阅读说明：本技术 检测血液中丙戊酸的方法 (Method for detecting valproic acid 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丙戊酸的方法包括：制备丙戊酸标准储备液、制备丙戊酸的标准中间液、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,拟合得到丙戊酸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-质谱联用仪检测对待测血液,计算出待测血液中的丙戊酸浓度。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,使得丙戊酸的识别更为准确,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 valproic acid in blood comprises the following steps: preparing a valproic acid standard stock solution, preparing a standard intermediate solution of valproic acid, 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 valproic acid as follows: y is 1 ＝a*x 1 + b; and treating the blood to be detected, detecting the blood to be detected by using a liquid chromatography-mass spectrometer, and calculating the concentration of valproic acid in the blood to be detected. In the inventionIn the method, the processing method of the blood sample to be detected and the selection of the internal standard substance enable the identification of the valproic acid to be more accurate, the analysis time to be short, the interference to be small, the internal standard quantification to be suitable, the specificity to be strong, the sensitivity to be high, meanwhile, all technical indexes such as the recovery rate, the detection limit and the precision to be in line with the requirements, thereby improving the accuracy of the detection result and eliminating the system error.)

1. A method for detecting valproic acid in blood, comprising: it comprises the following steps:

calibration of a standard solution

(a) Preparation of a Valproic acid Standard stock solution

Transferring the purchased certified valproic acid standard solution to a 2mL freezing storage tube to obtain 1mg/mL valproic acid standard stock solution, and storing at-80 ℃;

(b) preparation of a Standard intermediate for Valproic acid

Diluting the standard valproic acid stock solution by using a methanol diluent with the water content of 10-80% to prepare a standard intermediate solution of at least three kinds of valproic acid with the concentration of 2.5-160 mg/L of valproic acid, and storing the standard intermediate solution at-80 ℃;

(c) preparation of internal standard working solution

Diluting 1mg/mL valproic acid-d 6 stock solution by using methanol diluent with the water content of 10-80% to obtain internal standard working solution containing 10-50 mg/L valproic acid-d 6, and storing at-80 ℃;

(d) preparation of Standard solutions

Respectively transferring 10 mu L of the at least three concentrations of valproic acid standard intermediate solution, respectively adding 10 mu L of internal standard working solution and 180 mu L of methanol diluent with the water content of 70% into each valproic acid standard intermediate solution, and uniformly mixing by vortex at the rotating speed of 1000-2000 rpm for 30 s-1 min 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 valproic acid and valproic acid-d 6 of at least three standard solutions with different concentrations; respectively obtaining the chromatographic peak area of valproic acid and the chromatographic peak area of valproic acid-d 6 from the chromatogram of the standard solution with each concentration, and respectively using the chromatographic peak areas of valproic acid in the standard solutions with at least three different concentrationsThe ratio of the peak area of the chromatographic peak of valproic acid-d 6 is used as the ordinate y of the standard curve equation₁Taking the ratio of the concentration of valproic acid in the standard solution with at least three different concentrations to the concentration of valproic acid-d 6 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 valproic acid, 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 valproic acid-d 6 in the step (c) into a 1.5mL centrifuge tube, adding 10 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing for 30s at the rotating speed of 1500rpm, adding 80 mu L of a protein precipitation reagent into the centrifuge tube, carrying out vortex oscillation mixing for 3min at the rotating speed of 2000rpm so as to achieve the purpose of fully precipitating the protein, carrying out high-speed centrifugation for 10min at the rotating speed of 10000-15000rpm to obtain the supernatant, taking 50 mu L of the supernatant, and adding 50 mu L of a methanol solution with the water content of 70% to obtain a blood sample to be detected;

(III) detection of blood sample to be detected

Detecting the blood sample to be detected by using a liquid chromatography-mass spectrometer to obtain the chromatographic peak area of valproic acid in the blood sample to be detected and the chromatographic peak area of valproic acid-d 6; taking the ratio of the chromatographic peak area of the valproic acid in the blood sample to be detected to the chromatographic peak area of valproic acid-d 6 as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of the valproic acid in the blood sample to be detected to the concentration of the valproic acid-d 6 is calculated₁Since the concentration of valproic acid-d 6 in the blood sample is known, the concentration of valproic acid in the blood sample is calculated.

2. The method of detecting valproic acid in blood according to claim 1, wherein: in step (b), seven standard intermediate solutions containing different concentrations of valproic acid were prepared, which were standard intermediate solutions containing concentrations of valproic acid of 2.5, 5, 10, 20, 40, 80, and 160mg/L, respectively.

3. The method for detecting valproic acid in blood according to claim 2, wherein: in step (b), the methanol diluent is a methanol solution with 70% water content.

4. The method for detecting valproic acid in blood according to claim 3, wherein: in step (two), the protein precipitation reagent added is acetonitrile.

5. The method for detecting valproic acid in blood according to claim 4, wherein: in step (c), the valproic acid-d 6 stock solution was diluted with a 70% methanol dilution containing water to give an internal standard working solution containing 20mg/L valproic acid-d 6.

6. The method for detecting valproic acid in blood according to claim 5, wherein: the chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c18 reverse phase chromatography column; the column temperature is 20-40 ℃; the mobile phase is as follows: an aqueous solution containing 8mM ammonium acetate and a pure methanol solution, wherein the gradient elution time of the aqueous solution and the pure methanol solution is not less than 6.0 min; the flow rate of the mobile phase is 0.2-0.5 mL/min.

7. The method for detecting valproic acid 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 valproic acid in blood according to claim 7, wherein: the C8 reverse phase chromatographic column is Agilent Poroshell120 EC-C18.

9. The method for detecting valproic acid 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 valproic acid in blood.

Background

Valproic acid has unique chemical structural formula and pharmacological action, and is effective on various epilepsy such as petit mal, myoclonic epilepsy, focal seizure, grand mal and mixed epilepsy. The method is mainly used for various epileptic patients with ineffective antiepileptic drugs, and especially for the patients with epilepsy with small seizures, so the method has important clinical significance for monitoring valproic acid.

Currently, high performance liquid chromatography is generally used to test blood samples of patients taking valproic acid.

However, when the valproic acid content in a blood sample is detected by the above-mentioned method, the blood sample needs to be subjected to a pretreatment such as extraction. The detection time is long due to the long time of the extraction process, which results in low detection efficiency of the blood sample.

Disclosure of Invention

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

The method for detecting valproic acid in blood of the present invention comprises: it comprises the following steps:

calibration of a standard solution

(a) Preparation of a Valproic acid Standard stock solution

Transferring the purchased certified valproic acid standard solution to a 2mL freezing storage tube to obtain 1mg/mL valproic acid standard stock solution, and storing at-80 ℃;

(b) preparation of a Standard intermediate for Valproic acid

Diluting the standard valproic acid stock solution by using a methanol diluent with the water content of 10-80% to prepare a standard intermediate solution of at least three kinds of valproic acid with the concentration of 2.5-160 mg/L of valproic acid, and storing the standard intermediate solution at-80 ℃;

(c) preparation of internal standard working solution

Diluting 1mg/mL valproic acid-d 6 stock solution by using methanol diluent with the water content of 10-80% to obtain internal standard working solution containing 10-50 mg/L valproic acid-d 6, and storing at-80 ℃;

(d) preparation of Standard solutions

Respectively transferring 10 mu L of the at least three concentrations of valproic acid standard intermediate solution, respectively adding 10 mu L of internal standard working solution and 180 mu L of methanol diluent with the water content of 70% into each valproic acid standard intermediate solution, and uniformly mixing by vortex at the rotating speed of 1000-2000 rpm for 30 s-1 min 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 valproic acid and valproic acid-d 6 of at least three standard solutions with different concentrations; respectively obtaining the chromatographic peak area of valproic acid and the chromatographic peak area of valproic acid-d 6 from the chromatogram of the standard solution with each concentration, and respectively taking the ratio of the chromatographic peak area of valproic acid in the standard solutions with at least three different concentrations to the chromatographic peak area of valproic acid-d 6 as the ordinate y of a standard curve equation₁The ratio of the concentration of valproic acid to the concentration of valproic acid-d 6 in the standard solution of the above-mentioned at least three different concentrations was taken 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 valproic acid, 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 valproic acid-d 6 in the step (c) into a 1.5mL centrifuge tube, adding 10 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing for 30s at the rotating speed of 1500rpm, adding 80 mu L of a protein precipitation reagent into the centrifuge tube, carrying out vortex oscillation mixing for 3min at the rotating speed of 2000rpm so as to achieve the purpose of fully precipitating the protein, carrying out high-speed centrifugation for 10min at the rotating speed of 10000-15000rpm to obtain the supernatant, taking 50 mu L of the supernatant, and adding 50 mu L of a methanol solution with the water content of 70% to obtain a blood sample to be detected;

(III) detection of blood sample to be detected

Detecting the blood sample to be detected by using a liquid chromatography-mass spectrometer to obtain the chromatographic peak area of valproic acid in the blood sample to be detected and the chromatographic peak area of valproic acid-d 6; taking the ratio of the chromatographic peak area of the valproic acid in the blood sample to be detected to the chromatographic peak area of valproic acid-d 6 as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of the valproic acid in the blood sample to be detected to the concentration of the valproic acid-d 6 is calculated₁Since the concentration of valproic acid-d 6 in the blood sample is known, the concentration of valproic acid in the blood sample is calculated.

The method for detecting valproic acid in blood of the present invention comprises: in step (b), seven standard intermediate solutions containing different concentrations of valproic acid were prepared, which were standard intermediate solutions containing concentrations of valproic acid of 2.5, 5, 10, 20, 40, 80, and 160mg/L, respectively.

The method for detecting valproic acid in blood of the present invention comprises: in step (b), the methanol diluent is a methanol solution with 70% water content.

The method for detecting valproic acid in blood of the present invention comprises: in step (two), the protein precipitation reagent added is acetonitrile.

The method for detecting valproic acid in blood of the present invention comprises: in step (c), the valproic acid-d 6 stock solution was diluted with a 70% methanol dilution containing water to give an internal standard working solution containing 20mg/L valproic acid-d 6.

The method for detecting valproic acid in blood of the present invention comprises: the chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c18 reverse phase chromatography column; the column temperature is 20-40 ℃; the mobile phase is as follows: an aqueous solution containing 8mM ammonium acetate and a pure methanol solution, wherein the gradient elution time of the aqueous solution and the pure methanol solution is not less than 6.0 min; the flow rate of the mobile phase is 0.2-0.5 mL/min.

The method for detecting valproic acid in blood of the present invention comprises: 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 method for detecting valproic acid in blood of the present invention comprises: the C18 reverse phase chromatographic column is Agilent Poroshell120 EC-C18.

The method for detecting valproic acid in blood of the present invention comprises: 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 valproic acid standard solutions 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 valproic acid 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 the centrifuged supernatant, carrying out vortex oscillation mixing at a high rotating speed, adding a precipitated protein reagent, carrying out high-speed vortex oscillation mixing and high-speed centrifugation treatment again to uniformly mix the solution to obtain the supernatant with the interference substances removed, and adding a certain amount of diluent into the supernatant. The diluted solution is detected by using a liquid chromatography-mass spectrometer, and the concentration of the valproic acid in the blood sample to be detected is obtained based on the first detection result of the valproic acid and the standard curve equation of the valproic acid, so that the detection of the valproic acid in the blood is realized, and the detection time is effectively shortened.

2. According to the method, the processing method of the blood sample to be detected and the selection of the internal standard substance enable the identification of the valproic acid to be more accurate, the analysis time is short, the interference is small, the internal standard is suitable for quantification, 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 valproic acid in the blood is good in reproducibility, the sampling recovery rate is good, the accuracy of the detection result is improved, and the system error is eliminated.

Drawings

FIG. 1 is a chemical structural formula of VPA, valproic acid, according to an embodiment of the present invention;

FIG. 2 is a chromatogram of valproic acid and valproic acid-d 6 in a standard solution provided by an embodiment of the invention;

FIG. 3 is a mass spectrum of valproic acid and valproic acid-d 6 in a standard solution provided by an embodiment of the present invention;

FIG. 4 is a chromatogram of valproic acid and valproic acid-d 6 in a blood sample to be assayed according to an embodiment of the present invention;

FIG. 5 is a mass spectrum of valproic acid and valproic acid-d 6 in a blood sample to be tested according to an embodiment of the present invention.

Detailed Description

The technical scheme of the invention is clearly and completely described below with reference to the accompanying drawings.

The method for detecting valproic acid in blood comprises the following steps:

calibration of a standard solution

(a) Preparation of a Valproic acid Standard stock solution

Transferring the purchased certified valproic acid standard solution to a 2mL freezing storage tube to obtain 1mg/mL valproic acid standard stock solution, and storing at-80 ℃;

(b) preparation of a Standard intermediate for Valproic acid

Diluting the standard stock solution of valproic acid with a methanol diluent with the water content of 70 percent to prepare seven standard intermediate solutions of valproic acid with the valproic acid concentration of 2.5 mg/L-160 mg/L, wherein the seven standard intermediate solutions are the standard intermediate solutions with the valproic acid concentration of 2.5, 5, 10, 20, 40, 80 and 160mg/L respectively and are stored at the temperature of minus 80 ℃;

(c) preparation of internal standard working solution

Diluting 1mg/mL valproic acid-d 6 stock solution with 70% methanol diluent to obtain an internal standard working solution containing 20mg/L valproic acid-d 6, and storing at-80 deg.C;

(d) preparation of Standard solutions

Respectively transferring 10 mu L of the seven kinds of concentration valproic acid standard intermediate solutions, respectively adding 10 mu L of internal standard working solution and 180 mu L of methanol diluent with the water content of 70% into each kind of valproic acid standard intermediate solution, and uniformly mixing by vortex for 30 s-1 min at the rotating speed of 1000-2000 rpm to prepare seven kinds of standard solutions with different concentrations, wherein the internal standard substances contained in each kind of concentration 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 valproic acid and the valproic acid-d 6 of the standard solutions with seven different concentrations; respectively obtaining the chromatographic peak area of valproic acid and the chromatographic peak area of valproic acid-d 6 from the chromatogram of the standard solution with each concentration, and respectively taking the ratio of the chromatographic peak area of valproic acid in the seven standard solutions with different concentrations to the chromatographic peak area of valproic acid-d 6 as the ordinate y of a standard curve equation₁The ratio of the concentration of valproic acid in the seven standard solutions with different concentrations to the concentration of valproic acid-d 6 is taken as the abscissa x of the standard curve₁Performing linear regression on the seven detected data with different concentrations, and fitting to obtain a standard curve equation corresponding to valproic acid, 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 valproic acid-d 6 in the step (c) into a 1.5mL centrifuge tube, adding 10 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing for 30s at the rotating speed of 1500rpm, adding 80 mu L of the protein precipitation reagent acetonitrile into the centrifuge tube, carrying out vortex oscillation mixing for 3min at the rotating speed of 2000rpm so as to achieve the purpose of fully precipitating the protein, carrying out high-speed centrifugation for 10min at the rotating speed of 10000-15000rpm to obtain the supernatant, taking 50 mu L of the supernatant, and adding 50 mu L of a methanol solution with the water content of 70% to obtain a blood sample to be detected;

(III) detection of blood sample to be detected

Detecting the blood sample to be detected by using a liquid chromatography-mass spectrometer to obtain the chromatographic peak area of valproic acid in the blood sample to be detected and the chromatographic peak area of valproic acid-d 6; taking the ratio of the chromatographic peak area of the valproic acid in the blood sample to be detected to the chromatographic peak area of valproic acid-d 6 as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of the valproic acid in the blood sample to be detected to the concentration of the valproic acid-d 6 is calculated₁Since the concentration of valproic acid-d 6 in the blood sample is known, the concentration of valproic acid in the blood sample is calculated.

The chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the C18 reverse phase chromatographic column is an Agilent Poroshell120 EC-C18 reverse phase chromatographic column; the column temperature is 20-40 ℃; the mobile phase is as follows: an aqueous solution containing 8mM ammonium acetate and a pure methanol solution, wherein the volume ratio of the aqueous solution to the pure methanol solution is 45: 55 in 0.00min, the volume ratio of the aqueous solution to the pure methanol solution is 45: 55 in 1.60min, the volume ratio of the aqueous solution to the pure methanol solution is 20: 80 in 1.61min, the volume ratio of the aqueous solution to the pure methanol solution is 20: 80 in 3.00min, the volume ratio of the aqueous solution to the pure methanol solution is 45: 55 in 3.01min, the volume ratio of the aqueous solution to the pure methanol solution is 45: 55 in 6.00min, and the gradient elution time of the aqueous solution to the methanol solution is not less than 6.0 min; gradient elution time of the aqueous solution and the pure methanol solution is not less than 6.0 min; the flow rate of the mobile phase is 0.2-0.5 mL/min.

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.

Aiming at a liquid chromatogram-mass spectrum combination instrument used for detecting valproic acid 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-5000-3000V.

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 ℃.

The capillary voltage of-5000 to-3000V means any voltage of-5000V to-3000V, for example, -3000V, -3100V, -3300V, -3500V, -3900V, -4000V, -4200V, -4300V, -4500V, -4700V, -4900V, or-5000V.

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
							Valproic acid	143	143	200	100	0	7
Valproic acid-d 6	149	149	200	100	0	7

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 chromatogram of valproic acid and valproic acid-d 6 in a standard solution in the above example is shown in FIG. 2, and the mass spectrum is shown in FIG. 3; wherein, numeral 1 in fig. 2 is a chromatogram of valproic acid in the standard solution, and numeral 2 is a chromatogram of valproic acid-d 6 in the standard solution; in FIG. 3, numeral 1 is a mass spectrum of valproic acid in the standard solution, and numeral 2 is a mass spectrum of valproic acid-d 6 in the standard solution. The retention times of valproic acid and valproic acid-d 6 in the standard solution were 2.620min and 2.634min, respectively.

The chromatogram of valproic acid and valproic acid-d 6 in the blood sample to be tested in the above example is shown in FIG. 4, and the mass spectrum is shown in FIG. 5; wherein, the numeral identifier 1 in fig. 4 is a chromatogram of valproic acid in the blood sample to be tested, and the numeral identifier 2 is a chromatogram of valproic acid-d 6 in the blood sample to be tested; in fig. 5, numeral 1 is a mass spectrum of valproic acid in the blood sample to be tested, and numeral 2 is a mass spectrum of valproic acid-d 6 in the blood sample to be tested. The retention time of valproic acid and valproic acid-d 6 in the blood sample to be tested is 2.690min and 2.734min respectively.

As can be seen from the comparison of fig. 2, fig. 3, fig. 4 and fig. 5, the retention time of valproic acid in the blood sample to be tested and the retention time of valproic acid in the labeling solution are known, and valproic acid-d 6 is used as the internal standard substance, so that the identification of valproic acid is more accurate, the analysis time is short, the interference is small, the internal standard is suitable for quantification, the specificity is strong, and the accuracy and the sensitivity are high.

To demonstrate the linear relationship that exists between certain concentrations of valproic acid in the solutions of the present application, the following experiments were performed:

adding 10 mu L of internal standard working solution into the prepared standard working solution of valproic acid with each concentration, adding 180 mu L of methanol diluent with 70% of water content, uniformly mixing and injecting, detecting according to the processing method and the detection conditions, and drawing by using the peak area of the 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 valproic acid are as follows:

valproic acid

(1) Detection Line (LOD): 0.167 mg/L.

(2) Limit of quantitation (LOQ): 0.5 mg/L.

(3) Linear range: the linearity is good in the range of 2.5mg/L to 160mg/L, and the correlation coefficient R2 is more than 0.998.

The standard yeast mixed intermediate solution containing valproic acid is prepared into high, medium and low 3 concentrations respectively to carry out sample adding recovery rate experiments and precision experiments, detection is carried out according to the processing method and the detection conditions, 3 batches of samples are repeatedly analyzed and determined, and the recovery rate and precision of valproic acid are shown in the following table 2:

TABLE 2

Adding quantity of scalar	5mg/L	20mg/L	80mg/L
				Average recovery rate	100.96％	100.84％	100.50％
Precision RSD	1.69％	0.86％	0.58％

As shown in Table 2, the average recovery rate of valproic acid was 100.50-100.96% and the relative standard deviation was 0.58-1.69% in the range of 3 addition levels, i.e., low, medium and high.

By integrating the verification tests, the technical indexes such as detection limit, recovery rate and precision and the like of the method meet the requirements, the method for detecting the concentration of valproic acid in blood has good reproducibility and high sample recovery rate, and the accuracy of the detection result is improved.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.