CN109060972A - Application of the rabbit blood in preparation human disease's external diagnosis reagent case - Google Patents
Application of the rabbit blood in preparation human disease's external diagnosis reagent case Download PDFInfo
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The present invention provides a kind of human disease's external diagnosis reagent case or detection method, the kit includes rabbit heme, calibration object, quality-control product, the content for detecting drug in subject's human blood biological sample；Rabbit blood is wherein used to be diluted as matrix to calibration object or quality-control product, the blood biological sample and standard items or quality-control product of the subject people is tested and analyzed after extract liquor extracts by high performance liquid chromatography-tandem mass instrument.
Description
Cross reference to related applications
This application claims the preferential of the Chinese patent application sequence number 201810453042.9 submitted on May 16th, 2018
The equity of power, entire contents are incorporated herein by reference in their entirety described.
Technical field
The present invention relates to biomedicine fields, and in particular to blood testing class external diagnosis reagent case.
Background technique
Liquid Chromatography-Tandem Mass Spectrometry is widely used in new drug development, clinical diagnosis field, and can with one sample
Multiple drugs, specificity height are detected simultaneously not vulnerable to matrix and impurity effect；Accuracy, precision are high, and high sensitivity examines line
Property the wide equal outstanding advantages of range and it is very popular.At home and abroad have multiple clinical diagnosis examinations based on liquid chromatography mass spectrometric joint technology
Agent box gets the Green Light listing, is mainly used for the examination of newborn infant diseases and therapeutic drug monitoring.Dried blood spot (Dried Blood
Spot, DBS) sampling method is by peripheral blood sample collection on paper jam, it is a kind of convenient and fast clinical blood sample collection method,
DBS just has been used for the screening of neonatal phenylketonuria early in the 1960s.With same routine sampling method (such as blood
Clearly, whole blood, blood plasma) it compares, DBS has following apparent advantage: (1) sample collection procedure is simple, grasps without professional person
Make, without centrifugation and homogenization, it can be achieved that self-service sampling at home；(2) minimally invasive, blood-sample withdrawal amount is few (10 μ L-30 μ L), can room temperature fortune
Defeated, storage etc..In recent years, with the development of Automated inspection technology, dried blood spot technology is produced in viral DNA content, metabolism
Object content, Enzyme assay etc. have relatively broad application.
Dried blood spot specimen sample technology is combined with liquid phase tandem mass spectrometry, so that clinical examination sample collection is by original
Several milliliters or hundreds of microlitres be down to 10-30 microlitres, for infant, critical patient and other patients for needing frequently to take a blood sample
Blood clinical inspection has significant advantage.In addition, the clinical test method of dried blood spot liquid phase tandem mass spectrometry is relative to traditional
Based on the method for immune antiboidy capture, there are high sensitivity and better stability, may be implemented to multiple in trace sample
The quick analysis detection of drug ingedient.
Calibration object and quality-control product matrix in existing therapeutic drug monitoring kit are all homologous matrix (i.e. people's blood source
Whole blood, serum or plasma sample), such as the therapeutic drug monitoring kit based on dried blood spot technology.But the acquisition of people's blood exists
All be in world wide it is carefully controlled, recruitment, physical examination screening by stringent volunteer's early period；Meet ethics to want
It asks, carry out blood-borne venereal disease poison safety detection；Meanwhile the acquisition of whole blood, preservation, traffic condition are all very harsh, can produce
Raw high cost.Therefore, domestic IVD development & production enterprise can select quality-control product or calibration from overseas directly purchase finished product
Product, however other than at high cost, no product category also will limit the research and development of kit.These problems also greatly limit
The exploitation and production of such external diagnosis reagent case.
Therefore, calibration object and quality-control product source of people matrix can be replaced by needing to find, and with more highly sensitive specificity
Kit.Present invention seek to address that the problem.
Summary of the invention
The invention discloses application of the rabbit blood in preparation human disease's external diagnosis reagent or kit, the reagent includes
Matrix containing rabbit blood, the kit include the matrix containing rabbit blood, calibration object and/or quality-control product.
In one embodiment, the matrix containing rabbit blood in mentioned reagent or kit is for calibration object or Quality Control
The dilution of product.
In one embodiment, the kit further includes at least one of extract liquor and Filter Paper Dry Blood piece, such as described
Kit further includes extract liquor and Filter Paper Dry Blood piece or the kit further includes extract liquor or Filter Paper Dry Blood piece.
In one embodiment, those skilled in the art's content according to the present invention, can choose suitable extract liquor.Example
Such as, extract liquor can be by methanol and water configuration, preferably prepare by 1: 1 volume ratio, wherein the methanol preferably is selected from chromatography
Grade methanol, the water preferably are selected from ultrapure water.
In one embodiment, human disease's in-vitro diagnosis include detection human blood biological sample in drug, poisonous substance and
The content of at least one of endogenous object.
In one embodiment, the blood biological sample and calibration object or quality-control product of the subject people extracts by extract liquor
After taking, tested and analyzed by high performance liquid chromatography-tandem mass instrument.
In a specific embodiment, blood biological sample, calibration object and/or the quality-control product of the subject people above-mentioned
To drip manufactured Filter Paper Dry Blood piece.
In the present invention, the calibration object or quality-control product are, it is preferable to use dried blood spot, the dried blood spot are complete using new fresh rabbit
Blood, drug target/compound and card filter paper are prepared, and the card filter paper is selected from DMPK-A, DMPK-C or FTN-903 card
Any one in filter paper.
In a specific embodiment, foregoing pharmaceutical includes but is not limited at least one of voriconazole and Topiramate, this
Field technical staff it can be anticipated that drug, poisonous substance or endogenous analyte detection, also within the protection scope of the present invention.
In a specific embodiment, the rabbit blood is selected from rabbit plasma, rabbit anteserum, rabbit whole blood.
In a specific embodiment, the volumn concentration that the rabbit blood accounts for the reagent or the matrix, which is selected from, to be greater than
0 volume % to 100 volume % any values below, surplus is people's blood in matrix.For example, rabbit blood accounts for the reagent or the matrix
Volumn concentration be 80 volume % to 100 volume %, 50 volume % to 80 volume %, 10 volume % to 50 volume %, it is remaining
Amount is people's blood.It is preferred that it is 100 volume % that rabbit blood, which accounts for the reagent or the percentage composition of the matrix,.
On the other hand, the invention discloses a kind of human disease's external diagnosis reagent case, the kit include rabbit heme,
Calibration object, and/or quality-control product.
In one embodiment, the kit further includes at least one of extract liquor and Filter Paper Dry Blood piece, such as described
Kit further includes extract liquor and Filter Paper Dry Blood piece；The kit further includes extract liquor or Filter Paper Dry Blood piece.
In one embodiment, aforementioned agents box further includes specification.
It is recorded in the specification and uses rabbit blood, preferably rabbit whole blood, calibration object or quality-control product are diluted as matrix,
The blood biological sample of the subject people is examined after extract liquor extracts by high performance liquid chromatography-tandem mass instrument
Survey analysis.
In one embodiment, aforementioned agents box is for detecting in drug in human blood biological sample, poisonous substance and endogenous object
At least one content
In one embodiment, blood biological sample, calibration object and/or the quality-control product of the subject people is made of drop
Filter Paper Dry Blood piece, the dried blood spot are prepared using fresh rabbit whole blood, drug target/compound and card filter paper, the card
Any one of filter paper in DMPK-A, DMPK-C or FTN-903 card filter paper.
In one embodiment, the drug includes but is not limited at least one of voriconazole and Topiramate.
In a specific embodiment, the rabbit blood is selected from least one of rabbit plasma, rabbit anteserum and rabbit whole blood.
In a specific embodiment, the volumn concentration that the rabbit blood accounts for the reagent or the matrix, which is selected from, to be greater than
0 volume % to 100 volume % any values below, surplus is people's blood in matrix.For example, rabbit blood accounts for the reagent or the matrix
Volumn concentration be 80 volume % to 100 volume %, 50 volume % to 80 volume %, 10 volume % to 50 volume %, it is remaining
Amount is people's blood.It is preferred that it is 100 volume % that rabbit blood, which accounts for the reagent or the percentage composition of the matrix,.
In another aspect, the invention discloses foregoing kits in detection detection subject's human blood biological sample
Application in the content of at least one of drug, poisonous substance and endogenous object.For example, the kit detects subject people in detection
Application in blood biological sample in the content of drug, poisonous substance or endogenous object.
In another aspect, the invention discloses drug, poisonous substance and endogenous objects at least one in a kind of detection human blood biological sample
The method of the content of kind, which comprises the following steps:
Calibration object or quality-control product are diluted using rabbit heme.
The above method is further comprising the steps of:
Dried blood spot is made in blood biological sample and calibration object or the quality-control product drop of subject people, after extract liquor extracts,
It is tested and analyzed by high performance liquid chromatography-tandem mass instrument.
In a specific embodiment, the rabbit blood is selected from at least one of rabbit plasma, rabbit anteserum and rabbit whole blood.
In a specific embodiment, the volumn concentration that the rabbit blood accounts for the reagent or the matrix, which is selected from, to be greater than
0 volume % to 100 volume % any values below, surplus is people's blood in matrix.For example, rabbit blood accounts for the reagent or the matrix
Volumn concentration be 80 volume % to volume 100%, 50 volume % to 80 volume %, 10 volume % to 50 volume %, it is remaining
Amount is people's blood.It is preferred that it is 100 volume % that rabbit blood, which accounts for the reagent or the percentage composition of the matrix,.
Beneficial effect
Rabbit blood compares people's blood great advantage as the matrix of calibration object and quality-control product in clinical application:
Have found a kind of method that can replace people's heme using rabbit blood.The accuracy of this method, precision are compared with people's blood
The method of matrix is more excellent, for example, in dried blood spot-Mass Spectrometry detection method rabbit blood substitute people's blood, obtain compared with the better accuracy of people's blood,
Precision.
In addition, new fresh rabbit blood dried blood spot is examined, calibration object and quality-control product are low in cost, performance is stable, to human body environment's safety
Environmental protection, simple process, easily stored, transport and use, forbid being commercialized under the overall situation of people's blood transaction at home, rabbit blood at present
As the substitute of people's blood, the unavailable problem of raw material can solve, promote the exploitation of China's blood clinical testing product
And application.
Detailed description of the invention
Fig. 1 shows rabbit blood bare substrate chromatogram, wherein upper figure is the corresponding rabbit blood bare substrate of voriconazole medicine group
Liquid chromatogram；The following figure is the corresponding rabbit blood bare substrate liquid chromatogram of voriconazole internal standard group.
Fig. 2 shows the voriconazole compounds and interior target chromatogram of the lower limit of quantitation (LLOQ) that people's whole blood is prepared, wherein
Upper figure is the voriconazole compound liquid chromatogram of lower limit of quantitation (LLOQ), and the following figure is target liquid chromatogram in voriconazole
Figure.
Fig. 3 shows the voriconazole compound and interior target chromatogram of the upper limit of quantification (ULOQ) of people's whole blood preparation, wherein
Upper figure is the voriconazole compound liquid chromatogram of upper limit of quantification (ULOQ), and the following figure is target liquid chromatogram in voriconazole
Figure.
Fig. 4 shows the voriconazole compound and interior target chromatogram of the lower limit of quantitation (LLOQ) of rabbit whole blood preparation, wherein
Upper figure is the voriconazole compound liquid chromatogram of lower limit of quantitation (LLOQ), and the following figure is target liquid chromatogram in voriconazole
Figure.
Fig. 5 shows the voriconazole compound and interior target chromatogram of the upper limit of quantification (ULOQ) of rabbit whole blood preparation, wherein
Upper figure is the voriconazole compound liquid chromatogram of upper limit of quantification (ULOQ), and the following figure is target liquid chromatogram in voriconazole
Figure.
Fig. 6 is shown in voriconazole compound test using people's whole blood and rabbit whole blood as matrix linear curve comparison figure.
Fig. 7 shows people's blood bare substrate chromatogram, wherein upper figure is the corresponding people's blood bare substrate liquid of topiramate drug group
Phase chromatogram；The following figure is the corresponding people's blood bare substrate liquid chromatogram of Topiramate internal standard group.
Fig. 8 shows rabbit blood bare substrate chromatogram, wherein upper figure is the corresponding rabbit blood bare substrate liquid of topiramate drug group
Phase chromatogram；The following figure is the corresponding rabbit blood bare substrate liquid chromatogram of Topiramate internal standard group.
Fig. 9 shows the Topiramate compound and interior target chromatogram of the lower limit of quantitation (LLOQ) of people's whole blood preparation, wherein on
Figure is the Topiramate compound liquid chromatogram of lower limit of quantitation (LLOQ), and the following figure is target liquid chromatogram in Topiramate.
Figure 10 shows the Topiramate compound and interior target chromatogram of the upper limit of quantification (ULOQ) of people's whole blood preparation, wherein on
Figure is the Topiramate compound liquid chromatogram of upper limit of quantification (ULOQ), and the following figure is target liquid chromatogram in Topiramate.
Figure 11 shows the Topiramate compound and interior target chromatogram of the lower limit of quantitation (LLOQ) of rabbit whole blood preparation, wherein on
Figure is the Topiramate compound liquid chromatogram of lower limit of quantitation (LLOQ), and the following figure is target liquid chromatogram in Topiramate.
Figure 12 shows the Topiramate compound and interior target chromatogram of the upper limit of quantification (ULOQ) of rabbit whole blood preparation, wherein on
Figure is the Topiramate compound liquid chromatogram of upper limit of quantification (ULOQ), and the following figure is target liquid chromatogram in Topiramate.
Figure 13 is shown in Topiramate compound test using people's whole blood and rabbit whole blood as matrix linear curve comparison figure.
Specific embodiment
Below will be by specifically describing, the present invention is further illustrated.
Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention
Those of ordinary skill be generally understood identical meaning.
In the application, singular "one", "the" include plural reference, unless in addition context clearly provides.
Definition
Term " calibration object " or " standard items " as used herein alternatively, refer to standard article, mark as a kind of measurement
Standard is the standard content in assay.
Term " LLOQ " as used herein refers to for quantifying in a certain certain drug detection method lieutenant colonel's directrix curve
Lower limit.
Term " ULOQ " as used herein refers to for quantifying in a certain certain drug detection method lieutenant colonel's directrix curve
The upper limit.
Term " rabbit " as used herein refers to the general name of all categories of mammality Lagomorpha rabbit section subordinate, is divided into Ochotonidae
(pika) and rabbit section (rabbit and hare), the including but not limited to rabbit of wild type or saltant type such as transgenosis, specifically include,
Japan large ear rabbit, New Zealand White Rabbit, rabbit, Chinese White Rabbit etc..
Term " rabbit blood " as used herein refers to the blood of rabbit, including but not limited to serum, blood plasma, whole blood.Rabbit blood
Source or preparation method be it is various, can be the new blood that acquisition obtains, used after storing after being also possible to acquisition
Blood is also possible to the method acquisition of genetic engineering, and the rabbit blood that the method that those skilled in the art are expected obtains belongs to
In scope of the invention.
Term " rabbit whole blood " as used herein refers to whole blood of the rabbit blood after anticoagulation, including haemocyte and
The all the components of blood plasma.
Term " endogenous object " as used herein refers to the substance of non-allogene present in biological sample such as blood, example
Product or intermediate product as formed in former already present and metabolic process in body in biological sample, the hormone such as secreted
Deng." drug " and " poisonous substance " used herein belong to allogene.
Term " bias " as used herein refers to the deviation of measured value relative real value, belongs to systematic error.
Embodiment
It is further illustrated by the examples that follow the present invention.It provides embodiment to be for illustration purposes only, and should not be solved
It is interpreted as limiting the scope of the invention in any way or content.
Embodiment 1: the screening of school mark matrix
The detection of fresh whole blood HCT: HCT detection is carried out to fresh whole blood raw material using blood capillary tube method, same sample is adopted
Sample calculates average value after detecting twice.
Quality testing standard determines the blood raw material HCT range of different plant species 20%~60%.
By taking dried blood spot as an example, matrix of the whole blood from different plant species as calibration object and sample is used respectively, is compared
The fit standard curve of these different plant species source whole blood matrix calculates R2Value, and accuracy in computation, wherein accuracy bias≤
20% is acceptable candidate matrix.In screening, species include but is not limited to the anticoagulation (ox whole blood) of ox, pig it is anticoagulant
The species such as blood (pig whole blood), the anticoagulation (rabbit whole blood) of rabbit, people's whole blood.It is final to determine by the investigation of accuracy and precision
Specific species whole blood.
Main experimental procedure is as follows:
1) it is directed to the anticoagulation of different plant species, visually observes and screen no haemolysis first, without blood coagulation and without chyle exception
Fresh anticoagulated whole blood；
2) detect HCT value in these different plant species different batches anticoagulated whole bloods, wherein with people's whole blood, rabbit whole blood, pig whole blood,
For ox whole blood, detect respectively the fresh anticoagulant rabbit whole blood in 8 adult healthy rabbit sources HCT value and 8 volunteers
The HCT value of fresh anticoagulant people's whole blood, and compare the HCT of commercialization pig fresh anticoagulation and fresh ox anticoagulation different batches
Value.These people's whole bloods, rabbit whole blood, pig whole blood different batches HCT value between 20-60%, meet the requirements.
The interference component contained in different plant species blood is different, it is possible to will affect separation, the ion of drug liquid chromatogram
Change efficiency, responded and the accuracy and stability of interference medicament concentration mensuration to finally influence ion pair, in addition plasma protein
And other blood constitutents also will affect chromatography of the blood on filter paper, influence distribution of the drug in Blood piece to finally influence
The accuracy and stability of determination of drug concentration.The sensitivity and precision that can reach certain in detection method also need reality
Testing is proved.Therefore, after inventor also analyzes the whole blood in different plant species source as matrix, the accuracy of drug is detected.
The preparation of calibration object solution: weighing the drug target bulk pharmaceutical chemicals of corresponding weight with electronic balance, (this sentences Fu Likang
For azoles, national standard definite value is had been subjected to), dissoluble capacity bottle constant volume is carried out with the suitable solvent such as DMSO or methanol, is made
The proofreaded sample stock solution (SSC, 2 mg/mL).Gradient dilution is carried out to the proofreaded sample stock solution with rabbit whole blood, obtains 7 concentration
The calibration object (0.1,0.2,1,2,10,20,50 μ g/mL) of gradient.The voriconazole raw material of corresponding weight is weighed with electronic balance
Medicine carries out dissoluble capacity bottle constant volume with DMSO, prepares quality-control sample stock solution (SSQC, 2mg/mL).With rabbit blood to quality-control sample
Stock solution is diluted, and obtains the quality-control product (0.4,4,40 μ g/mL) of high, medium and low 3 concentration.
The preparation of dried blood spot: by the above-mentioned the proofreaded sample prepared and quality-control product by each blood cake 10-50 μ L liquid-transfering gun point
Blood cake processed.The dried blood spot of point sample is done for drying at room temperature at least 1 hour or (37 DEG C~50 DEG C) of baking oven under 100,000 clean area environment
Dry 15 minutes or more.Dried calibration object dried blood spot and quality-control product dried blood spot are sealed up for safekeeping with the packaging bag that sealing is protected from light respectively.
Every batch of calibration object and quality-control product are examined, is detected using liquid chromatography-tandem mass spectrometry technology, uses punch
The former piece that diameter is 3mm is beaten in calibration object and the dry blood cake center of quality-control product, is put into 96 deep-well plates, extraction containging interior traget is added
Liquid (methanol: water ratio be 1: 1) extraction, after albumen precipitation and centrifugation plus water redissolve, liquid phase-quadrupole rod tandem mass spectrometer into
Row detection.Specific method see the table below:
UPLC setting:
Chromatographic column: C18,21mm × 50mm, 1.7 μm of Waters ACQUITY UPLC BEH (SN:
02823821018242)
Mobile phase:
A:0.1 volume % aqueous formic acid (pure water 500mL+ formic acid 0.5mL is mixed)
B:0.1 volume % formic acid acetonitrile solution (acetonitrile 1000mL+ formic acid 1.0mL is mixed)
Gradient elution design parameter is shown in Table 1:
1. gradient elution table of table
MS setting:
Ion source: ESI collision energy (CE): 49eV removes cluster voltage (DP): 100eV
Quota ion:
Voriconazole: 350.1 > 127.1,
D3- voriconazole: 353.1 > 130.1
After quota ion peak is carried out integral calculation, the integrated value of 7 calibration object dried blood spots is carried out with least square method
Linear regression, R after Regressive calibration curve2It should be greater than 0.99, theoretical value and detected value bias should be less than 15%, LLOQ and be less than or wait
In 20%, inspection is repeated 6 times in batch, CV% value is less than or equal to 15%, LLOQ CV% value less than 20%.
By taking common species as an example, rabbit whole blood and people's whole blood, voriconazole drug stock solution, Whatman 903 are used respectively
Blood collecting card prepares the calibration object and quality-control product dried blood spot of voriconazole therapeutic drug monitoring kit.By according to kit standard
Sample process SOP is extracted, is precipitated, being redissolved, upper machine testing (combination of liquid chromatography-tandem quadrupole mass spectrometer).
It is fitted to calibration curve with calibration object test data prepared by rabbit whole blood (or other common species, such as the anticoagulation of ox, pig),
Record R2Value, and the test value of owner's blood sample is calculated, the accuracy in computation compared with theoretical value.Accuracy bias≤20% is
Acceptable.With different animals blood (rabbit blood provides fresh acquisition for oneself, and pig and ox whole blood are all commercial source) as substitution matrix
Prepare three people's blood samples of calibration curve Parallel testing (low quality-control sample LQC；Middle quality-control sample MQC；High quality-control sample HQC) in
Voriconazole concentration, and calculate the bias with theoretical value.The accuracy of (table 2) as the result is shown, pig whole blood is undesirable, partially
Leaning on value, more than 20%, and under low concentration, accuracy is lower under high, normal, basic Quality Control sample.And ox whole blood, although middle Quality Control sample
Accuracy meets the requirements under product and high quality-control sample, but in the detection of low quality-control sample, accuracy rate is not high, and bias is more than
20%.And it is smaller compared to the bias between the measured value and theoretical value of people's whole blood, rabbit blood is more stable, and the absolute value of bias is equal
Less than using people's whole blood as the detection of matrix.Three people's blood of calibration curve Parallel testing are prepared using different animals blood as substitution matrix
Sample (low quality-control sample LQC；Middle quality-control sample MQC；High quality-control sample HQC) in Topiramate concentration, and calculate with theoretical value
Bias.(table 3) as the result is shown, compared to for other species, rabbit blood bias under high concentration or middle concentration is minimum.As it can be seen that making
It uses rabbit whole blood as standard items or quality-control product matrix, and then detects the content value of drug in human blood sample product, accuracy is higher, tool
There is unexpected advantage.
Other than ox whole blood, pig whole blood, rabbit whole blood, the whole blood of other species such as horse and sheep is also compared, by essence
The experiment of density and accuracy, preferably (table 2 and table 3), sheep whole blood exists for the accuracy for as a result still showing rabbit whole blood and precision
The bias for measuring voriconazole or Topiramate is larger, and horse whole blood is larger in the bias of measurement Topiramate.
2. different animals blood of table substitutes matrix and makes this determination of voriconazole of standard curve determination proper manners concentration bias value
3. different animals blood of table substitutes Topiramate concentration bias value in matrix production standard curve determination proper manners sheet
Embodiment 2: the drug dried blood spot of rabbit whole blood preparation and the drug dried blood spot liquid chromatography Comparative result of people's whole blood preparation
(by taking voriconazole as an example)
The configuration of standard items and quality-control product used in experiment is as described in example 1 above, the preparation method and reality of dried blood spot
It applies consistent described in example 1.
The drug dried blood spot of rabbit whole blood preparation and the drug dried blood spot of people's whole blood preparation are connected after extraction through liquid phase
Mass spectrograph is detected, and the comparison of ion pair peak figure is as follows, and two kinds of matrix nothing at drug retention time 1.2 minutes significantly interferes with
Peak, for drug test peak shape without hangover, signal-to-noise ratio height is noiseless.
User's whole blood prepares LLOQ voriconazole compound or ULOQ voriconazole compound and corresponding internal standard, chromatography
Figure shows that drug retention time is 1.2 minutes (referring to figs. 2 and 3)；And compared to the liquid chromatogram of people's whole blood configuration drug, make
LLOQ voriconazole compound or ULOQ voriconazole compound are prepared with rabbit whole blood and corresponding internal standard, chromatogram are shown in medicine
Same without peak is significantly interfered at object retention time 1.2 minutes, drug test peak shape is without hangover, and signal-to-noise ratio height is noiseless (referring to fig. 4
And Fig. 5).Therefore, use rabbit whole blood substitution people's whole blood feasible as matrix this method of drug quality-control product or standard items.
Embodiment 3: rabbit whole blood and the drug calibration object linearity curve of people's whole blood preparation (by taking voriconazole as an example)
The configuration of standard items and quality-control product used in experiment is as described in example 1 above, the preparation method and reality of dried blood spot
It applies consistent described in example 1.
It is respectively that matrix does calibration curve with rabbit whole blood and people's whole blood, if Fig. 6 is shown, people's whole blood matrix voriconazole standard
The R of curve2It is 0.9954, linear equation y=2.18x-0.00787；The R of rabbit whole blood matrix voriconazole standard curve2For
0.9982, linear equation y=2.17x-0.0142；Illustrate the standard curve precision of rabbit whole blood substraturn approach better than people's whole blood
The conventional method of matrix.Inventor further detects and calculates each 6 people blood pharmaceutical samples of tri- concentration of LQC, MQC, HQC, meter
Calculate precision (CV%) and bias (Bias%).Three differences of standard curve determination prepared respectively with rabbit whole blood and people's whole blood are dense
Spend the accuracy and precision of (0.4 μ g/mL, 4 μ g/mL, 40 μ g/mL) people's blood quality-control product.
The accuracy and precision of 4 various concentration people's blood quality-control product of table
As can be known from Table 4, no matter drug is under low middle and high concentration, the bias of accuracy is respectively less than 20%, conforms to
It asks.But data under comparing various concentration are it can be found that in rabbit whole blood substraturn approach, for low concentration and high concentration medicine
Detection, it is more advantageous.Wherein, SD value is respectively less than the obtained numerical value of people's heme method respectively between accuracy bias and group.
For example, the accuracy rate of rabbit whole blood substraturn approach detection is 98.83% when drug concentration is down to 0.4 μ g/mL, it is significantly higher than people's blood
Substraturn approach.As it can be seen that rabbit whole blood substraturn approach, has more real advantage in detection people's blood drug concentration, particularly with
The drug test of low concentration can significantly improve the accuracy and precision of method, so that more acurrate in the detection of low concentration.
The clinical test method of dried blood spot liquid phase tandem mass spectrometry is relative to traditional side based on immune antiboidy capture
Method has high sensitivity and better stability, and the quick analysis inspection to drug ingedients multiple in trace sample may be implemented
It surveys.But matrix of the rabbit blood substitution people's blood as standard items or quality-control product is used, it is not limited to blood-sampling method, the i.e. use of rabbit blood
Way is not limited to dried blood spot technology.Those skilled in the art's reason will be understood that rabbit blood substitution people's blood as standard items or quality-control product
Matrix is used in all human disease's external diagnosis reagent cases or method comprising standard items and standard curve.On and
The range stated also belongs to the scope of the present invention.
Embodiment 4: the drug dried blood spot of rabbit whole blood preparation and the drug dried blood spot liquid chromatography Comparative result of people's whole blood preparation
(by taking Topiramate as an example)
Topiramate Sample pretreatment method is similarly methanol with the voriconazole in embodiment 1 and embodiment 2, extract liquor:
Water (50: 50) solution.It the use of liquid phase column is Waters ACQUITY UPLC BEH C18,1.7um, 2.1 × 50mm.Mobile phase:
A:0.1% formic acid acetonitrile solution, B:0.1 volume % aqueous formic acid, Parameters of gradient elution are shown in Table 5.
5. gradient elution table of table
Mass spectrometry method:
MS setting:
Ion source: ESI collision energy (CE): -66eV removes cluster voltage (DP): -70eV
Quota ion:
Topiramate: 338.1 > 78,
D12- Topiramate: 350.2 > 78
The same voriconazole of method, after quota ion peak integral, by analyte area/IS area to theoretical concentration
Value is returned with least square method, and weight is (1/x2), obtain regression equation, R2Value is greater than 0.99 for qualification.
User's whole blood prepares LLOQ Topiramate compound or ULOQ Topiramate compound and corresponding internal standard, and chromatogram is aobvious
Show that drug retention time is 1.05 minutes (referring to figs. 7 and 8)；And compared to the liquid chromatogram of people's whole blood configuration drug, it uses
Rabbit whole blood prepares LLOQ Topiramate and closes object or ULOQ Topiramate compound and corresponding internal standard, when chromatogram is shown in drug reservation
Between it is same without peak is significantly interfered at 1.05 minutes, drug test peak shape is without hangover, and signal-to-noise ratio height is noiseless (referring to Fig. 9 and figure
10).Meanwhile inventor also has detected the chromatogram of people's blood bare substrate and rabbit blood bare substrate, it can be seen that the equal noise of the two
Very little (Fig. 7 and Fig. 8).Therefore, to sum up, rabbit whole blood is used to substitute people's whole blood as the matrix of drug quality-control product or standard items
This method is feasible.
Further, by taking drug is Topiramate as an example, continue to investigate different animals blood substitution matrix production standard curve determination
Proper manners Chinese medicine object concentration and bias value.The data of table 6 show that the bias of ox whole blood and pig whole blood is all excessive, and complete compared to people
Blood, the bias very little of rabbit whole blood matrix compare people's whole blood especially in the detection of low concentration and high concentration, and accuracy is higher.?
In the accuracy and precision detection in people's whole blood matrix or rabbit whole blood matrix, the data of table 7 are shown various concentration quality-control product
When detecting the Topiramate of low concentration 1.5 μ g/mL and 16 μ g/mL, accuracy and precision are got well than people's whole blood substraturn approach,
This illustrates rabbit whole blood substraturn approach, and low concentration is more advantageous in the detection.In the detection of 160 μ g/mL of high concentration, rabbit whole blood
The bias of substraturn approach is -6.35, also complies with requirement.
6. different animals blood of table substitutes matrix production standard curve determination proper manners Chinese medicine object concentration and bias value
The accuracy and precision of 7. various concentration quality-control product of table
It is respectively that matrix does calibration curve with rabbit whole blood and people's whole blood, if Figure 13 is shown, people's whole blood matrix Topiramate standard
The R of curve2It is 0.9936, linear equation y=0.166x-0.00172；The R of rabbit whole blood matrix Topiramate standard curve2For
0.9976, linear equation y=0.155x-0.0161；Illustrate that the standard curve precision of rabbit whole blood substraturn approach is complete better than people
The conventional method of heme.
It is incorporated by reference into
The complete disclosure of herein cited each patent document and scientific literature is incorporated herein by reference for institute
Purposefully.
It is equivalent
The present invention can be implemented in other specific forms in the case where not departing from its essential characteristic.Therefore, aforementioned implementation
Example is considered illustrative, rather than the limitation to invention as described herein.The scope of the present invention is by appended claims
Book rather than indicated by aforementioned specification, and be intended to fall into all in the meaning and scope of the equivalents of the claims
Change is included therein.
Claims (15)
1. application of the rabbit blood in preparation human disease's external diagnosis reagent or kit, which is characterized in that the reagent includes containing
There is the matrix of rabbit blood, the kit includes the matrix containing rabbit blood, calibration object and/or quality-control product.
2. application as described in claim 1, which is characterized in that the kit further include extract liquor and Filter Paper Dry Blood piece extremely
Few one kind.
3. application as claimed in claim 1 or 2, which is characterized in that human disease's in-vitro diagnosis includes that detection human blood is raw
The content of at least one of drug, poisonous substance and endogenous object in object sample.
4. the application as described in any one of preceding claim, which is characterized in that the drug includes voriconazole and Topiramate
It is at least one.
5. the application as described in any one of preceding claim, which is characterized in that the rabbit blood is selected from rabbit plasma, rabbit anteserum and rabbit
At least one of whole blood.
6. the application as described in any one of preceding claim, which is characterized in that the rabbit blood accounts for the reagent or the matrix
Volumn concentration, which is selected from, is greater than 0 volume % to 100 volume % any values below, and surplus is people's blood in matrix.
7. a kind of human disease's external diagnosis reagent case, which is characterized in that the kit include rabbit heme, calibration object, and/or
Quality-control product.
8. kit as claimed in claim 7, which is characterized in that the kit further includes extract liquor and Filter Paper Dry Blood piece
It is at least one.
9. kit as claimed in claim 7 or 8, which is characterized in that it is complete that the rabbit blood is selected from rabbit plasma, rabbit anteserum and rabbit
At least one of blood.
10. kit as claimed in claim 7 or 8, which is characterized in that the rabbit blood accounts for the body of the reagent or the matrix
Product percentage composition, which is selected from, is greater than 0 volume % to 100 volume % any values below, and surplus is people's blood in matrix.
11. kit as claimed in claim 7 or 8 drug, poisonous substance and endogenous in detection subject's human blood biological sample
Application in the content of at least one of object.
12. the method for the content of at least one of drug, poisonous substance and endogenous object, special in a kind of detection human blood biological sample
Sign is, comprising the following steps:
Calibration object or quality-control product are diluted using rabbit heme.
13. method as claimed in claim 12, further comprising the steps of:
Dried blood spot is made in blood biological sample and calibration object or the quality-control product drop of subject people, after extract liquor extracts, passes through
High performance liquid chromatography-tandem mass instrument tests and analyzes.
14. method as described in claim 12 or 13, which is characterized in that it is complete that the rabbit blood is selected from rabbit plasma, rabbit anteserum and rabbit
At least one of blood.
15. method as described in claim 12 or 13, which is characterized in that the rabbit blood accounts for the body of the reagent or the matrix
Product percentage composition, which is selected from, is greater than 0 volume % to 100 volume % any values below, and surplus is people's blood in matrix.
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| CN109030672A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The kit and its application of four kinds of antifungal drugs are measured in dry blood cake simultaneously |
| CN110045037A (en) * | 2019-05-06 | 2019-07-23 | 上海药明康德医学检验所有限公司 | A kind of kit and detection method detecting nilotinib drug concentration in dry blood cake |
| CN111650296A (en) * | 2020-05-27 | 2020-09-11 | 苏州药明泽康生物科技有限公司 | Method for improving matrix of calibrator and quality control material in blood detection type in-vitro diagnostic kit and product |
| CN112213417A (en) * | 2020-09-16 | 2021-01-12 | 苏州药明泽康生物科技有限公司 | Kit and method for detecting concentration of mycophenolic acid medicine in dried blood spots |
| CN114414709A (en) * | 2021-12-21 | 2022-04-29 | 益诺思生物技术南通有限公司 | Method for detecting voriconazole in cornea |
| CN114527230A (en) * | 2022-02-28 | 2022-05-24 | 上海药明奥测医疗科技有限公司 | Method for detecting concentration of vitamin A and vitamin E in human dry blood spots |
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| CN109030672A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The kit and its application of four kinds of antifungal drugs are measured in dry blood cake simultaneously |
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| CN111650296A (en) * | 2020-05-27 | 2020-09-11 | 苏州药明泽康生物科技有限公司 | Method for improving matrix of calibrator and quality control material in blood detection type in-vitro diagnostic kit and product |
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| CN114527230A (en) * | 2022-02-28 | 2022-05-24 | 上海药明奥测医疗科技有限公司 | Method for detecting concentration of vitamin A and vitamin E in human dry blood spots |
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