CN110806449B - Method for simultaneously detecting content of clomipramine and N-norclomipramine in blood - Google Patents

Abstract

The invention provides a method for simultaneously detecting the contents of clomipramine and N-norclomipramine in blood. Preparing at least three standard working solutions containing a clomipramine standard substance with known concentration and an N-norclomipramine standard substance; mixing the standard working solution, the internal standard working solution and the blank blood sample, and performing sample pretreatment to obtain a standard solution; respectively detecting each standard solution by using a high performance liquid chromatograph to obtain a chromatogram of each standard solution; fitting to obtain a standard curve equation according to the chromatogram of each standard solution; mixing the internal standard working solution with a blood sample obtained by treating blood to be detected, and performing the same sample pretreatment to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the contents of clomipramine and N-norclomipramine in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation. The scheme can simultaneously detect the contents of clomipramine and N-norclomipramine in blood.

Description

Method for detecting clomipramine and N-desmethylclomipramine content in blood simultaneously

technical field

The invention relates to the technical field of clinical chemistry, in particular to a method for simultaneously detecting the contents of clomipramine and N-desmethylclomipramine in blood.

Background technique

Depression, also known as depressive disorder, is the main type of mood disorder with significant and persistent low mood as the main clinical feature. It can be seen clinically that the mood is not commensurate with the situation. The depression of mood can range from gloomy to distraught, low self-esteem and depression, even pessimistic world-weariness, and there may be suicide attempts or behaviors; even stupor; some cases have obvious anxiety and motor agitation; In severe cases, psychotic symptoms such as hallucinations and delusions may appear. Each episode of depression lasts at least 2 weeks, or even several years in the elderly. Most cases have a tendency to recur. Most of each episode can be relieved, and some may have residual symptoms or become chronic.

Clomipramine is a tricyclic antidepressant drug mainly used in the treatment of obsessive-compulsive disorder (OCD), and also commonly used in the treatment of obsessive-compulsive neurosis and phobic neurosis. The main function of clomipramine is to block the reuptake of serotonin in the central nervous system, and its active metabolite desmethylclomipramine can block the reuptake of norepinephrine in the central nervous system, thereby exerting antidepressant and anxiolytic effects , also has sedative and anticholinergic effects.

There are currently methods for the determination of clomipramine levels in blood. Since its metabolites are also pharmacologically active and will also affect the therapeutic effect, it is necessary to detect their active metabolites. Therefore, it is necessary to propose a method capable of simultaneously detecting the content of clomipramine and its metabolite N-desmethylclomipramine in blood.

Contents of the invention

The invention provides a method for simultaneously detecting the contents of clomipramine and N-desmethylclomipramine in blood, which can simultaneously detect the contents of clomipramine and N-desmethylclomipramine in blood.

In order to achieve the above object, the present invention is achieved through the following technical solutions:

The invention provides a method for simultaneously detecting the content of clomipramine and N-desmethylclomipramine in blood, comprising:

Prepare at least three standard working solutions, wherein the standard working solutions contain clomipramine standard substances and N-desmethylclomipramine standard substances with known concentrations, and the concentrations of the same standard substance in different standard working solutions are different;

Mix a certain amount of standard working solution, internal standard working solution and blank blood sample, and perform sample pretreatment to obtain a standard solution, wherein the internal standard working solution contains an internal standard with a known concentration;

Using a high performance liquid chromatograph, under certain detection conditions, respectively detect at least three standard solutions, and obtain the chromatograms of each standard solution;

According to the chromatogram of each standard solution, fitting obtains the standard curve equation of clomipramine and the standard curve equation of N-desmethylclomipramine;

Mixing a certain amount of internal standard working solution with the blood sample, performing the same sample pretreatment to obtain the sample to be tested, wherein the blood sample is obtained by processing the blood to be tested;

Using a high performance liquid chromatograph, detect the sample to be tested under the same detection conditions, and obtain the chromatogram of the sample to be tested;

According to the chromatogram of the sample to be tested and each standard curve equation obtained by fitting, the contents of clomipramine and N-desmethylclomipramine in the blood sample are calculated.

Preferably, the sample pretreatment includes the following A1 to A5:

A1: Vortex the mixture;

A2: Add methanol and vortex to mix;

A3: Add the extractant, vortex and mix, and centrifuge to obtain the supernatant;

A4: Pipette the supernatant into a centrifuge tube and dry it with nitrogen at room temperature;

A5: Add the reconstitution solution to the supernatant-dried centrifuge tube, vortex to mix, and centrifuge to obtain the supernatant.

Preferably, the A1 includes: vortex mixing the mixed solution at a speed of 1800-2800rpm for 1-2min;

The A2 includes: adding 80-120 μL of methanol, and vortex mixing at a speed of 1800-2800 rpm for 1-2 minutes;

The A3 includes: adding 800-1200 μL extractant, vortex mixing at a speed of 1800-2800 rpm for 15-25 min, and then centrifuging at a speed of 10000-15000 rpm for 3-7 min to obtain a supernatant;

The A4 includes: pipetting 700-1000 μL supernatant into a centrifuge tube, and blowing dry with nitrogen at room temperature;

Said A5 includes: adding 80-120 μL of reconstituted solution to a centrifuge tube dried with supernatant, vortexing at 1800-2800 rpm for 2-4 minutes, and then centrifuging at 10000-15000 rpm for 4-6 minutes to obtain the above Serum.

Preferably, the detection conditions include: a Waters Xbridge C18 chromatographic column, the length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the filler particle size is 3.5 μm.

Preferably, the detection conditions include: the column temperature is 50-60°C, the mobile phase A contains ammonium acetate with a concentration of 80-120mmol/L, formic acid with a volume ratio of 0.15-0.25%, and a volume ratio of 0.15-0.25% triethylamine in water, the mobile phase B is acetonitrile, the flow rate is 0.2-0.4mL/min, the injection volume is 20-40μL, and the analysis time is 12-13min.

Preferably, the detection conditions include: single-column double-pump elution mode;

The switching mode of the double pumps is: switch from the analysis pump to the clean pump at 10.7min, and switch from the clean pump to the analysis pump at 11.2min;

The elution mode is: the volume ratio of mobile phase A and mobile phase B is 59-61%: 41-39% in other elution time ranges other than 10.7-11.2min.

Preferably, the ultraviolet detector in the high performance liquid chromatograph is a DAD-3000 detector, the detection wavelength is 295-305nm, the collection frequency is 5Hz, and the bandwidth is 4nm.

Preferably, the in-line filter used by the high performance liquid chromatograph is SSI COLPRE-FILTER WATER 1/16 0.5M.

Preferably, the internal standard is iminostilbene.

Preferably, in the standard working solution, the concentration of the clomipramine standard substance is 500-15000ng/mL, the concentration of the N-desmethylclomipramine standard substance is 1000-30000ng/mL, and the diluent is 40-60 % methanol in water.

The invention provides a method for simultaneously detecting the contents of clomipramine and N-desmethylclomipramine in blood. Prepare at least three standard working solutions containing a known concentration of clomipramine standard and N-desmethylclomipramine standard; mix the standard working solution, internal standard working solution and blank blood sample for sample pretreatment Obtain the standard solution; Utilize high performance liquid chromatography to detect each standard solution respectively, obtain the chromatogram of each standard solution; According to the chromatogram of each standard solution, fit and obtain the standard curve equation; The obtained blood samples are mixed, and the same sample pretreatment is carried out to obtain the sample to be tested; the chromatogram of the sample to be tested is also detected; according to the chromatogram of the sample to be tested and each standard curve equation, the clomipramine and N in the blood sample are calculated. - Desmethylclomipramine content. The invention can simultaneously detect the contents of clomipramine and N-desmethylclomipramine in blood.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are For some embodiments of the present invention, those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is a flow chart of a method for simultaneously detecting the content of clomipramine and N-desmethylclomipramine in blood provided by an embodiment of the present invention;

Fig. 2 is the chemical structural formula of clomipramine provided by an embodiment of the present invention;

Fig. 3 is the chemical structural formula of N-desmethylclomipramine provided by an embodiment of the present invention;

Fig. 4 is the chromatogram of clomipramine standard substance, N-desmethylclomipramine standard substance and internal standard substance in a standard solution provided by an embodiment of the present invention;

Figure 5 is a chromatogram of clomipramine, N-desmethylclomipramine and an internal standard in a sample to be tested provided by an embodiment of the present invention;

Figure 6 is a linear relationship diagram of clomipramine provided by an embodiment of the present invention;

Fig. 7 is a linear relationship graph of N-desmethylclomipramine provided by an embodiment of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments It is a part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection of the present invention. scope.

It has been found through research that the extraction rates of the prototype (ie clomipramine), the metabolite of the prototype (ie N-desmethylclomipramine) and the internal standard are different during the blood sample pretreatment process. For example, when iminostilbene is used as an internal standard, the test results of the extraction rate are shown in Table 1 below.

Table 1

internal standard Metabolites prototype The peak area of the substance in the standard solution without pretreatment 2.1439 0.3644 0.242 The peak area of the substance in the standard solution during pretreatment 1.2254 0.2382 0.1979 Extraction rate 57.2% 65.4% 81.8%

In Table 1, without pretreatment, that is, standard working solution and internal standard working solution, the standard solution was directly prepared by dilution and loaded for analysis. For pretreatment, that is, after standard working solution, internal standard working solution plus blank blood, the same pretreatment operation as the blood sample pretreatment is performed to obtain the standard solution and load it for analysis.

Please refer to Table 1, the peak area of the internal standard substance is 2.1439 without pretreatment, and the peak area of the internal standard substance is 1.2254 when pretreatment is performed. It can be seen that the pretreatment process has a certain influence on the extraction of the internal standard substance, and the extraction rate was 57.2%.

Please refer to Table 1. The peak area of the metabolites without pretreatment is 0.3644, while the peak area of the internal standard is 0.2382 with pretreatment. It can be seen that the pretreatment process has a certain impact on the extraction of metabolites, and the extraction rate is 65.4 %.

Please refer to Table 1, the peak area of the prototype without pretreatment is 0.242, and the peak area of the internal standard is 0.1979 with pretreatment. It can be seen that the pretreatment process has a certain impact on the extraction of the prototype, and the extraction rate is 81.8%.

It can be seen that during the pretreatment process, the extraction rate of the internal standard and the metabolite, as well as the extraction rate of the internal standard and the prototype, are all different and quite different. Therefore, in order to correct the fixed value error caused by the inconsistency of the extraction rate of the prototype, metabolites and internal standards, the method of adding blood can be used to obtain the standard curve, so that the pretreatment process of preparing the standard solution according to the standard working solution, It is the same as the pretreatment process of preparing the sample to be tested based on the blood sample to ensure the accuracy of the test.

Based on the above, as shown in Figure 1, the embodiment of the present invention provides a method for simultaneously detecting the content of clomipramine and N-desmethylclomipramine in blood, which may include the following steps:

Step 101: Prepare at least three standard working solutions, wherein the standard working solutions contain clomipramine standard substances and N-desmethylclomipramine standard substances with known concentrations, and the concentration of the same standard substance in different standard working solutions different.

Step 102: Mix a certain amount of standard working solution, internal standard working solution and blank blood sample, and perform sample pretreatment to obtain a standard solution, wherein the internal standard working solution contains an internal standard with a known concentration.

In detail, the blank blood sample can usually be serum or plasma without clomipramine, N-desmethylclomipramine and internal standard substance, and the clomipramine, N-desmethylclomipramine and internal standard of the blank blood sample The content detection result of the internal standard should usually be not detected.

Step 103: Using a high performance liquid chromatograph, under certain detection conditions, detect at least three standard solutions respectively, and obtain the chromatograms of each standard solution.

Step 104: According to the chromatograms of each standard solution, the standard curve equation of clomipramine and the standard curve equation of N-desmethylclomipramine are obtained by fitting.

Step 105: Mix a certain amount of internal standard working solution with the blood sample, and perform the same sample pretreatment to obtain the sample to be tested, wherein the blood sample is obtained by processing the blood to be tested.

Step 106: Using a high-performance liquid chromatograph to detect the sample to be tested under the same detection conditions, and obtain a chromatogram of the sample to be tested.

Step 107: Calculate the content of clomipramine and N-desmethylclomipramine in the blood sample according to the chromatogram of the sample to be tested and each standard curve equation obtained by fitting.

The embodiment of the present invention uses the internal standard method to detect the content of clomipramine and N-desmethylclomipramine in the blood sample, so as to avoid the value deviation caused by the operation error in the pretreatment process, so the detection accuracy can be guaranteed .

Please refer to FIG. 2 and FIG. 3 , FIG. 2 shows the chemical structural formula of clomipramine, and FIG. 3 shows the chemical structural formula of N-desmethylclomipramine.

Usually, the establishment of the standard curve equation requires at least three coordinate points to ensure the accuracy of the established equation, so it is necessary to prepare at least three standard solutions, so that the obtained chromatograms can be tested according to each standard solution, and the fitting method can be obtained. Standard curve equation for clomipramine and standard curve equation for N-desmethylclomipramine.

In detail, taking clomipramine as an example, the fitted standard curve equation of clomipramine can usually be y=k×x+b. Wherein, these two variables of x, y can be respectively the peak area ratio of the standard substance of clomipramine and the internal standard in the chromatogram of each standard solution, and, in each standard solution, the standard of clomipramine Concentration ratio of product to internal standard. In this way, according to the chromatogram of the sample to be tested, the peak area ratio of clomipramine and the internal standard substance, and the concentration of the internal standard substance in the sample to be tested, the clomipramine in the sample to be tested can be calculated by substituting into the standard curve equation. The concentration of clomipramine was obtained to obtain the content of clomipramine in the blood sample. Of course, this realization is also applicable to N-desmethylclomipramine.

In the embodiment of the present invention, the blood sample can usually be serum or plasma obtained by processing the blood to be tested. After the blood to be tested is sampled, it can be processed accordingly to obtain a blood sample. For example, take at least 5 mL of the blood to be tested, centrifuge at a centrifugation speed of 3500 rpm for 10 min, and take the supernatant to obtain serum or plasma, that is, the above blood sample is obtained. Serum or plasma samples can be stored at -20°C until analysis.

After the blood sample is obtained, the sample to be tested is obtained through the same pretreatment, and the sample to be tested is detected under the same detection conditions to obtain its chromatogram. As mentioned above, based on the chromatogram and the fitted standard curve equation, the contents of clomipramine and N-desmethylclomipramine in the blood sample can be obtained.

In summary, the method for detecting the content of clomipramine and N-desmethylclomipramine in the blood provided by the embodiment of the present invention is to combine the internal standard method with high performance liquid chromatography, and pass the same The standard working solution and blood samples are processed in the pre-treatment process, which greatly reduces the interference factors, and has accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection results, low cost, and short analysis time, which is beneficial to Detection of high-throughput blood samples.

In detail, during the inter-laboratory comparison test of the British Government Chemist Laboratory (LGC), it was found that when the sample was pretreated, if there was no step of adding methanol before the extraction, the experimental results obtained had a large deviation from the true value, while If there is a step of adding methanol before the extraction, the deviation between the obtained experimental results and the true value is small. For specific experimental data, please refer to the following Table 2.

Table 2

In Table 2, before improvement means that there is no step of adding methanol before extraction in sample pretreatment, and after improvement means that there is a step of adding methanol before extraction in sample pretreatment. Among them, z'score≤2, the result is qualified.

Based on the above content, it is believed that the high protein binding rate should be the cause of the low extraction rate through analysis. Therefore, in the sample pretreatment process, it is preferable to add methanol before extraction. Therefore, in one embodiment of the present invention, the sample pretreatment includes the following A1 to A5:

A1: Vortex the mixture;

A2: Add methanol and vortex to mix;

A3: Add the extractant, vortex and mix, and centrifuge to obtain the supernatant;

A4: Pipette the supernatant into a centrifuge tube and dry it with nitrogen at room temperature;

A5: Add the reconstitution solution to the supernatant-dried centrifuge tube, vortex to mix, and centrifuge to obtain the supernatant.

In detail, the supernatant obtained by sample pretreatment of the standard working solution is the standard solution, and the supernatant obtained by the sample pretreatment of the blood sample is the sample to be tested.

In the embodiment of the present invention, methanol can be added before the extraction operation to realize the protein precipitation operation, so as to improve the accuracy of actual sample detection.

Preferably, in one embodiment of the present invention, the A1 includes: vortex mixing the mixed solution at a rotation speed of 1800-2800rpm for 1-2min;

The A2 includes: adding 80-120 μL of methanol, and vortex mixing at a speed of 1800-2800 rpm for 1-2 minutes;

The A3 includes: adding 800-1200 μL extractant, vortex mixing at a speed of 1800-2800 rpm for 15-25 min, and then centrifuging at a speed of 10000-15000 rpm for 3-7 min to obtain a supernatant;

The A4 includes: pipetting 700-1000 μL supernatant into a centrifuge tube, and blowing dry with nitrogen at room temperature;

Said A5 includes: adding 80-120 μL of reconstituted solution to a centrifuge tube dried with supernatant, vortexing at 1800-2800 rpm for 2-4 minutes, and then centrifuging at 10000-15000 rpm for 4-6 minutes to obtain the above Serum.

For example, the value of the vortex rotation speed can be 1800, 2000, 2200, 2400, 2600 or 2800; the value of the vortex time in A1 can be 1, 1.2, 1.5, 1.7 or 2; the value of the amount of methanol can be 80, 90, 100, 110 or 120; the value of vortex time in A2 can be 1, 1.2, 1.5, 1.7 or 2; the value of extractant dosage can be 800, 900, 1000, 1100 or 1200; A3 The value of the vortex time in A3 can be 15, 17, 20, 22 or 25; the value of the centrifugal speed can be 10000, 11000, 12000, 13000, 14000 or 15000; the value of the centrifugal time in A3 can be 3, 4 , 5, 6 or 7; the value of supernatant in A4 can be 700, 80, 900 or 1000; the value of complex solution can be 80, 90, 100, 110 or 120; the vortex time in A5 The value can be 2, 2.5, 3, 3.5 or 4; the value of centrifugation time in A5 can be 4, 4.5, 5, 5.5 or 6.

Preferably, the above extractant can be n-hexane, and the above complex solution can be 40-60% methanol aqueous solution. For example, in the complex solution, the percentage content of methanol can be 40%, 45%, 50%, 55% or 60%.

The embodiment of the present invention adopts the internal standard method to detect the content of clomipramine and N-desmethylclomipramine in the blood sample, and the above-mentioned pretreatment method can be used. error, so that more accurate quantification can be achieved. In addition, because the preprocessing method is relatively simple, it is beneficial to popularization and application of the embodiments of the present invention.

Based on the above, in one embodiment of the present invention, the standard working solution is used in an amount of 10 μL, the internal standard working solution is used in an amount of 10 μL, and the blank blood sample is used in an amount of 150-250 μL. The three are mixed for sample pretreatment. For example, the amount of the blank blood sample can be 150, 170, 200, 220 or 250.

Based on the above, in one embodiment of the present invention, the volume of the internal standard working solution is 10 μL, and the volume of the blood sample is 150-250 μL, and the two are mixed for sample pretreatment. For example, the amount of blood samples can be 150, 170, 200, 220 or 250.

In one embodiment of the present invention, the detection conditions include: a Waters Xbridge C18 chromatographic column, the length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the filler particle size is 3.5 μm.

In one embodiment of the present invention, the detection conditions include: the column temperature is 50-60°C, the mobile phase A is ammonium acetate containing a concentration of 80-120mmol/L, formic acid with a volume ratio of 0.15-0.25%, and a volume ratio of The water is 0.15-0.25% triethylamine, the mobile phase B is acetonitrile, the flow rate is 0.2-0.4mL/min, the injection volume is 20-40μL, and the analysis time is 12-13min.

For example, the value of column temperature can be 50, 52, 54, 56, 58 or 60; in mobile phase A, the value of ammonium acetate concentration can be 80, 90, 100, 110 or 120, formic acid and The value of the volume ratio of the amine can be 0.15, 0.17, 0.20, 0.22 or 0.25; the value of the flow rate can be 0.2, 0.25, 0.3, 0.35 or 0.4; 35 or 40; the value of analysis time can be 12, 12.2, 12.4, 12.6, 12.8 or 13.

In the embodiment of the present invention, based on the above detection conditions, the analysis time can be about 12.5 min, which is short and the analysis efficiency is improved.

In an embodiment of the present invention, the volume ratio of the mobile phase A to the mobile phase B is 59-61%:41-39% during the target analysis phase of the entire analysis time. For example, the volume ratio of mobile phase A and mobile phase B can be 59:41, 59.5:40.5, 60:40, 60.5:39.5 or 61:39.

In detail, the target substances to be analyzed include prototypes, metabolites and internal standards.

Based on the above, in one embodiment of the present invention, the detection conditions include: single-column double-pump elution mode;

The switching mode of the double pumps is: switch from the analysis pump to the clean pump at 10.7min, and switch from the clean pump to the analysis pump at 11.2min;

The elution mode is: the volume ratio of mobile phase A and mobile phase B is 59-61%: 41-39% in other elution time ranges other than 10.7-11.2min.

In the embodiment of the present invention, during the period of 10.7-11.2min, the mobile phase of the cleaning pump enters the chromatographic column to clean the chromatographic column. Before 10.7min and after 11.2min, the mobile phase of the analytical pump enters the chromatographic column. , for target analysis. Thus, for the analytical pump mobile phase, the volume ratio of mobile phase A to mobile phase B is 59-61%:41-39%. In one embodiment of the present invention, for the clean pump mobile phase, the volume ratio of mobile phase A and mobile phase B may be 40:60.

Compared with the single-pump gradient elution mode, the single-column dual-pump elution mode can minimize the analysis time under single-column conditions. At the same time, the amount of organic reagents used can be reduced to a certain extent.

Therefore, the embodiment of the present invention can shorten the analysis time, increase the detection throughput, and reduce the consumption of organic solvents.

In one embodiment of the present invention, the ultraviolet detector in the high performance liquid chromatograph is a DAD-3000 detector, the detection wavelength is 295-305nm, the collection frequency is 5Hz, and the bandwidth is 4nm. For example, the value of the detection wavelength can be 295, 300 or 305.

In one embodiment of the present invention, the in-line filter used in the high performance liquid chromatograph is SSI COLPRE-FILTER WATER 1/16 0.5M.

In one embodiment of the present invention, the internal standard is iminostilbene.

In detail, iminostilbene is an intermediate used in the synthesis of drugs, and basically does not exist in the patient's body. Compared with using psychotropic drugs or commonly used drugs with similar efficacy as internal standard, the use of iminostilbene as internal standard has great advantages, which can avoid the mutual interference brought by combined drugs to the analysis.

Based on the foregoing, in one embodiment of the present invention, in the standard working solution, the concentration of the clomipramine standard substance is 500-15000ng/mL, and the concentration of the N-desmethylclomipramine standard substance is 1000-30000ng /mL, the diluent is 40-60% methanol water solution.

In detail, the corresponding linear range can be set in combination with the population to be tested, the amount of blood to be tested, and the approximate content range of clomipramine and N-desmethylclomipramine in the human body, so as to ensure that most clinical samples The test result falls within the reportable range.

For example, preferably, in the above-mentioned at least three standard working solutions, the concentration of clomipramine standard can be 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, 6000ng/mL, 12000ng/mL, 15000ng/mL At least three in mL, the concentration of N-desmethylclomipramine standard can be in 1000ng/mL, 2000ng/mL, 4000ng/mL, 8000ng/mL, 12000ng/mL, 24000ng/mL, 30000ng/mL At least three. Preferably, the number of standard working solutions is seven.

In summary, the method for detecting the content of clomipramine and N-desmethylclomipramine in the blood provided by the embodiments of the present invention is to combine the internal standard method with high performance liquid chromatography, so that the interference factors are greatly reduced. It has the advantages of accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection results, low cost, and short analysis time, which is conducive to the detection of large-volume blood samples.

The embodiment of the present invention not only detects the prototype of the drug, but also detects its active metabolites at the same time. In this way, the detection method provided by the embodiment of the present invention can be used to detect clomipramine and its metabolite N in the patient's body during clinical treatment. -Monitoring the content of desmethylclomipramine, providing an experimental basis for the personalized administration of clomipramine and its metabolite N-desmethylclomipramine, and reducing the occurrence of toxic and side effects, so as to be more conducive to guiding patients medication.

The present invention will be described in detail through examples below, but the present invention is not limited to the following examples.

Example 1

The embodiment of the present invention is used to obtain the standard curve equation.

1.1 Preparation of standard stock solution

Standard stock solution: Accurately pipette 15 μL of 1.0 mg/mL clomipramine standard solution and 30 μL of 1.0 mg/mL N-desmethylclomipramine standard solution, put them in a 1.5 mL centrifuge tube, and use 50% Methanol aqueous solution was diluted to obtain a standard stock solution. In the obtained standard stock solution, the concentration of clomipramine standard substance is 15000ng/mL, and the concentration of N-desmethylclomipramine standard substance is 30000ng/mL.

1.2 Preparation of internal standard stock solution

Internal standard stock solution: Accurately weigh 5mg of iminostilbene standard substance into a 5mL volumetric flask, dissolve in methanol, and dilute to 5mL to obtain internal standard stock solution.

1.3 Instruments for testing

Thermo Fisher U3000 high performance liquid chromatograph.

1.4 Detection conditions

1.4.1 Columns

Waters Xbridge C ₁₈ chromatographic column, the length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the filler particle size is 3.5 μm.

1.4.2 Mobile phase

Mobile phase A: water containing ammonium acetate at a concentration of 100 mmol/L, formic acid at a volume ratio of 0.2%, and triethylamine at a volume ratio of 0.2%.

Mobile Phase B: Acetonitrile.

1.4.3 Elution method

Single column double pump elution mode.

The double-pump switching method is shown in Table 3 below. Within 0-10.7min and 11.2-12.5min, the volume ratio of mobile phase A to mobile phase B is 60:40; within 10.7-11.2min, the volume ratio of mobile phase A to mobile phase B is 40:60.

table 3

serial number time/min ValveLeft 1 {initial time} 10_1 2 10.700 1_2 3 11.200 10_1

In Table 3, 10_1 marks the analysis pump, and 1_2 marks the switch from the analysis pump to the cleaning pump.

The control of the cleaning pump is shown in Table 4 below.

Table 4

The controls for the assay pumps are shown in Table 5 below.

table 5

1.4.4 Others

The analysis time was 12.5 min; the column temperature was 55°C; the injection volume was 30 μL; the flow rate was 0.3 mL/min.

1.4.5 Detector

The ultraviolet detector is a DAD-3000 detector with a detection wavelength of 300nm, an acquisition frequency of 5Hz, and a bandwidth of 4nm.

1.4.6 Online filter

The in-line filter is SSI COL PRE-FILTER WATER 1/16 0.5M.

1.5 Preparation of standard working solution

Standard working solution: Take an appropriate amount of standard stock solution, dilute it with 50% methanol aqueous solution, and prepare the concentrations of clomipramine standard products as 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, 6000ng/mL , 12000ng/mL, 15000ng/mL, the concentration of N-desmethylclomipramine standard is 1000ng/mL, 2000ng/mL, 4000ng/mL, 8000ng/mL, 12000ng/mL, 24000ng/mL, 30000ng/mL standard working solution and stored at -80°C.

It can be seen that among the seven standard working solutions, the concentrations of the clomipramine standard substance and N-desmethylclomipramine standard substance are different and increase sequentially.

1.6 Preparation of internal standard working solution

Internal standard working solution: Take an appropriate amount of internal standard stock solution and dilute it with 50% aqueous methanol to obtain an internal standard working solution with an iminostilbene concentration of 20 μg/mL, and store the internal standard working solution at -80°C.

1.7 Preparation of standard solution

Use a pipette to pipette 10 μL of standard working solution, 10 μL of internal standard working solution and 190 μL of blank serum or plasma respectively, place them in 1.5 mL centrifuge tubes, vortex and mix at a speed of 2500 rpm for 1 min, add 100 μL of methanol, and After vortex mixing at 2500 rpm for 1 min, add 1000 μL of n-hexane, and vortex mixing at 2500 rpm for 20 min, then centrifuge at 14000 rpm for 5 min at high speed, pipette 900 μL of supernatant into another In a 1.5mL centrifuge tube, blow dry slowly with N2 at room temperature, add 100 μL of 50% methanol aqueous solution (methanol:water ratio 50:50) to the dry centrifuge tube, and then vortex mix at 2500rpm for 3min After that, centrifuge at a high speed of 14000rpm for 5min, and take the supernatant as the standard solution to be detected.

In this way, for seven standard working solutions, seven standard solutions can be obtained.

1.8 Detect standard solution and generate standard curve equation

After each standard solution is obtained, the seven standard solutions can be detected by using a high-performance liquid chromatograph, and the chromatograms of each standard solution can be obtained correspondingly.

Please refer to Fig. 4, Fig. 4 shows the chromatogram of clomipramine standard substance, N-desmethylclomipramine and internal standard substance (ie iminostilbene standard substance) in a standard solution.

Taking clomipramine as example, from the chromatogram of standard solution, can obtain the chromatographic peak area of clomipramine standard substance and the chromatographic peak area of iminostilbene standard substance, then combine the clomipramine standard in each standard solution The known concentration of product and iminostilbene standard can obtain the standard curve equation of clomipramine. Similarly, the standard curve equation of N-desmethylclomipramine can be obtained.

Please refer to Figure 6, Figure 6 shows the obtained linear relationship diagram of clomipramine, according to the linear relationship diagram, the standard curve equation of clomipramine can be obtained: Y=0.1789×X-0.2293, correlation coefficient R ² =0.99939 , Y is the concentration ratio of clomipramine and internal standard, X is the area ratio of clomipramine and internal standard.

It can be seen that the correlation coefficient R ² >0.9900 of clomipramine in the linear range of 25-750ng/mL indicates a good linear relationship, and the accuracy of calculating the content of clomipramine in blood samples based on the standard curve equation is high , the error is small.

Please refer to Fig. 7, Fig. 7 shows the obtained N-desmethylclomipramine linear relationship diagram, according to the linear relationship diagram, the standard curve equation of N-desmethylclomipramine can be obtained: Y=0.1713×X- 0.3935, correlation coefficient R ² =0.99927, Y is the concentration ratio of N-desmethylclomipramine and the internal standard, X is the area ratio of N-desmethylclomipramine and the internal standard.

It can be seen that the correlation coefficient R ² of N-desmethylclomipramine in the linear range of 50-1500ng/mL>0.9900, indicating that the linear relationship is good, based on the standard curve equation to calculate the N-desmethylclomipramine in blood samples When the content is clear, the accuracy is high and the error is small.

After the standard curve equation is obtained, the blood sample can be pretreated to obtain the sample to be tested, and then under the same detection conditions, the sample to be tested is tested, combined with the obtained standard curve equations, the clomiphene concentration in the blood sample can be obtained. Peramine and N-desmethylclomipramine content. 〃

Example 2

The embodiment of the present invention is used to detect the content of clomipramine and N-desmethylclomipramine in venous blood.

2.1 Obtaining blood samples

The blood sample is obtained by processing at least 5mL of blood to be tested. After the blood sample is obtained, it can be pre-treated to obtain a corresponding sample to be tested that can be directly loaded.

2.2 Blood sample pretreatment

Use a pipette gun to pipette 10 μL of internal standard working solution into a 1.5 mL centrifuge tube, then add 200 μL of blood sample, vortex mix at 2500 rpm for 1 min, add 100 μL of methanol, and vortex mix at 2500 rpm After homogenizing for 1 min, add 1000 μL of n-hexane, vortex and mix at 2500 rpm for 20 min, then centrifuge at 14000 rpm for 5 min at high speed; take 900 μL of supernatant and put it into another 1.5 mL centrifuge tube. Blow dry slowly with N2; add 100 μL of 50% methanol aqueous solution (methanol: water ratio 50:50) to the blow-dried centrifuge tube, then vortex mix at 2500 rpm for 3 min, and then vortex at 14000 rpm After centrifugation for 5 minutes, the supernatant obtained was the sample to be tested.

2.3 Detection of samples to be tested

Under the detection conditions of Example 1, the same high performance liquid chromatograph was used to detect the sample to be tested, and the chromatogram of the sample to be tested was obtained.

Please refer to FIG. 5. FIG. 5 shows the chromatograms of clomipramine, N-desmethylclomipramine and internal standard (ie iminostilbene standard) in the sample to be tested.

Please refer to Fig. 4 and Fig. 5, the retention time of clomipramine in the sample to be tested is consistent with the retention time of clomipramine standard substance in the standard solution, and the retention time of N-desmethylclomipramine in the sample to be tested is Consistent with the retention time of the N-desmethylclomipramine standard in the standard solution, using iminostilbene as the internal standard makes the identification of the target compound more accurate, the analysis time is short, the interference is small, the internal standard is quantitatively suitable, Strong specificity, high accuracy and sensitivity.

In addition, as mentioned above, in order to avoid the value deviation caused by the inconsistency between the extraction rate of the prototype, the extraction rate of metabolites and the extraction rate of the internal standard, the standard working solution and blood samples will undergo the same pretreatment process, so that after the same pretreatment Finally, in the chromatograms of the standard solution and the sample to be tested, there are usually only the chromatographic peaks of the prototype, metabolites and internal standards, and the solvent peaks in the elution stage of the chromatographic column are basically the same, and no other obvious impurities are found. Based on this, it also shows that the above-mentioned pretreatment method is more reasonable and does not introduce other substances in the matrix.

2.4 Calculation of the content of clomipramine and N-desmethylclomipramine in the sample to be tested

According to the chromatogram of the sample to be tested, the chromatographic peak areas of clomipramine, N-desmethylclomipramine and the internal standard, and the known concentration of the internal standard in the sample to be tested, substitute the above two standard curve equations , the content of clomipramine and N-desmethylclomipramine in the sample to be tested can be calculated.

Example 3

The embodiments of the present invention are used to determine the limit of quantification and limit of detection.

Select a standard solution with an appropriate concentration, and dilute it to different degrees with blank blood to prepare blood sample dilutions with different concentrations, and perform blood sample dilutions on these blood sample dilutions according to the blood sample pretreatment method and measurement conditions in Example 2. Determination.

After testing, it was found that the detection limit and quantification limit of clomipramine are as follows:

(1) Limit of detection (LOD): 6.25 ng/mL.

(2) Limit of quantification (LOQ): 12.5 ng/mL.

After testing, it was found that the detection limit and quantification limit of N-desmethylclomipramine are as follows:

(1) Limit of detection (LOD): 12.5 ng/mL.

(2) Limit of quantification (LOQ): 20.0 ng/mL.

As can be seen from this embodiment, the detection limit of clomipramine can be as low as 6.25ng/mL, and the limit of quantification can be as low as 12.5ng/mL, and the detection limit of N-desmethylclomipramine can be as low as 12.5ng/mL, quantitatively The limit can be as low as 20.0ng/mL, and the sensitivity is high, which can improve the parallelism of low concentration samples and enhance the accuracy.

Due to the high sensitivity, the embodiment of the present invention has wider requirements on the sample volume of the blood to be tested, thereby improving the overall accuracy of sample detection. In addition, the embodiments of the present invention can also accurately quantify biological samples with very low content of clomipramine and N-desmethylclomipramine, which ensures the high accuracy and wide applicability of the detection method.

Example 4

The embodiments of the present invention are used to determine recovery and precision.

Get standard working solution, be mixed with high, medium and low 3 kinds of concentrations, carry out sample addition recovery rate and precision experiment, measure by the detection method in the embodiment 2, analyze and measure 3 batches, clomipramine and N- The recovery and precision of norclomipramine are shown in Table 6.

Table 6

It can be seen that the average recovery rate of clomipramine and N-desmethylclomipramine in the low, medium and high ranges of three addition levels is 93.9% to 106.1%, with good reproducibility. Good, the precision is 1.1% to 2.7%, the accuracy of the test results is high, and the system error can be eliminated.

Comparative example 1

类药物浓度方法的研究，期刊号为：中国药理学通报，2015Apr；31(4):582～5。Simultaneous Determination of Clomipramine and Four Benzodiazepines in Plasma by Solid Phase Extraction-High Performance Liquid Chromatography

Research on the concentration method of similar drugs, the journal number is: China Pharmacology Bulletin, 2015Apr; 31(4):582～5.

1. Detection object

Comparative Example 1 can detect the content of clomipramine in the blood, but does not detect the content of N-desmethylclomipramine in the blood. However, the embodiment of the present invention can simultaneously detect the contents of clomipramine and N-desmethylclomipramine in blood.

In Comparative Example 1, only the blood concentration of the drug prototype was detected, and according to the consensus guidelines for blood drug monitoring of neuropsychopharmacological therapeutic drugs, its metabolites are also pharmacologically active and will also affect the therapeutic effect. The embodiment of the present invention not only detects the prototype of the drug, but also detects its active metabolites at the same time. In this way, the detection method provided by the embodiment of the present invention can be used to detect clomipramine and its metabolite N in the patient's body during clinical treatment. -Monitoring the content of desmethylclomipramine, providing an experimental basis for the personalized administration of clomipramine and its metabolite N-desmethylclomipramine, and reducing the occurrence of toxic and side effects, so as to be more conducive to guiding patients medication.

2. Sample pretreatment

(1) In Comparative Example 1, the sample pretreatments of the standard working solution and the blood sample are different, but in the embodiment of the present invention, the sample pretreatment of the standard working solution and the blood sample are consistent.

Since the pre-treatments are consistent, the embodiment of the present invention can correct the fixed value error caused by the inconsistency between the extraction rate of the prototype, the extraction rate of metabolites and the extraction rate of the internal standard, and improve the detection accuracy.

(2) In comparative example 1, the blood sample pretreatment process is as follows: take a solid phase extraction column BondElut-C1 (100mg, 1mL) (Agilent, USA), activate it with 5mL methanol and ultrapure water in turn; take 500μL plasma, add 1000μL Phosphate buffer (pH7.0), add 50 μL of internal standard working solution, vortex for 1 min to mix well, and then load the sample to the above C1 column; use 1 mL deionized water and 1 mL water/methanol mixture (80:20, V/ V) Rinse once and discard; elute with 2 mL of methanol, collect the eluate, dry it with nitrogen at 45°C, redissolve in 125 μL of mobile phase, and take the supernatant for sampling after centrifugation.

For the pretreatment process of the blood sample in the embodiment of the present invention, please refer to the relevant description above, and details are not repeated here.

It can be seen that although the internal standard method is also used in Comparative Example 1 for quantification, the pretreatment method is more complicated, and the determination result is likely to be inaccurate due to operational errors. The embodiment of the present invention also adopts the internal standard method, but the pretreatment method is relatively simple, and can avoid errors caused by operation, thereby enabling more accurate quantification.

(3) In Comparative Example 1, no methanol was added to precipitate protein before the extraction operation, but in the embodiment of the present invention, methanol was added to precipitate protein before the extraction operation.

Considering that a high protein binding rate should lead to a low extraction rate, and when there is no methanol step, the experimental results obtained deviate greatly from the true value, so the detection accuracy of the embodiment of the present invention is higher.

3. Blood requirement

In Comparative Example 1, the serum volume is relatively large, requiring 500 μL, while the blood sample volume in the embodiment of the present invention is relatively small, requiring 200 μL, so the amount of blood collected by patients can be reduced accordingly, and patient compliance can be improved.

4. Selection of internal standard

The internal standard used in Comparative Example 1 is diphenhydramine, which is suitable for the treatment of allergies and motion sickness. It is widely used, and it is easy to interfere with the sample analysis due to the combination of drugs.

However, the internal standard used in the examples of the present invention is iminostilbene, which is an intermediate used in drug synthesis and basically does not exist in the patient's body. Compared with using psychotropic drugs or commonly used drugs with similar efficacy as internal standards, it has great advantages, and can avoid mutual interference brought by combined drugs to the analysis.

5. Analysis time

In Comparative Example 1, clomipramine peaked at 13 minutes, and the analysis time was not less than 21 minutes. In the embodiment of the present invention, the retention time of N-desmethylclomipramine and clomipramine is between 3.75-5min, the retention time of the internal standard iminostilbene is about 10.3min, and the analysis time is 12.5min.

It can be seen that the analysis time of the embodiment of the present invention is significantly shortened, the analysis efficiency is greatly improved, and it is beneficial to the detection of large-throughput blood samples.

6. Limit of detection, limit of quantitation, recovery and precision

In Comparative Example 1, clomipramine was linear in the linear range of 5.0-200 μg/L, the minimum detection concentration was 5.5 μg/L, the limit of quantification was 18.5 μg/L, and the recovery rate of the method was 84.11%-87.78%. The standard deviation (RSD) ranged from 3.53% to 13.21%.

And in the embodiment of the present invention, clomipramine is linear in the linear range of 25-750ng/mL, the detection limit is 6.25ng/mL, the limit of quantification is 12.5ng/mL, and N-desmethylclomipramine is 50- It was linear in the linear range of 1500ng/mL, the limit of detection was 12.5ng/mL, the limit of quantification was 20.0ng/mL, the average recovery was 93.9%-106.1%, and the precision (RSD) was 1.1%-2.7%.

It can be seen that for clomipramine, the quantitative limit, recovery rate and precision of the examples of the present invention are all better than those of Comparative Example 1, and the detection limit is basically the same as that of Comparative Example 1. For N-desmethylclomipramine, the detection limit, quantification limit, recovery rate and precision of the embodiment of the present invention are all better.

Therefore, the detection limit and quantification limit of the embodiment of the present invention are lower, the sensitivity of the detection method is higher, the recovery rate and precision are better, so that the requirements for the sample size can be wider, and the overall accuracy of sample detection is higher.

7. Testing cost

Comparative Example 1 uses solid phase extraction combined with high performance liquid chromatography to achieve the detection purpose, while the embodiment of the present invention uses high performance liquid chromatography to achieve the detection purpose. Compared with the solid phase extraction method, the analysis cost of the embodiment of the present invention is lower.

In summary, compared with Comparative Example 1, the detection method for the content of clomipramine and N-desmethylclomipramine in the blood provided by the embodiment of the present invention has the advantages of simple sample pretreatment, short analysis time, internal standard The advantages of reasonable selection, high detection sensitivity, good recovery rate and precision, high detection accuracy, and low detection cost are conducive to the detection of a large number of samples, so they have more advantages in the field of clinical detection.

Finally, it should be noted that: the above descriptions are only preferred embodiments of the present invention, and are only used to illustrate the technical solutions of the present invention, and are not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims (5)

1. A method for simultaneously detecting the contents of clomipramine and N-norclomipramine in blood is characterized by comprising the following steps:

preparing at least three standard working solutions, wherein the standard working solutions contain clomipramine standard substances and N-norclomipramine standard substances with known concentrations, and the concentrations of the same standard substance in different standard working solutions are different; the diluent of the standard working solution is 40-60% methanol water solution;

mixing a certain amount of standard working solution, internal standard working solution and a blank blood sample, and performing sample pretreatment to obtain standard solution, wherein the internal standard working solution contains an internal standard substance with known concentration; the dosage of the standard working solution is 10 muL, the dosage of the internal standard working solution is 10 muL, the dosage of the blank blood sample is 150-250 muL,

respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions;

fitting to obtain a standard curve equation of clomipramine and a standard curve equation of N-norclomipramine according to the chromatogram of each standard solution;

mixing a certain amount of internal standard working solution with a blood sample, wherein the using amount of the internal standard working solution is 10 mu L, and the using amount of the blood sample is 150-250 mu L, and performing the same sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by treating blood to be detected;

detecting a sample to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the sample to be detected;

calculating the contents of clomipramine and N-norclomipramine in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting;

the sample pretreatment comprises the following A1 to A5:

a1 comprises: mixing the mixed solution at 1800-2800rpm for 1-2min;

a2 comprises: adding 80-120 μ L methanol, and mixing for 1-2min at rotation speed of 1800-2800 rpm;

a3 comprises the following steps: adding 800-1200 μ L of extractant, mixing uniformly at 1800-2800rpm for 15-25min, and centrifuging at 10000-15000rpm for 3-7min to obtain supernatant;

a4 comprises: transferring 700-1000 μ L of supernatant into a centrifuge tube, and blow-drying with nitrogen at normal temperature;

a5 comprises: adding 80-120 μ L of the complex solution into an upward blowing centrifugal tube, uniformly mixing at 1800-2800rpm for 2-4min in a vortex manner, and centrifuging at 10000-15000rpm for 4-6min to obtain a supernatant; the compound solution is 40-60% methanol water solution;

the detection conditions include: a Waters xbridge c18 column having a length of 150mm, an internal diameter of 2.1mm, and a packing particle size of 3.5 μm; the column temperature is 50-60 ℃, the mobile phase A is water containing ammonium acetate with the concentration of 80-120mmol/L, formic acid with the volume ratio of 0.15-0.25% and triethylamine with the volume ratio of 0.15-0.25%, the mobile phase B is acetonitrile, the flow rate is 0.2-0.4mL/min, the sample injection amount is 20-40 mu L, and the analysis time is 12-13 min; the detection wavelength is 295-305nm;

the detection conditions include: single column dual pump elution mode;

the double-pump switching mode is as follows: switching from the analysis pump to the cleaning pump at 10.7min, and switching from the cleaning pump to the analysis pump at 11.2 min;

the elution mode is as follows: the volume ratio of the mobile phase A to the mobile phase B is 59-61% in other elution time ranges except 10.7-11.2 min: 41 to 39 percent.

2. The method of claim 1, wherein the uv detector in the hplc is a DAD-3000 detector with a collection frequency of 5Hz and a bandwidth of 4nm.

3. The method of claim 1, wherein the in-line filter used in the hplc is SSI COL PRE-FILTERWATER 1/16 0.5m.

4. The method of claim 1, wherein the internal standard is iminostilbene.

5. The method according to any one of claims 1 to 4, wherein the concentration of clomipramine standard in the standard working fluid is between 500 and 15000ng/mL and the concentration of N-norclomipramine standard is between 1000 and 30000ng/mL.

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