CN115774067A - Detection kit for detecting antiepileptic drug in serum by high performance liquid chromatography tandem mass spectrometry and detection method thereof - Google Patents
Detection kit for detecting antiepileptic drug in serum by high performance liquid chromatography tandem mass spectrometry and detection method thereof Download PDFInfo
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Abstract
The invention provides a detection kit for detecting an antiepileptic drug in serum by high performance liquid chromatography tandem mass spectrometry and a detection method thereof, wherein pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin is used as matrixes of a calibrator and a quality control material, so that the interference of bovine serum or bovine serum albumin on the detection of valproic acid is avoided; the matrix is added with ethylenediamine-di-o-phenyl sodium acetate and the like as antioxidants, and rabbit serum is preferably adopted as the matrix, so that the stability of the calibrator and the quality control product is obviously improved; meanwhile, the internal standard is stored in the precipitator containing the ethylenediamine-di-o-phenyl sodium acetate, so that the internal standard correction, the protein precipitation and the target substance extraction can be realized by one-step operation when the internal standard is used for sample pretreatment, and the internal standard solution can be stored for a long time and can be taken at any time, thereby achieving the effect of prolonging the effective period of the kit product.
Description
Technical Field
The invention belongs to the technical field of biochemical analysis, and particularly relates to a detection kit for detecting an antiepileptic drug in serum by high performance liquid chromatography tandem mass spectrometry and a detection method thereof.
Background
In the treatment of epileptic seizures, antiepileptic drugs are of particular importance. Antiepileptic drugs can eliminate or alleviate seizures by two means, one is to affect central neurons to prevent or reduce their pathological overdischarge; secondly, the excitation threshold of normal brain tissue is improved, the spread of focus excitation is weakened, and the recurrence of epilepsy is prevented. The antiepileptic drug has influence on human memory, movement speed and the like, and the higher the blood concentration is, the more obvious the influence is. Therefore, the monitoring of the blood concentration of the antiepileptic drug is significant.
The antiepileptic drug can be used alone or in combination to enhance the therapeutic effect or relieve the toxic and side effects of the drug. Carbamazepine is a derivative of imino 1, 2-stilbene which is commonly used alone or in combination with other antiepileptic drugs for the treatment of psychomotor seizures (grand mal seizures), regional-mixed grand seizures. It can also be used for treating trigeminal neuralgia. Carbamazepine is also sometimes used to treat manic depression patients who are refractory to lithium therapy. There is a great difference in the individual response to carbamazepine due to the difference between absorption and metabolism.
Valproic acid (VPA: 2-valproic acid; sodium valproate) is a relatively new anticonvulsant drug, is mainly used for primary and secondary generalized epileptic seizures, is also effective in treating non-seizures, has obvious treatment effect on myoclonus, and is a treatment drug for chronic photosensitive epilepsy. VPA is often used in combination with other antiepileptic drugs, but recent studies have shown that VPA alone is also effective. Additional studies suggest that VPA is useful for treatment of affective disorders, especially in bipolar disorder patients who are not susceptible to lithium treatment. VPA is converted to metabolic complexes by β, ω oxidation and binding, some metabolites have significant anticonvulsant activity, while others may be associated with the development of drug toxic side effects. In addition, other antiepileptic drugs used simultaneously have a significant effect on VPA metabolism.
Phenytoin is generally selected for the treatment of generalized hyperkinetic seizures (grand mal seizures) and focal seizures. It can also be used for basic therapy of partial seizures (focal) or complex partial seizures (psychomotor, temporal lobe). Phenytoin has high binding force with plasma proteins, and unbound phenytoin has high pharmacological activity in a free form. Unbound phenytoin accounted for about 10% in individuals with normal albumin levels and normal renal function. The distinctive metabolic elimination characteristics of phenytoin make evaluation of the dose/concentration relationship relatively difficult. Significant differences in the absorption, metabolism and elimination of phenytoin have been reported between individuals. The relationship between drug dosage and pharmacological activity can be further complicated by disease states, drug management, and patient complications.
It can be seen that valproic acid, carbamazepine and phenytoin are all very frequently used, and the combined antiepileptic drugs can help doctors to determine the treatment dosage according to the individual difference of the patient to the drug response by monitoring the blood concentration of valproic acid, carbamazepine and phenytoin at the same time and adjusting the drug dosage to reduce the toxic and side effects, thereby controlling epileptic seizure and ensuring that the patient can achieve better compliance.
At present, a plurality of methods for measuring antiepileptic drugs in serum are reported at home and abroad, and the common methods comprise: chemiluminescence and chromatography, among the many methods, liquid tandem mass spectrometry is the preferred method for the determination of antiepileptic drugs. However, the problems of complex operation process, multiple interference factors, poor specificity, long analysis time and low detection flux generally exist in the conventional method, which are mainly caused by the complex sample pretreatment process and poor stability.
The existing antiepileptic drug sample pretreatment method mainly comprises protein precipitation, liquid-liquid extraction and the like, for example, CN111812216A, and adopts a mixed solution of methanol and acetonitrile as a protein precipitator to carry out pretreatment on a serum sample so as to detect the antiepileptic drug of the sample.
Therefore, an antiepileptic drug sample pretreatment method which has the advantages of simple operation, high treatment efficiency, high detection flux, high detection accuracy and sensitivity, low cost, small manual workload and the like is urgently needed, and the defects of the existing method can be overcome.
Disclosure of Invention
In order to solve the problems, the invention provides a detection kit for detecting an antiepileptic drug in serum by high performance liquid chromatography-tandem mass spectrometry and a detection method thereof, wherein pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin is used as a matrix of a calibrator and a quality control material, so that the interference of bovine serum or bovine serum albumin on the detection of valproic acid is avoided; the matrix is added with ethylenediamine-di-o-phenyl sodium acetate and the like as antioxidants, and rabbit serum is preferably adopted as the matrix, so that the stability of the calibrator and the quality control product is obviously improved; meanwhile, the internal standard is stored in the precipitator containing ethylenediamine-di-o-phenyl sodium acetate, so that the internal standard can be corrected, protein can be precipitated and a target substance can be extracted by one-step operation when the internal standard is used for sample pretreatment, and the internal standard solution can be stored for a long time and can be taken at any time, so that the effect of prolonging the effective period of the kit product is achieved.
On one hand, the invention provides a detection kit for detecting an antiepileptic drug in serum by high performance liquid chromatography tandem mass spectrometry, which comprises a standard substance and/or a quality control substance, wherein the standard substance and/or the quality control substance contains an additive, and the additive is any one or more of ethylenediamine diphenyl sodium acetate, ethylene diamine tetraacetic acid disodium, sodium gluconate and sodium citrate.
The antiepileptic drug provided by the invention comprises valproic acid, carbamazepine and phenytoin.
The inventor proves through a large number of experiments that the stability of the antiepileptic drug in the solution matrix, especially the valproic acid in the antiepileptic drug, is poor in oxidation and alkaline environments, for example, the content of the valproic acid in the detection kit is very unstable, and the problem of serious content reduction can occur after the detection kit is placed for several days at normal temperature, so that the detection accuracy and sensitivity can be obviously influenced, all samples, standards, internal standards, quality control products and the like for detecting the antiepileptic drug need to be prepared at present, great troubles are brought to actual operation, and the detection cost is high. Therefore, in order to more accurately detect the content of 3 antiepileptic drugs including valproic acid, carbamazepine and phenytoin in serum, the problem of stability of valproic acid in a solution matrix must be overcome first.
The screening of the valproic acid stabilizer needs to comprehensively consider the antioxidant capacity and the acid-base property of the valproic acid stabilizer, and a proper concentration is selected to achieve a better stabilizing effect. That is, in the standard or quality control product containing valproic acid, carbamazepine and phenytoin, an additive must be added so that the valproic acid is not oxidized while maintaining the most suitable acid-base property and concentration so that the content of valproic acid is kept as stable as possible.
The inventor surprisingly finds that the stability of the antiepileptic drug, especially the stability of valproic acid, can be obviously improved by adding ethylenediamine-dipolyphenyl sodium acetate (EDDHA), disodium ethylenediamine tetraacetate, sodium gluconate or sodium citrate into the standard substance or the quality control substance.
Further, the additive is sodium ethylenediamine-di-o-phenyl acetate.
Research proves that when the additive is ethylenediamine diphenyl sodium acetate (EDDHA), the effect of improving the stability is best.
Further, the standard substance and/or the quality control substance are prepared by adopting a matrix, the matrix is animal serum or animal serum albumin, the animal serum is not bovine serum, and the animal serum albumin is not bovine serum albumin.
Since human serum is not readily available and cannot be used as a substrate for the preparation of mass-produced kit products, there is a need to find suitable animal sera as a substrate.
The inventor finds that the detection result of valproic acid is very unstable due to obvious interference of bovine serum or bovine serum albumin on the detection of valproic acid; therefore, the matrices for the standard and quality control cannot be formulated with bovine serum or bovine serum albumin.
Further, the matrix is any one or more of pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin, and the concentration of the ethylenediamine diphthalic sodium acetate is 0.02-0.1mg/mL.
Pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin are used as a matrix, and additives in the matrix comprise one or more of ethylenediamine-di-o-phenyl sodium acetate, ethylene diamine tetraacetic acid disodium, sodium gluconate and sodium citrate, most preferably 0.02-0.1mg/mL of ethylenediamine-di-o-phenyl sodium acetate, so that the effects of improving the stability of a calibrator and a quality control product and prolonging the effective period of a product are achieved. By simulating a human body sample with appropriate and readily available animal serum, the matrix effect can be reduced, so that the detection result is more accurate and stable.
Further, the matrix is rabbit serum.
The research proves that the rabbit serum is adopted as the matrix, so that the stability of the valproic acid in the standard substance or the quality control substance is further improved, the matrix effect is low, the background noise is low, and the accurate detection of the valproic acid can be ensured.
Further, the detection kit further comprises an internal standard solution, wherein the internal standard solution contains a precipitator and a stabilizer, and the stabilizer is ethylenediamine diphenyl sodium acetate.
Further, the precipitant is methanol.
The valproic acid in the conventional internal standard liquid of the antiepileptic drug is unstable and must be prepared at each time, and in order to reduce workload and waste, the stabilizer is added into the internal standard liquid to keep the content of the valproic acid stable, so that the storage life of the internal standard liquid is prolonged.
The internal standard solution containing the ethylenediamine-di-o-phenyl sodium acetate prepared by the invention is very stable, can be stored for a long time and taken at any time, the shelf life reaches more than three years, and after three years, the content change of the valproic acid internal standard is still kept within 3%, so that the detection process is simpler, more convenient and more efficient, the detection cost is lower, and the detection result is more accurate and stable.
The method mixes the methanol, the ethylenediamine di-o-phenyl sodium acetate and the isotope labeled antiepileptic drug to obtain the internal standard solution, adds the internal standard solution into the sample to be mixed uniformly, can play a role of the internal standard, and can synchronously realize the internal standard correction, the protein precipitation separation and the target substance extraction in the sample.
Further, the internal standard contains an isotope-labeled antiepileptic drug; the standard substance is an antiepileptic drug with standard concentration; the quality control product is an antiepileptic drug containing two different levels of concentration.
Further, the antiepileptic drug is any one or more of valproic acid, carbamazepine or phenytoin.
In another aspect, the present invention provides a method for detecting antiepileptic drugs in serum, wherein the method employs the detection kit as described above for detection; the method comprises the steps of system applicability test, sample preparation, sample pretreatment and sample detection; wherein, the sample pretreatment comprises the following steps: and uniformly mixing the sample and the internal standard solution according to the ratio of 1: 7-1: 11, centrifuging, and taking the supernatant to perform high performance liquid chromatography tandem mass spectrometry.
In a further aspect, the present invention provides the use of sodium ethylenediamine diphthalate for the preparation of a stabilizer for a valproic acid solution.
In another aspect, the invention provides the use of ethylenediamine diphthalic sodium acetate as a stabilizer for the preparation of a solution of an antiepileptic drug which is any one or more of valproic acid, carbamazepine or phenytoin.
In still another aspect, the invention provides the use of rabbit serum for preparing a standard substance and/or a quality control substance for improving the stability of valproic acid.
Further, the standard substance and/or the quality control substance contain sodium ethylenediamine-di-o-phenyl acetate.
The invention has the following beneficial effects:
(1) The ethylenediamine-di-o-phenyl sodium acetate is added into the matrix of the standard substance and/or the quality control substance, so that the problem that the content of valproic acid in the antiepileptic drug is unstable and seriously reduced after being placed for several days at normal temperature can be effectively solved; the standard substance and/or the quality control substance can be stored for a long time, the content of the antiepileptic drug in the standard substance and/or the quality control substance is stable, the existing preparation is not needed, the effects of improving the stability of the calibrator and the quality control substance and prolonging the validity period of the product are achieved, and the accuracy and the sensitivity of detection can be obviously improved;
(2) The bovine serum or the bovine serum albumin is found to have obvious interference on the detection of the valproic acid, and the bovine serum or the bovine serum albumin can not be adopted to prepare a matrix; pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin can be selected as the matrixes of the calibrator and the quality control material, so that the matrix effect is reduced, and the detection result is more accurate and stable;
(3) The rabbit serum is selected as a matrix of a standard substance and/or a quality control substance, and the stability of valproic acid in the matrix can be further improved under the condition of adding ethylenediamine-diphenylphthalic sodium acetate, and the accuracy of valproic acid detection can be better ensured;
(4) The ethylenediamine di-o-phenyl sodium acetate is added into the internal standard solution, and internal standard correction, protein precipitation and target substance extraction are simultaneously realized through one-step operation, so that the accuracy, the sensitivity and the simplicity of operation of detection are improved, the content of the antiepileptic drug in the internal standard is stable, the internal standard can be stored for a long time, the quality guarantee period is more than three years, and the internal standard solution can be taken at any time;
(5) When a patient takes one or more than one medicine, different blood concentration can be detected by adopting a one-step sample pretreatment method, the repeated sampling times are reduced, the pain of the patient is reduced, the analysis cost is effectively reduced, the report range is wide, and the serum samples with different blood concentration levels can be accurately analyzed.
Drawings
FIG. 1 is a detected mass spectrum of valproic acid of standard 10 in example 1;
FIG. 2 is a spectrum of phenytoin detection mass spectrum of the standard 10 in example 1;
FIG. 3 is a carbamazepine detection mass spectrum of standard 10 of example 1;
FIG. 4 is the mass spectrum of bovine serum albumin as the matrix in valproic acid ion channel in example 3;
FIG. 5 is the mass spectrum of the bovine serum as the matrix in the valproic acid ion channel in example 3;
FIG. 6 is the mass spectrum of the detection of pig serum albumin as the matrix in valproic acid ion channel in example 3;
FIG. 7 is the mass spectrum of the detection of the porcine serum as the substrate in the valproic acid ion channel in example 3;
FIG. 8 is the mass spectrum of horse serum albumin as the matrix in valproic acid ion channel in example 3;
FIG. 9 is the mass spectrum of horse serum as the substrate in valproic acid ion channel in example 3;
FIG. 10 is the mass spectrum of rabbit serum as matrix in valproic acid ion channel in example 3.
Detailed Description
The invention will be described in further detail below with reference to the drawings and examples, which are intended to facilitate the understanding of the invention and are not intended to limit it in any way.
Example 1 sample preparation, pretreatment and testing
1. Sample preparation
1. Preparation of calibrator and quality control material
The antiepileptic drug valproic acid, carbamazepine and phenytoin standard substance are prepared into mixed solution for preparing standard substance working solution and quality control substance working solution. The working solution was added to rabbit serum containing 0.05mg/mL of ethylenediamine-diphenylphthalic sodium acetate to prepare 10 calibrator and 2 quality control samples of the respective concentrations shown in Table 1 (unit: μ g/mL):
table 1, 3 concentrations of standard substance and quality control substance of antiepileptic drug
| Sample (I) | Valproic acid | Carbamazepine | Phenytoin |
| Calibration article 1 | 4 | 0.2 | 0.5 |
| Calibration article 2 | 5 | 0.5 | 1.25 |
| Calibration article 3 | 10 | 1 | 2.5 |
| Calibration article 4 | 15 | 3 | 7.5 |
| Calibration article 5 | 25 | 5 | 12.5 |
| Calibration article 6 | 45 | 9 | 22.5 |
| Calibration article 7 | 75 | 15 | 37.5 |
| Calibration article 8 | 90 | 18 | 45 |
| Calibration article 9 | 120 | 24 | 60 |
| Calibration article 10 | 150 | 30 | 75 |
| Quality control product 1 | 20 | 2.4 | 4 |
| Quality control product 2 | 100 | 12 | 20 |
2. Preparation of internal standard liquid
(1) Internal standard stock solution
Preparing mixed internal standard stock solution, valproic acid-d 6 and carbamazepine-d 2, 15 the concentrations of N and phenytoin-d 10 were 200. Mu.g/mL, 20. Mu.g/mL and 50. Mu.g/mL, respectively.
(2) Precipitating agent
50mg of ethylenediamine-di-o-phenyl sodium acetate is weighed, dissolved in 100mL of purified water, 900mL of methanol is added, and the mixture is uniformly mixed to obtain the precipitator.
(3) Internal standard liquid
And (3) uniformly mixing the internal standard stock solution and the precipitant according to the proportion of 1.
3. Preparation of System suitability solutions
A system-compatible solution was prepared by mixing 100. Mu.L of a mixed standard solution containing 4. Mu.g/mL valproic acid, 0.2. Mu.g/mL carbamazepine, and 0.5. Mu.g/mL phenytoin with 49.9mL of a 50% aqueous methanol solution.
The system applicability test is that before sample detection, high performance liquid chromatography tandem mass spectrometry is used for detecting directly-injected 3 times of system applicability solution, and whether the high performance liquid chromatography tandem mass spectrometry system is normal or not is judged according to the retention time, response intensity and precision of a target substance.
2. Sample pretreatment
(1) Adding 20 mu L of sample into a 96-well plate or a centrifuge tube;
(2) Add 180. Mu.L of internal standard solution, vortex for 5 minutes, centrifuge at 4000rpm for 10 minutes, and take the supernatant for assay.
3. Sample detection
Taking the supernatant and detecting by high performance liquid chromatography tandem mass spectrometry. The specific analysis conditions were as follows: gradient elution is adopted for a liquid phase, wherein a mobile phase A is 0.1% formic acid-10% methanol water, and a mobile phase B is 0.1% formic acid methanol; the column was a C18 column, the mobile phase flow rate was 0.6mL/min, the column temperature was 30 ℃ and the elution gradient is shown in Table 2:
TABLE 2 elution gradient
| Time (min) | Mobile phase A% | Mobile phase B% |
| 0.0 | 90 | 10 |
| 0.4 | 90 | 10 |
| 1.3 | 5 | 95 |
| 1.4 | 90 | 10 |
| 2.0 | 90 | 10 |
Mass spectrometry uses electrospray ion source (ESI) and multiple reaction monitoring scan mode (MRM). The parent ion/daughter ion to mass-to-charge ratios (m/z) for the three antiepileptic drugs tested are shown in table 3:
TABLE 3 parent/daughter ion to Mass/Charge ratio
| Analyte | Q1 | Q3 |
| Valproic acid | 142.9 | 142.9 |
| Carbamazepine | 237 | 179 |
| Phenytoin | 253 | 104 |
Detection of 3 antiepileptic drugs can be determined by monitoring the detected ion pairs for a selected reaction, and the corresponding retention times, and quantitated by internal standards for each antiepileptic drug.
After the sample is separated by liquid chromatography, different antiepileptic drugs and matrix components are eluted at different times and are detected by a mass spectrum multiple reaction monitoring mode, so that the content of the antiepileptic drugs and the matrix components is detected. The mass spectrum of the calibrator 10 is shown in fig. 1-3, wherein fig. 1 is a mass spectrum of valproic acid, fig. 2 is a mass spectrum of phenytoin, and fig. 3 is a mass spectrum of carbamazepine. Therefore, according to the method provided by the embodiment, 3 antiepileptic drugs can be simultaneously and accurately detected, the 3 antiepileptic drugs all peak at the retention time of about 1.04 minutes, and the non-interfering specific detection is realized through three different ion channels.
Example 2 Effect of different additives on the stability of calibrators/quality controls
This example follows the method provided in example 1, and uses different types and concentrations of additives to formulate a matrix, which is left at 37 ℃ for 14 days to examine the effect of different additives on the stability of the calibrator/quality controller. The additives and their concentrations in the matrix are: 0.05mg/mL ethylenediamine-diphophyl sodium acetate, 0.1mg/mL ethylenediamine tetraacetic acid disodium salt, 0.05mg/mL sodium gluconate, 0.01mg/mL nitrilotriacetic acid sodium salt and 0.02mg/mL sodium citrate. Examination of the change in the valproic acid content of calibrator 1 is shown in Table 4:
TABLE 4 Effect of different additives on the stability of valproic acid in the base
| Additives and concentrations thereof | Change in valproic acid content |
| Without additives | -15.2％ |
| 0.05mg/mL sodium ethylenediamine dipentyl acetate | 0.5％ |
| 0.1mg/mL disodium edetate | -3.2％ |
| 0.05mg/mL sodium gluconate | -4.7％ |
| 0.01mg/mL nitrilotriacetic acid sodium salt | -11.3％ |
| 0.02mg/mL sodium citrate | -7.9％ |
The stability of valproic acid in oxidation and alkaline environments is poor, the screening of the stabilizer needs to comprehensively consider the oxidation resistance and the acidity and alkalinity of the valproic acid, and a proper concentration is selected to achieve a good stabilizing effect.
As can be seen from table 4, in the absence of the additive, the content of valproic acid decreased by 15.2% when calibrator 1 was left at 37 ℃ for 14 days; after a certain amount of additive is added, the proportion of the reduction of the content of the valproic acid can be obviously reduced, and the stability is obviously improved.
However, the effect of adding different additives on improving the stability of the calibrator/quality control product is obviously different, and although sodium citrate and sodium nitrilotriacetate also have certain antioxidant capacity, the pH values of the sodium citrate and the sodium nitrilotriacetate in the solution are both greater than 7 and are alkaline, the antioxidant effect of the sodium citrate and the sodium nitrilotriacetate is weakened, and therefore, the effect of improving the stability of valproic acid in the calibrator/quality control product is also slightly poor.
The content of valproic acid can be changed to be about +/-3% by adding 0.1mg/mL disodium ethylenediamine tetraacetic acid, and the stabilizing effect is good; the acid-base property of the sodium gluconate is similar to that of ethylenediamine-dipheny sodium acetate, but the oxidation resistance of the sodium gluconate is weaker than that of the ethylenediamine-dipheny sodium acetate, so that under the action of 0.05mg/mL of sodium gluconate, the content of valproic acid is reduced by 4.7%, and the degradation rate is still within the acceptable range of 10%.
Compared with the additives, the effect of adding 0.05mg/mL of ethylenediamine-diphenylacetic acid is particularly outstanding, and after the additive is placed at 37 ℃ for 14 days, the valproic acid is reduced by only 0.5%, so that the stabilizing effect is optimal. Therefore, ethylenediamine diphthalic acid sodium acetate is the most preferable additive for improving the stability of the antiepileptic drug calibrator/quality control product.
Example 3 interference of bovine serum and bovine serum albumin on detection of Valproic acid
In this embodiment, according to the method provided in embodiment 1, bovine serum, pig serum, horse serum, rabbit serum, pig serum albumin, horse serum albumin, and bovine serum albumin are used as blank matrices, and no antiepileptic drug is added to the blank matrices, and then mass spectrometry of valproic acid ion channels is performed, respectively, and the detection spectra are shown in fig. 4 to 10, where fig. 4 is a mass spectrum of bovine serum albumin as a matrix, fig. 5 is a mass spectrum of bovine serum as a matrix, fig. 6 is a mass spectrum of pig serum albumin as a matrix, fig. 7 is a mass spectrum of pig serum as a matrix, fig. 8 is a mass spectrum of horse serum albumin as a matrix, fig. 9 is a mass spectrum of horse serum as a matrix, and fig. 10 is a mass spectrum of rabbit serum as a matrix.
From the maps (fig. 4-10) of valproic acid ion channels under different matrixes, under the condition of a blank matrix, bovine serum and bovine serum albumin contain impurities which interfere the detection of valproic acid, and pig serum, pig serum albumin, horse serum albumin and rabbit serum do not contain the interference substances, so that the interference on valproic acid in a standard substance/a quality control substance can be avoided, and the kit matrix can be used; and the rabbit serum had the lowest background noise and the least interference to valproic acid.
In this embodiment, bovine serum, pig serum, horse serum, rabbit serum, pig serum albumin, horse serum albumin, bovine serum albumin, and human serum are respectively used as blank matrices to prepare the standard 1, the method provided in embodiment 1 is used to detect three anti-epileptic drugs, and the influence of the standard prepared by using different matrices on the detection accuracy of the three anti-epileptic drugs is examined, and the results are shown in table 5.
TABLE 5 influence of samples formulated with different matrices on the accuracy of detection of three antiepileptic drugs
| Substrate | Valproic acid content (μ g/mL) | Carbamazepine containingAmount (μ g/mL) | Phenytoin content (μ g/mL) |
| Bovine serum | 4.852 | 0.205 | 0.509 |
| Pig serum | 3.861 | 0.202 | 0.499 |
| Horse serum | 4.203 | 0.203 | 0.503 |
| Rabbit serum | 4.002 | 0.202 | 0.502 |
| Porcine serum albumin | 3.672 | 0.202 | 0.510 |
| Horse serum albumin | 3.885 | 0.198 | 0.504 |
| Bovine serum albumin | 4.963 | 0.203 | 0.507 |
| Human serum | 4.001 | 0.201 | 0.501 |
The content of valproic acid in the standard 1 is 4 mug/mL, the content of carbamazepine is 0.2 mug/mL, and the content of phenytoin is 0.5 mug/mL.
As can be seen from table 5, when the standard 1 prepared from different sera was used to detect three antiepileptic drugs, there was no obvious difference in the content changes of carbamazepine and phenytoin, but the results of valproic acid detection were different, so that it can be seen that when different sera were used as the substrate to prepare samples, the accuracy of valproic acid detection in the samples was greatly affected.
When the bovine serum or the bovine serum albumin is used as the matrix, the detection result of the valproic acid is obviously higher, and the bovine serum or the bovine serum albumin matrix can seriously interfere the detection of the valproic acid. When horse serum, horse serum protein, pig serum and pig serum protein are used as matrixes, certain errors exist in detection results of valproic acid, and the reason may be that certain matrix effects exist in the serum, so that accurate detection of valproic acid is influenced. When the rabbit serum is used as a matrix to prepare the standard substance 1, the valproic acid in the standard substance can be detected more accurately, and the detection result is most consistent with the detection result of human serum used as the matrix, so that the detection precision of the valproic acid is greatly ensured.
Example 4 Effect of different substrates on the stability of valproic acid
In this embodiment, the preparation and detection of the detection kit for antiepileptic drugs are performed according to the method provided in example 1, wherein the standard substance/quality control substance is prepared from serum or serum protein of an animal other than bovine serum, 0.05mg/mL ethylenediamine sodium diphosphate acetate is added, the standard substance/quality control substance is left at 37 ℃ for 14 days, and in the case of preparing the standard substance 1 with 0.05mg/mL ethylenediamine sodium diphosphate, the stability of valproic acid in different matrices is as shown in table 6:
TABLE 6 Effect of different substrates on the stability of valproic acid
| Substrate | Change in Valproic acid content |
| Pig serum | -1.0％ |
| Horse serum | -1.2％ |
| Rabbit serum | -0.5％ |
| Porcine serum albumin | -2.5％ |
| Horse serum albumin | -2.6％ |
As can be seen from Table 6, when different animal sera or animal serum proteins are adopted to prepare the standard substance 1, the stability of valproic acid is greatly different, when rabbit sera is adopted as a substrate, the stability of valproic acid is further improved, and when the rabbit sera contains 0.05mg/mL ethylenediamine-diphenyphenyl-sodium acetate, the valproic acid content is almost unchanged after the rabbit sera is placed for 14 days at 37 ℃; with other animal sera, more or less valproic acid loss still occurs. Therefore, rabbit serum is most preferably used for preparing a standard substance or a quality control substance, so that the stability of valproic acid can be remarkably improved.
Example 5 Effect of different internal standards on the stability of valproic acid internal standards
In order to improve the stability of the internal standard solution and prolong the storage life of the internal standard solution without being prepared each time, in this example, according to the method provided in example 1, 0.05mg/mL of sodium metabisulfite, 2, 0.05mg/mL of ethylenediamine-dipheny-l sodium acetate, and 3, the internal standard solution is prepared by using the precipitant without sodium metabisulfite and ethylenediamine-dipheny-l sodium acetate, and is placed at 37 ℃ for 7, 20 and 40 days, and the influence of the sodium metabisulfite and the ethylenediamine-dipheny-l sodium acetate on the quality guarantee period of the valproic acid internal standard (valproic acid-d 6) in the internal standard solution is examined.
TABLE 7 influence of different internal standard solutions on the stability of valproic acid internal standard
The results are shown in table 7, and it can be seen that the valproic acid internal standard content is significantly reduced with the increase of the standing time without adding sodium metabisulfite or sodium ethylenediamine dipentyl acetate; in the precipitator added with sodium metabisulfite or ethylenediamine-di-o-phenyl sodium acetate, the internal standard solution is placed for 40 days at 37 ℃, the content of the valproic acid internal standard is changed within 15 percent, and the stability is better. Particularly, when ethylenediamine-di-o-phenyl-sodium acetate was added, the internal standard solution was left at 37 ℃ for 40 days, and the change in the content of the valproic acid internal standard was only about 1%, resulting in very excellent stability.
In the embodiment, experiments further prove that the valproic acid internal standard content is reduced by more than 35% in the internal standard solution without the stabilizer after the internal standard solution is placed for 3 years at the temperature of 2-8 ℃; in the internal standard solution added with sodium metabisulfite, the content of the valproic acid internal standard is changed by about 20 percent; the content of the internal standard solution added with the ethylenediamine diphthalic sodium acetate still keeps stable, and the content change of the valproic acid internal standard is only about 3 percent. Therefore, the addition of the ethylenediamine-di-o-phenyl sodium acetate into the internal standard solution can obviously improve the stability of the internal standard solution, prolong the storage life, realize the storage for 3 years, realize the random use within 3 years and greatly reduce the labor and material costs.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected by one skilled in the art without departing from the spirit and scope of the invention, as defined in the appended claims.
Claims (10)
1. A detection kit for detecting an antiepileptic drug in serum by high performance liquid chromatography-tandem mass spectrometry is characterized by comprising a standard substance and/or a quality control substance, wherein the standard substance and/or the quality control substance contains an additive, and the additive is any one or more of ethylenediamine-di-o-phenyl sodium acetate, ethylene diamine tetraacetic acid disodium, sodium gluconate and sodium citrate.
2. The test kit of claim 1, wherein the additive is sodium ethylenediamine diphthalate.
3. The test kit of claim 2, wherein the standard substance and/or the quality control substance is prepared by using a matrix, the matrix is animal serum or animal serum albumin, the animal serum is not bovine serum, and the animal serum albumin is not bovine serum albumin.
4. The detection kit according to claim 3, wherein the matrix is any one or more of pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin, and the concentration of the ethylenediamine diphthalic acid sodium acetate is 0.02-0.1mg/mL.
5. The test kit of claim 4, wherein the matrix is rabbit serum; the detection kit further comprises an internal standard solution, wherein the internal standard solution contains a precipitator and a stabilizer, and the stabilizer is ethylenediamine-di-o-phenyl sodium acetate.
6. The test kit of claim 5, wherein the precipitating agent is methanol; the internal standard solution also contains an antiepileptic drug marked by an isotope.
7. The test kit of claim 6, wherein the standard substance is a drug containing a standard concentration of an antiepileptic drug; the quality control product contains antiepileptic drugs with two different levels of concentration; the antiepileptic drug is any one or more of valproic acid, carbamazepine or phenytoin.
8. A method for detecting antiepileptic drugs in serum, which comprises carrying out detection using the detection kit according to any one of claims 1 to 7; the method comprises the steps of system applicability test, sample preparation, sample pretreatment and sample detection; wherein, the sample pretreatment comprises the following steps: and uniformly mixing the sample and the internal standard solution according to the ratio of 1: 7-1: 11, centrifuging, and taking the supernatant to perform high performance liquid chromatography tandem mass spectrometry.
9. Use of sodium ethylenediamine-di-o-phenylacetate as a stabilizer for the preparation of a valproic acid solution.
10. The application of rabbit serum in preparing standard substance and/or quality control substance for improving the stability of valproic acid.
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| CN117129605B (en) * | 2023-10-25 | 2024-02-02 | 济南和合医学检验有限公司 | Method for detecting 11 antihypertensive drugs and 3 metabolites by liquid chromatography-tandem mass spectrometry |
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