A method for simultaneous detection of 6 drug concentrations in blood

Abstract

The application discloses a method for simultaneously detecting the concentration of six drugs in blood, comprising the following steps: S1, taking a blood sample, centrifuging, and adding methanol to precipitate protein; S2, preparing a series of concentration calibration solution, quality control sample and internal standard working solution; S3, adding the internal standard working solution to the supernatant 1 prepared in S1, vortex mixing, centrifuging, and taking the supernatant 2; S4, using high performance liquid chromatography-tandem mass spectrometry to detect the supernatant 2 obtained in S3 for the selected drugs, and performing mass spectrometry screening analysis on the basis of the retention time of the compound, two pairs or three pairs of parent ions / daughter ions; the drugs include one, multiple or all of fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram and escitalopram; the detection method provided by the application has the characteristics of simple and convenient sample pretreatment, small required sample volume, high sensitivity, high accuracy and high throughput.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and in particular relates to a method for simultaneously detecting the concentrations of six drugs in blood. Background Art

[0002] Depression is a psychiatric disorder characterized by a persistent depressed mood. Clinical manifestations include low mood, anxiety, retardation, and numerous somatic complaints, as well as loss of interest in pleasurable activities, feelings of guilt or worthlessness, and even, in severe cases, suicidal ideation and behavior. Selective somatic reuptake inhibitors (SSRIs) are widely used as the antidepressant of choice worldwide. Fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram, and escitalopram are known as the "golden six" of SSRIs. The mechanism of action of SSRIs is that they selectively inhibit 5-HT recycling, indirectly increasing 5-HT concentrations in the synaptic cleft. They are highly selective for 5-HT, with minimal effects on neurexin, dapaminergic (DA), histamine, and cholinergic neurons. They are well absorbed orally, have high bioavailability, are well tolerated, and offer excellent compliance, making them suitable for all forms of depression. Common side effects of SSRIs include gastrointestinal effects (nausea, vomiting, diarrhea), central nervous system side effects (anxiety, insomnia, dizziness), sexual dysfunction, and serotonin syndrome. SSRIs are antidepressants that are currently considered relatively safe in clinical practice. However, in actual clinical practice, it has been found that SSRIs are effective for some patients but not for others, and doctors often need to constantly change medications to find effective ones. If it is possible to predict in advance whether a specific drug will be effective for a patient, it will be very beneficial to the patient. Blood tests can predict the efficacy of antidepressants in patients with depression. Blood drug concentration tests can accurately detect the blood drug concentration of the drug after the patient has taken the drug for a period of time. It can even analyze the different metabolic components of the drug in the body and obtain quantitative test results, providing sufficient basis for adjusting the dosage of drugs with very narrow therapeutic windows (such as antidepressants). Therefore, it is of great significance to develop blood test methods that can help doctors carry out individualized treatment. Summary of the Invention

[0003] The present invention aims to provide a method for simultaneously detecting the concentrations of six drugs in blood, namely fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram, and escitalopram, so as to guide the rational clinical use of antidepressants from different perspectives. The detection method has the characteristics of simple and convenient sample pretreatment, small required sample volume, high sensitivity, high accuracy, and high throughput.

[0004] To this end, the present invention adopts the following technical solutions:

[0005] A method for simultaneously detecting the concentrations of six drugs in blood comprises the following steps:

[0006] S1. Take a blood sample, centrifuge it, add methanol to precipitate protein, vortex mix, centrifuge it again, and take the supernatant 1;

[0007] S2. Prepare a series of calibrant solutions, quality control samples, and internal standard working solutions;

[0008] S3, add internal standard working solution to the supernatant 1 prepared in S1, vortex mix, centrifuge, and take supernatant 2;

[0009] S4. Detecting the selected drug in the supernatant 2 obtained in step S3 using high performance liquid chromatography-tandem mass spectrometry, and performing mass spectrometry screening analysis based on compound retention time and two or three pairs of parent ions / daughter ions;

[0010] The drugs include one, more or all of fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram and escitalopram;

[0011] The analytical columns of the liquid chromatography were: Eclipse Plus C18 RRHD 2.1×50 mm, 1.8 μm;

[0012] The mobile phases are: mobile phase A: 0.01%-1% formic acid in water; mobile phase B: methanol;

[0013] Flow rate: 0.2-1.0 ml / min; column temperature: 25-50°C; injection volume: 1 μl.

[0014] Preferably, the elution gradient of the liquid chromatography is: mobile phase A + mobile phase B = 100%; 0-0.5 min, the volume of mobile phase A is maintained at 95%; 0.5-1.0 min, the volume of mobile phase A is decreased from 95% to 85%; 1.0-3.0 min, the volume of mobile phase A is decreased from 85% to 65%; 3.0-5.0 min, the volume of mobile phase A is decreased from 65% to 10%; 5.0-7.0 min, the volume of mobile phase A is maintained at 5%; 7.0-7.5 min, the volume of mobile phase A is increased from 5% to 95%; 7.5-10 min, the volume of mobile phase A is maintained at 95%; flow rate: 0.2-1.0 mL / min; column temperature: 30°C.

[0015] Preferably, the mass spectrometry conditions are as follows: ion source temperature: 440° C.; ion source nebulizer gas: 35 psi; ion source heating auxiliary gas: 35 psi; curtain gas: 20 psi; spray capillary voltage: 4000 V; and collision gas: 9.

[0016] Preferably, the centrifugation conditions in step S1 are: the first centrifugation is at 3000-8000 rpm for 3-20 min, and the second centrifugation is at 6000-12000 rpm for 5-15 min.

[0017] Preferably, the internal standard comprises fluoxetine-d6, sertraline-d4, fluvoxamine-d4, escitalopram-d4, paroxetine-d4, and citalopram-d4.

[0018] Preferably, the amount of methanol added to S1 is 5-8 times that of the blood sample.

[0019] Further preferably, the flow rate of the chromatographic column is 0.3-0.7 mL / min;

[0020] More preferably, it is 0.3 mL / min.

[0021] Preferably, the calibrator solution is a series of standard solutions of different concentrations prepared with methanol.

[0022] Preferably, the mobile phase A is an aqueous solution containing 0.1%-0.5% formic acid.

[0023] Preferably, in step S3, the centrifugal speed is 1500-5000 rpm, and the centrifugation is performed for 3-10 minutes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] In line with clinical testing needs, this method uses high-performance liquid chromatography-tandem mass spectrometry to test six drugs. After confirming parameters such as retention time and two parent / daughter ion pairs, the blood concentration of SSRIs, a psychotropic drug, is analyzed, meeting the timeliness requirements of actual testing. The use of stable isotope internal standards for calibration effectively avoids matrix effects, resulting in high detection accuracy and low residual rates. DETAILED DESCRIPTION

[0026] The following further describes the claims of the present invention in detail in conjunction with specific implementation methods, but does not constitute any limitation to the present invention. Any limited modifications made by anyone within the scope of protection of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0027] Example 1

[0028] This embodiment provides a method for preparing a standard working solution.

[0029] (1) Preparation of 6 drug standard stock solutions

[0030] Fluoxetine was accurately weighed into a 2 mL cryovial using a balance. 2 mL of a 7:3 methanol:water solution was added to dissolve the solution. 40 μL of formic acid (99.5% purity) was added and mixed to obtain a stock solution with a concentration of 1221.31 μg / mL.

[0031] Precision weigh the paroxetine standard into a 2 mL cryovial using a balance, add 0.5 mL of methanol to dissolve, then add 1 mL of a 1:1 methanol:water solution (100.0% purity), and mix thoroughly to obtain a stock solution with a concentration of 2500.00 μg / mL.

[0032] Sertraline standard was precisely weighed into a 2 mL cryovial using a balance, and 2 mL of a 7:3 methanol:water solution was added to dissolve the solution. 40 μL of formic acid (92.0% purity) was then added and mixed to obtain a stock solution with a concentration of 1627.68 μg / mL.

[0033] Fluvoxamine standard was precisely weighed on a balance into a 2 mL cryovial, 1 mL of methanol was added to dissolve, and then 0.5 mL of a 7:3 methanol:water solution (100.0% purity) was added and mixed to obtain a stock solution with a concentration of 1727.92 μg / mL.

[0034] Precision weigh the thalassopane standard into a 2 mL cryovial using a balance, dissolve it in 1.5 mL of methanol (purity 100.0%), and mix thoroughly to obtain a stock solution with a concentration of 992.13 μg / mL.

[0035] Escitalopram standard was accurately weighed on a balance into a 2 mL cryovial, 1.5 mL of a methanol:water = 7:3 solution (100.0% purity) was added, and mixed to obtain a stock solution with a concentration of 833.57 μg / mL.

[0036] (2) Standard intermediate solutions were prepared using a diluent of methanol containing 10% sodium metabisulfite and water in a ratio of 1:9. The standard intermediate solutions were mixed and diluted using a diluent of methanol containing 10% sodium metabisulfite and water in a ratio of 1:9 to obtain standard working solutions with 8 levels of concentration.

[0037] Example 2

[0038] This embodiment provides a method for preparing an internal standard working solution.

[0039] (1) Preparation of internal standard stock solution

[0040] Fluoxetine-d6 standard, 2.00 mg, 98.7% purity, was prepared by adding 1 mL of a 1:1 methanol:water solution, followed by 40 μL of formic acid, and finally 960 μL of a 7:3 methanol:water solution to obtain a stock solution of 987 μg / mL.

[0041] Sertraline-d6 standard (1.00 mg, 99.1% purity) was prepared by adding 0.5 mL of a 1:1 methanol:water solution, followed by 20 μL of formic acid, and finally 480 μL of a 7:3 methanol:water solution to obtain a stock solution of 991 μg / mL.

[0042] Fluvoxamine-d6 standard, 2.00 mg, 99.2% purity, was added to 1 mL of methanol, followed by 1 mL of a 7:3 methanol:water solution to obtain a stock solution with a concentration of 803.24 μg / mL.

[0043] Escitalopram-d6 standard, 2.00 mg, 97.8% purity, was added to 1 mL of methanol, followed by 1 mL of a 7:3 methanol:water solution to obtain a stock solution with a concentration of 815.73 μg / mL.

[0044] Paroxetine-d4 standard (1.00 mg, 99.3% purity) was added to 0.5 mL of methanol, followed by 0.5 mL of a 7:3 methanol:water solution to obtain a stock solution of 618.05 μg / mL.

[0045] Citalopram-d4 standard, specification: 1.00 mg, purity: 89.4%, was added to 0.5 mL of methanol, and then to 0.5 mL of a 7:3 methanol:water solution to obtain a stock solution with a concentration of 894 μg / mL.

[0046] (2) An internal standard intermediate solution was prepared using a diluent of methanol:water = 7:3. The internal standard stock solution / internal standard intermediate solution were mixed and diluted with a diluent of methanol:water = 7:3 to obtain an internal standard working solution.

[0047] Example 3

[0048] A method for simultaneously detecting the concentrations of six drugs in blood comprises the following steps:

[0049] S1. Take a blood sample, centrifuge at 5000 rpm for 10 min, add methanol to precipitate protein, vortex mix, centrifuge at 12000 rpm for 10 min, and collect supernatant 1;

[0050] S2. Prepare a series of calibrant solutions, quality control samples, and internal standard working solutions;

[0051] S3, add internal standard working solution to the supernatant 1 prepared in S1, vortex mix, centrifuge, and take supernatant 2;

[0052] S4. Detecting the selected drug in the supernatant 2 obtained in step S3 using high performance liquid chromatography-tandem mass spectrometry, and performing mass spectrometry screening analysis based on compound retention time and two or three pairs of parent ions / daughter ions;

[0053] The drugs include fluoxetine and paroxetine;

[0054] The analytical columns of the liquid chromatography were: Eclipse Plus C18 RRHD 2.1×50 mm, 1.8 μm;

[0055] The mobile phases are: mobile phase A: 0.1% formic acid in water; mobile phase B: methanol;

[0056] Flow rate: 0.3 ml / min; column temperature: 40°C; injection volume: 1 μl.

[0057] The elution gradient of the liquid chromatography is as follows: mobile phase A+mobile phase B=100%; from 0 to 0.5 min, the volume of mobile phase A is maintained at 95%; from 0.5 to 1.0 min, the volume of mobile phase A is gradually reduced from 95% to 85%; from 1.0 to 3.0 min, the volume of mobile phase A is gradually reduced from 85% to 65%; from 3.0 to 5.0 min, the volume of mobile phase A is gradually reduced from 65% to 10%; from 5.0 to 7.0 min, the volume of mobile phase A is maintained at 5%; from 7.0 to 7.5 min, the volume of mobile phase A is increased from 5% to 95%; from 7.5 to 10 min, the volume of mobile phase A is maintained at 95%; flow rate: 0.2-1.0 mL / min; column temperature: 30°C.

[0058] The mass spectrometry conditions are as follows: ion source temperature: 440° C.; ion source nebulizer gas: 35 psi; ion source heating auxiliary gas: 35 psi; curtain gas: 20 psi; spray capillary voltage: 4000 V; and collision gas: 9.

[0059] Example 4

[0060] A method for simultaneously detecting the concentrations of six drugs in blood comprises the following steps:

[0061] S1. Take a blood sample, centrifuge at 8000 rpm for 5 min, add methanol to precipitate protein, vortex mix, centrifuge at 10000 rpm for 10 min, and collect supernatant 1;

[0062] S2. Prepare a series of calibrant solutions, quality control samples, and internal standard working solutions;

[0063] S3, add internal standard working solution to the supernatant 1 prepared in S1, vortex mix, centrifuge, and take supernatant 2;

[0064] S4. Detecting the selected drug in the supernatant 2 obtained in step S3 using high performance liquid chromatography-tandem mass spectrometry, and performing mass spectrometry screening analysis based on compound retention time and two or three pairs of parent ions / daughter ions;

[0065] The drugs include sertraline and escitalopram;

[0066] The analytical columns of the liquid chromatography were: Eclipse Plus C18 RRHD 2.1×50 mm, 1.8 μm;

[0067] The mobile phases are: mobile phase A: 0.1% formic acid in water; mobile phase B: methanol;

[0068] Flow rate: 0.3 ml / min; column temperature: 40°C; injection volume: 1 μl.

[0069] The elution gradient of the liquid chromatography is as follows: mobile phase A+mobile phase B=100%; from 0 to 0.5 min, the volume of mobile phase A is maintained at 95%; from 0.5 to 1.0 min, the volume of mobile phase A is gradually reduced from 95% to 85%; from 1.0 to 3.0 min, the volume of mobile phase A is gradually reduced from 85% to 65%; from 3.0 to 5.0 min, the volume of mobile phase A is gradually reduced from 65% to 10%; from 5.0 to 7.0 min, the volume of mobile phase A is maintained at 5%; from 7.0 to 7.5 min, the volume of mobile phase A is increased from 5% to 95%; from 7.5 to 10 min, the volume of mobile phase A is maintained at 95%; flow rate: 0.2-1.0 mL / min; column temperature: 30°C.

[0070] The mass spectrometry conditions are as follows: ion source temperature: 440° C.; ion source nebulizer gas: 35 psi; ion source heating auxiliary gas: 35 psi; curtain gas: 20 psi; spray capillary voltage: 4000 V; and collision gas: 9.

[0071] Example 5

[0072] A method for simultaneously detecting the concentrations of six drugs in blood comprises the following steps:

[0073] S1. Take a blood sample, centrifuge at 7000 rpm for 8 min, add methanol to precipitate protein, vortex mix, centrifuge at 8000 rpm for 10 min, and collect supernatant 1;

[0074] S2. Prepare a series of calibrant solutions, quality control samples, and internal standard working solutions;

[0075] S3, add internal standard working solution to the supernatant 1 prepared in S1, vortex mix, centrifuge, and take supernatant 2;

[0076] S4. Detecting the selected drug in the supernatant 2 obtained in step S3 using high performance liquid chromatography-tandem mass spectrometry, and performing mass spectrometry screening analysis based on compound retention time and two or three pairs of parent ions / daughter ions;

[0077] The drugs include fluvoxamine and citalopram;

[0078] The analytical columns of the liquid chromatography were: Eclipse Plus C18 RRHD 2.1×50 mm, 1.8 μm;

[0079] The mobile phases are: mobile phase A: 0.1% formic acid in water; mobile phase B: methanol;

[0080] Flow rate: 0.3 ml / min; column temperature: 40°C; injection volume: 1 μl.

[0081] The elution gradient of the liquid chromatography is as follows: mobile phase A+mobile phase B=100%; from 0 to 0.5 min, the volume of mobile phase A is maintained at 95%; from 0.5 to 1.0 min, the volume of mobile phase A is gradually reduced from 95% to 85%; from 1.0 to 3.0 min, the volume of mobile phase A is gradually reduced from 85% to 65%; from 3.0 to 5.0 min, the volume of mobile phase A is gradually reduced from 65% to 10%; from 5.0 to 7.0 min, the volume of mobile phase A is maintained at 5%; from 7.0 to 7.5 min, the volume of mobile phase A is increased from 5% to 95%; from 7.5 to 10 min, the volume of mobile phase A is maintained at 95%; flow rate: 0.2-1.0 mL / min; column temperature: 30°C.

[0082] The mass spectrometry conditions are as follows: ion source temperature: 440° C.; ion source nebulizer gas: 35 psi; ion source heating auxiliary gas: 35 psi; curtain gas: 20 psi; spray capillary voltage: 4000 V; and collision gas: 9.

[0083] Comparative Example 1

[0084] Compared with Example 3, the difference is that the stabilizer in the pretreatment of the sample to be tested in step S1 is acetonitrile (precipitant), which correspondingly replaces the perchloric acid in the standard solution.

[0085] Comparative Example 2

[0086] Compared with Example 3, the difference is that the liquid chromatography mobile phase A is water, and the mobile phase B is 1% formic acid in acetonitrile.

[0087] (1) HPLC-MS test results

[0088] Table 1 Retention time and ion pair parameters of each drug

[0089]

[0090] (2) Matrix effect

[0091] Samples with varying matrix ratios (90%, 80%, 50%, 20%, and 0%) were added to the same high-concentration standard solution (concentration greater than or equal to 10× the concentration in the matrix sample). Five different matrix ratios were tested for each sample. The results showed that the RSD of the target area / internal standard area for the same matrix ratio across different matrices was ≤10%. The bias of the target area / internal standard area ratio ((X matrix - 0 matrix) / 0 matrix) for the same matrix ratio across different matrix ratios was less than 10%, meeting the requirements.

[0092] (3) The residual signals of fluoxetine and paroxetine were evaluated using the detection methods provided in Example 3 and Comparative Examples 1 and 2. The results are shown in Table 2:

[0093] Table 2 Residual rates of fluoxetine and paroxetine

[0094] Detection method Fluoxetine residual rate % Paroxetine residual rate % Example 3 9 11 Comparative Example 1 34 38 Comparative Example 2 29 25

[0095] The sample pretreatment method provided in the present disclosure, combined with liquid chromatography-mass spectrometry detection conditions, can accurately separate and detect the contents of six drugs, improve the detection accuracy of memantine, reduce the residual rate, avoid subsequent long cleaning time, and reduce the overall analysis time.

[0096] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting the concentrations of six drugs in blood, comprising the following steps: S1. Take a blood sample, centrifuge it, add methanol to precipitate protein, vortex mix, centrifuge it again, and take supernatant 1. The amount of methanol added is 5-8 times that of the blood sample; S2. Prepare a series of calibrant solutions, quality control samples, and internal standard working solutions, where the internal standards include fluoxetine-d6, sertraline-d4, fluvoxamine-d4, escitalopram-d4, paroxetine-d4, and citalopram-d4. The calibrant solutions are standard solutions of different concentrations prepared using methanol. S3, add internal standard working solution to the supernatant 1 prepared in S1, vortex mix, centrifuge, and take supernatant 2; S4. Detecting the selected drug in the supernatant 2 obtained in step S3 using high performance liquid chromatography-tandem mass spectrometry, and performing mass spectrometry screening analysis based on compound retention time and two or three pairs of parent ions / daughter ions; The drugs include one, more or all of fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram and escitalopram; The analytical columns of the liquid chromatography were: Eclipse Plus C18 RRHD 2.1×50 mm, 1.8 μm; The mobile phases are: mobile phase A: 0.01%-1% formic acid in water; mobile phase B: methanol; Flow rate: 0.2-1.0 ml / min; column temperature: 25-50°C; injection volume: 1 μl; The elution gradient of the liquid chromatography is: mobile phase A + mobile phase B = 100%; from 0 to 0.5 min, the volume of mobile phase A is maintained at 95%; from 0.5 to 1.0 min, the volume of mobile phase A is gradually reduced from 95% to 85%; From 1.0 to 3.0 min, the volume of mobile phase A was decreased from 85% to 65%; From 3.0 to 5.0 min, the volume of mobile phase A was decreased from 65% to 10%; From 5.0 to 7.0 minutes, the volume of mobile phase A was maintained at 5%; from 7.0 to 7.5 minutes, the volume of mobile phase A was increased from 5% to 95%; from 7.5 to 10 minutes, the volume of mobile phase A was maintained at 95%; Flow rate: 0.2-1.0 mL / min; Column temperature: 30°C; The mass spectrometry conditions are as follows: ion source temperature: 440° C.; ion source nebulizer gas: 35 psi; ion source heating auxiliary gas: 35 psi; curtain gas: 20 psi; and spray capillary voltage: 4000 V.

2. The method for detecting 43 drugs in blood by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The centrifugation conditions in step S1 are: the first centrifugation is at 3000-8000 rpm for 3-20 min, and the second centrifugation is at 6000-12000 rpm for 5-15 min.

3. The method for simultaneously detecting the concentrations of six drugs in blood according to claim 1, characterized in that: The flow rate of the chromatographic column is 0.3-0.7 mL / min.

4. The method for simultaneously detecting the concentrations of six drugs in blood according to claim 3, characterized in that: The flow rate of the chromatographic column was 0.3 mL / min.

5. The method for simultaneously detecting the concentrations of six drugs in blood according to claim 1, characterized in that: The mobile phase A is an aqueous solution containing 0.1%-0.5% formic acid.

6. The method for simultaneously detecting the concentrations of six drugs in blood according to claim 1, characterized in that: In step S3, the centrifugal speed is 1500-5000 rpm, and the centrifugation is 3-10 minutes.

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