CN109541106A - A kind of method that LC-MS measures frusemide concentration in blood plasma - Google Patents
A kind of method that LC-MS measures frusemide concentration in blood plasma Download PDFInfo
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Abstract
The invention discloses the methods of frusemide 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, provide foundation for the determination of plasma concentration of frusemide；The plasma standard curve linear range of this method is 10~2000ng/mL, and in batch and betweenrun precision RSD is respectively less than ± 15%, is suitable for measuring the concentration of frusemide 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 frusemide concentration in measurement blood plasma.
Background technique
Frusemide is a known compound, is had the following structure:
Frusemide is also known as furosemide, chemical name: 2- [(2- furfuryl) amino] -5- (sulfamoyl) -4- chlorobenzoic acid,
It is to act on the high-effect diuretics of Heng Shi loop ascending branch.It is clinically bad and be badly in need of the clinical setting of diuresis for diuretics curative effect,
It can be used for treating oedema, hypertension, acute pulmonary edema or brain edema caused by congestive heart failure, cirrhosis and kidney diaseases,
Certain poisonous substances eliminated through kidney are accelerated to drain.
Currently, the document report not yet about frusemide measuring method.
Summary of the invention
The purpose of the present invention is to provide the method for frusemide concentration in a kind of LC-MS measurement blood plasma, this method can be mentioned
Speed, the accuracy and sensitivity of high detection.
To achieve the above object, a method of frusemide concentration in measurement blood plasma, through height after plasma sample is preprocessed
Effect liquid phase chromatogram-tandem mass spectrum detects its concentration, specific method the following steps are included:
(1) plasma sample pre-processes:
Blood plasma is with K2EDTA is anti-coagulants, using frusemide-d5 as internal standard；The accurate blood that 100uL is added in 96 deep-well plates
Slurry samples, the volume ratio that 5uL is added is the methanol aqueous solution of 1:1, and the internal standard frusemide-d5 of the 1ng/uL of 5uL is added after mixing
The acetonitrile of 500uL is added in 96 deep-well plates in solution after mixing, vortex mixed 1min is centrifuged 10min in 20 DEG C with 3000rpm,
Take supernatant liquor 80uL into 96 deep-well plates that 420uL mixed organic solvents are housed, mixed organic solvents are water: acetonitrile: formic acid
It according to the mixture that volume ratio 95:5:0.2 is mixed to get, is vortexed and mixes, as test after being centrifuged 5min using 3000rpm in 20 DEG C
Sample is to be detected；
(2) Specimen Determination:
It takes in 10uL test sample injection high performance liquid chromatography-tandem mass instrument, frusemide and internal standard furan in test sample
The chromatographic peak of Sai meter -d5, and the frusemide concentration in the plasma sample is 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；Stream
Dynamic phase A: water/formic acid/1M ammonium acetate percent by volume is 100/0.2/0.1；Mobile phase B: acetonitrile/water/formic acid/1M ammonium acetate
Percent by volume be 80/20/0.2/0.1；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 10uL, analysis time 4min；
(4) mass spectroscopy, condition are as follows:
Ion source is electric spray ion source, and spray voltage is -4500V, and atomization temperature is 480 DEG C, and spray pressure power is
20Psi, auxiliary heating atmospheric pressure are 20Psi, and gas curtain atmospheric pressure is 20Psi, and collision atmospheric pressure is 8Psi, and cluster voltage is gone to distinguish
For the frusemide and frusemide-d5 of -25eV；Collision cell entrance potential is respectively the frusemide and frusemide-d5 of -10eV；Collision
Voltage is respectively the frusemide and frusemide-d5 of -20eV；Collision cell exit potential is respectively the frusemide and frusemide-of -10eV
d5；Negative-ion mode detection；Scanning mode is multiple reaction monitoring；
Ionic reaction for quantitative analysis is respectively as follows: m/z329.0 → m/z285.0, is frusemide；And m/z334.0
→ m/z290.0 is frusemide-d5.
Preferably, in the step (3) gradient elution program are as follows:
| Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 75 | 25 |
| 0.5 | 75 | 25 |
| 0.6 | 60 | 40 |
| 1.5 | 60 | 40 |
| 1.6 | 75 | 25 |
| 1.9 | 75 | 25 |
| 2.0 | 0 | 100 |
| 2.5 | 0 | 100 |
| 2.6 | 75 | 25 |
Preferably, in the step (2), using internal standard method, with the peak area ratio band of frusemide and internal standard frusemide-d5
Enter the concentration for the frusemide that calibration curve equation calculates in the plasma sample.
Preferably, the calibration curve equation foundation the following steps are included:
10 parts of 100uL blank plasma are taken to be placed in 96 deep-well plates, adding 5uL concentration respectively in the form of stock solution is respectively
0.2ng/ μ L, 0.4ng/ μ L, 1ng/ μ L, 2ng/ μ L, 10ng/ μ L, 24ng/ μ L, 30ng/ μ L, 40ng/ μ L frusemide solution extremely
Minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, in highest upper limit of quantification sample, respectively
Adding 5 μ l volume ratios is the methanol aqueous solution of 1:1 to blank sample and zero-dose sample, respectively to minimum lower limit of quantitation 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, 5uL is added in zero-dose sample in sample
1ng/uL internal standard frusemide-d5 solution, into blank sample be added 5uL volume ratio be 1:1 methanol aqueous solution, mix
Afterwards respectively into 10 parts of samples be added 500uL acetonitrile in 96 deep-well plates, vortex mixed 1min, in 20 DEG C with 3000rpm from
Heart 10min takes supernatant liquor 80uL into 96 deep-well plates that 420uL mixed organic solvents are housed, and mixed organic solvents are water: second
Nitrile: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.2 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；
Taken in 10uL standard sample injection high performance liquid chromatography-tandem mass instrument respectively, frusemide in test sample and
The chromatographic peak of internal standard frusemide-d5, and standard curve is obtained accordingly, with the concentration for calculating the frusemide in the blood plasma.
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, frusemide retention time is 2.430min or so,
Internal standard frusemide-d5 retention time is in 2.401min or so.The peak shape of frusemide and internal standard frusemide-d5 are good, and no miscellaneous peak is dry
Measurement is disturbed, baseline is steady；
(3) high sensitivity: blood plasma is minimum to be quantitatively limited to 10ng/mL, and the concentration of frusemide, sensitive in energy Accurate Determining blood plasma
Degree is high, high specificity；
(4) the method for the present invention quickly, it is accurate, high sensitivity, easy to operate, for frusemide determination of plasma concentration provide according to
According to.The plasma standard curve linear range of this method is 10~2000ng/mL, be respectively less than in batch with betweenrun precision RSD ±
15%.
Detailed description of the invention
Fig. 1 is canonical plotting of the frusemide that measures of HPLC-MS/MS method in human plasma；
Fig. 2 is that the HPLC-MS/MS of people's blank plasma schemes；
Fig. 3 is that frusemide and the HPLC-MS/MS figure of frusemide-d5 is added in people's blank plasma；
Fig. 4 adds the HPLC-MS/ of internal standard frusemide-d5 for plasma sample after the oral frusemide drug of health volunteer
MS figure.
Specific embodiment
Invention is further described in detail with reference to embodiments.
Embodiment: mankind K2The measurement of frusemide concentration in edta plasma
One, experimental material and analytical equipment
Frusemide (analyte): National Institute for Food and Drugs Control or identical, greater degree standard items
Frusemide-d5 (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 |
| Methanol | HPLC | J.T.Baker |
| Formic acid | ACS | Adamas |
| Ammonium acetate | HPLC | J.T.Baker |
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 |
| Binary pump (binary pump) | AC Pump | AB SCIEX |
| Degasser (degasser) | Degasser | AB SCIEX |
| Column oven (thermocolumn case) | AC Column oven | AB SCIEX |
| Autosampler (Autosampler) | AC Autosampler | AB SCIEX |
| Sample rack (sample rack) | Rack Changer | AB SCIEX |
| Mass spectrometer (mass spectrograph) | TRIPLE QUADTM6500+ | AB SCIEX |
| Data processor (data processor) | Analyst 1.6.3(software) | AB SCIEX |
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 100/0.2/0.1；Mobile phase B: acetonitrile/water/formic acid/1M ammonium acetate
Percent by volume be 80/20/0.2/0.1；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 10uL, analysis time 4min.
3 gradient elution program of table
| Step | Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 1 | 0 | 75 | 25 |
| 2 | 0.5 | 75 | 25 |
| 3 | 0.6 | 60 | 40 |
| 4 | 1.5 | 60 | 40 |
| 5 | 1.6 | 75 | 25 |
| 6 | 1.9 | 75 | 25 |
| 7 | 2.0 | 0 | 100 |
| 8 | 2.5 | 0 | 100 |
| 9 | 2.6 | 75 | 25 |
2, Mass Spectrometry Conditions
Ion source is electric spray ion source, and spray voltage is -4500V, and atomization temperature is 480 DEG C, and spray pressure power is
20Psi, auxiliary heating atmospheric pressure are 20Psi, and gas curtain atmospheric pressure is 20Psi, and collision atmospheric pressure is 8Psi, and cluster voltage is gone to distinguish
For the frusemide and frusemide-d5 of -25eV；Collision cell entrance potential is respectively the frusemide and frusemide-d5 of -10eV；Collision
Voltage is respectively the frusemide and frusemide-d5 of -20eV；Collision cell exit potential is respectively the frusemide and frusemide-of -10eV
d5；Negative-ion mode detection；Scanning mode is multiple reaction monitoring；Ionic reaction for quantitative analysis is respectively as follows: m/
Z329.0 → m/z285.0 is frusemide；It is frusemide-d5 with m/z334.0 → m/z290.0.
Three, experimentation
1, the preparation of frusemide standard solution
The preparation of frusemide standard solution: precision weighs frusemide (analyte) 0.005g, is placed in 10mL volumetric flask, adds
Enter the methanol aqueous solution that volume ratio is 1:1 and dissolve and be settled to scale, shake up, both obtains the frusemide stock solution of 500ng/uL, then
Frusemide standard solution is prepared for the molten successively dilution of methanol aqueous solution of 1:1 with volume ratio, specific diluted concentration see the table below 4:
4 frusemide standard solution compound concentration of table
| Source solution (ng/uL) | Source liquor capacity (uL) | Final volume (mL) | Ultimate density (ng/uL) |
| 500a | 800 | 10 | 40 |
| 500a | 640 | 10 | 32 |
| 500a | 600 | 10 | 30 |
| 500a | 480 | 10 | 24 |
| 500a | 400 | 10 | 20 |
| 500a | 200 | 10 | 10 |
| 40 | 500 | 10 | 2 |
| 40 | 250 | 10 | 1 |
| 40 | 100 | 10 | 0.4 |
| 40 | 50 | 10 | 0.2 |
| 40 | 150 | 10 | 0.6 |
A: it is directly prepared from frusemide (analyte)
Frusemide standard solution in be stored in when not in use plastic containers and (4 DEG C) of refrigerator preservation, volume can optionally according to
Ratio increases or decreases.
2, the preparation of frusemide-d5 internal standard standard solution
The preparation of frusemide-d5 internal standard standard solution: precision weighs frusemide-d5 (internal standard) 0.001g, is placed in 10mL appearance
In measuring bottle, the methanol aqueous solution that volume ratio is 1:1 is added and dissolves and be settled to scale, shakes up, both obtains the frusemide-of 100ng/uL
D5 stock solution, then for the molten dilution of methanol aqueous solution of 1:1, to be configured to concentration be that 1ng/uL frusemide-d5 internal standard is molten with volume ratio
Liquid, specific diluted concentration see the table below 5:
5 frusemide-d5 standard solution compound concentration of table
| Source solution (ng/uL) | Source liquor capacity (uL) | Final volume (mL) | Ultimate density (ng/uL) |
| 100a | 1000 | 100 | 1b |
A: it is directly prepared from frusemide-d5 (internal standard)
B: sample preparation steps are used for
For frusemide-d5 internal standard standard solution in being stored in plastic containers and (4 DEG C) of refrigerator preservations when not in use, volume is visual
It needs to increase or decrease 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 100uL 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 frusemide standard solution 5uL or dilute solution of degree prepare each sample and mix, and are made into the plasma containing drug of various concentration, press
" plasma sample pretreatment " operation.Calculate the ratio Y (Y=As/ of frusemide peak area As and internal standard frusemide-d5 peak area Ai
Ai), blood concentration X is made to return with peak area ratio Y and is calculated, the result is shown in Figure 1 and table 7.With mean ratio Y to blood concentration X
It does recurrence to calculate, obtains regression equation Y=0.0125+0.0193X, r=0.9990, weight coefficient W=1/X2, measure in this way
Frusemide blood concentration minimum quantitative limit are as follows: 10ng/mL.
6 frusemide standard curve compound concentration of table
B: the dilute solution of analyte: MeOH/H2O=50/50
Standard curve (n=13) of the frusemide that table 7HPLC-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 frusemide standard solution prepare 5 kinds of various concentrations plasma containing drug quality-control sample (LLOQ, QL, QLM, QM, QH) and one
Retinue standard curve, is operated by " plasma sample pretreatment ", and quality-control sample preparation is as shown in table 8 below.A batch and one are daily
Retinue standard curve is continuously done 3 days, and totally three batches, first and each concentration of second batch make 6 parts of samples respectively, and third batch is each
Concentration makees 16 parts of samples respectively, calculates the ratio Y of frusemide peak area As and internal standard frusemide-d5 peak area Ai, substitutes into the same day
Standard curve in acquire measured concentration, by measured concentration calculate batch in and betweenrun precision, measured concentration and concentration is added
Ratio is accuracy, the results are shown in Table 9.The result shows that in frusemide plasma sample batch, betweenrun precision, accuracy be less than ±
15% meets the requirements.
8 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 indicated and are stored in theoretical temperatures -80
℃.Volume can be increased or decreased optionally to scale.
Table 9HPLC-MS/MS method measure blood plasma in frusemide 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 frusemide analyte and internal standard frusemide-
The interference of d5.
It is dry at frusemide retention time meeting after six separate sources blank healthy human body plasma sample preparation analyses
Peak response is below the frusemide response of lower limit of quantitation sample in the standard curve of the analysis batch 20.0% is disturbed, the results are shown in Table
10.The result shows that the analysis method has specificity to the analysis of frusemide.
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 the table 11 in annex.
The result shows that the internal target analysis of the analysis method has selectivity.
Interference data comparison table of the 10 6 separate sources blank healthy human body blood plasma of table to frusemide 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 11 6 separate sources blank healthy human body blood plasma of table to frusemide-d5
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, to be analyzed
The retention time of object ", the region peak area are considered zero.
As can be seen that the blank plasma of different human body does not cause to do to the testing result of frusemide from table 10 and table 11
It disturbs.Therefore, this method can be used for detecting the frusemide concentration in different human body blood plasma.
6, human plasma sample detects
(1) the people's blank plasma for being not given to frusemide is taken, the accurate blank plasma that 100uL is added in 96 deep-well plates
Sample, the volume ratio that 10uL is added is the methanol aqueous solution of 1:1, and the acetonitrile of 500uL 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 80uL deep to 96 that 420uL mixed organic solvents are housed
In orifice plate, mixed organic solvents are water: acetonitrile: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.2, are vortexed and mix,
10uL 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 2.
(2) the people's blank plasma for being not given to frusemide is taken, the accurate blank plasma that 100uL is added in 96 deep-well plates
Sample is added the frusemide standard solution that 5uL concentration is 1ng/uL, the internal standard frusemide-d5 of the 1ng/uL of 5uL is added after mixing
The acetonitrile of 500uL is added in 96 deep-well plates in solution after mixing, vortex mixed 1min is centrifuged 10min in 20 DEG C with 3000rpm,
Take supernatant liquor 80uL into 96 deep-well plates that 420uL mixed organic solvents are housed, mixed organic solvents are water: acetonitrile: formic acid
It according to the mixture that volume ratio 95:5:0.2 is mixed to get, is vortexed and mixes, take 10uL sample after being centrifuged 5min in 20 DEG C with 3000rpm
Product carry out LC-MS/MS analysis, as a result as shown in Figure 3.
(3) blood plasma during 1~5h after the oral frusemide drug of acquisition health volunteer is accurate in 96 deep-well plates
The human plasma sample of the acquisition of 100uL is added, the volume ratio that 5uL is added is the methanol aqueous solution of 1:1, and 5uL is added after mixing
1ng/uL internal standard frusemide-d5 solution, after mixing be added 500uL acetonitrile in 96 deep-well plates, vortex mixed 1min, in
20 DEG C are centrifuged 10min with 3000rpm, take supernatant liquor 80uL into 96 deep-well plates that 420uL mixed organic solvents are housed, and mix
Organic solvent is water: acetonitrile: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.2, be vortexed mix, in 20 DEG C with
10uL sample is taken to carry out LC-MS/MS analysis after 3000rpm centrifugation 5min, as a result as shown in Figure 4.
In conclusion the present invention provides the measuring method of frusemide concentration in a kind of blood plasma of preprocess method simplicity,
Using two step organic extractant solution methods, it is suitable for conventional determining；It shows from the above analysis, the chromatographic condition used by this experiment
Under, frusemide retention time is 2.430min or so, internal standard frusemide-d5 retention time in 2.401min or so, frusemide and
The peak shape of internal standard frusemide-d5 is good, no miscellaneous peak interference measurement, and baseline is steady；This method specificity with higher, can be accurate
The concentration of the frusemide in blood plasma is measured, sensitivity is higher, and blood plasma is minimum to be quantitatively limited to 10ng/mL；Meanwhile the method for the present invention is fast
It is speed, accurate, high sensitivity, easy to operate, foundation is provided for the determination of plasma concentration of frusemide.The plasma standard curve of this method
The range of linearity is 10~2000ng/mL, and in batch and betweenrun precision RSD is respectively less than ± 15%.
Claims (4)
1. a kind of method of frusemide concentration in LC-MS measurement blood plasma, it is characterised in that: passed through after plasma sample is preprocessed
High performance liquid chromatography-tandem mass detects its concentration, specific method the following steps are included:
(1) plasma sample pre-processes:
Blood plasma is with K2EDTA is anti-coagulants, using frusemide-d5 as internal standard；The accurate blood plasma sample that 100uL is added in 96 deep-well plates
Product, the volume ratio that 5uL is added is the methanol aqueous solution of 1:1, and the internal standard frusemide-d5 solution of the 1ng/uL of 5uL is added after mixing,
The acetonitrile of 500uL is added after mixing in 96 deep-well plates, vortex mixed 1min is centrifuged 10min in 20 DEG C with 3000rpm, takes
Layer clear liquid 80uL into 96 deep-well plates that 420uL mixed organic solvents are housed, mixed organic solvents are water: acetonitrile: formic acid according to
The mixture that volume ratio 95:5:0.2 is mixed to get is vortexed and mixes, as test sample after being centrifuged 5min using 3000rpm in 20 DEG C
It is to be detected；
(2) Specimen Determination:
It takes in 10uL test sample injection high performance liquid chromatography-tandem mass instrument, frusemide and internal standard frusemide-in test sample
The chromatographic peak of d5, and the frusemide concentration in the plasma sample is 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 100/0.2/0.1；Mobile phase B: acetonitrile/water/formic acid/1M ammonium acetate body
Product percentage is 80/20/0.2/0.1；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 10uL, analysis time 4min；
(4) mass spectroscopy, condition are as follows:
Ion source is electric spray ion source, and spray voltage is -4500V, and atomization temperature is 480 DEG C, and spray pressure power is 20Psi,
Auxiliary heating atmospheric pressure is 20Psi, and gas curtain atmospheric pressure is 20Psi, and collision atmospheric pressure is 8Psi, and removing cluster voltage is respectively -25eV
Frusemide and frusemide-d5；Collision cell entrance potential is respectively the frusemide and frusemide-d5 of -10eV；Collision voltage difference
For the frusemide and frusemide-d5 of -20eV；Collision cell exit potential is respectively the frusemide and frusemide-d5 of -10eV；Bear from
Submode detection；Scanning mode is multiple reaction monitoring；
Ionic reaction for quantitative analysis is respectively as follows: m/z329.0 → m/z285.0, is frusemide；With m/z334.0 → m/
Z290.0 is frusemide-d5.
2. the method for frusemide concentration in a kind of LC-MS measurement blood plasma according to claim 1, it is characterised in that: institute
State the program of gradient elution in step (3) are as follows:
。
3. the method for frusemide concentration, feature exist in a kind of LC-MS measurement blood plasma according to claim 1 or 2
In: in the step (2), using internal standard method, standard curve side is brought into the peak area ratio of frusemide and internal standard frusemide-d5
Journey calculates the concentration of the frusemide in the plasma sample.
4. the method for frusemide concentration in a kind of LC-MS measurement blood plasma according to claim 3, it is characterised in that: institute
State the foundation of calibration curve equation the following steps are included:
10 parts of 100uL blank plasma are taken to be placed in 96 deep-well plates, adding 5uL concentration respectively in the form of stock solution is respectively
0.2ng/ μ L, 0.4ng/ μ L, 1ng/ μ L, 2ng/ μ L, 10ng/ μ L, 24ng/ μ L, 30ng/ μ L, 40ng/ μ L frusemide solution extremely
Minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, in highest upper limit of quantification sample, respectively
Adding 5 μ l volume ratios is the methanol aqueous solution of 1:1 to blank sample and zero-dose sample, respectively to minimum lower limit of quantitation 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, 5uL is added in zero-dose sample in sample
1ng/uL internal standard frusemide-d5 solution, into blank sample be added 5uL volume ratio be 1:1 methanol aqueous solution, mix
Afterwards respectively into 10 parts of samples be added 500uL acetonitrile in 96 deep-well plates, vortex mixed 1min, in 20 DEG C with 3000rpm from
Heart 10min takes supernatant liquor 80uL into 96 deep-well plates that 420uL mixed organic solvents are housed, and mixed organic solvents are water: second
Nitrile: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.2 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 10uL standard sample injection high performance liquid chromatography-tandem mass instrument respectively, frusemide and internal standard in test sample
The chromatographic peak of frusemide-d5, and standard curve is obtained accordingly, with the concentration for calculating the frusemide in the blood plasma.
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