Determination of losartan in human plasma by liquid chromatography-tandem mass spectrometry

Abstract

This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the concentration of the antihypertensive drug losartan in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The mobile phase of the LC comprises an aqueous formic acid solution and acetonitrile; the elution program of the LC includes: 60-80% acetonitrile by volume in the mobile phase for 0-1.4 min; and a flow rate of 0.6-1 mL / min. This invention provides a highly efficient and accurate method for detecting losartan drug concentration, enabling precise determination of the losartan concentration in plasma and ensuring the accuracy and reliability of the data.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the concentration of the antihypertensive drug losartan in human plasma using liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Losartan, a first-line antihypertensive drug in the angiotensin II receptor antagonist class, has shown significant efficacy in clinical treatment when combined with amlodipine, a calcium channel blocker. The detection of plasma concentrations of losartan, its active metabolite losartan carboxylic acid (EXP-3174), and amlodipine is a core component of pharmacokinetic and bioequivalence studies for these combination formulations. While methods for detecting single components are relatively mature, the simultaneous detection of multiple components in combination formulations requires consideration of the physicochemical properties of different drugs, facing multiple challenges such as matrix interference, extraction efficiency, and detection sensitivity. Therefore, developing efficient, sensitive, and widely applicable simultaneous detection methods has become an urgent need in the industry.

[0003] In the prior art, Chinese invention patent application CN114609287A discloses a method for the simultaneous detection of amlodipine, losartan, and losartan carboxylic acid in plasma using LC-MS. This method partially solves the problem of simultaneous extraction of multiple components by combining liquid-liquid extraction with protein precipitation as a pretreatment, and reduces costs compared to traditional solid-phase extraction technology. However, this method still has significant shortcomings: First, the pretreatment process is complex, requiring the mixed plasma to be processed in two parts separately. One part is used for protein precipitation extraction of losartan and metabolites with 0.1% formate acetonitrile, and the other part is alkalized and then extracted with ethyl acetate and evaporated to dryness. The operation steps are cumbersome and time-consuming. Second, the chromatographic conditions are complex to design, using a BEHC8 column and a gradient elution system containing ammonia. The control of mobile phase components is difficult, which is not conducive to the widespread application of the detection method. In addition, the nitrogen evaporation and other steps in the detection process can easily lead to the loss of target components, which may affect the stability and reproducibility of the detection results. In summary, existing multi-component detection methods still need improvement in terms of ease of operation, detection efficiency, and stability. There is an urgent need to develop an LC-MS / MS detection method with simpler pretreatment, more optimized chromatographic conditions, and higher detection sensitivity to meet the rapid and accurate detection requirements in clinical studies of amlodipine compound preparations. Summary of the Invention

[0004] The first aspect of this invention provides a method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry, comprising the following steps: preparing a standard curve sample, a quality control sample, a test sample, a blank sample, a zero concentration sample, and a sample with no internal standard for the upper limit of quantitation; and performing detection on the standard curve sample, the quality control sample, the test sample, the blank sample, the zero concentration sample, and the sample with no internal standard for the upper limit of quantitation using liquid chromatography-tandem mass spectrometry. The stationary phase of the column used in the liquid chromatography has an average particle size of 3-8 μm and an average pore size of 80-150 Å; the stationary phase comprises C18 chemical bonds; the mobile phase of the liquid chromatography comprises an aqueous solution of 0.1-1 wt% formic acid and acetonitrile; the elution program of the liquid chromatography includes: for 0-1.4 min, the volume content of acetonitrile in the mobile phase is 60-80%; the flow rate of the mobile phase is 0.6-1 mL / min.

[0005] Losartan, an angiotensin II receptor antagonist containing hydrophobic functional groups, exhibits specific retention in reversed-phase liquid chromatography (RP-HPLC) via hydrophobic interactions with a C18 chemically bonded stationary phase. However, existing losartan detection methods often employ stationary phase parameters that are poorly suited to the physicochemical properties of losartan, leading to high mass transfer resistance and slow molecular diffusion, resulting in low detection efficiency and prolonged detection time. This invention, however, limits the average particle size of the stationary phase to 3-8 μm. Compared to larger particle sizes, this particle size improves column efficiency and reduces the retention of losartan molecules on the stationary phase. The mass transfer resistance between the stationary and mobile phases accelerates molecular diffusion and elution rates. The average pore size of 80-150 Å is well-matched to the molecular volume of losartan, ensuring that losartan molecules can effectively enter the pores of the stationary phase and fully interact with the C18 bonded phase to achieve effective retention. This avoids the problems of insufficient molecular diffusion due to excessively small pore sizes and insufficient retention due to excessively large pore sizes. The hydrophobic retention characteristics of the C18 bonded phase, combined with the suitable particle size and pore size parameters, achieves efficient retention and rapid elution of losartan, thereby improving detection efficiency and shortening detection time.

[0006] Optionally, the stationary phase of the column used in the liquid chromatography has an average particle size of 5 μm and an average pore size of 100 Å. Optionally, the elution program of the liquid chromatography includes: during the period of 0-1.4 min, the volume content of acetonitrile in the mobile phase is 70%, and the flow rate of the mobile phase is 0.8 mL / min.

[0007] This invention employs a reversed-phase liquid chromatography system. Losartan is retained by the C18 stationary phase due to hydrophobic interactions. Acetonitrile, as a highly polar organic phase, is a highly efficient eluent in reversed-phase chromatography. An acetonitrile volume content of 60-80% provides sufficient elution strength, rapidly breaking the hydrophobic interaction between losartan and the C18 stationary phase, allowing losartan to quickly desorb from the stationary phase into the mobile phase. A flow rate of 0.6-1 mL / min is within a reasonable range that balances elution speed and separation effect. This avoids the problems of excessively slow flow rates leading to long residence time of losartan in the column and increased detection time, while also avoiding excessively fast flow rates causing excessive column pressure and reduced separation effect between losartan and plasma matrix impurities. The synergistic effect of the strong elution capacity of high-proportion acetonitrile and the suitable flow rate allows losartan to complete elution from the column and chromatographic signal acquisition within 1.4 min, further significantly shortening the detection time.

[0008] Losartan contains a protonable basic functional group in its chemical structure. A mobile phase consisting of a 0.1-1 wt% aqueous solution of formic acid and acetonitrile, where formic acid, as a volatile acid, provides protons to losartan, causing it to protonate and acquire a positive charge. On one hand, the protonated losartan interacts more stably with the C18 stationary phase, exhibiting more uniform retention behavior and effectively reducing peak broadening and tailing, thus improving peak symmetry and reproducibility. On the other hand, the addition of formic acid does not introduce non-volatile salts and has good compatibility with acetonitrile, avoiding column clogging and subsequent mass spectrometry ion source contamination, ensuring the stability of the detection process. Furthermore, the 0.1-1 wt% formic acid concentration range satisfies the requirement for sufficient protonation of losartan without causing hydrolysis of the bonded phase of the C18 stationary phase or structural instability of losartan due to excessively high acid concentrations. Therefore, while achieving rapid detection, this further improves the precision and accuracy of losartan concentration detection.

[0009] The standard curve sample was prepared from a standard curve solution, and the concentration of losartan in the standard curve solution ranged from 1 to 3000 ng / mL.

[0010] The standard curve solution contains 6-10 concentration gradients of losartan.

[0011] Optionally, the concentrations of losartan in the standard curve solutions are 1.5 ng / mL, 3 ng / mL, 7.5 ng / mL, 50 ng / mL, 250 ng / mL, 1000 ng / mL, 2400 ng / mL, and 3000 ng / mL, respectively.

[0012] The zero-concentration sample was prepared from an internal standard solution, wherein the concentration of losartan-d4 in the internal standard solution was 400-800 ng / mL.

[0013] The quality control sample was prepared from a quality control solution, wherein the concentration of losartan in the quality control solution ranged from 1 to 3000 ng / mL.

[0014] The concentrations of losartan in the quality control solutions were 1.5 ng / mL, 4.5 ng / mL, 70 ng / mL, 900 ng / mL, and 2250 ng / mL, respectively.

[0015] The test sample was prepared from a test solution, in which the concentration of losartan was 1000-3000 ng / mL.

[0016] The ionization mode of the mass spectrometer is: electrospray ionization source, positive ion mode, multiple reaction monitoring.

[0017] The ion source parameters of the mass spectrometer include the following parameters: collision gas 8.00 psi, curtain gas 40.00 psi, first ion source gas 30.00 psi, second ion source gas 60.00 psi, ion source spray voltage 5500.00 V, and ion source temperature 550.00 °C.

[0018] The ion parameters for the losartan reaction in the mass spectrometer were as follows: monitored ion pair 423.2 / 207.2, declustering voltage 110.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 27.00eV, and residence time 100.00msec.

[0019] The ionic parameters for the losartan-d4 reaction in the mass spectrometer were as follows: monitored ion pair 427.2 / 211.3, declustering voltage 110.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 27.00eV, and residence time 100.00msec.

[0020] Formic acid in the mobile phase protonates losartan, giving it a positive charge. This provides the material basis for using electrospray ionization (ESI) in positive ion mode for mass spectrometry, ensuring efficient ionization of losartan to generate quasi-molecular ions, a prerequisite for specific mass spectrometry detection. The specific mass spectrometry parameters defined in this invention are precisely designed for the molecular structure, ionization characteristics, and fragmentation patterns of protonated losartan. The ESI positive ion mode combined with multiple reaction monitoring (MRM) enables specific screening of target ions. Optimization of parameters such as the collision gas, curtain gas, and ion source gas ensures the ion transport efficiency and ion source stability of protonated losartan. The 110.00V declustering voltage set for losartan... Effective removal of solvated ions yields a pure precursor ion 423.2. A collision energy of 27.00 eV causes the precursor ion to undergo specific fragmentation, generating the characteristic daughter ion 207.2. The mass spectrometry parameters of the internal standard losartan-d4 are highly matched with those of losartan, and the characteristic ion pair 427.2 / 211.3 can achieve precise internal standard calibration. The multiple reaction monitoring mode only acquires specific precursor ion-daughter ion pairs of losartan and losartan-d4, effectively eliminating non-specific ion signal interference from other impurities in the plasma matrix. The synergistic effect of specific mass spectrometry parameters and the formic acid-acetonitrile mobile phase enables specific identification and detection of losartan in plasma, significantly improving the selectivity of detection.

[0021] Beneficial effects 1. The present invention limits the average particle size of the stationary phase used in the liquid chromatography column to 3-8 μm and the average pore size to 80-150 Å; the stationary phase includes C18 chemical bonds, which can improve detection efficiency and shorten detection time.

[0022] 2. The elution procedure of the liquid chromatography includes: the volume content of acetonitrile in the mobile phase is 60-80%; the flow rate of the mobile phase is 0.6-1 mL / min, which can further shorten the detection time to within 1.4 min.

[0023] 3. For liquid chromatography, the mobile phase can be an aqueous solution of 0.1-1 wt% formic acid and acetonitrile, which can further improve the precision and accuracy of detection while shortening the detection time.

[0024] 4. By limiting specific mass spectrometry parameters and using specific mobile phases, detection selectivity can be improved.

[0025] 5. The detection method of this invention meets the clinical requirements for precision and accuracy. Attached Figure Description

[0026] Figure 1 The conditions for liquid chromatography in Example 1 are as follows.

[0027] Figure 2 The conditions for mass spectrometry in Example 1 are (Q1: quadrupole mass analyzer 1; Q3: quadrupole mass analyzer 3).

[0028] Figure 3 The results of precision, accuracy and recovery tests for Example 1 are shown.

[0029] Figure 4 The results are from the selective test in Example 1.

[0030] Figure 5 The results are from the durability test of Example 1.

[0031] Figure 6 This is the ion scanning spectrum of losartan in Example 1.

[0032] Figure 7 This is the scanning spectrum of losartan-d4 daughter ions in Example 1.

[0033] Figure 8 The standard curve is obtained from the standard curve sample of Example 1. Detailed Implementation

[0034] Example 1 A method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry comprises the following steps: Losartan standard (losartan potassium, purchased from the National Institutes for Food and Drug Control, batch number: 100597-202104) and losartan-d4 standard (purchased from ISOREAG, batch number: 25Z769-A1) were respectively prepared with dimethyl sulfoxide to prepare 1.00 mg / mL losartan stock solution and losartan-d4 stock solution; the above losartan stock solution was diluted with 50% methanol to prepare standard curve sample working solution and quality control sample working solution; the above losartan-d4 stock solution was diluted with 50% methanol to prepare 500 ng / mL internal standard working solution.

[0035] The concentrations of losartan in the standard curve solutions were 1.5 ng / mL (lower limit of quantitation, LLOQ), 3 ng / mL, 7.5 ng / mL, 50 ng / mL, 250 ng / mL, 1000 ng / mL, 2400 ng / mL, and 3000 ng / mL (upper limit of quantitation, ULOQ).

[0036] The concentrations of losartan in the quality control solutions were 1.5 ng / mL (lowest quality control concentration, LLOQQC), 4.5 ng / mL (low quality control concentration, LQC), 70 ng / mL (medium quality control concentration, GMQC), 900 ng / mL (medium quality control concentration, MQC), and 2250 ng / mL (high quality control concentration, HQC).

[0037] The blank matrix is ​​blank human plasma collected from healthy volunteers, with K2EDTA as the anticoagulant.

[0038] Take the plasma to be tested (provided by Zibo Traditional Chinese Medicine Hospital) and dilute the quality control solution (quality control solution: 10000ng / mL, dilution factor: 5) to obtain a 2000ng / mL test solution; In a 2.2 mL 96-well polypropylene plate, under room temperature and white light conditions, 50 μL of standard curve solution, 50 μL of quality control solution, and 50 μL of test solution were mixed with 25 μL of internal standard solution to obtain 75 μL of standard curve sample, 75 μL of quality control sample, and 75 μL of test sample, respectively. 50 μL of blank matrix was mixed with 25 μL of methanol to obtain a 75 μL blank sample. 50 μL of blank matrix was mixed with 25 μL of internal standard solution to obtain a zero-concentration sample. 50 μL of the highest losartan concentration quality control solution HQC was mixed with 25 μL of methanol to obtain a 75 μL sample with the upper limit of quantitation (without internal standard). All the above samples were diluted with 400 μL of methanol, mixed thoroughly, and centrifuged at 3220 g for 5 min at 4 °C. 150 μL of the supernatant was transferred to another 96-well polypropylene plate, 150 μL of purified water was added, and the plate was placed in an autosampler for detection using liquid chromatography-tandem mass spectrometry. Chromatographic acquisition and peak integration were performed using ABSciex Analyst software (version 1.7.2). The standard curve was obtained using regression analysis with the Watson LIMS system (version 7.6.1), with the chromatographic response ratio of the analyte to the internal standard as the ordinate, and weighted averages (W=1 / x) calculated. 2 The least squares method uses linear regression between the concentration (x) of the analyte in plasma and the response ratio (y). The resulting regression equation (y=ax+b) is the standard curve. The drug concentration of the sample is calculated from the fitted standard curve equation. Figure 8 As shown, a = 0.00514; b = 0.000445; R 2 =0.9982.

[0039] The conditions for the liquid chromatography are as follows: Figure 1 As shown, the mass spectrometry conditions are as follows: Figure 2 As shown, the losartan ion scanning spectrum is as follows: Figure 6 As shown, the scan spectrum of losartan-d4 ions is as follows: Figure 7 As shown.

[0040] Performance testing methods and data 1. Precision and accuracy Intra-batch precision and accuracy Intra-batch precision and accuracy were assessed using quality control samples (LLOQQC, LQC, GMQC, MQC, and HQC), with six replicates for each concentration of quality control sample.

[0041] Precision was assessed by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and accuracy was assessed by calculating the deviation (Diff%) between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level.

[0042] Acceptance criteria: The deviation between the measured mean concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQQC, the coefficient of variation should not exceed 20.0%).

[0043] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples should have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQQC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level should meet the above standards.

[0044] Inter-batch precision and accuracy analysis Inter-batch precision and accuracy were assessed by examining at least three independent validation analysis batches (intra-batch precision and accuracy analysis batches, completed within at least two days) using freshly prepared quality control samples with a blank matrix.

[0045] The quality control samples used to calculate inter-batch precision and accuracy were derived from the quality control samples (LLOQQC, LQC, GMQC, MQC, and HQC) prepared to examine intra-batch precision and accuracy, with six replicates for each concentration level quality control sample per validation analysis batch.

[0046] Acceptance criteria: The deviation of the overall mean concentration of each quality control sample at each concentration level from its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%).

[0047] The overall coefficient of variation for the measured concentration of each quality control sample at each concentration level shall not exceed 15.0% (the coefficient of variation for LLOQQC shall not exceed 20.0%).

[0048] If an analytical batch fails to meet the acceptance criteria, three additional accuracy and precision analytical batches are tested to validate the methodology.

[0049] like Figure 3 As shown, Example 1 has high precision and accuracy.

[0050] 2. Extraction recovery rate Blank matrix from the same batch (or source) as the routine quality control samples (extracted samples, test samples) was used as blank samples. After extraction, the analyte and internal standard were added to the extract of the blank samples to prepare low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC). Six replicates of each concentration were used as reference samples.

[0051] Test samples are routine quality control samples or samples prepared using the same process, including low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC) (6 replicates for each concentration).

[0052] Analyte extraction recovery rate calculation: The peak area of ​​the analyte in each concentration of routine quality control sample (test sample) is divided by the average peak area of ​​the analyte in the reference sample of the same concentration.

[0053] Internal standard extraction recovery rate calculation: The peak area of ​​the internal standard in each routine quality control sample (test sample) is divided by the average peak area of ​​the internal standard in the reference sample.

[0054] Acceptance criteria: The overall coefficient of variation for analyte extraction does not exceed 15.0%; the coefficient of variation for internal standard extraction recovery does not exceed 15.0%.

[0055] If the independent extraction recovery of the analyte or internal standard does not meet the acceptance criteria, the extraction recovery of the analytical method can be evaluated by the extraction recovery after internal standard correction.

[0056] Extraction recovery rate calculation for internal standard correction: The ratio of the peak area of ​​the analyte to its internal standard in each concentration routine quality control sample (test sample) divided by the mean ratio of the peak area of ​​the analyte to its internal standard in the reference sample of the same concentration.

[0057] Acceptance criteria for extraction recovery after internal standard correction: The overall coefficient of variation of extraction recovery after internal standard correction shall not exceed 15.0%.

[0058] like Figure 3 As shown, Example 1 has a high extraction recovery rate.

[0059] 3. Selectivity Matrix selectivity (endogenous interference) Matrix selectivity was evaluated by examining blank biological matrices from at least six different individuals, a high-lipid matrix from one individual, and a hemolyzed matrix from one individual, with measurements of blank samples without internal standards and LLOQ-level samples, respectively.

[0060] High-lipid matrix: via commercially available or simulated hyperlipidemic plasma (1 ± 0.1 mg low-density lipoprotein (LDL) and 3 ± 0.3 mg triglycerides added to 1 mL of blank plasma).

[0061] Hemolysing matrix: Whole blood was frozen at -80°C for at least 30 minutes, thawed, vortexed for at least 1 minute, and then mixed with conventional blank matrix (1:49, v:v) to prepare hemolysing blank matrix.

[0062] Acceptance criteria: The response value of interfering components in the blank matrix at the analyte retention time shall not exceed 20.0% of the analyte response value of the LLOQ sample prepared with the same individual blank matrix; the response value at the internal standard retention time shall not exceed 5.0% of the internal standard response value of the LLOQ sample prepared with the same individual blank matrix.

[0063] If a blank matrix sample from a certain source (batch) does not meet the acceptance criteria, the same method will be used to evaluate the interference of three additional blank matrices from different sources (batches) on the analyte and internal standard. If a high-lipid matrix or hemolyzed matrix does not meet the acceptance criteria, the same method will be used to evaluate one additional high-lipid matrix or hemolyzed matrix from a different source.

[0064] Interference of the analyte to the internal standard Three samples containing only a single analyte and without an internal standard were prepared, processed, and analyzed in parallel to determine the upper limit of quantitation concentration.

[0065] Acceptance criteria: The average peak area of ​​the internal standard at the retention time of a sample containing only a single analyte should not exceed 5.0% of the average peak area of ​​the internal standard in the samples meeting the lower limit of quantitation of the standard curve in the same analytical batch.

[0066] Interference of internal standard with analyte Three samples containing only a single internal standard and without the analyte were prepared, processed, and analyzed in parallel. The concentration of the internal standard was the actual concentration used.

[0067] Acceptance criteria: The average peak area at the retention time of the analyte should not exceed 20.0% of the average peak area of ​​the analyte in the standard curve samples that meet the acceptance criteria in the same analytical batch.

[0068] Selectivity of analysis batch The selectivity of the analytical batch was evaluated using the first blank matrix sample and the first blank quality control sample of the analytical batch.

[0069] Acceptance criteria: The peak area of ​​the analyte in the detection channel of both blank samples does not exceed 20.0% of the average peak area of ​​the analyte in the samples with the limit of quantitation of the effective standard curve; the peak area of ​​the internal standard in the detection channel of the first blank matrix sample does not exceed 5.0% of the average peak area of ​​the internal standard in the samples with the limit of quantitation of the effective standard curve in the same analytical batch.

[0070] like Figure 4 As shown, Example 1 has a higher selectivity.

[0071] 4. Durability The precision was assessed by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, with samples injected by different analysts (Analyst A and Analyst B) or on different instruments (LC-MS / MS, two sets of instruments of the same model with the same parameters). The accuracy was assessed by calculating the deviation (Diff%) between the mean measured concentration of the quality control samples at each concentration level and their theoretical concentration.

[0072] Acceptance criteria: The deviation between the measured mean concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQQC, the coefficient of variation should not exceed 20.0%).

[0073] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples should have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQQC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level should meet the above standards.

[0074] The mass spectrometer model is TripleQuad5500+; the chromatography system includes a liquid phase pump (LC-30AD), a controller (CBM-20A), a degasser (DGU-20A5R(C), a column oven (CTO-20A), and an injection system (SIL-30ACMP).

[0075] like Figure 5 As shown, the durability test results of Example 1 meet the requirements.

Claims

1. A method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: Prepare standard curve samples, quality control samples, test samples, blank samples, zero concentration samples, and samples with upper limit of quantitation (UPQ) without internal standard; perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) on the standard curve samples, quality control samples, test samples, blank samples, zero concentration samples, and samples with upper limit of quantitation (UPQ) without internal standard. The stationary phase of the column used in the liquid chromatography has an average particle size of 3-8 μm and an average pore size of 80-150 Å; the stationary phase comprises C18 chemical bonds; the mobile phase of the liquid chromatography comprises an aqueous solution of 0.1-1 wt% formic acid and acetonitrile; the elution program of the liquid chromatography includes: for 0-1.4 min, the volume content of acetonitrile in the mobile phase is 60-80%; the flow rate of the mobile phase is 0.6-1 mL / min.

2. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The elution program for the liquid chromatography includes the following steps: from 0 to 1.4 min, the volume content of acetonitrile in the mobile phase is 70%, and the flow rate of the mobile phase is 0.8 mL / min.

3. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that, The standard curve sample was prepared from a standard curve solution, and the concentration of losartan in the standard curve solution ranged from 1 to 3000 ng / mL.

4. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1 or 3, characterized in that, The standard curve solution contains 6-10 concentration gradients of losartan.

5. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that, The concentrations of losartan in the standard curve solutions were 1.5 ng / mL, 3 ng / mL, 7.5 ng / mL, 50 ng / mL, 250 ng / mL, 1000 ng / mL, 2400 ng / mL, and 3000 ng / mL, respectively.

6. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The zero-concentration sample was prepared from an internal standard solution, wherein the concentration of losartan-d4 in the internal standard solution was 400-800 ng / mL.

7. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1 or 6, characterized in that, The ionization modes of the mass spectrometer are: electrospray ionization source, positive ion mode, and multiple reaction monitoring.

8. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 7, characterized in that, The ion source parameters of the mass spectrometer include the following parameters: Collision gas 8.00psi, curtain gas 40.00psi, first ion source gas 30.00psi, second ion source gas 60.00psi, ion source spray voltage 5500.00V, ion source temperature 550.00℃.

9. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 8, characterized in that, The ion parameters for the losartan reaction in the mass spectrometer were as follows: monitored ion pair 423.2 / 207.2, declustering voltage 110.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 27.00eV, and residence time 100.00msec.

10. The method for determining the concentration of the antihypertensive drug losartan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 9, characterized in that, The ionic parameters for the losartan-d4 reaction in the mass spectrometer were as follows: monitored ion pair 427.2 / 211.3, declustering voltage 110.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 27.00eV, and residence time 100.00msec.

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