CN112748206B - High performance liquid chromatography analysis method simultaneously suitable for determination of 24 antiepileptic drugs in human plasma - Google Patents
High performance liquid chromatography analysis method simultaneously suitable for determination of 24 antiepileptic drugs in human plasma Download PDFInfo
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Abstract
The invention relates to a high performance liquid chromatography analysis method simultaneously suitable for determination of 24 antiepileptic drugs in human plasma, which is used for grouping the types of the 24 antiepileptic drugs, performing chromatographic analysis under the basically same chromatographic conditions according to different gradient elution conditions, realizing the simultaneous satisfaction of the determination of the 24 antiepileptic drugs in the human plasma, adding an ultraviolet derivatization reagent into the drug to be derivatized for pre-column derivatization, so that the antiepileptic drug without ultraviolet absorption can be detected, the separation efficiency is high, and the selectivity is better.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a high performance liquid chromatography analysis method for simultaneously adapting to the determination of 24 antiepileptic drugs in human plasma, wherein the 24 antiepileptic drugs are respectively as follows: 1. piracetam; 2. levetiracetam; 3. ethosuximide; 4. primidone; 5. lacosamide; 6. (iii) suzuzine; 7. zonisamide; 8. b, busulfacetam; 9. phenobarbital; 10. oxcarbazepine; 11. licarbazepine; 12. sodium phenytoin; 13. clonazepam; 14. diazepam; 15. (ii) a spandex; 16. lamotrigine; 17. a felbamate; 18. rufinamide; 19. contest with guests; 20. carbamazepine; 21. perampanel; 22. vigabatrin; 23. pregabalin; 24. gabapentin.
Background
Epilepsy (epilepsy) is a common disease of the nervous system, a chronic disease in which neurons in the brain suddenly discharge abnormally, resulting in transient cerebral dysfunction, affecting about 7000 million people worldwide. In China, epilepsy has become the second most common disease of neurology, which is second to headache, and brings great pain to patients and family members.
According to the literature report, in the clinical application of the anti-epileptic, a doctor needs to know the blood concentration of the anti-epileptic in the shortest possible time before adjusting the medication, so that the accuracy and rapidness are the basic requirements of the clinical drug monitoring of the anti-epileptic.
At present, the therapeutic drug monitoring method of the antiepileptic drug mainly comprises an immunoassay method and a chromatography-spectroscopy analysis method.
The immunoassay method is a principle determination method by utilizing the in vitro reaction of the antiepileptic drug and a specific antibody thereof, and is widely adopted in the monitoring of the treatment drugs of the antiepileptic drug due to the characteristics of small sample amount, convenient and quick operation. However, the method must be used for researching antibodies aiming at each antiepileptic drug put on the market, and metabolites, structural analogs of the antiepileptic drug to be monitored and other drugs or foods taken by a patient can generate cross reaction with the antibodies, so that the measured result is often higher than the true blood concentration, and the adjustment of a medication scheme and a dosage is interfered.
The high selectivity and sensitivity chromatographic-combination technology based on the strong separating power of the chromatogram and the spectral detection is the main choice for measuring the antiepileptic drug in the biological sample. Such as Gas Chromatography (GC), high Performance Liquid Chromatography (HPLC), capillary Electrophoresis (CE), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). Because the instrument is relatively cheap, the accuracy and the reproducibility are good, HPLC has become the main choice for measuring the clinical blood concentration of the antiepileptic drug.
At present, more than 20 varieties are put into clinical use, hospital clinical pharmacy workers may face epileptic patients taking different medicines every day, if each medicine or a plurality of medicines need to establish an HPLC analysis method, the inconvenience in work is inevitably brought to the monitoring of the anti-epileptic medicines, the decision time for individualized medication and dosage adjustment of clinicians can be influenced, and the treatment quality of the epileptic is further influenced.
Therefore, establishing an HPLC analysis system (without changing the types of a fixed phase and a mobile phase) capable of meeting the determination of different antiepileptic drugs in plasma to adapt to the requirements of efficient and rapid extraction, separation and analysis of blood sample analysis in the monitoring of clinical treatment drugs becomes an urgent need of clinical pharmacy workers.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography analysis method which is simultaneously suitable for the determination of 24 antiepileptic drugs in human plasma on the basis of the prior art.
The technical scheme of the invention is as follows:
a high performance liquid chromatography analysis method simultaneously suitable for determination of 24 antiepileptic drugs in human plasma is provided, wherein the 24 antiepileptic drugs are divided into the following three groups, and are detected by using a high performance liquid chromatography method, and the specific chromatographic conditions are as follows: the chromatographic column is Eclipse Plus C 18 A column; taking a mobile phase A and a mobile phase B as a mixed mobile phase to carry out gradient elution, wherein the mobile phase A is acetonitrile, and the mobile phase B is a potassium dihydrogen phosphate solution;
the first group of 15 antiepileptic drugs are: piracetam; levetiracetam; ethosuximide; primidone; lacosamide; (iv) an thiazide; zonisamide; b, busulfacetam; phenobarbital; oxcarbazepine; licarbazepine; phenytoin sodium; clonazepam; diazepam; (ii) a spandex; the pretreated blood plasma containing the first group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,10% by volume A;2min,10% by weight of A;3min,28% by weight A;7.5min,28% A;8min,35% A;8.5min,45% by volume A;9.5min,55% A;11min,60% A;11.5min,65% A;20min,65% A.
The second group of 6 antiepileptic drugs are: lamotrigine; a felbamate; rufinamide; contest with guests; carbamazepine; perampanel; the pretreated blood plasma containing the second group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,20% by volume A;2min,20% by weight A;5min,30% by weight A;8min,60% A;10min,60% A;13min,60% by weight A;15min,20% A.
The third group of 3 antiepileptic drugs are: vigabatrin; pregabalin; gabapentin; the pretreated blood plasma containing the third group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,20% of A;2min,20% by weight A;3min,28% by weight A;7.5min,28% A;8min, 35. Percent A;8.5min,45% by volume A;9.5min,55% A;11min,60% A;11.5min,65% A;20min,65% A.
The detection method can simultaneously meet the requirements of efficient, rapid, simple and accurate detection of 24 antiepileptic drugs in human plasma, and can perform chromatographic analysis on different groups of drugs according to different gradient elution conditions, wherein other chromatographic conditions are the same (such as chromatographic columns, mobile phase types, column temperature and the like). In order to improve the chromatographic separation selectivity, when the invention is used for performing high performance liquid chromatography analysis on the 24 antiepileptic drugs, gradient elution is performed on a plurality of mixed mobile phases, such as acetonitrile-monopotassium phosphate, methanol-monopotassium phosphate, acetonitrile-0.1% formic acid water, acetonitrile-ammonium formate and acetonitrile-water, and as a result of experiments, the problems that the chromatographic separation selectivity is poor, a base line shifts during elution, peaks are not produced, the peak shape of a chromatographic peak is poor and the like exist in other mixed mobile phases under the same elution conditions except that acetonitrile-monopotassium phosphate is used as the mixed mobile phase are found. In order to obtain a better spectrum, when acetonitrile-potassium dihydrogen phosphate is used as a mixed mobile phase, the concentration of the potassium dihydrogen phosphate in the mobile phase B is limited to be 1-60 mM. In a preferred embodiment, the concentration of the potassium dihydrogen phosphate in the selected mobile phase B is 5-50 mM, and further preferably, the concentration of the potassium dihydrogen phosphate in the selected mobile phase B is 5mM, that is, acetonitrile-5 mM potassium dihydrogen phosphate solution is used as the mixed mobile phase.
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. In the invention, when the chromatographic conditions are explored, a large number of other chromatographic columns, such as Zorbax SB-C18, are tried, and the results show that the selectivity is poor and the effect is poor. The invention adopts acetonitrile-potassium dihydrogen phosphate solution, such as acetonitrile-5 mM potassium dihydrogen phosphate solution as a mixed mobile phase, and a chromatographic column is Eclipse Plus C 18 The column can simultaneously meet the requirements of efficient, quick, simple, convenient and accurate detection of 24 antiepileptic drugs in human plasma under the coordination of other conditions, and has high sensitivity, strong specificity and basically meets the requirements on accuracy and precision. In a preferred embodiment, eclipse Plus C 18 The column had a length of 150mm, a diameter of 4.6mm and a filler particle size of 3.5. Mu.m.
In a preferred embodiment, the above mentioned chromatographic conditions further comprise a detection wavelength of 205 to 215nm, preferably 210nm.
Further, the column temperature is 30 to 40 ℃, preferably 35 ℃.
Further, the injection volume is 5 to 20. Mu.L, for example, 5. Mu.L, 10. Mu.L, 15. Mu.L, or 20. Mu.L.
For the present invention, when gradient elution is performed, the flow rate of the mixed mobile phase in different gradient elution processes is limited, so as to obtain a better separation effect, and in a preferred embodiment, when gradient elution is performed on the first group of antiepileptic drugs, the flow rate is 0.8-1.2 mL/min, preferably 1.0mL/min. When the second group of antiepileptic drugs is subjected to gradient elution, the flow rate is 0.8-0.9 mL/min, preferably 0.85mL/min within 0-8 min, and the flow rate is 0.9-1.2 mL/min, preferably 1.0mL/min within 15min after 8 min. When the third group of antiepileptic drugs is subjected to gradient elution, the flow rate is 0.8-1.2 mL/min, and the preferred flow rate is 1.0mL/min.
In a preferred scheme, when 24 antiepileptic drugs in human plasma are detected by using a high performance liquid chromatography method, the specific chromatographic conditions are as follows:
the chromatographic column is Eclipse Plus C 18 Columns (150X 4.6mm,3.5 μm); gradient elution was carried out using acetonitrile (mobile phase A) -5mM potassium dihydrogen phosphate (mobile phase B) as a mixed mobile phase, the detection wavelength was 210nm, and the column temperature was 35 ℃. For three different groups of antiepileptic drugs, the gradient elution procedure was as follows:
the pretreated blood plasma containing the first group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,10% by volume A;2min,10% by weight of A;3min,28% by weight A;7.5min,28% by volume A;8min, 35. Percent A;8.5min,45% by volume A;9.5min,55% A;11min,60% by volume A;11.5min,65% A;20min,65% by weight of A; the gradient elution pattern is detailed in table 1.
TABLE 1 first group of parameters for mobile phase gradient elution of antiepileptic drugs
| Time (min) | Flow rate (mL/min) | ％A | ％B |
| 0.0 | 1.0 | 10 | 90 |
| 2.0 | 1.0 | 10 | 90 |
| 3.0 | 1.0 | 28 | 72 |
| 7.5 | 1.0 | 28 | 72 |
| 8.0 | 1.0 | 35 | 65 |
| 8.5 | 1.0 | 45 | 55 |
| 9.5 | 1.0 | 55 | 45 |
| 11.0 | 1.0 | 60 | 40 |
| 11.5 | 1.0 | 65 | 35 |
| 20.0 | 1.0 | 65 | 35 |
The pretreated blood plasma containing the second group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,20% by volume A;2min,20% by weight of A;5min,30% by weight A;8min,60% A;10min,60% A;13min,60% by weight A;15min,20% A; the gradient elution pattern is detailed in Table 2.
TABLE 2 second set of parameters for mobile phase gradient elution of antiepileptic drugs
The pretreated blood plasma containing the third group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,20% by volume A;2min,20% by weight A;3min,28% by weight of A;7.5min,28% by volume A;8min, 35. Percent A;8.5min,45% by volume A;9.5min,55% A;11min,60% by volume A;11.5min,65% A;20min,65% by volume A; the gradient elution pattern is detailed in Table 3.
TABLE 3 third group of antiepileptic drug mobile phase gradient elution parameters
| Time (min) | Flow rate (mL/min) | ％A | ％B |
| 0.0 | 1.0 | 20 | 80 |
| 2.0 | 1.0 | 20 | 80 |
| 3.0 | 1.0 | 28 | 72 |
| 7.5 | 1.0 | 28 | 72 |
| 8.0 | 1.0 | 35 | 65 |
| 8.5 | 1.0 | 45 | 55 |
| 9.5 | 1.0 | 55 | 45 |
| 11.0 | 1.0 | 60 | 40 |
| 11.5 | 1.0 | 65 | 35 |
| 20.0 | 1.0 | 65 | 35 |
The chemical structural formula of 24 antiepileptic drugs is shown in the literature:
the plasma referred to in the present invention is human plasma.
In the invention, acetonitrile and/or acetonitrile-water solution is selected as a solvent when a reference solution is prepared, and the method has higher selectivity and good peak shape of a chromatographic peak under the same chromatographic condition. In the process of chromatographic experiments, the inventors of the present application also tried to prepare reference solutions by using methanol, water, methanol-water and methanol-acetonitrile as solvents and perform detection under the same chromatographic conditions, and as a result, the inventors found that in the process of chromatographic analysis, the selectivity is poor, the peak shape is poor and the effect is poor. In a preferred embodiment, when three different groups of antiepileptic drugs are formulated, the solvent selected is acetonitrile and/or an acetonitrile-water solution, wherein the volume concentration of acetonitrile in the acetonitrile-water solution is 40 to 60%, preferably 50%, for example, 50% acetonitrile-water solution.
When 24 anti-epileptic drugs in human plasma are subjected to chromatographic analysis, three groups of different mixed reference substance solutions are prepared according to the following method:
for the first group of 15 antiepileptic drugs: respectively preparing piracetam, levetiracetam, ethosuximide, pamidone, lacosamide, suthiazide, zonisamide, brivaracetam, phenobarbital, oxcarbazepine, licarbazepine, phenytoin sodium, clonazepam, diazepam and spandex into mother solutions with the concentration of 2.0mg/mL by adopting acetonitrile-aqueous solution (preferably 50% acetonitrile-aqueous solution); respectively taking 750 mu L of mother liquor of the 15 antiepileptic drugs, adding 250 mu L of acetonitrile-water solution (preferably 50% acetonitrile-water solution) into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 1.5mg/mL, then taking 200 mu L of each intermediate solution, and uniformly mixing to obtain mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 100 mu g/mL, and the mixed solution is used as a first mixed reference solution. And taking 10 mu L of the first mixed reference substance solution, adding 190 mu L of blank plasma, and uniformly mixing to obtain a plasma solution with the concentration of each antiepileptic drug of 5 mu g/mL as a first mixed plasma solution.
For a second group of 6 antiepileptic drugs: preparing lamotrigine, felbamate, rufinamide, saigatene, carbamazepine and perampanel into mother liquor with the concentration of 1.0mg/mL by adopting acetonitrile-water solution (preferably 50% acetonitrile-water solution); taking 600 mu L of mother liquor of the 6 antiepileptic drugs respectively, adding 400 mu L of acetonitrile into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 600 mu g/mL, taking 200 mu L of each intermediate liquor respectively, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 100 mu g/mL, and the mixed solution is used as a second mixed reference solution. And taking 10 mu L of the second mixed reference substance solution, adding 190 mu L of blank plasma, and uniformly mixing to obtain a plasma solution with the concentration of each antiepileptic drug of 5 mu g/mL, wherein the plasma solution is used as a second mixed plasma solution.
For the third group of 3 anti-epileptic drugs: respectively preparing vigabatrin, pregabalin and gabapentin into mother liquor with the concentration of 1.0mg/mL by adopting acetonitrile-water solution (preferably 50% acetonitrile-water solution); taking 600 mu L of mother liquor of the 3 antiepileptic drugs respectively, adding 400 mu L of acetonitrile into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 600 mu g/mL, taking 200 mu L of each intermediate liquor respectively, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 200 mu g/mL, and the mixed solution is used as a third mixed reference solution. And taking 10 mu L of the third mixed reference substance solution, adding 190 mu L of blank plasma, and uniformly mixing to obtain a plasma solution with the concentration of each antiepileptic drug of 10 mu g/mL, wherein the plasma solution is used as a third mixed plasma solution.
For the purposes of the present invention, the pretreated plasma containing the first group of antiepileptic drugs described above is prepared as follows: adding the first mixed reference substance solution into the filtered blood plasma, uniformly mixing, adding methanol for precipitation, centrifuging, taking the supernatant, volatilizing, adding acetonitrile-water solution for redissolution, and injecting a sample.
In the reconstitution process, the acetonitrile-water solution is selected to have a volume concentration of acetonitrile of 40 to 60%, preferably 50%, for example, the reconstituted solution is a 50% acetonitrile-water solution.
In a preferred embodiment, the pretreated plasma containing the first group of antiepileptic drugs is prepared according to the following method: adding 10 μ L of the first mixed control solution into 190 μ L of blood plasma, mixing, adding 600 μ L of methanol for protein precipitation, centrifuging at 12000 speed for 10min at 4 deg.C, collecting 600 μ L of supernatant, volatilizing, adding 100 μ L of 50% acetonitrile-water for redissolution, and sampling.
For the purposes of the present invention, the pretreated blood plasma containing the first group of antiepileptic drugs mentioned above is prepared as follows: adding the first mixed reference substance solution into the filtered blood plasma, uniformly mixing, adding methanol for precipitation, centrifuging, collecting the supernatant, volatilizing, adding acetonitrile-water solution for redissolution, and injecting a sample.
In the reconstitution process, the acetonitrile-water solution is selected to have a volume concentration of acetonitrile of 40 to 60%, preferably 50%, for example, the reconstituted solution is a 50% acetonitrile-water solution.
In a preferred embodiment, the pretreated blood plasma containing the second group of antiepileptic drugs is prepared as follows: and (3) adding 10 mu L of second mixed reference substance solution into 190 mu L of blood plasma, uniformly mixing, adding 600 mu L of methanol for protein precipitation, centrifuging at 12000 rotation speed for 10min at 4 ℃, taking 600 mu L of supernate for volatilizing, adding 100 mu L of 50% acetonitrile-water for redissolution, and injecting a sample.
For the purposes of the present invention, the pretreated plasma containing the third group of antiepileptic drugs described above is prepared as follows: adding the third mixed reference substance solution into the filtered blood plasma, uniformly mixing, adding methanol for precipitation, centrifuging, taking the supernatant, adding 9-fluorenylmethoxycarbonyl chloride as a derivatization reagent, performing derivatization reaction for 10-60min at 40-80 ℃, volatilizing, adding acetonitrile-water solution for redissolution, and feeding a sample.
The temperature of the derivatization reaction can be, but is not limited to, 40 ℃,45 ℃,50 ℃,55 ℃,60 ℃,65 ℃, 70 ℃, 75 ℃ or 80 ℃, and in a preferred embodiment, the reaction temperature is 60 ℃ during the derivatization reaction.
The time for the derivatization reaction can be, but is not limited to, 10min, 20min, 30min, 40min, 50min or 60min, and in a preferred embodiment, the reaction time is 30min.
In the reconstitution process, the acetonitrile-water solution is selected to have a volume concentration of acetonitrile of 40 to 60%, preferably 50%, for example, the reconstituted solution is a 50% acetonitrile-water solution.
In a preferable scheme, 190 mu L of blood plasma is taken, 10 mu L of third mixed reference substance solution is added into the blood plasma, 600 mu L of methanol is added after uniform mixing for protein precipitation, the blood plasma is centrifuged for 10min at 12000 under the condition of 4 ℃, after centrifugation, 600 mu L of supernatant is taken, 200 mu L of 9-fluorenylmethoxycarbonyl chloride is added into the supernatant, the mixture is completely volatilized to dry after reaction for 30min in a water bath at 60 ℃, 100 mu L of 50% acetonitrile-water is added for redissolution, and then sample injection is carried out.
By adopting the technical scheme of the invention, the advantages are as follows:
(1) The invention uses acetonitrile-water solution as solvent to prepare the reference substance of 24 antiepileptic drugs, can improve the speed of peak generation in chromatographic analysis and the peak shape of chromatographic peak, and has high separation efficiency and better selectivity.
(2) The detection method can solve the problem that 24 anti-epileptic drugs cannot be distinguished due to similar structures and similar peak-out time.
(3) According to the method, 24 antiepileptic drugs cannot be taken by the same epileptic patient at the same time according to different types of the antiepileptic drugs, and chromatographic analysis is performed under the basically same chromatographic conditions according to different gradient elution conditions, so that the requirement for detecting the 24 antiepileptic drugs in human plasma is met at the same time, and the method is efficient, rapid, simple, convenient and accurate.
(4) Grouping whether the 24 anti-epileptic drugs have ultraviolet absorption performance, and adding an ultraviolet derivatization reagent into the drugs to be derivatized for pre-column derivatization so that the anti-epileptic drugs without ultraviolet absorption can be detected.
Drawings
FIG. 1 shows the use of Eclipse Plus C 18 (150X 4.6mm,3.5 μm) column, first mixed control chromatogram of a first set of 15 non-derivatized antiepileptic drugs using acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as the mixed mobile phase;
FIG. 2 shows the use of Eclipse Plus C 18 (150X 4.6mM,3.5 μm) column chromatography with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as mixed mobile phase to determine the color of the first set of 15 plasma samples of the antiepileptic drug without derivatizationA spectrogram;
FIG. 3 shows the use of Eclipse Plus C 18 (150X 4.6mm,3.5 μm) column, a second mixed control solution chromatogram of a second set of 6 anti-epileptic drugs without derivatization, using acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as the mixed mobile phase;
FIG. 4 shows the use of Eclipse Plus C 18 (150X 4.6mm,3.5 μm) column, chromatogram of plasma samples of a second set of 6 anti-epileptic drugs without derivatization, with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as mixed mobile phase;
FIG. 5 shows the use of Eclipse Plus C 18 (150X 4.6mm,3.5 μm) column, with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as the mixed mobile phase for the determination of 3 derived antiepileptic drug third mixed control solution chromatograms;
FIG. 6 shows the use of Eclipse Plus C 18 (150X 4.6mm,3.5 μm) column, chromatogram of plasma sample of 3 derived antiepileptic drugs measured with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as mixed mobile phase;
FIG. 7 shows the use of Eclipse Plus C 18 (150X 4.6mm,3.5 μm) column, chromatogram of a first mixed control solution of a first set of 15 anti-epileptic drugs without derivatization, with methanol (A) -5mM potassium dihydrogen phosphate solution (B) as the mixed mobile phase;
FIG. 8 is a chromatogram of a second mixed control solution of a second set of 6 antiepileptic drugs measured using a Zorbax SB-C18 (150X 4.6mm,5 μm) column with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as the mixed mobile phase;
FIG. 9 is a chromatogram of a third mixed control solution of a third set of 3 anti-epileptic drugs after derivatization using an Eclipse Plus C18 (150X 4.6mm,3.5 μm) column with acetonitrile (A) -water (B) as the mixed mobile phase;
in fig. 1, 2, and 7, 1: piracetam; 2. levetiracetam; 3: ethosuximide; 4: primidone; 5: lacosamide; 6: (iv) an thiazide; 7: zonisamide; 8: b, busulfacetam; 9: phenobarbital; 10: oxcarbazepine; 11: licarbazepine; 12: sodium phenytoin; 13: clonazepam; 14: diazepam; 15: (ii) a spandex;
in fig. 3 and 4 and fig. 8, 16: lamotrigine; 17: a felbamate; 18: rufinamide; 19: contest with guests; 20: carbamazepine; 21: perampanel;
in fig. 5 and 6 and fig. 9, 22: vigabatrin; 23: pregabalin; 24: gabapentin.
Detailed Description
The method for measuring 24 antiepileptic drugs according to the present invention is further illustrated by the following examples, which are not intended to limit the present invention in any way. The equipment or materials used in the examples are commercially available. The water used was 18.2. Omega. Ultra pure water, and the methanol and acetonitrile used were purchased from SEPSRV, inc. Plasma source: derived from normal human plasma.
Example 1
1. Chromatographic conditions
Using Eclipse Plus C 18 (150X 4.6mM,3.5 μm) column, gradient elution was carried out with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as a mixed mobile phase, and gradient elution was carried out as follows: 0min,10% by volume A;2min,10% by weight of A;3min,28% by weight A;7.5min,28% A;8min,35% A;8.5min,45% A;9.5min,55% A;11min,60% A;11.5min,65% A;20min,65% A, see Table 1 for details; the column temperature was 35 ℃ and the detection wavelength was 210nm.
2. Experimental procedures
(1) Preparation of a reference substance:
for the first group of antiepileptic drugs, 15 antiepileptic drugs (piracetam, levetiracetam, ethosuximide, pamidone, lacosamide, suthiazide, zonisamide, brivaracetam, phenobarbital, oxcarbazepine, licarbazepine, phenytoin sodium, clonazepam, diazepam, and spandex) were prepared into 2.0mg/mL mother solutions with 50% acetonitrile-water solutions, respectively. Respectively taking 750 mu L of the mother liquor of the 15 antiepileptic drugs, adding 250 mu L of 50% acetonitrile-water solution into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 1.5mg/mL of intermediate liquor, then taking 200 mu L of each intermediate liquor, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 100 mu g/mL, and the mixed solution is used as a first mixed reference solution.
(2) Sample processing
Treatment of plasma samples: adding 10 μ L of the first mixed control solution into 190 μ L of blood plasma, mixing, adding 600 μ L of methanol for protein precipitation, centrifuging at 12000 speed for 10min at 4 deg.C, collecting 600 μ L of supernatant, volatilizing, adding 100 μ L of 50% acetonitrile-water for redissolution, and injecting sample.
And respectively taking 10 mu L of the first reference substance solution and 10 mu L of the processed plasma sample, respectively injecting samples, and recording chromatograms which are shown in figure 1 and figure 2.
Example 2
1. Chromatographic conditions
Using Eclipse Plus C 18 (150X 4.6mM,3.5 μm) column, gradient elution was carried out with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as a mixed mobile phase, and gradient elution was carried out as follows: 0min,20% of A;2min,20% by weight of A;5min,30% by weight A;8min, 60. Percent A;10min,60% by volume A;13min,60% by weight A;15min,20% A, see Table 2; the column temperature is 35 ℃, the detection wavelength is 210nm, and the injection volume is 10 mu L.
2. Experimental procedures
(1) Preparing a reference substance:
for the second group of antiepileptic drugs, the formulation process of the control was as follows: 6 antiepileptic drugs (lamotrigine, felbamate, rufinamide, cycabene, carbamazepine and Perampanel) are prepared into 1.0mg/mL mother liquor respectively by adopting 50% acetonitrile-water solution.
And respectively taking 600 mu L of mother liquor of the 6 antiepileptic drugs, adding 400 mu L of acetonitrile into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 600 mu g/mL, taking 200 mu L of each intermediate solution, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 100 mu g/mL, and the mixed solution is used as a second mixed reference solution.
(2) Sample processing
Treatment of plasma samples: adding 10 μ L of the second mixed control solution into 190 μ L of blood plasma, mixing, adding 600 μ L of methanol for protein precipitation, centrifuging at 12000 speed for 10min at 4 deg.C, collecting 600 μ L of supernatant, volatilizing, adding 100 μ L of 50% acetonitrile-water for redissolution, and sampling.
And respectively taking 10 mu L of the second reference substance solution and the treated plasma sample, respectively injecting samples, and recording chromatograms which are shown in figures 3 and 4.
Example 3
1. Chromatographic conditions
Using Eclipse Plus C 18 (150X 4.6mM,3.5 μm) column, gradient elution was carried out with acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) as a mixed mobile phase, and gradient elution was carried out as follows: 0min,20% of A;2min,20% by weight A;3min,28% by weight A;7.5min,28% A;8min, 35. Percent A;8.5min,45% A;9.5min,55% A;11min,60% by volume A;11.5min,65% A;20min,65% A, see Table 3 for details; the column temperature is 35 ℃, the detection wavelength is 210nm, and the injection volume is 10 mu L.
2. Procedure of experiment
(1) Preparation of a reference substance:
for the third group of antiepileptic drugs, the preparation process of the reference substance was as follows: 3 antiepileptic drugs (vigabatrin; pregabalin; gabapentin) were prepared into 1.0mg/mL mother solutions with 50% acetonitrile-water solution, respectively.
Taking 600 mu L of mother liquor of each of the 3 antiepileptic drugs, respectively, adding 400 mu L of acetonitrile into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 600 mu g/mL, taking 200 mu L of each intermediate liquor, respectively, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 200 mu g/mL, and the mixed solution is used as a second mixed reference solution.
(2) Sample processing
Treatment of plasma samples: and (3) adding 10 mu L of third mixed reference substance solution into 190 mu L of blood plasma, uniformly mixing, adding 600 mu L of methanol for protein precipitation, centrifuging at 12000 for 10min at 4 ℃, centrifuging, taking 600 mu L of supernatant, adding 200 mu L of 9-fluorenylmethoxycarbonyl chloride, reacting in a water bath at 60 ℃ for 30min, completely volatilizing, adding 100 mu L of 50% acetonitrile-water for redissolution, and injecting.
And respectively taking 10 mu L of the third reference substance solution and the treated plasma sample, respectively injecting samples, and recording chromatograms which are shown in fig. 5 and fig. 6.
Comparative example 1
Referring to example 1, the mixed mobile phase acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) was replaced with methanol (A) -5mM potassium dihydrogen phosphate solution (B), and the specific chromatogram is shown in FIG. 7.
Comparative example 2
Referring to examples 1, 2 and 3, respectively, the mixed mobile phase acetonitrile (a) -5mM potassium dihydrogen phosphate solution (B) was replaced with acetonitrile (a) -0.1% formic acid solution (B).
Comparative example 3
Referring to examples 1 and 2, respectively, the mixed mobile phase acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) was replaced with acetonitrile (A) -10mM ammonium formate solution (B).
Comparative example 4
Referring to examples 1 and 2, respectively, the mixed mobile phase acetonitrile (A) -5mM potassium dihydrogen phosphate solution (B) was replaced with acetonitrile (A) -10mM ammonium formate solution (B).
Table 4 composition of mixed mobile phase in comparative examples 1 to 4
| Comparative example | Mixed mobile phase |
| Comparative example 1 | Methanol (A) -5mM potassium dihydrogen phosphate solution (B) |
| Comparative example 2 | Acetonitrile (A) -0.1% formic acid solution (B) |
| Comparative example 3 | Acetonitrile (A) -10mM ammonium formate solution (B) |
| Comparative example 4 | Acetonitrile (A) -Water (B) |
In comparative example 1, gradient elution was performed using methanol (a) -5mM potassium dihydrogen phosphate solution (B) as a mixed mobile phase, and the other conditions were the same as in example 1, and the chromatogram of the plasma sample of the first group of 15 antiepileptic drugs showed that the baseline shifted during the elution of the gradient elution, as shown in fig. 4, and the separation of the first group of 15 antiepileptic drugs was poor.
In comparative example 2, the water phase additive was 0.1% formic acid, and the mixed mobile phase was acetonitrile (a) -0.1% formic acid solution (B), and the other conditions were the same as in example 3, and it was found from the chromatogram that: the chromatographic peaks were slightly improved for the plasma samples of the derivatized third group of 3 antiepileptic drugs. However, plasma samples of the first and second groups of antiepileptic drugs that were not required to be derivatized, did not show peaks, and had shifted baselines and poor separation.
In comparative example 3, with acetonitrile (a) -10mM ammonium formate solution (B) as the mixed mobile phase, plasma samples of both the first and second groups of antiepileptic drugs, which did not require derivatization, appeared off-peak and baseline drifts, similar to the case of formic acid as the aqueous additive in comparative example 2.
In comparative example 4, acetonitrile (a) -water (B) was used as a mixed mobile phase for gradient elution, water was used as a water phase directly, and the chromatogram of the plasma sample of the third group of 3 derived antiepileptic drugs was shown in fig. 9, where the 3 antiepileptic drugs had poor chromatographic peak shape and poor separation effect during the elution process of gradient elution.
Comparative example 5
Referring to example 2, column Eclipse Plus C was run 18 The specific chromatogram is shown in FIG. 8, which is replaced by a chromatographic column Zorbax SB-C18. After replacement of the column, zo was foundDetermination of the second group of 6 anti-epileptic drugs and Eclipse Plus C by a rbax SB-C18 column 18 Compared with a chromatographic column, the chromatographic column has poor peak shape, and has the phenomena of unstable base line and trailing peak shape.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. A high performance liquid chromatography analysis method simultaneously suitable for determination of 24 antiepileptic drugs in human plasma is characterized in that the 24 antiepileptic drugs are divided into the following three groups, and the detection is carried out by using a high performance liquid chromatography method, wherein the specific chromatographic conditions are as follows: the chromatographic column is Eclipse Plus C 18 The length of the chromatographic column is 150mm, the diameter of the chromatographic column is 4.6mm, and the particle size of the filler is 3.5 mu m; the detection wavelength is 210 nm; the column temperature was 35 ℃; performing gradient elution by taking a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the mobile phase A is acetonitrile, and the mobile phase B is a 5mM potassium dihydrogen phosphate solution;
the first group of 15 antiepileptic drugs are: piracetam; levetiracetam; ethosuximide; primidone; lacosamide; (iv) an thiazide; zonisamide; b, busulfacetam; phenobarbital; oxcarbazepine; licarbazepine; sodium phenytoin; clonazepam; diazepam; (ii) a spandex; the pretreated plasma containing the first group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,10% A;2min,10% A;3min,28% A;7.5min,28% A;8min,35% A;8.5min,45% A;9.5min,55% A;11min,60% A;11.5min,65% A;20min,65% A; the flow rate is 1.0mL/min;
for the first group of 15 antiepileptic drugs: respectively preparing piracetam, levetiracetam, ethosuximide, primidone, lacosamide, suthiazide, zonisamide, brivaracetam, phenobarbital, oxcarbazepine, licarbazepine, phenytoin sodium, clonazepam, diazepam and spandex into mother liquor with the concentration of 2.0mg/mL by adopting acetonitrile-water solution; respectively taking 750 mu L of mother liquor of the 15 antiepileptic drugs, adding 250 mu L of acetonitrile-water solution into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 1.5mg/mL, then taking 200 mu L of each intermediate solution, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 100 mu g/mL, and the mixed solution is used as a first mixed reference solution;
the pretreated blood plasma containing the first group of antiepileptic drugs is prepared according to the following method: adding the first mixed reference substance solution into the filtered blood plasma, uniformly mixing, adding methanol for precipitation, centrifuging, taking the supernatant, volatilizing, adding acetonitrile-water solution for redissolution, and injecting a sample;
the second group of 6 antiepileptic drugs are: lamotrigine; a felbamate; rufinamide; adding guests for the contest; carbamazepine; perampanel; the pretreated plasma containing the second group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,20% A;2min,20% A;5min,30% A;8min,60% of A;10min,60% A;13min,60% A;15min,20% A; the flow rate is 0.85mL/min within 0-8 min, and the flow rate is 1.0mL/min within 15min after 8 min;
for a second group of 6 antiepileptic drugs: preparing lamotrigine, felbamate, rufinamide, saigatin, carbamazepine and perampanel into mother liquor with the concentration of 1.0mg/mL by adopting acetonitrile-water solution; respectively taking 600 mu L of mother liquor of the 6 antiepileptic drugs, adding 400 mu L of acetonitrile into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 600 mu g/mL, then taking 200 mu L of each intermediate liquor, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 100 mu g/mL, and taking the mixed solution as a second mixed reference solution;
the pretreated blood plasma containing the second group of antiepileptic drugs is prepared according to the following method: adding a second mixed reference substance solution into the filtered plasma, uniformly mixing, adding methanol for precipitation, centrifuging, taking supernatant, volatilizing, adding acetonitrile-water solution for redissolution, and injecting a sample;
the third group of 3 antiepileptic drugs are: vigabatrin; pregabalin; gabapentin; the pretreated plasma containing the third group of antiepileptic drugs is subjected to gradient elution according to the following process: 0min,20% A;2min,20% A;3min,28% of A;7.5min,28% A;8min,35% A;8.5min,45% A;9.5min,55% A;11min,60% A;11.5min,65% A;20min,65% A; the flow rate is 1.0mL/min;
for the third group of 3 antiepileptic drugs: respectively preparing vigabatrin, pregabalin and gabapentin into mother liquor with the concentration of 1.0mg/mL by adopting acetonitrile-water solution; respectively taking 600 mu L of mother liquor of the 3 antiepileptic drugs, adding 400 mu L of acetonitrile into the mother liquor to dilute the mother liquor until the concentration of each antiepileptic drug is 600 mu g/mL, then taking 200 mu L of each intermediate liquor, and uniformly mixing to obtain a mixed solution of each antiepileptic drug, wherein the concentration of each antiepileptic drug is 200 mu g/mL, and the mixed solution is used as a third mixed reference solution;
the pretreated blood plasma containing the third group of antiepileptic drugs is prepared according to the following method: and (3) adding a third mixed reference substance solution into the filtered blood plasma, uniformly mixing, adding methanol for precipitation, centrifuging, taking a supernatant, adding 9-fluorene methoxy carbonyl chloride serving as a derivatization reagent, performing derivatization reaction for 10-60min at 40-80 ℃, volatilizing, adding an acetonitrile-water solution for redissolution, and feeding a sample.
2. The high performance liquid chromatography analysis method for simultaneously adapting to determination of 24 anti-epileptic drugs in human plasma according to claim 1, characterized in that the sample injection volume is 5 to 20 μ L.
3. The high performance liquid chromatography analysis method simultaneously adapted to determination of 24 antiepileptic drugs in human plasma according to claim 2, characterized in that the sample injection volume is 10 μ L.
4. The HPLC analysis method for simultaneously adapting to the determination of 24 antiepileptic drugs in human plasma according to claim 1, wherein the reaction temperature is 60 ℃ during the derivatization reaction; the reaction time was 30min.
5. The HPLC analysis method for simultaneously adapting to the determination of 24 antiepileptic drugs in human plasma according to claim 1, 2, 3 or 4, wherein the volume concentration of acetonitrile in the selected acetonitrile-water solution is 40-60% during the reconstitution process.
6. The HPLC analysis method for simultaneous determination of 24 antiepileptic drugs in human plasma according to claim 5, wherein the volume concentration of acetonitrile in the selected acetonitrile-water solution is 50% during the reconstitution process.
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