Monitoring method and monitoring preparation for plasma concentration of amiodarone and application of monitoring preparation

Abstract

The invention relates to a method for monitoring the plasma concentration of amiodarone, a monitoring preparation and application thereof, and relates to the technical field of plasma concentration monitoring, and the monitoring method for the plasma concentration of amiodarone comprises the following steps: S1, preparing a solution of a to-be-detected product; s2, detecting the solution of the product to be detected by two-dimensional liquid chromatography; in the step S2, a one-dimensional eluent, a two-dimensional eluent and a cleaning solution are arranged to respond to a corresponding elution program, so that the solution of the product to be detected is pumped into a first chromatographic system, a primary product is pumped into a second chromatographic system, and an extraction column and an analysis column are cleaned; the flow velocity of the one-dimensional eluent is 0.8-2.0 mL / min, the flow velocity of the two-dimensional eluent is 0.9-1.1 mL / min, and the flow velocity of the cleaning solution is 0.05-0.4 mL / min; the one-dimensional eluent comprises a composition of methanol and water and a composition of acetonitrile and phosphoric acid water, the two-dimensional eluent comprises acetonitrile and phosphoric acid water, and the cleaning solution is water; s3, calculating the content of amiodarone in the to-be-detected product solution according to the typical spectrogram. The method has the characteristics that the plasma concentration of amiodarone can be rapidly monitored, the accuracy is high, and the operation is simple.

Description

Technical Field

[0001] This application relates to the field of blood drug concentration monitoring technology, and in particular to a method for monitoring amiodarone blood drug concentration, a monitoring preparation, and its application. Background Technology

[0002] Amiodarone is a class III antiarrhythmic drug. Its main electrophysiological effect is to prolong the action potential and effective refractory period of various myocardial tissues, which is beneficial for eliminating reentry excitation. This drug also has a mild non-competitive adrenergic receptor blocking effect, combining the characteristics of class I and class IV antiarrhythmic drugs, and can reduce sinoatrial node automaticity. Therefore, it has a wide range of clinical applications.

[0003] However, amiodarone has complex pharmacokinetic properties, a narrow therapeutic window, and dependence toxicity. Furthermore, individual differences in physiological function can lead to insufficient or excessive amiodarone blood concentrations. Therefore, monitoring amiodarone blood concentrations is essential to achieve precise and personalized medication, balancing its complex pharmacokinetics, narrow therapeutic window, and multi-systemic toxicity risks.

[0004] Currently, common methods for monitoring amiodarone as a therapeutic agent include high-performance thin-layer chromatography (HPLC), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). Among these methods, HPLC is generally the mainstream approach for monitoring amiodarone as a therapeutic agent.

[0005] Related document CN115639301A discloses a method for detecting amiodarone and desethylamiodarone in plasma, but this method involves cumbersome pretreatment and a long processing time. Related document CN110361484A discloses a blood amiodarone drug concentration monitoring kit and its detection method. This method uses multiple reagents to process the sample solution one by one, which is not only cumbersome but also suffers from low accuracy.

[0006] Therefore, there is an urgent need to provide a monitoring method that can rapidly monitor amiodarone blood concentration, is highly accurate, and is easy to operate. Summary of the Invention

[0007] This application addresses the shortcomings of existing technologies by providing a method, a monitoring preparation, and its application for monitoring amiodarone blood concentration. This method achieves rapid monitoring while being highly accurate and easy to operate.

[0008] Firstly, this application provides a method for monitoring amiodarone blood concentration, comprising the following steps:

[0009] S1. Prepare the solution to be tested;

[0010] S2. Two-dimensional liquid chromatography for the detection of the test solution;

[0011] S2 specifically includes:

[0012] S21. The sample solution is pumped into the extraction column of the first chromatographic system to complete the enrichment, purification and impurity removal of the target analyte, and to obtain the initial product.

[0013] S22. The initial product on the extraction column is pumped into the analytical column of the second chromatographic system for separation to obtain the analyte, and the extraction column is simultaneously rinsed and regenerated.

[0014] S23. Analyze the analyte and obtain a typical spectrum;

[0015] In S2, a one-dimensional eluent, a two-dimensional eluent, and a cleaning solution are set up to respond to the corresponding elution program, so as to pump the sample solution into the first chromatographic system, pump the initial product into the second chromatographic system, and clean the extraction column and the analytical column.

[0016] The flow rate of the one-dimensional eluent is 0.8–2.0 mL / min, the flow rate of the two-dimensional eluent is 0.9–1.1 mL / min, and the flow rate of the washing solution is 0.05–0.4 mL / min.

[0017] The one-dimensional eluent comprises a composition of methanol and water, and a composition of acetonitrile and aqueous phosphoric acid; the two-dimensional eluent comprises acetonitrile and aqueous phosphoric acid; and the cleaning solution is water.

[0018] S3. Calculate the content of amiodarone in the test solution based on the typical spectrum.

[0019] By adopting the above scheme, the flow rate and composition of the one-dimensional and two-dimensional eluents are designed according to the properties of amiodarone to match the elution program. At the same time, two-dimensional liquid chromatography is used to pre-treat and separate the sample through two tandem chromatographic systems, which greatly improves the monitoring efficiency and analytical accuracy of the target analyte amiodarone. It can effectively reduce interfering substances in the sample and improve the purity of the analyte, thereby enabling more accurate determination of the amiodarone content in plasma.

[0020] Furthermore, the elution procedure for the one-dimensional eluent is as follows:

[0021] The flow rate was 0.8 mL / min from 0 to 1 min, and the volume ratio of methanol to water in the one-dimensional eluent was 1:9.

[0022] 2–4 min, flow rate 2.0 mL / min, volume ratio of acetonitrile to phosphoric acid water in one-dimensional eluent 3:1;

[0023] The flow rate was 2.0 mL / min for 6–8 min, and the volume ratio of methanol to water in the one-dimensional eluent was 1:9.

[0024] The eluent was prepared for 8.1–12 min at a flow rate of 0.8 mL / min, with a methanol to water volume ratio of 1:9 in the one-dimensional eluent.

[0025] Using the above scheme, the sample solution is transported to the extraction column within 0–1 min for enrichment, purification, and impurity removal to form the initial product. During this period, a large volume of aqueous one-dimensional eluent (methanol and water at a ratio of 1:9) is used to wash away highly polar substances or water-soluble impurities. Subsequently, the initial product is transported to the analytical column through the cut-off valve and return valve within a 1–2 min window. At this point, within 2–4 min, a large volume of organic one-dimensional eluent (acetonitrile and phosphoric acid at a ratio of 3:1) is used to wash away endogenous impurities, low-polarity impurities, or lipid-soluble impurities on the extraction column. Then, the extraction column is washed again with methanol and water at a ratio of 1:9 to remove residual acetonitrile and phosphoric acid, ultimately restoring the extraction column to its initial conditions, preparing it for the next injection and analysis.

[0026] Furthermore, the elution procedure of the cleaning solution is as follows:

[0027] 0–1 min, flow rate 0.4 mL / min;

[0028] 2–8 min, flow rate 0.05 mL / min;

[0029] 8.1–12 min, flow rate 0.4 mL / min.

[0030] By adopting the above method, the flow rate of the cleaning solution is reduced at 2-8 minutes to reduce its dilution of the one-dimensional eluent, thus ensuring the cleaning effect of the one-dimensional eluent and cleaning the extraction column better.

[0031] Furthermore, the elution procedure for the two-dimensional eluent is as follows:

[0032] The flow rate was 1.0 mL / min for 0–12 min, and the volume ratio of acetonitrile to phosphoric acid water in the two-dimensional eluent was 3:1.

[0033] By adopting the above scheme, the two-dimensional eluent with a high proportion of organic phase can effectively cope with the hydrophobicity of amiodarone. At the same time, the use of phosphoric acid water can provide an acidic environment, which can protonate the diethylamino (tertiary amine) at the end of the amiodarone side chain to form a cation, thereby enhancing polarity, reducing hydrophobic interaction with the analytical column, shortening retention time and improving peak shape (reducing tailing). Under these conditions, the typical spectrum obtained has a good peak shape and a tailing factor of around 1.0. Moreover, compared with gradient elution, it can further reduce the detection time and achieve the purpose of rapid monitoring.

[0034] In summary, acetonitrile's high elution capacity and low viscosity complement the strong hydrophobicity of amiodarone, while phosphoric acid water adjusts polarity and stabilizes pH through protonated amino groups. Together, they achieve efficient and stable chromatographic separation. This combination balances solubility, separation efficiency, and detection compatibility, making it an ideal choice for amiodarone HPLC analysis. Furthermore, phosphoric acid water is easier to prepare and provides a more suitable acidic environment compared to potassium dihydrogen phosphate solution, reducing the likelihood of crystallization and instrument damage in the tubing.

[0035] Furthermore, the extraction column is an Outstanding SPE column with dimensions of 3.0 × 30 mm and a diameter of 25 μm; the analysis column is an Outstanding C18 column with dimensions of 4.6 mm × 150 mm and a diameter of 5 μm; and the column temperature of both the extraction column and the analysis column is 25–35 °C.

[0036] By adopting the above scheme, a suitable extraction column and analytical column are selected based on the characteristics of amiodarone.

[0037] Further, step S1 specifically includes:

[0038] S11. Dissolve amiodarone in acetonitrile to prepare a mother liquor;

[0039] S12. Take the mother liquor and dilute it with acetonitrile to obtain the standard solution;

[0040] S13. Mix the mother liquor with bovine serum, then add protein precipitant to dilute, centrifuge and take the supernatant, filter the supernatant to obtain blood sample solution.

[0041] S14. Take bovine serum and dilute it with protein precipitant. After centrifugation, take the supernatant and filter it to obtain a blank sample solution.

[0042] By adopting the above scheme, standard solutions and blood sample solutions are prepared sequentially using the mother liquor as the base material, and blank sample solutions are prepared accordingly based on the two solutions. The blood concentration of amiodarone is analyzed through multiple experimental groups to ensure that the monitoring results have high accuracy.

[0043] Furthermore, the concentration of amiodarone in the standard solution and the blood sample solution is the same and is 0.1 to 1 μg / ml.

[0044] By adopting the above method, the mother liquor is diluted and amiodarone is monitored at a concentration of 0.1–1 μg / ml to facilitate the delivery of the analyte and subsequent elution of the first and second chromatographic systems.

[0045] Furthermore, the protein precipitant is set as an acetonitrile solution.

[0046] By adopting the above scheme, the type of protein precipitant is kept consistent with that of the diluent, thus maintaining consistency in the variable parameters.

[0047] Secondly, this application provides a monitoring formulation for the above-mentioned method of monitoring amiodarone blood concentration, the monitoring formulation comprising a first formulation and a second formulation, the first formulation comprising a composition of methanol and water, and the second formulation comprising a composition of acetonitrile and phosphate water.

[0048] Thirdly, this application provides an application of the above-mentioned monitoring preparation in monitoring amiodarone blood concentration.

[0049] In summary, this application includes at least one of the following beneficial technical effects:

[0050] 1. This application employs two-dimensional liquid chromatography, which uses two tandem chromatographic systems for sample pretreatment and separation, greatly improving the monitoring efficiency and analytical accuracy of the target analyte amiodarone. It can effectively reduce interfering substances in the sample and improve the purity of the analyte, thereby enabling more accurate determination of the amiodarone content in plasma.

[0051] 2. This application sets up a one-dimensional eluent, a two-dimensional eluent, and a washing solution to correspond to the elution procedure, which ensures high-precision monitoring of amiodarone blood concentration while making the monitoring method simpler and faster.

[0052] 3. This application uses a combination of acetonitrile and phosphoric acid water to treat the sample solution. The high elution capacity and low viscosity of acetonitrile are well-suited to the strong hydrophobicity of amiodarone, while phosphoric acid water adjusts the polarity and stabilizes the pH through protonated amino groups. The two work together to achieve efficient and stable chromatographic separation. This combination takes into account solubility, separation efficiency and detection compatibility, making it an ideal choice for amiodarone HPLC analysis. Attached Figure Description

[0053] Figures 1a-1e This is the linear spectrum of the standard sample in this application;

[0054] Figure 2 This is the standard curve diagram in this application;

[0055] Figures 3a-3d This is a graph of the specificity experiment in this application;

[0056] Figures 4a-4f This is a graph of the reproducibility experiment in this application;

[0057] Figure 5a This is the chromatogram of the limit of quantitation experiment in this application;

[0058] Figure 5b This is the spectrum of the detection limit experiment in this application;

[0059] Figures 6a-6eThis is a graph from the stability test in this application;

[0060] Figures 7a-7d , Figures 8a-8d , Figures 9a-9d , Figures 10a-10b This is a graph of the spiked recovery experiment in this application;

[0061] Figure 10c This is a chromatogram of the content determination experiment in this application. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0063] Those skilled in the art will understand that, unless otherwise stated, the terms "the," "the," and "the foregoing" used in this application may also include plural forms. It should be further understood that the word "comprising" as used in the specification of this application means the presence of the stated features, steps, or operations, but does not exclude the presence or addition of one or more other features, integers, or steps.

[0064] Those skilled in the art will understand that, where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field; and where the manufacturers of the raw materials or instruments and equipment used are not specified, they are all conventional products that can be obtained commercially.

[0065] Those skilled in the art will understand that, unless otherwise stated in this application, when numerical ranges are given in the embodiments, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application, as well as the prior art known to those skilled in the art and the descriptions in this application, can be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made by means of methods, devices, and materials in the embodiments of this application.

[0066] A method for monitoring amiodarone blood concentration includes the following steps:

[0067] S1. Prepare the solution to be tested.

[0068] The test solution can be set in multiple ways, including standard solution, blood sample solution and blank sample solution. By setting multiple test solutions with variables, high accuracy can be ensured when analyzing amiodarone blood concentration.

[0069] Correspondingly, step S1 specifically includes:

[0070] S11. Dissolve amiodarone in acetonitrile to prepare a mother liquor.

[0071] In this embodiment, 5.33 mg of amiodarone was weighed and dissolved in acetonitrile, and the volume was adjusted to 5 ml in a volumetric flask to obtain a primary product of 1.066 mg / ml. Then, 0.5 ml of the 1.066 mg / ml primary product was transferred to a 10 ml volumetric flask using a standard pipette and adjusted to volume with acetonitrile to obtain a mother solution of 53.3 μg / ml.

[0072] Furthermore, using the mother liquor as the base, standard solutions and blood sample solutions were prepared, and blank sample solutions were prepared based on the standard solutions and blood sample solutions as a reference.

[0073] S12. Mix the mother liquor with pure water, then dilute with acetonitrile to obtain the standard solution;

[0074] S13. Mix the mother liquor with bovine serum, then add protein precipitant to dilute, centrifuge and take the supernatant, filter the supernatant to obtain blood sample solution.

[0075] S14. Take bovine serum and dilute it with protein precipitant. After centrifugation, take the supernatant and filter it to obtain a blank sample solution.

[0076] It should be noted that this embodiment does not specifically limit the specific parameters of centrifugation and filtration in S13 and S14 above. In one specific embodiment, the centrifugation process is specifically centrifuged at 10000 r / min for 10 min, and a 0.45 μm microporous membrane is used when filtering the supernatant; in other embodiments, the parameters of centrifugation and filtration can also be adjusted according to the actual situation to ensure the preparation of the test solution.

[0077] The concentration of amiodarone in the standard solution and blood sample solution was the same, and the amounts of purified water in the standard solution, bovine serum in the blood sample solution, and bovine serum in the blank sample solution were also consistent. In this embodiment, the protein precipitant was set as an acetonitrile solution. By controlling the concentration of amiodarone, the type of protein precipitant, and the amounts of purified water and bovine serum added, the variable parameters were kept consistent.

[0078] Furthermore, the concentration of amiodarone in the standard solution and blood sample solution is determined based on the drug window concentration of amiodarone. Referring to the drug window concentration of amiodarone as 0.5–5 μg / ml, this embodiment further dilutes it 10-fold based on this window concentration. Correspondingly, the concentration of amiodarone in the standard solution and blood sample solution is the same and is 0.1–1 μg / ml (preferably 0.533 μg / ml in this embodiment). Diluting the concentration of amiodarone facilitates monitoring of amiodarone and facilitates the delivery of the analyte and subsequent elution of the first and second chromatographic systems.

[0079] S2. Two-dimensional liquid chromatography (2LC) is used to detect the sample solution. Specific parameters for 2LC are shown in Table 1.

[0080] Table 1

[0081]

[0082]

[0083] S2 specifically includes:

[0084] S21. The sample solution is pumped into the extraction column of the first chromatographic system to complete the enrichment, purification and impurity removal of the target analyte, and to obtain the initial product.

[0085] S22. The initial product on the extraction column is pumped into the analytical column of the second chromatographic system for separation to obtain the analyte, and the extraction column is simultaneously rinsed and regenerated.

[0086] S23. Analyze the analyte to obtain a typical spectrum.

[0087] Correspondingly, in S2, a one-dimensional eluent, a two-dimensional eluent, and a cleaning solution are set to respond to the corresponding elution program to pump the sample solution into the first chromatographic system, pump the initial product into the second chromatographic system, and clean the extraction column and the analytical column.

[0088] In this embodiment, the extraction column is an Outstanding SPE column with dimensions of 3.0 × 30 mm and a diameter of 25 μm; the analysis column is an Outstanding C18 column with dimensions of 4.6 mm × 150 mm and a diameter of 5 μm; the column temperature of both the extraction and analysis columns is 25–35 °C (preferably 30 °C in this embodiment).

[0089] Further, the one-dimensional eluent comprises a composition of methanol and water, and a composition of acetonitrile and aqueous phosphoric acid; the two-dimensional eluent comprises acetonitrile and aqueous phosphoric acid; and the washing solution is water. The flow rate of the one-dimensional eluent is 0.8–2.0 mL / min, the flow rate of the two-dimensional eluent is 0.9–1.1 mL / min, and the flow rate of the washing solution is 0.05–0.4 mL / min.

[0090] Correspondingly, the elution procedure for the one-dimensional eluent is as follows:

[0091] The flow rate was 0.8 mL / min for 0–1 min, and the volume ratio of methanol to water in the one-dimensional eluent was 1:9.

[0092] The eluent was prepared for 2–4 min at a flow rate of 2.0 mL / min, with a volume ratio of acetonitrile to phosphoric acid water of 3:1 in the one-dimensional eluent.

[0093] The eluent was prepared for 6–8 min at a flow rate of 2.0 mL / min, with a methanol to water volume ratio of 1:9 in the one-dimensional eluent.

[0094] The eluent was prepared for 8.1–12 min at a flow rate of 0.8 mL / min, with a methanol to water volume ratio of 1:9 in the one-dimensional eluent.

[0095] Correspondingly, the elution procedure for the two-dimensional eluent is as follows:

[0096] The flow rate was 1.0 / min from 0 to 12 min, and the volume ratio of acetonitrile to phosphoric acid water in the two-dimensional eluent was 3:1.

[0097] Correspondingly, the elution procedure for the cleaning solution is as follows:

[0098] 0–1 min, flow rate 0.4 mL / min.

[0099] 2–8 min, flow rate 0.05 mL / min.

[0100] 8.1–12 min, flow rate 0.4 mL / min.

[0101] In the first chromatographic system, the analyte solution is transported to the extraction column within a 0–1 min window for enrichment, purification, and impurity removal to form the initial product. The 1 min enrichment, purification, and impurity removal time was determined based on the physicochemical properties of amiodarone and extensive experimental results. During this period, a large volume of aqueous one-dimensional eluent (1:9 ratio of methanol and water) washes away highly polar substances or water-soluble impurities from the analyte. Subsequently, the initial product is transported to the analytical column via a cut-off valve and return valve within a 1–2 min window.

[0102] It is also worth noting that the composition of the one-dimensional eluent is adjusted to be the same as that of the two-dimensional eluent between 2 and 4 minutes. This is because it can effectively clean the endogenous impurities, low-polarity impurities, or lipid-soluble impurities interfering with the extraction column (due to the interference of amiodarone by its deethylated derivative and its unique low polarity). Between 6 and 8 minutes, the extraction column is rinsed again with methanol and water in a 1:9 ratio to remove residual acetonitrile and phosphoric acid, ultimately restoring the extraction column to its initial conditions and preparing it for the next injection detection.

[0103] Correspondingly, the flow rate of the cleaning solution is reduced during 2-8 minutes to minimize its dilution of the one-dimensional eluent and ensure the cleaning effect of the one-dimensional eluent.

[0104] Furthermore, in the second chromatographic system, acetonitrile, which has a lower polarity than methanol, is selected to enhance the elution capacity, so as to rapidly elute the highly hydrophobic amiodarone. At the same time, it can further reduce the organic content in the eluent, reduce the column pressure (the viscosity of the mixture of acetonitrile and water is low), and improve the analytical efficiency and the separation efficiency of the chromatographic column.

[0105] Phosphoric acid water is used to provide an acidic environment, which protonates the diethylamino (tertiary amine) at the end of the amiodarone side chain to form a cation, thereby enhancing polarity, reducing hydrophobic interaction with the analytical column, shortening retention time, and improving peak shape (reducing tailing). Under these conditions, the typical spectrum obtained has a good peak shape and a tailing factor of around 1.0.

[0106] Meanwhile, the two-dimensional eluent uses isocratic elution, which further reduces detection time compared to gradient elution, achieving the goal of rapid monitoring. It should be noted that the flow rate of the two-dimensional eluent, in addition to 1.0 mL / min, can be adjusted according to the concentration of amiodarone, for example: 0.9 mL / min, 1.1 mL / min.

[0107] S3. Calculate the content of amiodarone in the test solution based on the typical spectrum.

[0108] Based on the peak area of ​​amiodarone corresponding to the standard solution, a standard curve of amiodarone was plotted. The peak area of ​​amiodarone corresponding to the serum sample solution was analyzed, and the content of amiodarone in the serum sample solution was calculated using the external standard method.

[0109] The external standard method is based on the formula: concentration of amiodarone in serum sample = peak area of ​​amiodarone in serum sample × concentration of amiodarone in standard × dilution factor / peak area of ​​amiodarone standard.

[0110] In one specific embodiment, a 0.533 μg / ml standard solution was transferred into a sample vial, and injection volumes of 8 μl, 20 μl, 40 μl, 80 μl, and 100 μl were obtained according to the procedure and chromatograms shown in Table 1, respectively, to obtain linear chromatograms of the standard. The linear chromatograms of the standard are shown below. Figures 1a-1e As shown in Table 2, the specific parameters of the solution are as follows.

[0111] Table 2

[0112] Injection volume μl Actual concentration μg / ml Corresponding blood drug concentration μg / ml Peak area mAU·min 8 0.08525 0.4264 11.031 20 0.2132 1.066 32.386 40 0.4264 2.132 67.544 80 0.8528 4.264 140.324 100 1.066 5.33 176.411

[0113] The monitored concentration range of amiodarone was 0.4264-5.33 μg / ml. Based on the target peak area in the chromatogram, a standard curve was fitted with the standard sample solution concentration as the x-axis and the peak area as the y-axis. The standard curve is shown below. Figure 2 As shown.

[0114] The curve equation is: y = 168.80640x - 3.71121, and the linear correlation coefficient is: R 2 =0.99997. This demonstrates that the monitoring method has high accuracy.

[0115] Based on this, further auxiliary experiments were conducted to verify the accuracy of the monitoring method.

[0116] Experiment 1: Specificity Experiment

[0117] Transfer the acetonitrile solution into the sample vial and inject the sample according to the procedure in Table 1. Inject three times consecutively, and the resulting blank test chromatogram is shown below. Figure 3a As shown. Similarly, by sequentially injecting the standard solution, blood sample solution, and blank sample solution, and following the same method sequence, the standard test chromatogram, blood sample test chromatogram, and blank test chromatogram are obtained sequentially, which verifies the specificity of the method. The standard test chromatogram is shown below. Figure 3b As shown, the blank blood sample test chromatogram is as follows: Figure 3c As shown, the blood sample test chromatogram is as follows: Figure 3d As shown.

[0118] Depend on Figures 3a-3d Analysis of typical spectra showed that no amiodarone interference peaks appeared in either the blank test spectrum or the blank blood sample test spectrum. The resolution of the impurity peak closest to amiodarone in the blood sample test spectrum was greater than 1.5, which verified that the two-dimensional liquid chromatography system and parameter settings have strong specificity, that is, this method has good applicability and can be used for subsequent blood sample monitoring and analysis.

[0119] Experiment 2: Reproducibility Experiment (Precision Experiment)

[0120] Transfer the 0.533 μg / ml standard solution into the sample vial and inject it according to the procedure in Table 1. Inject six times consecutively to obtain the standard solution chromatogram. Calculate the RSD value of the target peak area retention time and peak area in the six-injection chromatogram. Generally, the RSD value should be less than or equal to 2% to meet the requirements.

[0121] Spectrum of standard solution as follows Figures 4a-4f As shown, Figures 4a-4f The corresponding data is detailed in Table 3.

[0122] Table 3

[0123] Retention time / min Peak area / mAU·min First injection 10.228 81.898 Second injection 10.219 82.716 Third injection 10.214 82.774 Fourth injection 10.212 82.723 Fifth needle 10.211 82.931 Sixth needle 10.220 82.661 mean 10.219 82.617 RSD% 0.056 0.441

[0124] As shown in Table 3, the precision of this experiment meets the requirements.

[0125] Experiment 3: Limit of Quantitation Experiment

[0126] Transfer the 0.533 μg / ml standard solution into the sample vial and inject it according to the procedure shown in Table 1. Measure the chromatogram until the peak height of the target peak is 10 times the baseline noise. Record the peak area and calculate the corresponding drug mass based on the injection volume and concentration. This is the limit of quantitation.

[0127] The diagram is as follows Figure 5a As shown, Figure 5aThe corresponding data is detailed in Table 4.

[0128] Table 4

[0129] Concentration (μg / ml) 0.533 Injection volume / μl 30 Retention time / min 10.17 Peak area / (mAU·min) 1.731 mass number / μg 319.8 Peak height / mAU 0.129

[0130] It should be noted that the baseline noise in this experiment refers to the fluctuation of a relatively stable baseline within 10 minutes, i.e., the peak height of the baseline fluctuation. The corresponding baseline noise data is shown in Table 5.

[0131] Table 5

[0132] Channel Name noise drift absorbance at 242nm wavelength 0.0131 (mAU) 0.0073 (mAU / min)

[0133] Experiment 4: Detection Limit Experiment

[0134] Transfer the 0.533 μg / ml standard solution into the sample vial and inject it according to the procedure shown in Table 1. Measure the spectrum until the peak height of the target peak is three times the baseline noise. Record the peak area when the peak height is three times the baseline noise. Calculate the corresponding drug mass based on the injection volume and concentration; this is the detection limit.

[0135] The diagram is as follows Figure 5b As shown, Figure 5b The corresponding data is detailed in Table 6.

[0136] Table 6

[0137] Concentration (μg / ml) 0.533 Injection volume / μl 9 Retention time / min 10.143 Peak area / (mAU·min) 0.309 mass number / μg 95.94 Peak height / mAU 0.041

[0138] Experiment 5: Stability Experiment

[0139] Transfer a 0.533 μg / ml blood sample solution into a sample vial. Following the procedure shown in Table 1, administer one injection at 0h, 2h, 4h, 6h, and 8h respectively. The resulting blood sample chromatograms are shown below. Figures 6a-6e As shown, the target peak area RSD value is calculated based on the blood sample spectrum to determine the stability of the drug over a certain period of time. Figures 6a-6e The corresponding data is shown in Table 7.

[0140] Table 7

[0141]

[0142] As shown in Table 7, the stability of this experiment meets the requirements.

[0143] Experiment 6: Spike Recovery Experiment

[0144] Six blood sample solutions of different concentrations were prepared, with actual concentrations of C1 = 0.2133 μg / ml, C2 = 0.3198 μg / ml, C3 = 0.3731 μg / ml, C4 = 0.4264 μg / ml, C5 = 0.4797 μg / ml, and C6 = 0.533 μg / ml, respectively. These solutions were transferred into sample vials and injected according to the pre-set procedure. Two injections were performed for each concentration, with an injection volume of 50 μl. The resulting chromatograms are shown below. Figures 7a-7d , Figures 8a-8d , Figures 9a-9d As shown, the mean peak area of ​​the target peak for each concentration is calculated. Prepare one reference solution with a concentration of C. 标 =0.533 μg / ml, transferred into a sample vial, and injected according to the set procedure, two injections were performed, with an injection volume of 50 μl. The obtained chromatogram is shown below. Figure 10a , Figure 10b As shown, the average area of ​​the target peak is calculated. Based on the principle that the ratio of concentrations equals the ratio of peak areas, the following formula applies:

[0145]

[0146] The concentration C of the six blood samples was calculated separately. x1 C x2 C x3 C x4 C x5 C x6 The recovery rates of the six blood sample solutions were then obtained by dividing the measured concentration Cxi of the blood sample solution by the actual concentration Ci. The mean recovery rate and the RSD (n=6) of the recovery rate were calculated. The recovery rate should be between 95% and 105%.

[0147] Figures 7a-7d , Figures 8a-8d , Figures 9a-9d and Figure 10a , Figure 10b The corresponding data is detailed in Table 8.

[0148] Table 8

[0149]

[0150]

[0151] As shown in Table 7, the recovery rate meets the requirements.

[0152] Experiment 7: Content Determination Experiment

[0153] A sample solution with a known concentration of 0.533 μg / ml was prepared and injected according to the prescribed procedure. The injection volume was 50 μl, and two consecutive injections were performed. The resulting chromatogram of the sample is shown below. Figure 10c As shown.

[0154] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Identical components are represented by the same reference numerals. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for monitoring amiodarone blood concentration, characterized in that, Includes the following steps: S1. Prepare the solution to be tested; S2. Two-dimensional liquid chromatography for the detection of the test solution; S2 specifically includes: S21. The sample solution is pumped into the extraction column of the first chromatographic system to complete the enrichment, purification and impurity removal of the target analyte, and to obtain the initial product. S22. The initial product on the extraction column is pumped into the analytical column of the second chromatographic system for separation to obtain the analyte, and the extraction column is simultaneously rinsed and regenerated. S23. Analyze the analyte to obtain a typical spectrum; In S2, a one-dimensional eluent, a two-dimensional eluent, and a cleaning solution are set up to respond to the corresponding elution program, so as to pump the sample solution into the first chromatographic system, pump the initial product into the second chromatographic system, and clean the extraction column and the analytical column. The flow rate of the one-dimensional eluent is 0.8–2.0 mL / min, the flow rate of the two-dimensional eluent is 0.9–1.1 mL / min, and the flow rate of the washing solution is 0.05–0.4 mL / min. The one-dimensional eluent comprises a composition of methanol and water, and a composition of acetonitrile and aqueous phosphoric acid; the two-dimensional eluent comprises a composition of acetonitrile and aqueous phosphoric acid; and the cleaning solution is water. S3. Calculate the content of amiodarone in the test solution based on the typical spectrum.

2. The method for monitoring amiodarone blood concentration as described in claim 1, characterized in that, The elution procedure for the one-dimensional eluent is as follows: The flow rate was 0.8 mL / min from 0 to 1 min, and the volume ratio of methanol to water in the one-dimensional eluent was 1:

9. 2–4 min, flow rate 2.0 mL / min, volume ratio of acetonitrile to phosphoric acid water in one-dimensional eluent 3:1; The flow rate was 2.0 mL / min for 6–8 min, and the volume ratio of methanol to water in the one-dimensional eluent was 1:

9. The eluent was prepared for 8.1–12 min at a flow rate of 0.8 mL / min, with a methanol to water volume ratio of 1:9 in the one-dimensional eluent.

3. The method for monitoring amiodarone blood concentration as described in claim 2, characterized in that, The elution procedure for the cleaning solution is as follows: 0–1 min, flow rate 0.4 mL / min; 2–8 min, flow rate 0.05 mL / min; 8.1–12 min, flow rate 0.4 mL / min.

4. A method for monitoring amiodarone blood concentration as described in claim 1 or 2, characterized in that, The elution procedure for the two-dimensional eluent is as follows: The flow rate was 1.0 mL / min for 0–12 min, and the volume ratio of acetonitrile to phosphoric acid water in the two-dimensional eluent was 3:

1.

5. The method for monitoring amiodarone blood concentration as described in claim 1, characterized in that, The extraction column is an Outstanding SPE column with dimensions of 3.0 mm × 30 mm and a diameter of 25 μm; the analysis column is an Outstanding C18 column with dimensions of 4.6 mm × 150 mm and a diameter of 5 μm; the column temperature of both the extraction and analysis columns is 25–35 °C.

6. The method for monitoring amiodarone blood concentration as described in claim 1, characterized in that, S1 specifically includes: S11. Dissolve amiodarone in acetonitrile to prepare a mother liquor; S12. Mix the mother liquor with pure water, then dilute with acetonitrile to obtain the standard solution; S13. Mix the mother liquor with bovine serum, then add protein precipitant to dilute, centrifuge and take the supernatant, filter the supernatant to obtain blood sample solution. S14. Take bovine serum and dilute it with protein precipitant. After centrifugation, take the supernatant and filter it to obtain a blank sample solution.

7. The method for monitoring amiodarone blood concentration as described in claim 6, characterized in that, The concentrations of amiodarone in the standard solution and the blood sample solution are the same and are both 0.1–1 μg / ml.

8. The method for monitoring amiodarone blood concentration as described in claim 6, characterized in that, The protein precipitant is set as an acetonitrile solution.

9. A monitoring preparation, characterized in that, The method for monitoring amiodarone blood concentration according to any one of claims 1-8, wherein the monitoring preparation comprises a first preparation and a second preparation, the first preparation comprising a composition of methanol and water, and the second preparation comprising a composition of acetonitrile and aqueous phosphoric acid.

10. The application of the monitoring formulation according to claim 9 in monitoring amiodarone blood concentration.

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