CN115774067B - Detection kit for detecting antiepileptic drugs in serum by high performance liquid chromatography-tandem mass spectrometry and detection method thereof - Google Patents
Detection kit for detecting antiepileptic drugs 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 antiepileptic drugs in serum by high performance liquid chromatography tandem mass spectrometry and a detection method thereof, which adopt pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin as matrixes of calibrator and quality control product, so that interference of bovine serum or bovine serum albumin on valproic acid detection is avoided; the substrate is added with ethylenediamine di-o-sodium phenylacetate and the like as antioxidants, and rabbit serum is preferably adopted as the substrate, 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-sodium phenylacetate, so that the internal standard correction, protein precipitation and target substance extraction can be realized by one-step operation when the internal standard is used for sample pretreatment, and the internal standard liquid can be stored for a long time and can be taken and used at any time, thereby achieving the effect of prolonging the effective period of a kit product.
Description
Technical Field
The invention belongs to the technical field of biochemical analysis, and particularly relates to a detection kit for detecting antiepileptic drugs in serum by high performance liquid chromatography-tandem mass spectrometry and a detection method thereof.
Background
In the treatment of seizures, anti-epileptic drugs are of particular importance. Antiepileptic drugs can eliminate or alleviate epileptic seizures in two ways, one is to affect central neurons to prevent or reduce their pathological overdischarge; and secondly, the excitation threshold of normal brain tissues is improved, the diffusion of focus excitation is weakened, and the recurrence of epilepsy is prevented. The antiepileptic drugs have influence on memory, movement speed and the like of people, and the higher the blood concentration is, the more obvious the influence is. Therefore, the blood concentration monitoring of the antiepileptic drug is significant.
The antiepileptic drugs can be used singly or in combination to enhance the therapeutic effect of the drugs or reduce the toxic and side effects of the drugs. Carbamazepine is a derivative of imino 1, 2-stilbenes commonly used in the treatment of psychomotor seizures (epileptic seizure), localized-mixed seizures, alone or in combination with other antiepileptic drugs. It can be used for treating trigeminal neuralgia. Carbamazepine is also sometimes used to treat manic-depressive patients who are not refractory to lithium therapy. There is a great difference in individual responses to carbamazepine due to the difference between absorption and metabolism.
Valproic acid (VPA: 2-propyl valeric acid; sodium propyl valerate) is a relatively new anticonvulsant drug, is mainly used for primary and secondary systemic epileptic seizures, is also effective for treating seizure without seizures, has obvious treatment effect on myoclonus, and is a treatment drug for chronic photosensitive epileptic. VPA is often used in combination with other antiepileptic drugs, but recent studies have shown that VPA alone has good efficacy. Additional studies suggest that VPA is useful for treatment of affective disorders, especially in bipolar disorder patients that are insensitive to lithium therapy. VPA is converted to metabolic complexes by beta, omega oxidation and binding, some of which have significant anticonvulsant activity, while others may be associated with the development of toxic side effects of the drug. In addition, other antiepileptic drugs used simultaneously can have a significant effect on VPA metabolism.
Phenytoin is generally selected for the treatment of generalized macromotor seizures (epileptic large seizures) and focal epileptic seizures. It can also be used for the basic treatment of local seizures (focal) or complex local seizures (psychomotor, temporal lobe). The phenytoin has high binding force with plasma protein, and unbound phenytoin has higher pharmacological activity in free form. Unbound phenytoin is about 10% in individuals with normal albumin levels and normal renal function. The unusual metabolic elimination properties of phenytoin make evaluation of the dose/concentration relationship relatively difficult. Significant differences in phenytoin absorption, metabolism and elimination are reported between individuals. The relationship between the dosage of drug administration and pharmacological activity is further complicated by the effects of disease states, drug management, and patient complications.
It can be seen that valproic acid, carbamazepine and phenytoin are very commonly used, and the combined antiepileptic drugs are needed to monitor the blood concentration of valproic acid, carbamazepine and phenytoin at the same time, so that doctors can be helped to determine the therapeutic dose according to the individual difference of the patient in response to the drugs, and the drug dose is adjusted to reduce toxic and side effects, so that epileptic seizures are controlled, and better compliance of the patient can be ensured.
At present, a plurality of methods for measuring antiepileptic drugs in serum at home and abroad are reported, and common methods are as follows: chemiluminescence and chromatography, liquid tandem mass spectrometry are among the most preferred methods for determining antiepileptic drugs. However, the prior method has the problems of complex operation process, more interference factors, poor specificity, long analysis time and low detection flux, which are mainly caused by complex sample pretreatment process and poor stability.
The existing pretreatment methods of antiepileptic drugs mainly comprise protein precipitation, liquid-liquid extraction and the like, such as CN111812216A, and a serum sample is pretreated by adopting a mixed solution of methanol and acetonitrile as a protein precipitant, so that the antiepileptic drugs in the sample are detected, but the detection result is easy to be interfered, the error is large, and the detection sensitivity is low due to the problem of poor stability in the pretreatment process of the sample.
Therefore, there is an urgent need for an antiepileptic drug sample pretreatment method with the advantages of simple operation, high treatment efficiency, high detection flux, high detection accuracy and sensitivity, low cost, small manual workload and the like, so that the method can overcome the defects and the shortcomings of the existing methods.
Disclosure of Invention
In order to solve the problems, the invention provides a detection kit for detecting antiepileptic drugs 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 are used as matrixes of a calibrator and a quality control product, so that interference of bovine serum or bovine serum albumin on valproic acid detection is avoided; the substrate is added with ethylenediamine di-o-sodium phenylacetate and the like as antioxidants, and rabbit serum is preferably adopted as the substrate, 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-sodium phenylacetate, so that the internal standard correction, protein precipitation and target substance extraction can be realized by one-step operation when the internal standard is used for sample pretreatment, and the internal standard liquid can be stored for a long time and can be taken and used at any time, thereby achieving the effect of prolonging the effective period of a kit product.
In one aspect, the invention provides a detection kit for detecting antiepileptic drugs in serum by high performance liquid chromatography-tandem mass spectrometry, which comprises a standard product and/or a quality control product, wherein the standard product and/or the quality control product contains an additive, and the additive is any one or more of ethylenediamine di-o-sodium phenylacetate, ethylenediamine tetraacetic acid disodium, sodium gluconate and sodium citrate.
The antiepileptic drugs of the present invention include valproic acid, carbamazepine and phenytoin.
The inventor proves through a large number of experiments that the anti-epileptic drugs in solution matrixes, especially valproic acid in the anti-epileptic drugs, have poor stability under the oxidation and alkaline environments, such as a standard substance, a quality control substance and an internal standard in a detection kit, the content of the valproic acid in the anti-epileptic drugs is very unstable, the problem of serious content reduction occurs when the anti-epileptic drugs are placed for several days at normal temperature, the detection accuracy and sensitivity can be obviously influenced, all samples, standard substances, internal standard substances, quality control substances and the like for detecting the anti-epileptic drugs 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 of valproic acid, carbamazepine and phenytoin in serum, the stability problem of valproic acid in the solution matrix must first be overcome.
The valproic acid stabilizer is screened by comprehensively considering the antioxidant capacity and the acid-base property, and proper concentration is selected to achieve a good stabilizing effect. That is, in a standard or quality control product containing valproic acid, carbamazepine and phenytoin together, an additive must be added to keep the valproic acid from oxidizing while maintaining the most suitable acidity and alkalinity to keep the valproic acid content as stable as possible.
The inventors have surprisingly found that the addition of ethylenediamine di-o-phenyl sodium acetate (EDDHA), disodium ethylenediamine tetraacetate, sodium gluconate or sodium citrate to a standard or quality control product significantly improves the stability of the antiepileptic drug, in particular valproic acid.
Further, the additive is ethylenediamine di-o-sodium phenylacetate.
Research shows that when the additive is ethylenediamine di-o-sodium phenylacetate (EDDHA), the stability improving effect is the best.
Further, the standard and/or quality control is formulated with a matrix that is animal serum or animal serum albumin, the animal serum is not bovine serum, and the animal serum albumin is not bovine serum albumin.
Because human serum is not readily available and cannot be used as a substrate for the preparation of mass-produced kit products, it is desirable to find suitable animal serum as a substrate.
The inventor finds that the existence of obvious interference of bovine serum or bovine serum albumin on the detection of valproic acid can lead to the very unstable detection result of valproic acid; therefore, neither the standard nor the quality control substrate can 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 di-o-phenyl sodium acetate is 0.02-0.1mg/mL.
Pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin is used as a matrix, and additives in the matrix comprise one or more of ethylenediamine di-o-phenyl sodium acetate, ethylenediamine tetra-sodium acetate, sodium gluconate and sodium citrate, and 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 the product are achieved. The human body sample is simulated by using proper and easily obtained animal serum, so that the matrix effect can be reduced, and the detection result is more accurate and stable.
Further, the matrix is rabbit serum.
Researches prove that the adoption of rabbit serum as a matrix is favorable for further improving the stability of valproic acid in a standard product or a quality control product, and meanwhile, the matrix effect is low, the background noise is low, and the accurate detection of valproic acid can be ensured.
Further, the detection kit further comprises an internal standard solution, wherein the internal standard solution contains a precipitant and a stabilizer, and the stabilizer is ethylenediamine diphthalic sodium acetate.
Further, the precipitant is methanol.
The internal standard solution of the existing anti-epileptic drug is unstable in valproic acid, and the valproic acid needs to be prepared and used at present each time, so that the content of the valproic acid is kept stable by adding the stabilizer into the internal standard solution, and the shelf life of the internal standard solution is prolonged.
The internal standard solution containing the ethylenediamine di-o-sodium phenylacetate prepared by the invention is very stable, can be stored for a long time, can be taken along with use, has a shelf life of 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 efficient, the detection cost is lower, and the detection result is more accurate and stable.
The invention mixes methanol, ethylenediamine di-o-sodium phenylacetate and isotope labeled antiepileptic drugs to obtain an internal standard solution, and adds the internal standard solution into a sample to be uniformly mixed, thereby not only playing the role of the internal standard, but also synchronously realizing the internal standard correction, the precipitation separation of proteins and the extraction of target substances in the sample, and the solution system has better solubility to the antiepileptic drugs in the sample, does not need enrichment processes such as freeze drying or nitrogen blowing after liquid-liquid extraction, and can directly sample and detect after separating precipitate impurities, thereby simplifying the operation process, and the used reagent is a conventional chemical reagent with lower cost, and reducing the detection cost.
Further, the internal standard contains an isotope labeled anti-epileptic drug; the standard substance is an antiepileptic drug with standard concentration; the quality control product is an antiepileptic drug with two different levels of concentration.
Further, the antiepileptic drug is any one or more of valproic acid, carbamazepine or phenytoin.
In another aspect, the invention provides a method of detecting an antiepileptic drug in serum, said method employing a detection kit as described above; the method comprises the steps of system applicability test, sample preparation, sample pretreatment and sample detection; wherein the sample pretreatment comprises the steps of: and uniformly mixing the sample with the internal standard solution according to the ratio of 1:7-1:11, centrifuging, and taking the supernatant for high performance liquid chromatography-tandem mass spectrometry detection.
In yet another aspect, the present invention provides the use of sodium ethylenediamine di-o-phenylacetate for the preparation of a stabilizer for valproic acid solutions.
In yet another aspect, the present invention provides the use of sodium ethylenediamine di-o-phenylacetate in the preparation of a stabilizer for an antiepileptic drug solution, said antiepileptic drug being any one or more of valproic acid, carbamazepine or phenytoin.
In yet another aspect, the invention provides the use of rabbit serum for the preparation of a standard and/or quality control for improving valproic acid stability.
Further, the standard and/or quality control product contains ethylenediamine di-o-phenyl sodium acetate.
The invention has the following beneficial effects:
(1) The sodium ethylenediamine diphthalic 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 severely drops after the antiepileptic drug is placed for several days at normal temperature can be effectively solved; the standard substance and/or quality control substance can be stored for a long time, the content of the antiepileptic drug in the standard substance and/or 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 effective period of the product are achieved, and the accuracy and the sensitivity of detection can be obviously improved;
(2) The detection of valproic acid by bovine serum or bovine serum albumin is found to have obvious interference, and the matrix cannot be prepared by adopting the bovine serum or the bovine serum albumin; pig serum, horse serum, rabbit serum, pig serum albumin or horse serum albumin can be selected as the matrix of the calibrator and quality control product, so that the matrix effect is reduced, and the detection result is more accurate and stable;
(3) The rabbit serum is found to be used as a matrix of a standard product and/or a quality control product, and under the condition of adding ethylenediamine diphthalic sodium acetate, the stability of valproic acid in the matrix can be further improved, and the accuracy of valproic acid detection can be better ensured;
(4) The method has the advantages that the ethylenediamine di-o-sodium phenylacetate is added into the internal standard solution, and the internal standard correction, the protein precipitation and the target substance extraction are simultaneously realized through one-step operation, so that the detection accuracy, the detection sensitivity and the operation simplicity are improved, the content of the anti-epileptic 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 can be taken along with use;
(5) When a patient takes one or more medicines, the one-step sample pretreatment method can detect different blood concentration, reduce repeated sampling times, reduce pain of the patient, effectively reduce analysis cost, and can report a wide range and accurately analyze serum samples with different blood concentration levels.
Drawings
FIG. 1 is a spectrum of valproic acid detection of sample 10 of example 1;
FIG. 2 is a mass spectrum of phenytoin assay of sample 10 of example 1;
FIG. 3 is a carbamazepine assay of sample 10 of example 1;
FIG. 4 is a mass spectrum of bovine serum albumin as a matrix in valproic acid ion channel in example 3;
FIG. 5 is a mass spectrum of the sample of bovine serum as a matrix in valproic acid ion channel in example 3;
FIG. 6 is a graph showing the detection mass spectrum of porcine serum albumin as a matrix in valproic acid ion channel in example 3;
FIG. 7 is a mass spectrum of pig serum as a matrix in valproic acid ion channel in example 3;
FIG. 8 is a mass spectrum of the horse serum albumin as a matrix in valproic acid ion channel in example 3;
FIG. 9 is a mass spectrum of the horse serum as a matrix in valproic acid ion channel in example 3;
FIG. 10 is a mass spectrum of the sample of example 3 in which rabbit serum was used as the substrate in valproic acid ion channel.
Detailed Description
The invention will be described in further detail below with reference to the drawings and examples, it being noted that the examples described below are intended to facilitate an understanding of the invention and are not intended to limit the invention in any way.
Example 1 sample preparation, pretreatment and detection
1. Sample preparation
1. Preparation of calibration material and quality control material
The anti-epileptic drug valproic acid, carbamazepine and phenytoin standard products are prepared into mixed solution which is used for preparing standard product working solution and quality control product working solution. The working solution is taken and added into rabbit serum containing 0.05mg/mL ethylenediamine di-o-phenyl sodium acetate to prepare 10 series concentration calibrator and 2 series concentration quality control product, the concentrations of which are shown in table 1 (unit: mu g/mL):
table 1, 3 standards and quality control concentrations of antiepileptic drugs
| Sample of | Valproic acid | Carbamazepine (Kamahalanobis) | Phenytoin |
| Calibration material 1 | 4 | 0.2 | 0.5 |
| Calibration material 2 | 5 | 0.5 | 1.25 |
| Calibrator 3 | 10 | 1 | 2.5 |
| Calibration material 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 article 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) Precipitant
50mg of ethylenediamine di-o-sodium phenylacetate was weighed, dissolved in 100mL of purified water, and 900mL of methanol was added thereto, followed by mixing to obtain a precipitant.
(3) Internal standard liquid
And uniformly mixing the internal standard stock solution and the precipitant in a ratio of 1:100 to obtain the internal standard solution.
3. Formulation of System applicability solution
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 was mixed with 49.9mL of a 50% aqueous methanol solution to prepare a system applicability solution.
The system applicability test is to directly sample the system applicability solution for 3 times by using high performance liquid chromatography tandem mass spectrometry detection before sample detection, and judge whether the high performance liquid chromatography tandem mass spectrometry system is normal or not according to the retention time, the response strength and the precision of the target substance.
2. Sample pretreatment
(1) Taking 20 mu L of sample, and adding the sample into a 96-well plate or a centrifuge tube;
(2) 180 μl of the internal standard solution was added, vortexed for 5 minutes, centrifuged at 4000rpm for 10 minutes, and the supernatant was taken for testing.
3. Sample detection
And taking the supernatant, and detecting by using high performance liquid chromatography tandem mass spectrometry. The specific analysis conditions were as follows: the liquid phase adopts gradient elution, the mobile phase A is 0.1% formic acid-10% methanol water, and the mobile phase B is 0.1% formic acid methanol; the chromatographic column is a C18 chromatographic column, the flow rate of the mobile phase is 0.6mL/min, the column temperature is 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 employs electrospray ion sources (ESI) and multiple reaction monitoring scan mode (MRM). The parent ion/daughter ion pair mass to charge ratios (m/z) of the three antiepileptic drugs used for detection are shown in table 3:
TABLE 3 parent ion/child ion to mass to charge ratio
| Analyte(s) | Q1 | Q3 |
| Valproic acid | 142.9 | 142.9 |
| Carbamazepine (Kamahalanobis) | 237 | 179 |
| Phenytoin | 253 | 104 |
Detection of 3 antiepileptic drugs can be determined by selecting pairs of ions detected by reaction monitoring, and corresponding retention times, and quantified by internal standards for the various antiepileptic drugs.
After the sample is separated by liquid chromatography, different antiepileptic drugs and matrix components are eluted at different times and detected by a mass spectrum multi-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 to 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. It can be seen that according to the method provided by the embodiment, 3 antiepileptic drugs can be detected accurately at the same time, the 3 antiepileptic drugs all show peaks at a retention time of about 1.04 minutes, and the specificity detection without mutual interference is realized through three different ion channels.
Example 2 Effect of different additives on stability of calibrant/quality control
This example was prepared according to the method provided in example 1, and the substrates were formulated with different types and concentrations of additives, respectively, and left at 37 ℃ for 14 days to examine the effect of the different additives on the stability of the calibrator/quality control. The concentrations of each additive and its in the matrix are: 0.05mg/mL of ethylenediamine di-o-sodium phenylacetate, 0.1mg/mL of disodium ethylenediamine tetraacetate, 0.05mg/mL of sodium gluconate, 0.01mg/mL of sodium nitrilotriacetate, and 0.02mg/mL of sodium citrate. Examination of the changes in valproic acid content in calibrator 1 is shown in table 4:
TABLE 4 influence of different additives on the stability of valproic acid in a matrix
| Additive and its concentration | Valproic acid content variation |
| No additive | -15.2％ |
| 0.05mg/mL sodium ethylenediamine di-o-phenylacetate | 0.5％ |
| 0.1mg/mL disodium edetate | -3.2％ |
| 0.05mg/mL sodium gluconate | -4.7％ |
| 0.01mg/mL sodium nitrilotriacetate | -11.3％ |
| Sodium citrate 0.02mg/mL | -7.9％ |
Valproic acid has poor stability in oxidizing and alkaline environments, and stabilizer screening needs to comprehensively consider the oxidation resistance and the acid-base property of the valproic acid, and proper concentration is selected to achieve a good stabilizing effect.
As can be seen from table 4, in the absence of additives, calibrator 1 was left at 37 ℃ for 14 days, the valproic acid content was reduced by 15.2%; after a certain amount of additive is added, the proportion of the content of the valproic acid is 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 sodium citrate and sodium nitrilotriacetate have certain antioxidant capacity, but the pH value of the sodium citrate and sodium nitrilotriacetate in the solution is more than 7, and the sodium nitrilotriacetate presents alkalinity, so that the antioxidant effect is weakened, and the effect on improving the stability of valproic acid in the calibrator/quality control product is slightly poorer.
The content of the valproic acid can be changed by about +/-3% by adding 0.1mg/mL of disodium ethylenediamine tetraacetate, and the stabilizing effect is good; the acidity and alkalinity of sodium gluconate are similar to those of ethylenediamine di-o-phenyl sodium acetate, but the oxidation resistance of sodium gluconate is weaker than that of sodium ethylenediamine di-o-phenyl sodium acetate, so that the content of valproic acid is slightly reduced by 4.7% under the action of 0.05mg/mL sodium gluconate, and the degradation rate is still within an acceptable range of 10%.
Compared with the above additives, the effect of adding 0.05mg/mL ethylenediamine diphthalic sodium acetate is outstanding, and the valproic acid is reduced by 0.5% after the mixture is placed for 14 days at 37 ℃, so that the stabilizing effect is optimal. Thus, ethylenediamine di-o-phenyl sodium acetate is the most preferred additive for improving the stability of antiepileptic drug calibrator/quality control.
EXAMPLE 3 interference of bovine serum and bovine serum Albumin on valproic acid detection
According to the method provided in example 1, bovine serum, porcine serum, horse serum, rabbit serum, porcine serum albumin, horse serum albumin and bovine serum albumin are respectively used as blank matrixes, antiepileptic drugs are not added in the blank matrixes, then mass spectrometry detection of valproic acid ion channels is respectively carried out, the detection patterns are shown in fig. 4-10, wherein 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 porcine serum albumin as a matrix, fig. 7 is a mass spectrum of porcine 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.
As can be seen from the maps of valproic acid ion channels under different matrixes (figures 4-10), under the condition of a blank matrix, bovine serum and bovine serum albumin contain impurities which interfere with valproic acid detection, and pig serum, pig serum albumin, horse serum albumin and rabbit serum do not contain the interfering substances, so that valproic acid in a standard substance/quality control substance is not interfered, and the valproic acid can be used as a matrix of a kit; and wherein rabbit serum has minimal background noise and minimal interference to valproic acid.
In this example, bovine serum, porcine serum, horse serum, rabbit serum, porcine serum albumin, horse serum albumin, bovine serum albumin, and human serum were used as blank substrates to prepare standard 1, and the method provided in example 1 was used to detect three anti-epileptic drugs, and the effect of the standard prepared with different substrates on the detection accuracy of the three anti-epileptic drugs was examined, and the results are shown in table 5.
TABLE 5 influence of samples formulated with different matrices on the detection accuracy of three antiepileptic drugs
| Substrate | Valproic acid content (μg/mL) | Carbamazepine content (μg/mL) | Phenytoin content (μg/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 |
| Pig 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 valproic acid content in the standard 1 was 4. Mu.g/mL, carbamazepine content was 0.2. Mu.g/mL, and phenytoin content was 0.5. Mu.g/mL.
As can be seen from table 5, when three antiepileptic drugs are detected by using standard product 1 prepared from different serum, there is no obvious difference between carbamazepine and phenytoin content, and the results of valproic acid detection are different, so that samples are prepared from different serum as a matrix, and the accuracy of valproic acid detection in the samples is greatly affected.
When bovine serum or bovine serum albumin is used as a matrix, the detection result of valproic acid is obviously higher, and the matrix of bovine serum or bovine serum albumin has serious interference on the detection of valproic acid. When horse serum, horse serum protein, pig serum and pig serum albumin are used as matrixes, a certain error exists in the detection result of the valproic acid, and the reason is probably that the matrixes effect exists in the serum, so that the accurate detection of the valproic acid is influenced. When the rabbit serum is used as a matrix to prepare the standard product 1, the valproic acid in the standard product can be detected more accurately, and the detection result is the most consistent with the detection result of the human serum as the matrix, so that the precision of valproic acid detection is greatly ensured.
Example 4 Effect of different matrices on valproic acid stability
In this example, the preparation and detection of the detection kit of the antiepileptic drug were performed according to the method provided in example 1, wherein the standard substance/quality control substance was prepared by using animal serum or animal serum protein except bovine serum, and 0.05mg/mL ethylenediamine di-o-phenyl sodium acetate was added, and the mixture was left at 37 ℃ for 14 days, and when the standard substance 1 was prepared by using different substrates with 0.05mg/mL ethylenediamine di-o-phenyl sodium acetate as an additive, the stability of valproic acid in the different substrates was as shown in table 6:
TABLE 6 influence of different matrices on valproic acid stability
| Substrate | Valproic acid content variation |
| Pig serum | -1.0％ |
| Horse serum | -1.2％ |
| Rabbit serum | -0.5％ |
| Pig serum albumin | -2.5％ |
| Horse serum albumin | -2.6％ |
As can be seen from Table 6, when different animal serum or animal serum proteins are adopted to prepare the standard product 1, the stability of valproic acid is greatly different, when rabbit serum is adopted as a matrix, the stability of valproic acid is further improved, and when the sample contains 0.05mg/mL ethylenediamine diphthalic sodium acetate as an additive, the sample is placed for 14 days at 37 ℃, and the valproic acid content is hardly changed; 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, and the stability of valproic acid can be remarkably improved.
EXAMPLE 5 Effect of different internal Standard fluids on valproic acid internal Standard stability
In order to improve the stability of the internal standard solution and prolong the storage period of the internal standard solution, the method provided in the embodiment 1 is not used for each preparation, 1mg/mL sodium metabisulfite is added, 2mg/mL sodium ethylenediamine diphthalic acetate is added, 3 mg/mL sodium ethylenediamine diphthalic acetate is added, the internal standard solution is prepared without adding sodium metabisulfite and sodium ethylenediamine diphthalic acetate precipitants, and the influence of sodium metabisulfite and sodium ethylenediamine diphthalic acetate on the shelf life of valproic acid internal standard (valproic acid-d 6) in the internal standard solution is examined under the condition of 37 ℃ for 7 days, 20 days and 40 days.
TABLE 7 influence of different internal standard solutions on valproic acid internal standard stability
The results are shown in Table 7, and it can be seen that the content of the valproic acid internal standard is obviously reduced along with the extension of the standing time under the condition that sodium metabisulfite or ethylenediamine di-o-phenyl sodium acetate is not added; in the precipitant added with sodium metabisulfite or ethylenediamine di-o-sodium phenylacetate, the internal standard solution is placed at 37 ℃ for 40 days, the content of valproic acid internal standard is changed within 15%, and the stability is good. In particular, when ethylenediamine di-o-sodium phenylacetate was added, the internal standard solution was left at 37℃for 40 days, and the content of valproic acid as an internal standard was changed by about 1%, resulting in excellent stability.
Experiments prove that the content of the valproic acid internal standard in the internal standard liquid without the stabilizer is reduced by more than 35 percent after the internal standard liquid 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 di-o-sodium phenylacetate still keeps stable, and the content change of the valproic acid internal standard is only about 3 percent. The sodium ethylenediamine diphthalic acetate is added into the internal standard solution, so that the stability of the internal standard solution can be obviously improved, the storage life is prolonged, the internal standard solution can be placed for 3 years, the internal standard solution can be taken and used in 3 years, and the labor and material cost is greatly reduced.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Claims (9)
1. Use of sodium ethylenediamine di-o-phenylacetate for the preparation of a stabilizer for valproic acid solutions.
2. The use according to claim 1, wherein the valproic acid solution comprises any one or more of a standard, a quality control or an internal standard for detecting valproic acid content in serum, wherein the standard, quality control or internal standard comprises sodium ethylenediamine diphenyoacetate.
3. The use according to claim 2, wherein the standard and/or quality control is formulated with a matrix that is animal serum or animal serum albumin, the animal serum not being bovine serum, the animal serum albumin not being bovine serum albumin.
4. The use according to claim 3, wherein the matrix is any one or more of porcine serum, equine serum, rabbit serum, porcine serum albumin or equine serum albumin, and the concentration of ethylenediamine diphthalic sodium acetate is 0.02-0.1mg/mL.
5. The use according to claim 4, wherein the substrate is rabbit serum; the internal standard solution contains a precipitator and a stabilizer, wherein the stabilizer is ethylenediamine di-o-phenyl sodium acetate.
6. The use according to claim 5, wherein the precipitant is methanol; the internal standard solution also contains an isotope labeled anti-epileptic drug.
7. The use of claim 6, wherein the standard comprises a standard concentration of valproic acid; the quality control product contains valproic acid with different levels of concentration.
8. The use according to claim 7, wherein the method for detecting valproic acid content in serum samples using standard, quality control or internal standard fluid comprises system suitability testing, sample preparation, sample pretreatment and sample detection; wherein the sample pretreatment comprises the steps of: and uniformly mixing the sample with the internal standard solution according to the ratio of 1: 7~1:11, centrifuging, and taking the supernatant for high performance liquid chromatography-tandem mass spectrometry detection.
9. The rabbit serum is used for preparing a standard substance and/or a quality control substance for improving the stability of valproic acid.
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