CN109374768A - A kind of method of hypoxanthine content in measurement SHUXUETONG ZHUSHEYE - Google Patents
A kind of method of hypoxanthine content in measurement SHUXUETONG ZHUSHEYE Download PDFInfo
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- CN109374768A CN109374768A CN201811338059.6A CN201811338059A CN109374768A CN 109374768 A CN109374768 A CN 109374768A CN 201811338059 A CN201811338059 A CN 201811338059A CN 109374768 A CN109374768 A CN 109374768A
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- hypoxanthine
- ismipur
- solution
- methanol
- mark
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Abstract
The present invention relates to a kind of methods of hypoxanthine content in measurement SHUXUETONG ZHUSHEYE, Ismipur inner mark solution is added in SHUXUETONG ZHUSHEYE to be measured and hypoxanthine standard solution is configured to mark-on sample, using Liquid Chromatography/Mass Spectrometry, carry out multiple-reaction monitoring MRM scanning, using mark-on hypoxanthine final concentration X as abscissa, target analytes and interior target peak area ratio Y are ordinate, carry out linear regression, obtain regression equation；Then Ismipur inner mark solution is added in SHUXUETONG ZHUSHEYE to be measured, using Liquid Chromatography/Mass Spectrometry, multiple-reaction monitoring MRM scanning is carried out, target analytes and interior target peak area ratio are obtained, hypoxanthine content in SHUXUETONG ZHUSHEYE is obtained according to above-mentioned regression equation.The detection method that the present invention establishes can fast and accurately detect hypoxanthic content in SHUXUETONG ZHUSHEYE, effectively reduce the interference of matrix effect.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical fields, are related to a kind of quantitative analysis method, more particularly to a kind of accurate survey
Determine the method for hypoxanthine content in SHUXUETONG ZHUSHEYE.
Background technique
SHUXUETONG ZHUSHEYE is formed by leech and pheretima prescription, has effects that activating microcirculation and removing stasis medicinal, clearing and activating the channels and collaterals, is used for hemostasis
Hinder ischemia apoplexy apoplex involving the channels and collaterals acute stage caused by network symptoms include hemiplegia, dispute skew, dysphasia.Hypoxanthine is
One of the main intermediate product of purine bases metabolic process in organism has bioactivity, can enter cell through cell membrane
It is interior, the activity of a variety of enzymes is improved, participates in adjusting some important physiological functions of human body, wherein it is particularly evident to Cardiovascular,
Therefore assay index components of the hypoxanthine as SHUXUETONG ZHUSHEYE are selected.
Currently, hypoxanthic assay is using high performance liquid chromatography-ultraviolet method in SHUXUETONG ZHUSHEYE
(HPLC-UV).It is found through preliminary experiment, if when the hypoxanthine in SHUXUETONG ZHUSHEYE fails and other structures class in chromatography
As nucleotide base when being kept completely separate, due to the ultraviolet maximum absorption wavelength λ of chaff interferentmaxIt is close with hypoxanthine, therefore contain
Amount measurement result will and true value have certain deviation.And if measured using Liquid Chromatography/Mass Spectrometry (LC-MS/MS), it is not necessarily to color
Spectrum is kept completely separate i.e. using the unique mass resolving power of mass spectrum, fast to secondary Huang using more reactive ions monitoring (MRM) mode
Purine is scanned, quick, exclusive, delicately detect and dredge blood to effectively reduce the interference that other nucleotide bases may cause
Hypoxanthic content in logical injection.
But mass spectrum is as a kind of mass flow rate sensitive detector, only drawback is that quantitative result more or less will receive matrix effect
Influence.In order to eliminate the interference of sample substrate, generallys use chromatography and remove matrix online, or use liquid-liquid extraction, consolidate
Phase extraction etc. carries out removal of impurities pre-treatment to sample.Aforesaid operations or higher to the chromatographic isolation skill requirement of staff,
Operating procedure is many and diverse, larger workload.Therefore, it is more economical, effective to develop one kind, and is suitable for Accurate Determining Shu Xue-tong
Hypoxanthic detection method is particularly important in injection.
Summary of the invention
The technical problem to be solved in the present invention is to provide it is a kind of measurement SHUXUETONG ZHUSHEYE in hypoxanthine content method,
At least to realize the purpose for improving measurement accuracy and specificity.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
A kind of method of hypoxanthine content in measurement SHUXUETONG ZHUSHEYE, comprising:
Step 1 prepares Ismipur inner mark solution and a series of hypoxanthine standard solutions；
Ismipur inner mark solution and hypoxanthine standard solution is added in step 2 in SHUXUETONG ZHUSHEYE to be measured
It is configured to mark-on sample, using Liquid Chromatography/Mass Spectrometry, carries out multiple-reaction monitoring MRM scanning, is cross with mark-on hypoxanthine final concentration X
Coordinate, target analytes and interior target peak area ratio Y are ordinate, carry out linear regression, obtain regression equation；
Ismipur inner mark solution is added in step 3 in SHUXUETONG ZHUSHEYE to be measured, using Liquid Chromatography/Mass Spectrometry, carries out
Multiple-reaction monitoring MRM scanning, obtains target analytes and interior target peak area ratio, obtains Shu Xue-tong according to above-mentioned regression equation
Hypoxanthine content in injection；
In step 2 and step 3, liquid phase chromatogram condition are as follows: analytical column is Waters Acquity UPLC BEH C18
(100mm × 2.1mm, 1.7 μm), guard column are Waters Acquity UPLC BEH C18(5mm × 2.1mm, 1.7 μm), stream
Dynamic phase A phase is methanol, and B phase is 0.1% aqueous formic acid, and gradient elution, flow velocity 0.3ml/min, column temperature is 40 DEG C, sample volume
For 2 μ l；
Mass Spectrometer Method condition are as follows: Negative electrospray ionization ESI (-) detection pattern；Capillary voltage (Capillary):
0.5KV；Orifice potential (Cone): 30V；Spray pressure power (Nebuliser): 7psi；Desolvation temperature (Desolvation
Temp): 400 DEG C；Desolventizing gas flow (Desolvation Gas Flow): 800Lh-1；Taper hole throughput (Cone Gas
Flow): 150Lh-1, collision gas argon flow (Collision Gas Flow): 0.25mlmin-1；
The parameter of multiple-reaction monitoring MRM scan method is as follows:
Hypoxanthine: parent ion mass number (Q1): 134.9；Product ion mass number (Q3): 92.0；Residence time (Dewll
Time): 0.129s；Orifice potential (Cone Voltage): 30V；Collision energy (Collision Energy): 12V；
Ismipur: parent ion mass number (Q1): 151.0；Product ion mass number (Q3): 92.1；Residence time
(Dewll Time): 0.129s；Orifice potential (Cone Voltage): 56V；Collision energy (Collision Energy):
16V。
Further, preparation Ismipur inner mark solution described in step 1, comprising steps of Ismipur is weighed,
Add methanol dissolution, dilution, the Ismipur methanol stock solution of 250 μ g/ml is made；Ismipur stock solution is pipetted, first is used
- 0.2% aqueous formic acid of alcohol (2:8) dissolution, dilution, are made the Ismipur inner mark solution of 125 μ g/ml.
Further, preparation hypoxanthine standard solution described in step 1 adds comprising steps of weighing hypoxanthine
Make it completely dissolved, let cool in 60 DEG C of ultrasounds after methanol, then plus methanol dilution 100 μ g/ml hypoxanthine methanol stock solutions are made；
Hypoxanthine stock solution 1.00ml is pipetted, the hypoxanthine mark of 50 μ g/ml is diluted to -0.2% aqueous formic acid of methanol (2:8)
Quasi- solution.
Further, in step 2, mark-on sample is prepared, comprising steps of pipetting totally 6 parts of 100 μ l of SHUXUETONG ZHUSHEYE, respectively
The 100 μ l of Ismipur inner mark solution that concentration is 125 μ g/ml is added, is separately added into the hypoxanthine mark that concentration is 50 μ g/ml
Quasi- 0 μ l of product solution, 100 μ l, 200 μ l, 300 μ l, 400 μ l, 500 μ l；Then with -0.2% aqueous formic acid of diluent methanol (2:
8) volume is supplied to 1.00ml, is vortexed, is mixed；Respectively take 100 μ l, then with appropriate -0.2% aqueous formic acid of diluent methanol (2:
8) 10 times are diluted, is vortexed, mixes；200 μ l are taken respectively, are diluted 5 times with -0.2% aqueous formic acid of methanol (2:8), are made containing interior
Mark 250ng/ml, containing added with the final concentration of 0ng/ml, 100ng/ml of hypoxanthine, 200ng/ml, 300ng/ml, 400ng/ml,
The mark-on test solution of 500ng/ml.
Further, gradient elution program are as follows: 0~0.5min (15%A, 85%B)；0.5~0.6min (95%A, 5%
B), 0.6~1.0min (95%A, 5%B)；1.0~1.1min (5%A, 95%B)；1.1~3.5min (5%A, 95%B).
Standard addition method is to be intended to survey the pure material of component to be added in sample to be tested, and component pure material to be surveyed is added in measurement
The peak area (or peak height) of front and back component to be surveyed, thus the method for calculating the content of component to be surveyed in the sample.The present invention establishes
Detection method multiple-reaction monitoring MRM scanning by Liquid Chromatography/Mass Spectrometry, is carried out using standard addition method, can be fast and accurately
Hypoxanthic content in SHUXUETONG ZHUSHEYE is detected, effectively reduces the interference of matrix effect, this method specificity is good, sensitivity
Height, precision, accuracy, extraction recovery, matrix effect and stability have reached the requirement of Pharmaceutical Analysis.
Detailed description of the invention
Fig. 1 is hypoxanthine and interior target MRM chromatogram in SHUXUETONG ZHUSHEYE.
In figure, A is blank physiological saline, and B is the standard solution of containing the internal standard, and C is that the Shu Xue-tong test sample of containing the internal standard is molten
Liquid.Peak 1 is hypoxanthine glycosides, and peak 2 is Ismipur.
Specific embodiment
The method of hypoxanthine content, packet in a kind of measurement SHUXUETONG ZHUSHEYE that typical embodiment provides of the present invention
It includes:
Step 1 prepares Ismipur inner mark solution and a series of hypoxanthine standard solutions；
Ismipur inner mark solution and hypoxanthine standard solution is added in step 2 in SHUXUETONG ZHUSHEYE to be measured
It is configured to mark-on sample, using Liquid Chromatography/Mass Spectrometry, carries out multiple-reaction monitoring MRM scanning, is cross with mark-on hypoxanthine final concentration X
Coordinate, target analytes and interior target peak area ratio Y are ordinate, carry out linear regression, obtain regression equation；
Ismipur inner mark solution is added in step 3 in SHUXUETONG ZHUSHEYE to be measured, using Liquid Chromatography/Mass Spectrometry, carries out
Multiple-reaction monitoring MRM scanning, obtains target analytes and interior target peak area ratio, obtains Shu Xue-tong according to above-mentioned regression equation
Hypoxanthine content in injection；
In step 2 and step 3, the ginseng of liquid phase chromatogram condition, Mass Spectrometer Method condition and multiple-reaction monitoring MRM scan method
Number is identical.
Liquid phase chromatogram condition are as follows: analytical column is Waters Acquity UPLC BEH C18(100mm×2.1mm,1.7μ
M), guard column is WatersAcquity UPLC BEH C18(5mm × 2.1mm, 1.7 μm), mobile phase A is mutually methanol, and B phase is
0.1% aqueous formic acid, gradient elution, flow velocity 0.3ml/min, column temperature are 40 DEG C, and sample volume is 2 μ l；Preferably, gradient is washed
De- program are as follows: 0~0.5min (15%A, 85%B)；0.5~0.6min (95%A, 5%B), 0.6~1.0min (95%A, 5%
B)；1.0~1.1min (5%A, 95%B)；1.1~3.5min (5%A, 95%B).
Mass Spectrometer Method condition are as follows: Negative electrospray ionization ESI (-) detection pattern；Capillary voltage (Capillary):
0.5KV；Orifice potential (Cone): 30V；Spray pressure power (Nebuliser): 7psi；Desolvation temperature (Desolvation
Temp): 400 DEG C；Desolventizing gas flow (Desolvation Gas Flow): 800Lh-1；Taper hole throughput (Cone Gas
Flow): 150Lh-1, collision gas argon flow (Collision Gas Flow): 0.25mlmin-1；
The parameter of multiple-reaction monitoring MRM scan method is as follows:
Hypoxanthine: parent ion mass number (Q1): 134.9；Product ion mass number (Q3): 92.0；Residence time (Dewll
Time): 0.129s；Orifice potential (Cone Voltage): 30V；Collision energy (Collision Energy): 12V；
Ismipur: parent ion mass number (Q1): 151.0；Product ion mass number (Q3): 92.1；Residence time
(Dewll Time): 0.129s；Orifice potential (Cone Voltage): 56V；Collision energy (Collision Energy):
16V。
In some opposite specific embodiments,
Preparation Ismipur inner mark solution described in step 1 adds methanol molten comprising steps of weighing Ismipur
Solution, dilution, are made the Ismipur methanol stock solution of 250 μ g/ml；Ismipur stock solution is pipetted, with methanol -0.2%
The Ismipur inner mark solution of 125 μ g/ml is made in aqueous formic acid (2:8) dissolution, dilution.
Preparation hypoxanthine standard solution described in step 1 adds after methanol in 60 comprising steps of weigh hypoxanthine
DEG C ultrasound makes it completely dissolved, and lets cool, then plus methanol dilution 100 μ g/ml hypoxanthine methanol stock solutions are made；It is fast to pipette time Huang
Purine stock solution 1.00ml is diluted to the hypoxanthine standard solution of 50 μ g/ml with -0.2% aqueous formic acid of methanol (2:8).
In step 2, mark-on sample is prepared comprising steps of pipetting totally 6 parts of 100 μ l of SHUXUETONG ZHUSHEYE, concentration is respectively added
For the 100 μ l of Ismipur inner mark solution of 125 μ g/ml, it is separately added into the hypoxanthine standard solution that concentration is 50 μ g/ml
0μl,100μl,200μl,300μl,400μl,500μl；Then body is supplied with -0.2% aqueous formic acid of diluent methanol (2:8)
Product is vortexed, mixes to 1.00ml；100 μ l are respectively taken, then dilute 10 with appropriate -0.2% aqueous formic acid of diluent methanol (2:8)
Times, it is vortexed, mixes；200 μ l are taken respectively, dilute 5 times with -0.2% aqueous formic acid of methanol (2:8) and containing the internal standard 250ng/ is made
Ml, contain added with hypoxanthine final concentration of 0ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml
Mark-on Shu Xue-tong test solution.
Claimed technical solution is described further below by some embodiments.Unless otherwise specifically
Illustrate, material therefor, reagent can be obtained from the commercially produced product of this field in the present invention.
Embodiment 1
The preparation of sample
The preparation of stock solution: precision weighs hypoxanthine, adds methanol appropriate, in 60 DEG C of ultrasound 30min, keeps it completely molten
Solution, let cool, then plus methanol dilution 100 μ g/ml hypoxanthine methanol stock solutions are made.Another precision weighs Ismipur, adds first
Appropriate alcohol, vortex 2min, makes it completely dissolved, and adds methanol dilution that the Ismipur methanol stock solution of 250 μ g/ml is made.
The preparation of standard solution: precision pipettes hypoxanthine stock solution 1.00ml, with -0.2% aqueous formic acid of methanol
The hypoxanthine standard solution that (2:8) is diluted to 50 μ g/ml is spare；Another precision pipettes Ismipur stock solution, with methanol-
The Ismipur inner mark solution that 0.2% aqueous formic acid (2:8) is diluted to 125 μ g/ml is spare.
The preparation of mark-on sample: precision pipettes totally 6 parts of 100 μ l of SHUXUETONG ZHUSHEYE, and each concentration that is added is 125 μ g/ml's
100 μ l of Ismipur inner mark solution, be separately added into concentration be 0 μ l of hypoxanthine standard solution of 50 μ g/ml, 100 μ l,
200μl,300μl,400μl,500μl；Then volume is supplied extremely with -0.2% aqueous formic acid of diluent methanol (2:8)
1.00ml is vortexed, mixes；100 μ l are respectively taken, then dilute 10 times with appropriate -0.2% aqueous formic acid of diluent methanol (2:8), whirlpool
Rotation mixes；Take 200 μ l respectively, with -0.2% aqueous formic acid of methanol (2:8) dilute 5 times be made containing the internal standard 250ng/ml, containing plus
There is the Shu Xue-tong of hypoxanthine final concentration of 0ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml
Test solution.
Using Liquid Chromatography/Mass Spectrometry, multiple-reaction monitoring MRM scanning is carried out:
Liquid phase chromatogram condition are as follows: analytical column is Waters Acquity UPLC BEH C18(100mm×2.1mm,1.7μ
M), guard column is WatersAcquity UPLC BEH C18(5mm × 2.1mm, 1.7 μm), mobile phase A is mutually methanol, and B phase is
0.1% aqueous formic acid, gradient elution, flow velocity 0.3ml/min, column temperature are 40 DEG C, and sample volume is 2 μ l；
Mass Spectrometer Method condition are as follows: Negative electrospray ionization ESI (-) detection pattern；Capillary voltage (Capillary):
0.5KV；Orifice potential (Cone): 30V；Spray pressure power (Nebuliser): 7psi；Desolvation temperature (Desolvation
Temp): 400 DEG C；Desolventizing gas flow (Desolvation Gas Flow): 800Lh-1；Taper hole throughput (Cone Gas
Flow): 150Lh-1, collision gas argon flow (Collision Gas Flow): 0.25mlmin-1；
The parameter of multiple-reaction monitoring MRM scan method is as follows:
Hypoxanthine: parent ion mass number (Q1): 134.9；Product ion mass number (Q3): 92.0；Residence time (Dewll
Time): 0.129s；Orifice potential (Cone Voltage): 30V；Collision energy (Collision Energy): 12V；
Ismipur: parent ion mass number (Q1): 151.0；Product ion mass number (Q3): 92.1；Residence time
(Dewll Time): 0.129s；Orifice potential (Cone Voltage): 56V；Collision energy (Collision Energy):
16V。
The foundation of mark-on curve
Taking lot number is 17040811 SHUXUETONG ZHUSHEYE, is divided into 6 parts, and time Huang for being separately added into internal standard and different volumes is fast
Purine standard items are made containing added with hypoxanthine final concentration of 0ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/
The mark-on SHUXUETONG ZHUSHEYE (containing the internal standard 250ng/ml) of ml, 500ng/ml, 2 μ l of sample introduction measurement.It is dense eventually with mark-on hypoxanthine
Spending (X) is abscissa, and target analytes and interior target peak area ratio (Y) are ordinate, weight 1/X2, linear regression is carried out,
Obtain regression equation Y=5.664 × 10-3X+1.447, r=0.9998.Measuring minimal detectable concentration simultaneously is 0.3ng/ml, minimum
Quantitative concentrations are 1ng/ml.
The SHUXUETONG ZHUSHEYE to be measured of multiple lot numbers is separately taken, Ismipur inner mark solution is added, according to above-mentioned recurrence side
Journey acquires hypoxanthine content in SHUXUETONG ZHUSHEYE.
Standard Addition Method for Determination hypoxanthine content
The SHUXUETONG ZHUSHEYE of 20 batches is taken, sample introduction measurement is as a result as follows.
Specificity
Under the conditions of the present embodiment, physiological saline, standard solution, test solution difference sample introduction measurement are taken, secondary Huang is fast
Purine, Ismipur chromatographic peak profile are good, measure noiseless.
Precision is investigated
Taking lot number is 17040811 SHUXUETONG ZHUSHEYE, and hypoxanthine standard items and internal standard is added, is made final concentration of
Low (100ng/ml), in (300ng/ml), high (500ng/ml) mark-on SHUXUETONG ZHUSHEYE (containing the internal standard 250ng/ml), respectively
It is in a few days measured 6 times in same, and takes the mark-on SHUXUETONG ZHUSHEYE of above-mentioned basic, normal, high three kinds of concentration, respectively at 1d, 2d, 3d sample introduction
Analysis records and analyzes object and internal standard peak area, calculates its peak area ratio (AAnalyte/AInternal standard) and RSD.The result shows that basic, normal, high
Three kinds of concentration is in a few days respectively less than 1% with day to day precision RSD value.
1 withinday precision test result of table
Table interior Precision test result on the 2nd
Stability test
Taking lot number is 17040811 SHUXUETONG ZHUSHEYE, and hypoxanthine standard items and internal standard is added, and is made containing added with secondary
Xanthine is final concentration of 0, low (100ng/ml), in (300ng/ml), high (500ng/ml) mark-on SHUXUETONG ZHUSHEYE (containing interior
Mark 250ng/ml), respectively at be placed at room temperature for 0,2,4,8,12,24, sample introduction is analyzed after 48h, record and analyze object and internal standard peak area,
Calculate its peak area ratio (AAnalyte/AInternal standard) and RSD.The result shows that its RSD value is respectively less than 1%, illustrate that solution is being placed at room temperature for
Stablize in 48h.
The stability of 3 Shuxuetong injection liquor of table
Sample recovery rate
Taking lot number is 17040811 SHUXUETONG ZHUSHEYE, and hypoxanthine standard items and internal standard is added, and is made containing added with secondary
Xanthine final concentration of low (100ng/ml), in (300ng/ml), high (500ng/ml) mark-on SHUXUETONG ZHUSHEYE (containing the internal standard
250ng/ml) each 6 parts, sample introduction measurement calculates the rate of recovery with measured value/mark-on value × 100%, the results showed that, it is average to recycle
Rate is 99.2%.
4 sample recovery rate test result of table
Reproducibility is investigated
Taking lot number is 17040811 SHUXUETONG ZHUSHEYE, and hypoxanthine standard items and internal standard is added, prepares 6 parts of confessions respectively
Test sample solution, sample introduction measurement.The result shows that repeated RSD is less than 3%.
5 repetitive test result of table
In conclusion detection method established by the present invention can fast and accurately detect in SHUXUETONG ZHUSHEYE it is time yellow fast
The content of purine, this method specificity is good, high sensitivity, and precision, accuracy, extraction recovery, matrix effect and stability are equal
The requirement of Pharmaceutical Analysis is reached.Above embodiments are to be used to explain the present invention embodiment, without departing from present subject matter
Range, the scope of the present invention are not limited by the embodiment.
Claims (5)
1. a kind of method of hypoxanthine content in measurement SHUXUETONG ZHUSHEYE characterized by comprising
Step 1 prepares Ismipur inner mark solution and a series of hypoxanthine standard solutions；
Step 2, is added Ismipur inner mark solution in SHUXUETONG ZHUSHEYE to be measured and hypoxanthine standard solution is prepared
Multiple-reaction monitoring MRM scanning is carried out using Liquid Chromatography/Mass Spectrometry at mark-on sample, with mark-on hypoxanthine final concentration X for horizontal seat
Mark, target analytes and interior target peak area ratio Y are ordinate, carry out linear regression, obtain regression equation；
Ismipur inner mark solution is added in step 3 in SHUXUETONG ZHUSHEYE to be measured, using Liquid Chromatography/Mass Spectrometry, how anti-carries out
MRM scanning should be monitored, target analytes and interior target peak area ratio are obtained, Shuxuetong injection is obtained according to above-mentioned regression equation
Hypoxanthine content in liquid；
In step 2 and step 3, liquid phase chromatogram condition are as follows: analytical column is Waters Acquity UPLC BEH C18(100mm
× 2.1mm, 1.7 μm), guard column is Waters Acquity UPLC BEH C18(5mm × 2.1mm, 1.7 μm), mobile phase A phase
For methanol, B phase is 0.1% aqueous formic acid, and gradient elution, flow velocity 0.3ml/min, column temperature is 40 DEG C, and sample volume is 2 μ l；
Mass Spectrometer Method condition are as follows: Negative electrospray ionization ESI (-) detection pattern；Capillary voltage (Capillary): 0.5KV；Cone
Hole voltage (Cone): 30V；Spray pressure power (Nebuliser): 7psi；Desolvation temperature (Desolvation Temp):
400℃；Desolventizing gas flow (Desolvation Gas Flow): 800Lh-1；Taper hole throughput (Cone Gas Flow):
150L·h-1, collision gas argon flow (Collision Gas Flow): 0.25mlmin-1；
The parameter of multiple-reaction monitoring MRM scan method is as follows:
Hypoxanthine: parent ion mass number (Q1): 134.9；Product ion mass number (Q3): 92.0；Residence time (Dewll
Time): 0.129s；Orifice potential (Cone Voltage): 30V；Collision energy (Collision Energy): 12V；
Ismipur: parent ion mass number (Q1): 151.0；Product ion mass number (Q3): 92.1；Residence time (Dewll
Time): 0.129s；Orifice potential (Cone Voltage): 56V；Collision energy (Collision Energy): 16V.
2. according to the method described in claim 1, it is characterized by: preparation Ismipur inner mark solution described in step 1,
Comprising steps of weighing Ismipur, adds methanol dissolution, dilution, the Ismipur methanol stock solution of 250 μ g/ml is made；It moves
Ismipur stock solution is taken, with -0.2% aqueous formic acid of methanol (2:8) dissolution, dilution, the 6- sulfydryl of 125 μ g/ml is made
Purine inner mark solution.
3. according to the method described in claim 2, it is characterized by: preparation hypoxanthine standard solution described in step 1,
Comprising steps of weighing hypoxanthine, make it completely dissolved, let cool in 60 DEG C of ultrasounds after adding methanol, then plus methanol dilution be made 100
μ g/ml hypoxanthine methanol stock solution；Hypoxanthine stock solution 1.00ml is pipetted, with -0.2% aqueous formic acid of methanol (2:8)
It is diluted to the hypoxanthine standard solution of 50 μ g/ml.
4. according to the method described in claim 3, it is characterized by: mark-on sample is prepared, comprising steps of pipetting in step 2
Totally 6 parts of 100 μ l of SHUXUETONG ZHUSHEYE, it is each that the 100 μ l of Ismipur inner mark solution that concentration is 125 μ g/ml is added, it is separately added into
Concentration is 0 μ l of hypoxanthine standard solution, 100 μ l, 200 μ l, 300 μ l, 400 μ l, the 500 μ l of 50 μ g/ml；Then with dilution
- 0.2% aqueous formic acid of agent methanol (2:8) is diluted to 1.00ml, is vortexed, mixes；Respectively take 100 μ l, then with appropriate diluent first
- 0.2% aqueous formic acid of alcohol (2:8) dilutes 10 times, is vortexed, mixes；200 μ l are taken respectively, with -0.2% aqueous formic acid of methanol
(2:8) dilutes 5 times and containing the internal standard 250ng/ml is made, containing added with hypoxanthine final concentration of 0ng/ml, 100ng/ml, 200ng/
The mark-on solution of ml, 300ng/ml, 400ng/ml, 500ng/ml.
5. method according to any of claims 1-4, it is characterised in that: gradient elution program are as follows: 0~0.5min
(15%A, 85%B)；0.5~0.6min (95%A, 5%B), 0.6~1.0min (95%A, 5%B)；1.0~1.1min (5%
A, 95%B)；1.1~3.5min (5%A, 95%B).
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| CN112557527A (en) * | 2020-11-17 | 2021-03-26 | 牡丹江友搏药业有限责任公司 | Method for measuring polypeptide content in Shuxuetong injection |
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| CN112557527A (en) * | 2020-11-17 | 2021-03-26 | 牡丹江友搏药业有限责任公司 | Method for measuring polypeptide content in Shuxuetong injection |
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