CN115078611A - Mobile phase formula of immunosuppressant for liquid chromatography-mass spectrometry and detection method - Google Patents
Mobile phase formula of immunosuppressant for liquid chromatography-mass spectrometry and detection method Download PDFInfo
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- CN115078611A CN115078611A CN202210532094.1A CN202210532094A CN115078611A CN 115078611 A CN115078611 A CN 115078611A CN 202210532094 A CN202210532094 A CN 202210532094A CN 115078611 A CN115078611 A CN 115078611A
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- immunosuppressant
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- 229960003444 immunosuppressant agent Drugs 0.000 title claims abstract description 20
- 230000001861 immunosuppressant effect Effects 0.000 title claims abstract description 20
- 239000003018 immunosuppressive agent Substances 0.000 title claims abstract description 20
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 title abstract description 33
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- 239000003960 organic solvent Substances 0.000 claims abstract description 11
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical group N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 10
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- 238000000034 method Methods 0.000 claims abstract description 8
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- 238000009472 formulation Methods 0.000 claims description 13
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- 210000004369 blood Anatomy 0.000 claims description 7
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- HKVAMNSJSFKALM-KENIYLLDSA-N everolimus-d4 Chemical compound C1[C@@H](OC)[C@H](OC([2H])([2H])C([2H])(O)[2H])CC[C@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 HKVAMNSJSFKALM-KENIYLLDSA-N 0.000 description 2
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Abstract
The invention relates to a mobile phase formula of an immunosuppressant for liquid chromatography-mass spectrometry and a detection method, wherein the formula comprises a mobile phase A and a mobile phase B, and the mobile phase A is a buffered saline solution; the mobile phase B is a buffer salt organic solvent; the buffer salt is ammonium acetate or ammonium formate; the concentration of the buffer salt solution is 2mmol/L-12 mmol/L. The detection time is optimized by optimizing the mobile phase and the buffer salt in the mobile phase, so that the detection time is shortened to 3.5min, the detection efficiency is greatly improved, and the method is simple, effective, good in linearity, practical and reliable, and is suitable for rapid detection of a large number of clinical biological samples.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a mobile phase formula of an immunosuppressant for liquid chromatography-mass spectrometry and a detection method.
Background
Two drugs of cyclosporine A and everolimus are mainly used for rejection reaction of organ transplantation and autoimmune reaction diseases clinically. In addition to organ transplantation, cyclosporin a can be loaded onto recombinant lipoprotein for the treatment of craniocerebral injury, and everolimus can be loaded onto H-ferritin nanocages for the treatment of breast cancer and the like. However, both of these drugs have the defects of large individual difference of pharmacokinetics, low therapeutic index, narrow effective blood concentration range and the like, and need to be applied to individual administration to ensure the safety and effectiveness of drug treatment.
The currently common detection methods include an immunoassay method and a chromatography method, wherein the liquid chromatography tandem mass spectrometry method has the advantages of strong specificity, high accuracy, rapid analysis, high sensitivity and the like, so that most clinical laboratories use the detection method as a gold standard for monitoring therapeutic drugs. The LC-MS/MS combines the high separation capacity of LC and the accurate and sensitive qualitative and quantitative capacity of MS, has extremely high specificity, accuracy, sensitivity, rapidness and low quantitative limit, and is particularly suitable for the quantitative analysis of trace drugs.
In LC-MS/MS quantitative analysis, chromatographic conditions and mass spectral parameters are usually optimized for higher detection sensitivity and accuracy. The choice of the proportions and concentration adjustments of the mobile phase and mobile phase additives (buffer salts) can, among other things, influence the chromatographic peak separation, peak shape, and thus sensitivity by influencing ion formation in the ion source.
The mobile phase of the high performance liquid chromatography can be composed of one solvent or two or more solvents, and different solvents have different elution capacities on the target substance. In practical application, several solvents are often mixed together according to a proper proportion, the solvent components and proportions are different, the polarities are different, the dissolving capacity to a measured object is different, the separation effect is naturally different, and the peak emergence time is correspondingly changed.
The buffer salt in the mobile phase is a regulator in chromatographic analysis and has the functions of regulating pH value, modifying peak type and better separating chromatographic peaks. But too large a buffer salt concentration can result in increased column pressure, decreased column efficiency, abnormal retention times, and the like. Therefore, it is important to optimize the type and concentration of the buffer salts in the mobile phase.
The application aims to establish a novel mobile phase formula of the immunosuppressant for the liquid chromatography-mass spectrometry.
Disclosure of Invention
To achieve the above objects and other advantages and in accordance with the purpose of the invention, a first object of the present invention is to provide a mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry, the formulation comprising a mobile phase a and a mobile phase B, the mobile phase a being a buffered saline solution; the mobile phase B is a buffer salt organic solvent; the buffer salt is ammonium acetate or ammonium formate; the concentration of the buffer salt solution is 2mmol/L-12 mmol/L.
Preferably, the concentration of the buffered salt solution in the mobile phase is 4 mmol/L.
Preferably, the organic solvent is a mixture of at least two of methanol, acetonitrile, methanol and isopropanol.
Preferably, the organic solvent is a mixture of methanol and isopropanol.
Preferably, the volume ratio of the methanol to the isopropanol is 4: 6.
Preferably, the mobile phase flow rate is 0.5-1.5 mL/min.
Preferably, the mobile phase flow rate is 0.8 mL/min.
Preferably, both the mobile phase a and the mobile phase B comprise formic acid.
Preferably, the formic acid accounts for 0.1% of the weight of the mobile phase a or the mobile phase B.
The second purpose of the invention is to provide a method for detecting the inhibitor in blood, which adopts the mobile phase formula for detecting the immunosuppressant by the liquid chromatography-mass spectrometry to detect the everolimus and the cyclosporine A in the blood.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a mobile phase formula of an immunosuppressant for a liquid chromatography-mass spectrometry and a detection method, wherein the formula comprises a mobile phase A and a mobile phase B, and the mobile phase A is a buffered saline solution; the mobile phase B is a buffer salt organic solvent; the buffer salt is ammonium acetate or ammonium formate; the concentration of the buffer salt solution is 2mmol/L-12 mmol/L. The detection time is optimized by optimizing the mobile phase and the buffer salt in the mobile phase, so that the detection time is shortened to 3.5min, the detection efficiency is greatly improved, and the method is simple, effective, good in linearity, practical and reliable, and is suitable for rapid detection of a large number of clinical biological samples.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings. The detailed description of the present invention is given in detail by the following examples and the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a chromatogram of everolimus and cyclosporine A from example 1;
FIG. 2 is a chromatogram of everolimus and cyclosporine A in example 2;
FIG. 3 is a chromatogram of everolimus and cyclosporine A from example 3;
FIG. 4 is a chromatogram of everolimus and cyclosporine A from example 4;
FIG. 5 is a chromatogram of everolimus and cyclosporine A from example 5;
FIG. 6 is a chromatogram of everolimus and cyclosporine A from example 6;
FIG. 7 is a chromatogram of everolimus and cyclosporine A from example 7;
FIG. 8 is a chromatogram of everolimus and cyclosporine A of example 8;
FIG. 9 is a chromatogram of everolimus and cyclosporine A of example 9;
FIG. 10 is a comparison of the reduction in the detection time in example 10;
FIG. 11 is a line graph of everolimus and cyclosporin A in example 10.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
In the following examples, the equipment and reagents used are as follows:
(1) the instrument comprises the following steps:
EClassical 3200 series high performance liquid chromatography: dalianyirit analytical instruments, Inc.; HTQ-2020 series triple quadrupole mass spectrometry: tianjin national medical science and technology development Co., Ltd.
A chromatographic column (GL Sciences 5020 and 01765Inertsil ODS-35 um 4.6X 100mm) with octadecyl and high-purity spherical silica gel as a filler, the column temperature is 55-65 ℃, and the flow rate of a mobile phase is 0.5-1.5 mL/min.
(2) Reagent:
TABLE 1 reagent list
Formic acid (i.e., FA), ammonium formate was purchased from Fisher Chemical company with 98% formic acid purity and 99% ammonium formate purity.
Methanol and isopropanol are purchased from Kancoded science and technology Limited
The high-purity water is first-grade water which conforms to International GB/T6682-2008.
A mobile phase formula of an immunosuppressant for liquid chromatography-mass spectrometry comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a buffered saline solution; the mobile phase B is a buffer salt organic solvent; the buffer salt is ammonium acetate or ammonium formate; the concentration of the buffer salt solution is 2mmol/L-12 mmol/L. According to the invention, the detection accuracy of the mobile phase in actual detection can be improved by optimizing the mobile phase and the mobile phase additive buffer salt. Preferably, the buffered salt solution is ammonium formate, and the use of a mobile phase in which ammonium formate is buffered salt solution gives better detection results.
In some embodiments, the concentration of the buffered salt solution in the mobile phase is 4mmol/L, which provides better detection results than other concentrations.
In some embodiments, the organic solvent is a mixture of at least two of methanol, acetonitrile, methanol, and isopropanol.
In some embodiments, the organic solvent is a mixture of methanol and isopropanol, and the mixture has better detection effect than a single methanol solution or isopropanol solution.
In some embodiments, the volume ratio of methanol to isopropanol is 4: 6.
In some embodiments, both mobile phase a and mobile phase B include formic acid, which controls the PH of the mobile phase and facilitates ionization of the positive ions.
In some embodiments, the weight ratio of formic acid to mobile phase a or mobile phase B is 0.1% to ensure that a good detection result is obtained during actual detection.
The HPLC conditions are shown in Table 2: the flow rate of the mobile phase is 0.5-1.5 mL/min; preferably, the flow rate of the mobile phase is 0.8 mL/min; the column temperature is 55-65 ℃, and preferably 60 ℃; by setting the flow rate of the mobile phase to 0.8 mL/min. The column temperature is 60 ℃ to ensure that a better detection result is finally obtained.
TABLE 2 gradient setup parameter Table
The optimized tandem mass spectrum conditions are as follows: an ion source: electrospray ion source (ESI +); the detection mode is as follows: multiple Reaction Monitoring (MRM); air curtain gas (CUR)10psi, spray gas (GS1)55psi, heated gas (GS2)60psi, Temperature (TEM)450 ℃, spray high pressure (IS)5500V, and impinging gas (CAD)13 psi. The mass spectrum parameters of parent ion, daughter ion, residence time, cone hole voltage, collision energy, etc. of each compound are shown in table 3.
TABLE 3 Mass Spectrometry parameters Table
The detection method is realized by the following steps:
a. taking appropriate amount of everolimus, everolimus-d 4, cyclosporine A and cyclosporine A-d 12, dissolving with methanol to prepare a mixed sample solution with everolimus concentration of 400ng/mL, everolimus-d 4 concentration of 200ng/mL, cyclosporine A concentration of 4000ng/mL and cyclosporine A-d 12 concentration of 4000 ng/mL;
b. setting the flow rate of the mobile phase at 0.5-1.5mL/min and the column temperature at 55-65 ℃;
c. b, injecting 20 mu L of the sample solution of the step a into a mass spectrum to finish detection;
wherein:
high performance liquid chromatograph: EClassical 3200 high performance liquid chromatograph, a large liangliter analyzer ltd;
mass spectrometry: HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medical science and technology development Limited;
a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 um 4.6 × 100 mm;
column temperature: 60 ℃;
flow rate: 0.8 mL/min.
The detection method provided by the invention combines the high separation capability of LC and the accurate and sensitive qualitative and quantitative capability of MS, and has the advantages of high sensitivity, good repeatability and stable and reliable result.
The specific implementation mode is as follows:
the optimal detection method of the invention is determined by the following comparative experiments:
example 1
Instruments and conditions: EClassical 3200 high performance liquid chromatograph, a large liangliter analyzer ltd; HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medicine science and technology development Limited; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 μm 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 4mmol/L ammonium acetate water with 0.1% FA, and phase B was 4mmol/L ammonium acetate methanol with 0.1% FA.
As a result: as shown in figure 1, the everolimus has satisfactory peak shape, and the cyclosporine A has poor peak shape.
Example 2
Instruments and conditions: EClassical 3200 HPLC (large Lily Elite Analyzer Co., Ltd.); HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medical science and technology development Limited; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 μm 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 4mmol/L ammonium acetate water containing 0.1% FA and phase B was 4mmol/L ammonium acetate acetonitrile containing 0.1% FA.
As a result: referring to the attached figure 2, the everolimus peak shape meets the requirement, and the cyclosporine A peak shape is still poor.
Example 3
Instruments and conditions: EClassical 3200 high performance liquid chromatograph, a large liangliter analyzer ltd; HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medical science and technology development Limited; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 μm 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 10mmol/L ammonium formate water with 0.1% FA, phase B was 10mmol/L ammonium formate methanol: isopropanol 9: 1 contains 0.1% FA.
As a result: as shown in the attached figure 3, the everolimus peak shape meets the requirement, and the cyclosporine A peak shape is improved. Both retention times are superior to example 2, indicating that varying the mobile phase additive and mobile phase composition has an effect on the peak shape.
Example 4
Instruments and conditions: EClassical 3200 high performance liquid chromatograph, a large liangliter analyzer ltd; HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medical science and technology development Limited; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 μm 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 10mmol/L ammonium formate water containing 0.1% FA, phase B was 10mmol/L ammonium formate methanol: isopropanol 7: 3 contains 0.1% FA.
As a result: as shown in figure 4, the peak shapes of everolimus and cyclosporine A meet the requirements. Both retention times and peak intensities were better than example 3.
Example 5
Instruments and conditions: EClassical 3200 high performance liquid chromatograph, a large liangliter analyzer ltd; HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medical science and technology development Limited; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 μm 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 10mmol/L ammonium formate water containing 0.1% FA, phase B was 10mmol/L ammonium formate methanol: 1-isopropyl alcohol: 1 contains 0.1% FA.
As a result: as shown in figure 5, the shapes of everolimus and cyclosporine A peaks meet the requirements, the peak intensity is higher than that of example 4, and the retention time is slightly prolonged.
Example 6
Instruments and conditions: EClassical 3200 high performance liquid chromatograph, a large liangliter analyzer ltd; HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medical science and technology development Limited; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 μm 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 4mmol/L ammonium formate water with 0.1% FA, phase B was 4mmol/L ammonium formate methanol: isopropyl alcohol ═ 4:6 contains 0.1% FA.
As a result: referring to the attached figure 6, the shapes of everolimus and cyclosporine A peaks meet the requirements, and the peak intensity and retention time are better than those of the example 5. Example 6 reduces the salt concentration and extends the useful life of the column.
Example 7
Instruments and conditions: EClassical 3200 high performance liquid chromatograph, a large liangliter analyzer ltd; HTQ-2020 series triple quadrupole mass spectrometry of Tianjin national medical science and technology development Limited; and (3) chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 um 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 4mmol/L ammonium acetate water with 0.1% FA, phase B was 4mmol/L ammonium acetate methanol: isopropyl alcohol ═ 4:6 contains 0.1% FA.
As a result: referring to the attached figure 7, the comparative experiment, in which the salt was replaced by ammonium acetate, showed that the peak shapes of everolimus and cyclosporine A were satisfactory and the peak intensities were lower than those of example 6.
By comparison of the above examples, the optimum conditions were those described in example 6. To further verify this idea, comparative experiments with buffered salts were performed on AB Sciex API 4000.
Example 8
Instruments and conditions: shimadzu high performance liquid chromatograph LC-20 AXR; AB Sciex API 4000 triple quadrupole mass spectrometer; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 um 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 4mmol/L ammonium formate water with 0.1% FA, phase B was 4mmol/L ammonium formate methanol: isopropyl alcohol ═ 4:6 contains 0.1% FA.
As a result: referring to the attached figure 8, the peak shapes of everolimus and cyclosporine A meet the requirements.
Example 9
Instruments and conditions: shimadzu high performance liquid chromatograph; AB API 4000 triple quadrupole mass spectrometer; a chromatographic column: GL Sciences 5020-01765Inertsil ODS-35 um 4.6 × 100 mm; column temperature: 60 ℃; flow rate: 0.8 mL/min; mobile phase: phase A was 4mmol/L ammonium acetate water with 0.1% FA, phase B was 4mmol/L ammonium acetate methanol: isopropyl alcohol ═ 4:6 contains 0.1% FA.
As a result: see fig. 9, retention time is greater than example 8 and peak intensity is lower than example 8.
From the results of examples 8 and 9, it can be seen that for everolimus and cyclosporine a, the chromatographic peak intensity and retention time are better when the buffering salt in the mobile phase is ammonium formate.
Example 10
Instruments and conditions: same as in example 6.
As a result: by observing chromatographic peaks, the detection time is shortened from 5min to 3.5min, and the detection efficiency is improved.
Example 11
Instruments and conditions: same as in example 6.
The method comprises the following steps: weighing 1mg of standard everolimus, cyclosporine A and internal standards of the standard everolimus and the cyclosporine A respectively, and diluting with methanol to obtain everolimus with the concentrations of 0.5ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 50ng/mL, 70ng/mL and 100ng/mL respectively; the concentration of the cyclosporine A is 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 500ng/mL, 700ng/mL, 1000ng/mL respectively; the concentration of everolimus internal standard is 200ng/mL, and the concentration of cyclosporine A internal standard is 4000 ng/mL.
As a result: referring to fig. 11, linear regression analysis was performed using everolimus or cyclosporine a concentration (ng/mL) as abscissa and (peak area/internal standard peak area) as ordinate to obtain everolimus linear regression equation y of 0.1441x-0.0617, correlation coefficient r of 0.9974, cyclosporine a linear regression equation y of 0.0211x-0.0019, and correlation coefficient r of 0.9979. The linear relation of everolimus in the range of 0.5ng/mL-100ng/mL is good, and the linear relation of cyclosporine A in the range of 5ng/mL-1000ng/mL is good.
The results are summarized in Table 4 below.
TABLE 4 summary of the results
The invention also relates to a method for detecting the inhibitor in blood, which adopts the formula of the mobile phase of the immunosuppressant for the liquid chromatography-mass spectrometry to detect the everolimus and the cyclosporine A in the blood.
Claims (10)
1. A mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry, wherein the formulation comprises a mobile phase a and a mobile phase B, the mobile phase a is a buffered saline solution; the mobile phase B is a buffer salt organic solvent; the buffer salt is ammonium acetate or ammonium formate; the concentration of the buffer salt solution is 2mmol/L-12 mmol/L.
2. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry of claim 1, wherein the concentration of the buffered salt solution in the mobile phase is 4 mmol/L.
3. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry according to claim 1, wherein the organic solvent is a mixture of at least two of methanol, acetonitrile, methanol and isopropanol.
4. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry according to claim 3, wherein the organic solvent is a mixture of methanol and isopropanol.
5. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry of claim 4, wherein the volume ratio of methanol to isopropanol is 4: 6.
6. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry of claim 1, wherein the mobile phase flow rate is 0.5-1.5 mL/min.
7. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry of claim 6, wherein the mobile phase flow rate is 0.8 mL/min.
8. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry of claim 1, wherein both mobile phase a and mobile phase B comprise formic acid.
9. The mobile phase formulation of an immunosuppressant for liquid chromatography-mass spectrometry of claim 8, wherein formic acid is present in an amount of 0.1% by weight of mobile phase a or mobile phase B.
10. A method for detecting an inhibitor in blood, which comprises detecting everolimus and cyclosporine a in blood using the mobile phase formulation for detecting an immunosuppressant according to any one of claims 1 to 9.
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