Method for determining blood concentration of triflurazine in human blood and liquid chromatograph

Abstract

The invention provides a method for determining the blood concentration of triflurazine in human blood and a liquid chromatograph. The method comprises the following steps: preparing a plurality of standard working solutions with the concentration in a range of 25-150ng / ml; testing the standard working solutions with different concentrations by using a liquid chromatograph, and fitting a standard curve equation of the standard working solutions; centrifuging the serum of the blood sample to be detected, and taking the centrifuged supernatant as the sample to be detected; the method comprises the following steps: testing a to-be-tested sample by using a liquid chromatograph, obtaining a chromatogram of a blank sample, and calculating the blood concentration of the triflurazine in the to-be-tested sample by combining the chromatogram of the to-be-tested sample and the standard curvilinear equation. According to the method for measuring the blood concentration of the triflurazine in the human blood, interferents in a sample can be effectively reduced and the purity of an analyte can be improved by adjusting the proportion of the mobile phase, so that the detection efficiency and the analysis accuracy of the blood concentration of the triflurazine are improved.

Description

Technical Field

[0001] The present application relates to the field of medical blood drug concentration detection, and in particular to a method for determining the blood drug concentration of trifluoperazine in human blood and a liquid chromatograph. Background Art

[0002] Trifluoperazine is an antipsychotic drug primarily used clinically to treat symptoms such as agitation, hallucinations, and delusions caused by conditions like schizophrenia and manic-depressive illness. The total concentration of the drug in plasma after absorption is the blood concentration, and the intensity of the drug's effect is positively correlated with the blood concentration. The patient's blood concentration should be controlled within a safe range. Levels above this safe and effective range can cause toxic side effects or even severe poisoning, while levels below this range have no therapeutic effect.

[0003] Chromatography and mass spectrometry are the primary methods used in China and abroad to measure trifluoperazine concentrations in human blood. However, these methods suffer from complex pre-treatment processes, resulting in time-consuming and labor-intensive processes and limited clinical application. Currently, immunoassays, fluorescence polarization immunoassays (FPIAs), homogeneous enzyme immunoassays (EMITs), and spectroscopy are commonly used to measure trifluoperazine concentrations in clinical settings. However, limitations in sensitivity and specificity have led to a relative decline in their application.

[0004] In view of this, it is necessary to design a method for determining the blood concentration of trifluoperazine in human blood and a liquid chromatograph to solve one of the above problems. Summary of the Invention

[0005] The present application provides a method for determining the blood concentration of trifluoperazine in human blood and a liquid chromatograph. This method has the advantages of fast analysis and detection speed, simple operation, high detection sensitivity, good reproducibility, and meets clinical application requirements.

[0006] In order to achieve the above objectives, the technical solutions provided by this application are as follows: The present application provides a method for determining the blood concentration of trifluoperazine in human blood, wherein the method comprises: Prepare standard working solutions by dissolving trifluoperazine standard in pure methanol and diluting to obtain several standard working solutions with concentrations ranging from 25 to 150 ng / ml; Use liquid chromatography to test standard working solutions of different concentrations and fit the standard curve equation of the standard working solution; Prepare the sample to be tested, centrifuge the serum of the blood sample to be tested, and take the supernatant after centrifugation as the sample to be tested; The sample to be tested is tested using a liquid chromatograph, specifically including: The mobile phase of the extraction column was methanol in phase A and pure water in phase B, with a volume ratio of phase A to phase B of (10-30):(70-90). The mobile phase of the analytical column was a mixture of acetonitrile and potassium dihydrogen phosphate solution in phase C, and pure water in phase D, with a volume ratio of phase C to phase D of 94:6. The flow rate of the first constant flow pump was 0.5 ml / min, the flow rate of the second constant flow pump was 0.7 ml / min, and the flow rate of the third constant flow pump was 1.7 ml / min. The elution procedure corresponding to the extraction column is: At 0-2 min, the volume ratio of phase A to phase B was (10-30): (70-90); At 2-4 minutes, the volumes of phase A and phase B are (10-30):(70-90), the flow rate of the first constant flow pump is changed to 1.7 ml / min, the flow rate of the third constant flow pump is changed to 0.1 ml / min, the six-way valve is switched, and the flow direction of the second constant flow pump is reversed to elute the extraction column; At 4-5 min, the volumes of phase A and phase B are (10-30):(70-90); At 5-5.1 min, the volume of phase A and phase B became 90:10; 5. At 1-8 min, the volume of phase A and phase B is 90:10; At 8-9 min, the volumes of phase A and phase B changed to (10-30): (70-90); At 9-9.1 minutes, the volumes of phase A and phase B change to (10-30):(70-90), the flow rate of the first constant flow pump changes to 0.5 ml / min, and the flow rate of the third constant flow pump changes to 1.7 ml / min; preparations are made for the next sample to be tested; At 9.1-11 min, the volume ratio of phase A to phase B becomes (10-30): (70-90); The data analysis specifically includes: obtaining a blank sample chromatogram, and calculating the blood concentration of trifluoperazine in the test sample in combination with the chromatogram of the test sample and the standard curve equation.

[0007] Furthermore, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution in phase C is (40-50): (50-60), wherein the concentration of potassium dihydrogen phosphate is 0.06 mol / L.

[0008] Furthermore, each liter of potassium dihydrogen phosphate solution contains 5 ml of triethylamine, and the pH value of phase C is 3-7.

[0009] Furthermore, 5 mg of trifluoperazine standard was accurately weighed, dissolved in pure methanol and diluted to 10 mL to obtain a primary standard stock solution with a concentration of 0.5 mg / ml. The primary standard stock solution was then diluted with pure methanol to obtain a secondary standard stock solution with a concentration of 5 ug / ml. 3ul, 6ul, 9ul, 12ul, 15ul, and 18ul of secondary standard stock solution were measured respectively, and 600uL of pure water was added to obtain mixed solutions of different concentrations. Then, 897ul, 894ul, 891ul, 888ul, 885ul, and 882ul of sample release agent were added to the corresponding mixed solutions to obtain 6 standard working solutions of different concentrations.

[0010] Furthermore, a liquid chromatograph is used to test standard working solutions of different concentrations to obtain chromatograms of standard working solutions of different concentrations. The concentration of each standard working solution is set as the horizontal coordinate X, and the peak area of ​​the chromatogram corresponding to each concentration is set as the vertical coordinate Y. Linear regression fitting is performed to obtain the corresponding standard curve unary equation y=a*x+b, and a and b are obtained.

[0011] Furthermore, preparing the sample to be tested also includes: first centrifuging the blood after taking the medicine to obtain serum, then adding the sample release agent acetonitrile to the extracted serum, and successively performing vortex treatment and high-speed centrifugation, and then taking the supernatant after centrifugation as the sample to be tested, and the volume of the added sample release agent is not less than the volume of the extracted serum.

[0012] Furthermore, the high-speed centrifugation speed is 10000 rpm and the time is 10 min.

[0013] Furthermore, the blank sample chromatogram is obtained by testing a blank sample, and preparing the blank sample includes: first centrifuging normal blood to obtain normal serum, then adding the sample release agent acetonitrile to the extracted normal serum, and sequentially performing vortex treatment and high-speed centrifugation treatment, and then taking the supernatant after centrifugation as the blank sample.

[0014] Furthermore, the column temperature was 35°C.

[0015] The present application also provides a liquid chromatograph using the above-mentioned method for determining the blood concentration of trifluoperazine in human blood, wherein the liquid chromatograph includes an extraction column and an analytical column, the extraction column model is MFPH-1, and the parameters are: 4.0mm*20mm, 5um; the analytical column model is C18, and the parameters are: 4.6mm*100mm, 5um.

[0016] Compared with the prior art, the beneficial effect of the present application is that the method for determining the blood concentration of trifluoperazine in human blood of the present application can effectively reduce the interference in the sample by adjusting the ratio of the mobile phase, improve the purity of the analyte, and enhance the detection efficiency and analysis accuracy of the blood concentration of trifluoperazine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a structural schematic diagram of the liquid chromatograph of this application.

[0018] Figure 2 The present invention is a flow chart of an embodiment of a method for determining the concentration of trifluoperazine in human blood.

[0019] Figure 3 This is the chromatogram of the standard working solution in Example 1 of the present application.

[0020] Figure 4 yes Figure 3 The standard working curve of trifluoperazine peak area and concentration in the chromatogram of the standard working solution was fitted.

[0021] Figure 5 This is the chromatogram of the standard working solution with a blood drug concentration of 75 ng / ml in Example 1.

[0022] Figure 6 This is the chromatogram of the blood sample with a blood drug concentration of 75 ng / ml in Example 1.

[0023] Figure 7 is the chromatogram of the blank sample in Example 1.

[0024] Figure 8 1 is a chromatogram of six standard working solutions with a blood drug concentration of 75 ng / ml in Example 1.

[0025] Figure 9 This is a chromatogram of the low-concentration standard working solution in the detection limit verification experiment in Example 1.

[0026] Figure 10 This is a chromatogram of the low-concentration standard working solution in the quantitative limit verification experiment in Example 1.

[0027] Figure 11 It is a chromatogram of the standard working solution of the comparative example with a blood drug concentration of 75 ng / ml.

[0028] Among them, 1-mobile phase system, 11-first constant flow pump, 12-second constant flow pump, 13-third constant flow pump, 14-six-way valve, 2-injection system, 3-separation system, 31-extraction column, 32-analytical column, 4-detection system, 5-control system. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts should fall within the scope of protection of this application.

[0030] In the various figures of the present application, for the sake of convenience, some dimensions of structures or parts are exaggerated relative to other structural parts. Therefore, they are only used to illustrate the basic structure of the subject matter of the present application.

[0031] The present application provides a method for determining the concentration of trifluoperazine in human blood and a liquid chromatograph for measuring the concentration of trifluoperazine in human blood using the method. Figure 1 As shown, the liquid chromatograph includes a mobile phase system 1 , a sample injection system 2 , a separation system 3 , a detection system 4 and a control system 5 .

[0032] The separation system 3 includes an extraction column 31 and an analysis column 32, wherein the extraction column 31 is mainly used to enrich trifluoperazine. The extraction column 31 in this application is the MF PH-1 model of Hunan Innovation General Instrument Co., Ltd., and its parameters are a column inner diameter of 4.0 mm, a column length of 20 mm, and a filler particle size of 5 μm.

[0033] The analytical column 32 is mainly used for fine separation of the trifluoperazine substance transferred from the extraction column 31. The finely separated trifluoperazine substance enters the detection system 4. The analytical column 32 in this application is C18 from Hunan Innovation General Instrument Co., Ltd., and its parameters are a column inner diameter of 4.6 mm, a column length of 100 mm, and a filler particle size of 5 μm.

[0034] The mobile phase system 1 includes a first constant flow pump 11 for communicating with the injection system 2, a second constant flow pump 12 for communicating with the analytical column 32, a third constant flow pump 13 for diluting the sample, and a six-way valve 14 connected to each pump. The first constant flow pump 11, the second constant flow pump 12 and the third constant flow pump 13 are all quaternary constant flow pumps.

[0035] The first constant flow pump 11 provides mobile phase for the extraction column 31, the second constant flow pump 12 provides mobile phase for the analysis column 31, and the third constant flow pump 13 is connected to the injection system 2, and its main function is to dilute the injected sample and reduce the organic phase ratio and impurity concentration in the sample.

[0036] Different valves of the six-way valve 14 are respectively connected to the separation system 3 and each constant flow pump. By changing the valve position of the six-way valve 14, the flow direction of the liquid is changed, thereby controlling the completion of the entire test process.

[0037] The detection system 4 includes an ultraviolet detector, which is used to convert the collected component signals of trifluoperazine into electrical signals and then transmit them to the control system 5.

[0038] like Figure 2As shown, the method includes: preparing a standard working solution; testing the standard working solution of different concentrations using a liquid chromatograph and fitting a standard curve equation for the standard working solution; preparing a sample to be tested; setting the liquid phase parameters of the liquid chromatograph and then testing the sample; and then performing data analysis, obtaining a blank sample chromatogram, combining the chromatogram of the sample to be tested and the standard curve equation to calculate the blood concentration of trifluoperazine in the sample to be tested. By adjusting the ratio of the mobile phase, the method of the present application can effectively reduce interfering substances in the sample, improve the purity of the analyte, and enhance the detection efficiency and analysis accuracy of trifluoperazine blood concentration.

[0039] Furthermore, preparing the standard working solution includes dissolving a trifluoperazine standard in pure methanol to obtain a primary standard stock solution, further diluting the solution with pure methanol to obtain a secondary standard stock solution, and then further diluting the solution with pure water and a sample release agent to obtain several standard working solutions with concentrations ranging from 25 to 150 ng / ml, which are used to obtain a calibration curve equation. The concentration of trifluoperazine in the sample to be tested is approximately within the range of 25 to 150 ng / ml, and a more accurate concentration can be obtained based on the calibration curve equation.

[0040] Specifically, 5 mg of trifluoperazine standard was accurately weighed and placed into a 10 mL volumetric flask. The solution was dissolved in pure methanol and diluted to 10 mL to obtain a primary standard stock solution with a concentration of 0.5 mg / mL. The primary standard stock solution was then diluted with pure methanol to obtain a secondary standard stock solution with a concentration of 5 ug / mL. Using a pipette, 3 μl, 6 μl, 9 μl, 12 μl, 15 μl, and 18 μl of the secondary standard stock solution were pipetted into corresponding sterile EP tubes. 600 μl of pure water was added to each solution. 897 μl, 894 μl, 891 μl, 888 μl, 885 μl, and 882 μl of the sample release agent (acetonitrile) were added to the corresponding solutions into sterile EP tubes. The solution was vortexed for 1 minute and then centrifuged at high speed (10,000 rpm for 10 minutes) to obtain trifluoperazine standard working solutions with a concentration range of 25-150 ng / mL.

[0041] It is understandable that since the blood drug concentration of the blood sample is tested using a standard working curve prepared using standard working solutions of various concentrations, the process of preparing the blood sample is 600 ul of serum plus 900 ul of sample release agent, which is diluted 2.5 times. Therefore, the concentration of trifluoperazine in the standard working solution needs to be converted into the concentration of trifluoperazine in the blood sample, and the standard working solution of each concentration is multiplied by 2.5, that is, the concentrations of the six trifluoperazine standard working solutions are: 25 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 125 ng / ml, and 150 ng / ml, respectively.

[0042] Use a liquid chromatograph to test standard working solutions of different concentrations to obtain chromatograms of standard working solutions of different concentrations. Set the concentration of each standard working solution as the horizontal coordinate X, and the peak area of ​​the chromatogram corresponding to each concentration as the vertical coordinate Y, and perform linear regression fitting to obtain the corresponding standard curve equation y=a*x+b, and obtain a and b. Among them, the closer a is to 1, the better the linearity of the standard curve equation, and the more accurate the concentration of the sample to be tested obtained by the standard curve equation.

[0043] To prepare the test sample, blood from a patient taking trifluoperazine is first centrifuged to obtain serum. Acetonitrile, a sample-releasing agent, is then added to the extracted serum to facilitate precipitation of macromolecules such as proteins in the blood sample. The sample is then vortexed and centrifuged at high speed. The supernatant after centrifugation is then used as the test sample. It is understood that blood sampling occurs after the patient has reached steady-state levels after oral trifluoperazine administration.

[0044] Preferably, the volume of the added sample release agent is not less than the volume of the extracted serum, so that trifluoperazine can be fully released, thereby improving the accuracy of the test results.

[0045] In this example, 600 μl of serum after centrifugation was taken, 900 μl of sample release agent was added, and the mixture was thoroughly mixed and centrifuged to obtain the supernatant after centrifugation as the test sample. A vortex was used for thorough mixing, the vortex lasted for 1 min, and high-speed centrifugation (10,000 rpm, 10 min) was performed.

[0046] For serum that has not been used or cannot be tested in time, it should be stored at -20℃. When it is to be used again, it should be thawed into liquid at room temperature, shaken or vortexed, and 600ul of serum should be transferred with a pipette. 900ul of sample release agent (acetonitrile) should be added. After vortexing for 1min, high-speed centrifugation (10000rpm, 10min) should be carried out, and the supernatant should be taken as the sample to be tested.

[0047] When using this test method for the first time, prepare a blank sample and obtain a blank sample chromatogram. The blank sample has the same base as the test sample, lacking only the target substance, trifluoperazine. Subtracting the blank sample's chromatogram from the test sample's chromatogram during subsequent data analysis can reduce interference from interfering substances. If a blank sample chromatogram is stored in the liquid chromatograph, use the blank sample chromatogram directly after the test sample is completed.

[0048] In this application, a blank sample was prepared by extracting blood from a healthy individual who had not taken trifluoperazine. 600 μl of serum from healthy individuals was added to 900 μl of a sample release agent, thoroughly mixed using a vortexer, and then centrifuged to obtain the supernatant, which served as the blank sample. The vortexing lasted for 1 minute, followed by high-speed centrifugation (10,000 rpm for 10 minutes). Using the same processing method as the test sample can reduce test errors caused by sample processing.

[0049] Before testing the sample using the liquid chromatograph, the test parameters of the liquid chromatograph are set first, specifically, the temperature of the column oven is set to 35° C. to ensure that the temperature of the extraction column 31 and the analysis column 32 is kept at a constant temperature of 35° C.

[0050] Phase A of the mobile phase of the extraction column 31 is methanol and phase B is pure water, and the volume ratio of phase A to phase B is set to (10-30): (70-90), that is, the volume of phase A is (10-30)% and the volume of phase B is (70-90)%.

[0051] Phase C of the mobile phase of the analytical column 32 is a mixture of acetonitrile and potassium dihydrogen phosphate solution, and phase D is pure water. The volume ratio of acetonitrile to potassium dihydrogen phosphate solution in phase C is set to (40-50): (50-60). Preferably, the volume of acetonitrile accounts for 50%, the volume of potassium dihydrogen phosphate solution is 50%, and the concentration of potassium dihydrogen phosphate is 0.06 mol / L. Acetonitrile, as an organic solvent, has low viscosity and good solubility, which can increase the transmission speed of the substance in the analytical column 32, thereby reducing the analysis time and ensuring high separation efficiency. In addition, the premixed solution of acetonitrile and potassium dihydrogen phosphate can optimize the peak shape and separation of the chromatographic peak, making the analysis results more accurate and reliable.

[0052] Preferably, each liter of potassium dihydrogen phosphate solution contains 5 ml of triethylamine to improve separation, adjust pH value, reduce tailing of alkaline components in the sample, etc.

[0053] Preferably, the pH value of phase C is adjusted to 3-7 by phosphoric acid, preferably, the pH value of phase C is 3, to maintain the chemical balance and stability of the reaction system. The buffer resists external acid-base interference through the dissociation balance of its buffer pair and maintains the pH stability of the system.

[0054] In the elution procedure, the volume of phase C is 94% and the volume of phase D is 6%, so that the acetonitrile concentration in the final mixed liquid entering the analytical column 32 is 94%×50%=47%, and the concentration of other mixed liquids is 94%×50%+6%=53%, so that the strength of the organic phase entering the analytical column 32 is stable, thereby maintaining a stable pH value of the mixed liquid.

[0055] Set the flow rate of the first constant flow pump 11 to 0.5 ml / min, the flow rate of the second constant flow pump 12 to 0.7 ml / min, and the flow rate of the third constant flow pump 13 to 1.7 ml / min. Start the test according to the elution program corresponding to the extraction column 31, as follows: At 0 min, the liquid chromatograph is in the initial state, the state of the mobile phase is the set state, and the flow rate of the pump remains unchanged. Specifically, the volume of phase A is (10-30)%, the volume of phase B is (70-90)%, the flow rate of the first constant flow pump 11 is 0.5 ml / min, and the flow rate of the third constant flow pump 13 is 1.7 ml / min. During the entire elution process, the flow rate of the second constant flow pump 12 is always 0.7 ml / min.

[0056] During the 0-2 minute period, the volumes of phase A and phase B are (10-30):(70-90), specifically: the volume of phase A is (10-30)%, and the volume of phase B is (70-90)%. The flow rate of the first constant flow pump 11 is 0.5 ml / min, and the flow rate of the third constant flow pump 13 is 1.7 ml / min. This is the process of enriching trifluoperazine in the extraction column 31. This process has a low elution intensity, maintaining the retention of trifluoperazine on the extraction column 31 while flushing water-soluble impurities. The flow rate of the first constant flow pump 11 is relatively low to prevent high flow rates from disturbing the adsorption of trifluoperazine in the sample to be tested.

[0057] At 2-4 minutes, the volumes of phase A and phase B are (10-30):(70-90), and the flow rate of the first constant flow pump 11 becomes 1.7 ml / min. Increasing the flow rate of the first constant flow pump 11 can improve the efficiency of eluting trifluoperazine from the extraction column 31. The flow rate of the third constant flow pump 13 becomes 0.1 ml / min, and the total flow rate is kept slightly different to avoid interference caused by sudden changes in system pressure. The six-way valve is switched, and the flow of the second constant flow pump 12 is eluted in the opposite direction of the extraction column 31; trifluoperazine begins to be transferred to the analytical column 32.

[0058] For 4-5 minutes, the volume ratio of phase A to phase B is maintained at (10-30):(70-90); the volume of phase A is (10-30)% and the volume of phase B is (70-90)%. This maintains a weak elution environment to prevent the loss of the target compound, trifluoperazine. The first constant flow pump 11 continues to maintain a high flow rate, ensuring that impurities in the column pores are completely pushed out, thereby completely removing any residual impurities.

[0059] At 5-5.1 minutes, the volume ratio of phase A to phase B begins to change to 90:10, and the flow rates of the first constant flow pump 11 and the third constant flow pump 13 remain unchanged.

[0060] At 5-8 minutes, the volume of phase A and phase B becomes 90:10, that is, the volume of phase A is 90%, and the volume of phase B is 10%. The ratio of phase A to phase B is changed, and the target trifluoperazine on the extraction column 31 is transferred to the analysis column 32. The extraction column 31 is in the activation stage. The flow rate of the first constant flow pump 11 is changed to 1.7 ml / min, and the flow rate of the third constant flow pump 13 is changed to 0.1 ml / min to fully flush the extraction column 31.

[0061] At 8-8.1 minutes, the volumes of phase A and phase B become (10-30):(70-90); the flow rates of the first constant flow pump 11 and the third constant flow pump 13 remain unchanged.

[0062] At 8.1-9 minutes, the volumes of phase A and phase B become (10-30):(70-90), that is, the volume of phase A is (10-30)%, and the volume of phase B is (70-90)%. The flow rate of the first constant flow pump 11 is 1.7 ml / min, and the flow rate of the third constant flow pump 13 is 0.1 ml / min. At this stage, the analytical column 32 is in the process of accurately separating the target substance trifluoperazine, and the extraction column 31 is still in the activation stage.

[0063] At 9-9.1 minutes, the volumes of phase A and phase B change to (10-30): (70-90), the flow rate of the first constant flow pump 11 changes to 0.5 ml / min, and the flow rate of the third constant flow pump 13 changes to 1.7 ml / min; preparation for the next sample to be tested begins; At 9.1-11 minutes, the volume ratio of phase A to phase B becomes (10-30): (70-90), and the extraction column continues to be flushed, in the preparation stage for the next generation of test samples. At this time, the chromatogram of the sample to be tested has been collected, and the test process of the sample to be tested is completed.

[0064] At 11-13 minutes, the volume ratio of phase A to phase B is (10-30): (70-90), the flow rate of the first constant flow pump 11 is 0.5 ml / min, the flow rate of the third constant flow pump 13 is 1.7 ml / min, and the next sample to be tested begins to be collected.

[0065] The elution program in this application dynamically achieves the balance between enrichment and elution through a gradient program to achieve the sensitivity and specificity goals of the method.

[0066] The following detailed description uses a blood sample as an example. The blood sample preparation process is as follows: 600 μl of normal serum is added to 9 μl of the secondary standard stock solution, followed by 891 μl of the sample release agent, acetonitrile, to a final volume of 1500 μl. Vortex and high-speed centrifuge, and the supernatant is measured as the blood sample. Vortex for at least 1 minute. The high-speed centrifugation parameters are the same as those for the serum centrifugation described above. This means that the blood sample is processed using the same method as the test sample to minimize errors caused by sample handling.

[0067] In Example 1, the elution procedure is as follows: the volume of phase A is 10%, the volume of phase B is 90%, and other parameters are shown in Table 1: Table 1

[0068] Before testing blood samples, a standard curve equation is established. Liquid chromatograph is used to test standard working solutions of different concentrations. The chromatograms corresponding to different concentrations are as follows: Figure 3 As shown in Table 2, the peak area of ​​trifluoperazine was obtained by analysis, and the trifluoperazine standard curve Y=0.2116x-1.4305 was drawn, and the correlation coefficient was 0.9995. Figure 4 As shown, the curve was then used to calculate the content of trifluoperazine in subsequent blood samples.

[0069] Table 2

[0070] The specificity test process of this method is as follows: according to the parameters of Example 1, a liquid chromatograph is used to detect the standard working solution with a concentration of 75 ng / ml, a blood sample, and a blank sample, and the results are as follows: Figure 5 The chromatogram of the standard working solution is shown in Figure 6 The blood sample chromatogram shown, Figure 7 The chromatogram of the blank sample shown is obtained by Figures 5 to 7 As shown, the separation degree of the impurity peak closest to trifluoperazine in the blood sample chromatogram is greater than 1.5, and the retention time of the trifluoperazine peak in the chromatograms of the standard working solution and the blood sample is consistent. There is no interfering peak of trifluoperazine at the corresponding position in the chromatogram of the blank sample. The above verifies that this method has strong specificity and good performance in testing the blood concentration of trifluoperazine.

[0071] The verification process of the reproducibility experiment of this method is as follows: take the standard working solution with a corresponding blood drug concentration of 200 ng / ml, transfer it to six injection bottles, and continuously test six identical samples according to the parameters of Example 1 to obtain six standard working solution chromatograms, as shown in Table 3 and Figure 8 shown.

[0072] Table 3

[0073] From Table 3 and Figure 8 As shown in the figure, the relative standard deviation of the peak area of ​​trifluoperazine in the standard working solution tested six times is 1.0508%, and the reproducibility of this method is good.

[0074] The accuracy of the method was verified by placing the treated blood samples into six sample bottles, testing the six samples according to the parameters of Example 1. The peak areas of trifluoperazine in the chromatograms of the six blood samples are shown in Table 4. The RSD values ​​of the six trifluoperazine peak areas were calculated to be 0.2760%, indicating that the method had good accuracy.

[0075] Table 4

[0076] Among them, the recovery rate of this method is 101.50%, which is calculated by dividing the average value of the trifluoperazine peak area in six blood samples by the average value of the trifluoperazine peak area in six standard working solutions. That is, the concentration of trifluoperazine in the standard working solution is the same as the concentration of trifluoperazine in the blood sample. Based on the measured results, the recovery rate measured by this method is between 95% and 105%, indicating that the method is reliable and applicable.

[0077] The verification process of the detection limit experiment of this method is as follows: prepare a standard working solution with a lower concentration, and then test it according to the parameters of Example 1 until the peak height is 3 times the baseline noise. At this time, record the peak area, and the corresponding concentration at this time is: 2.15ng / ml. Figure 9 shown.

[0078] The verification process of the quantitative limit experiment of this method is as follows: prepare a standard working solution with a lower concentration, and then test it according to the parameters of Example 1 until the measured peak height is 10 times the baseline noise. At this time, the peak area is recorded. The corresponding concentration at this time is: 7.17ng / ml. Figure 10 shown.

[0079] In Example 2, the volume ratio of mobile phase A to phase B of the extraction column 31 was changed to 20:80, and other parameters remained unchanged. The detailed parameters of the elution program are shown in Table 5. The standard working solution corresponding to a blood drug concentration of 75 ng / ml and a blood sample were tested. The test results are shown in Table 7. The recovery rate of Example 2 was 102.50%. The recovery rate measured in Example 2 was between 95% and 105%. This method is accurate and reliable for testing the concentration of trifluoperazine in the blood.

[0080] Table 5

[0081] In Example 3, the volume ratio of mobile phase A to phase B of the extraction column 31 was changed to 30:70, and other parameters remained unchanged. The detailed parameters of the elution program are shown in Table 6. The standard working solution corresponding to a blood drug concentration of 75 ng / ml and a blood sample were tested. The test results are shown in Table 7. The recovery rate of Example 3 was 102.65%. The recovery rate measured in Example 3 was between 95% and 105%. This method is accurate and reliable for testing the concentration of trifluoperazine in the blood.

[0082] Table 6

[0083] Table 7

[0084] Comparative Example 1: The parameters of the mobile phase of the extraction column 31 are the same as those of Example 1. In this example, only the mobile phase of the analytical column 32 is changed. The ratio of acetonitrile to potassium dihydrogen phosphate aqueous solution is 20:80. The flow rate of the second constant flow pump 12 is still 0.8 ml / min, and the injection volume is 300 ul. The wavelength of the ultraviolet detector is set to 260 nm. The target peak cannot be separated within 10 min. Figure 11 As shown, the optimal ratio of the mobile phase acetonitrile to the potassium dihydrogen phosphate aqueous solution in the analytical column 32 of this method is (40-50): (50-60).

[0085] In summary, the method for determining the blood concentration of trifluoperazine in human blood of the present application can effectively reduce the interference in the sample, improve the purity of the analyte, and enhance the detection efficiency and analysis accuracy of the blood concentration of trifluoperazine by adjusting the ratio of the mobile phase.

[0086] It should be understood that although this specification is described according to embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0087] The series of detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present application and are not intended to limit the scope of protection of the present application. Any equivalent embodiments or changes that do not deviate from the technical spirit of the present application should be included in the scope of protection of the present application.

Claims

1. A method for determining the concentration of trifluoperazine in human blood, characterized in that: The method comprises: Prepare standard working solutions by dissolving trifluoperazine standard in pure methanol and diluting to obtain several standard working solutions with concentrations ranging from 25 to 150 ng / ml; Use liquid chromatography to test standard working solutions of different concentrations and fit the standard curve equation of the standard working solution; Prepare the sample to be tested, centrifuge the serum of the blood sample to be tested, and take the supernatant after centrifugation as the sample to be tested; The sample to be tested is tested using a liquid chromatograph, specifically including: The mobile phase of the extraction column was methanol in phase A and pure water in phase B, with the volume ratio of phase A to phase B set to (10-30):(70-90). The mobile phase of the analytical column was a mixture of acetonitrile and potassium dihydrogen phosphate solution in phase C, and pure water in phase D, with the volume ratio of phase C to phase D set to 94:

6. The flow rate of the first constant flow pump was 0.5 ml / min, the flow rate of the second constant flow pump was 0.7 ml / min, and the flow rate of the third constant flow pump was 1.7 ml / min. The elution procedure corresponding to the extraction column is: At 0-2 min, the volume ratio of phase A to phase B was (10-30): (70-90); At 2-4 minutes, the volumes of phase A and phase B are (10-30):(70-90), the flow rate of the first constant flow pump is changed to 1.7 ml / min, the flow rate of the third constant flow pump is changed to 0.1 ml / min, the six-way valve is switched, and the flow direction of the second constant flow pump is reversed to elute the extraction column; At 4-5 min, the volumes of phase A and phase B are (10-30):(70-90); At 5-5.1 min, the volume of phase A and phase B became 90:10; 5. At 1-8 min, the volume of phase A and phase B is 90:10; At 8-9 min, the volumes of phase A and phase B changed to (10-30): (70-90); At 9-9.1 minutes, the volumes of phase A and phase B change to (10-30):(70-90), the flow rate of the first constant flow pump changes to 0.5 ml / min, and the flow rate of the third constant flow pump changes to 1.7 ml / min; preparations are made for the next sample to be tested; The data analysis specifically includes: obtaining a blank sample chromatogram, and calculating the blood concentration of trifluoperazine in the test sample in combination with the chromatogram of the test sample and the standard curve equation.

2. The method for determining the concentration of trifluoperazine in human blood according to claim 1, wherein: The volume ratio of acetonitrile to potassium dihydrogen phosphate solution in phase C is (40-50): (50-60), wherein the concentration of potassium dihydrogen phosphate is 0.06 mol / L.

3. The method for determining the concentration of trifluoperazine in human blood according to claim 2, wherein: Each liter of potassium dihydrogen phosphate solution contains 5 ml of triethylamine, and the pH value of phase C is 3-7.

4. The method for determining the concentration of trifluoperazine in human blood according to claim 1, wherein: Accurately weigh 5 mg of trifluoperazine standard, dissolve it in pure methanol and dilute to 10 mL to obtain a primary standard stock solution with a concentration of 0.5 mg / mL. Then dilute the primary standard stock solution with pure methanol to obtain a secondary standard stock solution with a concentration of 5 μg / mL. 3ul, 6ul, 9ul, 12ul, 15ul, and 18ul of secondary standard stock solution were measured respectively, and 600uL of pure water was added to obtain mixed solutions of different concentrations. Then, 897ul, 894ul, 891ul, 888ul, 885ul, and 882ul of sample release agent were added to the corresponding mixed solutions to obtain 6 standard working solutions of different concentrations.

5. The method for determining the concentration of trifluoperazine in human blood according to claim 1, wherein: Use liquid chromatography to test standard working solutions of different concentrations to obtain chromatograms of standard working solutions of different concentrations. Let the concentration of each standard working solution be the horizontal coordinate X, and the peak area of ​​the chromatogram corresponding to each concentration be the vertical coordinate Y, and perform linear regression fitting to obtain the corresponding standard curve unary equation y=a*x+b, and calculate a and b.

6. The method for determining the concentration of trifluoperazine in human blood according to claim 1, wherein: The preparation of the sample to be tested also includes: first centrifuging the blood after taking the medicine to obtain serum, then adding the sample release agent acetonitrile to the extracted serum, and sequentially performing vortex treatment and high-speed centrifugation, and then taking the supernatant after centrifugation as the sample to be tested, and the volume of the added sample release agent is not less than the volume of the extracted serum.

7. The method for determining the concentration of trifluoperazine in human blood according to claim 6, wherein: The high-speed centrifugation speed was 10000 rpm and the time was 10 min.

8. The method for determining the concentration of trifluoperazine in human blood according to any one of claims 1 to 7, wherein: The blank sample chromatogram is obtained by testing a blank sample. The preparation of the blank sample includes: first centrifuging normal blood to obtain normal serum, then adding a sample release agent, acetonitrile, to the extracted normal serum, and sequentially performing vortex treatment and high-speed centrifugation, and then taking the supernatant after centrifugation as the blank sample.

9. The method for measuring the concentration of trifluoperazine in human blood according to any one of claims 1 to 7, wherein: The column temperature was 35°C.

10. A liquid chromatograph using the method for determining the concentration of trifluoperazine in human blood according to any one of claims 1 to 9, characterized in that: The liquid chromatograph includes an extraction column and an analysis column. The extraction column model is MFPH-1, and the parameters are: 4.0mm*20mm, 5um; the analysis column model is C18, and the parameters are: 4.6mm*100mm, 5um.

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