
Contain 0.1% formic acid and 10mM ammonium formate in the mobile phase A, Mobile phase B is obtained using propylene glycol and acetonitrile according to volume ratio 9: 1
Mixed solution make solvent, which contains 0.1% formic acid and 10mM ammonium formate.
4. metabonomic analysis methods as described in claims 1 to 3, it is characterised in that: the third step is further specially
Instrument initial data is extracted and is aligned after being tested and analyzed, the data for obtaining noiseless interference, can be used for statisticalling analyze.It adopts
Data are analyzed with orthogonal partial least squares discriminant analysis (OPLS-DA) and independent sample T inspection, wherein OPLS-DA is pressed
Difference metabolin is excavated according to the VIP value of OPLS, is examined according to independent sample T and is excavated respectively with VIP value, respectively excavate one group of difference
2 groups of difference lipid moleculars are obtained in metabolin, are compared by intersecting, and obtain shared difference lipid molecular.Again to shared lipid point
Son does correlation analysis with patient's Olanzapine, aripipazole drug concentration and biochemical indicator (ALT, AST, GGT, TC, TG, LDL), leads to
The identical property of pathology for crossing analysed for relevance result and clinical symptoms is verified, and metabolic disorder prediction lipid molecular is obtained.
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