CN109541109A - The method of Silodosin and KMD-3213G concentration in a kind of LC-MS measurement blood plasma - Google Patents
The method of Silodosin and KMD-3213G concentration in a kind of LC-MS measurement blood plasma Download PDFInfo
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- G—PHYSICS
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- 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
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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
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Abstract
The invention discloses the methods of Silodosin and KMD-3213G concentration in a kind of LC-MS measurement blood plasma first to take sample to be tested using LC-MS system measurement, a certain amount of mixed organic solvents are added to be extracted twice, after pretreatment, through chromatography post separation, detected with mass detector.The method of the present invention quickly, it is accurate, high sensitivity, easy to operate, the determination of plasma concentration for Silodosin and KMD-3213G provides foundation；The plasma standard curve Silodosin quantification range of this method is 0.2~60ng/mL, and KMD-3213G quantification range is 0.2~40ng/mL, and in batch and betweenrun precision RSD is respectively less than ± 15%, is suitable for measuring the concentration of Silodosin and KMD-3213G in blood plasma.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of measuring method of drug, in particular to a kind of liquid matter connection
With the method for Silodosin and KMD-3213G concentration in measurement blood plasma.
Background technique
Silodosin (Silodosin, also known as silodosin) be raw (Kissei) the drugmaker research and development of Japanese tangerine α 1- by
Body antagonist can be used for treating and benign prostatic hyperplasis or loose relevant symptom.Japanese Ju Sheng drugmaker and the one or three
(Daiichi Sankyo) drugmaker cooperation application altogether, and listing approval is obtained for the first time in Japan in May, 2006, it uses
Trade name is Urief.Then, tangerine is raw also licenses to U.S. Hua Sheng (Watson) drugmaker for Silodosin altogether with the one or three,
The latter lists in the approval of 2008 Nian8Yue Huo Food and Drug Adminstration of the US (FDA) in the U.S..By the pharmacy altogether of Japan the one or three
The Silodosin capsule preparations of company's import are also in 2011 in Discussion on Chinese Listed.
The chemical name of Silodosin (Silodosin) are as follows: 2,3- dihydro -1- (3- hydroxypropyl) -5- [(2R) -2-
[2- [2- (2,2,2- trifluoro ethoxy) phenoxy group] ethylamino-] propyl] -1H- indoles -7- formamide, the following institute of structural formula
Show:
Silodosin, which shrinks urethral smooth muscle, has selective inhibitory, is clinically used for treatment benign prostate and increases
It is raw.Silodosin oral absorption is good, and linear pharmacokinetics within the scope of therapeutic dose, tmax is about 2.6-3.5h, Absolute oral
Availability is about 32%, apparent volume of distribution 49.5L, plasma protein binding rate 97%.The drink of moderate appropriate fat and heat
Food can influence its absorption, reduce ρ max and AUC by about 18%-43% and 4%-9% respectively.Silodosin by with glucose
The number of ways metabolism such as aldehydic acid combines, ethyl alcohol and acetaldehyde dehydrogenase, Cytochrome P450 3A4 (CYP3A4), main metabolites
It is that glucuronide conjugate (KMD-3213G) is formed by glucosyl transferase 2B7 (UGT2B7) directly effect.It takes simultaneously
Silodosin and UGT2B7 inhibitor such as probenecid, valproic acid and Fluconazole can be such that Silodosin blood concentration increases.In vitro
Experiments have shown that KMD-3213G is active metabolite, half-life period is longer than Silodosin, and AUC is about 4 times of Silodosin.Through
After being orally ingested the Silodosin 10d of radioisotope 14 C label, from urinating and defecating, the discharge radioactive material rate of recovery be respectively may be about
33.5% and 54.9%.After intravenous injection, plasma clearance is about 10Lh-1.Silodosin has excellent medicinal valence
Value, currently, the document report not yet about Silodosin and KMD-3213G measuring method.
Summary of the invention
The purpose of the present invention is to provide Silodosin and KMD-3213G concentration in a kind of LC-MS measurement blood plasma
The speed of detection, accuracy and sensitivity can be improved in method, this method.
To achieve the above object, a method of Silodosin and KMD-3213G concentration in measurement blood plasma, plasma sample warp
Detect its concentration through high performance liquid chromatography-tandem mass after pretreatment, specific method the following steps are included:
(1) plasma sample pre-processes:
Blood plasma is with K2EDTA is anti-coagulants, using Silodosin-d4 as internal standard；It is accurate in 96 deep-well plates to be added 200uL's
Plasma sample, the volume ratio that 10uL is added is the acetonitrile solution of 1:1, and the internal standard match of the 0.05ng/uL of 10uL is added after mixing
The more octyl- d4 solution in Lip river, after mixing be added 500uL acetonitrile in 96 deep-well plates, vortex mixed 1min, in 20 DEG C with 3000rpm from
Heart 10min takes supernatant liquor 150uL into 96 deep-well plates that 350uL mixed organic solvents are housed, and mixed organic solvents are water:
Formic acid: the mixture that 1M ammonium acetate is mixed to get according to volume ratio 100:0.1:0.1, be vortexed mix, in 20 DEG C with 3000rpm from
It is to be detected as test sample after heart 5min；
(2) Specimen Determination:
It takes in 15uL test sample injection high performance liquid chromatography-tandem mass instrument, Silodosin, KMD- in test sample
The chromatographic peak of 3213G and internal standard Silodosin-d4, and Silodosin and KMD-3213G in the plasma sample are calculated accordingly
Concentration；
(3) liquid chromatogram measuring, condition are as follows:
Chromatographic column: Agilent ZORBAX XDB-C18,5um, column specification is 50 × 2.1mm；Chromatographic column temperature: 40 DEG C；Stream
Dynamic phase A: water/formic acid/1M ammonium acetate percent by volume is 100/0.1/0.1；Mobile phase B: acetonitrile/water/formic acid/1M ammonium acetate
Percent by volume be 85/15/0.1/0.1；Washing lotion: the percent by volume of acetonitrile/water is 50/50；Autosampler temperature is
15℃；Gradient elution, flow velocity 0.4mL/min, sample volume 15uL, analysis time 3.5min；
(4) mass spectroscopy, condition are as follows:
Ion source is electric spray ion source, and spray voltage 5000V, atomization temperature is 450 DEG C, and spray pressure power is
40Psi, auxiliary heating atmospheric pressure are 40Psi, and gas curtain atmospheric pressure is 20Psi, and collision atmospheric pressure is Low, go the cluster voltage to be respectively
The Silodosin and Silodosin-d4 of 60eV, removing cluster voltage is the KMD-3213G of 40eV；Collision cell entrance potential is respectively
The Silodosin and Silodosin-d4 of 10eV, collision cell entrance potential are the KMD-3213G of 7eV；Collision voltage is respectively 35eV
Silodosin, KMD-3213G and Silodosin-d4；Collision cell exit potential be respectively 14eV Silodosin and celo it is more
Octyl- d4, collision cell exit potential are the KMD-3213G of 12eV；Positive ion mode detection；Scanning mode is multiple reaction monitoring；
Ionic reaction for quantitative analysis is respectively as follows: m/z496.3 → m/z200.1, is Silodosin；And m/
Z672.4 → m/z479.2 is KMD-3213G；It is Silodosin-d4 with m/z500.3 → m/z200.1.
Preferably, in the step (3) gradient elution program are as follows:
| Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 1.1 | 60 | 40 |
| 1.2 | 80 | 20 |
| 3.5 | 80 | 20 |
Preferably, in the step (2), using internal standard method, with Silodosin, KMD-3213G and internal standard Silodosin-d4
Peak area ratio bring the concentration of Silodosin and KMD-3213G that calibration curve equation calculates in the plasma sample into.
Preferably, the calibration curve equation foundation the following steps are included:
It takes 10 parts of 200uL blank plasma to be placed in 96 deep-well plates, adds 10uL concentration difference respectively in the form of stock solution
For 0.004ng/ μ L, 0.01ng/ μ L, 0.02ng/ μ L, 0.1ng/ μ L, 0.2ng/ μ L, 0.4ng/ μ L, 0.8ng/ μ L, 1.2ng/ μ
The Silodosin solution of L is fixed to minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest
Measure in upper limit sample, then added respectively in the form of stock solution 10uL concentration be respectively 0.004ng/ μ L, 0.01ng/ μ L,
0.02ng/ μ L, 0.04ng/ μ L, 0.1ng/ μ L, 0.2ng/ μ L, 0.6ng/ μ L, 0.8ng/ μ L KMD-3213G solution to minimum
Lower limit of quantitation sample, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, in highest upper limit of quantification sample, adds standard specimen 1 respectively
10 μ l volume ratios are the acetonitrile solution of 1:1 to blank sample and zero-dose sample, respectively to minimum lower limit of quantitation sample after mixing
Standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest upper limit of quantification sample, 10uL is added in zero-dose sample in sheet
0.05ug/uL internal standard Silodosin-d4 solution, into blank sample be added 10uL volume ratio be 1:1 aqueous acetonitrile
Liquid, is added 500uL acetonitrile in 96 deep-well plates after mixing, vortex mixed 1min is centrifuged 10min in 20 DEG C with 3000rpm, takes
For supernatant liquor 150uL into 96 deep-well plates that 350uL mixed organic solvents are housed, mixed organic solvents are water: formic acid: 1M vinegar
The mixture that sour ammonium is mixed to get according to volume ratio 100:0.1:0.1 is vortexed and mixes, after 20 DEG C are centrifuged 5min with 3000rpm
It is to be detected respectively as 10 parts of standard samples；
It is taken in 15uL standard sample injection high performance liquid chromatography-tandem mass instrument respectively, Silodosin in test sample,
The chromatographic peak of KMD-3213G and internal standard Silodosin-d4, and standard curve is obtained accordingly, for calculating in the blood plasma
The concentration of Silodosin and KMD-3213G.
Compared with prior art, the invention has the following advantages that
(1) preprocess method is easy, two step organic extractant solutions, is suitable for conventional determining；
(2) specificity is strong: under chromatographic condition used by this experiment, Silodosin retention time is 1.571min left
The right side, KMD-3213G retention time are 1.309 or so, and internal standard Silodosin-d4 retention time is in 1.561min or so.Celo is more
The peak shape of pungent, KMD-3213G and internal standard Silodosin-d4 are good, no miscellaneous peak interference measurement, and baseline is steady；
(3) high sensitivity: blood plasma is minimum to be quantitatively limited to 0.2ng/mL, the concentration of Silodosin in energy Accurate Determining blood plasma,
High sensitivity, high specificity；
(4) the method for the present invention quickly, it is accurate, high sensitivity, easy to operate, provided for the determination of plasma concentration of Silodosin
Foundation.The plasma standard curve Silodosin quantification range of this method is 0.2~60ng/mL, and KMD-3213G quantification range is
0.2~40ng/mL, in batch and betweenrun precision RSD is respectively less than ± 15%.
Detailed description of the invention
Fig. 1 is the canonical plotting of HPLC-MS/MS the method Silodosin and KMD-3213G that measure in human plasma；
Fig. 2 is that the HPLC-MS/MS of people's blank plasma schemes；
Fig. 3 is the HPLC-MS/MS figure that Silodosin, KMD-3213G and Silodosin-d4 is added in people's blank plasma；
Fig. 4 is that plasma sample adds internal standard Silodosin-d4's after health volunteer takes orally Silodosin drug
HPLC-MS/MS figure.
Specific embodiment
Invention is further described in detail with reference to embodiments.
Embodiment: mankind K2The measurement of Silodosin concentration in edta plasma
One, experimental material and analytical equipment
Silodosin (analyte 1): Toronto Research Chemicals or identical, greater degree standard items
KMD-3213G (analyte 2): TLC Pharmaceutical Standards or identical, greater degree standard
Product
Silodosin-d4 (internal standard): TLC Pharmaceutical Standards or identical, greater degree standard items
It see the table below 1 using reagent:
1 reagent detail of table
| Reagent name | Rank | Manufacturer |
| Acetonitrile | HPLC | J.T.Baker |
| Ammonium acetate | HPLC | J.T.Baker |
| Formic acid | ACS | Adamas |
Note: the reagent of same levels or higher level also can be used
It see the table below 2 using analytical equipment:
Table 2 uses device inventory
| Component | Type | Manufacturer |
| Binarypump (binary pump) | ACPump | ABSCIEX |
| Degasser (degasser) | Degasser | ABSCIEX |
| Columnoven (thermocolumn case) | ACColumnoven | ABSCIEX |
| Autosampler (Autosampler) | ACAutosampler | ABSCIEX |
| Samplerack (sample rack) | RackChanger | ABSCIEX |
| Massspectrometer (mass spectrograph) | QTRAPR6500+ | ABSCIEX |
| Dataprocessor (data processor) | Analyst1.6.3(software) | ABSCIEX |
Identical LC/MS/MS system can also be used.
Two, liquid matter condition
1, liquid phase chromatogram condition
Chromatographic column: Agilent ZORBAX XDB-C18,5um, column specification is 50 × 2.1mm；Chromatographic column temperature: 40 DEG C；Stream
Dynamic phase A: water/formic acid/1M ammonium acetate percent by volume is 100/0.1/0.1；Mobile phase B: acetonitrile/water/formic acid/1M ammonium acetate
Percent by volume be 85/15/0.1/0.1；Washing lotion: the percent by volume of acetonitrile/water is 50/50；Autosampler temperature is
15℃；Gradient elution, flow velocity 0.4mL/min, sample volume 15uL, analysis time 3.5min.
3 gradient elution program of table
| Step | Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 1 | 0 | 80 | 20 |
| 2 | 1.1 | 60 | 40 |
| 3 | 1.2 | 80 | 20 |
| 4 | 3.5 | 80 | 20 |
2, Mass Spectrometry Conditions
Ion source is electric spray ion source, and spray voltage 5000V, atomization temperature is 450 DEG C, and spray pressure power is
40Psi, auxiliary heating atmospheric pressure are 40Psi, and gas curtain atmospheric pressure is 20Psi, and collision atmospheric pressure is Low, go the cluster voltage to be respectively
The Silodosin and Silodosin-d4 of 60eV, removing cluster voltage is the KMD-3213G of 40eV；Collision cell entrance potential is respectively
The Silodosin and Silodosin-d4 of 10eV, collision cell entrance potential are the KMD-3213G of 7eV；Collision voltage is respectively 35eV
Silodosin, KMD-3213G and Silodosin-d4；Collision cell exit potential be respectively 14eV Silodosin and celo it is more
Octyl- d4, collision cell exit potential are the KMD-3213G of 12eV；Positive ion mode detection；Scanning mode is multiple reaction monitoring；
Ionic reaction for quantitative analysis is respectively as follows: m/z496.3 → m/z200.1, is Silodosin；With m/z672.4 → m/
Z479.2 is KMD-3213G；It is Silodosin-d4 with m/z500.3 → m/z200.1.
Three, experimentation
1, the preparation of Silodosin and KMD-3213G standard solution
The preparation of Silodosin and KMD-3213G standard solution: precision weighs Silodosin (analyte 1) respectively
0.001g, KMD-3213G (analyte 2) 0.001g, is respectively placed in 10mL volumetric flask, and the acetonitrile water that volume ratio is 1:1 is added
Solution dissolves and is settled to scale, shakes up, both 100ng/uL Silodosin stock solution and KMD-3213G stock solution, then with
Volume ratio is that molten successively dilute of the acetonitrile solution of 1:1 prepares Silodosin and KMD-3213G standard solution respectively, specific to dilute
Concentration see the table below 4:
4 Silodosin of table and KMD-3213G standard solution compound concentration
A: it is directly prepared from Silodosin (analyte 1) and KMD-3213G (analyte 2)
Silodosin and KMD-3213G standard solution in being stored in plastic containers and (2~8 DEG C) of refrigerator guarantors when not in use
It deposits, volume can be increased or decreased optionally to scale.
2, the preparation of Silodosin-d4 internal standard standard solution
The preparation of Silodosin-d4 internal standard standard solution: precision weighs Silodosin-d4 (internal standard) 0.001g, is placed in
In 10mL volumetric flask, the acetonitrile solution that volume ratio is 1:1 is added and dissolves and be settled to scale, shakes up, has both obtained 100ng/uL's
Silodosin-d4 stock solution, then for the molten dilution of acetonitrile solution of 1:1, to be configured to concentration be 0.05ng/uL celo with volume ratio
More octyl- d4 inner mark solutions, specific diluted concentration see the table below 5:
5 Silodosin-d4 standard solution compound concentration of table
| Source solution (ng/uL) | Source liquor capacity (uL) | Final volume (mL) | Ultimate density (ng/uL) |
| 100a | 50 | 100 | 0.05b |
A: it is directly prepared from Silodosin-d4 (internal standard)
B: sample preparation steps are used for
Silodosin-d4 internal standard standard solution in be stored in when not in use plastic containers and (2~8 DEG C) of refrigerator preservation, body
Product can be increased or decreased optionally to scale.
3, linear test
Blank plasma is put into water-bath in room temperature environment to thaw；10 parts of blank plasma of transfer 200uL are into 96 deep-well plates
(each standard curve sample, blank sample -00 and zero-dose sample -0), according to listed by the following table 6, accurate addition is different dense respectively
The Silodosin and KMD-3213G mixed standard solution 10uL or dilute solution of degree prepare each sample and mix, and are made into not
With the plasma containing drug of concentration, operated by " plasma sample pretreatment ".Calculate Silodosin and KMD-3213G peak area As and internal standard
The ratio Y (Y=As/Ai) of Silodosin-d4 peak area Ai makees blood concentration X with peak area ratio Y to return calculating, as a result
See Fig. 1, table 7 and table 8.Recurrence is done to blood concentration X with mean ratio Y to calculate, and obtains Silodosin regression equation Y=0.477X-
0.0024, r=0.9997, KMD-3213G regression equation Y=0.363X-0.00998, r=0.9997, weight coefficient W=1/
X2, the minimum quantitative limit of the blood concentration of the Silodosin and KMD-3213G that measure in this way are as follows: 0.2ng/mL.
6 Silodosin of table and KMD-3213G standard curve compound concentration
B: the dilute solution of analyte: ACN/H2O=50/50
Standard curve (n=6) of the Silodosin that table 7HPLC-MS/MS method measures in human plasma
Standard curve (n=8) of the KMD-3213G that table 8HPLC-MS/MS method measures in human plasma
4, accuracy and precision
Blank plasma is put into water-bath in room temperature environment to thaw；Shift proper volume blank plasma to container appropriate simultaneously
Add Silodosin and KMD-3213G standard solution prepare 5 kinds of various concentrations plasma containing drug quality-control sample (LLOQ, QL, QLM,
QM, QH) and a retinue standard curve, it is operated by " plasma sample pretreatment ", quality-control sample preparation is as shown in table 9 below.Daily
A batch and a retinue standard curve are done, is continuously done 3 days, totally three batches, first and each concentration of second batch make 6 parts of samples respectively
Product, each concentration of third batch make 15 parts of samples respectively, calculate Silodosin peak area As, KMD-3213G peak area As and internal standard
The ratio Y of Silodosin-d4 peak area Ai is substituted into the standard curve on the same day and is acquired measured concentration, is calculated and is criticized by measured concentration
Interior and betweenrun precision, measured concentration and the ratio that concentration is added are accuracy, the results are shown in Table 10 and table 11.The result shows that
Silodosin and KMD-3213G plasma sample criticize interior, betweenrun precision, accuracy is less than ± 15% and meets the requirements.
9 quality-control sample compound concentration of table
A: final volume=source liquor capacity+Plasma volumes
According to needed for each analysis batch, enough volumes are dispensed into the specimen bottle bottle indicated and are stored in theoretical temperatures-
20℃.Volume can be increased or decreased optionally to scale.
Table 10HPLC-MS/MS method measure blood plasma in Silodosin batch in, batch between precision and accuracy
Table 11HPLC-MS/MS method measure blood plasma in KMD-3213G batch in, batch between precision and accuracy
5, interference
Six different blank plasma samples distinguish source difference healthy human body, by six different blank plasma samples in same
Analysis batch is prepared and is analyzed according to sample preparation steps, to evaluate different blank plasmas to Silodosin analyte, KMD-3213G
The interference of analyte and internal standard Silodosin-d4.
After six separate sources blank healthy human body plasma sample preparation analyses, meeting at Silodosin retention time
Interference peak response is below the 20.0% of the Silodosin response of lower limit of quantitation sample in the standard curve of the analysis batch, as a result
It is shown in Table 12.The result shows that the analysis method has specificity to the analysis of Silodosin.
After six separate sources blank healthy human body plasma sample preparation analyses, meeting at KMD-3213G retention time
Interference peak response be below lower limit of quantitation sample in the standard curve of the analysis batch KMD-3213G response 20.0%, knot
Fruit is shown in Table 13.The result shows that the analysis method has specificity to the analysis of KMD-3213G.
After six separate sources blank human normal plasma sample preparation analyses, meeting the Interference Peaks at internal standard retention time
Response is below the 20.0% of the internal standard response of lower limit of quantitation sample in the standard curve of the analysis batch, sees in annex
Table 14.The result shows that the internal target analysis of the analysis method has selectivity.
Interference data comparison table of the 12 6 separate sources blank healthy human body blood plasma of table to Silodosin analyte
A: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve) × 100.0%≤
20.0% b: when " no significant peak value can be integrated (or without peak) " or " retention time of peak area, which is not met in sample, divides
Analyse the retention time of object ", which is considered zero.
Interference data comparison table of the 13 6 separate sources blank healthy human body blood plasma of table to KMD-3213G
A: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve) × 100.0%≤
20.0% b: when " no significant peak value can be integrated (or without peak) " or " retention time of peak area, which is not met in sample, divides
Analyse the retention time of object ", which is considered zero.
Interference data comparison table of the 14 6 separate sources blank healthy human body blood plasma of table to internal standard Silodosin-d4
A: analyte peak area (selective sample)/internal standard peak area (LLOQ of standard curve) × 100.0%≤
20.0%
B: when " no significant peak value can be integrated (or without peak) " or " retention time of peak area, which is not met in sample, divides
Analyse the retention time of object ", which is considered zero.
As can be seen that the blank plasma of different human body is to Silodosin and KMD-3213G from table 12, table 13 and table 14
Testing result do not interfere.Therefore, this method can be used for detecting Silodosin and KMD- in different human body blood plasma
The concentration of 3213G.
6, human plasma sample detects
(1) the people's blank plasma for being not given to Silodosin is taken, the accurate blank blood that 200uL is added in 96 deep-well plates
Slurry samples, the volume ratio that 20uL is added is the acetonitrile solution of 1:1, and 500uL acetonitrile is added after mixing in 96 deep-well plates, is vortexed
1min is mixed, 10min is centrifuged with 3000rpm in 20 DEG C, takes supernatant liquor 150uL to being equipped with the 96 of 350uL mixed organic solvents
In deep-well plates, mixed organic solvents are water: formic acid: the mixing that 1M ammonium acetate is mixed to get according to volume ratio 100:0.1:0.1
Object is vortexed and mixes, and takes 15uL sample to carry out LC-MS/MS analysis after being centrifuged 5min in 20 DEG C with 3000rpm, as a result such as Fig. 2 institute
Show.
(2) the people's blank plasma for being not given to Silodosin is taken, the accurate blank blood that 200uL is added in 96 deep-well plates
The Silodosin standard solution that 5uL concentration is 0.05ng/uL is added in slurry samples, and the KMD- that 5uL concentration is 0.05ng/uL is added
3213G standard solution is added the internal standard Silodosin-d4 solution of the 0.05ng/uL of 10uL, 500uL is added after mixing after mixing
Acetonitrile is in 96 deep-well plates, vortex mixed 1min, is centrifuged 10min in 20 DEG C with 3000rpm, takes supernatant liquor 150uL to being equipped with
In 96 deep-well plates of 350uL mixed organic solvents, mixed organic solvents are water: formic acid: 1M ammonium acetate is according to volume ratio 100:
The mixture that 0.1:0.1 is mixed to get is vortexed and mixes, and takes 15uL sample to carry out LC- after being centrifuged 5min in 20 DEG C with 3000rpm
MS/MS analysis, as a result as shown in Figure 3.
(3) acquisition health volunteer takes orally the blood plasma after Silodosin drug, accurate in 96 deep-well plates that 200uL is added
Acquisition human plasma sample, the volume ratio that 10uL is added is the acetonitrile solution of 1:1, and 10uL is added after mixing
500uL acetonitrile is added in 96 deep-well plates in the internal standard Silodosin-d4 solution of 0.05ng/uL after mixing, vortex mixed 1min,
10min is centrifuged with 3000rpm in 20 DEG C, takes supernatant liquor 150uL into 96 deep-well plates that 350uL mixed organic solvents are housed,
Mixed organic solvents are water: formic acid: the mixture that 1M ammonium acetate is mixed to get according to volume ratio 100:0.1:0.1, are vortexed and mix,
15uL sample is taken to carry out LC-MS/MS analysis after being centrifuged 5min in 20 DEG C with 3000rpm, as a result as shown in Figure 4.
In conclusion the present invention provides the measurement sides of Silodosin concentration in a kind of blood plasma of preprocess method simplicity
Method is suitable for conventional determining using two step organic extractant solution methods；Result is it is found that used by this experiment from the above analysis
Under chromatographic condition, Silodosin retention time is 1.571min or so, and KMD-3213G retention time is 1.309 or so, internal standard match
For the more octyl- d4 retention times in Lip river in 1.561min or so, the peak shape of Silodosin, KMD-3213G and internal standard Silodosin-d4 is good
Well, no miscellaneous peak interference measurement, baseline are steady；This method specificity with higher, can Silodosin in Accurate Determining blood plasma
Concentration, sensitivity is higher, and blood plasma is minimum to be quantitatively limited to 0.2ng/mL；Meanwhile the method for the present invention quickly, accurate, high sensitivity,
Easy to operate, the determination of plasma concentration for Silodosin and KMD-3213G provides foundation.The plasma standard curve celo of this method
Mostly pungent quantification range is 0.2~60ng/mL, and KMD-3213G quantification range is 0.2~40ng/mL, batch interior and betweenrun precision
RSD is respectively less than ± 15%.
Claims (4)
1. a kind of method of Silodosin and KMD-3213G concentration in LC-MS measurement blood plasma, it is characterised in that: plasma sample
Detect its concentration through high performance liquid chromatography-tandem mass after preprocessed, specific method the following steps are included:
(1) plasma sample pre-processes:
Blood plasma is with K2EDTA is anti-coagulants, using Silodosin-d4 as internal standard；The accurate blood plasma sample that 200uL is added in 96 deep-well plates
Product, the volume ratio that 10uL is added is the acetonitrile solution of 1:1, and the internal standard Silodosin-of the 0.05ng/uL of 10uL is added after mixing
500uL acetonitrile is added in 96 deep-well plates in d4 solution after mixing, vortex mixed 1min is centrifuged 10min in 20 DEG C with 3000rpm,
Take supernatant liquor 150uL into 96 deep-well plates that 350uL mixed organic solvents are housed, mixed organic solvents are water: formic acid: 1M vinegar
The mixture that sour ammonium is mixed to get according to volume ratio 100:0.1:0.1 is vortexed and mixes, after 20 DEG C are centrifuged 5min with 3000rpm
It is to be detected as test sample；
(2) Specimen Determination:
It takes in 15uL test sample injection high performance liquid chromatography-tandem mass instrument, Silodosin, KMD-3213G in test sample
With the chromatographic peak of internal standard Silodosin-d4, and the Silodosin in the plasma sample and KMD-3213G concentration are calculated accordingly；
(3) liquid chromatogram measuring, condition are as follows:
Chromatographic column: Agilent ZORBAX XDB-C18,5um, column specification is 50 × 2.1mm；Chromatographic column temperature: 40 DEG C；Mobile phase
A: water/formic acid/1M ammonium acetate percent by volume is 100/0.1/0.1；Mobile phase B: acetonitrile/water/formic acid/1M ammonium acetate body
Product percentage is 85/15/0.1/0.1；Washing lotion: the percent by volume of acetonitrile/water is 50/50；Autosampler temperature is 15 DEG C；
Gradient elution, flow velocity 0.4mL/min, sample volume 15uL, analysis time 3.5min；
(4) mass spectroscopy, condition are as follows:
Ion source is electric spray ion source, and spray voltage 5000V, atomization temperature is 450 DEG C, and spray pressure power is 40Psi, auxiliary
Helping heating atmospheric pressure is 40Psi, and gas curtain atmospheric pressure is 20Psi, and collision atmospheric pressure is Low, and removing cluster voltage is respectively the match of 60eV
Luo Duoxin and Silodosin-d4, removing cluster voltage is the KMD-3213G of 40eV；Collision cell entrance potential is respectively the celo of 10eV
Mostly pungent and Silodosin-d4, collision cell entrance potential are the KMD-3213G of 7eV；Collision voltage is respectively that the celo of 35eV is more
Pungent, KMD-3213G and Silodosin-d4；Collision cell exit potential is respectively the Silodosin and Silodosin-d4 of 14eV, is touched
Hit the KMD-3213G that room exit potential is 12eV；Positive ion mode detection；Scanning mode is multiple reaction monitoring；
Ionic reaction for quantitative analysis is respectively as follows: m/z496.3 → m/z200.1, is Silodosin；With m/z672.4 →
M/z479.2 is KMD-3213G；It is Silodosin-d4 with m/z500.3 → m/z200.1.
2. the method for Silodosin and KMD-3213G concentration in a kind of LC-MS measurement blood plasma according to claim 1,
It is characterized by: in the step (3) gradient elution program are as follows:
。
3. the side of Silodosin and KMD-3213G concentration in a kind of LC-MS measurement blood plasma according to claim 1 or 2
Method, it is characterised in that: in the step (2), using internal standard method, with Silodosin, KMD-3213G and internal standard Silodosin-d4
Peak area ratio bring the concentration of Silodosin and KMD-3213G that calibration curve equation calculates in the plasma sample into.
4. the method for Silodosin and KMD-3213G concentration in a kind of LC-MS measurement blood plasma according to claim 3,
It is characterized by: the foundation of the calibration curve equation the following steps are included:
10 parts of 200uL blank plasma are taken to be placed in 96 deep-well plates, adding 10uL concentration respectively in the form of stock solution is respectively
0.004ng/ μ L, 0.01ng/ μ L, 0.02ng/ μ L, 0.1ng/ μ L, 0.2ng/ μ L, 0.4ng/ μ L, 0.8ng/ μ L, 1.2ng/ μ L
Silodosin solution to minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest quantifies
It limits in sample, then adding 10uL concentration respectively in the form of stock solution is respectively 0.004ng/ μ L, 0.01ng/ μ L, 0.02ng/ μ
L, the KMD-3213G solution of 0.04ng/ μ L, 0.1ng/ μ L, 0.2ng/ μ L, 0.6ng/ μ L, 0.8ng/ μ L are to minimum lower limit of quantitation
Sample, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, in highest upper limit of quantification sample, adds 10 μ l volumes at standard specimen 1 respectively
Than the acetonitrile solution for 1:1 to blank sample and zero-dose sample, after mixing respectively to minimum lower limit of quantitation sample, standard specimen 1,
Standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest upper limit of quantification sample, the 0.05ug/ that 10uL is added in zero-dose sample
Internal standard Silodosin-d4 the solution of uL, the volume ratio that 10uL is added into blank sample is the acetonitrile solution of 1:1, after mixing
500uL acetonitrile is added in 96 deep-well plates, vortex mixed 1min is centrifuged 10min in 20 DEG C with 3000rpm, takes supernatant liquor
For 150uL into 96 deep-well plates that 350uL mixed organic solvents are housed, mixed organic solvents are water: formic acid: 1M ammonium acetate is according to body
The mixture that product is mixed to get than 100:0.1:0.1 is vortexed and mixes, as 10 after being centrifuged 5min using 3000rpm in 20 DEG C
Part standard sample is to be detected；
It is taken in 15uL standard sample injection high performance liquid chromatography-tandem mass instrument respectively, Silodosin, KMD- in test sample
The chromatographic peak of 3213G and internal standard Silodosin-d4, and standard curve is obtained accordingly, for calculating the celo in the blood plasma
Mostly pungent and KMD-3213G concentration.
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