Robust lamotrigine serum therapeutic drug monitoring high performance liquid chromatography determination method

Abstract

The invention discloses a stable lamotrigine serum therapeutic drug monitoring high performance liquid chromatography determination method, and belongs to the field of medical examination. According to the determination method, glacial acetonitrile is used as a serum sample pretreatment reagent and is combined with an acetonitrile-ammonium acetate mobile phase system, so that sample pretreatment is simpler and more efficient, and the method is good in specificity. According to the determination method, accurate quantification can be completed by an external standard method, and the experimental operation and the calculation process are further simplified. Through sigma performance verification and national laboratory quality evaluation, the detection method is stable in performance, and is suitable for routine lamotrigine serum treatment drug monitoring in clinical laboratories.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical testing, and relates to a robust high performance liquid chromatography (HPLC) determination method for serum therapeutic drug monitoring of lamotrigine. More specifically, on the basis of methodological verification, the six sigma (σ) quality management technique is adopted and combined with the external quality assessment of the National Center for Clinical Laboratories to further evaluate the robustness of its test performance. The invention has been successfully applied to clinical therapeutic drug monitoring and can accurately determine the serum lamotrigine concentration of patients. Background Art

[0002] Lamotrigine is a new type of broad-spectrum antiepileptic drug of benzotriazine class. Clinical studies have shown that this drug can not only be used for epilepsy, but also be used as an emotional stabilizer and an atypical antipsychotic drug for the treatment of bipolar disorder. The therapeutic concentration range of this drug for epilepsy treatment is 3 - 14 μg / mL, and as an emotional stabilizer, its recommended therapeutic concentration range is 1 - 6 μg / mL. There are large individual differences in this drug, which are affected by various factors such as concomitant medications and drug gene polymorphisms. Conducting serum therapeutic drug monitoring of lamotrigine can better guide individualized medication for patients.

[0003] Chromatography is the main technical means for conducting therapeutic drug monitoring, such as: high performance liquid chromatography (HPLC) method, liquid chromatography - mass spectrometry technology, etc. For example, the liquid chromatography - tandem mass spectrometry technology developed by Jia Ziqiang, Wang Xiongwei, Li Long, etc. (Comparative Document 1: Detection method and kit for antiepileptic drugs in blood samples, CN 116500158 A; Comparative Document 2: Quality control product, kit and method for detecting the concentration of antiepileptic drugs in human blood matrix, CN 117825592 B; Comparative Document 3: A method for detecting the concentration of antiepileptic drugs in clinical samples, CN 117214341 A) can all achieve accurate quantification of the lamotrigine concentration in plasma or serum matrix, and its lower limit of quantification is between 0.5 μg / mL and 1.0 μg / mL. Compared with liquid chromatography - mass spectrometry technology, the HPLC method can fully meet the sensitivity requirements (0.5 μg / mL - 1.0 μg / mL) for steady - state serum lamotrigine concentration. For example, the multi - dimensional online solid - phase extraction liquid chromatography technology developed by Shi Gongming, etc. (Comparative Document 4: A kit for monitoring the concentration of lamotrigine drug in blood and its detection method, CN110361488A;), Rosa The lamotrigine blood drug concentration determination method reported by Deng et al. (Comparative Document 5: Development and application of an HPLC-DAD technique for human plasma concentration monitoring of perampanel and lamotrigine in drug-resistant epileptic patients, Journal of Chromatography B 1162 (2021) 122491) and the blood drug concentration method developed by Jiao Zheng et al. (Comparative Document 6: A method for determining the blood drug concentration of antiepileptic drugs). The HPLC technique has a higher popularity and lower technical requirements for operators, making it easier to promote and apply the invention technology. Therefore, the present invention aims to improve the existing HPLC determination technology for serum lamotrigine therapeutic drug monitoring to make it more economical, simple, fast, and robust. To achieve this technical improvement, the present invention mainly optimizes the serum sample pretreatment method, regression curve calculation method, and quality control technology. Summary of the Invention

[0004] The purpose of the present invention is to provide a robust HPLC determination method for serum lamotrigine therapeutic drug monitoring.

[0005] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: A robust HPLC determination method for serum lamotrigine therapeutic drug monitoring, including:

[0006] (1) Preparation of working solutions:

[0007] Take a lamotrigine reference substance, dissolve it in methanol and make up the volume to obtain a stock solution, and store it in a medical refrigerator at -25°C.

[0008] Preparation of quality control working solutions: Take the stock solution, dissolve it in methanol with a volume fraction of 50% to obtain a quality control working solution with a concentration of 1000 μg / mL; take the high-concentration quality control solution, dissolve it in methanol with a volume fraction of 50% to obtain a medium-concentration quality control working solution with a concentration of 500 μg / mL; take the medium-concentration quality control solution, dissolve it in methanol with a volume fraction of 50% to obtain a low-concentration quality control working solution with a concentration of 50 μg / mL.

[0009] Linear working solution: Accurately pipette 0.64 mL of the stock solution, add methanol solution with a volume fraction of 50% to 1 mL, and shake well to obtain C1 with a concentration of 1280 μg / mL. Dilute it successively in a ratio of 1:2 to obtain C2 with a concentration of 640 μg / mL, C3 with a concentration of 320 μg / mL, C4 with a concentration of 160 μg / mL, C5 with a concentration of 80 μg / mL, C6 with a concentration of 40 μg / mL, and C7 with a concentration of 20 μg / mL;

[0010] (2) Chromatographic conditions: Chromatographic column: Hypersil ODS2; 4.6 mm × 250 mm, 5 μm; Mobile phase: acetonitrile - ammonium acetate solution, isocratic elution; Flow rate: 0.8 mL / min, injection volume 10 μL, column temperature 35 °C, elution duration 10 min, detection wavelength λ is set at 310 nm;

[0011] (3) Preparation of clinical quality control products and calibration curve samples: Pipette 5 μL of the quality control solution into 100 μL of blank serum matrix with a pipette to prepare high, medium, and low level clinical quality control products with serum drug concentrations of 50 μg / mL, 25 μg / mL, and 2.5 μg / mL respectively; Prepare calibration curve samples with serum drug concentrations of 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL, and 64.0 μg / mL respectively;

[0012] (4) Pretreatment of serum samples: Use acetonitrile stored at -25 °C as the pretreatment reagent for serum samples; Take 100 μL of patient serum or clinical quality control products and calibration curve samples, add ice-cold acetonitrile at -20 °C to -25 °C in a volume ratio of 1:3, and vortex for 2 min; Centrifuge the above samples at 15000 g for 10 min to separate the supernatant, and then centrifuge again; Take 100 μL of the supernatant and transfer it to an injection vial as the sample to be measured.

[0013] Due to the application of the above technical solution, the advantages of the present invention compared with the prior art are:

[0014] 1. The present invention preferably uses ice acetonitrile (-25°C to -20°C) as a reagent for the pretreatment of serum samples. Compared with the ethyl acetate and dichloromethane extraction methods used in Comparative Document 5, Comparative Document 6 (Sánchez-Sellero, Inés et al., Determination of lamotrigine in human plasma by HPLC-PDA. Application to forensic samples. Forensic Sci Med Pathol. 2024 Apr 10.) and Comparative Document 8 (Wu Yan et al., Indoor quality control assessment of lamotrigine therapeutic drug monitoring based on Westgard multi-rule theory, Chinese Journal of Modern Applied Pharmacy, 2019, Vol. 36, No. 19: 166), the pretreatment steps are more simplified, and at the same time, the damage caused by the volatilization of organic reagents due to nitrogen purging to the environment and the human body is avoided. The present invention preferably uses ice acetonitrile as a precipitant and combines it with an acetonitrile-ammonium acetate mobile phase system (pH = 6.59), and the detection wavelength is preferably 310 nm. Compared with the representative chromatograms attached in Comparative Document 4, Comparative Document 9 (Xue-ping Zhu et al., The establish of the HPLC method to examine the plasma concentration of lamotrigine and oxcarbazepine, Pakistan journal of pharmaceutical sciences, 2015 Vol. 28, No. 3 (Suppl)), Comparative Document 10 (Xu Yichao et al., Simultaneous determination of the blood drug concentrations of lamotrigine and its active metabolite in human plasma by high performance liquid chromatography and its clinical application analysis, Chinese Journal of Hospital Pharmacy, 2019, Vol. 39, No. 2), not only can the endogenous interference be effectively reduced, the adverse effects of the ultraviolet end absorption of the methanol system on quantification can be reduced, making the chromatographic baseline more stable, but also the analysis time can be further shortened to within 10 min (attached Figure 1 ). The present invention has also compared with acetonitrile, methanol and ethyl acetate widely used in the comparative documents, and confirmed that ice acetonitrile can better reduce the interference of solvent peaks. Therefore, the present invention has successively investigated the effect of the above precipitant on removing endogenous interference using the acetonitrile-ammonium acetate system and the methanol-ammonium acetate system, and both have confirmed that using ice acetonitrile as the precipitant and acetonitrile-ammonium acetate as the mobile phase system can make the endogenous interference smaller. The specific effects of the above improvements are all concentratedly shown in the attached Figure 2 . The preferably used ice acetonitrile precipitant and acetonitrile-ammonium acetate mobile phase system of the present invention have also been successfully used for the therapeutic drug monitoring of lamotrigine in the serum of clinical patients. It can be seen that other drugs and their endogenous substances that the patients may take have not interfered with the retention time (6.1 min) and peak area of lamotrigine (attachedFigure 3 )。It is worth noting that ice acetonitrile (-25°C to -20°C) can be obtained only by cryogenic freezing of acetonitrile. The detection system used is the most common HPLC tandem UV detector (UV), which is more conducive to the popularization and application of the present invention.

[0015] 2. The present invention preferably establishes a lamotrigine calibration curve for serum matrix based on the external standard method. Compared with the internal standard calibration methods used in Comparative Document 1 (internal standard calibrator: isotope internal standard), Comparative Document 2 (isotope internal standard), Comparative Document 3 (isotope), Comparative Document 5 (entacapone), Comparative Document 6 (chlorzoxazone), Comparative Document 7 (chloramphenicol), Comparative Document 8 (ornidazole), and Comparative Document 9 (chlorzoxazone), the present invention can still achieve a good linear relationship (R 2 > 0.99) and fully meet the technical requirements of the "Chinese Pharmacopoeia (2020 Edition)" for the standard curve.

[0016] 3. Therapeutic drug monitoring belongs to the technical category of quantitative determination in clinical laboratories. Its determination technology not only requires methodological investigation but also a strict quality control system to objectively evaluate the robustness of the detection performance. Internal quality control is the basic prerequisite to ensure the robustness of the detection performance, and external quality assessment is the objective proof to evaluate the robustness degree of the detection performance. Therefore, based on the methodological investigation, the present invention establishes a more objective Westgard multi-rule internal quality control system. Compared with all the comparative documents of the present invention, Comparative Document 8 does not explain the rationality of the internal quality control rules . The sigma index (σ) of the HPLC determination method disclosed in the present invention reaches or approaches the detection performance level of the ultra-high performance liquid chromatography (UPLC) for lamotrigine blood drug concentration (4 ≤ σ < 5) disclosed in Comparative Document 11. Compared with all the comparative documents of the present invention, the present invention is an HPLC determination method for serum lamotrigine therapeutic drug monitoring that has publicly participated in the 2024 annual external quality assessment program of the National Health Commission (the serum lamotrigine therapeutic drug monitoring was first carried out in 2024) and passed with full marks. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Specificity investigation of the high performance liquid chromatography determination method for serum lamotrigine therapeutic drug monitoring; A, reference substance solution; B, blank serum; C, blank serum matrix plus control substance at the lowest quantitation limit.

[0018] Figure 2Representative chromatograms under combinations of different precipitants and different mobile phase systems. A, acetonitrile; B, methanol; C, ethyl acetate; 1, acetonitrile-ammonium acetate system; 2, methanol-ammonium acetate system.

[0019] Figure 3 High performance liquid chromatogram of serum therapeutic drug monitoring for patients taking lamotrigine.

[0020] Figure 4 Evaluation results of participating in the external quality assessment program (first time) of serum therapeutic drug monitoring (lamotrigine) in 2024 using the present invention.

[0021] Figure 5 Evaluation results of participating in the external quality assessment program (second time) of serum therapeutic drug monitoring (lamotrigine) in 2024 using the present invention. Detailed implementation manners

[0022] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this embodiment.

[0023] Please refer to Figures 1-5 Note that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in the art to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they do not have technical essential significance. Any modification of the structure, change of the proportional relationship, or adjustment of the size. The following embodiments are provided to better understand the present invention, rather than to limit the present invention. The experimental materials used in the following embodiments are all obtained from conventional consumables and biochemical reagent stores unless otherwise specified.

[0024] Example 1: A robust high performance liquid chromatography method for serum therapeutic drug monitoring of lamotrigine

[0025] One of the objectives of the present invention is to simplify its analysis steps based on existing public documents or literature research, including but not limited to: mobile phase composition, serum sample pretreatment, etc.

[0026] Another objective of the present invention is to conduct method validation with reference to the guiding principles of "Chinese Pharmacopoeia (2020)" 9012. At the same time, an in-house quality control system is established to evaluate the precision of the technology of the present invention, and participating in the external quality assessment provides verification of the inspection ability for the present invention, providing a sufficient and objective reference basis for the robustness of the method.

[0027] Another objective of the present invention is to use this method to monitor the blood drug concentration of real clinical patients taking lamotrigine.

[0028] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0029] Establishment of an HPLC assay for serum therapeutic drug monitoring of lamotrigine, including the following steps:

[0030] S1. Instrumentation: Waters e2695 high performance liquid chromatograph equipped with a 2489 UV / Vis detector (Waters Corporation, USA); VX-200 vortex oscillator (Labnet International, Inc., USA); AB135-5 electronic balance (Mettler Toledo, Shanghai); Eppendorf 5430R high-speed refrigerated centrifuge (Eppendorf, Germany); pipettes of various specifications (Eppendorf, Germany).

[0031] S2. Preparation of working solutions: ① Weigh accurately 20 mg of lamotrigine reference standard, dissolve it in methanol and dilute to a stock solution with a concentration of 2000 μg / mL, and store it in a refrigerator at -25 °C; ② Preparation of quality control working solutions: Take 2.5 mL of the stock solution, dilute it to 5 mL with 50% methanol solution (v / v) to obtain a high-concentration quality control working solution with a concentration of 1000 μg / mL; Take 2.5 mL of the high-concentration quality control solution, dilute it to 5 mL with 50% methanol solution (v / v) to obtain a medium-concentration quality control working solution with a concentration of 500 μg / mL; Take 1 mL of the medium-concentration quality control solution, dilute it to 10 mL with 50% methanol solution (v / v) to obtain a low-concentration quality control working solution with a concentration of 50 μg / mL; ③ Linear working solutions: Accurately pipette 0.64 mL of the stock solution, add 50% methanol solution (v / v) to 1 mL, shake well to obtain C1 (1280 μg / mL), and successively dilute it by a factor of two to obtain C2 (640 μg / mL), C3 (320 μg / mL), C4 (160 μg / mL), C5 (80 μg / mL), C6 (40 μg / mL) and C7 (20 μg / mL).

[0032] S3. Preparation of blank serum matrix: Take the serum of patients who have not taken lamotrigine (not taken lamotrigine, from health examination centers, department of neurology, intensive care unit, department of organ transplantation, n≥6), gently shake and mix evenly, and filter through a 0.45 μm microporous membrane to obtain it.

[0033] S4. Preparation of clinical quality control products and calibration curve samples: Pipette 5 μL of the quality control solution into 100 μL of blank human serum matrix with a pipette to prepare high-, medium- and low-level clinical quality control products with serum lamotrigine concentrations of 50 μg / mL, 25 μg / mL and 2.5 μg / mL respectively. Similarly, prepare calibration curve samples with serum lamotrigine concentrations of 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL and 64.0 μg / mL respectively. Perform linear regression on the serum lamotrigine concentration and the lamotrigine peak area in the calibration curve samples to establish a calibration curve (equation).

[0034] S5. Serum sample pretreatment: Acetonitrile stored at -25°C (ice acetonitrile, -20°C to -25°C) is used as the serum sample pretreatment reagent. Take 100 μL of patient serum or clinical quality control product and calibration curve sample, add 300 μL of the serum sample pretreatment reagent according to the volume ratio of 1:3, and vortex for 2 min. The above samples are centrifuged to separate the supernatant at 15000 g for 10 min and then centrifuged again under the same conditions. Take 100 μL of the supernatant and put it into the sample vial as the sample to be tested.

[0035] S6. Preferably, the most common octadecylsilane-bonded silica gel is used as the packing type in the present invention, with the specific model being Hypersil ODS2 (4.6 mm × 250 mm, 5 μm), the column temperature is 35°C; the simplified mobile phase is an acetonitrile-ammonium acetate solution (30 mM) system, isocratic elution (30:70, v / v), the elution time is 10 min, the flow rate is 0.8 mL / min; the detection wavelength λ is set at 310 nm, and the injection volume is 10 μL for HPLC determination.

[0036] S7. Using the established weighted calibration curve, substitute the peak area of lamotrigine in the sample to be tested into the calibration curve equation to calculate the serum lamotrigine concentration.

[0037] Methodology verification process:

[0038] S8. The retention time of the lamotrigine reference solution (10 μg / mL) in the specificity experiment is 6.1 min (attached Figure 1 A); blank serum (attached Figure 1 B), blank serum + lamotrigine reference (concentration below the lower limit of quantitation, 1.0 μg / mL) (attached Figure 1 C) and patient serum (attached Figure 2 ), are pretreated according to item S5 and analyzed according to item S6. The endogenous substances of lamotrigine have no obvious interference on the peak time and peak area of lamotrigine, and the detection method has high specificity.

[0039] S9. Investigation of linear relationship: The prepared concentrations of blank serum and lamotrigine standard solution are 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL and 64.0 μg / mL of quality control serum containing lamotrigine. Pretreat according to item S5 and analyze according to item S6. Perform linear regression by weighted (1 / Y 2 ) least squares method, and the regression equation is Y = 5746X + 471.1 (n = 4). The linear relationship of the blood drug concentration is good in the range of 1.0 - 64 μg / mL, and the correlation coefficient > 0.99 (the results are shown in Table 1).

[0040] S7. Precision test: Prepare simulated drug-containing sera with concentrations of 2.5 μg / mL, 25.0 μg / mL, and 50.0 μg / mL using blank serum and lamotrigine standard solution, with 5 replicates for each concentration. Perform pretreatment according to item S5 and analysis according to item S6. Intra-batch precision: Determine 3 batches within the 1st day; Inter-batch precision: Determine 1 batch per day for 3 days. The results show that the relative standard deviations (RSD) within and between batches are both less than 15%, meeting the requirements of the "Pharmacopoeia of the People's Republic of China (2020 Edition)" for bioanalytical methods (the results are shown in Table 2).

[0041] S9. Accuracy test: Prepare drug-containing sera with concentrations of 2.5 μg / mL, 25.0 μg / mL, and 50.0 μg / mL using blank serum and lamotrigine standard solution, with 5 replicates for each concentration. Perform pretreatment according to item S5 and analysis according to item S6. Intra-batch accuracy: Determine 3 batches within the 1st day; Inter-batch accuracy: Determine 1 batch per day for 3 days. The results show that the means within and between batches are within 15% of the labeled values of the quality control samples, meeting the requirements of the "Pharmacopoeia of the People's Republic of China (2020 Edition)" for bioanalytical methods (the results are shown in Table 2).

[0042] S10. Recovery experiment: Prepare 5 drug-containing sera with concentrations of 2.5 μg / mL, 25.0 μg / mL, and 50 μg / mL each using blank serum and lamotrigine standard solution as controls. Separately, take blank serum, perform pretreatment according to item S5, and then add the same lamotrigine standard solution as in the control group to each, with 5 replicates for each concentration. The remaining treatment methods are the same as the control group, and detect under item S6 to calculate the recovery rate. Under the established sample preparation method, the average extraction recovery rate is 91.2% - 95.8%, and the relative standard deviation of the measured values is within 3.92%. The recoveries of lamotrigine at different concentrations are consistent, accurate, and have good reproducibility (the results are shown in Table 2).

[0043] Table 1 Investigation of the linear relationship of the determination method for serum therapeutic drug monitoring of lamotrigine

[0044]

[0045] Note: C7: 1.0 μg / mL; C6: 2.0 μg / mL; C5: 4.0 μg / mL; C4: 8.0 μg / mL; C3: 16.0 μg / mL; C2: 32.0 μg / mL; C1: 64.0 μg / mL.

[0046] Table 2 Accuracy, precision, and extraction recovery rate of the determination method for serum therapeutic drug monitoring of lamotrigine

[0047]

[0048] Note: a. Unit: μg / mL; RE: Relative error; RSD: Relative standard deviation

[0049] S10. Blank serum for stability test and lamotrigine standard solution were used to prepare drug-containing sera with concentrations of 1 μg / mL, 2.5 μg / mL, 25.0 μg / mL, and 50 μg / mL, with 5 replicates for each concentration. The stability of the samples after being repeatedly frozen and thawed 3 times, placed at room temperature for 6 h, placed in an autoinjector for 24 h, placed in a refrigerator for 7 d and 14 d was determined respectively, and pretreatment was carried out according to item S5 and analysis was carried out according to item S6. The results showed that the deviation of the mean value of each concentration in the stability experiment carried out from the labeled concentration was within 15%, meeting the requirements of the "Pharmacopoeia of the People's Republic of China (2020 Edition)" for bioanalytical methods (the results are shown in Table 3).

[0050] Table 3 Stability of the determination method for serum therapeutic drug monitoring of lamotrigine

[0051]

[0052]

[0053] a. Unit: μg / mL.

[0054] S11. Performance verification: The detection data of three-level serum quality control products during the methodological verification of the present invention were collected. Taking the acceptable range (target value ±20%) of the National Center for Clinical Laboratory as the acceptable standard, after data cleaning (removing outliers beyond ±1 / 2TEa), the sigmas of the three-level quality control were calculated to be 4.07, 4.12, and 4.07 respectively according to σ=(TEa - |Bias|) / CV%, indicating that the method system has good analytical performance (Table 4). The 1-3s / 2 / 3-2s / R-4s / 3-1s multi-rule quality control scheme (n = 3, r = 1) was used as its in-house quality control rule, and this scheme can ensure that the error detection rate (P ed )≥0.90 and the false out-of-control probability (P fr )≤0.05, effectively ensuring the reliability of the detection results. To ensure the reliability of the test results of each laboratory, the National Center for Clinical Laboratory launched an inter-laboratory quality assessment program for serum therapeutic drug monitoring of lamotrigine in 2024. The present invention obtained full marks in both national inter-laboratory quality assessments for serum therapeutic drug monitoring participated in the same year (Appendix Figure 4 and Figure 5 ), fully verifying the robustness of the method. In addition, the method developed by the present invention has currently been successfully applied to the serum therapeutic drug monitoring of clinical patients (Table 5 and Figure 3 ).

[0055] Table 4 Sigma metric values of the method for serum therapeutic drug monitoring of lamotrigine

[0056]

[0057] Note: a. The unit is μg / mL; CV is the coefficient of variation; TEa is the maximum allowable total error specified by the National Clinical Laboratory Center.

[0058] Table 5 Results of serum therapeutic drug monitoring of lamotrigine in patients

[0059]

[0060] Accompanying standard curve: aY=5200X+1236; bY=5269X+1949; cY=5357X+430.9

[0061] Example 2: A robust HPLC method for lamotrigine serum therapeutic drug monitoring

[0062] A robust HPLC assay for the therapeutic drug monitoring of lamotrigine in serum comprising:

[0063] (1) Preparation of working solution:

[0064] Take the lamotrigine reference substance, dilute it with methanol to obtain the stock solution, and store it in a -25℃ medical refrigerator;

[0065] Preparation of quality control working solution: Take the stock solution and dilute it with 50% methanol solution to obtain a quality control working solution with a concentration of 1000μg / mL; take the high-concentration quality control solution and dilute it with 50% methanol solution to obtain a medium-concentration quality control working solution with a concentration of 500μg / mL; take the medium-concentration quality control solution and dilute it with 50% methanol solution to obtain a low-concentration quality control working solution with a concentration of 50μg / mL;

[0066] Linear working solution: accurately pipette 0.64 mL of the stock solution, add 50% methanol solution to 1 mL, shake well to obtain C1 with a concentration of 1280 μg / mL, and dilute in this ratio to obtain 640 μg / mL of C2, 320 μg / mL of C3, 160 μg / mL of C4, 80 μg / mL of C5, 40 μg / mL of C6, and 20 μg / mL of C7;

[0067] (2) Chromatographic conditions: Chromatographic column: Hypersil ODS2; 4.6 mm × 250 mm, 5 μm; Mobile phase: acetonitrile-ammonium acetate solution, isocratic elution; Flow rate: 0.8 mL / min, injection volume 10 μL, column temperature 35 °C, elution time 10 min, detection wavelength λ set to 310 nm;

[0068] (3) Preparation of clinical quality control products and calibration curve samples: Use a pipette to aspirate 5 μL of the quality control solution into 100 μL of blank serum matrix to prepare high, medium, and low-level clinical quality control products with serum drug concentrations of 50 μg / mL, 25 μg / mL, and 2.5 μg / mL, respectively; prepare calibration curve samples with serum drug concentrations of 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL, and 64.0 μg / mL.

[0069] (4) Pretreatment of serum samples: Use acetonitrile stored at -25°C as the reagent for serum sample pretreatment; take 100 μL of patient serum or clinical quality control products and calibration curve samples, add ice acetonitrile at -20°C to -25°C according to a volume ratio of 1:3, and vortex for 2 min; centrifuge the above samples at 15000 g for 10 min to separate the supernatant, and then continue secondary centrifugation; take 100 μL of the supernatant and place it in an injection vial as the sample to be measured.

[0070] The above are only preferred embodiments for explaining the present invention and are not intended to limit the present invention in any form. Therefore, any modifications or changes to the present invention made in the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A robust HPLC assay for lamotrigine serum therapeutic drug monitoring, characterized in that: include: (1) Preparation of working solution: Take the lamotrigine reference substance, dilute it with methanol to obtain the stock solution, and store it in a -25℃ medical refrigerator; Preparation of quality control working solution: Take the stock solution and dilute it with 50% methanol solution to obtain a quality control working solution with a concentration of 1000μg / mL; take the high-concentration quality control solution and dilute it with 50% methanol solution to obtain a medium-concentration quality control working solution with a concentration of 500μg / mL; take the medium-concentration quality control solution and dilute it with 50% methanol solution to obtain a low-concentration quality control working solution with a concentration of 50μg / mL; Linear working solution: accurately pipette 0.64 mL of the stock solution, add 50% methanol solution to 1 mL, shake well to obtain C1 with a concentration of 1280 μg / mL, and dilute in this ratio to obtain 640 μg / mL of C2, 320 μg / mL of C3, 160 μg / mL of C4, 80 μg / mL of C5, 40 μg / mL of C6, and 20 μg / mL of C7; (2) Chromatographic conditions: Chromatographic column: Hypersil ODS2; 4.6 mm × 250 mm, 5 μm; Mobile phase: acetonitrile-ammonium acetate solution, isocratic elution; Flow rate: 0.8 mL / min, injection volume 10 μL, column temperature 35 °C, elution time 10 min, detection wavelength λ set to 310 nm; (3) Preparation of clinical quality control products and calibration curve samples: Use a pipette to draw 5 μL of the quality control solution into 100 μL of blank serum matrix to prepare high, medium and low clinical quality control products with serum drug concentrations of 50 μg / mL, 25 μg / mL and 2.5 μg / mL, respectively; prepare calibration curve samples with serum drug concentrations of 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL and 64.0 μg / mL, respectively; (4) Serum sample pretreatment: Use acetonitrile stored at -25°C as the serum sample pretreatment reagent; take 100 μL of patient serum or clinical quality control product or calibration curve sample, add -20°C to -25°C ice acetonitrile at a volume ratio of 1:3, and vortex for 2 min; the above samples are centrifuged at 15000g × 10 min and the supernatant is separated and centrifuged twice; 100 μL of the supernatant is separated and placed in a sampling bottle as the sample to be tested.

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