A HPLC-MS / MS-based method for detecting the concentration of four SARS-CoV-2 drugs

Abstract

The invention discloses a method for detecting the concentration of four SARS-CoV-2 drugs based on HPLC-MS / MS, wherein the four SARS-CoV-2 drugs are ritonavir, namatevir, azithromycin and baricitinib, and the method comprises: obtaining standard curve working solutions of the four drugs respectively by an external standard method; detecting the sample test solution by HPLC-MS / MS, and quantifying the drug content by using the standard curve, wherein the chromatographic column is ACE Excel 2 C18–PFP; mobile phase A is 0.01% formic acid aqueous solution; mobile phase B is 0.01% formic acid acetonitrile solution; strong wash solution is 80% methanol; gradient elution time is 3 min. The invention is a highly sensitive and specific detection method based on HPLC-MS / MS: the method requires a short time, only 3 min, and greatly saves time for experiments with large sample sizes.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS. Background Art

[0002] SARS-CoV-2 drugs refer to drugs used in the treatment of SARS-CoV-2 infection. Currently, the drugs approved for the treatment of SARS-CoV-2 infection in China include domestically produced azithromycin tablets, imported namatevir tablets / ritonavir tablets combination package (Paxlovid), and monoravir capsules.

[0003] In the World Health Organization's updated SARS-CoV-2 treatment guidelines, it is strongly recommended that for patients with severe or critical SARS-CoV-2, glucocorticoids are combined with baricitinib, and baricitinib alone is used to treat pneumonia caused by SARS-CoV-2 infection in hospitalized adults and pediatric patients 2 years or older who require supplemental oxygen, non-invasive or invasive mechanical ventilation, or extracorporeal membrane oxygenation.

[0004] Baricitinib inhibits the occurrence of this inflammatory storm by inhibiting related immune responses. In addition, it can effectively prevent SARS-CoV-2 from entering normal cells by binding to AAK1. However, when using baricitinib tablets to treat diseases, the risks of medication should also be considered, because the treatment of baricitinib tablets is related to an increased incidence of infection. If baricitinib tablets are used in combination with methotrexate, the risk of infection may increase, so the blood concentration of baricitinib needs to be monitored.

[0005] Azvudine tablets were originally used to treat acquired immunodeficiency syndrome, or AIDS. Later, it was found that azivudine could fight SARS-CoV-2. Although SARS-CoV-2 is different from HIV, both are RNA viruses that use RNA as their genetic material. Studies have found that azivudine can inhibit viral RNA-dependent RNA polymerase (RdRp) by inhibiting nucleoside analogs, specifically acting on SARS-CoV-2 RdRp, thereby inhibiting viral replication.

[0006] Because each person's physical constitution is different, adverse symptoms of the nervous system may also occur, such as dizziness, headache, drowsiness, mental fatigue, etc. In clinical trials, some subjects also experienced urinary system side effects such as increased urine protein and blood uric acid, so the blood concentration of azithromycin also needs to be monitored.

[0007] For special populations such as pregnant women, infants and young children, existing antiviral drugs should speed up relevant clinical trials for these special populations, accumulate more evidence-based medicine, and provide safe treatment for these special populations as soon as possible. At present, the detection of SARS-CoV-2 drug concentration mainly includes high-performance liquid chromatography and immunoassay: the advantages of these methods are high sensitivity, but the existing high-performance liquid chromatography has problems such as complex pretreatment, large minimum quantitative limit, long analysis time, and consumption of a large amount of organic solvents; there is also the problem of high cost: deuterated internal standards are used to correct matrix effects and other influences. The cost of such internal standards is high, which will directly lead to high costs for the entire method. These disadvantages make it less suitable for clinical emergency applications.

[0008] Patent CN116593601 A "Method for Simultaneous Determination of Drug Concentrations of Nematevir and Ritonavir in Human Serum" uses the internal standard method to measure only two drugs, and the total running time is 5 minutes, which is a long total running time.

[0009] Therefore, it is necessary to provide a quantitative detection method that is widely used, low-cost, rapid and simple. Summary of the invention

[0010] In order to solve at least one of the above problems, the present invention provides a method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS, which can simultaneously detect the concentrations of four SARS-CoV-2 drugs in serum / plasma. The method only takes 3 minutes to detect and has high accuracy and precision.

[0011] In order to achieve the above object, the present invention adopts the following technical means:

[0012] The first aspect of the present invention provides a method for detecting the concentration of four SARS-CoV-2 drugs based on HPLC-MS / MS, wherein the four SARS-CoV-2 drugs are ritonavir, nematevir, azithromycin and baricitinib, and the detection method comprises the following steps:

[0013] S1. Prepare the single standard stock solutions of ritonavir, namatevir, azithromycin and baricitinib, and the mixed standard stock solutions of ritonavir, azithromycin and baricitinib, respectively, and dilute them step by step with blank matrix to obtain the standard curve working solution;

[0014] S2, adding methanol solution to the sample to be tested, vortexing and extracting, taking the supernatant after centrifugation, and obtaining the sample solution to be tested;

[0015] S3. Use HPLC-MS / MS to obtain a standard curve of the target test substance using an external standard method, use HPLC-MS / MS to detect the sample solution, and use the standard curve to quantify the drug content:

[0016] Among them, the chromatographic conditions for HPLC-MS / MS detection are as follows:

[0017] Column: ACE Excel 2 C18–PFP, 2.1 mm × 100 mm;

[0018] Mobile phase A: 0.01% formic acid in water;

[0019] Mobile phase B: 0.01% formic acid in acetonitrile;

[0020] Strong wash solution: 80% methanol;

[0021] The flow rate was 0.4 mL / min and the column temperature was 40 °C;

[0022] In some embodiments, 20 μL is injected.

[0023] Gradient elution was used with a gradient elution time of 3 min;

[0024] In some embodiments of the present invention, the gradient elution method is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; the gradient elution time is 3 min and then stops, wherein:

[0025] At the beginning -1.0 min, the volume fraction of mobile phase A was 85%;

[0026] 1.0-1.5 min, the volume fraction of mobile phase A decreased from 85% to 15%;

[0027] 1.5-2.0 min The volume fraction of mobile phase A was maintained at 15%;

[0028] From 2.0 min to 2.20 min, the volume fraction of mobile phase A increased from 15% to 85%;

[0029] The volume fraction of mobile phase A was maintained at 85% from 2.20 to 3.0 min.

[0030] The mass spectrometry conditions for HPLC-MS / MS detection are as follows:

[0031] Ion source: electrospray ion source, positive ion mode;

[0032] Spray capillary voltage: 3.0 KV;

[0033] Desolvation temperature: 450°C;

[0034] Desolvation gas flow rate: 900 L / Hr;

[0035] Ion source temperature: 150°C;

[0036] Desolvation gas pressure: 8 bar;

[0037] Collision gas pressure: 0.7 bar;

[0038] Scan mode: MRM.

[0039] In some embodiments of the present invention, the quantitative ion pair information of each compound in the mass spectrometry conditions detected by HPLC-MS / MS is as follows:

[0040] Ritonavir quantitative ion pair: 721.9>296.1; cone voltage: 40; collision energy: 18;

[0041] Nematevir quantitative ion pair: 500.1>182.7; cone voltage: 30; collision energy: 32;

[0042] Azefidine quantitative ion pair: 286.9>112.6; cone voltage: 22; collision energy: 16;

[0043] Baricitinib quantitative ion pair: 372.4>105.6; cone voltage: 36; collision energy: 56.

[0044] In some embodiments of the present invention, the residence time corresponding to the above compounds in the detection is automatic.

[0045] In some embodiments of the present invention, the single-label stock solution of ritonavir is prepared using a methanol-DMSO solution with a volume ratio of 9:1; the single-label stock solution of namatevir is prepared using a methanol-DMSO solution with a volume ratio of 4:1; and the single-label stock solutions of azithromycin and baricitinib are prepared using a methanol solution.

[0046] In some specific embodiments of the present invention, the preparation of the single-label stock solution of ritonavir is as follows: 9.98 mg of ritonavir is weighed, and the volume is adjusted to 10 mL with a methanol DMSO solution with a volume ratio of 9:1. The accurate concentration is calculated according to the actual weighing result and the purity: 0.9829 mg / mL;

[0047] The preparation of the single-label stock solution of Nematevir is as follows: weigh 4.76 mg of Nematevir, and dilute to 10 mL with a methanol-DMSO solution with a volume ratio of 4:1. The accurate concentration is calculated based on the actual weighing result and purity: 0.4755 mg / mL;

[0048] The preparation of single standard stock solution of azithromycin is as follows: weigh 1 mg of azithromycin, dilute to 1 mL with methanol solution, and calculate the exact concentration based on the actual weighing result and purity: 0.9800 mg / mL;

[0049] The preparation of the single-label stock solution of baricitinib is as follows: weigh 5.64 mg of baricitinib, dilute to 10 mL with methanol solution, and calculate the exact concentration based on the actual weighing result and purity: 0.5623 mg / mL.

[0050] In some embodiments of the present invention, the volume ratio of ritonavir, azvudine and baricitinib in the mixed standard stock solution is 1:1:2.5, and the dilution solution is methanol.

[0051] In some specific embodiments of the present invention, the mixed standard stock solution is prepared as follows: 100 μL, 100 μL and 250 μL of ritonavir, azithromycin and baricitinib are respectively transferred and the volume is made up to 1 mL with methanol. The accurate concentrations of the obtained mixed standard stock solution calculated according to the actual weighing results and purity are: 39315.2 ng / mL, 39200.0 ng / mL and 56225.2 ng / mL.

[0052] In some embodiments of the present invention, in the highest concentration W1 of the standard curve working solution, the volume ratio of the mixed standard stock solution to the namatevir single standard is 2:1.

[0053] In some embodiments of the present invention, the standard curve working solution is diluted stepwise with a blank matrix and W1 to obtain W2-W10: the volume ratios of W1 and the blank matrix in W2-W6 are 4:1, 3:2, 1:1, 2:3; 1:4 respectively; the volume ratio of W1 and the blank matrix in W7 is 1:9; W8 and W9 are prepared using W7 and the blank matrix at a volume ratio of 1:1 and 1:4 respectively, and W10 is prepared using W9 and the blank matrix at a volume ratio of 1:9.

[0054] In some specific embodiments of the present invention, the highest concentration W1 of the standard curve working solution is prepared as follows: 100 μL of the mixed standard stock solution and 50 μL of the single standard of Nematevir are respectively transferred, and the volume is fixed to 5000 μL with a blank matrix; W2 is prepared using 400 μL of W1 and 100 μL of blank matrix; W3 is prepared using 300 μL of W1 and 200 μL of blank matrix; W4 is prepared using 300 μL of W1 and 300 μL of blank matrix; W 5 was prepared using 200 μL of W1 and 300 μL of blank matrix; W6 was prepared using 100 μL of W1 and 400 μL of blank matrix; W7 was prepared using 100 μL of W1 and 900 μL of blank matrix; W8 was prepared using 300 μL of W7 and 300 μL of blank matrix; W9 was prepared using 100 μL of W7 and 400 μL of blank matrix; W10 was prepared using 100 μL of W9 and 900 μL of blank matrix.

[0055] In some embodiments of the present invention, the step of using quality control products for quality control is also included. The preparation of the quality control products is as follows: the volume ratios of W1 and blank matrix in high-concentration quality control, medium-concentration quality control, and low-concentration quality control are 4:1, 3:5, and 1:9, respectively; and the volume ratio of W1 in high-concentration quality control, medium-concentration quality control, and low-concentration quality control is 8:3:1.

[0056] In some specific embodiments of the present invention, high-concentration quality control is prepared with 2000 μL of W1 and 500 μL of blank matrix, medium-concentration quality control is prepared with 750 μL of W1 and 17500 μL of blank matrix, and low-concentration quality control is prepared with 250 μL of W1 and 2250 μL of blank matrix.

[0057] In some embodiments of the present invention, the step of re-testing the high-concentration sample having a first quantitative concentration higher than the highest concentration of the standard curve by multiple dilution is also included.

[0058] In some embodiments of the present invention, the sample to be tested is a serum or plasma sample.

[0059] In some specific embodiments of the present invention, the step of storing the prepared single standard stock solution, mixed standard stock solution, standard curve working solution and quality control sample solution is further included: sealing and storing in a -80°C refrigerator.

[0060] In some embodiments of the present invention, the corresponding single standard stock solutions, mixed standard stock solutions, standard curve working solutions and quality control sample solution preparation schemes of ritonavir, namatevir, azithromycin and baricitinib in this application can be used to independently form a detection scheme for any one or a combination of any two or a combination of any three of ritonavir, namatevir, azithromycin and baricitinib, and non-target detection drugs can be removed accordingly.

[0061] A kit for detecting the concentrations of four SARS-CoV-2 drugs in serum / plasma based on the HPLC-MS / MS method, comprising a sample processing reagent; standard substances of four substances, namely ritonavir, nematevir, azithromycin and baricitinib, and a mobile phase; the sample processing reagent comprises methanol and DMSO; mobile phase A in the mobile phase comprises a 0.01% formic acid aqueous solution; and mobile phase B comprises a 0.01% formic acid acetonitrile solution.

[0062] Preparation method of the kit: Use standard substances and sample treatment reagents to prepare single standard stock solution, mixed standard stock solution, standard curve working solution and quality control sample solution. The specific operations are the same as those in the detection method.

[0063] Beneficial effects of the present invention

[0064] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for detecting the concentrations of four SARS-CoV-2 drugs in serum / plasma based on HPLC-MS / MS, which can quickly, quantitatively, and simultaneously detect the concentrations of four drugs. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) performs rapid and accurate quantitative analysis of the concentrations of new coronavirus drugs in serum / plasma. By coupling a triple quadrupole mass spectrometer, fragmented ions are detected by secondary mass spectrometry according to the mass-to-charge ratio (m / z), thereby achieving two detections with higher sensitivity and high specificity.

[0065] The detection time of this method is short, only 3 minutes, which greatly saves time, and also has the characteristics of high detection accuracy and good precision. At the same time, the invention reduces the amount of organic solvent used, further reducing pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 shows a linear relationship diagram of ritonavir of the present invention;

[0067] Figure 2 A linear relationship diagram of Namatevir of the present invention is shown;

[0068] Figure 3 A linear relationship diagram of azithromycin of the present invention is shown;

[0069] Figure 4 A linear relationship diagram of baricitinib of the present invention is shown;

[0070] Figure 5 A comparison diagram of the detection peaks of four drugs under the mobile phase of solution (1) of the present invention is shown;

[0071] Figure 6 A comparison diagram of the detection peaks of four drugs under the mobile phase of solution (2) of the present invention is shown;

[0072] Figure 7 A comparison diagram of the detection peaks of four drugs under the mobile phase of solution (3) of the present invention is shown. DETAILED DESCRIPTION

[0073] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0075] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0076] Example 1

[0077] 1. Equipment

[0078] Waters Xevo TQD triple quadrupole mass spectrometer (USA, Waters), including the Accurity UPLC system;

[0079] KQ-500E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China);

[0080] 1-14KS / 3-18KS desktop high-speed refrigerated centrifuge (SIGMA, Germany);

[0081] EVortex-Genie2 vortex mixer (Scientific Industries Company, USA);

[0082] Cascada I ultrapure water preparation system [Pall Forte Bioanalytical Instruments (Shanghai) Co., Ltd., Canada];

[0083] QUINTIX125D-1CN electronic balance [Germany, Sartorius Scientific Instruments (Beijing) Co., Ltd.];

[0084] 250 μL 96-well plates and sealing mats were purchased from Bonner Aeger;

[0085] 1.5 mL centrifuge tubes were purchased from Axygen;

[0086] Pipette (EPPENDORF, Germany).

[0087] 2. Reagents and consumables

[0088] Ritonavir standard (Ritonavir, source: Shanghai MacLean Biochemical Technology Co., Ltd., batch number: R833163-50mg, content 98.45%);

[0089] Nirmatrelvir standard (source: TLC, batch number: N-148001, content 99.9%);

[0090] Azvudine standard (Azvudine, source: GLY, batch number: ZG-62461, content 98%);

[0091] Baricitinib standard (Baricitinib, source: CER, batch number: ZC-49231, content 99.69%);

[0092] Acetonitrile (HPLC grade, Merck) Methanol (HPLC grade, Merck);

[0093] Formic acid (HPLC grade, Aladdin, China).

[0094] 3. Sample

[0095] 1. Sampling requirements

[0096] For plasma samples, use vacuum negative pressure blood collection tubes containing EDTA anticoagulant (purple) to collect plasma, and for serum samples, use vacuum negative pressure blood collection tubes with yellow caps (separation gel) to collect serum;

[0097] After collecting the blood sample with EDTAK2 anticoagulant tube, turn the anticoagulant tube upside down to mix the blood and anticoagulant thoroughly;

[0098] Plasma must be separated and packaged within 2 hours by centrifugal sedimentation (4000 rpm, 10 min);

[0099] Whether it is serum or plasma, exclude lipemia samples (the spiked recovery rate of lipemia samples is low).

[0100] Ritonavir and baricitinib can accept hemolyzed samples, while namatevir and azithromycin do not accept hemolyzed samples (the sample spike recovery rate is relatively high).

[0101] Data were collected 1 hour after baricitinib administration (peak) or before the next dose (trough).

[0102] 2. Sample quantity

[0103] Venous blood sampling is required on an empty stomach; the blood collection volume is 0.5-3.0 mL; press the puncture site with a dry cotton ball and ask the subject to press for 2-3 minutes;

[0104] 3. Storage and transportation of samples

[0105] Plasma must be separated and packaged within 2 hours by centrifugal sedimentation (4000 rpm for 10 min);

[0106] After collection, serum or plasma samples were stored at 4°C.

[0107] 4. Testing process

[0108] 1. Preparation of analytical instruments

[0109] (1) Chromatographic conditions

[0110] Column: ACE Excel 2 C18–PFP, 2.1 mm × 100 mm;

[0111] Mobile phase A: 0.01% formic acid in water; Mobile phase B: 0.01% formic acid in acetonitrile;

[0112] Strong wash solution: 80% methanol;

[0113] Flow rate 0.4 mL / min,

[0114] The column temperature was 40°C.

[0115] Inject 20 μL,

[0116] Gradient elution was used, and the elution program is shown in Table 1.

[0117] Table 1 Gradient elution program

[0118]

[0119] (2) Mass spectrometry conditions

[0120] Ion source: electrospray ionization (ESI), positive ion mode;

[0121] Spray capillary voltage (Capillary): 3.0 KV;

[0122] Desolvation Temp: 450℃;

[0123] Desolvation gas flow rate: 900 L / Hr;

[0124] Ion source temperature (Source Temp): 150°C;

[0125] Desolvation gas pressure (pressure reduction table): 8 bar;

[0126] Collision gas pressure (decompression table): 0.7 bar;

[0127] Scan mode: MRM.

[0128] The quantitative ion pair information of each compound is shown in Table 2.

[0129] Table 2 Quantitative ion pair information

[0130]

[0131] 2. Preparation of calibration solution

[0132] (1) Preparation of single standard stock solution

[0133] The preparation method of single-label stock solution is shown in Table 3 below.

[0134] The single-label stock solution was sealed and stored in a -80°C refrigerator after use.

[0135] Table 3 Preparation of single standard stock solution

[0136]

[0137] (2) Preparation of mixed standard stock solution M0

[0138] The preparation method is shown in Table 4 below. After preparation, the mixture is sealed and stored in a -80°C refrigerator for future use.

[0139] Table 4 Preparation of mixed standard stock solution M0

[0140]

[0141] 3. Sample pretreatment

[0142] (1) Preparation of serum and plasma standard curve working solution

[0143] The blank matrix was filtered human serum; W1 was diluted stepwise with the blank matrix to obtain standard curve working solutions of various concentrations. The preparation method was shown in Table 5, the working solution concentrations were shown in Table 6, and the solutions were sealed and stored in a -80°C refrigerator.

[0144] Table 5 Preparation of standard curve working solution

[0145]

[0146] Table 6 Concentrations of serum and plasma standard curve working solutions

[0147]

[0148] (2) Preparation of quality control samples

[0149] W1 was diluted with blank matrix to obtain high concentration quality control (HQC), medium concentration quality control (MQC) and low concentration quality control (LQC) samples, respectively. The preparation methods are shown in Table 7, and the concentrations of quality control samples are shown in Table 8. After preparation, 100 μL of each sample was dispensed into 1.5 mL centrifuge tubes and sealed and stored in a -80°C refrigerator.

[0150] Table 7 Preparation of quality control samples

[0151]

[0152] Table 8 Concentration of quality control samples

[0153]

[0154] Note: The actual concentration is based on the measured target value.

[0155] (3) Serum and plasma sample preparation process

[0156] Accurately pipette 100 μL of standard solution, quality control sample and blank matrix into clean and numbered 1.5 mL centrifuge tubes (use orange 96-well centrifuge tube rack).

[0157] 300 μL of methanol solution was added, and the mixture was vortexed at 2000 rpm using an MTV-100 multi-tube vortex mixer for 5 min.

[0158] Centrifuge for 5 min at 8°C and at a speed of not less than 13,000 rpm using a Sigma centrifuge (24 / 48 wells). Use a pipette to transfer 150 μL of the supernatant to a 96-well sample plate (note that the pipette tip is immersed in the solution to prevent the inhalation of precipitation), and freeze at -20°C for mass spectrometry injection.

[0159] (4) Pretreatment of high concentration samples

[0160] If the sample concentration exceeds the highest point concentration of the calibration curve, dilute it with a blank matrix when retesting, and estimate the appropriate dilution factor based on the result of the first test: 2x, 5x, 10x, etc., and measure again after dilution.

[0161] (5) For samples with concentrations lower than the lowest point of the calibration curve, the actual values ​​shall be reported.

[0162] 5. Results and Data Analysis

[0163] (1) The standard curve working solution was tested using HPLC-MS / MS, with the chromatographic peak area of ​​the target analyte as the ordinate and the concentration of the target analyte in the standard curve working solution as the abscissa. The standard curve equations corresponding to the target analyte were fitted and shown in Table 9 below.

[0164] Table 9 Standard curve equations for target analytes

[0165]

[0166] Standard curve requirements: Use 8-9 points of standard solution to make a quantitative curve, which must include the concentration points of the lowest and highest limits of quantitation. 2>0.99, requiring that the deviation of the LLOQ concentration point should be within ±20%, the deviation of the other concentration points should be within ±15%, and the CV<20%.

[0167] (2) Four different clinical samples, Sample 1, Sample 2, Sample 3 and Sample 4, sent by the laboratory were tested using HPLC-MS / MS. Based on the test results, the content of the target analyte in the test samples was calculated using the corresponding standard curve equation. The results are shown in Table 10 below.

[0168] Table 10 Test results of four samples

[0169]

[0170] The sample 5 containing four test compounds in the laboratory combination was tested by HPLC-MS / MS, and the content of the target analyte in the test sample was calculated according to the test results. The results are shown in Table 11 below. Table 11 Test results of four drugs in the combined sample

[0171]

[0172] (4) Accuracy and precision verification

[0173] Accuracy experiment: The inter-group accuracy was obtained by analyzing two samples of different concentrations for at least three days (six parallel samples were made for each concentration point), as shown in Table 12.

[0174] The qualified standard requires that the average measured concentration at each level be within 15% of the theoretical value, that is, the accuracy range is between 85%-115%.

[0175] Table 12 Accuracy data of quality control samples tested for three days

[0176]

[0177] Note: LQC is low concentration quality control; MQC is medium concentration quality control; HQC is high concentration quality control

[0178] Precision: Three different concentrations of QC were measured at one time, and the number of samples for each concentration was required to be no less than 5 (n=6). Among them, the intra-group precision was obtained from the process of measuring high and low different concentrations of QC at one time, and the number of samples for each concentration was required to be no less than 5. The inter-group accuracy was obtained by analyzing three high and low concentration QC samples for at least three days. The qualified standard requires that the CV of each level of concentration QC sample is ≤15%, and the verification results are shown in 13.

[0179] Table 13 Precision verification results

[0180]

[0181] Note: LQC is low concentration quality control; MQC is medium concentration quality control; HQC is high concentration quality control.

[0182] The results showed that the accuracy and precision of the method of the present invention met the requirements for quantitative analysis of biological samples.

[0183] Example 2 Optimization of chromatographic conditions for detection

[0184] During the testing of the four drugs in this application, it was found that the peak shape of azithromycin was not good, and the chromatographic conditions were optimized.

[0185] The other conditions of the chromatography were the same as those in Example 1, except that the mobile phase was different.

[0186] Scheme (1) Mobile phase A: 0.01% formic acid in water; Mobile phase B: 0.1% formic acid in (acetonitrile: methanol = 1:1) solution.

[0187] Scheme (2) Mobile phase A: 0.01% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile.

[0188] Scheme (3) Mobile phase A: 0.01% formic acid in water; mobile phase B: 0.01% formic acid in acetonitrile.

[0189] The detection results of the four drugs obtained by the above scheme (1) are as follows Figure 5 As shown; the detection results of the four drugs obtained by scheme (2) are as follows Figure 6 As shown; the detection results of the four drugs obtained by scheme (3) are as follows Figure 7 shown.

[0190] The results showed that when tested under the conditions of Scheme (1) and Scheme (2), the peak shape of azvudine was poor, but when tested under the conditions of Scheme (3), the peak shape of azvudine was greatly improved.

[0191] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A method for detecting the concentration of four SARS-CoV-2 drugs based on HPLC-MS / MS, characterized in that the four SARS-CoV-2 drugs are ritonavir, namatevir, azithromycin and baricitinib, and the detection method comprises the following steps: S1. Prepare the single standard stock solutions of ritonavir, namatevir, azithromycin and baricitinib, and the mixed standard stock solutions of ritonavir, azithromycin and baricitinib, respectively, and dilute them step by step with blank matrix to obtain the standard curve working solution; S2, adding methanol solution to the sample to be tested, vortexing and extracting, taking the supernatant after centrifugation, and obtaining the sample solution to be tested; S3. Use HPLC-MS / MS to obtain a standard curve of the target test substance using an external standard method, use HPLC-MS / MS to detect the sample solution, and use the standard curve to quantify the drug content: Among them, the chromatographic conditions for HPLC-MS / MS detection are as follows: Column: ACE Excel 2 C18–PFP, 2.1 mm × 100 mm; Mobile phase A: 0.01% formic acid in water; Mobile phase B: 0.01% formic acid in acetonitrile; Strong wash solution: 80% methanol; The flow rate was 0.4 mL / min and the column temperature was 40 °C; Gradient elution was used with a gradient elution time of 3 min; The mass spectrometry conditions for HPLC-MS / MS detection are as follows: Ion source: electrospray ion source, positive ion mode; Spray capillary voltage: 3.0 KV; Desolvation temperature: 450°C; Desolvation gas flow rate: 900 L / Hr; Ion source temperature: 150°C; Desolvation gas pressure: 8 bar; Collision gas pressure: 0.7 bar; Scan mode: MRM; The gradient elution method is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; the gradient elution time is 3 minutes and then stops, wherein: At the beginning -1.0 min, the volume fraction of mobile phase A was 85%; 1.0-1.5 min, the volume fraction of mobile phase A decreased from 85% to 15%; 1.5-2.0 min The volume fraction of mobile phase A was maintained at 15%; From 2.0 min to 2.20 min, the volume fraction of mobile phase A increased from 15% to 85%; The volume fraction of mobile phase A was maintained at 85% from 2.20 to 3.0 min.

2. The method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS according to claim 1, characterized in that: The quantitative ion pair information of each compound in the mass spectrometry conditions of the HPLC-MS / MS detection is as follows: Ritonavir quantitative ion pair: 721.9>296.1; cone voltage: 40; collision energy: 18; Nematevir quantitative ion pair: 500.1>182.7; cone voltage: 30; collision energy: 32; Azefidine quantitative ion pair: 286.9>112.6; cone voltage: 22; collision energy: 16; Baricitinib quantitative ion pair: 372.4>105.6; cone voltage: 36; collision energy:

56.

3. The method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS according to claim 1, characterized in that: The single-label stock solution of ritonavir is prepared using a methanol-DMSO solution with a volume ratio of 9:1; the single-label stock solution of namatevir is prepared using a methanol-DMSO solution with a volume ratio of 4:1; the single-label stock solutions of azithromycin and baricitinib are prepared using a methanol solution.

4. The method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS according to claim 1, characterized in that: The volume ratio of ritonavir, azithromycin and baricitinib in the mixed standard stock solution is 1:1:2.5, and the dilution solution is methanol.

5. The method for detecting the concentration of four SARS-CoV-2 drugs based on HPLC-MS / MS according to claim 1, characterized in that: In the highest concentration W1 of the standard curve working solution, the volume ratio of the mixed standard stock solution to the single standard of Namatevir is 2:

1.

6. The method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS according to claim 5, characterized in that: The standard curve working solution is diluted stepwise with blank matrix and W1 to obtain W2-W10:W2-W6, in which the volume ratios of W1 and blank matrix are 4:1, 3:2, 1:1, and 2:3 respectively; 1:4; the volume ratio of W1 to blank matrix in W7 is 1:9; W8 and W9 are prepared with W7 and blank matrix at a volume ratio of 1:1 and 1:4 respectively, and W10 is prepared with W9 and blank matrix at a volume ratio of 1:

9.

7. The method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS according to claim 5, characterized in that: It also includes the step of using quality control products for quality control. The preparation of the quality control products is as follows: the volume ratios of W1 and blank matrix in high-concentration quality control, medium-concentration quality control, and low-concentration quality control are 4:1, 3:5, and 1:9, respectively; and the volume ratio of W1 in high-concentration quality control, medium-concentration quality control, and low-concentration quality control is 8:3:

1.

8. The method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS according to claim 1, characterized in that: The method also includes the step of re-testing the high-concentration sample whose first quantitative concentration is higher than the highest concentration of the standard curve by multiple dilution.

9. A method for detecting the concentrations of four SARS-CoV-2 drugs based on HPLC-MS / MS according to any one of claims 1 to 8, characterized in that: The sample to be tested is a serum or plasma sample.

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