A HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and its metabolites in plasma

Abstract

The application discloses a kind of HPLC-MSMS method for simultaneously determining valproic acid, olanzapine and its metabolites in plasma, which comprises the following steps: S1. pretreatment of plasma samples with acetonitrile protein precipitation method;S2. HPLC-MSMS method is used to detect plasma samples, and qualitative and / or quantitative analysis of valproic acid, olanzapine and its metabolites in plasma is carried out simultaneously;Wherein, Agilent Poroshell 120EC-C18 column is selected as the chromatographic column;Electrospray ion source is used, and the mobile phase is A phase: formic acid water, B phase: acetonitrile;By optimizing the conditions of liquid phase and mass spectrometry, valproic acid, olanzapine and its metabolites are simultaneously determined by one method.The method has been applied to detect plasma samples of olanzapine and valproic acid combination, and olanzapine alone, and the measured results all meet the linear range.

Description

Technical Field

[0001] This invention relates to the field of detection method technology, specifically to an HPLC-MSMS method for the simultaneous determination of valproic acid, olanzapine and their metabolites in plasma. Background Technology

[0002] Studies have shown that olanzapine ranks among the top five most frequently used antipsychotic drugs, and compared with traditional antipsychotic drugs, novel atypical antipsychotics have better efficacy and milder adverse reactions. Now, the use of novel atypical antipsychotics such as olanzapine in combination with other drugs has been approved by the FDA, with the aim of improving efficacy and reducing adverse reactions[2].

[0003] Olanzapine has an antagonistic effect by binding to dopamine receptors, serotonin receptors and cholinergic receptors[3]. Clinically, it is mainly used to treat schizophrenia or affective disorders, and sometimes to treat depression. Valproic acid increases the activity of γ-aminobutyric acid by affecting the central inhibitory neurotransmitters[4]. Clinically, it is mainly used to treat epilepsy and mania. The combined use of olanzapine and valproic acid is often used to treat refractory epilepsy, bipolar disorder[5].

[0004] According to the research on the combined use of olanzapine and valproic acid by Pu Qixia et al. [6], the combined use of the two drugs can enhance the efficacy and reduce adverse reactions. However, most mental patients need to take the drugs for a long time, and olanzapine and valproic acid have problems such as a narrow therapeutic window, effective concentration close to the toxic concentration, large individual differences in drug use, and many adverse reactions [7]. At the same time, valproic acid will also affect the metabolism of olanzapine during the drug use process, making the drug use process difficult. Therefore, monitoring the blood drug concentration of psychotropic drugs is of great clinical significance for the rational drug use safety of patients.

[0005] References mentioned in the background art:

[0006] [1] Wang Jun, Liu Xiuping, Zhao Xiaoyan, et al. Survey on daily use of psychotropic drugs in psychiatric outpatient clinics [J]. Chinese Journal of Drug Dependence, 2015, 24(04):276-279.

[0007] [2] Jiang Lixin, Chen Yongxin, Zhang Ruiling. Time-point survey of psychotropic drugs in 964 hospitalized psychiatric patients [J]. Occupational Health, 2014, 30(15):2198-2200.

[0008] [3] Ding Yiduo, Liu Shuaibin, Miao Liyan. Comparison of dissolution of olanzapine tablets from different manufacturers [J]. Anti-infective Pharmacy, 2012, 9(04):290-292.

[0009] [4] Zhang Yongqiang, Xue Ranran, Wang Xubo, et al. Research progress on the treatment of bipolar disorder with sodium valproate [J]. Contemporary Medical Forum, 2017, 15(19):20-22.

[0010] [5] Lin Xiaoyong, Hao Kairong, Zhan Jiaxian. Study on blood drug concentration and effect of sodium valproate combined with olanzapine in the treatment of bipolar disorder in adolescents [J]. Sino-Foreign Medical Research, 2021, 19(25):65-67.

[0011] [6] Pu Qixia, Wu Haibo, Huang Xiong, et al. Comparative analysis of olanzapine combined with sodium valproate in the treatment of refractory schizophrenia [J]. Modern Biomedical Progress, 2013, 13(18):3503-3506.

[0012] [7] Peng Qingping, Song Cangsang, Li Xingde, et al. Research progress on monitoring of valproic acid therapeutic drugs [J]. Drug Evaluation Research, 2021, 44(05):1111-1116. Summary of the Invention

[0013] In view of this, in order to solve the above-mentioned technical problems, the purpose of this invention is to propose a rapid, convenient and cost-effective HPLC-MS / MS method for the simultaneous determination of valproic acid, olanzapine and their metabolites in plasma. This method has been applied to the detection of plasma samples taken in combination with olanzapine and valproic acid, as well as those taken alone with olanzapine, and the results obtained all meet the linear range.

[0014] The technical solution adopted is as follows:

[0015] An HPLC-MSMS method for the simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma includes the following steps:

[0016] S1. Plasma samples were pretreated using acetonitrile protein precipitation.

[0017] S2. Plasma samples were analyzed using HPLC-MSMS to simultaneously perform qualitative and / or quantitative analysis of valproic acid, olanzapine and their metabolites in plasma.

[0018] The chromatographic column used was an Agilent Poroshell 120EC-C18 column; an electrospray ionization source was used; the mobile phase was phase A: formic acid and water, and phase B: acetonitrile; through optimization of the liquid chromatography and mass spectrometry conditions, a method was developed to simultaneously determine valproic acid, olanzapine and their metabolites.

[0019] Further, in S1, the specific steps for pretreatment of plasma samples using the acetonitrile protein precipitation method are as follows: accurately pipette 200 μL of plasma sample into a 1.5 mL centrifuge tube, add 10 μL of olanzapine internal standard working solution and 10 μL of valproic acid internal standard working solution, add 800 μL of acetonitrile solution to the mixed solution, vortex for 3 min, centrifuge at 12700 r / min for 10 min, and take 200 μL of supernatant into a 96-well plate.

[0020] Furthermore, in S2, the chromatographic conditions were as follows: the selected Agilent Poroshell 120EC-C18 column was 2.1 mm × 50 mm and 2.7 μm; the mobile phase was phase A: 0.1% formic acid water (volume concentration); phase B: acetonitrile; the flow rate was 0.4 mL / min; the column temperature was maintained at 35℃ ± 2℃; and the injection volume was 5 μL.

[0021] Furthermore, the mass spectrometry conditions were as follows: the mass spectrometer used an electrospray ionization source, with positive ion mode for 0-2 min and negative ion mode from 2 min to the end of detection; the drying gas flow rate was 11 L / min; the drying gas temperature was 350℃; the nebulizing gas pressure was 35 Psi; and the capillary voltage was set to 4000V for positive ion mode and 3500V for negative ion mode.

[0022] Furthermore, the valproic acid, olanzapine and their metabolites are the following five analytes: valproic acid, olanzapine, 2-hydroxymethylolanzapine, olanzapine-10N-glucuronic acid, and N-norolanzapine.

[0023] Furthermore, the olanzapine internal standard working solution is prepared by the following method: accurately weigh 1 mg olanzapine-D3 and add 1 mL of methanol to prepare a 1 mg / mL olanzapine-D3 stock solution. Take 10 μL of the olanzapine-D3 stock solution into a 10 mL volumetric flask, add methanol to make up to volume, and prepare an olanzapine-D3 internal standard working solution with a concentration of 1000 ng / mL.

[0024] Furthermore, the valproic acid internal standard working solution was prepared by the following method: 100 mg of valproic acid-D15 was accurately weighed into a 10 mL volumetric flask, and methanol was added to dilute to the final volume to prepare a 10 mg / mL valproic acid-D15 stock solution. 1 mL of the valproic acid-D15 stock solution was then placed into another 10 mL volumetric flask, and methanol was added to dilute to the final volume to prepare a 1000 μg / mL valproic acid-D15 internal standard working solution.

[0025] Furthermore, in S2, olanzapine and its metabolites elute at 0.4-0.6 min, while valproic acid elutes at 3.1-3.2 min. By implementing segmented mass spectrometry acquisition, olanzapine, its metabolites, and internal standard are acquired using positive ion mode within 0-2 min, and valproic acid and its internal standard are acquired within 2-4.5 min. This allows for the simultaneous use of positive and negative ion acquisition modes in the same analytical method, enabling the simultaneous determination of valproic acid, olanzapine, and their metabolites.

[0026] The beneficial effects of this invention are as follows:

[0027] The total time for this method is 4.5 min. Olanzapine, its metabolites, and valproic acid showed good linearity within the standard curve (r² ≥ 0.99). Intra-batch and inter-batch precision and accuracy (LLOQ control) were all within the specified range. Matrix effect, recovery, and dilution effect all met the detection requirements. Stability under five different conditions was within the detection range. Peak shapes for each analyte were good, and endogenous substances in blank plasma did not interfere with detection. No significant residues were observed with this method.

[0028] This invention establishes a rapid, convenient, and cost-effective HPLC-MS / MS method for the simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma. This method has been applied to the detection of plasma samples from patients taking olanzapine and valproic acid in combination, as well as from patients taking olanzapine alone, and the results obtained all met the linear range. Attached Figure Description

[0029] Figure 1 Calibration curves for five analytes.

[0030] Figure 2 TIC plots (I) for LLOQ-spiked blank plasma and (II) for blank plasma containing 5 analytes and 2 internal standards.

[0031] Figure 3 The extraction chromatograms are shown for five analytes and two internal standards, including LLOQ (I) and blank plasma (II), in plasma spiked with LLOQ (II). Detailed Implementation

[0032] The present invention will be described in detail below through specific embodiments. However, the uses and purposes of these exemplary embodiments are only for illustrating the present invention and do not constitute any limitation on the actual protection scope of the present invention, nor are they intended to limit the protection scope of the present invention to this.

[0033] 1. An HPLC-MSMS method for the simultaneous determination of valproic acid, olanzapine and their metabolites in plasma according to the present invention includes the following steps:

[0034] S1. Plasma samples were pretreated using acetonitrile protein precipitation.

[0035] S2. Plasma samples were analyzed using HPLC-MSMS to simultaneously perform qualitative and / or quantitative analysis of valproic acid, olanzapine and their metabolites in plasma.

[0036] The chromatographic column used was an Agilent Poroshell 120EC-C18 column; an electrospray ionization source was used; the mobile phase was phase A: formic acid and water, and phase B: acetonitrile; through optimization of the liquid chromatography and mass spectrometry conditions, a method was developed to simultaneously determine valproic acid, olanzapine and their metabolites.

[0037] Finally, the established method was used to assess drug concentrations in clinical samples and verify the practicality of the testing method.

[0038] The specific implementation is as follows:

[0039] 2 Materials and Methods

[0040] 2.1 Reagents

[0041] The reagents used in this study are shown in Table 2-1.

[0042] Table 2-1 Reagent Information Table

[0043]

[0044] 2.2 Instruments

[0045] Table 2-2 Instrument Information Table

[0046]

[0047] 2.3 Chromatographic and Mass Spectrometric Conditions

[0048] 2.3.1 Chromatographic conditions

[0049] An Agilent Poroshell 120EC-C18 column (2.1 mm × 50 mm, 2.7 μm) was used. The mobile phase was phase A: 0.1 vol% formic acid in water; phase B: acetonitrile. The flow rate was 0.4 mL / min; the column temperature was maintained at approximately 35℃ (±2℃); and the injection volume was 5 μL per injection.

[0050] The gradient elution process of the chromatographic column is shown in Table 2-3, and the change in the ratio of phase A to phase B during the operation is reflected.

[0051] Table 2-3 Gradient Elution Procedure Table

[0052]

[0053] 2.3.2 Mass Spectrometry Conditions

[0054] Mass spectrometry was performed using an electrospray ionization (ESI) source with MRM. The system was in positive ion mode for 0-2 min and negative ion mode from 2 min until the end of detection. The drying gas flow rate was 11 L / min; the drying gas temperature was 350℃; the nebulizing gas pressure was 35 Psi; and the capillary voltage was set to 4000 V for positive ion mode and 3500 V for negative ion mode. The relevant parameters for the five analytes and two internal standards, olanzapine-D3 and valproic acid-D15, are shown in Table 2-4.

[0055] Table 2-4 Relevant parameters of 5 analytes and 2 internal standards olanzapine-D3 and valproic acid-D15

[0056]

[0057] 2.4 Solution Preparation

[0058] 2.4.1 Reference Standard Working Solution

[0059] Accurately weigh appropriate amounts of olanzapine and N-norolanzapine, and dilute to volume with methanol to prepare stock solutions with a concentration of 10 mg / mL, which are then stored at -20℃. Accurately measure 4, 4, 400, and 400 μL of the stock solutions of olanzapine, N-norolanzapine, olanzapine-10N-glucuronide, and 2-hydroxymethylolanzapine into the same 10 mL volumetric flask, and dilute to volume with methanol to prepare a mixed standard solution with a concentration of 4 μg / mL, which is then stored at -20℃. Accurately measure 50, 25, 20, 10, 10, 10, and 10 μL of the olanzapine and olanzapine metabolite mixed standard solution into different 1.5 mL EP tubes, and add 50, 75, 140, 150, 310, 630, and 1270 μL of methanol, respectively, to prepare standard curve control solutions. The working solution of olanzapine has a mass concentration range of 31.25-4000 ng / mL.

[0060] Accurately measure an appropriate amount of valproic acid solution into a volumetric flask, and dilute to a final volume of 10 mg / mL with methanol. Store at -20°C. Accurately measure an appropriate amount of the valproic acid stock solution, and dilute to a final volume of 4 mg / mL with methanol. Perform serial dilutions to prepare the valproic acid standard curve reference solution. The working solution of valproic acid corresponds to a mass concentration of 31.25–4000 μg / mL.

[0061] 2.4.2 Preparation of working solution for quality control products

[0062] High-concentration quality control (QC-H) is 75 vol of the highest concentration point of the standard curve; medium-concentration quality control (QC-M) is 20 vol of the high-concentration quality control; and low-concentration quality control (QC-L) is 20 vol of the medium-concentration quality control (QC-M).

[0063] Accurately measure 150, 30, and 15 μL of olanzapine standard solution into 1.5 mL EP tubes, respectively, and add 50, 170, and 485 μL of methanol to prepare high-concentration, medium-concentration, and low-concentration quality control working solutions, respectively. The final concentrations of the olanzapine quality control working solutions are 3000 ng / mL for high-concentration quality control (QC-H), 600 ng / mL for medium-concentration quality control (QC-M), and 120 ng / mL for low-concentration quality control (QC-L).

[0064] Accurately measure 375 μL of valproic acid standard solution into a 1.5 mL EP tube, add 125 μL of methanol solution and mix well to obtain the high-concentration quality control working solution. Accurately measure 100 μL of the high-concentration quality control working solution into another 1.5 mL EP tube, add 400 μL of methanol solution and mix well to obtain the medium-concentration quality control working solution. Accurately measure 100 μL of the medium-concentration quality control working solution into another 1.5 mL EP tube, add 400 μL of methanol solution and mix well to obtain the low-concentration quality control working solution. The final concentrations of the valproic acid quality control working solutions are: 3000 μg / mL for high-concentration quality control (QC-H), 600 μg / mL for medium-concentration quality control (QC-M), and 120 μg / mL for low-concentration quality control (QC-L).

[0065] 2.4.3 Internal Standard Working Solution

[0066] Accurately weigh 1 mg of olanzapine-D3 and add it to 1 mL of methanol to prepare a 1 mg / mL olanzapine-D3 stock solution. Take 10 μL of the olanzapine-D3 stock solution into a 10 mL volumetric flask, add methanol to make up to volume, and prepare an olanzapine-D3 internal standard working solution with a concentration of 1000 ng / mL.

[0067] Accurately weigh 100 mg of valproic acid-D15 into a 10 mL volumetric flask, add methanol to dilute to volume, and prepare a 10 mg / mL valproic acid-D15 stock solution. Take 1 mL of the valproic acid-D15 stock solution into another 10 mL volumetric flask, add methanol to dilute to volume, and prepare a 1000 μg / mL valproic acid-D15 internal standard working solution.

[0068] 2.5 Sample Pretreatment Methods

[0069] 2.5.1 Pretreatment methods for standard curve samples and quality control samples

[0070] The sample pretreatment method for the standard curve is as follows: Take 180 μL of blank plasma and place it in a 1.5 mL centrifuge tube. Accurately measure 10 μL of the series of working solutions containing the standard curve concentrations of each analyte, 10 μL each of olanzapine and valproic acid internal standard working solutions, add 800 μL of acetonitrile solution to the mixed solution, vortex for 3 min, centrifuge at 12700 r / min for 10 min, and take 200 μL of the supernatant for analysis in a 96-well plate.

[0071] The pretreatment method for quality control samples is as follows: Take 180 μL of blank plasma and place it in a 1.5 mL centrifuge tube. Accurately measure 10 μL of a series of working solutions containing the quality control concentrations of each analyte, 10 μL each of olanzapine and valproic acid internal standard working solutions, add 800 μL of acetonitrile solution to the mixed solution, vortex for 3 min, centrifuge at 12700 r / min for 10 min, and take 200 μL of the supernatant for analysis in a 96-well plate.

[0072] The standard curve concentration points and quality control concentrations of olanzapine, its metabolites, and valproic acid are shown in Table 2-5.

[0073] Table 2-5 Standard curve concentration points and quality control concentrations of olanzapine, its metabolites, and valproic acid

[0074]

[0075] The standard curves for olanzapine, its metabolites, and valproic acid (i.e., the five analytes) are shown in [reference needed]. Figure 1 As shown.

[0076] 2.5.2 Plasma Sample Pretreatment Methods

[0077] Accurately pipette 200 μL of plasma sample into a 1.5 mL centrifuge tube, add 10 μL of olanzapine internal standard working solution and 10 μL of valproic acid internal standard working solution, add 800 μL of acetonitrile solution to the mixed solution, vortex for 3 min, centrifuge at 12700 r / min for 10 min, and take 200 μL of supernatant for analysis in a 96-well plate.

[0078] 2.6 Verification of the method for valproic acid and olanzapine

[0079] The methodological validation of this invention was based on the 2020 edition of the Chinese Pharmacopoeia's Guidelines for Quantitative Analysis of Biological Samples and the Technical Guidelines for Clinical Pharmacokinetic Studies of Chemical Drugs. [8] The prescribed method design requirements were analyzed. Methodological validation of this invention included: linear range and lower limit of quantitation, accuracy and precision, stability (including stability at room temperature for 6 hours, stability after preparation at room temperature for 48 hours, repeated freeze-thaw stability after three cycles of freezing at -20°C and thawing at 20°C, and long-term stability after storage at -20°C and -80°C for 30 days), selectivity, matrix effect, extraction recovery, dilution effect, and residual effect.

[0080] 2.6.1 Linear range and lower limit of quantitation

[0081] Eight standard curve samples at different concentrations were prepared and injected according to the procedure outlined in "2.5.1 Preparation and Pretreatment of Standard Curve Samples and Quality Control Samples" in ascending order of concentration. The linearity of the method was evaluated using independent calibration curves from six plasma samples processed over three days. Calibration curves for the analyte were generated by plotting the peak area ratio (analyte / internal standard) against the theoretical concentration. The regression equation was calculated using a weighting factor of "1 / X²".

[0082] Prepare 8 parallel standard samples as the minimum concentration for the standard curve. The requirements are S / N≥10, RSD≤20%, and LLOQ is considered unqualified if it is not within this range.

[0083] 2.6.2 Accuracy and Precision

[0084] Accuracy refers to the degree of closeness between the test result and the labeled concentration of the analyte, and is expressed by the formula: (Measured value / True value) × 100%. Precision refers to the degree of closeness between the results obtained from repeated determinations of the analyte. LLOQ, QC-L, QC-M, and QC-H are prepared using blank plasma samples. Five parallel samples are prepared for each concentration level, and three batches are measured consecutively over three days using the same procedures. Intra-batch and inter-batch accuracy and precision are calculated based on the standard curve for each batch.

[0085] For QC-L, QC-M, and QC-H quality control samples, the intra-batch and inter-batch RSD of precision should not exceed 15%, and the mean accuracy should be within ±15% of the labeled value; similarly, for LLOQ, the intra-batch and inter-batch RSD of precision should not exceed 20%, and the mean accuracy should be within ±20% of the labeled value.

[0086] 2.6.3 Stability

[0087] This invention primarily tests the stability of samples under five different conditions: stability at room temperature for six hours, stability after preparation at room temperature for forty-eight hours, repeated freeze-thaw stability after three cycles of freezing at -20°C and thawing at 20°C, and long-term stability after storage at -20°C and -80°C for 30 days. For each condition, QC-L, QC-M, and QC-H formulations were prepared, with five replicates prepared for each concentration level. Samples were immediately injected for analysis after processing and under the tested storage conditions. A standard curve was prepared for each test. Quality control samples were analyzed based on the prepared standard curves, and the results were compared with the labeled values. The mean concentration for each test condition should deviate from the labeled value by no more than 15%.

[0088] 2.6.4 Selectivity

[0089] Six blank control matrix samples from different sources (batch numbers) without added valproic acid, olanzapine, olanzapine metabolites, or internal standards were collected and evaluated against LLOQ samples prepared with the corresponding matrices. Separate analyses and interferences were performed according to "2.5.2 Plasma Sample Pretreatment Methods".

[0090] If the peak area of ​​the detected analyte is less than or equal to 20.0% of the peak area of ​​the analyte in the LLOQ sample prepared with the corresponding matrix, and the peak area of ​​the internal standard is less than or equal to 5.0% of the peak area of ​​the LLOQ sample prepared with the corresponding matrix, then the interference is considered to have no significant effect on the experiment, and the interference experiment meets the method validation requirements.

[0091] 2.6.5 Matrix effect and extraction recovery

[0092] Prepare QC-L, QC-M, and QC-H samples separately. For each concentration level, prepare six parallel samples using the same method. After processing according to "2.5.1 Preparation and Pretreatment Methods for Standard Curve Samples and Quality Control Samples", inject the samples and record the peak area as A. Take blank plasma, process it, add working solution of reference standard, and prepare QC-L, QC-M, and QC-H samples. For each concentration level, prepare six parallel samples using the same method. Inject the samples and record the peak area as B. Take working solution of reference standard at the same concentration level, without processing, and inject it directly. Record the peak area as C.

[0093] The calculation formula is:

[0094]

[0095] The RSD of the matrix effect and extraction recovery calculated from 6 parallel assays should not exceed 15% for normalized matrix effect and extraction recovery.

[0096] 2.6.6 Dilution effect and residual effect

[0097] The sample with the highest concentration of 5 on the standard curve was diluted 10-fold with blank plasma and processed according to the method described in section "2.5.1 Preparation and Pretreatment Methods of Standard Curve Samples and Quality Control Samples" before injection analysis to evaluate the dilution effect. The precision and accuracy of the diluted sample should be within ±15% of the labeled concentration, with RSD ≤ 15%.

[0098] Immediately after injecting the sample at the peak of the standard curve, inject a blank sample to determine the peak areas of the analyte and internal standard, which is used to assess residual effects. The peak areas of the analyte and its internal standard in the blank sample should not exceed 20% of the LLOQ.

[0099] 2.6.7 Clinical Application

[0100] The drug concentration in the collected plasma samples was determined using the HPLC-MS / MS method according to "2.5.2 Plasma Sample Pretreatment Method".

[0101] 3 Results

[0102] This invention establishes an HPLC-MS / MS method for determining valproic acid, olanzapine and their metabolites in plasma.

[0103] The development steps are as follows: using a laboratory liquid chromatography-mass spectrometry (LC-MS) instrument to explore the mass spectrometry parameters of the analytes, optimize chromatographic conditions, and improve the detection method; validating the established method according to the relevant requirements for method validation and the actual situation of daily testing; and using the established method to detect the drug concentration in clinical samples to evaluate the applicability of the method.

[0104] 3.1 Optimization of Liquid Chromatography and Mass Spectrometry Conditions

[0105] Valproic acid and olanzapine were detected using negative and positive ion modes of an ESI source, respectively. Simultaneous detection of both compounds requires optimization of chromatographic conditions to separate them, and the simultaneous use of both positive and negative ion modes is crucial. Therefore, optimization of liquid chromatography and mass spectrometry (LC-MS) conditions is particularly important. Our LC-MS optimization is as follows: by optimizing the LC elution program, olanzapine and its metabolites elute at 0.4–0.6 min, while valproic acid elutes at 3.1–3.2 min. By implementing segmented mass spectrometry acquisition, olanzapine, its metabolites, and internal standard are acquired using positive ion mode within 0–2 min (excluding zero values), and valproic acid and its internal standard are acquired within 2–4.5 min (excluding two values). This allows for the simultaneous use of positive and negative ion acquisition modes within the same analytical method, enabling the simultaneous determination of valproic acid, olanzapine, and their metabolites, providing technical support for exploring the interaction between valproic acid and olanzapine.

[0106] 3.2 Results of Methodological Validation

[0107] 3.2.1 Linear range and lower limit of quantitation

[0108] Olanzapine, its metabolites, and valproic acid showed good linearity within the linear range (r² > 0.99). The linear regression equations for olanzapine, its metabolites, and valproic acid are shown in Table 3-1.

[0109] Table 3-1 Linear regression equations for olanzapine, its metabolites, and valproic acid

[0110]

[0111]

[0112] 3.2.2 Accuracy and Precision

[0113] The results showed that the intra-assay precision (RSD) for valproic acid, olanzapine, and its metabolites determined by this method was all LLOQ < 20%, and QC-L, QC-M, and QC-H < 15%; the intra-assay accuracy for LLOQ was 80.91%–115.81%, and for QC-L, QC-M, and QC-H it was 85.33%–114.32%. The inter-assay precision (RSD) was all LLOQ < 20%, and for QC-L, QC-M, and QC-H it was < 15%; the inter-assay accuracy for LLOQ was 92.78%–108.97%, and for QC-L, QC-M, and QC-H it was 87.67%–112.16%. The intra-assay and inter-assay accuracy and precision of olanzapine, its metabolites, and valproic acid are shown in Table 3-2.

[0114] Table 3-2 Intra-assay and inter-assay accuracy and precision of olanzapine, its metabolites, and valproic acid

[0115]

[0116] 3.2.3 Stability

[0117] The results are shown in the table below. The stability of each analyte ranged from 85.39% to 114.97%, with RSDs ranging from 1.42% to 14.86%. The stability of olanzapine, its metabolites, and valproic acid is shown in Table 3-3.

[0118] Table 3-3 Stability of Olanzapine, its metabolites, and valproic acid

[0119]

[0120] 3.2.4 Selectivity

[0121] Chromatograms of LLOQ samples and blank plasma samples containing 5 analytes and 2 internal standards are shown below. Figure 2 and Figure 3 As shown, the peak shapes of each analyte were good, and endogenous substances in the blank plasma did not interfere with the detection.

[0122] Figure 2 TIC plots (I) for LLOQ-spiked blank plasma and (II) for blank plasma containing 5 analytes and 2 internal standards;

[0123] Figure 3 The extraction chromatograms are shown for five analytes and two internal standards, including LLOQ (I) and blank plasma (II), in plasma spiked with LLOQ (II).

[0124] 3.2.5 Matrix effect and extraction recovery rate

[0125] Method validation showed that the matrix effect of internal standard normalized was 85.48%–110.90%, with an RSD of 0.54%–13.56%; the recovery rate was 85.26%–102.83%, with an RSD of 1.81%–10.83%. The matrix effect and recovery rate of olanzapine and its metabolites are shown in Tables 3–4.

[0126] Table 3-4 Matrix effects and recoveries of olanzapine and its metabolites

[0127]

[0128] 3.2.6 Dilution effect and residual effect

[0129] The RSD of the dilution effect was <15%, with an accuracy ranging from 102.38% to 114.02%. No significant residues were observed with this method.

[0130] 3.3 Test results of clinical samples

[0131] After verification, this method was used in 66 cases of combined olanzapine and valproic acid treatment and 19 cases of olanzapine alone treatment. The test results are shown in Tables 3-5. All the results were within the linear range, indicating that this method can be used for clinical blood drug concentration detection.

[0132] Table 3-5 Range of Clinical Sample Test Results

[0133]

[0134] 4 Discussion

[0135] This invention establishes a rapid, convenient, and cost-effective HPLC-MS / MS method for the simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma. By optimizing the liquid chromatography and mass spectrometry conditions, the method enables the separation and analysis of valproic acid, olanzapine, and their metabolites within 4.5 minutes using both positive and negative ion modes, providing methodological support for clinical investigation of the interaction between olanzapine and valproic acid.

[0136] This invention also targets the olanzapine metabolite, 2-hydroxymethylolanzapine. [9] Olanzapine-10N-glucuronide, N-norlanzapine [10-12] Blood drug concentration determination was performed. Currently, there is limited research on the above three olanzapine metabolites. This invention fills the research gap on olanzapine metabolites, including 2-hydroxymethylolanzapine, olanzapine-10N-glucuronic acid, and N-norolanzapine.

[0137] 5. Conclusion

[0138] In summary, this invention establishes an HPLC-MS / MS method for determining valproic acid, olanzapine, and their metabolites in plasma, and the method has been validated, with all results meeting the requirements. This method uses acetonitrile protein precipitation, which is simple and rapid. Furthermore, by optimizing chromatographic conditions, using an electrospray ionization source (ESI source) and multiple reaction monitoring (MRM) mode to simultaneously determine positive and negative ions, valproic acid, olanzapine, and their metabolites can be separated and analyzed within 4.5 min, meeting the requirements for rapid detection of clinical samples.

[0139] This method has been applied to the detection of plasma samples from patients taking olanzapine and valproic acid in combination, as well as from patients taking olanzapine alone, and the results obtained all met the linear range.

[0140] Appendix 1: References involved in the specific implementation methods:

[0141] [8] Technical Guidelines for Clinical Pharmacokinetic Studies of Chemical Drugs [J].

[0142] [9]Saar E, Gerostamoulos D, Drummer OH, et al. Identification of2-hydroxymethyl-olanzapine as a novel degradation product of olanzapine[J]. Forensic Sci Int, 2012, 220(1-3).

[0143]

[10] Li Wenbiao, Zhai Yimin, Wang Chuanyue, et al. Study on high performance liquid chromatography electrochemical detection characteristics of clozapine, norclozapine and olanzapine [J]. Chromatography, 2000(06):550-553.

[0144]

[11] Ma, Botao. Establishment of liquid chromatography-tandem mass spectrometry detection method for olanzapine and N-norolanzapine in plasma and analysis of factors affecting plasma concentration [D]. Peking University Health Science Center, 2021.

[0145]

[12] Shao Hongxia, Wan Changliang. Determination of the concentrations of olanzapine, clozapine and N-norclozapine in human serum by online column switching high performance liquid chromatography [J]. Northern Pharmacy, 2016, 13(06):1-3.

[0146] Appendix 2: Abbreviations, Full English Names, and Full Chinese Names

[0147] HPLC-MS / MS High performance liquid chromatography-Tandem mass spectrometry

[0148] ESI Electron spray ionization source

[0149] LLOQ (Lower Limit of Quantification)

[0150] QC Quality control samples

[0151] SD Relative standard deviation

[0152] MRM (Multi-reaction monitoring)

[0153] RT Ret time retention time

[0154] RSD (Relative Standard Deviation)

[0155] CE Collision energy (collision voltage)

[0156] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. An HPLC-MSMS method for the simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma, characterized in that, Includes the following steps: S1. Plasma samples were pretreated using acetonitrile protein precipitation. S2. Plasma samples were analyzed using HPLC-MSMS to simultaneously perform qualitative and / or quantitative analysis of valproic acid, olanzapine, and their metabolites; the valproic acid, olanzapine, and their metabolites were the following five analytes: valproic acid, olanzapine, 2-hydroxymethylolanzapine, olanzapine-10N-glucuronide, and N-norolanzapine. The chromatographic conditions were as follows: the Agilent Poroshell 120 EC-C18 column used was 2.1 mm × 50 mm, 2.7 μm; the mobile phase was phase A: 0.1% formic acid in water; phase B: acetonitrile; the mass spectrometry conditions were as follows: the mass spectrometer used an electrospray ionization source, with positive ion mode for 0-2 min and negative ion mode from 2 min to the end of detection; the drying gas flow rate was 11 L / min; the drying gas temperature was 350 ℃; the nebulizer gas pressure was 35 Psi; the capillary voltage was set to 4000 V for positive ion mode and 3500 V for negative ion mode. The gradient elution procedure is as follows: Valproic acid and olanzapine were detected using negative and positive ion modes of an electrospray ionization source, respectively. By optimizing the liquid chromatography elution program, olanzapine and its metabolites eluted at 0.4–0.6 min, while valproic acid eluted at 3.1–3.2 min. By implementing segmented mass spectrometry acquisition, olanzapine, its metabolites, and the internal standard were acquired using positive ion mode within 0–2 min (excluding zero values), and valproic acid and the internal standard were acquired within 2–4.5 min (excluding two values). Through optimization of the liquid chromatography and mass spectrometry conditions, a single method was achieved to simultaneously determine valproic acid, olanzapine, and their metabolites.

2. The HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma according to claim 1, characterized in that, In S1, the specific steps for pretreatment of plasma samples using the acetonitrile protein precipitation method are as follows: accurately pipette 200 µL of plasma sample into a 1.5 mL centrifuge tube, add 10 µL of olanzapine internal standard working solution and 10 µL of valproic acid internal standard working solution, add 800 µL of acetonitrile solution to the mixed solution, vortex for 3 min, centrifuge at 12700 r / min for 10 min, and take 200 µL of supernatant into a 96-well plate.

3. The HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma according to claim 1, characterized in that, In S2, the chromatographic conditions were: flow rate 0.4 mL / min; column temperature maintained at 35 ℃ ± 2 ℃; and injection volume 5 µL per injection.

4. The HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma according to claim 2, characterized in that, The olanzapine internal standard working solution was prepared by the following method: 1 mg olanzapine-D3 was accurately weighed and added to 1 mL of methanol to prepare a 1 mg / mL olanzapine-D3 stock solution. 10 µL of the olanzapine-D3 stock solution was placed in a 10 mL volumetric flask and diluted with methanol to prepare an olanzapine-D3 internal standard working solution with a concentration of 1000 ng / mL.

5. The HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine, and their metabolites in plasma according to claim 2, characterized in that, The valproic acid internal standard working solution was prepared as follows: 100 mg of valproic acid-D15 was accurately weighed into a 10 mL volumetric flask, and methanol was added to dilute to the final volume to prepare a 10 mg / mL valproic acid-D15 stock solution. 1 mL of the valproic acid-D15 stock solution was then placed into another 10 mL volumetric flask and diluted to the final volume with methanol to prepare a 1000 µg / mL valproic acid-D15 internal standard working solution.

Images