Method for quantitative analysis of acid-suppressing drugs by liquid chromatography-tandem mass spectrometry

Abstract

The present application provides a method for quantitatively analyzing acid-suppressing drugs by a high-performance liquid chromatography-tandem mass spectrometry device, comprising: preparing a sample solution containing the acid-suppressing drug; injecting the sample solution into the high-performance liquid chromatography-tandem mass spectrometry device to obtain a mass chromatogram; and quantitatively analyzing the acid-suppressing drug using an internal standard method according to the mass chromatogram, wherein the acid-suppressing drug is selected from: esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and vonoprazan fumarate; using a mixed mobile phase consisting of mobile phase A and mobile phase B for gradient elution in the high-performance liquid chromatography, wherein mobile phase A is an acetonitrile solution containing formic acid or a water-acetonitrile mixture containing ammonia and ammonium acetate (water: acetonitrile volume ratio: 1:99-10:90); and mobile phase B is selected from an aqueous solution containing ammonium acetate, an aqueous solution containing formic acid and ammonium acetate, an aqueous solution containing ammonia and ammonium acetate, and an aqueous solution containing ammonia and ammonium formate.

Description

Technical Field

[0001] The present invention belongs to the fields of biochemistry and pharmaceutical analytical chemistry, and in particular relates to a method for quantitatively analyzing acid-suppressing drugs by using a liquid chromatography-tandem mass spectrometry device. Background Art

[0002] Liquid chromatography-mass spectrometry is primarily used in drug metabolism, pharmacokinetics, clinical pharmacology, and natural drug development. It offers advantages such as high sensitivity, high specificity, excellent reproducibility, accurate quantification, a wide linear range, and simple data processing. High-performance liquid chromatography effectively separates the components of an analyte, while mass spectrometry allows for individual analysis of these components. Liquid chromatography-mass spectrometry exploits the differences in distribution and adsorption coefficients between the mobile and stationary phases of a chromatographic column. The mobile phase introduces the sample into the chromatographic column for separation. After passing through an interface device, the different ion fragments behave differently in different electric and / or magnetic fields. The mass analyzer separates the ions according to their mass-to-charge ratio (m / z), resulting in a mass spectrum arranged in order of mass. By analyzing and processing the mass spectrum, qualitative and quantitative analysis of the sample can be obtained.

[0003] Acid-related diseases (ARDs) are a general term for common conditions worldwide, including gastroesophageal reflux disease (GERD), peptic ulcers (PUs), and medication-related ARDs. These conditions are often caused by excessive gastric acid secretion, making inhibiting gastric acid secretion key to treating acid-related diseases. Peptic ulcer bleeding (PUB) is the most common complication of PUs, and peptic ulcer diagnosis and treatment guidelines recognize proton pump inhibitors (PPIs) as effective in preventing and treating upper gastrointestinal bleeding.

[0004] Acid-suppressing drugs include proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs).

[0005] Currently, proton pump inhibitors (PPIs) approved for gastrointestinal bleeding in China include omeprazole, lansoprazole, pantoprazole, rabeprazole, esomeprazole, and ilaprazole. Short-term use of acid-suppressing drugs, such as PPIs, for peptic ulcer bleeding is generally well tolerated and effective in most patients. However, PPIs also have limitations in clinical application. Most PPIs are prodrugs with a half-life of 1–2 hours, resulting in a short duration of action and a high risk of nocturnal acid breakthrough due to insufficient gastric acid suppression. Furthermore, PPIs are primarily metabolized by CYP2C19, an enzyme whose activity exhibits significant genetic polymorphism in the human population, resulting in significant inter-individual variability in the acid-suppressing effects of PPIs. Therefore, there remains an unmet clinical need for pharmacological treatment of gastrointestinal bleeding.

[0006] Potassium-competitive acid blockers (P-CABs) are a new class of acid-suppressing drugs that can ＋ / K ＋ -ATPase K ＋ The binding site is reversible and it is not a prodrug, so it does not require acid activation. It has inhibitory effects on both resting and active proton pumps and has a stronger acid-suppressing effect clinically.

[0007] Developed by Takeda Pharmaceutical, vonoprazan fumarate tablets became the first P-CAB approved for the Chinese market in December 2019. Currently, new acid-suppressing P-CABs are only available in oral formulations, which cannot meet the clinical needs of patients with peptic ulcer bleeding who are fasting or have swallowing difficulties and require rapid onset of action. Shandong New Era Pharmaceutical Co., Ltd.'s vonoprazan fumarate injection is a modified version of vonoprazan fumarate tablets.

[0008] Therefore, there is an urgent need in this field to develop a rapid, highly sensitive and highly stable quantitative analysis method to quantitatively analyze the above-mentioned acid-suppressing drugs to meet the needs of clinical pharmacokinetic research. Summary of the Invention

[0009] To achieve the above-mentioned object of the invention, the present invention provides a method for quantitatively analyzing acid-suppressing drugs by liquid chromatography-tandem mass spectrometry, the method comprising the following steps:

[0010] 1) Prepare a sample solution containing an acid-suppressing drug;

[0011] 2) injecting the sample solution into a high performance liquid chromatography-tandem mass spectrometry device to obtain a mass chromatogram; and

[0012] 3) performing quantitative analysis of the acid-suppressing drug using an internal standard method according to the mass chromatogram,

[0013] Wherein, the acid-suppressing drug is at least one selected from the group consisting of: esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and vonoprazan fumarate;

[0014] The high performance liquid chromatography uses a mixed mobile phase consisting of mobile phase A and mobile phase B for gradient elution.

[0015] wherein the mobile phase A is: an acetonitrile solution containing 0.1% to 0.5% by mass of formic acid, or a water-acetonitrile mixture containing 0.1% to 0.5% by mass of ammonia and 5 mmol / L to 20 mmol / L of ammonium acetate, wherein the volume ratio of water to acetonitrile in the water-acetonitrile mixture is in the range of 1:99 to 10:90; and

[0016] The mobile phase B is one selected from the following: an aqueous solution containing 5 mmol / L to 20 mmol / L ammonium acetate, an aqueous solution containing 0.1 mass % to 0.5 mass % formic acid and 5 mmol / L to 20 mmol / L ammonium acetate, an aqueous solution containing 0.1 mass % to 0.5 mass % ammonia and 5 mmol / L to 20 mmol / L ammonium acetate, and an aqueous solution containing 0.1 mass % to 0.3 mass % ammonia and 5 mmol / L to 20 mmol / L ammonium formate.

[0017] Preferably, the mobile phase A is an acetonitrile solution containing 0.2% by mass of formic acid. Preferably, the mobile phase B is an aqueous solution containing 0.1% by mass of ammonia and 10 mmol / L ammonium formate.

[0018] In some embodiments, the gradient elution program is as follows:

[0019] 0.00 to 0.50 min, 80 vol% to 80 vol% mobile phase B;

[0020] 0.50 to 1.50 minutes, 80% to 70% mobile phase B;

[0021] 1.50 to 2.30 minutes, 70% to 70% mobile phase B by volume;

[0022] 2.30 to 2.50 minutes, 70% to 5% mobile phase B;

[0023] 2.50 to 3.50 minutes, 5% to 5% mobile phase B by volume;

[0024] 3.50 to 3.60 minutes, 5% to 80% mobile phase B;

[0025] 3.60 to 4.50 minutes, 80 vol% to 80 vol% mobile phase B;

[0026] The total amount of mobile phase A and mobile phase B is 100% by volume.

[0027] In some embodiments, the internal standards are D3-vonoprazan fumarate and D3-omeprazole.

[0028] In some embodiments, the chromatographic column in the high performance liquid chromatography is a reverse phase chromatographic column, preferably a chromatographic column packed with octadecylsilane bonded silica gel. More preferably, the chromatographic column is an ACQUITY UPLC® BEH C18 (50×2.1 mm, 1.7 µm).

[0029] In some embodiments, the injection volume of the sample solution is 5 to 20 μL, preferably 10 μL.

[0030] In some embodiments, the flow rate of the mixed mobile phase is in the range of 0.2 to 0.5 mL / min, preferably in the range of 0.3 to 0.5 mL / min, and more preferably 0.4 mL / min.

[0031] In some embodiments, the column temperature in the HPLC is in the range of 35°C to 45°C, preferably 40°C.

[0032] In some embodiments, the acid-suppressing drug is from the subject's plasma.

[0033] In some embodiments, the preparation of the sample solution containing the acid-suppressing drug comprises: adding an internal standard working solution to the subject's plasma sample, shaking the resulting mixture, then centrifuging it, taking the supernatant and adding a reconstitution solution consisting of aqueous formic acid and acetonitrile thereto, and mixing.

[0034] In some embodiments, the internal standard working solution is prepared by dissolving the internal standards D3-vonoprazan fumarate and D3-omeprazole in dimethyl sulfoxide to obtain an internal standard stock solution, and then diluting the internal standard stock solution with acetonitrile.

[0035] In some embodiments, the reconstitution solution consists of 0.1 mass % formic acid aqueous solution and acetonitrile, and the volume ratio of acetonitrile to formic acid aqueous solution in the reconstitution solution is in the range of 5:95 to 20:80, more preferably 10:90.

[0036] In some preferred embodiments, the volume ratio of the plasma sample to the internal standard working solution is in the range of 1:3 to 1:5, preferably 1:4. In some preferred embodiments, the volume ratio of the supernatant to the reconstitution solution is in the range of 1:3 to 1:5, preferably 1:4.

[0037] In some embodiments, the mass spectrometry conditions include: using a triple quadrupole mass spectrometer in a positive ion mode with an electrospray ionization source and performing multiple reaction monitoring scanning detection.

[0038] Preferably, in the tandem mass spectrometer, the capillary voltage is 3.5 kV and the cone voltage is in the range of 30 to 60 V.

[0039] Preferably, in the tandem mass spectrometer, the cone voltage of esomeprazole is 50 V, the cone voltage of rabeprazole is 40 V, the cone voltage of ilaprazole is 30 V, the cone voltage of lansoprazole is 30 V, the cone voltage of pantoprazole is 30 V, the cone voltage of vonoprazan fumarate is 30 V, the cone voltage of D3-omeprazole is 60 V, and the cone voltage of D3-vonoprazan fumarate is 30 V. Preferably, the collision energy of esomeprazole is 12 eV, the collision energy of rabeprazole is 10 eV, the collision energy of ilaprazole is 10 eV, the collision energy of lansoprazole is 12 eV, the collision energy of pantoprazole is 20 eV, the collision energy of vonoprazan fumarate is 15 eV, the collision energy of D3-omeprazole is 15 eV, and the collision energy of D3-vonoprazan fumarate is 15 eV.

[0040] Preferably, in the tandem mass spectrometry, the quantitative ion pair of esomeprazole is m / z 346.3→198.1, the quantitative ion pair of rabeprazole is m / z 360.2→242.1, the quantitative ion pair of ilaprazole is m / z 367.2→184.0, the quantitative ion pair of lansoprazole is m / z 370.2→252.0, the quantitative ion pair of pantoprazole is m / z 384.2→200.2, the quantitative ion pair of vonoprazan fumarate is m / z 346.1→315.1, the quantitative ion pair of D3-omeprazole is m / z 349.2→198.1, and the quantitative ion pair of D3-vonoprazan fumarate is m / z 349.1→315.1.

[0041] In some embodiments, the detection method further comprises preparing one or more of an acid-suppressing drug stock solution, a standard curve working solution, a standard curve plasma sample solution, an internal standard stock solution, an internal standard working solution, a quality control working solution, and a quality control plasma sample solution.

[0042] In some embodiments, the method for preparing the acid-suppressing drug stock solution comprises: weighing an acid-suppressing drug standard and dissolving it in dimethyl sulfoxide. In some preferred embodiments, the concentration of the acid-suppressing drug stock solution is 0.5 mg / mL.

[0043] In some embodiments, the method for preparing a standard curve working solution comprises: taking an appropriate amount of the acid-suppressing drug stock solution and further diluting it with a 1M NaOH aqueous solution to prepare standard curve working solutions of varying concentrations. In some preferred embodiments, for vonoprazan fumarate, the concentrations of the standard curve working solution are 50, 125, 500, 2500, 5000, 12500, 25000, and 50000 ng / mL, and for any of esomeprazole, rabeprazole, ilaprazole, lansoprazole, and pantoprazole, the concentrations of the standard curve working solution are 20, 50, 200, 1000, 2000, 5000, 10000, and 20000 ng / mL.

[0044] In some embodiments, the method for preparing a standard curve plasma sample solution comprises: taking standard curve working solutions of different concentrations, diluting them with blank plasma, and ultimately obtaining a series of standard curve plasma sample solutions. In some preferred embodiments, for vonoprazan fumarate, the concentrations of the standard curve plasma sample solutions are 0.5, 1.25, 5, 25, 50, 125, 250, and 500 ng / mL, and for any one of esomeprazole, rabeprazole, ilaprazole, lansoprazole, and pantoprazole, the concentrations of the standard curve plasma sample solutions are 0.2, 0.5, 2, 10, 20, 50, 100, and 200 ng / mL.

[0045] In some embodiments, a method for preparing a quality control working solution comprises: taking an appropriate amount of a stock solution of an acid-suppressing drug and further diluting it with a 1M NaOH aqueous solution to prepare quality control working solutions of varying concentrations. In some preferred embodiments, for vonoprazan fumarate, the concentrations of the quality control working solution are 50, 100, 4,000, and 40,000 ng / mL, and for any of esomeprazole, rabeprazole, ilaprazole, lansoprazole, and pantoprazole, the concentrations of the quality control working solution are 20, 40, 1,600, and 16,000 ng / mL.

[0046] In some embodiments, the method for preparing a quality control plasma sample solution comprises: taking a certain amount of standard curve working solution of different concentrations, diluting it with blank plasma, and preparing the quality control sample solution. In some preferred embodiments, for vonoprazan fumarate, the concentration of the quality control plasma sample solution is 0.5, 1, 40, 400, and 4000 ng / mL, and for any of esomeprazole, rabeprazole, ilaprazole, lansoprazole, and pantoprazole, the concentration of the quality control plasma sample solution is 0.2, 0.4, 16, 160, and 1600 ng / mL.

[0047] In some embodiments, the preparation method of the internal standard stock solution comprises: weighing the internal standard standard and dissolving it in dimethyl sulfoxide. In some preferred embodiments, the concentration of the internal standard stock solution is 0.5 mg / mL.

[0048] In some embodiments, the internal standard working solution is prepared by diluting an appropriate amount of the internal standard stock solution with acetonitrile. In some preferred embodiments, the internal standard working solution contains 20 ng / mL of D3-omeprazole and 50 ng / mL of D3-vonoprazan fumarate.

[0049] Beneficial technical effects of the present invention

[0050] (1) This paper establishes for the first time a rapid, stable and sensitive UPLC-MS / MS method for the determination of acid-suppressing drugs in human plasma. This method is also applicable to the quantitative analysis of six acid-suppressing drugs: esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and vonoprazan fumarate.

[0051] (2) The chemical structures of the six compounds, esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate, all contain nitrogen atoms. The present invention employs the positive ionization mode of an electrospray ionization (ESI) source, which provides improved ionization performance and a stable response. Furthermore, the present invention optimizes source / gas and compound parameters (including capillary voltage and cone voltage) to obtain the most suitable ionization conditions.

[0052] (3) Since benzene rings are present in the chemical structures of esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate, reversed-phase chromatography is preferred. The present inventors optimized various chromatographic conditions (including chromatographic column, mobile phase composition, gradient elution procedure, etc.) to obtain good chromatographic peak shape, improve the sensitivity of the method, and significantly shorten the analysis time, thereby improving the analysis efficiency.

[0053] (3) The present inventors found that under the detection conditions described in the present invention, the matrix effect of plasma samples was negligible when the protein precipitation method was used to treat the plasma samples, and clear chromatographic peaks were obtained with good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1A-1F The esomeprazole ( Figure 1A ), Rabeprazole ( Figure 1B ), Ilaprazole ( Figure 1C )、Lansoprazole( Figure 1D )、Pantoprazole( Figure 1E ) and vonoprazan fumarate ( Figure 1FRepresentative mass chromatograms of double blank (DB) plasma samples.

[0055] Figure 2A-2F The esomeprazole ( Figure 2A ), Rabeprazole ( Figure 2B ), Ilaprazole ( Figure 2C )、Lansoprazole( Figure 2D )、Pantoprazole( Figure 2E ) and vonoprazan fumarate ( Figure 2F Representative mass chromatograms of plasma samples with lower limit of quantification (LLOQ).

[0056] Figure 3A-3F The esomeprazole ( Figure 3A ), Rabeprazole ( Figure 3B ), Ilaprazole ( Figure 3C )、Lansoprazole( Figure 3D )、Pantoprazole( Figure 3E ) and vonoprazan fumarate ( Figure 3F ) is a typical standard curve.

[0057] Note: Figure 1A-1F and Figure 2A-2F The vertical axis of each mass chromatogram in the figure represents the relative intensity of the instrument signal. DETAILED DESCRIPTION

[0058] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0059] The following describes specific embodiments of the technical solutions of the present invention, but those skilled in the art will understand that the following specific embodiments are only some examples of various solutions for implementing the present invention, and they should not be understood as limiting the scope of the present invention in any way.

[0060] To provide a more concise description, the term "about" is not used in the quantitative data herein. It should be understood that, regardless of whether the term "about" is explicitly used, each numerical value given herein includes not only the actual value given (a given value), but also is meant to include approximate values ​​of such given value based on reasonable inferences by a person of ordinary skill in the art, including equivalent values ​​and approximate values ​​of such given value due to experimental and / or measurement conditions. The approximate value is preferably ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, 2%, or ±1% based on the given value.

[0061] In some embodiments, the numerical ranges and numerical parameters setting forth the broad scope of some embodiments of the present invention are approximate and should be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques. Although the numerical values ​​set forth in the specific examples are reported as precisely as possible, the numerical values ​​presented in some embodiments of the present invention may contain certain errors that are inevitably caused by the standard deviation in testing measurements.

[0062] 1. Chemicals and Reagents

[0063] Esomeprazole (purity 99.68%), rabeprazole (purity 97.25%), ilaprazole (purity 99.65%), lansoprazole (purity 99.1%), pantoprazole (purity 99.97%), and D3-omeprazole (deuterated internal standard, purity 99.5%) were purchased from Beijing Putian Tongchuang Biotechnology Co., Ltd.; vonoprazan fumarate (purity 99.6%) was purchased from TLC Pharmaceutical Standards; and D3-vonoprazan fumarate (deuterated internal standard, purity 97.89%) was purchased from Shenzhen Zhenqiang Biotechnology Co., Ltd.

[0064] HPLC-grade acetonitrile was purchased from Honeywell Burdick & Jackson (ML, USA); analytical-grade formic acid was purchased from Sigma-Aldrich Chemicals (MO, USA); analytical-grade dimethyl sulfoxide was purchased from Beijing Chemical Reagent Company (Beijing, China); analytical-grade ammonium formate and analytical-grade ammonia were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China); and blank plasma was provided by Peking Union Medical College Hospital (Beijing, China). Deionized water was purified using a Milli-Q system (Millipore, Bedford, MA, USA).

[0065] 2. Chromatographic and mass spectrometry conditions

[0066] 2.1 Chromatographic conditions

[0067] Chromatographic columns

[0068] Different types of chromatographic columns were selected, and method parameters such as mobile phase type, gradient conditions, and flow rate were adjusted according to the detection results. The detection performance of the test solution in different types of chromatographic columns was investigated. Specific information on the chromatographic columns is shown in Table 1 below:

[0069] Table 1: Column Information

[0070]

[0071] The results showed that using an ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column resulted in better peak shape, higher response, and improved resolution.

[0072] Mobile phase composition

[0073] Mobile phase A (organic phase)

[0074] An aqueous solution of 10 mmol / L ammonium formate containing 0.1% by mass of ammonia was selected as mobile phase B (aqueous phase), while acetonitrile, methanol, and an acetonitrile-methanol mixture (volume ratio 1:1) were selected as mobile phase A (organic phase) for detection, and chromatograms were recorded.

[0075] Results showed that methanol as mobile phase A (organic phase) exhibited slightly poor retention, while acetonitrile exhibited superior elution and very low background noise. Therefore, an acetonitrile system was selected as mobile phase A. Acetonitrile systems supplemented with 0.1% to 0.5% formic acid and 10 mmol / L ammonium acetate were also tested. The results showed that the addition of formic acid further improved resolution. Therefore, an acetonitrile solution containing 0.2% formic acid was preferred as mobile phase A.

[0076] Mobile phase B (aqueous phase)

[0077] An acetonitrile solution containing 0.2 mass% formic acid was selected as mobile phase A (organic phase), and an aqueous solution containing 0.1 mass%-0.5 mass% formic acid, an aqueous solution containing 10 mmol / L ammonium acetate, an aqueous solution containing 0.1 mass%-0.5 mass% formic acid, an aqueous solution containing 10 mmol / L ammonium acetate, an aqueous solution containing 0.1 mass%-0.5 mass% ammonia and 10 mmol / L ammonium acetate, and an aqueous solution containing 5 mmol / L-20 mmol / L ammonium formate and 0.1 mass%-0.3 mass% ammonia were selected as mobile phase B (aqueous phase) for detection, and chromatograms were recorded.

[0078] The results showed that using an aqueous formic acid solution as mobile phase B resulted in poor peak separation, while using an aqueous ammonium acetate solution resulted in significant carryover. However, using an ammonium formate solution resulted in better peak separation. Furthermore, it was found that adding 0.1% ammonia to mobile phase B further enhanced the carryover removal effect. Therefore, an aqueous solution containing 0.1% ammonia and 10 mmol / L ammonium formate was the preferred mobile phase B.

[0079] flow rate

[0080] To investigate the effect of flow rate on the detection effect, flow rates of 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, and 0.5 mL / min were selected for detection, with other experimental conditions remaining the same.

[0081] The results showed that the peak elution time at flow rates of 0.2 mL / min and 0.3 mL / min was later, and the chromatographic column retention effect was stronger; the peak elution time at a flow rate of 0.4 mL / min was moderate, and the chromatographic peak separation effect was better. Therefore, the flow rate of 0.4 mL / min was preferred.

[0082] Therefore, the following chromatographic conditions were finally determined:

[0083] Chromatographic separation was performed using an ACQUITY UPLC system (Waters, MA, USA).

[0084] Chromatographic column: ACQUITY UPLC BEH® C18 column (2.1×50 mm, 1.7 µm).

[0085] Mobile phase A: acetonitrile solution containing 0.2% by mass of formic acid;

[0086] Mobile phase B: aqueous solution containing 0.1% ammonia and 10 mmol / L ammonium formate

[0087] Gradient elution program:

[0088]

[0089] Column temperature: 40°C

[0090] Autosampler temperature: 10°C

[0091] Injection volume: 10 μL

[0092] Flow rate: 0.4 mL / min

[0093] To reduce carryover, the autosampler syringe and injection valve were cleaned with a 50% acetonitrile solution containing 1% formic acid (strong wash) or a 50% acetonitrile solution containing 0.5% formic acid (weak wash).

[0094] 2.2 Mass spectrometry conditions

[0095] Mass spectrometry analysis was performed using a Xevo TQS triple quadrupole mass spectrometer (Waters, MA, USA) in the positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) scanning detection.

[0096] Adjust the desolvation gas flow rate, ion source temperature, capillary voltage, collision gas flow rate, cone voltage, collision energy and other parameters to determine the following conditions:

[0097] The quantitative ion pair of the compound esomeprazole is: m / z 346.3→198.1,

[0098] Quantitative ion pair of rabeprazole: m / z 360.2→242.1,

[0099] Quantitative ion pair for ilaprazole: m / z 367.2→184.0,

[0100] Quantitative ion pair for lansoprazole: m / z 370.2→252.0,

[0101] Quantitative ion pair for pantoprazole: m / z 384.2→200.2,

[0102] Quantitative ion transition of vonoprazan fumarate: m / z 346.1→315.1;

[0103] The quantitative ion of D3-vonoprazan fumarate is: m / z 349.1→315.1,

[0104] The quantitative ion of D3-omeprazole is: m / z 349.2→198.1.

[0105] The desolvation gas flow rate was 1000 L / Hr; the ion source temperature was 500°C; the capillary voltage was 3.5 kV; and the collision gas flow rate was 0.25 mL·min -1 .

[0106] Specific optimization parameters are shown in Table 2 below:

[0107] Table 2: Tandem mass spectrometry parameters

[0108]

[0109] 3. Preparation of Stock Solutions, Calibration Standards, and Quality Control Samples

[0110] (1) Preparation of acid-suppressing drug stock solution (0.5 mg / mL): Accurately weigh the standard samples of acid-suppressing drugs esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate, transfer them to a volumetric flask, and dissolve them in dimethyl sulfoxide to the same volume. The stock solution is used to prepare the standard curve working solution and quality control (QC) working solution.

[0111] (2) Standard curve working solution: Take an appropriate amount of acid-suppressing drug stock solution (0.5 mg / mL) and dilute it with diluent (which is prepared by 1 M NaOH aqueous solution and water in a volume ratio of 1:60) to prepare standard curve working solutions of different concentrations (the concentrations are 50 / 20, 125 / 50, 500 / 200, 2500 / 1000, 5000 / 2000, 12500 / 5000, 25000 / 10000, 50000 / 20000 ng / mL, respectively, where in the above concentrations A / B, A represents the concentration of vonoprazan fumarate, and B represents the concentration of esomeprazole, rabeprazole, ilaprazole, lansoprazole or pantoprazole).

[0112] (3) Standard curve plasma sample solution: Take a certain amount of standard curve working solution of different concentrations and dilute it with blank plasma to finally obtain a series of standard curve plasma sample solutions (standard curve samples with concentrations of 0.5 / 0.2, 1.25 / 0.5, 5 / 2, 25 / 10, 50 / 20, 125 / 50, 250 / 100 and 500 / 200 ng / mL, respectively. In the above concentrations A / B, A represents the concentration of vonoprazan fumarate, and B represents the concentration of esomeprazole, rabeprazole, ilaprazole, lansoprazole or pantoprazole).

[0113] (4) Quality control (QCs) working solution: Take an appropriate amount of acid-suppressing drug stock solution (0.5 mg / mL) and further dilute it with diluent (which is prepared by 1 M NaOH aqueous solution and water in a volume ratio of 1:60) to prepare quality control (QCs) working solutions of different concentrations (concentrations are 50 / 20, 100 / 40, 4000 / 1600, and 40000 / 16000 ng / mL, respectively).

[0114] (5) Quality control plasma sample solution: Take a certain amount of standard curve working solution with different concentrations and dilute it with blank plasma to prepare quality control plasma sample solution (the concentrations are 0.5 / 0.2, 1 / 0.4, 40 / 16, 400 / 160, 4000 / 1600 ng / mL, respectively, recorded as lower limit of quantification (LLOQ), low concentration quality control (LQC), medium concentration quality control (MQC), high concentration quality control (HQC) and dilution quality control (DQC)).

[0115] (6) Internal standard stock solution (0.5 mg / mL): Accurately weigh the internal standard substances, i.e., D3-omeprazole and D3-vonoprazan fumarate, transfer them into a volumetric flask, dissolve them in dimethyl sulfoxide and make up to volume.

[0116] (7) Internal standard working solution: Take an appropriate amount of internal standard stock solution and dilute it with acetonitrile solution to obtain an internal standard working solution containing 20 ng / mL of D3-omeprazole and 50 ng / mL of D3-vonoprazan fumarate.

[0117] 4. Plasma Sample Pretreatment Method

[0118] When preparing plasma samples, the inventors tested liquid-liquid extraction, solid-phase extraction, and protein precipitation methods for pretreatment. Liquid-liquid extraction led to chemical contamination and low sensitivity. Solid-phase extraction, due to its complex operation, consumes significant time and costs. However, under the detection conditions described in the present invention, protein precipitation demonstrated negligible matrix effects, resulting in clear chromatographic peaks with excellent reproducibility.

[0119] Methanol and acetonitrile were used as protein precipitation reagents to prepare plasma samples, and the chromatographic and mass spectrometric conditions were used as described above. The results showed that acetonitrile was used as the precipitant, resulting in good chromatographic peaks, so acetonitrile was selected as the precipitant.

[0120] The specific method for processing the plasma sample in the present invention is as follows:

[0121] A 50 µL plasma sample was placed in an EP tube. Protein was precipitated by adding 200 µL of internal standard working solution (D3-omeprazole 20 ng / mL, D3-vonoprazan fumarate 50 ng / mL, in acetonitrile). The sample was shaken for 1 minute and then centrifuged at 13,300 rpm for 10 minutes. A 50 µL supernatant was placed in a new EP tube. 200 µL of reconstitution solution (composed of acetonitrile and 0.1% formic acid in water, in a 1:9 volume ratio) was added. Mix thoroughly before injection.

[0122] Example 1: Selectivity

[0123] 1. Methods

[0124] Six human plasma blank matrices from different individuals, one hyperlipidemic plasma blank matrix, and one 2% hemolyzed plasma blank matrix were prepared. For each acid-suppressing drug, one double blank (DB) plasma sample and one lower limit of quantitation (LLOQ) plasma sample were prepared using each blank matrix source, for a total of 16 samples. The results of the DB plasma sample were compared with the LLOQ plasma sample to evaluate the selectivity of the method.

[0125] Acceptance criteria: If there is a certain interference at the corresponding retention time of the analyte in the DB plasma sample, this interference should not be greater than 20% of the average peak area of ​​the analyte in the LLOQ plasma sample; when the internal standard method is used for quantification, the chromatographic peak area at the corresponding retention time of the internal standard should not be greater than 5% of the average peak area of ​​the internal standard in the standard curve and quality control samples.

[0126] 2. Results

[0127] According to the LC-MS / MS conditions selected in the present invention, mass chromatograms of DB plasma samples and LLOQ plasma samples from 8 different sources were measured. Figure 1A-1F Representative profiles of DB plasma samples for esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate are shown, respectively. Figure 2A-2F Representative profiles of LLOQ plasma samples are shown for esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate, respectively.

[0128] Table 3 below summarizes the peak areas of representative DB plasma samples and LLOQ plasma samples of esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate at the retention time (RT) of each compound.

[0129] Table 3: Selectivity

[0130]

[0131] Depend on Figure 1A-1F and Figure 2A-2F As shown in Table 3, under the experimental conditions of the present invention, the plasma matrix had no interference with the separation and determination of the analyte and the internal standard.

[0132] Therefore, the method of the present invention meets the above acceptance criteria.

[0133] Example 2: Linear Relationship

[0134] 1. Methods

[0135] Two sets of standard curve samples containing 8 concentration points were processed and measured. The measurements of these two sets of standard curve samples were placed at the beginning and end of each analytical batch. The peak area ratio of esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and internal standard D3-omeprazole was set as Y and the concentration was set as X for fitting. The peak area ratio of vonoprazan fumarate to D3-vonoprazan fumarate was set as Y and the concentration was set as X for fitting. The weight coefficient was 1 / X. 2 The standard curve regression equation is Y = aX + b. Each analytical batch includes one double blank sample (DB) and one blank sample (BK) to ensure that the blank matrix and the added reagents do not interfere with the detection of the compound.

[0136] Acceptance criteria: good linearity, R ≥ 0.990 or R 2≥0.980; the back-calculated concentration of the calibration standards should be within 85%-115% of the labeled value, and the lower limit of quantification should be within 80%-120%. At least 75% of the calibration standards (at least 6 valid concentrations) should meet these requirements for measurement accuracy; at least 50% of the calibration standards at the same concentration point should meet these requirements.

[0137] 2. Results

[0138] The following Tables 4-1 to 4-6 respectively give the back-calculated concentration results of plasma samples of the standard curves of esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and vonoprazan fumarate.

[0139] Table 4-1: Back-calculated concentrations of the standard curve (esomeprazole)

[0140]

[0141] Table 4-2: Back-calculated concentrations of the standard curve (rabeprazole)

[0142]

[0143] Table 4-3: Back-calculated concentrations of the standard curve (ilaprazole)

[0144]

[0145] Table 4-4: Back-calculated concentrations of the standard curve (lansoprazole)

[0146]

[0147] Table 4-5: Back-calculated concentrations of the standard curve (pantoprazole)

[0148]

[0149] Table 4-6: Back-calculated concentrations of the standard curve (vonoprazan fumarate)

[0150]

[0151] Figure 3A-3F The typical standard curves of esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and vonoprazan fumarate measured under the conditions of the present invention are shown respectively. The linear relationships of the standard curves of various acid-suppressing drugs are summarized in Table 5 below.

[0152] Table 5: Linear relationship

[0153]

[0154] According to the test results, for all batches of samples, the concentration of vonoprazan fumarate has a good linear relationship with the peak area ratio in the range of 0.50-500 ng / mL, and the concentration of esomeprazole, rabeprazole, ilaprazole, lansoprazole and pantoprazole has a good linear relationship with the peak area ratio in the range of 0.20-200 ng / mL. For all standard curves, R 2 The values ​​are all between 0.990-1.000.

[0155] Example 3: Precision and Accuracy

[0156] 1. Methods

[0157] Each batch of precision and accuracy validation should use independently prepared, freshly prepared standard curves and QC samples. Precision and accuracy should not fail two consecutive analytical batches and must pass at least two consecutive analytical batches, performed on at least two days. Six quality control samples should be collected at each of the LLOQ, low-level quality control (LQC), medium-level quality control (MQC), and high-level quality control (HQC) concentration levels to assess intra- and inter-batch precision and accuracy. Intra-batch precision and accuracy should be assessed within a single batch, and inter-batch precision and accuracy should be assessed between three consecutive batches.

[0158] Acceptance criteria for intra-batch / inter-batch accuracy: The mean of the measured values ​​of the quality control samples at the low, medium, and high concentration levels should be within 85%-115% of the labeled value, and at least 67% of the sample values ​​of the six quality control samples at each concentration level should be within the range of 85%-115% of the labeled value. For LLOQ samples, the mean of the measured values ​​should be within 80%-120% of the labeled value, and at least 67% of the sample values ​​should be within the range of 80%-120% of the labeled value. Acceptance criteria for intra-batch / inter-batch precision: The coefficient of variation within and between batches should generally not exceed 15%, and the coefficient of variation of the lower limit of quantification should not exceed 20%.

[0159] 2. Results

[0160] The test results showed that the accuracy and precision values ​​of the method of the present invention met the specified acceptance criteria, indicating that the method of the present invention is reliable and reproducible for the determination of the compounds esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and vonoprazan fumarate.

[0161] Example 4: Extraction recovery

[0162] 1. Methods

[0163] Different amounts of standard substances were added to a certain amount of blank plasma matrix to prepare validation samples of low, medium and high concentrations, and then extracted. The blank plasma matrix was extracted, and the solution was used as a solvent to prepare a reference solution of the same concentration. The response values ​​of the two were compared to calculate the extraction recovery of the method.

[0164] Internal standard extraction recovery: A validation sample was prepared by adding a predetermined amount of internal standard (equivalent to the amount used in sample preparation) to blank plasma matrix and extracting the sample. The internal standard extraction recovery was calculated by comparing the response of an internal standard solution of the same concentration prepared using the blank plasma matrix extraction solution as the solvent.

[0165] Acceptance criteria: The absolute value of the extraction recovery rate of low, medium and high concentration quality control samples should not exceed 115%, the CV% of the peak area at each concentration level should be less than 15%, and the CV% of the extraction recovery rate at the three concentration levels should not exceed 20%.

[0166] 2. Results

[0167] The following Tables 6-1 and 6-2 summarize the extraction recoveries of the analytes and internal standards, respectively.

[0168] Table 6-1: Extraction recovery of analytes

[0169]

[0170] Note:

[0171] LQC: Low concentration quality control sample

[0172] MQC: Medium concentration quality control sample

[0173] HQC: High concentration quality control sample

[0174] %CV: Precision or coefficient of variation

[0175] Table 6-2: Internal standard extraction recovery

[0176]

[0177] From the above results, it can be seen that the extraction recovery of the method of the present invention meets the prescribed acceptance criteria.

[0178] Example 5: Matrix Effect

[0179] 1. Methods

[0180] The effects of different matrix sources on the assay were evaluated at the LQC and HQC concentration levels using six blank matrices of human plasma samples from different individuals, one blank matrix of hyperlipidemic plasma, and one blank matrix of 2% hemolyzed plasma. The matrix factor for each analyte and internal standard was calculated by ratioing the peak area in the presence of matrix (measured by blank matrix extraction followed by addition of analyte and internal standard) to the corresponding peak area in the absence of matrix (pure solutions of analyte and internal standard). The internal standard-normalized matrix factor (Absolute MF%) was further calculated by dividing the matrix factor of the analyte (Analyte MF%) by the matrix factor of the internal standard (IS MF%). Matrix effects for compounds and internal standards were considered satisfactory if the inter-individual variability was less than 15%.

[0181] 2. Results

[0182] The results of the matrix effects for each acid suppressive drug are summarized in Table 7 below.

[0183] Table 7: Matrix Effects

[0184]

[0185] From the above results, it can be seen that the matrix effect of the method of the present invention meets the above requirements.

[0186] Example 6: Stability

[0187] 1. Methods

[0188] Analyte stability was assessed by analyzing LQC samples (n = 3) and HQC samples (n = 3) under various storage, processing, and analytical conditions. Stock solution stability was assessed by comparing peak areas between freshly prepared solutions and storage solutions (24 hours at room temperature and 104 days at -30°C). Working solution stability was assessed by comparing peak areas between freshly prepared solutions and working solutions (24 hours at room temperature and 15 days at -80°C). Analyte stability in plasma was tested under various conditions. To assess short-term and long-term stability, samples were stored at room temperature for 24 hours, at -20°C for 12 days, and at -80°C for 12 days. Extracted samples were placed in an autosampler (10°C) for 48 hours and subsequently analyzed using a freshly prepared standard curve to assess autosampler stability. Additionally, prepared samples were stored in a refrigerator (10°C) for 48 hours and then reinjected to assess replicate stability. For freeze-thaw stability, the samples were frozen at -80 °C for more than 12 h and then thawed at room temperature for at least 2 h for a total of 4 cycles.

[0189] For whole blood stability assessment, samples at LQC and 1 / 4 HQC concentrations were prepared from freshly collected whole blood and then divided into two groups (Group A and Group B). Group A plasma samples were obtained by immediate centrifugation of the QC samples, while Group B plasma samples were centrifuged after 2 hours at room temperature. The average of the ratios of the analyte peak area to the internal standard peak area for each concentration in Group A was considered the labeled value. The Group B average must be within ±15% of the Group A average, with an RSD% not exceeding 15%.

[0190] 2. Results

[0191] This study investigated the stability of esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate in solution and plasma under different storage and handling conditions during routine analysis.

[0192] 1) Stock solution stability: The stock solutions of the analytes and internal standards showed good stability when stored at -80°C for 104 days. The stock solutions of the analytes and internal standards showed good stability when stored at room temperature for 24 hours.

[0193] 2) Working solution stability: The analyte and internal standard working solutions were stable when stored at -80°C for 15 days. The analyte and internal standard working solutions were stable when stored at room temperature for 24 hours.

[0194] 3) Stability during biological sample pretreatment (short-term stability): The analyte in plasma remains stable after being placed at room temperature for 24 hours.

[0195] 4) Freeze-thaw stability: The analyte in plasma can withstand at least four freeze-thaw cycles (-80°C to room temperature).

[0196] 5) Post-preparation sample stability: The analytes in the plasma after sample pretreatment were stored in an autosampler (10°C) for 48 hours and quantified using a freshly prepared standard curve. The accuracy and precision met the requirements.

[0197] 6) Repeated injection reproducibility: The analyte in plasma after sample pretreatment was stored at 10°C for 48 hours and then repeatedly injected, and the accuracy and precision met the requirements.

[0198] 7) Long-term cryopreservation stability: The analyte in plasma remains stable after being stored at -80°C for 12 days and at -20°C for 12 days.

[0199] 8) Stability of the analyte in the matrix before centrifugation: The analyte in whole blood remains stable after being placed at room temperature for 2 hours.

[0200] Example 7: Dilution reliability

[0201] 1. Methods

[0202] Dilution reliability was assessed to ensure that samples with concentrations above the standard curve range could be diluted and accurately measured. High-concentration diluted quality control samples (esomeprazole, rabeprazole, ilaprazole, lansoprazole, and pantoprazole at 1600 ng / mL, and vonoprazan fumarate at 4000 ng / mL) were prepared and diluted 10-fold (n=6) with the same blank biomatrix dilution. Quantification was performed using the accompanying standard curve, with the dilution factor being the dilution factor. Sample concentrations after dilution should be within the labeled concentration range. The concentrations were measured and compared to the labeled value. The %CV for the six diluted quality control samples was less than 15%, and the concentrations obtained by multiplying the measured values ​​by the dilution factor should be within 85%-115% of the labeled value for the diluted quality control.

[0203] 2. Results

[0204] The results regarding the dilution reliability of each acid-suppressing drug are summarized in Table 8 below.

[0205] Table 8: Dilution reliability

[0206]

[0207] The above results show that the dilution reliability of the method of the present invention meets the specified acceptance criteria when the concentration of the diluted quality control sample is diluted 10 times. These results show that samples with concentrations above the ULOQ can be reliably diluted 10 times for testing.

[0208] Example 8: Residual Effect

[0209] 1. Methods

[0210] At least one DB sample should be injected after the first standard curve ULOQ sample of each analytical batch to evaluate whether there is carryover of high-concentration samples in the blank plasma sample.

[0211] Acceptance criteria: The peak area of ​​the analyte in the DB sample after the ULOQ sample should not exceed 20% of the mean peak area of ​​the analyte in the LLOQ sample, and the peak area of ​​the internal standard in the DB sample should not exceed 5% of the mean peak area of ​​the internal standard in the LLOQ sample.

[0212] 2. Results

[0213] As a result, in the blank sample after the high concentration sample (ULOQ), the residue at the retention time of the analyte was less than 20.0% of the mean peak area of ​​the analyte in the LLOQ samples of the same batch. Therefore, the method of the present invention meets the above acceptance criteria.

[0214] As can be seen from the above description, the drug quantitative analysis method of the present invention is applicable to six acid-suppressing drugs: esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole, and vonoprazan fumarate. The method of the present invention is simple to operate, highly sensitive, stable, and has high analytical efficiency, and is of great significance for the clinical pharmacokinetic studies of these acid-suppressing drugs.

[0215] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this disclosure should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the appended claims.

Claims

1. A method for quantitatively analyzing acid-suppressing drugs by liquid chromatography-tandem mass spectrometry, the method comprising the following steps: 1) Preparing a sample solution containing an acid-suppressing drug from a subject's plasma sample; 2) injecting the sample solution into a high performance liquid chromatography-tandem mass spectrometry device to obtain a mass chromatogram; and 3) performing quantitative analysis of the acid-suppressing drug using an internal standard method according to the mass chromatogram, Wherein step 1) further comprises pre-treating the plasma sample by protein precipitation method, Wherein, the acid-suppressing drugs are esomeprazole, rabeprazole, ilaprazole, lansoprazole, pantoprazole and vonoprazan fumarate; The chromatographic column in the high performance liquid chromatography is a chromatographic column filled with octadecylsilane bonded silica gel. The high performance liquid chromatography uses a mixed mobile phase consisting of mobile phase A and mobile phase B for gradient elution. wherein the mobile phase A is an acetonitrile solution containing 0.1% to 0.5% by mass of formic acid; and The mobile phase B is an aqueous solution containing 0.1% to 0.3% ammonia and 5 mmol / L to 20 mmol / L ammonium formate. The gradient elution procedure is as follows: 0.00 to 0.50 min, 80 vol% to 80 vol% mobile phase B; 0.50 to 1.50 minutes, 80% to 70% mobile phase B; 1.50 to 2.30 minutes, 70% to 70% mobile phase B by volume; 2.30 to 2.50 minutes, 70% to 5% mobile phase B; 2.50 to 3.50 minutes, 5% to 5% mobile phase B by volume; 3.50 to 3.60 minutes, 5% to 80% mobile phase B; 3.60 to 4.50 minutes, 80 vol% to 80 vol% mobile phase B; The total amount of mobile phase A and mobile phase B is 100% by volume.

2. The method according to claim 1, wherein the mobile phase A is an acetonitrile solution containing 0.2% by mass of formic acid; and / or the mobile phase B is an aqueous solution containing 0.1% by mass of ammonia and 10 mmol / L ammonium formate.

3. The method according to claim 1, wherein the internal standard substances are D3-vonoprazan fumarate and D3-omeprazole.

4. The method according to claim 1, wherein step 1) comprises: An internal standard working solution was added to the plasma sample of the subject, the resulting mixture was shaken and then centrifuged, and a reconstitution solution consisting of aqueous formic acid solution and acetonitrile was added to the supernatant and mixed.

5. The method according to claim 4, wherein the internal standard working solution is prepared by dissolving the internal standards D3-vonoprazan fumarate and D3-omeprazole in dimethyl sulfoxide to obtain an internal standard stock solution, and then diluting the internal standard stock solution with acetonitrile.

6. The method according to claim 4, wherein the reconstitution solution consists of a 0.1 mass % formic acid aqueous solution and acetonitrile, and the volume ratio of acetonitrile to formic acid aqueous solution in the reconstitution solution is in the range of 5:95 to 20:

80.

7. method according to claim 6, the volume ratio of acetonitrile and formic acid aqueous solution in wherein said complex solution is 10:

90.

8. The method of claim 1, wherein the mass spectrometry conditions include: A triple quadrupole mass spectrometer was used with an electrospray ionization source in positive ion mode and multiple reaction monitoring detection.

9. The method according to claim 8, wherein the quantitative ion pair of esomeprazole is m / z 346.3→198.1, the quantitative ion pair of rabeprazole is m / z 360.2→242.1, the quantitative ion pair of ilaprazole is m / z 367.2→184.0, the quantitative ion pair of lansoprazole is m / z 370.2→252.0, the quantitative ion pair of pantoprazole is m / z 384.2→200.2, and the quantitative ion pair of vonoprazan fumarate is m / z 346.1→315.

1.

10. The method of claim 1, wherein in the tandem mass spectrometer, the collision energy of esomeprazole is 12 eV, the collision energy of rabeprazole is 10 eV, the collision energy of ilaprazole is 10 eV, the collision energy of lansoprazole is 12 eV, the collision energy of pantoprazole is 20 eV, and the collision energy of vonoprazan fumarate is 15 eV.

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