CN110806449B

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

Info

Publication number: CN110806449B
Application number: CN201911206887.9A
Authority: CN
Inventors: 张国成; 贾永娟; 倪君君
Original assignee: Ji'nan Hehe Medical Inspection Co ltd
Current assignee: Ji'nan Hehe Medical Inspection Co ltd
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2023-03-28
Anticipated expiration: 2039-11-29
Also published as: CN110806449A

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 simultaneously detecting content of clomipramine and N-norclomipramine in blood

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-norclomipramine in blood.

Background

Depression, also known as depressive disorder, is characterized clinically by a marked and persistent depression in the mood, the main type of mood disorder. The low mood is not matched with the situation in clinic, the depression of the mood can be from sultriness to sadness, and the self-declining depression and even the pessimism are taken away, and suicide attempts or behaviors can be caused; even the occurrence of stupor; in some cases, there is significant anxiety and motor agitation; in severe cases, psychotic symptoms such as hallucinations and delusions may occur. Depression persists for at least 2 weeks in each episode, with the long-term or even years being prone to recurrent episodes in most cases, with most remitting, and some residual symptoms or becoming chronic.

Clomipramine is a tricyclic antidepressant drug used mainly in the treatment of obsessive-compulsive disorder (OCD) and also in the treatment of obsessive-compulsive disorder, phobic neurosis. The clomipramine mainly has the function of blocking reuptake of 5-hydroxytryptamine in the central nervous system, and the active metabolite norclomipramine can block reuptake of norepinephrine in the central nervous system, thereby playing the roles of depression resistance and anxiety resistance and also having the functions of sedation and anticholinergic.

Methods for determining the level of clomipramine in blood are available. Since the metabolites also have pharmacological activity and influence the treatment effect, it is necessary to detect the active metabolites. Therefore, there is a need for a method capable of simultaneously detecting the levels of clomipramine and its metabolite, N-norclomipramine, in blood.

Disclosure of Invention

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

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention provides a method for simultaneously detecting the contents of clomipramine and N-norclomipramine in blood, which comprises 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;

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;

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, 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;

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 obtained by fitting.

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

a1: vortex and mix the mixed solution evenly;

a2: adding methanol, and mixing by vortex;

a3: adding an extracting agent, uniformly mixing by vortex, and centrifuging to obtain a supernatant;

a4: taking the supernatant fluid in a centrifuge tube, and drying the supernatant fluid by using nitrogen at normal temperature;

a5: adding the complex solution into the centrifugal tube blown to the upper part, uniformly mixing by vortex, and centrifuging to obtain a supernatant.

Preferably, said A1 comprises: mixing the mixed solution at 1800-2800rpm for 1-2min;

the A2 comprises: adding 80-120 μ L methanol, and mixing at 1800-2800rpm for 1-2min;

the A3 comprises: 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;

the A4 comprises: transferring 700-1000 μ L of supernatant into a centrifuge tube, and blow-drying with nitrogen at normal temperature;

the A5 comprises: adding 80-120 μ L of the complex solution into the upper clean blow-dried centrifuge tube, mixing uniformly at 1800-2800rpm for 2-4min, and centrifuging at 10000-15000rpm for 4-6min to obtain supernatant.

Preferably, the detection conditions include: a Waters Xbridge C18 column with a length of 150mm, an internal diameter of 2.1mm and a packing particle size of 3.5. Mu.m.

Preferably, the detection condition includes: 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 12-13min.

Preferably, the detection condition includes: 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.

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

Preferably, the on-line filter used by the high performance liquid chromatograph is SSI COLPRE-FILTER WATER 1/16.5M.

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-norclomipramine standard substance is 1000-30000ng/mL, and the diluent is 40-60% methanol aqueous solution.

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 invention can simultaneously detect the contents of clomipramine and N-norclomipramine in blood.

Drawings

In order to more clearly illustrate the embodiments or technical solutions of the present invention, the drawings used in the embodiments or technical solutions in the prior art are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a flow chart of a method for simultaneously detecting the levels of clomipramine and N-norclomipramine in blood according to an embodiment of the present invention;

FIG. 2 is a chemical structural formula of clomipramine provided in accordance with an embodiment of the present invention;

FIG. 3 is a chemical structural formula of N-norclomipramine provided in accordance with an embodiment of the present invention;

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

FIG. 5 is a chromatogram of clomipramine, N-norclomipramine and an internal standard in a test sample according to an embodiment of the present invention;

FIG. 6 is a graph of the linear relationship of clomipramine provided in accordance with an embodiment of the present invention;

FIG. 7 is a graph of the linear relationship of N-norclomipramine provided in accordance with an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.

It was found that the prototypes (i.e., clomipramine), metabolites of the prototypes (i.e., N-norclomipramine), and internal standards differ in their extraction rates during the pretreatment of blood samples. For example, the results of the extraction rate test using iminostilbene as an internal standard are shown in table 1 below.

TABLE 1

	Internal standard substance	Metabolites	Prototype
				Peak area of substance in calibration solution without pretreatment	2.1439	0.3644	0.242
Peak area of substance in standard solution at pretreatment	1.2254	0.2382	0.1979
				Extraction rate	57.2％	65.4％	81.8％

In table 1, standard solutions, i.e., standard working solutions and internal standard working solutions, were directly prepared by dilution and analyzed by loading. And (4) performing pretreatment, namely adding blank blood into the standard working solution and the internal standard working solution, performing pretreatment operation which is the same as that of blood sample pretreatment, obtaining a standard solution, and loading and analyzing the standard solution.

Referring to table 1, the peak area of the internal standard substance before pretreatment is 2.1439, and the peak area of the internal standard substance before pretreatment is 1.2254, it can be seen that the pretreatment process has a certain influence on the extraction of the internal standard substance, and the extraction rate is 57.2%.

Referring to table 1, the peak area of the metabolite before pretreatment was 0.3644, and the peak area of the internal standard during pretreatment was 0.2382, it can be seen that the pretreatment process has a certain effect on the extraction of the metabolite, and the extraction rate is 65.4%.

Referring to table 1, the peak area of the prototype before pretreatment was 0.242, and the peak area of the internal standard substance before pretreatment was 0.1979, it can be seen that the pretreatment process has a certain effect on the extraction of the prototype, and the extraction rate is 81.8%.

It can be seen that the extraction rate of the internal standard substance and the extraction rate of the metabolite, and the extraction rate of the internal standard substance and the extraction rate of the prototype are different and have larger difference in the pretreatment process. Therefore, in order to correct the fixed value error caused by the inconsistent extraction rates of the prototype, the metabolite and the internal standard substance, a blood adding pretreatment mode can be adopted for obtaining the standard curve, so that the pretreatment process for preparing the standard solution according to the standard working solution is the same as the pretreatment process for preparing the sample to be detected according to the blood sample, and the detection accuracy is ensured.

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

step 101: preparing at least three standard working solutions, wherein the standard working solutions contain clomipramine standard substances and N-demethylclomipramine standard substances with known concentrations, and the concentrations of the same standard substance in different standard working solutions are different.

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

In detail, the blank blood sample may be typically serum or plasma without clomipramine, N-norclomipramine and an internal standard substance, and the detection result of the contents of clomipramine, N-norclomipramine and an internal standard substance of the blank blood sample should be typically undetected.

Step 103: and respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain the chromatograms of the standard solutions.

Step 104: and 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.

Step 105: and mixing a certain amount of internal standard working solution with the blood sample, 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.

Step 106: and detecting the 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.

Step 107: 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 obtained by fitting.

The embodiment of the invention uses an internal standard method to detect the contents of clomipramine and N-norclomipramine in the blood sample so as to avoid constant value deviation caused by misoperation in the pretreatment process, thereby ensuring the detection accuracy.

Referring to fig. 2 and 3, fig. 2 shows the chemical structural formula of clomipramine, and fig. 3 shows the chemical structural formula of N-norclomipramine.

Usually, at least three coordinate points are needed for establishing the standard curve equation so as to ensure the accuracy of the established equation, and at least three standard solutions are prepared, so that the standard curve equation of clomipramine and the standard curve equation of N-norclomipramine can be fitted according to the chromatogram obtained by detecting each standard solution.

In detail, taking clomipramine as an example, the standard curve equation of clomipramine obtained by fitting may be generally y = k × x + b. The two variables x and y can be the peak area ratio of the standard substance and the internal standard substance of clomipramine in the chromatogram of each standard solution, and the concentration ratio of the standard substance and the internal standard substance of the clomipramine in each standard solution. Therefore, the concentration of the clomipramine in the sample to be detected can be calculated by substituting the peak area ratio of the clomipramine to the internal standard substance and the concentration of the internal standard substance in the sample to be detected in the chromatogram of the sample to be detected into the standard curve equation, so that the content of the clomipramine in the blood sample is obtained. Of course, this implementation is equally applicable to N-norclomipramine.

In the embodiment of the present invention, the blood sample may be serum or plasma, and is obtained by processing blood to be tested. After the blood to be detected is sampled, corresponding treatment can be carried out to obtain a blood sample. For example, at least 5mL of blood to be detected is taken, the blood is centrifuged for 10min at a centrifugal speed of 3500rpm, and the supernatant is taken to obtain serum or plasma, namely the blood sample. Serum or plasma samples can be stored frozen at-20 ℃ until ready for analysis.

After the blood sample is obtained, the sample to be detected is obtained through the same pretreatment, and the sample to be detected is detected under the same detection condition to obtain a chromatogram map of the sample to be detected. Based on the chromatogram and the standard curve equation obtained by fitting, the contents of clomipramine and N-norclomipramine in the blood sample can be obtained as described above.

In summary, the method for detecting the content of clomipramine and N-norclomipramine in blood provided by the embodiment of the invention combines an internal standard method and a high performance liquid chromatography, and processes a standard working solution and a blood sample through the same pretreatment process, so that interference factors are greatly reduced, and the method has the advantages of accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection result, low cost and short analysis time, and is beneficial to the detection of a large-flux blood sample.

In detail, in the case of performing an inter-laboratory comparison test in the british government chemist Laboratory (LGC), it was found that when a sample was pretreated, if there was no methanol addition step before extraction, the obtained experimental results were more deviated from the true values, and if there was a methanol addition step before extraction, the obtained experimental results were less deviated from the true values, and for specific experimental data, see table 2 below.

TABLE 2

In table 2, before the improvement, no methanol addition step was performed before the extraction in the sample pretreatment, and after the improvement, a methanol addition step was performed before the extraction in the sample pretreatment. Wherein z' score is less than or equal to 2, and the result is qualified.

Based on the above, it is considered that the higher protein binding rate should be the cause of the lower extraction rate by analysis, and therefore, it is preferable to perform a methanol addition step before extraction in the sample pretreatment process. Therefore, in one embodiment of the present invention, the performing the sample pretreatment includes the following A1 to A5:

a1: vortex and mix the mixed solution evenly;

a2: adding methanol, and mixing by vortex;

a5: adding the complex solution into a centrifugal tube blown to be dry, uniformly mixing in a vortex mode, and centrifuging to obtain a supernatant.

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

In the embodiment of the 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: mixing the mixed solution at 1800-2800rpm for 1-2min;

the A2 comprises: adding 80-120 μ L methanol, and mixing for 1-2min at rotation speed of 1800-2800 rpm;

the A5 comprises: adding 80-120 μ L of the complex solution into the upper clean blow-dried centrifuge tube, mixing for 2-4min at 1800-2800rpm, and centrifuging at 10000-15000rpm for 4-6min to obtain supernatant.

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

Preferably, the extractant may be n-hexane, and the redissolution may be a 40-60% aqueous methanol solution. For example, the percentage of methanol in the reconstituted solution may be 40%, 45%, 50%, 55%, or 60%.

The embodiment of the invention adopts an internal standard method to detect the contents of clomipramine and N-norclomipramine in the blood sample, can use the pretreatment mode, has simple operation, can avoid errors caused by operation, and can quantify more accurately. In addition, the pretreatment method is simple, so that the method is beneficial to popularization and application of the embodiment of the invention.

Based on the above, in one embodiment of the present invention, the standard working solution is used in an amount of 10 μ L, the internal standard working solution is used in an amount of 10 μ L, and the blank blood sample is used in an amount of 150 to 250 μ L, which are mixed together for sample pretreatment. For example, the amount of blank blood sample may take on a value of 150, 170, 200, 220, or 250.

Based on the above, in one embodiment of the present invention, the amount of the internal standard working solution is 10 μ L, the amount of the blood sample is 150-250 μ L, and the two are mixed for sample pre-treatment. For example, the blood sample volume can take on a value of 150, 170, 200, 220, or 250.

In one embodiment of the present invention, the detection condition includes: a Waters Xbridge C18 column with a length of 150mm, an internal diameter of 2.1mm and a packing particle size of 3.5. Mu.m.

In one embodiment of the present invention, the detection condition includes: 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 12-13min.

For example, the column temperature can take on a value of 50, 52, 54, 56, 58, or 60; in the mobile phase A, the concentration of ammonium acetate can be 80, 90, 100, 110 or 120, and the volume ratio of formic acid to triethylamine can be 0.15, 0.17, 0.20, 0.22 or 0.25; the flow rate can take the value of 0.2, 0.25, 0.3, 0.35 or 0.4; the value of the sample volume can be 20, 25, 30, 35 or 40; the value of the analysis time may be 12, 12.2, 12.4, 12.6, 12.8 or 13.

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

In one embodiment of the present invention, the volume ratio of mobile phase a to mobile phase B is 59-61% during the target analysis phase of the total analysis time: 41 to 39 percent. For example, the volume ratio of mobile phase a and mobile phase B may be 59, 59.5, 60, 40, 60.5.

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

Based on the above, in one embodiment of the present invention, the detection condition includes: single column dual pump elution mode;

In the embodiment of the invention, the time period of 10.7-11.2min is the time period for cleaning the chromatographic column by cleaning the mobile phase of the pump, and the time period of 10.7min and 11.2min is the time period for analyzing the mobile phase of the pump to enter the chromatographic column for analyzing the target substances. Thus, for the analysis pump mobile phase, the volume ratio of mobile phase a to mobile phase B is 59-61%:41 to 39 percent. In one embodiment of the present invention, for a clean pump mobile phase, the volume ratio of mobile phase a and mobile phase B may be 40.

Compared with the single-pump gradient elution mode, the single-column double-pump elution mode can reduce the analysis time to the maximum extent under the condition of a single column. At the same time, the amount of organic reagent used can be reduced to some extent.

Therefore, the embodiment of the invention can shorten the analysis time, improve the detection flux and reduce the using amount of the organic solvent.

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

In one embodiment of the invention, the on-line filter used by the HPLC is SSI COL PRE-FILTER WATER 1/16.5M.

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

In detail, iminostilbene is an intermediate for drug synthesis, and is not substantially present in patients. Compared with the use of mental drugs or common drugs with similar efficacies as internal standards, the selection of iminostilbene as the internal standard has great advantages, and can avoid mutual interference brought to analysis by combined drugs.

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

In detail, the corresponding linear range can be set by combining the detected population, the dosage of the blood to be detected and the approximate content range of the clomipramine and the N-norclomipramine in the human body, so as to ensure that most of the detection results of the clinical samples fall within the reportable range.

For example, in the at least three standard working solutions, the concentration of the clomipramine standard substance can be at least three of 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, 6000ng/mL, 12000ng/mL and 15000ng/mL, and the concentration of the N-norclomipramine standard substance can be at least three of 1000ng/mL, 2000ng/mL, 4000ng/mL, 8000ng/mL, 12000ng/mL, 24000ng/mL and 30000ng/mL. Preferably, the number of standard working fluids is 7.

In summary, the method for detecting the content of clomipramine and N-norclomipramine in blood provided by the embodiment of the invention combines an internal standard method with a high performance liquid chromatography, so that interference factors are greatly reduced, and the method has the advantages of accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection result, low cost, short analysis time and contribution to the detection of a large-flux blood sample.

The embodiment of the invention not only detects the prototype of the drug, but also simultaneously detects the active metabolite of the drug, thus, the detection method provided by the embodiment of the invention can be utilized to monitor the content of clomipramine and the metabolite N-norclomipramine thereof in the body of a patient in clinical treatment, and provide an experimental basis for personalized administration of the clomipramine and the metabolite N-norclomipramine thereof and reduction of toxic and side reactions, thereby being more beneficial to guiding the administration of the patient.

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

Example 1

The embodiment of the invention is used for obtaining the standard curve equation.

1.1 preparation of Standard stock solutions

Standard stock solutions: precisely transferring 15 mu L of 1.0mg/mL clomipramine standard solution and 30 mu L of 1.0mg/mL N-norclomipramine standard solution into a 1.5mL centrifuge tube, and diluting with 50% methanol aqueous solution to obtain a standard stock solution. In the obtained standard stock solution, the concentration of the clomipramine standard substance is 15000ng/mL, and the concentration of the N-norclomipramine standard substance is 30000ng/mL.

1.2 preparation of stock solutions for internal standards

Internal standard stock solution: accurately weighing 5mg of iminostilbene standard substance in a 5mL volumetric flask, dissolving the iminostilbene standard substance in methanol, and fixing the volume to 5mL to obtain an internal standard stock solution.

1.3 Instrument for detection

Saimeifei U3000 high performance liquid chromatograph.

1.4 detection conditions

1.4.1 chromatography columns

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

1.4.2 Mobile phase

A mobile phase A: water containing ammonium acetate with the concentration of 100mmol/L, formic acid with the volume ratio of 0.2 percent and triethylamine with the volume ratio of 0.2 percent.

Mobile phase B: and (3) acetonitrile.

1.4.3 elution mode

Single column dual pump elution mode.

The dual pump switching scheme is shown in table 3 below. The volume ratio of the mobile phase A to the mobile phase B is 60 to 10.7min and 11.2 to 12.5min; the volume ratio of the mobile phase A to the mobile phase B is 40.

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\ u 1 identifies the analysis pump and 1_2 identifies 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 control of the analysis pump is shown in table 5 below.

TABLE 5

1.4.4 other

The analysis time is 12.5min; the column temperature was 55 ℃; the sample injection amount is 30 mu L; the flow rate was 0.3mL/min.

1.4.5 Detector

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

1.4.6 in-line Filter

The in-line filter was SSI COL PRE-FILTER WATER 1/16.5M.

1.5 preparation of Standard working solution

Standard working solution: taking a proper amount of standard stock solution, diluting the stock solution by using 50% methanol aqueous solution to prepare standard working solution with the concentration of clomipramine standard substances of 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, 6000ng/mL, 12000ng/mL and 15000ng/mL respectively, and the concentration of N-norclomipramine standard substances of 1000ng/mL, 2000ng/mL, 4000ng/mL, 8000ng/mL, 12000ng/mL, 24000ng/mL and 30000ng/mL respectively, and storing the standard working solution at the temperature of-80 ℃.

It can be seen that the concentrations of the clomipramine standard and the N-norclomipramine standard in the seven standard working solutions are different and gradually increased.

1.6 preparation of internal standard working solution

Internal standard working solution: taking a proper amount of internal standard stock solution, diluting with 50% methanol water solution to obtain internal standard working solution with iminostilbene concentration of 20 mug/mL, and storing the internal standard working solution at-80 ℃.

1.7 preparation of Standard solution

Transferring 10 mu L of standard working solution, 10 mu L of internal standard working solution and 190 mu L of blank serum or plasma by using a pipette, respectively placing the standard working solution, the internal standard working solution and the blank serum or plasma in 1.5mL centrifuge tubes, performing vortex mixing for 1min at the rotation speed of 2500rpm, adding 100 mu L of methanol, performing vortex mixing for 1min at the rotation speed of 2500rpm, adding 1000 mu L of N-hexane, performing vortex mixing for 20min at the rotation speed of 2500rpm, performing high-speed centrifugation for 5min at the rotation speed of 14000rpm, transferring 900 mu L of supernatant into another 1.5mL centrifuge tube, performing slow blow-drying at normal temperature by using N2, adding 100 mu L of 50% methanol-water solution (methanol: water is 50) into a blow-dried centrifuge tube, performing vortex mixing for 3min at the rotation speed of 2500rpm, performing high-speed centrifugation for 5min at the rotation speed of 14000rpm, and taking the supernatant as a standard solution to be detected.

Thus, seven standard solutions can be obtained for seven standard working solutions.

1.8 detecting the standard solution to generate a standard curve equation

After obtaining each standard solution, the high performance liquid chromatograph can be used for respectively detecting the seven standard solutions, and the chromatogram of each standard solution is correspondingly obtained.

Referring to fig. 4, fig. 4 shows chromatograms of clomipramine standard, N-norclomipramine, and an internal standard (i.e., iminostilbene standard) in a standard solution.

Taking clomipramine as an example, the chromatographic peak area of the clomipramine standard substance and the chromatographic peak area of the iminostilbene standard substance can be obtained from the chromatogram of the standard solution, and then the known concentrations of the clomipramine standard substance and the iminostilbene standard substance in each standard solution are combined to obtain the standard curve equation of the clomipramine. Similarly, a standard curve equation of the N-demethylclomipramine can be obtained.

Referring to fig. 6, fig. 6 shows a linear relationship diagram of clomipramine obtained, from which a standard curve equation of clomipramine can be obtained: y =0.1789 XX-0.2293, correlation coefficient R ² =0.99939, y is the concentration ratio of clomipramine to the internal standard, and X is the area ratio of clomipramine to the internal standard.

As can be seen, clomipramine has a correlation coefficient R in the linear range of 25-750ng/mL ² The standard curve equation is more than 0.9900, the linear relation is good, and when the content of the clomipramine in the blood sample is calculated based on the standard curve equation, the accuracy is high and the error is small.

Referring to FIG. 7, FIG. 7 shows the linear relationship of the obtained N-norclomipramine, from which the standard curve equation of N-norclomipramine can be obtained: y =0.1713 XX-0.3935, correlation coefficient R ² =0.99927, Y is the concentration ratio of N-norclomipramine to the internal standard, and X is the area ratio of N-norclomipramine to the internal standard.

It can be seen that the coefficient of correlation R of N-norclomipramine is in the linear range of 50-1500ng/mL ² The linear relation is good when the standard curve equation is more than 0.9900, and the accuracy is high and the error is small when the content of the N-demethylclomipramine in the blood sample is calculated based on the standard curve equation.

After the standard curve equations are obtained, the blood sample can be pretreated to obtain a sample to be detected, the sample to be detected is detected under the same detection conditions, and the contents of the clomipramine and the N-norclomipramine in the blood sample can be obtained by combining the obtained standard curve equations. "A", "an

Example 2

The embodiment of the invention is used for detecting the content of clomipramine and N-norclomipramine in venous blood.

2.1 obtaining blood samples

The blood sample is obtained by treating at least 5mL of blood to be tested. After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded.

2.2 blood sample pretreatment

Transferring 10 mu L of internal standard working solution into a 1.5mL centrifuge tube by using a liquid transfer gun, then adding 200 mu L of blood sample, carrying out vortex mixing for 1min at the rotating speed of 2500rpm, adding 100 mu L of methanol, carrying out vortex mixing for 1min at the rotating speed of 2500rpm, adding 1000 mu L of n-hexane, carrying out vortex mixing for 20min at the rotating speed of 2500rpm, and then carrying out high-speed centrifugation for 5min at the rotating speed of 14000 rpm; putting 900 mu L of supernatant into another 1.5mL centrifuge tube, and slowly drying by N2 at normal temperature; adding 100 mu L of 50% methanol water solution (methanol: water is 50).

2.3 detection of samples to be tested

Under the detection conditions of example 1, the same high performance liquid chromatograph is used to detect the sample to be detected, and the chromatogram of the sample to be detected is obtained.

Referring to fig. 5, fig. 5 shows chromatograms of clomipramine, N-norclomipramine and an internal standard (i.e., iminostilbene standard) in a sample to be tested.

Referring to fig. 4 and 5, the retention time of clomipramine in the sample to be detected is consistent with that of the clomipramine standard in the standard solution, the retention time of N-norclomipramine in the sample to be detected is consistent with that of the N-norclomipramine standard in the standard solution, and iminostilbene is used as an internal standard substance, so that the identification of the target compound is more accurate, the analysis time is short, the interference is small, the internal standard is appropriate in quantification, the specificity is strong, and the accuracy and the sensitivity are high.

In addition, as mentioned above, in order to avoid the fixed value deviation caused by the inconsistency between the prototype extraction rate, the metabolite extraction rate and the internal standard extraction rate, the standard working solution and the blood sample are subjected to the same pretreatment process, so that after the same pretreatment, chromatograms of the standard solution and the sample to be detected generally only have chromatographic peaks of the prototype, the metabolite and the internal standard, solvent peaks at the elution stage of the chromatographic column are also basically consistent, and no other obvious impurities are found. Based on this, it is also demonstrated that the above pretreatment method is reasonable and that other substances are not introduced into the substrate.

2.4 calculation of Clomipramine and N-norclomipramine content in samples to be tested

Substituting the chromatographic peak areas of the clomipramine, the N-norclomipramine and the internal standard substance in the chromatogram of the sample to be detected and the known concentration of the internal standard substance in the sample to be detected into the 2 standard curve equations to calculate the contents of the clomipramine and the N-norclomipramine in the sample to be detected.

Example 3

The embodiment of the invention is used for determining the quantitative limit and the detection limit.

The appropriate concentration of standard solution is selected, blank blood is diluted to different degrees, so as to prepare blood sample diluents with different concentrations, and the blood sample diluents are measured according to the blood sample pretreatment mode and the measurement conditions in example 2.

The detection limit and the quantitative limit of the clomipramine are shown as follows:

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

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

The detection and quantification limits of the N-norclomipramine are shown as follows:

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

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

According to the embodiment, the detection limit of clomipramine can be as low as 6.25ng/mL, the quantification limit can be as low as 12.5ng/mL, the detection limit of N-norclomipramine can be as low as 12.5ng/mL, the quantification limit can be as low as 20.0ng/mL, the sensitivity is high, the parallelism of low-concentration samples can be improved, and the accuracy is enhanced.

Due to the high sensitivity, the requirement of the sample volume of the blood to be detected in the embodiment of the invention can be wider, so that the overall accuracy of sample detection is improved. In addition, the embodiment of the invention can accurately quantify the biological samples with very low clomipramine and N-norclomipramine contents, and ensures the high accuracy and wide applicability of the detection method.

Example 4

The embodiment of the invention is used for measuring the recovery rate and the precision.

The standard working solution was prepared to have high, medium and low concentrations, and the sample recovery and precision experiments were performed according to the method of example 2, and the recovery and precision of clomipramine and N-norclomipramine were measured for 3 batches and analyzed as shown in table 6.

TABLE 6

It can be seen that the clomipramine and the N-norclomipramine have the average recovery rate of 93.9-106.1%, good reproducibility, good sample-adding recovery rate, 1.1-2.7% precision, high accuracy of detection results and capability of eliminating system errors within the range of 3 addition levels of low, medium and high.

Comparative example 1

Method for simultaneously measuring clomipramine and 4 benzodiazepines in blood plasma by solid phase extraction-high performance liquid chromatography

The research of the concentration method of the quasi-drug is published as follows: chinese pharmacological report, 2015Apr;31 (4): 582-5.

1. Detecting an object

Comparative example 1 the amount of clomipramine in blood was detected, but the amount of N-norclomipramine in blood was not detected. The embodiment of the invention can simultaneously detect the contents of clomipramine and N-norclomipramine in blood.

Comparative example 1 only measured the blood concentration of proto-drug, while its metabolites also had pharmacological activity, as described in accordance with the consensus guidelines for blood monitoring of neuropsychiatric drugs, which also affected the therapeutic effect. The embodiment of the invention not only detects the prototype of the drug, but also simultaneously detects the active metabolite of the drug, thus, the detection method provided by the embodiment of the invention can be utilized to monitor the content of clomipramine and the metabolite N-norclomipramine thereof in the body of a patient in clinical treatment, and provide an experimental basis for personalized administration of the clomipramine and the metabolite N-norclomipramine thereof and reduction of toxic and side reactions, thereby being more beneficial to guiding the administration of the patient.

2. Sample pretreatment

(1) In comparative example 1, the sample pretreatments of the standard working solution and the blood sample were different, whereas in the present example, the sample pretreatments of the standard working solution and the blood sample were kept the same.

Because the pre-treatment is consistent, the embodiment of the invention can correct the fixed value errors caused by inconsistent prototype extraction rate, metabolite extraction rate and internal standard extraction rate, and improve the detection accuracy.

(2) In comparative example 1, the blood sample pretreatment process was: a solid phase extraction column BondElut-C1 (100mg, 1mL) (Agilent, U.S.A.) was taken and activated with 5mL of methanol and ultrapure water in this order; adding 1000 μ L phosphate buffer solution (pH7.0) into 500 μ L plasma, adding 50 μ L internal standard working solution, vortexing for 1min, mixing, and loading to the C1 column; leaching with 1mL of deionized water and 1mL of water/methanol mixed solution (80: 20, V/V) for 1 time respectively, and discarding; eluting with 2mL of methanol, collecting the eluent, drying the eluent at 45 ℃ with nitrogen, redissolving the eluent in 125 mu L of mobile phase, centrifuging the mobile phase, and taking the supernatant for sample injection.

The blood sample pretreatment process according to the embodiment of the present invention refers to the related description above, and is not repeated herein.

It can be seen that, although comparative example 1 also adopts the internal standard method for quantification, the pretreatment method is complicated, and the fixed value result is easily inaccurate due to operation errors. The embodiment of the invention also adopts an internal standard method, but the pretreatment method is relatively simple, and can avoid errors caused by operation, thereby being capable of quantifying more accurately.

(3) In comparative example 1, methanol was not added to perform the protein precipitation operation before the extraction operation, whereas in the present example, methanol was added to precipitate proteins before the extraction operation.

The detection accuracy of the embodiment of the invention is higher considering that higher protein binding rate can result in lower extraction rate and the deviation of the obtained experimental result and a true value is larger without a methanol step.

3. Blood demand

In comparative example 1, the amount of serum is larger and needs 500 μ L, while the amount of blood sample in the embodiment of the invention is smaller and needs 200 μ L, so that the blood collection amount of the patient can be correspondingly reduced, and the compliance of the patient can be improved.

4. Selection of internal standard substance

The internal standard used in the comparative example 1 is diphenhydramine, is suitable for treating allergy and carsickness, is relatively universal in application, and is easy to interfere with sample analysis due to combined medication.

The internal standard used in the embodiment of the invention is iminostilbene, which is an intermediate for drug synthesis and basically does not exist in a patient body. Compared with the use of mental drugs or common drugs with similar efficacies as internal standards, the method has great advantages and can avoid mutual interference brought to analysis by combined drugs.

5. Analysis time

In comparative example 1, clomipramine peaked at 13min, the analysis time was not less than 21min. In the present example, the retention time of N-norclomipramine and clomipramine is between 3.75-5min, the retention time of the internal standard substance iminostilbene is about 10.3min, and the analysis time is 12.5min.

Therefore, the analysis time of the embodiment of the invention is obviously shortened, the analysis efficiency is greatly improved, and the detection of a large-flux blood sample is facilitated.

6. Detection limit, quantitative limit, recovery rate and precision

In comparative example 1, clomipramine was linear in the linear range of 5.0 to 200. Mu.g/L, the lowest detected concentration was 5.5. Mu.g/L, the limit of quantitation was 18.5. Mu.g/L, the process recovery rate was 84.11% to 87.78%, and the Relative Standard Deviation (RSD) was 3.53% to 13.21%.

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

It can be seen that the limit of quantitation, the recovery rate and the precision of the embodiment of the invention are all better than those of the comparative example 1, and the detection limit is basically consistent with that of the comparative example 1. For N-norclomipramine, the detection limit, the quantification limit, the recovery rate and the precision of the embodiment of the invention are better.

Therefore, the detection limit and the quantification limit of the embodiment of the invention are lower, the sensitivity of the detection method is higher, the recovery rate and the precision are better, the requirement on the sample amount can be wider, and the overall accuracy of the sample detection is higher.

7. Detection cost

Comparative example 1 used solid phase extraction in combination with high performance liquid chromatography for detection purposes, whereas the examples of the present invention used high performance liquid chromatography for detection purposes. Compared with a solid phase extraction method, the analysis cost of the embodiment of the invention is lower.

In conclusion, compared with the comparative example 1, the method for detecting the content of clomipramine and N-demethylclomipramine in blood provided by the embodiment of the invention has the advantages of simple sample pretreatment, short analysis time, reasonable internal standard selection, high detection sensitivity, good recovery rate and precision, high detection accuracy, low detection cost and the like, is beneficial to detection of a large number of samples, and has more advantages in the field of clinical detection.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

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,

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;

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;

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.