A kit for detecting antiepileptic drugs in serum by high performance liquid chromatography tandem mass spectrometry and a detection method thereof

Abstract

The application provides a kit for detecting antiepileptic drugs in serum by high performance liquid chromatography tandem mass spectrometry and a detection method thereof, adopts pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin as the matrix of calibrators and quality control samples, avoids the interference of bovine serum or bovine serum albumin on the detection of valproic acid, adds ethylenediamine sodium di-o-phenylacetate as an antioxidant in the matrix, and preferably adopts rabbit serum as the matrix, so that the stability of the calibrators and the quality control samples is significantly improved; meanwhile, the internal standard is stored in a precipitant containing ethylenediamine sodium di-o-phenylacetate, so that the internal standard correction, protein precipitation and target substance extraction can be realized by one-step operation for sample pretreatment, the internal standard solution can be stored for a long time and used as needed, and the effective period of the kit product is prolonged.

Description

[0001] This application is a divisional application of parent application 2022114676103, filed on November 22, 2022. Technical Field

[0002] This invention belongs to the field of biochemical analysis technology, specifically, it relates to a detection kit and method for detecting antiepileptic drugs in serum by high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0003] In the treatment of epileptic seizures, antiepileptic drugs play a particularly important role. Antiepileptic drugs can eliminate or reduce seizures in two ways: first, by affecting central neurons to prevent or reduce their pathological excessive discharge; and second, by raising the excitation threshold of normal brain tissue, weakening the spread of excitation from the lesion, and preventing epileptic recurrence. Antiepileptic drugs also affect memory, motor speed, and other functions; the higher the blood drug concentration, the more pronounced the effect. Therefore, monitoring the blood concentration of antiepileptic drugs is of great significance.

[0004] Antiepileptic drugs can be used alone or in combination to enhance efficacy or reduce side effects. Carbamazepine, a derivative of imino-1,2-stilbene, is commonly used alone or in combination with other antiepileptic drugs for the treatment of psychomotor seizures (grand mal seizures), focal seizures, and focal-mixed grand mal seizures. It can also be used to treat trigeminal neuralgia. Carbamazepine is sometimes used to treat patients with bipolar disorder who are unresponsive to lithium therapy. Due to differences in absorption and metabolism, individual responses to carbamazepine vary considerably.

[0005] Valproic acid (VPA: 2-propylvalerate; sodium valproate) is a relatively new anticonvulsant drug, primarily used for primary and secondary generalized epilepsy. It is also effective in treating apoptotic epilepsy, particularly myoclonus, and is used to treat chronic photosensitive epilepsy. VPA is often used in combination with other antiepileptic drugs, but recent studies have shown that VPA can also be used alone with good efficacy. Other studies suggest that VPA is useful in treating affective disorders, especially in patients with bipolar disorder who are insensitive to lithium therapy. Through β- and ω-oxidation and conjugation, VPA is converted into metabolic complexes. Some metabolites have significant anticonvulsant activity, while others may be associated with drug toxicity. Furthermore, other antiepileptic drugs used concurrently can significantly affect VPA metabolism.

[0006] Phenytoin is commonly used to treat generalized tonic-clonic seizures (GMS) and focal seizures. It can also be used to treat basic focal seizures (focal) or complex focal seizures (psychomotor, temporal lobe). Phenytoin has a high binding affinity to plasma proteins, and unbound phenytoin, in its free form, has higher pharmacological activity. In individuals with normal albumin levels and renal function, approximately 10% of phenytoin is unbound. Phenytoin's unique metabolic elimination characteristics make dose / concentration evaluation relatively difficult. Significant differences have been reported in the absorption, metabolism, and elimination of phenytoin among individuals. The relationship between drug dosage and pharmacological activity is further complicated by disease state, drug administration, and patient complications.

[0007] It is evident that valproic acid, carbamazepine, and phenytoin are all commonly used antiepileptic drugs that can be used in combination. Simultaneous monitoring of the blood concentrations of valproic acid, carbamazepine, and phenytoin is necessary to help physicians determine the treatment dosage based on individual differences in patients' drug responses, adjust the drug dosage to reduce toxic side effects, thereby controlling epileptic seizures and ensuring good patient adherence.

[0008] Currently, numerous methods for determining antiepileptic drugs in serum have been reported both domestically and internationally. Common methods include chemiluminescence and chromatography. Among these methods, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the preferred method for determining antiepileptic drugs. However, previous methods generally suffer from complex operation, numerous interfering factors, poor specificity, long analysis time, and low throughput. These issues are mainly due to the cumbersome sample pretreatment process and poor stability.

[0009] Existing sample pretreatment methods for antiepileptic drugs mainly include protein precipitation and liquid-liquid extraction. For example, CN111812216A uses a mixed solution of methanol and acetonitrile as a protein precipitant to pretreat serum samples for the detection of antiepileptic drugs. However, due to the poor stability of the sample pretreatment process, the detection results are easily affected by interference, resulting in large errors and low detection sensitivity.

[0010] Therefore, there is an urgent need for a sample pretreatment method for antiepileptic drugs that is simple to operate, has high processing efficiency, high detection throughput, high detection accuracy and sensitivity, low cost, and low manual workload, so as to overcome the above-mentioned shortcomings and defects of existing methods. Summary of the Invention

[0011] To address the aforementioned issues, this invention provides a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) kit and method for detecting antiepileptic drugs in serum. The kit uses porcine serum, horse serum, rabbit serum, porcine serum albumin, or horse serum albumin as the matrix for calibrators and quality controls, avoiding interference from bovine serum or bovine serum albumin in the detection of valproic acid. Adding sodium ethylenediamine di-o-phenylacetate (EDDDI) as an antioxidant to the matrix, with rabbit serum being the preferred matrix, significantly improves the stability of the calibrators and quality controls. Furthermore, by storing the internal standard in a precipitant containing EDDDI, the sample pretreatment process can achieve internal standard calibration, protein precipitation, and target substance extraction in a single step. This allows for long-term storage of the internal standard solution, enabling immediate use and extending the shelf life of the kit.

[0012] On one hand, the present invention provides a detection kit for detecting antiepileptic drugs in serum by high performance liquid chromatography-tandem mass spectrometry. The kit contains standards and / or quality control products, and the standards and / or quality control products contain additives, wherein the additives are any one or more of ethylenediamine di-o-phenylacetic acid sodium salt, ethylenediaminetetraacetic acid disodium salt, sodium gluconate, and sodium citrate.

[0013] The antiepileptic drugs described in this invention include valproic acid, carbamazepine, and phenytoin.

[0014] The inventors have demonstrated through extensive experimentation that antiepileptic drugs in solution matrices, especially valproic acid, exhibit poor stability under oxidizing and alkaline conditions. For example, the valproic acid content in standards, quality controls, and internal standards within the test kits is highly unstable, showing a significant decrease after just a few days at room temperature. This substantially affects the accuracy and sensitivity of the detection, necessitating the preparation of all samples, standards, internal standards, and quality controls used for antiepileptic drug detection on a fresh-for-use basis, causing considerable inconvenience and high costs in practical operations. Therefore, to more accurately detect the levels of valproic acid, carbamazepine, and phenytoin—three antiepileptic drugs—in serum, the stability issue of valproic acid in the solution matrix must first be overcome.

[0015] The selection of valproic acid stabilizers requires comprehensive consideration of their antioxidant capacity, acidity, and alkalinity, and the selection of an appropriate concentration to achieve a good stabilizing effect. In other words, in standards or quality control products containing valproic acid, carbamazepine, and phenytoin, an additive must be added to prevent valproic acid oxidation while maintaining the most suitable acidity, alkalinity, and concentration to maximize the stability of valproic acid content.

[0016] The inventors made a surprising discovery that adding sodium ethylenediamine di-o-phenylacetate (EDDHA), disodium ethylenediaminetetraacetate, sodium gluconate, or sodium citrate to standards or quality control products can significantly improve the stability of antiepileptic drugs, especially the stability of valproic acid.

[0017] Furthermore, the additive is sodium ethylenediamine di-o-phenylacetate.

[0018] Studies have shown that when the additive is sodium ethylenediamine di-o-phenylacetate (EDDHA), the effect on improving stability is the best.

[0019] Furthermore, the standards and / or quality control samples are prepared using a matrix, which is animal serum or animal serum albumin, wherein the animal serum is not bovine serum and the animal serum albumin is not bovine serum albumin.

[0020] Since human serum is not readily available and cannot be used as a matrix for the preparation of mass-produced reagent kits, it is necessary to find suitable animal serum as a matrix.

[0021] The inventors discovered that bovine serum or bovine serum albumin significantly interferes with the detection of valproic acid, leading to highly unstable detection results; therefore, bovine serum or bovine serum albumin cannot be used to prepare the matrices for standards and quality control products.

[0022] Further, the matrix is ​​any one or more of porcine serum, horse serum, rabbit serum, porcine serum albumin, or horse serum albumin, and the concentration of sodium ethylenediamine di-o-phenylacetate is 0.02-0.1 mg / mL.

[0023] Porcine serum, horse serum, rabbit serum, porcine serum albumin, or horse serum albumin are used as the matrix. Additives in the matrix include one or more of sodium ethylenediaminetetraacetate (EDTA), disodium ethylenediaminetetraacetate (EDTA), sodium gluconate, and sodium citrate, with EDTA at a concentration of 0.02-0.1 mg / mL being the most preferred, to improve the stability of calibrators and quality control samples and extend product shelf life. Using suitable and readily available animal serum to simulate human samples can reduce matrix effects, resulting in more accurate and stable test results.

[0024] Furthermore, the matrix is ​​rabbit serum.

[0025] Studies have shown that using rabbit serum as a matrix helps to further improve the stability of valproic acid in standards or quality control products. At the same time, the matrix effect is low and the background noise is low, which can ensure the accurate detection of valproic acid.

[0026] Furthermore, the test kit also includes an internal standard solution containing a precipitant and a stabilizer, wherein the stabilizer is sodium ethylenediamine di-o-phenylacetate.

[0027] Furthermore, the precipitant is methanol.

[0028] Existing internal standard solutions for antiepileptic drugs require fresh preparation and use each time due to the instability of valproic acid. To reduce workload and waste, this invention extends the shelf life of the internal standard solution by adding a stabilizer to maintain the stability of the valproic acid content.

[0029] The internal standard solution containing sodium di-o-phenylacetate containing ethylenediamine is very stable and can be stored for a long time. It can be used as needed and has a shelf life of more than three years. After three years, the content of valproic acid internal standard in it still remains within 3%, making the detection process simpler and more efficient, the detection cost lower, and the detection results more accurate and stable.

[0030] This invention mixes methanol, sodium ethylenediamine di-o-phenylacetate, and isotopically labeled antiepileptic drugs to obtain an internal standard solution. This solution is then added to the sample and mixed thoroughly. This not only serves as an internal standard but also simultaneously achieves internal standard correction, protein precipitation and separation, and target substance extraction. Furthermore, this solution system exhibits good solubility for the antiepileptic drugs in the sample, eliminating the need for enrichment processes such as freeze-drying or liquid-liquid extraction followed by nitrogen blowing. After separating the precipitate impurities, the sample can be directly injected for detection, simplifying the operation process. Moreover, the reagents used are low-cost conventional chemical reagents, reducing detection costs.

[0031] Furthermore, the internal standard contains an isotopically labeled antiepileptic drug; the standard is an antiepileptic drug with a standard concentration; and the quality control contains an antiepileptic drug with two different concentrations.

[0032] Furthermore, the antiepileptic drug is any one or more of valproic acid, carbamazepine, or phenytoin.

[0033] On the other hand, the present invention provides a method for detecting antiepileptic drugs in serum, wherein the method uses the detection kit described above for detection; including system suitability testing, sample preparation, sample pretreatment and sample detection; wherein the sample pretreatment includes the following steps: taking the sample and internal standard solution at a ratio of 1:7 to 1:11, mixing them, centrifuging, and taking the supernatant for high performance liquid chromatography-tandem mass spectrometry detection.

[0034] In another aspect, the present invention provides the use of sodium ethylenediamine di-o-phenylacetate as a stabilizer in the preparation of valproic acid solutions.

[0035] In another aspect, the present invention provides the use of sodium ethylenediamine di-o-phenylacetate as a stabilizer in the preparation of antiepileptic drug solutions, wherein the antiepileptic drug is any one or more of valproic acid, carbamazepine, or phenytoin.

[0036] In another aspect, the present invention provides the use of rabbit serum in the preparation of standards and / or quality control products to improve the stability of valproic acid.

[0037] Furthermore, the standards and / or quality control products contain sodium ethylenediamine di-o-phenylacetate.

[0038] The present invention has the following beneficial effects:

[0039] (1) Adding sodium ethylenediamine di-o-phenylacetate to the matrix of the standard and / or quality control products can effectively solve the problem of unstable valproic acid content in antiepileptic drugs, which will cause a serious decrease in content after a few days at room temperature; it can enable the standard and / or quality control products to be stored for a long time, with stable antiepileptic drug content, without the need for fresh preparation, thereby improving the stability of calibrators and quality control products, extending the product shelf life, and significantly improving the accuracy and sensitivity of detection;

[0040] (2) It was found that bovine serum or bovine serum albumin significantly interfered with the detection of valproic acid, and bovine serum or bovine serum albumin could not be used to prepare the matrix; porcine serum, horse serum, rabbit serum, porcine serum albumin or horse serum albumin could be used as the matrix for calibrators and quality control products to reduce the matrix effect and make the detection results more accurate and stable.

[0041] (3) It was found that using rabbit serum as the matrix for standards and / or quality control products, with the addition of sodium ethylenediamine di-o-phenylacetate, can further improve the stability of valproic acid in the matrix and better ensure the accuracy of valproic acid detection.

[0042] (4) Add sodium ethylenediamine di-o-phenylacetate to the internal standard solution to achieve internal standard calibration, protein precipitation and target substance extraction in one step, thereby improving the accuracy, sensitivity and ease of operation of the detection, and making the content of antiepileptic drugs in the internal standard stable, which can be stored for a long time with a shelf life of more than three years and can be taken out as needed.

[0043] (5) When a patient is taking one or more drugs, a one-step sample pretreatment method can be used to detect different blood drug concentrations, reduce the number of repeated samplings, reduce patient suffering, effectively reduce analysis costs, and provide a wide range of reports. It can accurately analyze serum samples with different blood drug concentration levels. Attached Figure Description

[0044] Figure 1 This is the mass spectrum of valproic acid detection for standard sample 10 in Example 1;

[0045] Figure 2 The mass spectrum of phenytoin detection for standard sample 10 in Example 1;

[0046] Figure 3 The mass spectrum of carbamazepine detection in standard sample 10 in Example 1;

[0047] Figure 4 The mass spectrum of bovine serum albumin as a matrix in the valproate ion channel in Example 3 is shown.

[0048] Figure 5 The mass spectrum of bovine serum as a matrix in the valproate ion channel in Example 3 is shown.

[0049] Figure 6 This is the mass spectrum of porcine serum albumin as a matrix in the valproate ion channel in Example 3;

[0050] Figure 7 The mass spectrum of the detection of porcine serum as a matrix in the valproate ion channel in Example 3 is shown.

[0051] Figure 8 The mass spectrum of valproate ion channel detection using horse serum albumin as a matrix in Example 3 is shown.

[0052] Figure 9 The mass spectrum of horse serum as a matrix in the valproate ion channel in Example 3 is shown.

[0053] Figure 10 This is a mass spectrum of rabbit serum as a matrix detected in the valproate ion channel in Example 3. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0055] Example 1: Sample Preparation, Pretreatment, and Detection

[0056] I. Sample Preparation

[0057] 1. Preparation of calibrators and quality control samples

[0058] A mixed solution was prepared using the antiepileptic drugs valproic acid, carbamazepine, and phenytoin standards to prepare standard working solutions and quality control working solutions. The above working solution was added to rabbit serum containing 0.05 mg / mL ethylenediamine di-o-phenylacetic acid sodium to prepare calibrators with 10 series concentrations and quality control solutions with 2 series concentrations, as shown in Table 1 (unit: μg / mL).

[0059] Table 1. Concentrations of Standards and Quality Control Samples for Three Antiepileptic Drugs

[0060] Calibrator 1 4 0.2 0.5 Calibrator 2 5 0.5 1.25 Calibrator 3 10 1 2.5 Calibrator 4 15 3 7.5 Calibrator 5 25 5 12.5 Calibrator 6 45 9 22.5 Calibrator 7 75 15 37.5 Calibrator 8 90 18 45 Calibrator 9 120 24 60 Calibrator 10 150 30 75 Quality control product 1 20 2.4 4 Quality control product 2 100 12 20

[0061] 2. Preparation of internal standard solution

[0062] (1) Internal standard stock solution

[0063] Prepare a mixed internal standard stock solution: valproic acid-d6, carbamazepine-d2. 15The concentrations of N and phenytoin-d10 were 200 μg / mL, 20 μg / mL, and 50 μg / mL, respectively.

[0064] (2) Precipitator

[0065] Weigh 50 mg of sodium ethylenediamine di-o-phenylacetate, dissolve it in 100 mL of purified water, add 900 mL of methanol, and mix well to obtain a precipitant.

[0066] (3) Internal standard solution

[0067] Take the internal standard stock solution and the precipitant and mix them at a ratio of 1:100 to obtain the internal standard solution.

[0068] 3. Preparation of system suitability solutions

[0069] Take 100 μL of a mixed standard solution containing 4 μg / mL valproic acid, 0.2 μg / mL carbamazepine and 0.5 μg / mL phenytoin, and mix it with 49.9 mL of 50% methanol aqueous solution to prepare a system suitability solution.

[0070] The system suitability test involves using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to test the system suitability solution three times before sample detection. The retention time, response intensity, and precision of the target substance are used to determine whether the HPLC-MS / MS system is functioning properly.

[0071] II. Sample Pretreatment

[0072] (1) Take 20 μL of sample and add it to a 96-well plate or centrifuge tube;

[0073] (2) Add 180 μL of internal standard solution, vortex for 5 minutes, centrifuge at 4000 rpm for 10 minutes, and take the supernatant for testing.

[0074] III. Sample Testing

[0075] The supernatant was collected and analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Specific analytical conditions were as follows: gradient elution was used in the liquid phase; mobile phase A was 0.1% formic acid-10% methanol-water, and mobile phase B was 0.1% formic acid-methanol; the chromatographic column was a C18 column; the mobile phase flow rate was 0.6 mL / min; the column temperature was 30℃; and the elution gradient is shown in Table 2.

[0076] Table 2. Elution gradient

[0077] 0.0 90 10 0.4 90 10 1.3 5 95 1.4 90 10 2.0 90 10

[0078] Mass spectrometry was performed using an electrospray ionization (ESI) source and multiple reaction monitoring (MRM) mode. The mass-to-charge ratio (m / z) of the parent ion / daughter ion pairs for the three antiepileptic drugs used in the detection is shown in Table 3.

[0079] Table 3. Mass-to-charge ratio of parent ion / daughter ion

[0080] valproic acid 142.9 142.9 Carbamazepine 237 179 Phenytoin 253 104

[0081] The detection of three antiepileptic drugs can be determined by selecting the ion pairs detected by reaction monitoring and their corresponding retention times, and quantification can be performed using internal standards for each antiepileptic drug.

[0082] After separation by liquid chromatography, different antiepileptic drugs and matrix components were eluted at different times and detected by mass spectrometry in multiple reaction monitoring mode, thereby determining their content. The mass spectrum of calibrator 10 is shown below. Figures 1-3 As shown, where Figure 1 This is the mass spectrum of valproic acid. Figure 2 This is the mass spectrum of phenytoin. Figure 3 This is the mass spectrum of carbamazepine. As can be seen, the method provided in this embodiment can accurately detect three antiepileptic drugs simultaneously. All three drugs elute at a retention time of approximately 1.04 minutes, achieving specific detection without interference through three different ion channels.

[0083] Example 2: Effect of different additives on the stability of calibrators / quality control samples

[0084] This embodiment follows the method provided in Example 1, and uses different types and concentrations of additives to prepare the matrix. The matrix is ​​then placed at 37°C for 14 days to investigate the effect of different additives on the stability of the calibrators / quality control samples. The additives and their concentrations in the matrix are: 0.05 mg / mL sodium ethylenediamine di-o-phenylacetate, 0.1 mg / mL disodium ethylenediaminetetraacetate, 0.05 mg / mL sodium gluconate, 0.01 mg / mL sodium triacetate, and 0.02 mg / mL sodium citrate. The changes in valproic acid content in calibrator 1 are shown in Table 4.

[0085] Table 4. Effects of different additives on the stability of valproic acid in the matrix

[0086] No additives -15.2％ 0.05 mg / mL sodium ethylenediamine di-o-phenylacetate 0.5％ 0.1 mg / mL disodium ethylenediaminetetraacetate -3.2％ 0.05 mg / mL sodium gluconate -4.7％ 0.01 mg / mL sodium triacetate -11.3％ 0.02 mg / mL sodium citrate -7.9％

[0087] Valproic acid has poor stability in oxidizing and alkaline environments. The selection of stabilizers should take into account their antioxidant capacity and acidity / alkalinity, and select an appropriate concentration to achieve a better stabilizing effect.

[0088] As shown in Table 4, without additives, the content of valproic acid in calibrator 1 decreased by 15.2% after being placed at 37°C for 14 days; however, after adding a certain amount of additives, the proportion of valproic acid content decreases significantly, and the stability is significantly improved.

[0089] However, the effects of adding different additives on improving the stability of calibrators / quality control products vary significantly. Although sodium citrate and sodium triacetate also have certain antioxidant capabilities, their pH values ​​in solution are both greater than 7, indicating alkalinity. As a result, their antioxidant effects are weakened, and therefore their effect on improving the stability of valproic acid in calibrators / quality control products is also slightly worse.

[0090] Adding 0.1 mg / mL disodium ethylenediaminetetraacetate (EDTA) can stabilize the valproic acid content by about ±3%, which is quite good. Sodium gluconate has similar acidity and alkalinity to sodium ethylenediamine di-o-phenylacetate (EDTA), but its antioxidant capacity is weaker. Therefore, with the help of 0.05 mg / mL sodium gluconate, the valproic acid content decreased slightly by 4.7%, and the degradation rate is still within the acceptable range of 10%.

[0091] Compared to the additives mentioned above, the addition of 0.05 mg / mL sodium ethylenediamine di-o-phenylacetate showed a particularly outstanding effect. After being placed at 37°C for 14 days, valproic acid decreased by only 0.5%, demonstrating the best stabilization effect. Therefore, sodium ethylenediamine di-o-phenylacetate is the preferred additive for improving the stability of antiepileptic drug calibrators / quality control products.

[0092] Example 3: Interference of bovine serum and bovine serum albumin on valproic acid detection

[0093] This embodiment follows the method provided in Example 1, using bovine serum, porcine serum, horse serum, rabbit serum, porcine serum albumin, horse serum albumin, and bovine serum albumin as blank matrices, respectively. No antiepileptic drugs were added to the blank matrices. Mass spectrometry detection of the valproate ion channel was then performed, and the detection spectra are shown below. Figures 4-10 ,in Figure 4 This is a mass spectrum of bovine serum albumin as the matrix. Figure 5 The mass spectrum is for bovine serum as the matrix. Figure 6 This is a mass spectrum of porcine serum albumin as the matrix. Figure 7 The mass spectrum is for porcine serum as the matrix. Figure 8 This is a mass spectrum of equine serum albumin as a matrix. Figure 9 The mass spectrum is for horse serum as the matrix. Figure 10 The mass spectrum is for rabbit serum as the matrix.

[0094] Maps of valproate ion channels under different matrices ( Figures 4-10 As can be seen, in the case of a blank matrix, bovine serum and bovine serum albumin contain impurities that interfere with the detection of valproic acid, while porcine serum, porcine serum albumin, horse serum, horse serum albumin, and rabbit serum do not contain this interfering substance and will not interfere with valproic acid in the standard / quality control products, and can be used as a reagent kit matrix; moreover, rabbit serum has the lowest background noise and the least interference with valproic acid.

[0095] In this embodiment, standard 1 was prepared using bovine serum, porcine serum, horse serum, rabbit serum, porcine serum albumin, horse serum albumin, bovine serum albumin, and human serum as blank matrices. The method provided in Example 1 was used to detect the three antiepileptic drugs, and the effect of using different matrices to prepare the standard on the detection accuracy of the three antiepileptic drugs was investigated. The results are shown in Table 5.

[0096] Table 5. Effects of samples prepared with different matrices on the detection accuracy of three antiepileptic drugs.

[0097]

[0098]

[0099] The content of valproic acid in standard 1 was 4 μg / mL, the content of carbamazepine was 0.2 μg / mL, and the content of phenytoin was 0.5 μg / mL.

[0100] As shown in Table 5, when using standard 1 prepared with different serums to detect the three antiepileptic drugs, the changes in carbamazepine and phenytoin content were not significantly different, but the results of valproic acid detection were different. This shows that using different serums as the matrix to prepare samples can have a significant impact on the accuracy of valproic acid detection in the samples.

[0101] When bovine serum or bovine serum albumin (BSAP) was used as the matrix, the detection results for valproic acid were significantly elevated, indicating that the bovine serum or BSAP matrix significantly interfered with the detection of valproic acid. When horse serum, horse SSAP, porcine serum, or porcine SSAP were used as the matrix, the detection results for valproic acid also showed some error. This may be because these sera also exhibited a matrix effect, affecting the accurate detection of valproic acid. However, when rabbit serum was used as the matrix to prepare standard 1, valproic acid was detected more accurately, showing the best agreement with the detection results using human serum as the matrix, greatly ensuring the precision of valproic acid detection.

[0102] Example 4: Effect of different matrices on the stability of valproic acid

[0103] This embodiment follows the method provided in Example 1 to prepare and test the antiepileptic drug detection kit. The standards / controls are prepared using animal serum (excluding bovine serum) or animal serum proteins, with the addition of 0.05 mg / mL sodium ethylenediamine di-o-phenylacetate. The kits are then incubated at 37°C for 14 days. Table 6 shows the stability of valproic acid in different matrices when preparing standard 1 using different matrices, with the addition of 0.05 mg / mL sodium ethylenediamine di-o-phenylacetate.

[0104] Table 6. Effects of different matrices on the stability of valproic acid

[0105] porcine serum -1.0％ Horse serum -1.2％ rabbit serum -0.5％ porcine serum albumin -2.5％ Horse serum albumin -2.6％

[0106] As shown in Table 6, the stability of valproic acid varies significantly when different animal sera or serum proteins are used to prepare standard 1. Using rabbit serum as a matrix helps to further improve the stability of valproic acid. Even with the additive 0.05 mg / mL sodium ethylenediamine di-o-phenylacetate, the valproic acid content remained almost unchanged after 14 days at 37°C. However, using other animal sera still resulted in some degree of valproic acid loss. Therefore, rabbit serum is the optimal choice for preparing standards or quality control products, as it significantly improves the stability of valproic acid.

[0107] Example 5: Effect of different internal standard solutions on the stability of valproic acid internal standard

[0108] To improve the stability and extend the shelf life of the internal standard solution, eliminating the need for fresh preparation each time, this embodiment follows the method provided in Example 1, using 1) sodium metabisulfite with 0.05 mg / mL added, 2) sodium ethylenediamine di-o-phenylacetate with 0.05 mg / mL added, and 3) without adding sodium metabisulfite and sodium ethylenediamine di-o-phenylacetate as precipitants to prepare the internal standard solution. The solutions were then placed at 37°C for 7, 20, and 40 days to investigate the effects of sodium metabisulfite and sodium ethylenediamine di-o-phenylacetate on the shelf life of valproic acid internal standard (valproic acid-d6) in the internal standard solution.

[0109] Table 7. Effects of different internal standard solutions on the stability of valproic acid internal standard

[0110]

[0111] The results are shown in Table 7. It can be seen that without the addition of sodium metabisulfite or sodium ethylenediamine di-o-phenylacetate, the content of valproic acid as the internal standard decreased significantly with prolonged storage time. However, in the precipitant containing sodium metabisulfite or sodium ethylenediamine di-o-phenylacetate, the content of valproic acid as the internal standard solution remained within 15% after 40 days at 37°C, demonstrating good stability. Especially with the addition of sodium ethylenediamine di-o-phenylacetate, the content of valproic acid as the internal standard solution remained only about 1% after 40 days at 37°C, exhibiting excellent stability.

[0112] This embodiment further verified through experiments that, at 2-8℃, after 3 years of storage, the content of valproic acid in the internal standard solution without stabilizer decreased by more than 35%; in the internal standard solution with added sodium metabisulfite, the content of valproic acid changed by about 20%; while the content of the internal standard solution with added ethylenediamine di-o-phenylacetic acid remained stable, with a change of only about 3%. It is evident that adding ethylenediamine di-o-phenylacetic acid to the internal standard solution can significantly improve its stability and extend its shelf life, allowing it to be stored for 3 years and used immediately within those 3 years, greatly reducing labor and material costs.

[0113] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A detection kit for detecting antiepileptic drugs in serum using high performance liquid chromatography-tandem mass spectrometry, characterized in that, It contains a standard and / or a quality control product, wherein the standard and / or quality control product is rabbit serum and contains sodium ethylenediamine di-o-phenylacetate; it also includes an internal standard solution containing a precipitant and a stabilizer, wherein the stabilizer is sodium ethylenediamine di-o-phenylacetate; and the antiepileptic drug is valproic acid.

2. The detection kit as described in claim 1, characterized in that, The concentration of the sodium ethylenediamine di-o-phenylacetate is 0.02-0.1 mg / mL.

3. The detection kit as described in claim 2, characterized in that, The precipitant is methanol; the internal standard solution also contains isotopically labeled antiepileptic drugs.

4. The detection kit as described in claim 3, characterized in that, The standard is an antiepileptic drug containing a standard concentration; the quality control is an antiepileptic drug containing two different concentrations, high and low.

5. A method for detecting antiepileptic drugs in serum, characterized in that, The method is for non-disease diagnostic purposes and uses the detection kit as described in any one of claims 1 to 4 for detection; it includes system suitability testing, sample preparation, sample pretreatment and sample detection; wherein, the sample pretreatment includes the following steps: take the sample and internal standard solution, mix them at a ratio of 1:7 to 1:11, centrifuge, take the supernatant for high performance liquid chromatography-tandem mass spectrometry detection, and the antiepileptic drug is valproic acid.

6. The use of rabbit serum in the preparation of standards and / or quality control products to improve the stability of valproic acid, characterized in that, The standard and / or quality control products contain an additive, namely sodium ethylenediamine di-o-phenylacetate.

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