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

Abstract

The invention provides a method for determining the blood concentration of norzepam 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 80-800ng / 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 plasma concentration of norzepam 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 norzepam 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 norzepam are improved.

Description

Technical Field

[0001] This application relates to the field of medical blood drug concentration detection, and particularly to a method for determining the blood drug concentration of nordazepam in human blood and a liquid chromatograph. Background Art

[0002] Nordazepam has effects such as anti-anxiety, sedation, muscle relaxation, and anti-convulsion, and has the characteristics of fast and complete oral absorption. The total concentration in plasma after drug absorption is the blood drug concentration. The intensity of drug action is positively correlated with the blood drug concentration. The blood drug concentration of patients should be controlled within a safe range. If it is greater than the safe and effective range, toxic side effects or even severe poisoning reactions will occur. If it is less than the safe and effective range, there will be no therapeutic effect.

[0003] For the detection of the blood drug concentration of nordazepam in human blood, chromatography and mass spectrometry are mainly used at home and abroad. These methods have deficiencies in that the pretreatment is relatively complex, resulting in time-consuming and laborious work, and less clinical practical application. Currently, the clinical testing methods for the blood drug concentration of nordazepam are immunoassay, fluorescence polarization immunoassay (FPIA method), homogeneous enzyme immunoassay (EMIT), spectrometry, etc., but their applications have been relatively reduced due to limitations such as sensitivity and specificity.

[0004] Chinese Patent Application (CN114414707A) discloses a method and kit for detecting 19 drugs and their metabolites in blood by liquid chromatography-tandem mass spectrometry. However, this method detects a large number of drug types, and there are risks such as complex preparation process of standard solutions and interference in the testing process.

[0005] In view of this, it is necessary to design a method for determining the blood drug concentration of nordazepam in human blood and a liquid chromatograph to solve one of the above problems. Summary of the Invention

[0006] This application provides a method for determining the blood drug concentration of nordazepam 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 meeting clinical applications.

[0007] To achieve the above object, the technical solutions provided by this application are as follows: This application provides a method for determining the blood drug concentration of nordazepam in human blood, wherein the method includes: Prepare a standard working solution, dissolve the nordazepam standard with pure methanol, and dilute to obtain several standard working solutions with concentrations in the range of 80 - 800 ng / ml; Use a liquid chromatograph to test standard working solutions with different concentrations, and fit the standard curve equation of the standard working solutions; Prepare a 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; Use a liquid chromatograph to test the sample to be tested, specifically including: The mobile phase of the extraction column is phase A methanol and phase B pure water. Set the volume ratio of phase A to phase B as (10 - 30):(70 - 90). The mobile phase of the analytical column is phase C acetonitrile and phase D pure water. Set the volume ratio of phase C to phase D as (40 - 50):(50 - 60). Among them, phase C is an acetonitrile solution with a concentration of 50%. The flow rate of the first constant flow pump is 0.3 ml / min, the flow rate of the second constant flow pump is 0.8 ml / min, and the flow rate of the third constant flow pump is 1.5 ml / min; The elution program corresponding to the extraction column is: From 0 to 3 min, the volume ratio of phase A to phase B is (10 - 30):(70 - 90); From 3 to 4 min, the volume of phase A to phase B becomes 10:90. The flow rate of the first constant flow pump becomes 1.5 ml / min, the flow rate of the third constant flow pump becomes 0.1 ml / min, switch the six-way valve, and the mobile phase of the second constant flow pump elutes the extraction column in the reverse direction; From 4 to 6 min, the volume of phase A to phase B is 10:90; From 6 to 6.1 min, the volume ratio of phase A to phase B becomes 90:10; From 6.1 to 8.5 min, the volume ratio of phase A to phase B is 90:10; From 8.5 to 8.6 min, the volume ratio of phase A to phase B becomes (10 - 30):(70 - 90). The flow rate of the first constant flow pump becomes 0.3 ml / min, and the flow rate of the third constant flow pump becomes 1.5 ml / min to prepare for the next sample to be tested; From 8.6 to 11 min, the volume ratio of phase A to phase B is (10 - 30):(70 - 90); Data analysis, specifically including: Obtain the chromatogram of the blank sample, and calculate the blood drug concentration of nordazepam in the sample to be tested by combining the chromatogram of the sample to be tested and the standard curve equation.

[0008] Furthermore, the concentration of phase C acetonitrile solution is 50%, the volume of phase C is 80%, and the volume of phase D is 20%.

[0009] Furthermore, the preparation of the above standard working solution specifically includes: Accurately weigh 5 mg of nordazepam reference standard, dissolve it with pure methanol and make up the volume 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 4 μg / ml; Measure 12 μL, 15 μL, 30 μL, 60 μL, 90 μL, and 120 μL of the secondary standard stock solution respectively, and add 600 μL of pure water to each to obtain mixed solutions with different concentrations. Then, add 888 μL, 885 μL, 870 μL, 840 μL, 810 μL, and 780 μL of the sample releasing agent to the corresponding mixed solutions to obtain 6 standard working solutions with different concentrations.

[0010] Furthermore, use a liquid chromatograph to test the standard working solutions with different concentrations, obtain the chromatograms of the standard working solutions with different concentrations. Let the concentration of each standard working solution be the abscissa X, and the peak area of the chromatogram corresponding to each concentration be the ordinate Y, and perform linear regression fitting to obtain the corresponding standard curve linear equation y = a * x + b, and obtain a and b.

[0011] Furthermore, the preparation of the sample to be tested also includes: centrifuging the blood after taking the medicine to obtain serum, then adding acetonitrile as the sample releasing agent to the extracted serum, and performing vortex treatment and high-speed centrifugation treatment in sequence, and then taking the supernatant after centrifugation as the sample to be tested.

[0012] Furthermore, the volume of the added sample releasing agent is not less than the volume of the extracted serum.

[0013] Furthermore, the rotation speed of the high-speed centrifugation is 10000 rpm and the time is 10 min.

[0014] Furthermore, the blank sample chromatogram is obtained by testing the blank sample. The preparation of the blank sample includes: centrifuging normal blood to obtain normal serum, then adding acetonitrile as the sample releasing agent to the extracted normal serum, and performing vortex treatment and high-speed centrifugation treatment in sequence, and then taking the supernatant after centrifugation as the blank sample.

[0015] Furthermore, the column temperature is 30 °C.

[0016] This application also provides a liquid chromatograph using the above method for measuring the blood drug concentration of nordazepam in human blood. Among them, the liquid chromatograph includes an extraction column and an analytical column. The model of the extraction column is MFPh-1, and the parameters are: 4.0 mm * 20 mm, 5 μm; the model of the analytical column is C18, and the parameters are: 4.6 mm * 100 mm, 5 μm.

[0017] Compared with the prior art, the beneficial effect of this application is that: by using the method for measuring the blood drug concentration of nordazepam in human blood of this application and adjusting the proportion of the mobile phase, the interference substances in the sample can be effectively reduced, the purity of the analyte can be improved, and the detection efficiency and analysis accuracy of the blood drug concentration of nordazepam can be enhanced. Description of the Drawings

[0018] Figure 1It is a schematic structural diagram of the liquid chromatograph of the present application.

[0019] Figure 2 It is a flowchart of an embodiment of the method for measuring the blood drug concentration of nordazepam in human blood in the present application.

[0020] Figure 3 It is the chromatogram of the standard working solution in Example 1 of the present application.

[0021] Figure 4 It is Figure 3 The standard working curve obtained by fitting the peak area and concentration of nordazepam in the chromatogram of the standard working solution in

[0022] Figure 5 It is the chromatogram of the standard working solution with a blood drug concentration of 200 ng / ml in Example 1.

[0023] Figure 6 It is the chromatogram of the blood sample with a blood drug concentration of 200 ng / ml in Example 1.

[0024] Figure 7 It is the chromatogram of the blank sample in Example 1.

[0025] Figure 8 It is the chromatograms of six standard working solutions with a blood drug concentration of 200 ng / ml in Example 1.

[0026] Figure 9 It is the chromatogram of the low-concentration standard working solution in the experiment for verifying the detection limit in Example 1.

[0027] Figure 10 It is the chromatogram of the low-concentration standard working solution in the experiment for verifying the quantitation limit in Example 1.

[0028] Figure 11 It is the chromatogram of the standard working solution with a blood drug concentration of 200 ng / ml in the comparative example.

[0029] Among them, 1 - mobile phase system, 11 - first constant flow pump, 12 - second constant flow pump, 13 - third constant flow pump, 14 - six-port valve, 2 - injection system, 3 - separation system, 31 - extraction column, 32 - analytical column, 4 - detection system, 5 - control system. Detailed implementation manners

[0030] To enable those skilled in the art to better understand the technical solutions in this application, the following will clearly and completely describe the technical solutions in the embodiments of this application in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application.

[0031] In the various diagrams of this application, for the convenience of illustration, certain dimensions of the structure or part are exaggerated relative to other structural parts. Therefore, it is only used to illustrate the basic structure of the subject matter of this application.

[0032] This application provides a method for determining the blood drug concentration of nordazepam in human blood and a liquid chromatograph for testing the blood drug concentration of nordazepam in human blood using this method. As Figure 1 shown, the liquid chromatograph includes a mobile phase system 1, an injection system 2, a separation system 3, a detection system 4, and a control system 5.

[0033] The separation system 3 includes an extraction column 31 and an analytical column 32. Among them, the extraction column 31 is mainly used for enriching nordazepam substances and preliminary separation. The extraction column 31 in this application is of 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 packing particle size of 5 μm.

[0034] The analytical column 32 is mainly used for fine separation of the nordazepam substances transferred from the extraction column 31. The finely separated nordazepam substances enter the detection system 4. Since the mobile phase of the analytical column 32 matches the analytical column 32, the elution ability is stronger. The analytical column 32 in this application is C18 of 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 packing particle size of 5 μm.

[0035] Compared with a liquid chromatograph with only one chromatographic column, the liquid chromatograph of this application has an extraction column 31 and an analytical column 32, and respectively matches suitable mobile phases. The processes of enriching and analyzing nordazepam substances are separated, and the functions of the extraction column 31 and the analytical column 32 are refined, which can effectively remove other substances, making the detection results more accurate. Moreover, using two chromatographic columns to enrich and analyze nordazepam substances respectively can effectively improve the testing efficiency.

[0036] 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-port 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.

[0037] The first constant flow pump 11 provides the mobile phase for the extraction column 31, the second constant flow pump 12 provides the mobile phase for the analytical column 31, and the third constant flow pump 13 is connected to the injection system 2. Its main function is to dilute the injected sample, reduce the proportion of the organic phase and the impurity concentration in the sample.

[0038] Different valves of the six-port valve 14 are respectively connected to the separation system 3 and each pump. By changing the valve position of the six-port valve 14, the liquid flow direction is changed, thereby controlling the completion of the entire testing process.

[0039] The detection system 4 includes an ultraviolet detector. The ultraviolet detector is used to convert the collected component signal of nordazepam into an electrical signal and then transmit it to the control system 5.

[0040] As Figure 2 shown, the method includes: preparing a standard working solution; testing standard working solutions with different concentrations using a liquid chromatograph and fitting a standard curve equation for the standard working solutions; 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. By obtaining the chromatogram of a blank sample, combining the chromatogram of the sample to be tested and the standard curve equation, the blood drug concentration of nordazepam in the sample to be tested is calculated. The method of the present application can effectively reduce the interfering substances in the sample, improve the purity of the analyte, and enhance the detection efficiency and analysis accuracy of the blood drug concentration of nordazepam by adjusting the proportion of the mobile phase.

[0041] Further, the preparation of the standard working solution includes: dissolving the nordazepam standard product with pure methanol to obtain a primary standard stock solution, further diluting it with pure methanol to obtain a secondary standard stock solution, and then further diluting it with pure water and a sample releasing agent to obtain several standard working solutions with concentrations in the range of 80 - 800 ng / ml for obtaining the standard curve equation. The concentration of nordazepam in the sample to be tested is approximately in the range of 80 - 800 ng / ml, and a more accurate concentration can be obtained according to the standard curve equation.

[0042] Specifically, accurately weigh 5 mg of nordazepam reference standard and place it in a 10 mL volumetric flask. Dissolve it with pure methanol and make up the volume 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 4 μg / mL. Pipette 12 μL, 15 μL, 30 μL, 60 μL, 90 μL, and 120 μL of the secondary standard stock solution into corresponding sterile EP tubes, add 600 μL of pure water to each tube, and add 888 μL, 885 μL, 870 μL, 840 μL, 810 μL, and 780 μL of sample release agent (acetonitrile) to the corresponding solutions in the sterile EP tubes. Vortex for 1 min using a vortex mixer, and then centrifuge at high speed (10000 rpm, 10 min) to obtain standard working solutions with a concentration range of 80 - 800 ng / mL.

[0043] It can be understood that since the standard working curve made from standard working solutions of various concentrations is used to test the blood drug concentration of blood samples, the process of preparing blood samples is adding 600 μL of serum and 900 μL of sample release agent, which is diluted 2.5 times. Therefore, the concentration of nordazepam in the standard working solutions needs to be converted to the concentration of nordazepam in blood samples, and each concentration of the standard working solutions is multiplied by 2.5. That is, the concentrations of these six nordazepam standard working solutions are: 80 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, and 800 ng / mL.

[0044] Use a liquid chromatograph 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 abscissa X, and the peak area of the chromatogram corresponding to each concentration be the ordinate 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 test sample obtained through the standard curve equation.

[0045] When preparing the test sample, first centrifuge the blood taken from the patient after taking the nordazepam drug to obtain serum, then add the sample release agent acetonitrile to the extracted serum, and perform vortex treatment and high-speed centrifugation in sequence. Then take the supernatant after centrifugation as the test sample. It can be understood that the blood sampling time is after the patient reaches a steady state after oral administration of nordazepam.

[0046] Preferably, the volume of the added sample release agent is not less than the volume of the extracted serum, which can fully release nordazepam and improve the accuracy of the test results.

[0047] In this embodiment, 600 μl of centrifuged serum is taken, 900 μl of sample releasing agent is added, and after thorough mixing, centrifugation is performed to obtain the supernatant after centrifugation as the sample to be tested. Among them, a vortex mixer is used for thorough mixing, with vortexing lasting for 1 minute, and high-speed centrifugation (10000 rpm, 10 minutes).

[0048] For the unused or untested serum in time, it is stored in an environment of -20 °C. When it needs to be used again, it is thawed at room temperature into a liquid state, shaken or vortexed, 600 μl of serum is pipetted using a pipette, 900 μl of sample releasing agent (acetonitrile) is added, after vortexing for 1 minute, high-speed centrifugation (10000 rpm, 10 minutes) is carried out, and the upper clear liquid is taken as the sample to be tested.

[0049] When this test method is used for the first time, a blank sample needs to be prepared to obtain the chromatogram of the blank sample. The matrix of the blank sample is the same as that of the sample to be tested, only lacking the target substance nordazepam. In subsequent data analysis, subtracting the chromatogram of the blank sample from the chromatogram of the sample to be tested can reduce the interference of interfering substances. If the chromatogram of the blank sample is stored in the liquid chromatograph, the chromatogram of the blank sample is directly used after the test of the sample to be tested is completed.

[0050] In this application, the blank sample is realized by extracting the blood of normal people who have not taken nordazepam drugs. 600 μl of the serum of normal people's blood is taken, 900 μl of sample releasing agent is added, and thorough mixing is carried out using a vortex mixer, and then centrifugation is performed to obtain the supernatant after centrifugation as the blank sample. Among them, vortexing lasts for 1 minute, and high-speed centrifugation (10000 rpm, 10 minutes) is carried out. Using the same processing method as the sample to be tested can reduce the test error caused by sample processing.

[0051] Before testing the sample to be tested using a liquid chromatograph, the test parameters of the liquid chromatograph are first set, specifically including setting the temperature of the column oven to 30 °C to ensure that the temperature of the extraction column 31 and the analytical column 32 is at a constant temperature of 30 °C.

[0052] The mobile phase of the extraction column 31 is phase A methanol and phase B pure water, and the volume ratio of phase A to phase B is set to (10 - 30):(70 - 90), that is, the volume ratio of phase A is (10 - 30)%, and the volume ratio of phase B is (70 - 90)%.

[0053] The mobile phase of the analytical column 32 is phase C acetonitrile and phase D pure water, and the volume ratio of phase C to phase D is set to (40 - 50):(50 - 60). As an organic solvent, acetonitrile has a low viscosity and good solubility, which can improve the transmission speed of substances in the chromatographic column, thereby reducing the analysis time and ensuring a high separation efficiency.

[0054] In this application, Phase C is a solution of 50% acetonitrile. In the elution program, the volume of Phase C is 80%, and the volume of Phase D is 20%. By calculation, the volume of pure acetonitrile is 50%×80% + 0×20% = 0.4, and the volume of pure water is 50%×80% + 20%×100% = 0.6. That is, the volume ratio of pure acetonitrile to water is 40:60. This application simplifies the gradient program by premixing 50% acetonitrile, flexibly realizes the fine regulation of acetonitrile concentration, meets the separation requirements, and avoids the risk of solvent mutation caused by directly mixing high-concentration acetonitrile.

[0055] Set the flow rate of the first constant flow pump 11 to 0.3 ml / min, the flow rate of the second constant flow pump 12 to 0.8 ml / min, and the flow rate of the third constant flow pump 13 to 1.5 ml / min. Start the test according to the elution program corresponding to the following extraction column 31, specifically as follows: The elution program corresponding to the extraction column 31 is 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 0.3 ml / min, and the flow rate of the third constant flow pump 13 is 1.5 ml / min.

[0056] From 0 to 3 min, the volume ratio of Phase A to Phase B is (10 - 30):(70 - 90). 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 0.3 ml / min, and the flow rate of the third constant flow pump 13 is 1.5 ml / min. At this time, it is the process of the extraction column 31 enriching nordazepam.

[0057] From 3 to 4 min, the volume ratio of Phase A to Phase B becomes 10:90. The volume of Phase A is 10%, and the volume of Phase B is 90%. The flow rate of the first constant flow pump 11 becomes 1.5 ml / min. Increasing the flow rate of the first constant flow pump 11 can improve the efficiency of washing nordazepam from the extraction column 31. The flow rate of the second constant flow pump 12 remains unchanged, and the flow rate of the third constant flow pump 13 becomes 0.1 ml / min to reduce the interference of the water in the third constant flow pump 13 on the target substance nordazepam. Switch the valve of the six-port valve 14, and the mobile phase of the second constant flow pump 12 elutes the extraction column 31 in the reverse direction and transfers nordazepam to the analytical column 32.

[0058] From 4 to 6 min, the mobile phase of the first constant flow pump 11 remains unchanged. The volume ratio of Phase A to Phase B is 10:90. The volume of Phase A is 10%, and the volume of Phase B is 90%. The flow rates of the first constant flow pump 11 and the third constant flow pump 13 also remain unchanged. At this stage, the extraction column 31 is in the activation stage, and the analytical column 32 is in the process of precisely separating the target substance nordazepam.

[0059] At 6 - 6.1 min, change the mobile phase ratio of the first constant flow pump 11, and the volumes of phase A and phase B become 90:10; the volume of phase A is 90% and the volume of phase B is 10%. The flow rates of the first constant flow pump 11 and the third constant flow pump 13 also remain unchanged.

[0060] At 6.1 - 8.5 min, the volumes of phase A and phase B are 90:10; the volume of phase A is 90% and the volume of phase B is 10%. Increase the volume ratio of methanol in phase A to enhance the activation effect of the extraction column 31 and avoid interference with the next sample to be measured. At this time, the analytical column 32 is still in the process of precisely separating the target nor diazepam.

[0061] At 8.5 - 8.6 min, the volume ratio of phase A to phase B changes to (10 - 30):(70 - 90), the flow rate of the first constant flow pump 11 becomes 0.3 ml / min, the flow rate of the third constant flow pump 13 becomes 1.5 ml / min, and the six - way valve 14 resets to prepare for the next sample to be measured.

[0062] At 8.6 - 11 min, the volume ratio of phase A to phase B is (10 - 30):(70 - 90), in the preparation stage for the next sample to be measured. At this time, the chromatogram of this sample to be measured has been collected, and the test process of this sample to be measured ends.

[0063] At 11 - 14 min, 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 0.3 ml / min, the flow rate of the third constant flow pump 13 is 1.5 ml / min, and the collection of the next sample to be measured begins.

[0064] 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.

[0065] Taking the detection of blood samples as an example, the production process of blood samples is as follows: Take 600 ul of serum from normal blood, add 30 ul of the secondary standard stock solution, and then add 870 ul of sample release agent acetonitrile. After vortexing and high - speed centrifugation, measure the supernatant as the blood sample. Vortex for at least 1 min, and the parameters of high - speed centrifugation are the same as those for high - speed centrifuging serum above, that is, the blood sample is processed in the same way as the sample to be measured to reduce errors caused by the sample processing process.

[0066] In Example 1, the elution program is specifically as follows: the volume of phase A is 30%, the volume of phase B is 70%, and other parameters are shown in Table 1: Table 1

[0067] Before testing the blood sample, establish a standard curve equation. Use a liquid chromatograph to test standard working solutions of different concentrations respectively. The chromatograms corresponding to different concentrations are as shown in Figure 3 . The peak areas are shown in Table 2. By analyzing, obtain the peak area of nordazepam, and draw the standard curve of nordazepam Y = 0.2194x - 2.4274, with a correlation coefficient of 0.9994, as shown in Figure 4 . Then use the curve to calculate the content of nordazepam in subsequent blood samples.

[0068] Table 2

[0069] The verification process of the specificity experiment of this method is as follows: According to the parameters in Example 1, use a liquid chromatograph to detect the standard working solution, blood sample, and blank sample at a concentration of 200 ng / ml in sequence, and continuously test each twice respectively, and obtain the chromatogram of the standard working solution as shown in Figure 5 , the chromatogram of the blood sample as shown in Figure 6 , and the chromatogram of the blank sample as shown in Figure 7 . As shown in Figures 5 to 7 , the resolution of the impurity peak closest to nordazepam in the blood sample chromatogram is greater than 1.5. At the same time, the retention time corresponding to the nordazepam peak in the chromatograms of the standard working solution and the blood sample is consistent, and there is no interfering impurity peak of nordazepam at the corresponding position in the chromatogram of the blank sample. The above verifies that this method has strong specificity and good performance for testing the blood drug concentration of nordazepam.

[0070] 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 vials, and continuously test 6 identical samples according to the parameters in Example 1 to obtain 6 chromatograms of the standard working solution, as shown in Table 3 and Figure 8 .

[0071] Table 3

[0072] As shown in Table 3 and Figure 8 , the relative standard deviation of the peak areas of nordazepam in the six tests is 0.3623%, and the precision of this method is good.

[0073] The verification process of the accuracy experiment of this method is as follows: Put the processed blood samples into six sample bottles respectively, and test the above six samples according to the parameters in Example 1. The peak areas of nordazepam in the chromatograms of the six blood samples are shown in Table 4. Calculate the RSD value of the six peak areas of nordazepam to be 0.2760%, and the accuracy of this method is good.

[0074] Table 4

[0075] Among them, the recovery rate of this method is 100.55%, and the calculation method is the average value of the peak areas of nordazepam in six blood samples divided by the average value of the peak areas of nordazepam in six standard working solutions. That is, the concentration of nordazepam in the standard working solution is the same as that in the blood sample. According to the measured results for comparison, the recovery rate measured by this method is between 95% and 105%, indicating that this method is reliable and applicable.

[0076] The verification process of the detection limit experiment for this method is as follows: Prepare a standard working solution with a relatively low concentration, and then test according to the parameters in Example 1 until the measured peak height is 3 times the baseline noise. At this time, record the peak area, and the corresponding concentration is: 4.38 ng / ml. As Figure 9 shown.

[0077] The verification process of the quantitation limit experiment for this method is as follows: Prepare a standard working solution with a relatively low concentration, and then test according to the parameters in Example 1 until the measured peak height is 10 times the baseline noise. At this time, record the peak area, and the corresponding concentration is: 14.60 ng / ml, as Figure 10 shown.

[0078] Example 2: Change the volume ratio of mobile phase A to mobile phase B in extraction column 31 to 20:80, with other parameters unchanged. The parameters of the detailed elution program are shown in Table 5. Test the standard working solution with a corresponding blood drug concentration of 200 ng / ml and the blood sample. The test results are shown in Table 7. The recovery rate of Example 2 is 101.91%. The recovery rate measured in Example 2 is between 95% and 105%. This method can accurately and reliably measure the concentration of nordazepam in blood.

[0079] Table 5

[0080] Example 3: Change the volume ratio of mobile phase A to mobile phase B in extraction column 31 to 10:90, with other parameters unchanged. The parameters of the detailed elution program are shown in Table 6. Test the standard working solution with a corresponding blood drug concentration of 200 ng / ml and the blood sample. The test results are shown in Table 7. The recovery rate of Example 3 is 103.93%. The recovery rate measured in Example 3 is between 95% and 105%. This method can accurately and reliably measure the concentration of nordazepam in blood.

[0081] Table 6

[0083] Comparative Example 1: The parameters of the mobile phase of the extraction column 31 are the same as those in Example 1. In this example, only the mobile phase of the analytical column 32 is changed. The volume ratio of acetonitrile to pure water is 10:90, the flow rate of the second constant flow pump 12 is still 0.8 ml / min, and the injection volume is 100 μl; the wavelength of the ultraviolet detector is set to 240 nm; the target peak cannot be separated within 10 minutes, as shown in Figure 11 shown. Therefore, the optimal volume ratio of acetonitrile to pure water in the mobile phase of the analytical column 32 in this method is (40 - 50):(50 - 60).

[0084] Table 7

[0085] In summary, for the method for determining the blood drug concentration of nordazepam in human blood in this application, by adjusting the ratio of the mobile phase, the interference substances in the sample can be effectively reduced, the purity of the analyte can be improved, and the detection efficiency and analysis accuracy of the blood drug concentration of nordazepam can be enhanced.

[0086] It should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and 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 only specific descriptions of the feasible embodiments of this application, and are not used to limit the protection scope of this application. Any equivalent embodiments or changes made without departing from the technical spirit of this application should be included in the protection scope of this application.

Claims

1. A method for determining the blood drug concentration of nordazepam in human blood, characterized in that, The method includes: Preparing a standard working solution, dissolving the nordazepam standard with pure methanol and diluting to obtain several standard working solutions with concentrations in the range of 80 - 800 ng / ml; Testing the standard working solutions with different concentrations using a liquid chromatograph and fitting the standard curve equation of the standard working solutions; Preparing a sample to be tested, centrifuging the serum of the blood sample to be tested, and taking the supernatant after centrifugation as the sample to be tested; Testing the sample to be tested using a liquid chromatograph, specifically including: The mobile phase of the extraction column is phase A methanol and phase B pure water, setting the volume ratio of phase A to phase B as (10 - 30):(70 - 90), the mobile phase of the analytical column is phase C acetonitrile and phase D pure water, setting the volume ratio of phase C to phase D as (40 - 50):(50 - 60), where phase C is an acetonitrile solution with a concentration of 50%; the flow rate of the first constant flow pump is 0.3 ml / min, the flow rate of the second constant flow pump is 0.8 ml / min, and the flow rate of the third constant flow pump is 1.5 ml / min; The elution program corresponding to the extraction column is: From 0 - 3 min, the volume ratio of phase A to phase B is (10 - 30):(70 - 90); From 3 - 4 min, the volume of phase A and phase B becomes 10:90, the flow rate of the first constant flow pump becomes 1.5 ml / min, the flow rate of the third constant flow pump becomes 0.1 ml / min, switch the six - way valve, and the mobile phase of the second constant flow pump elutes the extraction column in the reverse direction; From 4 - 6 min, the volume of phase A and phase B is 10:90; From 6 - 6.1 min, the volume of phase A and phase B becomes 90:10; From 6.1 - 8.5 min, the volume of phase A and phase B is 90:10; From 8.5 - 8.6 min, the volume ratio of phase A to phase B becomes (10 - 30):(70 - 90), the flow rate of the first constant flow pump becomes 0.3 ml / min, the flow rate of the third constant flow pump becomes 1.5 ml / min, preparing for the next sample to be tested; From 8.6 - 11 min, the volume ratio of phase A to phase B is (10 - 30):(70 - 90); Data analysis, specifically including: obtaining the chromatogram of the blank sample, and calculating the blood drug concentration of nordazepam in the sample to be tested by combining the chromatogram of the sample to be tested and the standard curve equation.

2. The method for determining the blood drug concentration of nordazepam in human blood according to claim 1, wherein The volume of phase C is 80%, and the volume of phase D is 20%.

3. The method for determining the blood drug concentration of nordazepam in human blood according to claim 1, characterized in that, The above preparation of the standard working solution specifically includes: Accurately weighing 5 mg of the nordazepam standard, dissolving it with pure methanol and making the volume up to 10 mL to obtain a primary standard stock solution with a concentration of 0.5 mg / ml, and then diluting the primary standard stock solution with pure methanol to obtain a secondary standard stock solution with a concentration of 4 μg / ml; Respectively measuring volumes of 12 μl, 15 μl, 30 μl, 60 μl, 90 μl, 120 μl of the secondary standard stock solution, adding 600 μL of pure water to each to obtain mixed solutions with different concentrations, and then adding 888 μl, 885 μl, 870 μl, 840 μl, 810 μl, 780 μl of the sample release agent to the corresponding mixed solutions to obtain 6 standard working solutions with different concentrations.

4. The method for determining the blood drug concentration of nordazepam in human blood according to claim 1, characterized in that, The standard working solutions with different concentrations were tested using a liquid chromatograph to obtain the chromatograms of the standard working solutions with different concentrations. Let the concentration of each standard working solution be the abscissa X, and the peak area of the chromatogram corresponding to each concentration be the ordinate Y, and linear regression fitting was performed to obtain the corresponding standard curve linear equation y = a*x + b, and a and b were obtained.

5. The method for determining the blood drug concentration of nordazepam in human blood according to claim 1, characterized in that, The preparation of the sample to be tested further includes: the blood after taking the medicine is first centrifuged to obtain serum, then sample releasing agent acetonitrile is added to the extracted serum, and vortex treatment and high-speed centrifugation treatment are carried out in sequence, and then the supernatant after centrifugation is taken as the sample to be tested.

6. The method for determining the blood drug concentration of nordazepam in human blood according to claim 5, characterized in that, The volume of the added sample releasing agent is not less than the volume of the extracted serum.

7. The method for determining the blood drug concentration of nordazepam in human blood according to claim 5, characterized in that, The rotation speed of high-speed centrifugation is 10000 rpm, and the time is 10 min.

8. The method for determining the blood drug concentration of nordazepam in human blood according to any one of claims 1 to 7, characterized in that, The blank sample chromatogram is obtained by testing the blank sample. The preparation of the blank sample includes: normal blood is first centrifuged to obtain normal serum, then sample releasing agent acetonitrile is added to the extracted normal serum, and vortex treatment and high-speed centrifugation treatment are carried out in sequence, and then the supernatant after centrifugation is taken as the blank sample.

9. The method for determining the blood drug concentration of nordazepam in human blood according to any one of claims 1 to 7, characterized in that, The column temperature is 30 °C.

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

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