CN107655982A - A kind of method of Sebivo content in detection blood plasma - Google Patents
A kind of method of Sebivo content in detection blood plasma Download PDFInfo
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Abstract
The invention belongs to field of medical examination, is related to the method that Sebivo content in blood plasma is detected with liquid chromatography tandem mass spectrometry.Testing sample is not required to dry up through nitrogen in this method, and supernatant sample introduction is taken after direct dilution in acetonitrile/protein precipitation, is eluted and is separated in chromatographic column through mobile phase, is detected with tandem mass spectrum detector;The Sebivo of deuterium 3 (D3 LdT) is used in this method as internal standard, blood sample adds quantitative acetonitrile precipitation albumen, and for Sebivo without obvious degradation, the degree of accuracy of ensuring method improves the security of operator simultaneously after inactivation of viruses in plasma sample processing.It is 10 10000ng/mL to determine Sebivo and interior target concentration, quantitative linearity scope using tandem mass spectrometry in this method, can meet the requirement of human pharmacokineticses research.This method sample is few, and pretreatment is simple, quick, sensitive, only needs universal equipment and reagent, analytical cycle is short, and cost is low, the monitoring of adjustment and conventional blood concentration suitable for hepatitis B patient therapeutic scheme.
Description
Technical field
The invention belongs to technical field of medical examination, it is related to the analysis determining method of internal medicine, and in particular to one kind liquid
The method of Sebivo content, the Liquid Chromatography-Tandem Mass Spectrometry method are applied to detection in phase chromatographic tandem mass spectrography detection blood plasma
The content of Sebivo in hepatitis B patient blood plasma.
Background technology
Data shows that hepatitis type B virus (HBV) infection is health problem universal in global range.According to WHO, entirely
The people of ball about 2,000,000,000 once infected HBV, wherein 3.5 hundred million people are Patients with Chronic HBV Infection, there are about 1,000,000 people every year and dies from HBV infection institute
Hepatic failure, hepatic sclerosis and the liver cancer of cause;The national Hepatitis B With Its Epidemics investigation result carried out according to 06 year Ministry of Public Health of China pushes away
Calculate, the people of the existing Patients with Chronic HBV Infection in China about 93,000,000, wherein chronic hepatitis B (CHB) patient about 20,000,000.
Sebivo (Telbivudine, LdT) is that the left-handed nucleosides of artificial synthesized thymidine is similar
Thing, granted on 2 14th, 2007 SFDA, in April, 2007 is in Discussion on Chinese Listed.Research reports Sebivo due to higher disease
Malicious response rate, HBeAg Virus mutations rate, medication not by feed influenceed and zoopery find without carcinogenic, teratogenesis, mutagenesis
Effect is set to pregnant B classes medicine by FDA Food and Drug Administration, so as to the Anti-HBV drugs commonly used as hepatitis B patient.
The effect through cell kinase is converted into the triphosphoric acid of activity for than husband after studies have shown that Sebivo oral absorption
Fixed, the latter can suppress the activity of HBV archaeal dna polymerases, and mix viral DNA, cause the synthesis of viral DNA chain to terminate, final to suppress
Hbv replication;It is absorbed by the body rapidly after Sebivo is oral, bioavilability more than 40%;Daily oral Sebivo
600mg, reach blood peak concentration of drug within about 1 hour upon administration, there is the elimination half-life period up to 40 to 50 hours, up to steady after 5~7 days
State, blood concentration of the steady state plasma concentration about than single dose administration is high by 50% or so, slight accumulation, impaired renal function in vivo be present
Patient need to carry out dosage adjustment.
The assay method in document on Sebivo drug concentration has two kinds of LC-MS/MS and HPLC-UV at present, relates to
And the research of Sebivo pharmacokinetics, but specific detection method and Method validation there is not yet report in detail.In addition, from
The detection method learned in document is respectively present following some deficiencies or shortcoming:(1) cumbersome, the detection method reported is led to
Often nitrogen drying enrichment is rebuild to improve sensitivity again in sample pre-treatments, it is necessary to after albumen precipitation;(2) Sebivo I/
II clinical trial phases are prompted, Sebivo 600mg/d maximum body-fluid concentration be between 0.20~6.84 μ g/mL, it is and current
Narrower 0.01~5.00 μ g/mL of the detection method range of linearity of report, in view of to blood concentration situations such as clinical drug combination
Influence, therefore, current quantitative detection range can not meet that clinical practice determines needs rapidly;(2) the quantitative inspection of HPLC-UV methods
Survey scope is 0.1~10.00 μ g/mL, can not meet Sebivo low concentration point in human plasma while improving upper limit of detection
Measure requires that (3) HPLC-UV methods measure Sebivo and interior target retention time are respectively 4.9min and 7.7min, during analysis
Between it is longer while big to mobile phase requirement;(4) precision is poor;In a few days with day to day precision about 10.6%;(5) due to
Sebivo is used for the CHB patient for treating HBV, HCV cross-infection, based on the angle of protection measure operator's safety, pushes away in the industry
Recommending the blood plasma in preceding processing needs heat inactivation viral (58 DEG C, 40min), but the operating procedure has the concentration for influenceing Sebivo
Factor, on the other hand, further checking of still needing.
Present situation based on prior art, present inventor intend providing a kind of safe operation, simple and convenient sample treatment, fast
The method of Sebivo content in fast, sensitive, good, high specificity the analysis detection blood plasma of selectivity, beneficial to development Sebivo
Inside pharmacokinetic, to patient carry out dosage adjustment basis.
The content of the invention
The defects of purpose of the present invention is supplement and overcomes prior art and deficiency, there is provided at a kind of safe operation, sample
Manage easy, quick, sensitive, to be available for Sebivo blood concentration in clinical batches measure human plasma method, more particularly to one
The method that kind detects Sebivo content in blood plasma with liquid chromatography tandem mass spectrometry.
This method has the characteristics that safe operation, simplicity, quick, blood plasma dosage is few, available for clinical Sebivo blood medicine
Concentration monitor and the reference adjusted as dosage.
The method that Sebivo content in blood plasma is detected with liquid chromatography tandem mass spectrometry of the present invention, it is characterised in that
Testing sample is not required to through nitrogen gas blow dry step, supernatant sample introduction is taken after direct dilution in acetonitrile/protein precipitation, through mobile phase in chromatogram
Post elution separation, is detected with tandem mass spectrum detector;It includes step:
1) sample pretreatment
Testing sample is taken, internal standard working solution is added, adds quantitative organic solvent protein precipitation, after centrifugation, take supernatant
40min is heated in 58 DEG C of water-baths, it is to be measured;
2) sample separates
Using universal liquid phase chromatographic column, its filler is high intensity silica gel particle C18, and efficient liquid phase system is using universal
High-pressure pump and injector, using mixed liquor of the acidic aqueous solution containing 0.1% aqueous formic acid (A) and acetonitrile (B) as mobile phase,
Gradient elution;
Gradient elution:0-4.0min A:B is from (95:5, v/v) (20 are arrived:80, v/v), 4.0-4.1min A:B is from (20:
80, v/v) (95 are arrived:5, v/v), 4.1-6.0min A:B(95:5,v/v);Flow velocity:0.4mL/min;
3) sample detection
Sebivo and interior target peak area are detected using universal tandem mass spectrum detector;
Mass spectrum ionizes mode:Electric spray ion source;Polarity:Anionic textiles;Scan mode:More reactive ion detection scannings
(MRM);Ion channel selects:LdT:M/z 243.10 → 127.10, D3-LdT:m/z 246.10→130.10.
In the present invention, deuterium 3- Sebivos (D3-LdT) are used in the blood sample treatments of step 1) as internal standard, testing sample warp
Acetonitrile precipitation albumen simply pre-processes restrovirus inactivation;In embodiments of the invention, it is described in be designated as deuterium 3- Sebivos
(D3-LdT), protein precipitant is acetonitrile, and organic solvent is acetonitrile;
In the tandem mass spectrometry of step 3) of the present invention, swept using electric spray ion source and the more reactive ion detections of anion
Retouch, ion channel m/z 243.10 → 127.10 detects LdT peak area, and m/z 246.10 → 130.10 detects D3-LdT peak
Area, concentration is converted into calibration curve equation.
Sebivo blood in clinical batches human plasma sample have detected using liquid chromatography tandem mass spectrometry in the present invention
Concentration, in the steps below:
1) sample pretreatment
The μ L of testing sample 100 are taken in centrifuge tube, add 40 μ L internal standards working solutions (containing thymidine phosphorylase), DL
10s, 1h is incubated in 37 DEG C to digest the endogenous thymidine that may be influenceed Sebivo and determine, adds 200 μ L acetonitrile precipitations
Albumen, DL 10s, 13000rpm centrifugation 5min, the μ L of Aspirate supernatant 200 heats 40min in 58 DEG C, to be measured;
Deuterium 3- Sebivos (D3-Telbivudine, D3-LdT) are designated as in described;
After the heated inactivation of viruses of plasma sample, Sebivo ensure that the accuracy of method without obvious degradation;
2) sample separates
Using universal liquid-phase chromatographic column, its filler is high intensity silica gel particle C18, and efficient liquid phase system is using general
Type high-pressure pump and injector, mobile phase are made up of 0.1% aqueous formic acid and acetonitrile, gradient elution;
3) sample detection
Sebivo and interior target peak area are detected using universal tandem mass spectrum detector;
Mass spectrum ionizes mode:Electric spray ion source;Polarity:Positive ion detection;Scan mode:More reactive ion detection scannings
(MRM);Ion channel selects:LdT:M/z 243.10 → 127.10, D3-LdT:m/z 246.10→130.10;Collision gas:Nitrogen
Gas;
Instrument parameter is preferably set to:Interface voltage:0.25kV;Atomization gas flow velocity:3.0L/min;Heat gas velocity:
8.0L/min;Dry gas stream speed:12.0L/min;Interface temperature:250℃;DL temperature:150℃;Heating block temperature:350℃;
LdT and D3-LdT collision energy is respectively -10.0 and -9.0;Residence time:100ms;
4) concentration calculates
Using sample concentration as X-axis, the ratio of LdT peak areas (As) and internal standard peak area (Ai) is Y-axis, is weighted recurrence
Analysis, the concentration value of unknown blood sample is released using calibration curve method (in standard curve range).
Testing result shows, this method quick and precisely, easy to operate, high sensitivity, the range of linearity it is wide, the method rate of recovery is steady
Fixed, precision in the daytime and in a few days is respectively less than 10%, suitable for routine clinical monitoring, is adjusted suitable for the dosage of hepatitis B patient.
Compared with prior art, the contribution of this method is:Sample pretreating method is simplified, is carried out for measure thing
Experimental condition optimization:Analogue is used as internal standard;Sample pretreatment process eliminates nitrogen drying concentration step, shortens
Analysis time;Using high intensity silica gel particle C18 as chromatographic column filler, mobile phase condition is optimized;It is water-soluble using 0.1% formic acid
The mixed liquor of liquid and acetonitrile is as mobile phase, gradient elution;This hair return demonstrate heat inactivation virus processing after replace than
Husband is fixed without obvious degradation, there is the security for being suitable for and improving operator.
This method has advantages below:
1) internal standard selects:Using analogue D3-LdT, chromatographic behavior is similar for internal standard, and chromatographic peak appearance time connects
Closely, the matrix effect interference being subject to is close, ensures the accurate of quality control process.
2) preprocess method:One step organic solvent precipitation of protein, it is easy, inexpensive, suitable for conventional detection;Employ 58
DEG C water-bath 40 minutes is demonstrated the pre-treatment step and will not be caused LdT degraded with inactivation of viruses, ensure that the accurate of method
The security of operation is ensure that while property.
3) separation condition:It is high intensity silica gel particle C18 chromatographic columns as stationary phase, acetonitrile and formic acid that separation, which uses filler,
The mixed liquor of the aqueous solution gradient elution, is not disturbed as mobile phase by endogenous material matrix effect.
4) condition determination:Measure is detected using universal tandem mass spectrum, and this method is not required to chromatographic isolation sample and internal standard, letter
Change analytical procedure, substantially increase the efficiency of clinical blood concentration detection.
5) sampling quantity is few:Determining a sample only needs 100 μ L plasma samples.
6) high sensitivity:By using different ionic reaction passages for sample and internal standard, avoid sample internal standard mutual
Interference and the interference of endogenous material, obtain optimal response signal.The minimum of LdT is quantitatively limited to 10ng/mL.
7) high specificity:Endogenous material and conventional drug combination are not disturbed measure.
8) range of linearity is wide:LdT setting-out lines are good, and concentration range is 10~10000ng/mL.
Brief description of the drawings:
Fig. 1:Typical chromatogram:The blank plasma of LdT health volunteer is not used, wherein, figure A is LdT:m/z
243.10 → 127.10, figure B are internal standard D3-LdT:m/z 246.10→130.10.
Fig. 2:Typical chromatogram:Blank plasma adds LdT and internal standard standard items (concentration is 10ng/mL) wherein, and figure A is
LdT:M/z 243.10 → 127.10, figure B are internal standard D3-LdT:m/z 246.10→130.10.
Fig. 3:One chronic hepatitis B patient L122 determines chromatogram using LdT 600mg/d blood sample, wherein, scheme A
For LdT:M/z 243.10 → 127.10, figure B are internal standard D3-LdT:m/z 246.10→130.10;LdT blood concentrations are
198.3ng/mL, blood sampling time are up to 12 hours after the medication of stable state.
Fig. 4:One chronic hepatitis B patient L122 determines chromatogram using LdT 300mg/d blood sample, wherein, figure
A is LdT:M/z 243.10 → 127.10, figure B are internal standard D3-LdT:m/z 246.10→130.10;LdT blood concentrations are
358.3ng/mL, blood sampling time are up to 12 hours after the medication of stable state.
Embodiment
Embodiment 1:
Chromatographic condition
Japanese Shimadzu LCMS8050 systems:SIL-30AC automatic samplers (1), LC-30AD infusion pumps (2), DGU-
20A5On-line degassing instrument (1), CBM-20A controllers (1), CTO-30A column ovens (1).Data acquisition and procession software:
LabSolutions Ver.5.56SP1.Shimadzu Inertsil Sustain C18,3.0 × 100mm, 3 μm.Column temperature:40℃.Stream
Dynamic phase:0.1% aqueous formic acid (A) and acetonitrile (B), gradient elution:0-4.0min A:B is from (95:5, v/v) (20 are arrived:80,v/
V), 4.0-4.1min A:B is from (20:80, v/v) (95 are arrived:5, v/v), 4.1-6.0minA:B(95:5,v/v).Flow velocity:
0.4mL/min。
Mass Spectrometry Conditions:
Shimadzu, Japan's LCMS8050 mass spectrographs.Mass spectrum ionizes mode:Electric spray ion source;Polarity:Cation
Detection;Scan mode:More reactive ion detection scannings (MRM);Ion channel selects:LdT:M/z 243.10 → 127.10, D3-
LdT:m/z 246.10→130.10;Collision gas:Nitrogen.
Instrument parameter is preferably set to:Interface voltage:0.25kV;Atomization gas flow velocity:3.0L/min;Heat gas velocity:
8.0L/min;Dry gas stream speed:12.0L/min;Interface temperature:250℃;DL temperature:150℃;Heating block temperature:350℃;
LdT and D3-LdT collision energy is respectively -10.0 and -9.0;Residence time:100ms.
Plasma sample pre-processes
100 μ L sample of blood are taken in centrifuge tube, add 40 μ L internal standard working solutions, vortex 10s, be placed in 37 DEG C of water-baths be incubated with
Digestion may influence the interference of the endogenous thymidine of LdT measure, be taken out after being incubated 1h, add 200 μ L acetonitrile precipitation albumen,
Vortex 1min, and 5min, the μ L of Aspirate supernatant 200 are centrifuged in 13000rpm, it is placed in 58 DEG C of water-baths and heats 40min to inactivate disease
Poison, it is to be measured;Internal standard method is with peak area quantification.
Specificity
(1) interference of endogenous material:Two parts of blank plasma is taken, it (is 10ng/ that a copy of it, which adds LdT and D3-LdT,
ML), portion is not added with LdT and D3-LdT, is measured according to above-mentioned sample pretreatment and assay method, does not find plasma endogenous
Material is to LdT and interior indicates interference;(2) interference of the internal standard to LdT:Two parts of blank plasma is taken, a copy of it adds D3-LdT
(10ng/mL), another adds LdT and D3-LdT (being 10ng/mL), and Treatment Analysis, does not find internal standard as stated above
Compound has interference to LdT measure;(3) matrix effect:6 parts of blank plasmas are taken, are obtained according to above-mentioned sample pretreatment containing matrix
Supernatant, a certain amount of standard solution is added, the result and the results contrast of chemicals standard solution of sample introduction measure, is not sent out
Existing matrix effect has interference to LdT measure;(4) common drug combination:Aldoforwe ester, SNMC, polyene phosphatidyl
Choline, reduced glutathione, diammonium glycyrrhizinate, bicyclic alcohols, silymarin, magnesium isoglycyrrhetate etc. do not interfere with to measure;LdT
It is 2.3min with interior target typical color spectrum retention time, whole chromatography process time is 6min.
Linear test
It is appropriate that precision weighs LdT standard items, is dissolved with pure water, is diluted to series of working liquids, adds appropriate blank people blood
Slurry, it is respectively 10,20,50,100,200,500,1000,2000,5000 and 10000ng/mL to be configured to plasma concentration containing LdT
Standard blood sample.The μ L of blood plasma 100 are taken, are operated by " plasma sample pretreatment " method.Internal standard method is with component to be measured and interior target peak face
Product weights (1/X) linear regression than (Y) and concentration of component to be measured (X), and the range of linearity is 10~10000ng/mL, minimum quantitative
10ng/mL is limited to, LdT standard curve regression equation is respectively:Y=(0.000263052) X+ (- 3.11010e-005), r=
0.9999。
The degree of accuracy and precision
Precision weighs that LdT standard items are appropriate, and pure water dissolves and is diluted to series of working liquids, adds appropriate blank people blood
Slurry, is configured to the Serial blood that plasma concentration containing LdT is respectively 30,400 and 8000ng/mL, the μ L of blood plasma 100 is respectively taken, by " blood plasma
Sample pretreatment " method operates, and investigates preci-sion and accuracy in a few days and in the daytime.Its actual measurement is calculated according to equation of linear regression
Concentration, calculate actual measurement average value and the relative standard deviation (RSD) of every kind of concentration;As a result show LdT in a few days and day to day precision
Respectively less than 10%;Table 1 show LdT in a few days, day to day precision and the degree of accuracy.
This method is used for the measure of LdT in plasma of patients with chronic hepatitis B, and Fig. 3 is shown using LdT 600mg/d's
Chronic hepatitis B patient L122 is treated 8 months, and the blood sample measure chromatogram of 12 hours, LdT concentration are after medication
2219.64ng/mL, it is significantly higher than document report;In view of the patient has the malaise symptoms such as DOMS, serum creatine kinase is higher than
7 times of Upper Limit of Normal Value, with reference to above-mentioned inspection result, the therapeutic scheme of the patient is adjusted to LdT 300mg/d by clinician.
Embodiment 2:
Chromatographic condition
Japanese Shimadzu LCMS8050 systems:SIL-30AC automatic samplers (1), LC-30AD infusion pumps (2), DGU-
20A5On-line degassing instrument (1), CBM-20A controllers (1), CTO-30A column ovens (1).Data acquisition and procession software:
LabSolutions Ver.5.56SP1.Shimadzu Inertsil Sustain C18,3.0 × 100mm, 3 μm.Column temperature:40℃.Stream
Dynamic phase:0.1% aqueous formic acid (A) and acetonitrile (B), gradient elution:0-4.0min A:B is from (95:5, v/v) (20 are arrived:80,v/
V), 4.0-4.1min A:B is from (20:80, v/v) (95 are arrived:5, v/v), 4.1-6.0minA:B(95:5,v/v).Flow velocity:
0.4mL/min。
Mass Spectrometry Conditions:
Shimadzu, Japan's LCMS8050 mass spectrographs.Mass spectrum ionizes mode:Electric spray ion source;Polarity:Cation
Detection;Scan mode:More reactive ion detection scannings (MRM);Ion channel selects:LdT:M/z 243.10 → 127.10, D3-
LdT:m/z 246.10→130.10;Collision gas:Nitrogen.
Instrument parameter is preferably set to:Interface voltage:0.25kV;Atomization gas flow velocity:3.0L/min;Heat gas velocity:
8.0L/min;Dry gas stream speed:12.0L/min;Interface temperature:250℃;DL temperature:150℃;Heating block temperature:350℃;
LdT and D3-LdT collision energy is respectively -10.0 and -9.0;Residence time:100ms.
Plasma sample pre-processes
100 μ L sample of blood are taken in centrifuge tube, add 40 μ L internal standard working solutions, vortex 10s, be placed in 37 DEG C of water-baths be incubated with
Digestion may influence the interference of the endogenous thymidine of LdT measure, be taken out after being incubated 1h, add 200 μ L acetonitrile precipitation albumen,
Vortex 1min, and 5min, the μ L of Aspirate supernatant 200 are centrifuged in 13000rpm, it is placed in 58 DEG C of water-baths and heats 40min to inactivate disease
Poison, it is to be measured.Internal standard method is with peak area quantification.
Specificity
(1) interference of endogenous material:Two parts of blank plasma is taken, it (is 10ng/ that a copy of it, which adds LdT and D3-LdT,
ML), portion is not added with LdT and D3-LdT, is measured according to above-mentioned sample pretreatment and assay method, does not find plasma endogenous
Material is to LdT and interior indicates interference;(2) interference of the internal standard to LdT:Two parts of blank plasma is taken, a copy of it adds D3-LdT
(10ng/mL), another adds LdT and D3-LdT (being 10ng/mL), and Treatment Analysis, does not find internal standard as stated above
Compound has interference to LdT measure;(3) matrix effect:6 parts of blank plasmas are taken, are obtained according to above-mentioned sample pretreatment containing matrix
Supernatant, a certain amount of standard solution is added, the result and the results contrast of chemicals standard solution of sample introduction measure, is not sent out
Existing matrix effect has interference to LdT measure;(4) common drug combination:Aldoforwe ester, SNMC, polyene phosphatidyl
Choline, reduced glutathione, diammonium glycyrrhizinate, bicyclic alcohols, silymarin, magnesium isoglycyrrhetate etc. do not interfere with to measure;LdT
It is 2.3min with interior target typical color spectrum retention time, whole chromatography process time is 6min.
Linear test
It is appropriate that precision weighs LdT standard items, is dissolved with pure water, is diluted to series of working liquids, adds appropriate blank people blood
Slurry, it is respectively 10,20,50,100,200,500,1000,2000,5000 and 10000ng/mL to be configured to plasma concentration containing LdT
Standard blood sample.The μ L of blood plasma 100 are taken, are operated by " plasma sample pretreatment " method.Internal standard method is with component to be measured and interior target peak face
Product weights (1/X) linear regression than (Y) and concentration of component to be measured (X), and the range of linearity is 10~10000ng/mL, minimum quantitative
10ng/mL is limited to, LdT standard curve regression equation is respectively:Y=(0.000256315) X+ (- 0.000141726), r=
0.9999。
The degree of accuracy and precision
Precision weighs that LdT standard items are appropriate, and pure water dissolves and is diluted to series of working liquids, adds appropriate blank people blood
Slurry, is configured to the Serial blood that plasma concentration containing LdT is respectively 30,400 and 8000ng/mL, the μ L of blood plasma 100 is respectively taken, by " blood plasma
Sample pretreatment " method operates, and investigates preci-sion and accuracy in a few days and in the daytime.Its actual measurement is calculated according to equation of linear regression
Concentration, calculate actual measurement average value and the relative standard deviation (RSD) of every kind of concentration, the results showed that LdT in a few days and day to day precision
Respectively less than 10%;Table 2 show LdT in a few days, day to day precision and the degree of accuracy.
Fig. 4 shows that above-mentioned patient halves LdT dosage to 300mg/d, the blood sample measure color of 12 hours after treating 4 months
Spectrogram, LdT concentration are 1133.27ng/mL.
In the embodiment 1 of table 1 LdT in a few days, day to day precision and the degree of accuracy
In the embodiment 2 of table 2 LdT in a few days, day to day precision and the degree of accuracy
Claims (3)
- A kind of 1. method for detecting Sebivo content in blood plasma, it is characterised in that testing sample is not required to through nitrogen gas blow dry step, Supernatant sample introduction is taken after direct dilution in acetonitrile/protein precipitation, elutes and separates in chromatographic column through mobile phase, with tandem mass spectrum detector Detection;It includes step:1) sample pretreatmentTake testing sample, add internal standard working solution, add quantitative organic solvent protein precipitation, after centrifugation, Aspirate supernatant in 40min is heated in 58 DEG C of water-baths, it is to be measured;2) sample separatesUsing universal liquid-phase chromatographic column, chromatographic condition is, its filler is high intensity silica gel particle C18, efficient liquid phase system Using universal high-pressure pump and injector, using mixed liquor of the acidic aqueous solution containing 0.1% aqueous formic acid (A) and acetonitrile (B) As mobile phase, gradient elution;0-4.0min A:B is from (95:5, v/v) (20 are arrived:80, v/v), 4.0-4.1min A:B from (20:80, v/v) (95 are arrived:5, v/v), 4.1-6.0min A:B(95:5,v/v);Flow velocity:0.4mL/min;3) sample detectionSebivo and interior target peak area are detected using universal tandem mass spectrum detector, are converted into calibration curve equation dense Degree;Mass spectrum ionizes mode:Electric spray ion source;Polarity:Anionic textiles;Scan mode:More reactive ion detection scannings (MRM);Ion channel selects:LdT:M/z 243.10 → 127.10, D3-LdT:m/z 246.10→130.10.
- 2. the method for Sebivo content in detection blood plasma according to claim 1, it is characterized in that described step 1) In, it is described in be designated as deuterium 3- Sebivos (D3-LdT), protein precipitant is acetonitrile, and organic solvent is acetonitrile;Testing sample Through the pretreatment restrovirus inactivation of acetonitrile precipitation albumen.
- 3. the method for Sebivo content in detection blood plasma according to claim 1, it is characterized in that, described testing sample Blood plasma selected from hepatitis B patient.
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