阅读说明：本技术 检测血液中苯妥英钠的方法 (Method for detecting phenytoin sodium in blood ) 是由 雒琴 贾永娟 倪君君 于 2019-11-12 设计创作，主要内容包括：本发明的检测血液中苯妥英钠的方法包括：制备苯妥英钠标准储备液、制备苯妥英钠的标准中间液、制备内标工作液、制备标准溶液和利用液相色谱-质谱联用仪检测标准溶液,拟合得到苯妥英钠对应的标准曲线方程为：y<Sub>1</Sub>＝a*x<Sub>1</Sub>+b；对待测血液进行处理,利用液相色谱-质谱联用仪检测待测血液,计算出待测血液中的苯妥英钠浓度。在本发明中,对待测血液样品的处理方法以及内标物的选择,使得苯妥英钠的识别更为准确,分析时间短、干扰小,内标定量适宜特异性强、灵敏度高,同时,回收率,检测限和精密度等各项技术指标均符合要求,从而提高检测结果的准确度,消除系统误差。(The method for detecting the phenytoin sodium in the blood comprises the following steps: preparing a phenytoin sodium standard stock solution, preparing a standard intermediate solution of phenytoin sodium, preparing an internal standard working solution, preparing a standard solution, detecting the standard solution by using a liquid chromatography-mass spectrometer, and fitting to obtain a standard curve equation corresponding to the phenytoin sodium, wherein the standard curve equation is as follows: y is 1 ＝a*x 1 + b; treating the blood to be detected, detecting the blood to be detected by using a liquid chromatogram-mass spectrum combined instrument, and calculating the sodium phenytoin concentration in the blood to be detected. In the invention, the processing method of the blood sample to be detected and the selection of the internal standard substance enable the identification of the phenytoin sodium to be more accurate, the analysis time is short, the interference is small, the internal standard quantification is suitable, the specificity is strong, the sensitivity is high, and meanwhile, all technical indexes such as the recovery rate, the detection limit and the precision meet the requirements, thereby improving the accuracy of the detection result and eliminating the system error.)

1. A method for detecting phenytoin sodium in blood is characterized by comprising the following steps: it comprises the following steps:

calibration of a standard solution

(a) Preparation of Standard stock solution of sodium phenytoin

Accurately weighing 10.0mg of phenytoin sodium standard, placing the phenytoin sodium standard in a 5mL volumetric flask, dissolving the phenytoin sodium standard in a methanol solution with the water content of 0-30%, and fixing the volume to 5mL to obtain a phenytoin sodium standard stock solution, and storing the phenytoin sodium standard stock solution at-80 ℃;

(b) standard intermediate liquid for preparing phenytoin sodium

Diluting the standard stock solution of the phenytoin sodium by using a methanol diluent with the water content of 0-30 percent to prepare a standard intermediate solution of at least three phenytoin sodium containing 12.5-400 mg/L of phenytoin sodium, and storing the standard intermediate solution at-80 ℃;

(c) preparation of internal standard working solution

Diluting the cycloheptat stock solution by using methanol diluent with the water content of 0-30% to obtain internal standard working solution containing 10-50 mg/L of cycloheptat, and storing at-80 ℃;

(d) preparation of Standard solutions

Respectively transferring 10 mu L of the at least three concentrations of phenytoin sodium standard intermediate solution, respectively adding 10 mu L of internal standard working solution and 1090 mu L of methanol into each phenytoin sodium standard intermediate solution, uniformly mixing for 30s-1min at the rotating speed of 1000-2000 rpm, taking 100 mu L of supernatant, adding 400 mu L of methanol, and uniformly mixing for 30s-1min at the rotating speed of 1000-2000 rpm to prepare at least three different concentrations of standard solutions, wherein the internal standard substances contained in each concentration of standard solution have the same concentration;

(e) fitting standard curve equation

Detecting the standard solution with each concentration by using a liquid chromatography-mass spectrometer to obtain the maps of the phenytoin sodium and the cycloheptat of at least three standard solutions with different concentrations; obtaining the chromatographic peak area of phenytoin sodium and the chromatographic peak area of cycloheptat from the spectrum of the standard solution with each concentration, and taking the ratio of the chromatographic peak area of phenytoin sodium to the chromatographic peak area of cycloheptat in the standard solutions with at least three different concentrations as the ordinate y of a standard curve equation₁The ratio of the concentration of phenytoin sodium to the concentration of cycloheptat in the above standard solutions of at least three different concentrations is used as the abscissa x of the standard curve₁Performing linear regression on the detected data with at least three different concentrations, and fitting to obtain a standard curve equation corresponding to the phenytoin sodium, wherein the standard curve equation is as follows: y is₁＝a*x₁+ b and obtaining the weighting coefficients a and b;

(II) treatment of blood samples to be tested

Taking at least 2mL of blood to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, extracting supernatant of the blood to be detected, and storing at-20 ℃;

transferring 10 mu L of the internal standard working solution of the cycloheptat in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing for 30s-1min at the rotating speed of 1500-plus-2000 rpm, adding 1000 mu L of the extract into the centrifuge tube, carrying out vortex oscillation mixing for 5-10min at the rotating speed of 1200-plus-2000 rpm, then carrying out high-speed centrifugation for 5-10min at the rotating speed of 1000-plus-150000 rpm, taking the supernatant, slowly drying the supernatant by using nitrogen, adding 500 mu L of a complex solution, carrying out vortex oscillation mixing for 1-3min at the rotating speed of 1500-plus-2500 rpm, and obtaining the supernatant which is the blood sample to be detected;

(III) detection of blood sample to be detected

Detecting the blood sample to be detected by using a liquid chromatogram-mass spectrometer to obtain the chromatographic peak area of phenytoin sodium and the chromatographic peak area of cycloheptat in the blood sample to be detected; taking the ratio of the chromatographic peak area of the phenytoin sodium in the blood sample to be detected to the chromatographic peak area of the cycloheptat as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of the phenytoin sodium in the blood sample to be detected to the concentration of the cycloheptat is calculated₁Since the concentration of cycloheptat in the blood sample to be tested is known, the concentration of phenytoin sodium in the blood sample to be tested is calculated.

2. The method of claim 1, wherein the method comprises: in step (b), seven kinds of standard intermediate solutions containing sodium phenytoin at different concentrations were prepared, which were standard intermediate solutions containing sodium phenytoin at concentrations of 12.5, 25, 50, 100, 150, 200, and 400mg/L, respectively.

3. The method for detecting sodium phenytoin in blood according to claim 2, wherein: in the step (b) and the step (d), the methanol diluent is a methanol solution with the water content of 30%, and in the step (a), the phenytoin sodium standard substance is dissolved by pure methanol.

4. The method for detecting sodium phenytoin in blood according to claim 3, wherein: in the step (II), the extract is ethyl acetate; the composite solution is pure methanol solution.

5. The method for detecting sodium phenytoin in blood according to claim 4, wherein: in step (c), the cycloheptat stock solution was diluted with a pure methanol diluent to give an internal standard working solution containing 10mg/L cycloheptat.

6. The method for detecting sodium phenytoin in blood according to claim 5, wherein: the chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c8 reverse phase chromatography column; the column temperature is 20-40 ℃; the mobile phase is as follows: the method comprises the following steps of (1) preparing an aqueous solution containing 0.1% formic acid and a pure methanol solution, wherein the volume ratio of the aqueous solution to the pure methanol solution is 45: 55, and the isocratic elution time of the aqueous solution to the methanol solution is not less than 2.5 min; the flow rate of the mobile phase is 0.2-0.8 mL/min.

7. The method for detecting sodium phenytoin in blood according to claim 6, wherein: the mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; the ion source parameters include: the air flow is 8-50L/min, the spraying amount is 20-50L/min, the air temperature is 300-400 ℃, and the capillary tube voltage is 3000-5000V.

8. The method for detecting sodium phenytoin in blood according to claim 7, wherein: the C8 reverse chromatographic column is Agilent Zorbax XDB-C8.

9. The method for detecting sodium phenytoin in blood according to claim 8, wherein: the on-line filter of the liquid chromatography-mass spectrometer is SSI COL PRE-FILTER WATER 1/160.5M.

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for detecting phenytoin sodium in blood.

Background

Phenytoin sodium is widely used clinically for the treatment of generalized tonic-catalepsy seizures, complex partial seizures (psychomotor seizures, temporal lobe epilepsy), simple partial seizures (focal seizures) and status epilepticus. It can also be used for treating trigeminal neuralgia, recessive dystrophic epidermolysis bullosa, paroxysmal chorea athetosis, paroxysmal dyskinesia (behavior disorder including irritability, anxiety and insomnia), myotonia, heart conduction disorder caused by excessive tricyclic antidepressant, etc. It is also suitable for ventricular and supraventricular arrhythmia caused by digitalis poisoning, and has poor curative effect on arrhythmia caused by other reasons. Therefore, the method has important clinical significance for monitoring the phenytoin sodium.

Currently, high performance liquid chromatography is commonly used to test blood samples of patients taking phenytoin sodium.

However, when the sodium phenytoin content in a blood sample is measured by the above-mentioned method, the blood sample needs to be subjected to a pretreatment such as precipitation of proteins. Because the precipitated protein cannot be well treated to remove impurities, the detection time is long, and the detection efficiency of the blood sample is low.

Disclosure of Invention

The invention aims to provide a method for detecting sodium phenytoin in blood, which can improve the detection efficiency of a blood sample.

In order to achieve the purpose of the invention, the following technical scheme is adopted in the application:

the invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: it comprises the following steps:

calibration of a standard solution

(a) Preparation of Standard stock solution of sodium phenytoin

Accurately weighing 10.0mg of phenytoin sodium standard, placing the phenytoin sodium standard in a 5mL volumetric flask, dissolving the phenytoin sodium standard in a methanol solution with the water content of 0-30%, and fixing the volume to 5mL to obtain a phenytoin sodium standard stock solution, and storing the phenytoin sodium standard stock solution at-80 ℃;

(b) standard intermediate liquid for preparing phenytoin sodium

Diluting the standard stock solution of the phenytoin sodium by using a methanol diluent with the water content of 0-30 percent to prepare a standard intermediate solution of at least three phenytoin sodium containing 12.5-400 mg/L of phenytoin sodium, and storing the standard intermediate solution at-80 ℃;

(c) preparation of internal standard working solution

Diluting the cycloheptat stock solution by using methanol diluent with the water content of 0-30% to obtain internal standard working solution containing 10-50 mg/L of cycloheptat, and storing at-80 ℃;

(d) preparation of Standard solutions

Respectively transferring 10 mu L of the at least three concentrations of phenytoin sodium standard intermediate solution, respectively adding 10 mu L of internal standard working solution and 1090 mu L of methanol into each phenytoin sodium standard intermediate solution, uniformly mixing for 30s-1min at the rotating speed of 1000-2000 rpm, taking 100 mu L of supernatant, adding 400 mu L of methanol, and uniformly mixing for 30s-1min at the rotating speed of 1000-2000 rpm to prepare at least three different concentrations of standard solutions, wherein the internal standard substances contained in each concentration of standard solution have the same concentration;

(e) fitting standard curve equation

Detecting the standard solution with each concentration by using a liquid chromatography-mass spectrometer to obtain the maps of the phenytoin sodium and the cycloheptat of at least three standard solutions with different concentrations; obtaining the chromatographic peak area of phenytoin sodium and the chromatographic peak area of cycloheptat from the spectrum of the standard solution with each concentration, and taking the ratio of the chromatographic peak area of phenytoin sodium to the chromatographic peak area of cycloheptat in the standard solutions with at least three different concentrations as the ordinate y of a standard curve equation₁The ratio of the concentration of phenytoin sodium to the concentration of cycloheptat in the above standard solutions of at least three different concentrations is used as the abscissa x of the standard curve₁Performing linear regression on the detected data with at least three different concentrations, and fitting to obtain a standard curve equation corresponding to the phenytoin sodium, wherein the standard curve equation is as follows: y is₁＝a*x₁+ b and obtaining the weighting coefficients a and b;

(II) treatment of blood samples to be tested

Taking at least 2mL of blood to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, extracting supernatant of the blood to be detected, and storing at-20 ℃;

transferring 10 mu L of the internal standard working solution of the cycloheptat in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing for 30s-1min at the rotating speed of 1500-plus-2000 rpm, adding 1000 mu L of the extract into the centrifuge tube, carrying out vortex oscillation mixing for 5-10min at the rotating speed of 1200-plus-2000 rpm, then carrying out high-speed centrifugation for 5-10min at the rotating speed of 1000-plus-150000 rpm, taking the supernatant, slowly drying the supernatant by using nitrogen, adding 500 mu L of a complex solution, carrying out vortex oscillation mixing for 1-3min at the rotating speed of 1500-plus-2500 rpm, and obtaining the supernatant which is the blood sample to be detected;

(III) detection of blood sample to be detected

Detecting the blood sample to be detected by using a liquid chromatogram-mass spectrometer to obtain the chromatographic peak area of phenytoin sodium and the chromatographic peak area of cycloheptat in the blood sample to be detected; taking the ratio of the chromatographic peak area of the phenytoin sodium in the blood sample to be detected to the chromatographic peak area of the cycloheptat as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of the phenytoin sodium in the blood sample to be detected to the concentration of the cycloheptat is calculated₁Since the concentration of cycloheptat in the blood sample to be tested is known, the concentration of phenytoin sodium in the blood sample to be tested is calculated.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: in step (b), seven kinds of standard intermediate solutions containing sodium phenytoin at different concentrations were prepared, which were standard intermediate solutions containing sodium phenytoin at concentrations of 12.5, 25, 50, 100, 150, 200, and 400mg/L, respectively.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: in the step (b) and the step (d), the methanol diluent is a pure methanol solution, and in the step (a), the phenytoin sodium standard substance is dissolved by using pure methanol.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: in the step (II), the extract is ethyl acetate; the composite solution is pure methanol solution.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: in step (c), the cycloheptat stock solution was diluted with a pure methanol diluent to give an internal standard working solution containing 10mg/L cycloheptat.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: the chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c8 reverse phase chromatography column; the column temperature is 20-40 ℃; the mobile phase is as follows: the method comprises the following steps of (1) preparing an aqueous solution containing 0.1% formic acid and a pure methanol solution, wherein the volume ratio of the aqueous solution to the pure methanol solution is 45: 55, and the isocratic elution time of the aqueous solution to the methanol solution is not less than 2.5 min; the flow rate of the mobile phase is 0.2-0.8 mL/min.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: the mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; the ion source parameters include: the air flow is 8-50L/min, the spraying amount is 20-50L/min, the air temperature is 300-400 ℃, and the capillary tube voltage is 3000-5000V.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: the C8 reverse chromatographic column is Agilent Zorbax XDB-C8.

The invention discloses a method for detecting phenytoin sodium in blood, which comprises the following steps: the on-line filter of the liquid chromatography-mass spectrometer is SSI COL PRE-FILTER WATER 1/160.5M.

The invention has the following beneficial effects:

1. in the invention, standard solutions with at least three concentrations are respectively detected by using a liquid chromatography-mass spectrometer, wherein the standard solutions are standard solutions of phenytoin sodium with internal standard substances, and the internal standard substances in the standard solutions with at least three concentrations have consistent concentrations; respectively fitting a standard curve equation of the phenytoin sodium according to the detection results of the standard solutions with at least three concentrations; and (2) carrying out high-speed centrifugation treatment on blood to be detected, adding internal standard substance working solution with the same amount as that in the standard solution into centrifuged supernatant, carrying out vortex oscillation mixing at a high rotating speed, adding an extracting agent, carrying out high-speed vortex oscillation mixing and high-speed centrifugation treatment again, taking supernatant nitrogen to blow dry slowly, adding complex solution, carrying out high-speed vortex oscillation mixing and high-speed centrifugation treatment, so that the solution is uniformly mixed, and obtaining the supernatant with the interference substances removed. The supernatant is detected by using a liquid chromatography-mass spectrometer, and the concentration of the phenytoin sodium in the blood sample to be detected is obtained based on the first detection result of the phenytoin sodium and the standard curve equation of the phenytoin sodium, so that the phenytoin sodium in the blood is detected, and the detection time is effectively shortened.

2. According to the method, the identification of the phenytoin sodium is more accurate, the analysis time is short, the interference is small, the internal standard quantification is suitable, the specificity is strong, the sensitivity is high, meanwhile, all technical indexes such as the recovery rate, the detection limit and the precision meet the requirements, meanwhile, the method for detecting the phenytoin sodium in the blood has good reproducibility and good sample adding recovery rate, so that the accuracy of the detection result is improved, and the system error is eliminated.

Drawings

FIG. 1 is a chemical structural formula of sodium phenytoin PhT provided by the present invention;

FIG. 2 is a chromatogram of phenytoin sodium and cycloheptat in a standard solution provided by the present invention;

FIG. 3 is a mass spectrum of phenytoin sodium and cycloheptat in the standard solution provided by the present invention;

FIG. 4 is a chromatogram of phenytoin sodium and cycloheptat in a blood sample to be tested provided by the present invention;

FIG. 5 is a mass spectrum of phenytoin sodium and cycloheptat in a blood sample to be tested according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings.

The method for detecting the phenytoin sodium in the blood comprises the following steps:

calibration of a standard solution

(a) Preparation of Standard stock solution of sodium phenytoin

Accurately weighing 10.0mg of phenytoin sodium standard, placing the phenytoin sodium standard in a 5mL volumetric flask, dissolving the phenytoin sodium standard in a pure methanol solution, and fixing the volume to 5mL to obtain phenytoin sodium standard stock solution, and storing the phenytoin sodium standard stock solution at the temperature of minus 80 ℃;

(b) standard intermediate liquid for preparing phenytoin sodium

Diluting the standard stock solution of phenytoin sodium with methanol diluent with water content of 30% to prepare seven standard intermediate solutions of phenytoin sodium containing phenytoin sodium with concentration of 12.5mg/L-400mg/L, wherein the seven standard intermediate solutions are standard intermediate solutions containing phenytoin sodium with concentration of 12.5, 25, 50, 100, 150, 200 and 400mg/L respectively, and storing at-80 ℃;

(c) preparation of internal standard working solution

Diluting the cycloheptat stock solution with pure methanol diluent to obtain an internal standard working solution containing 10mg/L of cycloheptat, and storing at-80 ℃;

(d) preparation of Standard solutions

Respectively transferring 10 mu L of the seven phenytoin sodium standard intermediate solutions with different concentrations, respectively adding 10 mu L of internal standard working solution and 1090 mu L of methanol into each phenytoin sodium standard intermediate solution, uniformly mixing the internal standard working solution and 1090 mu L of methanol in a vortex manner at the rotating speed of 1000-2000 rpm for 30s-1min, taking 100 mu L of supernatant, adding 400 mu L of methanol in the supernatant, and uniformly mixing the internal standard working solution and 1090 mu L of methanol in a vortex manner at the rotating speed of 1000-2000 rpm for 30s-1min to prepare at least three standard solutions with different concentrations, wherein the internal standard substances contained in each standard solution with different concentrations have the same concentration;

(e) fitting standard curve equation

Detecting the standard solution with each concentration by using a liquid chromatography-mass spectrometer to obtain the maps of the phenytoin sodium and the cycloheptat of at least three standard solutions with different concentrations; obtaining the chromatographic peak area of phenytoin sodium and the chromatographic peak area of cycloheptat from the spectrum of the standard solution with each concentration, and taking the ratio of the chromatographic peak area of phenytoin sodium to the chromatographic peak area of cycloheptat in the standard solutions with at least three different concentrations as the ordinate y of a standard curve equation₁The ratio of the concentration of phenytoin sodium to the concentration of cycloheptat in the above standard solutions of at least three different concentrations is used as the abscissa x of the standard curve₁Performing linear regression on the detected data with at least three different concentrations, and fitting to obtain a standard curve equation corresponding to the phenytoin sodium, wherein the standard curve equation is as follows: y is₁＝a*x₁+ b and obtaining the weighting coefficients a and b;

(II) treatment of blood samples to be tested

Taking at least 2mL of blood to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, extracting supernatant of the blood to be detected, and storing at-20 ℃;

transferring 10 mu L of the internal standard working solution of the cycloheptatrite in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing for 30s-1min at the rotating speed of 1500-plus-2000 rpm, then adding 1000 mu L of ethyl acetate extract into the centrifuge tube, carrying out vortex oscillation mixing for 5-10min at the rotating speed of 1200-plus-2000 rpm, then carrying out high-speed centrifugation for 5-10min at the rotating speed of 1000-plus-150000 rpm, taking the supernatant, slowly drying the supernatant by using nitrogen, adding 100 mu L of pure methanol solution complex solution, and carrying out vortex oscillation mixing for 1-3min at the rotating speed of 1500-plus-2500 rpm to obtain the supernatant which is the blood sample to be detected;

(III) detection of blood sample to be detected

Detecting the blood sample to be detected by using a liquid chromatogram-mass spectrometer to obtain the chromatographic peak area of phenytoin sodium and the chromatographic peak area of cycloheptat in the blood sample to be detected; taking the ratio of the chromatographic peak area of the phenytoin sodium in the blood sample to be detected to the chromatographic peak area of the cycloheptat as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of the phenytoin sodium in the blood sample to be detected to the concentration of the cycloheptat is calculated₁Since the concentration of cycloheptat in the blood sample to be tested is known, the concentration of phenytoin sodium in the blood sample to be tested is calculated.

The chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the C8 reverse chromatographic column is Agilent Zorbax XDB-C8 reverse chromatographic column; the column temperature is 20-40 ℃; the mobile phase is as follows: the method comprises the following steps of (1) preparing an aqueous solution containing 0.1% formic acid and a pure methanol solution, wherein the volume ratio of the aqueous solution to the pure methanol solution is 45: 55, and the isocratic elution time of the aqueous solution and the methanol solution is not less than 2.5 min; the flow rate of the mobile phase is 0.2-0.8 mL/min.

The mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; the ion source parameters include: the gas flow is 11L/min, the spray flow is 30L/min, the gas temperature is 350 ℃, and the capillary voltage is 3000V.

The on-line filter of the LC-MS is SSI COL PRE-FILTER WATER 1/160.5M.

It is understood that the sample size may be any volume in the range of 0.1. mu.L to 30. mu.L, such as 0.1. mu.L, 0.2. mu.L, 0.5. mu.L, 1. mu.L, 5. mu.L, 10. mu.L, 15. mu.L, 20. mu.L, 25. mu.L and 30. mu.L.

In terms of column temperature, 20 to 40 ℃ means any temperature of 20 ℃ to 40 ℃, for example, 20 ℃, 21 ℃, 22.5 ℃, 23 ℃, 24 ℃, 25 ℃, 27.5 ℃, 29 ℃, 30.5 ℃, 33 ℃, 34 ℃, 35 ℃, 37 ℃, 38.5 ℃, 39 ℃, 40 ℃ and the like, and the detection results are substantially consistent at any temperature within the range.

For a liquid chromatography-mass spectrometer used for detecting phenytoin sodium in blood to be detected, the mass spectrum conditions comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; the ion source parameters include: the air flow is 8-50L/min, the spraying amount is 20-50L/min, the air temperature is 300-400 ℃, and the capillary tube voltage is 3000-5000V.

For the gas flow, 8-50L/min refers to any flow rate from 8L/min to 50L/min, such as 8L/min, 8.5L/min, 9L/min, 11L/min, 14L/min, 19L/min, 24L/min, 28.5L/min, 35L/min, 38.5L/min, 42L/min, 44L/min, 47.5L/min, 49L/min, 50L/min, and the like.

For the spray amount, 20-50L/min refers to any flow rate of 20L/min to 50L/min, such as 20L/min, 22.5L/min, 26L/min, 30L/min, 32L/min, 35.5L/min, 36L/min, 40L/min, 43L/min, 46.5L/min, 50L/min, and the like.

For the gas temperature, 300-400 ℃ refers to any temperature of 300-400 ℃, such as 300 ℃, 320 ℃, 350 ℃, 380 ℃ and 400 ℃.

For the capillary voltage, 3000 to 5000V means any voltage of 3000V to 5000V, for example, 3000V, 3100V, 3300V, 3500V, 3900V, 4000V, 4200V, 4300V, 4500V, 4700V, 4900V, 5000V, and the like.

Further, the mass spectrum parameters of the lc-ms can be further shown in table 1 below:

TABLE 1

Name of substance	Parent ion	Daughter ions	Dwell	Fragmentor	CE	CAV
							Phenytoin sodium	253.1	182.1	200	115	15	7
Phenytoin sodium	253.1	225.1	200	115	7	7
							Cycloheptat	238.1	193.1	200	115	17	3
Cycloheptat	238.1	115.1	200	115	35	5

In the above table, Dwell represents the scan time, Fragmentor represents the cracking voltage, CE represents the collision voltage, and CAV represents the collision cell acceleration voltage. The ion pair 253.1-182.1 of phenytoin sodium is used for quantification, and the ion pair 253.1-225.1 is used for qualitative determination. The ion pair of cycloheptamide 238.1-193.1 was used quantitatively, and the ion pair of 238.1-115.1 was used qualitatively. FIG. 2 shows the chromatogram of phenytoin sodium and cycloheptat in a standard solution of one concentration, and FIG. 3 shows the mass spectrum of phenytoin sodium and cycloheptat in a standard solution of one concentration. Wherein, the number label 1 in fig. 2 is the chromatogram of 253.1-182.1 quantitative ion pair and 253.1-225.1 qualitative ion pair of phenytoin sodium in the standard solution from top to bottom, and the number label 2 is the chromatogram of 238.1-115.1 qualitative ion pair and 238.1-193.1 quantitative ion pair of cycloheptat in the standard solution from top to bottom; in fig. 3, the number 1 is a mass spectrum of 253.1-182.1 quantitative ion pairs and 253.1-225.1 qualitative ion pairs of phenytoin sodium in the standard solution from top to bottom, and the number 2 is a mass spectrum of 238.1-115.1 qualitative ion pairs and 238.1-193.1 quantitative ion pairs of cycloheptat in the standard solution from top to bottom. The retention time of phenytoin sodium and cycloheptat in the standard solution is 0.978min and 1.487min respectively.

The chromatogram of the phenytoin sodium and cycloheptat in the blood sample to be detected in the above embodiment is shown in fig. 4, and the mass spectrum is shown in fig. 5; in fig. 4, numeral 1 indicates a chromatogram of 253.1-182.1 quantitative ion pairs and 253.1-225.1 qualitative ion pairs of phenytoin sodium in the blood sample to be detected in sequence from top to bottom, and numeral 2 indicates a chromatogram of 238.1-115.1 qualitative ion pairs and 238.1-193.1 quantitative ion pairs of cycloheptat in the blood sample to be detected; in fig. 5, the numeral 1 indicates the mass spectra of 253.1-182.1 quantitative ion pairs and 253.1-225.1 qualitative ion pairs of phenytoin sodium in the blood sample to be detected in sequence from top to bottom, and the numeral 2 indicates the mass spectra of 238.1-115.1 qualitative ion pairs and 238.1-193.1 quantitative ion pairs of cycloheptat in the blood sample to be detected. The retention time of the phenytoin sodium and the cycloheptat in the blood sample to be detected is 1.093min and 1.507min respectively.

As can be seen from the comparison of fig. 2, fig. 3, fig. 4 and fig. 5, the retention time of the phenytoin sodium in the blood sample to be tested and the retention time of the phenytoin sodium in the labeling solution are known, and the cycloheptamide is used as the internal standard substance, so that the identification of the phenytoin sodium is more accurate, the analysis time is short, the interference is small, the internal standard quantification is suitable, the specificity is strong, and the accuracy and the sensitivity are high.

In order to prove the linear relationship existing in the solution containing a certain concentration of phenytoin sodium, the following experiments are carried out: adding 10 mu L of internal standard working solution into the prepared standard working solution of the phenytoin sodium with each concentration, adding 1090 mu L of methanol, uniformly mixing and injecting samples, carrying out treatment and detection according to the method, and drawing a graph by using the peak area of a quantitative ion chromatographic peak and the concentration to obtain a standard curve, wherein the result shows that the linear range and the quantitative limit of the phenytoin sodium are as follows:

phenytoin sodium

(1) Detection Line (LOD): 0.25 ng/mL.

(2) Limit of quantitation (LOQ): 1.25 ng/mL.

(3) Linear range: the linearity is good in the range of 1250ng/mL to 40000ng/mL, and the correlation coefficient R2 is larger than 0.999.