A detection method and application of ultra-high performance liquid chromatography tandem mass spectrometry

Abstract

The present invention discloses a detection method and application of ultra-high performance liquid chromatography-tandem mass spectrometry. The method of the present invention adopts ultra-high performance liquid chromatography-tandem mass spectrometry, first with 0.1% (V / V) formic acid aqueous solution and methanol as mobile phase gradient elution, and then in electrospray positive ion mode, using multiple reaction monitoring mode and secondary full scan analysis scanning. The linearity of three antiviral drug components, namatevir, monoclavir and daclatasvir, detected by the detection method of the present invention is good; the average recovery rate of spiked is 80% to 91.6%, and the precision is less than 5%. The method of the present invention is sensitive, rapid and reliable, and can be used for the qualitative and quantitative analysis of the drug components namatevir, monoclavir and daclatasvir in preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a detection method and application of ultra-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Currently, there is no national standard or literature report on the liquid chromatography-mass spectrometry detection method for the simultaneous detection of nematevir, monoclavir, and daclatasvir. Therefore, there is an urgent need to establish qualitative and quantitative methods for detecting the above-mentioned antiviral drugs. Summary of the Invention

[0003] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a technical means for simultaneous qualitative and quantitative analysis of namatevir, ritonavir, and daclatasvir by ultra-high performance liquid chromatography-tandem mass spectrometry, thereby providing technical support for the effective supervision of drug safety.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] The first aspect of the present invention provides a detection method using ultra-high performance liquid chromatography tandem mass spectrometry.

[0006] The second aspect of the present invention proposes an application of a detection method of ultra-high performance liquid chromatography tandem mass spectrometry.

[0007] According to a first aspect of the present invention, a detection method of ultra-high performance liquid chromatography tandem mass spectrometry is proposed, the method comprising the following steps:

[0008] (1) preparing a mixed standard solution of namatevir, monoclavir, and daclatasvir;

[0009] (2) using ultra-performance liquid chromatography-tandem mass spectrometry to analyze the mixed standard solution and the pre-treated test sample solution, and calculating the contents of namatevir, monoclavir, and daclatasvir in the test sample according to the standard curve; wherein the mass spectrometry conditions include:

[0010] Ionization mode: electrospray ionization source (ESI), positive ion mode scanning, multiple reaction monitoring (MRM) mode excitation secondary mass spectrometry full scan (MS2);

[0011] Curtain air (CUR): 20-40 psi;

[0012] Collision gas (CAD): Medium;

[0013] Ionization voltage (IS): 4500~5500V;

[0014] Ion source temperature (TEM): 400-600°C;

[0015] Atomizing gas (GS1): 40-60 psi;

[0016] Auxiliary gas (GS2): 40-60 psi;

[0017] Declustering voltage (DP): 90-120V;

[0018] Entry voltage (EP): 10.0V;

[0019] Collision energy (CE): 20-80V;

[0020] Collision cell exit voltage (CXP): 10-15 V;

[0021] Secondary mass spectrometry full scan range (amu): 50~750.

[0022] In some embodiments of the present invention, the concentration of the mixed standard solution of namatevir, monolavir and daclatasvir in (1) is 0.6 ng / mL to 80 ng / mL, that is, the concentrations of namatevir, monolavir and daclatasvir in the mixed standard solution are the same, and the concentration of any one of them is 0.6 ng / mL to 80 ng / mL.

[0023] In some embodiments of the present invention, the specific preparation of the mixed standard sample in (1) includes:

[0024] S1: Prepare standard stock solutions of namatevir, monoclavir, and daclatasvir using methanol;

[0025] S2: Prepare a mixed intermediate standard solution using the standard stock solution described in S1, and dilute with methanol to the required concentration to obtain a mixed standard solution.

[0026] In some preferred embodiments of the present invention, the specific preparation of the mixed standard sample in (1) includes:

[0027] S1: Accurately weigh 10 mg each of nematevir, monoclavir, and daclatasvir reference substances, place them in 10 mL volumetric flasks, dissolve and dilute with methanol to the mark to prepare a 1 mg / mL standard stock solution, and store at -20°C;

[0028] S2: Accurately pipette each standard stock solution separately, prepare a 1 μg / mL mixed intermediate standard solution with methanol, and store it at -20°C; when using, restore the mixed intermediate standard solution to room temperature (15°C ~ 30°C), dilute it with methanol to 0.6 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 80 ng / mL to obtain mixed standard samples, which should be prepared before use.

[0029] In some preferred embodiments of the present invention, the ionization mode is: electrospray ionization source (ESI), positive ion mode scanning, multiple reaction monitoring (MRM) mode excitation secondary mass spectrometry full scan (MS2);

[0030] Curtain air (CUR): 30.0psi;

[0031] Collision gas (CAD): Medium;

[0032] Ionization voltage (IS): 5500.0 V;

[0033] Ion source temperature (TEM): 550.0°C;

[0034] Atomizing gas (GS1): 55 psi;

[0035] Auxiliary gas (GS2): 55psi;

[0036] Declustering voltage (DP): 120.0 V;

[0037] Entry voltage (EP): 10.0V;

[0038] Collision energy (CE): 20-80V;

[0039] Collision cell exit voltage (CXP): 13.0 V;

[0040] Secondary mass spectrometry full scan range (amu): 50~750.

[0041] In some preferred embodiments of the present invention, the pretreatment of the test sample solution in (2) includes: mixing the test sample with methanol, vortexing, ultrasonicating, constant volume, and filtering with a filter membrane.

[0042] In some preferred embodiments of the present invention, the pretreatment of the test sample solution in (2) includes: accurately weighing 0.200 g of the test sample and placing it in a 50 mL stoppered centrifuge tube, adding 30 mL of methanol, vortexing for 30 s, ultrasonicating at 70 kHz and 250 W for 15 min, cooling to room temperature (15°C to 30°C), and then adjusting the volume to 50 mL. Before use, the solution is filtered through a 0.22 μm nylon filter membrane.

[0043] In some preferred embodiments of the present invention, the elution system of the ultra-high performance liquid chromatography described in (2) is a gradient elution of methanol-formic acid aqueous solution; further a gradient elution of methanol-0.05-0.1% (V / V) formic acid aqueous solution; further a gradient elution of methanol-0.1% (V / V) formic acid aqueous solution.

[0044] In some preferred embodiments of the present invention, the ultra-high performance liquid chromatography conditions in (2) include:

[0045] Chromatographic column: C18 column;

[0046] Mobile phase: Phase A is formic acid aqueous solution, phase B is methanol;

[0047] Gradient elution program: 0-2 min, 95% A; 2-4 min, 95%-10% A; 4-7 min, 10% A; 7-10 min, 95% A;

[0048] Flow rate: 0.2-0.4 mL / min;

[0049] Column temperature: 30-45°C;

[0050] Injection volume: 2-20 μL.

[0051] In some more preferred embodiments of the present invention, the ultra-high performance liquid chromatography conditions in (2) include:

[0052] Chromatographic column: C18 column;

[0053] Mobile phase: Phase A is formic acid aqueous solution, phase B is methanol;

[0054] Gradient elution program: 0-2 min, 95% A; 2-4 min, 95%-10% A; 4-7 min, 10% A; 7-10 min, 95% A;

[0055] Flow rate: 0.3 mL / min;

[0056] Column temperature: 40°C;

[0057] Injection volume: 2 μL.

[0058] In some preferred embodiments of the present invention, the corresponding mass spectrometry parameters of namatevir, ritonavir and monolavir are:

[0059]

[0060] Where * is the quantification product ion.

[0061] In some preferred embodiments of the present invention, the specifications of the C18 chromatographic column in (2) are: column length 100 mm, column inner diameter 2.1 mm, and filler particle size 2.6 μm.

[0062] In some preferred embodiments of the present invention, the C18 chromatographic column in (2) is a CNWShell C18 Core-Shell chromatographic column.

[0063] In some preferred embodiments of the present invention, the ultra-high performance liquid chromatograph is LC-30AD manufactured by Shimadzu Corporation of Japan.

[0064] In some preferred embodiments of the present invention, the mass spectrometer model is QTrap5500 produced by SCIEX Corporation of the United States.

[0065] The second aspect of the present invention provides an application of the ultra-high performance liquid chromatography-tandem mass spectrometry detection method described in the first aspect in analyzing and determining at least one of namatevir, monoclavir, and daclatasvir.

[0066] In some embodiments of the present invention, the application is to analyze and determine at least one of namatevir, monoclavir and daclatasvir in a drug.

[0067] In some embodiments of the present invention, the drug is an antiviral drug.

[0068] The beneficial effects of the present invention are:

[0069] The present invention establishes a method for detecting three components, namely, nematevir, monolavir, and daclatasvir, by using ultra-performance liquid chromatography-tandem mass spectrometry. The method has high sensitivity, strong reliability, a simple and rapid analysis process, and good linearity for the three components. The average recovery rate (n=6) of spiked samples is 80% to 91.6%, and the relative standard deviation (RSD) is less than 5%.

[0070] The present invention can meet the detection requirements of simultaneously determining three antiviral components, and can complete qualitative and quantitative analysis in 10 minutes, providing technical support for the supervision of pharmaceutical preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Extracted ion chromatograms of spiked solutions of namatevir, monoclavir, and daclatasvir samples using methanol and ultrapure water as mobile phases;

[0072] Figure 2 Extracted ion chromatograms of spiked solutions of namatevir, monoclavir, and daclatasvir samples using acetonitrile and ultrapure water as mobile phases;

[0073] Figure 3 Extracted ion chromatograms of spiked solutions of namatevir, monoclavir, and daclatasvir samples using methanol and ultrapure water containing 0.1% (V / V) formic acid as the mobile phase;

[0074] Figure 4 Extracted ion chromatograms of spiked solutions of namatevir, monoclavir, and daclatasvir samples using methanol and ultrapure water solution containing 10 mmol of ammonium acetate as the mobile phase;

[0075] Figure 5The following are the MS spectra of namatevir, monoclavir, and daclatasvir samples;

[0076] Figure 6 This is the extracted ion chromatogram of daclatasvir in sample 1 solution;

[0077] Figure 7 This is the extracted ion chromatogram of daclatasvir in the reference solution;

[0078] Figure 8 This is the extracted ion chromatogram of Namatevir in sample 1 solution;

[0079] Figure 9 This is the extracted ion chromatogram of namatevir in the reference solution;

[0080] Figure 10 This is the secondary mass spectrum of daclatasvir in sample 1 solution;

[0081] Figure 11 The second mass spectrum of daclatasvir in the reference solution. DETAILED DESCRIPTION

[0082] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0083] Example

[0084] 1. Instruments, Reagents, and Materials

[0085] An LC-30AD liquid chromatograph (Shimadzu Corporation, Japan); a QTrap 5500 mass spectrometer equipped with an electrospray ionization (ESI) source (SCIEX, USA); and a Mili-Q Advantage A10 ultrapure water system (Millipore, USA) were used. Methanol and acetonitrile (chromatographic grade, Merck, Germany); formic acid (chromatographic grade, Tianjin Komeiou Co., Ltd.); and ammonium acetate (analytical grade, Tianjin Komeiou Co., Ltd.) were used in the experiments. Milli-Q ultrapure water was used. Namatevir (99.69%) was purchased from Shanghai Bichen Biochemical Technology Co., Ltd.; monoclavir (99% purity) and daclatasvir dihydrochloride (99% purity) were purchased from Aladdin Co., Ltd.

[0086] 1.1 Preparation of reference solution (mixed standard solution)

[0087] Accurately weigh 10 mg of Nirmatrelvir, Molnupiravir, and Daclatasvir reference substances, place them in 10 mL volumetric flasks, dissolve and dilute with methanol to the mark, prepare a 1 mg / mL standard stock solution, and store at -20 °C.

[0088] Accurately pipette an appropriate amount of each standard stock solution and prepare a 1 μg / mL mixed intermediate standard solution with methanol (i.e., the concentrations of nematevir, monoclavir, and daclatasvir in the mixed intermediate standard solution are all 1 μg / mL). Store at -20°C. Before use, return the mixed intermediate standard solution to room temperature and dilute it with methanol to 0.6 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 80 ng / mL mixed standard solutions. Prepare them immediately before use.

[0089] 1.2 Sample preparation

[0090] After the tablet sample is ground and mixed evenly, 0.2 g (accurate to 0.001 g) is accurately weighed and placed in a 50 mL stoppered centrifuge tube. 30 mL of methanol is added and the sample is vortexed for 30 s. Ultrasonic extraction (70 kHz, 250 W) is performed at room temperature for 15 min. After cooling to room temperature, the volume is adjusted to 50 mL and filtered through a 0.22 μm nylon filter membrane for LC-MS / MS analysis.

[0091] 1.3 Chromatographic conditions

[0092] CNWShell C18 Core-Shell chromatographic column (100 mm*2.1 mm, 2.6 μm); mobile phase: phase A is 0.1% (v / v) formic acid aqueous solution, and phase B is methanol.

[0093] Gradient elution program: 0-2 min, 95% A;

[0094] 2-4 min, 95%-10% A;

[0095] 4-7 min, 10% A;

[0096] 7-10 min, 95% A;

[0097] Flow rate: 0.3 mL / min;

[0098] Column temperature: 40°C;

[0099] Injection volume: 2 μL.

[0100] 1.4 Mass spectrometry conditions

[0101] Electrospray ionization (ESI) source, positive ion mode scanning, multiple reaction monitoring (MRM) mode excitation secondary mass spectrometry full scan (MS2); curtain gas (CUR): 30.0 psi; collision gas (CAD): Medium; ionization voltage (IS): 5500.0 V; ion source temperature (TEM): 550.0°C; nebulizer gas (GS1): 55 psi; auxiliary gas (GS2): 55 psi; declustering voltage (DP): 120.0 V; entrance voltage (EP): 10.0 V; collision energy (CE): 20, 30, or 50 V; collision cell exit voltage (CXP): 13.0 V; secondary mass spectrometry full scan range (amu): 50-750. The corresponding mass spectrometry conditions for namatevir, monoclavir, and daclatasvir are shown in Table 1:

[0102] Table 1

[0103]

[0104]

[0105] Where * is the quantification product ion.

[0106] 2 Results and Discussion

[0107] 2.1 Optimization of extraction solvent for sample pretreatment

[0108] The extraction efficiency of six extraction solvents was investigated: methanol, 80% (v / v) methanol-water solution, 50% (v / v) methanol-water solution, acetonitrile, 80% (v / v) acetonitrile-water solution, and 50% (v / v) acetonitrile-water solution. The results showed that the presence of ultrapure water in the extraction solvent increased the number of co-extractables in the sample solution and enhanced the ionization of daclatasvir. Comparing the extraction efficiency of methanol and acetonitrile revealed that the recovery rates of the three chemical components after methanol extraction were closer to 100%. Therefore, methanol was selected as the sample extraction solvent. The results are shown in Table 2:

[0109] Table 2

[0110]

[0111] 2.2 Optimization of analytical conditions

[0112] 2.2.1 Chromatographic conditions

[0113] The three drug ingredients, nematevir, monoclavir and daclatasvir, all have alkaline properties. It is not easy to obtain a good peak shape in a neutral mobile phase system, such as methanol-water or acetonitrile-water. At the same time, adding an appropriate amount of volatile organic acid to the mobile phase system can enhance the ion response intensity and improve the chromatographic peak shape. Four mobile phase systems, methanol-water, acetonitrile-water, methanol-0.1% (V / V) formic acid water, and methanol-10mmol ammonium acetate aqueous solution, were investigated, and all water was ultrapure water. The quantitative ion extraction ion current chromatograms of the sample spiked solutions (20ng / mL) of the three components corresponding to the above four mobile systems are shown in Figures 1 to 4 , Figures 1 to 4 Number 1 is the quantitative ion peak of daclatasvir, number 2 is the quantitative ion peak of monoclavir, and number 3 is the quantitative ion peak of namatevir.

[0114] Compared to acetonitrile, methanol exhibited superior retention, separation, and response for the target compounds. Adding 0.1% (v / v) formic acid to the aqueous phase improved both chromatographic peak shape and ion response. However, adding 10 mM ammonium acetate to the aqueous phase reduced ion response. Therefore, a methanol-0.1% (v / v) formic acid solution mobile phase was ultimately selected.

[0115] 2.2.2 Mass spectrometry conditions

[0116] The structures of the three drug ingredients, namatevir, monoclavir, and daclatasvir, all contain amino or amine groups, which are easy to form [M+H] in the positive ion mode. + Quasi-molecular ion peak. The mass spectrometry conditions were optimized using a 50 ng / mL mixed standard solution and ESI + As the ionization mode, the full scan mode and the multiple reaction monitoring mode were used through the flow syringe pump system to obtain the target compound's parent ion, daughter ion and optimal collision energy and other mass spectrometric parameters. The optimized mass spectrometric conditions corresponding to namatevir, monoclavir and daclatasvir are shown in Table 1. The secondary mass spectra of the three drug components are shown in Figure 5 .

[0117] 2.3 Methodological Investigation

[0118] 2.3.1 Linear range and detection limit

[0119] A series of mixed standard solutions (0.6 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 80 ng / mL) were prepared according to the procedure in Example 1.1. Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was performed on these mixed standard solutions according to the conditions of Examples 1.3 and 1.4. Calibration curves for the three drug components were plotted, with the concentration of the drug component to be tested (x) in the mixed standard solution as the abscissa and the peak area of ​​the quantitative ion of the drug component to be tested (y) as the ordinate. The limit of detection (LOD) and limit of quantification (LOQ) of the method were calculated using a signal-to-noise ratio of 3 times (S / N ≥ 3) and a signal-to-noise ratio of 10 times (S / N ≥ 10), respectively. The specific results are shown in Table 3:

[0120] Table 3

[0121]

[0122] 2.3.2 Repeatability and precision of the method

[0123] Eighteen blank matrix samples (tablets without nematevir, monoclavir, and daclatasvir) were spiked with mixed standard solutions at three concentration levels (low, medium, and high) (LOQ, 2xLOQ, and 10xLOQ). (For example, the low-concentration mixed standard solution contained 2 ng / mL nematevir, 2 ng / mL monoclavir, and 5 ng / mL daclatasvir). Six replicates were prepared for each concentration level to assess the repeatability and precision of the method. The average recoveries of the three drug components ranged from 80% to 91.6%, with relative standard deviations (RSDs) of less than 5%, demonstrating good repeatability and precision. The results are shown in Table 4.

[0124] Table 4

[0125]

[0126] Test example

[0127] The ultra-high performance liquid chromatography-tandem mass spectrometry detection method established in the examples was used, and the operations and conditions of Examples 1.1 to 1.4 were referred to for detection and analysis of five samples of the generic drug Primovir tablets labeled as containing 150 mg / tablet of Nematevir.

[0128] Qualitative analysis standard: Under the same instrument and conditions, the sample solution and the mixed standard solution are tested. If two pairs of ion pair chromatographic peaks of the target detection object exist at the same retention time (within the variation range of ±2%), and when the concentration difference of the target detection component in the sample solution and the reference solution is within ±10%, it is determined that the obtained secondary mass spectra are consistent, and the sample solution can be qualitatively identified as containing the target detection component.

[0129] The results showed that no namatevir was detected in the 5 samples, but daclatasvir was detected in all of them. Taking sample 1 as an example, the chromatogram of daclatasvir extracted ion current chromatogram in sample 1 is shown in Figure 6 The reference extracted ion chromatogram is shown in Figure 7 ; The extracted ion chromatogram of Namatevir in sample 1 is shown in Figure 8 The reference extracted ion chromatogram is shown in Figure 9 ; The secondary spectrum of Daclatasvir in sample 1 is shown in Figure 10 , and its secondary spectrum is shown in Figure 11 .

[0130] The quantitative ion peak areas of the components in the sample spectra were substituted into the corresponding standard curves in Example 2.3.1 to calculate the content of the corresponding components. The daclatasvir content in the five samples tested was found to be in the range of 7.4 mg / pill to 10.9 mg / pill. The relevant information is shown in Table 5:

[0131] Table 5

[0132]

[0133]

[0134] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A detection method of ultra-high performance liquid chromatography tandem mass spectrometry, characterized in that: The detection method is used to simultaneously detect namatevir, monoclavir and daclatasvir; the detection method comprises the following steps: (1) Prepare a mixed standard solution of namatevir, monoclavir, and daclatasvir; (2) Using ultra-high performance liquid chromatography tandem mass spectrometry to analyze the mixed standard solution and the pre-treated test sample solution, and calculating the contents of namatevir, monoclavir and daclatasvir in the test sample according to the standard curve; wherein the mass spectrometry conditions include: Ionization mode: electrospray ionization source, positive ion mode scanning, multiple reaction monitoring mode excitation secondary mass spectrometry full scan; Curtain gas: 20~40 psi; Collision gas: Medium; Ionization voltage: 4500~5500 V; Ion source temperature: 400~600℃; Atomizing gas: 40~60 psi; Auxiliary gas: 40~60 psi; Declustering voltage: 90~120 V; Inlet voltage: 10.0 V; Collision chamber exit voltage: 10-15 V; Secondary mass spectrometry full scan range: 50~750; The conditions of the ultra-high performance liquid chromatography include: Chromatographic column: C18 column; Mobile phase: Phase A is formic acid aqueous solution, phase B is methanol; Gradient elution program: 0-2 min, 95% A; 2-4 min, 95%-10% A; 4-7 min, 10% A; 7-10 min, 95% A; The corresponding mass spectrometry parameters of namatevir, monoclavir and daclatasvir are: in, is the quantitative daughter ion.

2. The detection method of ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The elution system of the ultra-high performance liquid chromatography described in (2) is a methanol-formic acid aqueous solution gradient elution.

3. The detection method of ultra-high performance liquid chromatography tandem mass spectrometry according to claim 2, characterized in that: The conditions of the ultra-high performance liquid chromatography include: Flow rate: 0.2~0.4 mL / min; Column temperature: 30~45℃; Injection volume: 2~20 μL.

4. The detection method of ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The concentration of the mixed standard solution of namatevir, monoclavir and daclatasvir described in (1) is 0.6 ng / mL~80 ng / mL.

5. The detection method of ultra-high performance liquid chromatography tandem mass spectrometry according to claim 2, characterized in that: The elution system of the ultra-high performance liquid chromatography described in (2) is a gradient elution of methanol-0.05~0.1% (V / V) formic acid aqueous solution.

6. The detection method of ultra-high performance liquid chromatography tandem mass spectrometry according to claim 3, characterized in that: The specifications of the C18 chromatographic column are: column length 100 mm, column inner diameter 2.1 mm, and filler particle size 2.6 μm.

7. The detection method of ultra-high performance liquid chromatography tandem mass spectrometry according to claim 4, characterized in that: The specific preparation of the mixed standard solution includes: S1: Prepare standard stock solutions of namatevir, monoclavir, and daclatasvir using methanol; S2: Prepare a mixed intermediate standard solution using the standard stock solution described in S1, and dilute with methanol to the required concentration to obtain a mixed standard solution.

8. The detection method of ultra-high performance liquid chromatography tandem mass spectrometry according to claim 5, characterized in that: The pre-treatment of the test sample solution includes: mixing the test sample with methanol, vortexing, ultrasonicating, constant volume, and membrane filtering.

9. Use of the ultra-high performance liquid chromatography-tandem mass spectrometry detection method according to any one of claims 1 to 8 for the simultaneous analysis and determination of nematevir, monoclavir and daclatasvir.

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