Liquid chromatography-tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid-phase extraction

Abstract

The invention provides a liquid chromatography-tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid-phase extraction. The liquid chromatography-tandem mass spectrometry detection method is used for pretreatment and quantitative analysis of a target object in serum. According to the method, a magnetic solid-phase extraction filler is controlled by a magnetic rod to be transferred, dispersed, adsorbed, leached and eluted between a water phase and an organic phase for multiple times, and interference of phospholipid and protein in serum is removed. And compared with traditional SPE, the problems of inter-hole difference, filler swelling and the like are avoided, automatic batch processing can be achieved, and the consumable cost is lower. And the eluent can be directly injected to a mass spectrum, so that a high-flux and high-sensitivity detection result can be quickly obtained. The method has the advantages of being simple and convenient to operate, high in selectivity and recovery rate, capable of effectively reducing the matrix effect and improving the instrument stability and the like, and is suitable for clinical monitoring and pharmacological research of various mental drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical mass spectrometry in vitro diagnostic reagent research and development, and in particular to a liquid chromatography tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction. Background Art

[0002] Schizophrenia is a common and serious mental disorder, with a prevalence of about 1% of the total disease burden and an annual growth rate of 1%. Since schizophrenia is a chronic disease, patients usually need long-term medication, or even lifelong medication, so it is crucial to choose drugs with good efficacy and few adverse reactions. Antipsychotic drugs, also known as strong tranquilizers or neuroleptics, are a class of drugs used to treat schizophrenia and other psychotic disorders. At the usual therapeutic dose, it does not affect the patient's intelligence and consciousness, but can effectively control the patient's psychomotor excitement, hallucinations, delusions, hostility, thinking disorders and abnormal behavior and other mental symptoms. Antidepressants refer to a group of psychotropic drugs mainly used to treat mental illnesses with depression as a prominent symptom. The difference from stimulants is that they can only eliminate the depressive symptoms of depressed patients, but cannot improve the mood of normal people.

[0003] To date, more than 200 psychotropic drugs have been developed and used in clinical practice. These psychotropic drugs have generated huge medical value and economic benefits. However, due to the metabolic differences among individual patients, the therapeutic effects of these drugs on many patients are not satisfactory. Therefore, therapeutic drug monitoring (TDM) has received more and more attention. TDM titrates the dose of individual patients by quantitatively measuring the concentration of drugs in plasma or serum in order to obtain better efficacy and better tolerance, while also reducing the risk of poisoning. Among the chemical detection methods of TDM, liquid chromatography tandem mass spectrometry (LC-MS / MS) has the characteristics of strong specificity, high accuracy, short analysis time, high sensitivity, and high throughput, and has gradually become the main force in drug concentration analysis. There are also methods for detecting psychotropic drugs based on LC-MS / MS in the prior art. For example, Chinese patent CN106168610B discloses a method for "determining the concentration of clozapine in plasma by high performance liquid chromatography-mass spectrometry", which provides a method for detecting clozapine psychotropic drugs by using high performance liquid chromatography-mass spectrometry and using risperidone as an internal standard. This method is specifically for a specific psychotropic drug - clozapine, and is not suitable for the simultaneous measurement of multiple psychotropic drugs; Chinese patent CN109655568B discloses a method for "simultaneous determination of 35 psychotropic drugs by high performance liquid chromatography-mass spectrometry and a kit", in which methyl alcohol is used. The method of protein precipitation with alcohol is used for sample pretreatment. This method has the characteristics of low cost and few operation steps, but this method has poor ability to remove endogenous interferences, and is easy to clog the chromatographic column and contaminate the mass spectrometer, greatly increasing the maintenance cost of the instrument; in order to reduce the cost of the instrument, there are also some methods that use liquid chromatography with ultraviolet detector as a detection instrument, such as Chinese patent CN112305140A discloses: "A method and application for detecting psychotropic drugs and their metabolites in vivo", in which sodium hydroxide aqueous solution and methyl tert-butyl ether are used to extract the sample twice by liquid-liquid extraction, and the supernatant is taken after centrifugation for nitrogen blowing and redissolution. In the method, an ultraviolet detector is used for sample detection. In order to achieve sufficient chromatographic separation of the target object, the chromatographic separation time in the patent is 70 minutes.

[0004] In summary, the main defects of the process of psychotropic drug detection in the prior art are:

[0005] First, the throughput is low. Most existing methods measure a single drug or several drugs simultaneously, which affects the detection efficiency.

[0006] Second, the degree of automation is low. Most of the existing methods are protein precipitation and liquid-liquid extraction. In order to concentrate the sample to improve the sensitivity, some methods require the supernatant to be nitrogen-dried and then redissolved, which greatly increases the workload of the experimenters.

[0007] Third, the analysis time is long. The presence of other substances in the blood sample increases the difficulty of chromatographic separation. Among the various existing testing methods, since the detectors used have no specific detection capabilities for the target, it is necessary to ensure that the chromatographic separation completely achieves baseline separation. The analysis time for some samples can even reach 70 minutes per sample.

[0008] Fourth, the sensitivity is low. In the process of simultaneously detecting multiple drugs, there is a problem of relatively low sensitivity of multiple targets, which affects the accuracy of low-concentration sample detection.

[0009] Therefore, it is necessary to provide a sample pretreatment solution for psychotropic drugs that is simple to operate, high-throughput, highly automated, highly sensitive, more reliable, and more efficient to solve the above problems. Summary of the invention

[0010] According to the technical problems raised above, a liquid chromatography tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction is provided. The present invention mainly adopts magnetic solid phase extraction technology to pre-treat psychotropic drugs in serum. The experimental mechanism is matrix dispersed solid phase extraction. In the experiment, a magnetic rod is used to transfer the magnetic solid phase extraction filler. The whole experimental process is easy to realize automation, while retaining the advantages of high selectivity and high purification rate of solid phase extraction, overcoming the shortcomings of existing methods, thereby improving the efficiency of clinical mass spectrometry psychotropic drug sample processing and ensuring reliable and stable detection results.

[0011] The technical means adopted by the present invention are as follows:

[0012] A liquid chromatography-tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction comprises the following steps:

[0013] S1. Prepare internal standard solution: dilute haloperidol-d4 standard, paliperidone-d4 standard, risperidone-d6 standard, clozapine-d3 standard, chlorpromazine hydrochloride-d6 standard, quetiapine fumarate-d8 standard, aripiprazole-d8 standard, ziprasidone hydrochloride-d8 standard, sulpiride-d5 standard, and olanzapine-d3 standard with methanol to the preset concentration for later use;

[0014] S2. Sample pretreatment: Use magnetic solid phase extraction instrument to transfer magnetic solid phase extraction filler;

[0015] (1) Pipette 50-200 μL of sample into a clean sample tube, add 10 μL of internal standard solution, add 400 μL of aqueous solution, and mix at 1000 rpm for 2 min;

[0016] (2) Add 1-4 mg of magnetic solid phase extraction filler to a clean sample tube, activate the filler with 300 μL of acetonitrile, and transfer the filler to 400 μL of water using a magnetic solid phase extraction instrument for filler balance;

[0017] (3) Transfer the filler to the sample mixed in (1) above and mix for 120 seconds;

[0018] (4) Transfer the filler to 400 μL of aqueous solution using a magnetic solid phase extraction instrument and mix for 60 seconds;

[0019] (5) Transfer the filler from step (4) to 400 μL acetonitrile using a magnetic solid phase extraction instrument and mix for 60 seconds;

[0020] (6) The filler from step (5) was transferred into 100 μL of acetonitrile / water (60 / 40, v / v) solution containing 1% formic acid using a magnetic solid phase extraction instrument and mixed for 60 seconds;

[0021] (7) Transferring the filler to the solution in step (1) using a magnetic solid phase extraction instrument, and discarding the filler;

[0022] (8) directly injecting the solution in step (6) for testing;

[0023] S3. Instrument detection: Liquid chromatography tandem mass spectrometry is used to detect the above samples.

[0024] Furthermore, in the step S1, the above standard samples are diluted with methanol to obtain 0.4 μg / mL haloperidol-d4, 0.8 μg / mL paliperidone-d4, 0.8 μg / mL risperidone-d6, 8 μg / mL clozapine-d3, 4 μg / mL chlorpromazine hydrochloride-d6, 15 μg / mL quetiapine fumarate-d8, 10 μg / mL aripiprazole-d8, 6 μg / mL ziprasidone hydrochloride-d8, 8 μg / mL sulpiride-d5, and 0.8 μg / mL olanzapine-d3.

[0025] Furthermore, in step S3, the liquid chromatography conditions are:

[0026] 1) Chromatographic column: Fenome C18 column (Kinetex 2.6μm C18 50×3mm);

[0027] 2) Mobile phase: 0.1% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B);

[0028] 3) Gradient elution program: the initial ratio is 30% phase B, which is maintained for 0.5 min, increased to 50% in 0.5 min, increased to 90% phase B in 3.5 min, decreased to the initial ratio of 30% B in 0.5 min, and maintained for 0.5 min;

[0029] 4) Flow rate: 0.6 mL / min; injection volume: 2 μL; column temperature: 40°C.

[0030] Furthermore, in step S3, the mass spectrometry conditions are:

[0031] 1) Ion source: electrospray ionization (ESI);

[0032] 2) Detection method: multiple reaction monitoring (MRM);

[0033] 3) Curtain gas (CUR) 20 psi, nebulizer (GS1) 55 psi, auxiliary heating gas (GS2) 60 psi, temperature (TEM) 450 °C, ionization voltage (IS) 5500 V, collision gas (CAD) 12 psi;

[0034] 4) Detect the mass spectrometry parameters of parent ion, daughter ion, residence time, cone voltage, and collision energy of each compound.

[0035] Furthermore, the sample is serum, and the magnetic solid phase extraction is based on the mechanism of matrix-dispersed solid phase extraction, which uses a magnetic rod to transfer magnetic solid phase extraction fillers between different solutions, thereby achieving rapid and accurate extraction of psychotropic drugs in serum.

[0036] The present invention uses magnetic solid phase extraction technology to extract psychotropic drugs from biological sample serum quickly and effectively, overcoming the shortcomings of traditional solid phase extraction (SPE) technology, such as large pore differences, difficulty in automation, and high cost, while retaining the advantages of solid phase extraction technology, such as good purification effect and high recovery rate of target extraction. In subsequent liquid chromatography-tandem mass spectrometry detection, high-sensitivity and high-throughput analysis results can be obtained.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) Dispersed adsorption increases the contact area, improves the adsorption rate and enrichment effect, and shortens the equilibrium time;

[0039] (2) Avoid errors such as SPE flow rate differences and filler swelling, and reduce mass spectrometry detection variability;

[0040] (3) Remove endogenous phospholipids and protein interference through elution, improve sample cleanliness, and reduce matrix effects and instrument contamination risks;

[0041] (4) Rapid separation and transfer of the analytes by applying an external magnetic field, facilitating automated pre-processing;

[0042] (5) The common amino groups of psychotropic drugs can be specifically adsorbed by magnetic solid phase extraction fillers, which can efficiently extract psychotropic drugs and improve sample processing efficiency;

[0043] (6) Utilize the high-throughput and high-sensitivity detection capabilities of mass spectrometry to improve sample detection throughput and sensitivity;

[0044] (7) The magnetic solid phase extraction filler used is at the mg level, and the price of consumables is low, which can reduce the cost of experimental consumables.

[0045] Based on the above reasons, the present invention can not only significantly improve the recovery rate and detection accuracy of psychotropic drugs in serum, but also greatly reduce the difficulty and cost of operation, and is easy to realize automation and large-scale application. It has significant clinical and scientific research application value and can be widely promoted in the technical field of mass spectrometry in vitro diagnostic reagent research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0047] Figure 1 The chromatograms are of the target substances, including: (a) aripiprazole chromatogram; (b) haloperidol chromatogram; (c) ziprasidone hydrochloride chromatogram; (d) olanzapine chromatogram; (e) risperidone chromatogram; (f) quetiapine fumarate chromatogram; (g) clozapine chromatogram; (h) sulpiride chromatogram; (i) chlorpromazine hydrochloride chromatogram; and (j) paliperidone chromatogram.

[0048] Figure 2 Aripiprazole linear graph.

[0049] Figure 3 is the linear graph of haloperidol.

[0050] Figure 4 It is a linear graph of ziprasidone hydrochloride.

[0051] Figure 5 is the linear graph of olanzapine.

[0052] Figure 6 is the linear graph of risperidone.

[0053] Figure 7 is a linear graph for quetiapine fumarate.

[0054] Figure 8 This is the linear graph for clozapine.

[0055] Fig. 9 This is a linear graph of Sulpiride.

[0056] Fig.10 This is a linear graph for chlorpromazine hydrochloride.

[0057] Fig.11 is the linear graph of paliperidone.

[0058] Fig.12 Schematic diagram of magnetic filler transfer. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] like Figures 1 to 12 As shown, the present invention discloses a liquid chromatography tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction, which specifically comprises the following steps:

[0061] First, prepare the internal standard solution: dilute the haloperidol-d4 standard with methanol to obtain a haloperidol-d4 concentration of 0.4 μg / mL, dilute the paliperidone-d4 standard with methanol to obtain a paliperidone-d4 concentration of 0.8 μg / mL, dilute the risperidone-d6 standard with methanol to obtain a risperidone-d6 concentration of 0.8 μg / mL, dilute the clozapine-d3 standard with methanol to obtain a clozapine-d3 concentration of 8 μg / mL, and dilute the chlorpromazine hydrochloride-d6 standard with methanol to obtain a chlorpromazine hydrochloride-d6 concentration of 4 μg / mL. mL, the quetiapine fumarate-d8 standard was diluted with methanol to obtain a quetiapine fumarate-d8 concentration of 15μg / mL, the aripiprazole-d8 standard was diluted with methanol to obtain an aripiprazole-d8 concentration of 10μg / mL, the ziprasidone hydrochloride-d8 standard was diluted with methanol to obtain a ziprasidone hydrochloride-d8 concentration of 6μg / mL, the sulpiride-d5 standard was diluted with methanol to obtain a sulpiride-d5 concentration of 8μg / mL, and the olanzapine-d3 standard was diluted with methanol to obtain an olanzapine-d3 concentration of 0.8μg / mL. Table 1 shows the standard working curves of the above standards.

[0062] Table 1 Standard working curve

[0063] drug Linear range S1 S2 S3 S4 S5 S6 S7 S8 1 Paliperidone 10–150 ng / mL 10 20 40 60 80 100 120 150 2 Risperidone 10–150 ng / mL 10 20 40 60 80 100 120 150 3 Clozapine 100~1500 ng / mL 100 200 400 600 800 1000 1200 1500 4 Chlorpromazine hydrochloride 10~1000 ng / mL 10 20 50 100 200 400 800 1000 5 Quetiapine Fumarate 50~1500 ng / mL 50 100 150 200 400 600 800 1500 6 Olanzapine 10–150 ng / mL 10 20 40 60 80 100 120 150 7 Aripiprazole 50~1500 ng / mL 50 100 150 200 400 600 800 1500 8 Ziracil Copper Hydrochloride 10–500 ng / mL 10 20 50 100 200 300 400 500 9 Haloperidol 1–100 ng / mL 1 2 5 10 20 40 80 100 10 Sulpiride 100~1500 ng / mL 100 200 400 600 800 1000 1200 1500

[0064] Then, the sample pretreatment process is carried out. Specifically, a magnetic solid phase extraction instrument is used in the experiment to transfer the magnetic solid phase extraction filler. The magnetic solid phase extraction technology is based on the experimental mechanism of matrix dispersed solid phase extraction. The magnetic solid phase extraction filler is transferred between different solutions using a magnetic rod, thereby achieving rapid and accurate extraction of psychotropic drugs in serum.

[0065] (1) Pipette 90 μL of sample into a clean sample tube, add 10 μL of internal standard solution, add 400 μL of aqueous solution, and mix at 1000 rpm for 2 min.

[0066] (2) Add 3 mg of magnetic solid phase extraction filler to a clean sample tube, activate the filler with 300 μL of acetonitrile, and transfer the filler to 400 μL of water using a magnetic solid phase extraction instrument for filler balance.

[0067] (3) Transfer the filler to the sample mixed in (1) above and mix for 120 seconds.

[0068] (4) The filler was transferred to 400 μL of aqueous solution using a magnetic solid phase extraction instrument and mixed for 60 seconds.

[0069] (5) The filler material from step (4) was transferred to 400 μL of acetonitrile using a magnetic solid phase extraction instrument and mixed for 60 seconds.

[0070] (6) The filler from step (5) was transferred into 100 μL of acetonitrile / water (60 / 40, v / v) solution containing 1% formic acid using a magnetic solid phase extraction instrument and mixed for 60 seconds.

[0071] (7) Transfer the filler material to the solution in step (1) using a magnetic solid phase extraction instrument and discard the filler material.

[0072] (8) Directly inject the solution from step (6) into the test tube.

[0073] like Fig.12As shown in the figure, the above process is briefly described as follows: the present invention uses a magnetic bar (purple long column) to manipulate the transfer of magnetic microbeads (small particles in the figure) between different solutions. The left part represents the "collecting magnetic beads (sample)" stage: the magnetic bar is inserted into the tube containing magnetic microbeads and sample solution. When the magnetic bar moves downward and applies an external magnetic field, the microbeads will be adsorbed to the lower end of the magnetic bar. Subsequently, the magnetic bar is removed from the sample tube and transferred to another tube. The arrow "transfer" in the middle indicates that the microbeads adsorbed with the target object are moved from the sample solution to a new solution (such as a washing solution or an eluent). In the right part of the "release sample" stage, the magnetic bar moves upward, which means turning off or weakening the magnetic force, so that the microbeads are released from the magnetic bar into a new solution, thereby completing the washing or elution step. That is, when the magnetic bar approaches or is inserted, the microbeads are absorbed to achieve separation from the mother solution; transferred to the washing solution or the eluent; the magnetic field is weakened to release the microbeads, so that the microbeads are fully in contact with the solution, and the impurities are removed or the target object is eluted. Repeat this process many times to obtain the purified or enriched target substance.

[0074] Finally, the instrument detection method is as follows:

[0075] (1) Liquid chromatography conditions

[0076] 1) Chromatographic column: Fenome C18 column (Kinetex 2.6μm C18 50×3mm);

[0077] 2) Mobile phase: 0.1% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B);

[0078] 3) Gradient elution program: the initial ratio is 30% phase B, which is maintained for 0.5 min, increased to 50% in 0.5 min, increased to 90% phase B in 3.5 min, decreased to the initial ratio of 30% B in 0.5 min, and maintained for 0.5 min;

[0079] 4) Flow rate: 0.6 mL / min; injection volume: 2 μL; column temperature: 40°C.

[0080] (2) Mass spectrometry conditions

[0081] 1) Ion source: electrospray ionization (ESI)

[0082] 2) Detection method: Multiple reaction monitoring (MRM)

[0083] 3) Curtain gas (CUR) 20 psi, nebulizer (GS1) 55 psi, auxiliary heating gas (GS2) 60 psi, temperature (TEM) 450 °C, ionization voltage (IS) 5500 V, collision gas (CAD) 12 psi;

[0084] 4) The mass spectrometry parameters such as parent ion, daughter ion, dwell time, cone voltage, collision energy, etc. of each compound are shown in Table 2.

[0085] Table 2 Mass spectrometry acquisition parameters

[0086]

[0087] In order to verify the reliability and accuracy of the method of the present invention, experimental evaluations of a number of key performance indicators were carried out, including matrix effect evaluation, quantitative limit, linear range, precision and correctness.

[0088] First, the matrix effect evaluation is performed by comparing the recovery and interference of the target in different matrix samples (such as serum, urine, etc.), and the anti-interference ability of this method in practical applications is evaluated. Secondly, in order to ensure the accuracy of the quantitative results, the quantitative limit (LOQ) and linear range of the method are determined to verify its quantitative ability at low concentration levels and reliability in the high concentration range. The precision test evaluates the stability of the method under the same experimental conditions by repeating the experiment, and the accuracy is verified by comparing with known standard samples. These experimental data will fully support the widespread application of the method of the present invention in clinical and scientific research, and ensure that it has enough detection accuracy and stability in actual operation.

[0089] Matrix effect evaluation

[0090] In the experiment, the matrix effect was evaluated by the sample treatment and addition method, that is, MF = response of the blank matrix added to the analyte after extraction / response of the analyte in the pure solvent. Internal standard normalized MF = analyte MF / internal standard MF, when the internal standard normalized MF range is 0.8-1.2, it is acceptable. Three concentrations of each analyte were selected according to the principle of low, medium and high, and 5 samples of each concentration were measured. The results are shown in Tables 3-1 to 3-10, indicating that there is no influence of matrix effect within the acceptable range.

[0091] Table 3-1 Internal standard normalization of haloperidol

[0092]

[0093] Table 3-2 Internal standard normalization of olanzapine

[0094]

[0095] Table 3-3 Internal standard normalization of Sulpiride

[0096]

[0097] Table 3-4 Internal standard normalization of aripiprazole

[0098]

[0099] Table 3-5 Internal standard normalization of Ziprasidone hydrochloride

[0100]

[0101] Table 3-6 Internal standard normalization of risperidone

[0102]

[0103] Table 3-7 Internal standard normalization of clozapine

[0104]

[0105] Table 3-8 Internal standard normalization of chlorpromazine hydrochloride

[0106]

[0107] Table 3-9 Internal standard normalization of paliperidone

[0108]

[0109] Table 3-10 Internal standard normalization of quetiapine fumarate

[0110]

[0111] Linear range

[0112] Determine the detection range according to the clinical detection requirements of the drug, set the concentration gradient within the detection range, detect the calibration material of the gradient concentration, fit the ratio of the target analyte and internal standard peak area with the gradient concentration, and obtain the working curve. The fitting curve and engineering curve equation are shown in the figure below. Figure 2-Figure 11 As shown, the linearity of the fitting is good, and the r values ​​are all above 0.99. The linear range of the target is shown in Table 4.

[0113] Table 4 Linear range and linear relationship of target

[0114] Target Linear range ng / mL Linear correlation coefficient Aripiprazole 50-1500 0.9945 Haloperidol 1-100 0.9978 Paliperidone 10-150 0.9953 Risperidone 10-150 0.9954 Clozapine 100-1500 0.9908 Chlorpromazine hydrochloride 10-1000 0.995 Quetiapine Fumarate 50-1500 0.9933 Olanzapine 10-150 0.9948 Ziprasidone hydrochloride 10-500 0.9976 Sulpiride 100-1500 0.9935

[0115] Lower limit of quantitation

[0116] According to the clinical testing requirements of 10 psychotropic drugs and the sensitivity of the instrument, the quantitative lower limit of the method is: aripiprazole 50ng / mL, haloperidol 1ng / mL, chlorpromazine hydrochloride 10ng / mL, ziprasidone hydrochloride 10ng / mL, olanzapine 10ng / mL, paliperidone 10ng / mL, risperidone 10ng / mL, quetiapine fumarate 50ng / mL, clozapine 100ng / mL, sulpiride 100ng / mL. The test was performed 12 times in a row, and the deviation between the test value and the theoretical value and the coefficient of variation CV value of the test result were calculated. The test results are shown in Table 5. It can be seen from the results that the deviation between the test value and the theoretical value of the 10 psychotropic drugs is within 15%, and the coefficient of variation of the parallel samples tested is within 15%, which meets the requirements of clinical testing methodology.

[0117] Table 5 Quantitative lower limit sample test results

[0118]

[0119] Precision

[0120] The precision of the method was evaluated by selecting three concentrations: low, medium and high. Each batch of samples of each concentration was processed at least 5 times, and three batches were measured continuously. The total number of samples was not less than 45. The intra-batch precision, inter-batch precision and total precision of each concentration sample were evaluated respectively. The results are shown in Tables 6-1 to 6-10.

[0121] Table 6-1 Method precision of aripiprazole

[0122]

[0123] Table 6-2 Method precision of haloperidol

[0124]

[0125] Table 6-3 Method precision of Ziprasidone hydrochloride

[0126]

[0127] Table 6-4 Method precision of olanzapine

[0128]

[0129] Table 6-5 Method precision of risperidone

[0130]

[0131] Table 6-6 Method precision of quetiapine

[0132]

[0133] Table 6-7 Method precision of clozapine

[0134]

[0135] Table 6-8 Method precision of Sulpiride

[0136]

[0137] Table 6-9 Method precision of chlorpromazine

[0138]

[0139] Table 6-10 Method precision of paliperidone

[0140]

[0141] From the data results, we can see that the intra-batch precision, inter-batch precision and total precision of the three concentrations of low, medium and high are all within 15%, which meets the requirements.

[0142] Accuracy

[0143] The accuracy of the method was evaluated by the recovery rate of spiked real mixed human serum. Three parallel samples were tested for each concentration, and each sample was tested three times. The recovery rate and the coefficient of variation of the test results were calculated. The results are shown in Tables 7-1 to 7-10. From the data results, it can be seen that the deviation between the test value and the theoretical value of the spiked sample is within the range of ±15%.

[0144] Table 7-1 Method recovery of aripiprazole

[0145]

[0146] Table 7-2 Method recovery of haloperidol

[0147]

[0148] Table 7-3 Recovery rate of Ziprasidone hydrochloride

[0149]

[0150] Table 7-4 Method recovery of olanzapine

[0151]

[0152] Table 7-5 Method recovery of risperidone

[0153]

[0154] Table 7-6 Method recovery of quetiapine fumarate

[0155]

[0156] Table 7-7 Method recovery rate of clozapine

[0157]

[0158] Table 7-8 Method recovery rate of Sulpiride

[0159]

[0160] Table 7-9 Method recovery of chlorpromazine hydrochloride

[0161]

[0162] Table 7-10 Method recovery of paliperidone

[0163]

[0164] Through the verification of the above series of detection experiments, it can be proved that the method of the present invention has high efficiency and accuracy in extracting psychotropic drugs in serum samples. The experimental results show that the method has good matrix adaptability, can effectively reduce the matrix effect and improve the recovery rate of the target. The test results of the lower limit of quantification and the linear range show that the method can also achieve accurate detection at concentrations as low as nanograms, and maintain a good linear relationship in a wide concentration range. The precision experiment shows that the variability of the method in repeated experiments is extremely low and has a high degree of stability, while the correctness test ensures the accuracy of the measurement results. Based on the above experimental data, the method not only improves the efficiency of psychotropic drug detection, but also ensures the reliability and stability of the results, and has a wide range of clinical applications and scientific research value.

[0165] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, a person of ordinary skill in the art should understand that any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A liquid chromatography tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction, characterized in that The following steps are included: S1. Prepare internal standard solution: dilute haloperidol-d4 standard, paliperidone-d4 standard, risperidone-d6 standard, clozapine-d3 standard, chlorpromazine hydrochloride-d6 standard, quetiapine fumarate-d8 standard, aripiprazole-d8 standard, ziprasidone hydrochloride-d8 standard, sulpiride-d5 standard, and olanzapine-d3 standard with methanol to the preset concentration for later use; S2. Sample pretreatment: Use magnetic solid phase extraction instrument to transfer magnetic solid phase extraction filler; (1) Pipette 50-200 μL of sample into a clean sample tube, add 10 μL of internal standard solution, add 400 μL of aqueous solution, and mix at 1000 rpm for 2 min; (2) Add 1-4 mg of magnetic solid phase extraction filler to a clean sample tube, activate the filler with 300 μL of acetonitrile, and transfer the filler to 400 μL of water using a magnetic solid phase extraction instrument for filler balance; (3) Transfer the filler to the sample mixed in (1) above and mix for 120 seconds; (4) Transfer the filler to 400 μL of aqueous solution using a magnetic solid phase extraction instrument and mix for 60 seconds; (5) Transfer the filler from step (4) to 400 μL acetonitrile using a magnetic solid phase extraction instrument and mix for 60 seconds; (6) Transfer the filler from step (5) to 100 μL of acetonitrile / water (60 / 40, v / v) solution containing 1% formic acid using a magnetic solid phase extraction instrument and mix for 60 seconds; (7) Transferring the filler to the solution in step (1) using a magnetic solid phase extraction instrument, and discarding the filler; (8) directly injecting the solution in step (6) for testing; S3. Instrument detection: Liquid chromatography tandem mass spectrometry is used to detect the above samples.

2. The liquid chromatography-tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction according to claim 1, characterized in that: In the step S1, the above standard samples are diluted with methanol to obtain 0.4 μg / mL haloperidol-d4, 0.8 μg / mL paliperidone-d4, 0.8 μg / mL risperidone-d6, 8 μg / mL clozapine-d3, 4 μg / mL chlorpromazine hydrochloride-d6, 15 μg / mL quetiapine fumarate-d8, 10 μg / mL aripiprazole-d8, 6 μg / mL ziprasidone hydrochloride-d8, 8 μg / mL sulpiride-d5, and 0.8 μg / mL olanzapine-d3.

3. The liquid chromatography tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction according to claim 1, characterized in that: In step S3, the liquid chromatography conditions are: 1) Chromatographic column: Fenome C18 column; 2) Mobile phase: 0.1% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B); 3) Gradient elution program: the initial ratio is 30% phase B, which is maintained for 0.5 min, increased to 50% in 0.5 min, increased to 90% phase B in 3.5 min, decreased to the initial ratio of 30% B in 0.5 min, and maintained for 0.5 min; 4) Flow rate: 0.6 mL / min; injection volume: 2 μL; column temperature: 40°C.

4. The liquid chromatography-tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction according to claim 1, characterized in that: In step S3, the mass spectrometry conditions are: 1) Ion source: electrospray ion source; 2) Detection method: multiple reaction monitoring; 3) Curtain gas 20psi, nebulizer 55psi, auxiliary heating gas 60psi, temperature 450°C, ionization voltage 5500V, collision gas 12psi; 4) Detect the mass spectrometry parameters of parent ion, daughter ion, residence time, cone voltage, and collision energy of each compound.

5. The liquid chromatography-tandem mass spectrometry detection method for psychotropic drugs based on magnetic solid phase extraction according to claim 2, characterized in that: The sample is serum, and the magnetic solid phase extraction is based on the mechanism of matrix dispersed solid phase extraction. A magnetic rod is used to transfer magnetic solid phase extraction fillers between different solutions, thereby achieving rapid and accurate extraction of psychotropic drugs in serum.

Images