阅读说明：本技术 检测血液中艾司西酞普兰的方法 (Method for detecting escitalopram 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艾司西酞普兰的方法包括：制备艾司西酞普兰标准储备液、制备艾司西酞普兰的标准中间液、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,拟合得到艾司西酞普兰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艾司西酞普兰浓度。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,使得艾司西酞普兰的识别更为准确,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 escitalopram in blood comprises the following steps: preparing standard escitalopram stock solution, preparing standard intermediate solution of escitalopram, preparing internal standard working solution, preparing standard solution, detecting the standard solution by using a liquid chromatography-mass spectrometer, and fitting to obtain a standard curve equation corresponding to the escitalopram, wherein the standard curve equation comprises the following steps: y is 1 ＝a*x 1 + b; processing the blood to be detected, detecting the blood to be detected by using a liquid chromatogram-mass spectrometer, and calculating the blood to be detectedThe escitalopram concentration in the blood was measured. 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 escitalopram 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 the detection of escitalopram in blood, characterized in that: it comprises the following steps:

calibration of a standard solution

(a) Preparation of escitalopram Standard stock solution

Accurately weighing 10mg of escitalopram standard, placing the escitalopram standard in a 10mL volumetric flask, dissolving the escitalopram standard in a methanol solution with the water content of 0-40%, and fixing the volume to 10mL to obtain an escitalopram standard stock solution, and storing the standard stock solution at-80 ℃;

(b) standard intermediate solution for the preparation of escitalopram

Diluting the standard escitalopram stock solution with a methanol diluent with the water content of 10-80% to prepare a standard intermediate solution containing at least three escitalopram with the concentration of 25-1500 ng/mL, and storing at-80 ℃;

(c) preparation of internal standard working solution

Diluting 1mg/mL escitalopram-d 6 stock solution by using methanol diluent with the water content of 10-80% to obtain an internal standard working solution containing 100-400 ng/mL escitalopram-d 6, and storing at-80 ℃;

(d) preparation of Standard solutions

Transferring 10 mu L of the escitalopram standard intermediate solution with at least three concentrations, respectively adding 10 mu L of internal standard working solution and 390 mu L of methanol diluent with the water content of 10-80% into each escitalopram standard intermediate solution, and uniformly mixing for 30 s-1 min by vortex at the rotating speed of 1000-2000 rpm 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 spectra of the escitalopram and escitalopram-d 6 of at least three standard solutions with different concentrations; respectively obtaining the area of the chromatographic peak of escitalopram and the area of the chromatographic peak of escitalopram-d 6 from the spectrum of the standard solution with each concentration, and respectively taking the ratio of the area of the chromatographic peak of escitalopram in the at least three standard solutions with different concentrations to the area of the chromatographic peak of escitalopram-d 6 as the ordinate y of a standard curve equation₁The ratio of the concentration of escitalopram in the standard solution of the above-mentioned at least three different concentrations to the concentration of escitalopram-d 6 is taken as the abscissa x of the standard curve₁Performing linear regression on the detected data of at least three different concentrations, and fitting to obtain a standard curve equation corresponding to escitalopram, 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 escitalopram-d 6 obtained in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, performing vortex oscillation mixing at the rotating speed of 1500rpm for 30s, adding 1-3 times of the volume of the supernatant of the blood to be detected into the centrifuge tube, performing vortex oscillation mixing at the rotating speed of 2000rpm for 3min, and performing high-speed centrifugation at the rotating speed of 12000rpm for 10min to obtain the supernatant, namely 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 chromatography-mass spectrometer to obtain the area of a chromatographic peak of escitalopram in the blood sample to be detected and the area of a chromatographic peak of escitalopram-d 6; taking the ratio of the chromatographic peak area of escitalopram in the blood sample to be detected to the chromatographic peak area of escitalopram-d 6 as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of escitalopram in the blood sample to be tested to the concentration of escitalopram-d 6 is calculated₁Since the concentration of escitalopram-d 6 in the blood sample to be tested is known, the escitalopram concentration in the blood sample to be tested is calculated.

2. The method for the detection of escitalopram in blood according to claim 1, wherein: in step (b), seven standard intermediate solutions containing escitalopram at different concentrations were prepared, which were standard intermediate solutions containing escitalopram at concentrations of 25, 50, 100, 250, 500, 1000, 1500ng/mL, respectively.

3. Method for the detection of escitalopram in blood according to claim 2, characterised in that: in step (b) and step (d), the methanol diluent is a methanol solution with a water content of 30%, and in step (a), the escitalopram standard is dissolved in pure methanol.

4. Method for the detection of escitalopram in blood according to claim 3, characterised in that: in the second step, the volume of the added precipitated protein reagent acetonitrile is 3 times of the volume of the supernatant of the blood to be detected.

5. Method for the detection of escitalopram in blood according to claim 4, characterised in that: in step (c), the escitalopram-d 6 stock solution was diluted with a methanol diluent containing 30% water to obtain an internal standard working solution containing escitalopram-d 6 at a concentration of 300 ng/mL.

6. Method for the detection of escitalopram in blood according to claim 5, characterised in that: 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 volume ratio of the ammonium acetate aqueous solution to the methanol solution is 4: 6, and the isocratic elution time of the ammonium acetate aqueous solution and the methanol solution is not less than 2.0 min; the flow rate of the mobile phase is 0.2-0.8 mL/min.

7. Method for the detection of escitalopram in blood according to claim 6, characterised in that: 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 3000-4000V.

8. Method for the detection of escitalopram in blood according to claim 7, characterised in that: the C8 reverse chromatographic column is Agilent Zorbax XDB-C8.

9. Method for the detection of escitalopram in blood according to claim 8, characterised in that: 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 escitalopram in blood.

Background

Escitalopram is the levorotatory enantiomer of racemic citalopram, belongs to a selective 5-hydroxytryptamine reuptake inhibitor, and has the action mechanism of enhancing the 5-hydroxytryptamine (5-HT) performance of the central nervous system and inhibiting the reuptake of 5-HT. Is widely applied to the treatment of Major Depressive Disorder (MDD) and generalized anxiety clinically, so the method has important clinical significance for the monitoring of escitalopram.

At present, the blood samples of patients taking escitalopram are usually tested by high performance liquid chromatography. However, the detection of escitalopram content in a blood sample by the above-mentioned method requires a pre-treatment, e.g. an extraction, of the blood sample. 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 escitalopram 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 relates to a method for detecting escitalopram in blood, which comprises the following steps: it comprises the following steps:

calibration of a standard solution

(a) Preparation of escitalopram Standard stock solution

Accurately weighing 10mg of escitalopram standard, placing the escitalopram standard in a 10mL volumetric flask, dissolving the escitalopram standard in a methanol solution with the water content of 0-40%, and fixing the volume to 10mL to obtain an escitalopram standard stock solution, and storing the standard stock solution at-80 ℃;

(b) standard intermediate solution for the preparation of escitalopram

Diluting the standard escitalopram stock solution with a methanol diluent with the water content of 10-80% to prepare a standard intermediate solution containing at least three escitalopram with the concentration of 25-1500 ng/mL, and storing at-80 ℃;

(c) preparation of internal standard working solution

Diluting 1mg/mL escitalopram-d 6 stock solution by using methanol diluent with the water content of 10-80% to obtain an internal standard working solution containing 100-400 ng/mL escitalopram-d 6, and storing at-80 ℃;

(d) preparation of Standard solutions

Transferring 10 mu L of the escitalopram standard intermediate solution with at least three concentrations, respectively adding 10 mu L of internal standard working solution and 390 mu L of methanol diluent with the water content of 10-80% into each escitalopram standard intermediate solution, and uniformly mixing for 30 s-1 min by vortex at the rotating speed of 1000-2000 rpm 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 spectra of the escitalopram and escitalopram-d 6 of at least three standard solutions with different concentrations; respectively obtaining the area of the chromatographic peak of escitalopram and the area of the chromatographic peak of escitalopram-d 6 from the spectrum of the standard solution with each concentration, and respectively taking the ratio of the area of the chromatographic peak of escitalopram in the at least three standard solutions with different concentrations to the area of the chromatographic peak of escitalopram-d 6 as the ordinate y of a standard curve equation₁The ratio of the concentration of escitalopram in the standard solution of the above-mentioned at least three different concentrations to the concentration of escitalopram-d 6 is taken as the abscissa x of the standard curve₁Performing linear regression on the detected data of at least three different concentrations, and fitting to obtain a standard curve equation corresponding to escitalopram, 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 escitalopram-d 6 obtained in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, performing vortex oscillation mixing at the rotating speed of 1500rpm for 30s, adding 1-3 times of the volume of the supernatant of the blood to be detected into the centrifuge tube, performing vortex oscillation mixing at the rotating speed of 2000rpm for 3min, and performing high-speed centrifugation at the rotating speed of 12000rpm for 10min to obtain the supernatant, namely 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 chromatography-mass spectrometer to obtain the area of a chromatographic peak of escitalopram in the blood sample to be detected and the area of a chromatographic peak of escitalopram-d 6; taking the ratio of the chromatographic peak area of escitalopram in the blood sample to be detected to the chromatographic peak area of escitalopram-d 6 as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of escitalopram in the blood sample to be tested to the concentration of escitalopram-d 6 is calculated₁Since the concentration of escitalopram-d 6 in the blood sample to be tested is known, the escitalopram concentration in the blood sample to be tested is calculated.

The invention relates to a method for detecting escitalopram in blood, which comprises the following steps: in step (b), seven standard intermediate solutions containing escitalopram at different concentrations were prepared, which were standard intermediate solutions containing escitalopram at concentrations of 25, 50, 100, 250, 500, 1000, 1500ng/mL, respectively.

The invention relates to a method for detecting escitalopram in blood, which comprises the following steps: in step (b) and step (d), the methanol diluent is a methanol solution with a water content of 30%, and in step (a), the escitalopram standard is dissolved in pure methanol.

The invention relates to a method for detecting escitalopram in blood, which comprises the following steps: in the second step, the added precipitated protein reagent acetonitrile is 3 times of the volume of the supernatant of the blood to be detected.

The invention relates to a method for detecting escitalopram in blood, which comprises the following steps: in step (c), the escitalopram-d 6 stock solution was diluted with a methanol diluent containing 30% water to obtain an internal standard working solution containing escitalopram-d 6 at a concentration of 300 ng/mL.

The invention relates to a method for detecting escitalopram 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 volume ratio of the ammonium acetate aqueous solution to the methanol solution is 4: 6, and the isocratic elution time of the ammonium acetate aqueous solution and the methanol solution is not less than 2.0 min; the flow rate of the mobile phase is 0.2-0.8 mL/min.

The invention relates to a method for detecting escitalopram 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 3000-4000V.

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

The invention relates to a method for detecting escitalopram 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 escitalopram with an internal standard substance, and the concentrations of the internal standard substances in the standard solutions with at least three concentrations are consistent; respectively fitting a standard curve equation of escitalopram according to the detection results of the standard solutions with at least three concentrations; and (3) carrying out high-speed centrifugation treatment on the 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, and carrying out high-speed vortex oscillation mixing and high-speed centrifugation treatment again to uniformly mix the solution to obtain the supernatant from which the interferents are removed. The liquid chromatogram-mass spectrum combination instrument is used for detecting the supernatant, and the concentration of the escitalopram in the blood sample to be detected is obtained based on the first detection result of the escitalopram and the standard curve equation of the escitalopram, so that the detection of the escitalopram 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 escitalopram 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, meanwhile, all technical indexes such as the recovery rate, the detection limit and the precision meet the requirements, and meanwhile, the method for detecting escitalopram in blood is good in reproducibility and good in sample adding recovery rate, so that the accuracy of the detection result is improved, and the system error is eliminated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a chemical structural formula of escitalopram ESC of the present invention;

figure 2 is a chromatogram of escitalopram and escitalopram-d 6 in a standard solution according to the invention;

figure 3 is a mass spectrum of escitalopram and escitalopram-d 6 in a standard solution according to the invention;

FIG. 4 is a chromatogram of escitalopram and escitalopram-d 6 from a blood sample to be tested according to the invention;

figure 5 is a mass spectrum of escitalopram and escitalopram-d 6 in a blood sample to be tested according to the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.

The method for detecting escitalopram in blood comprises the following steps:

calibration of a standard solution

(a) Preparation of escitalopram Standard stock solution

Accurately weighing 10mg of escitalopram standard, placing the escitalopram standard in a 10mL volumetric flask, dissolving the escitalopram standard in a pure methanol solution, fixing the volume to 10mL to obtain an escitalopram standard stock solution, and storing the standard stock solution at the temperature of minus 80 ℃;

(b) standard intermediate solution for the preparation of escitalopram

Diluting the escitalopram standard stock solution with a methanol diluent with the water content of 30 percent to prepare seven escitalopram standard intermediate solutions containing escitalopram with the concentration of 25ng/mL-1500ng/mL, wherein the seven escitalopram standard intermediate solutions respectively contain the concentration of 25, 50, 100, 250, 500, 1000 and 1500ng/mL escitalopram and are stored at the temperature of-80 ℃;

(c) preparation of internal standard working solution

Diluting escitalopram-d 6 stock solution with methanol diluent with water content of 30% to obtain an internal standard working solution containing 300ng/mL escitalopram-d 6, and storing at-80 ℃;

(d) preparation of Standard solutions

Transferring 10 mu L of the seven standard escitalopram intermediate solutions with different concentrations, respectively adding 10 mu L of an internal standard working solution and 390 mu L of a methanol diluent with the water content of 30 percent into each standard escitalopram intermediate solution, and uniformly mixing for 30 s-1 min in a vortex manner at the rotating speed of 1000-2000 rpm to prepare seven 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 spectra of escitalopram and escitalopram-d 6 of the standard solutions with seven different concentrations; respectively obtaining the area of the chromatographic peak of escitalopram and the area of the chromatographic peak of escitalopram-d 6 from the spectrum of the standard solution with each concentration, and respectively taking the ratio of the area of the chromatographic peak of escitalopram in the seven standard solutions with different concentrations to the area of the chromatographic peak of escitalopram-d 6 as the ordinate y of a standard curve equation₁The ratio of the concentration of escitalopram to the concentration of escitalopram-d 6 in the above seven standard solutions of different concentrations 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 escitalopram, 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 escitalopram-d 6 obtained in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, performing vortex oscillation mixing at the rotating speed of 1500rpm for 30s, adding 3 times of the volume of the precipitated protein reagent acetonitrile of the supernatant of the blood to be detected into the centrifuge tube, performing vortex oscillation mixing at the rotating speed of 2000rpm for 3min, and performing high-speed centrifugation at the rotating speed of 12000rpm for 10min to obtain the supernatant, namely 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 escitalopram in the blood sample to be detected and the area of the peakPeak area of the chromatographic peak of escitalopram-d 6; taking the ratio of the chromatographic peak area of escitalopram in the blood sample to be detected to the chromatographic peak area of escitalopram-d 6 as y₁Substituting into y obtained in step (e)₁＝a*x₁In the formula + b, the ratio x of the concentration of escitalopram in the blood sample to be tested to the concentration of escitalopram-d 6 is calculated₁Since the concentration of escitalopram-d 6 in the blood sample to be tested is known, the escitalopram concentration in the blood sample to be tested is calculated.

The chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c8 reverse phase chromatography columns, for example: agilent Zorbax XDB-C8; the column temperature is 20-40 ℃; the mobile phase is as follows: the volume ratio of the ammonium acetate aqueous solution to the methanol solution is 4: 6, and the isocratic elution time of the ammonium acetate aqueous solution and the methanol solution is not less than 2.0 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 12L/min, the spray amount is 30L/min, the gas temperature is 350 ℃, and the capillary voltage is 3500V.

In the embodiment of the invention, the internal standard substance and the precipitated protein reagent in the same amount as those in the standard solution are added into the blood sample to be detected, high-speed centrifugation is carried out, and the centrifuged supernatant is taken, so that escitalopram in the blood sample to be detected can be retained to the maximum extent, and the interferents are removed.

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 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, 3000-4000V means any voltage of 3000V to 4000V, for example, 3000V, 3100V, 3300V, 3500V, 3900V, 4000V, 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	Fragmcntor	CE	CAV
							Escitalopram	325.2	262.1	200	125	18	7
Escitalopram	325.2	109.0	200	125	26	7
							Escitalopram-d 6	331.2	262.1	200	125	18	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. Escitalopram 325.2-262.1 ion pair was used quantitatively and 325.2-109.0 ion pair was used qualitatively.

Generally, the preparation mode of the mixed standard solution directly affects the accuracy of detection, and the preparation mode of the standard solution selected by the embodiment of the invention can ensure the accuracy of the detection result.

The chromatograms of escitalopram and escitalopram-d 6 in a standard solution of one concentration from the above examples are shown in figure 2 and the mass spectrum is shown in figure 3. Wherein, the number label 1 in the figure 2 is the chromatogram of the qualitative ion pair 325.2-109.0 and the quantitative ion pair 325.2-262.1 of escitalopram in the standard solution from top to bottom, and the number label 2 is the chromatogram of escitalopram-d 6 in the standard solution; the number label 1 in figure 3 is the mass spectrum of qualitative ion pair 325.2-109.0 and quantitative ion pair 325.2-262.1 of escitalopram in the standard solution, and the number label 2 is the mass spectrum of escitalopram-d 6 in the standard solution, from top to bottom. The retention times of escitalopram and escitalopram-d 6 in the standard solution were 1.093min and 1.070min, respectively.

Further, the on-line filter used by the liquid chromatography-mass spectrometer is as follows: SI COL PRE-FILTER WATER 1/160.5M.

The chromatograms of escitalopram and escitalopram-d 6 in the blood sample to be tested in the above embodiments are shown in fig. 4, and the mass spectrograms are shown in fig. 5; the numerical identifier 1 in the figure 4 is the chromatogram of the qualitative ion pair 325.2-109.0 and the quantitative ion pair 325.2-262.1 of escitalopram in the blood sample to be detected in turn from top to bottom, and the numerical identifier 2 is the chromatogram of escitalopram-d 6 in the blood sample to be detected; the numerical identifier 1 in fig. 5 is a mass spectrum of qualitative ion pair 325.2-109.0 and quantitative ion pair 325.2-262.1 of escitalopram in the blood sample to be tested in sequence from top to bottom, and the numerical identifier 2 is a mass spectrum of escitalopram-d 6 in the blood sample to be tested. The retention time of escitalopram and escitalopram-d 6 in the blood sample to be tested is 1.095min and 1.060min respectively.

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

To demonstrate the linear relationship existing for escitalopram at a certain concentration in the solutions of the present application, the following experiments were carried out: adding 10 mu L of internal standard working solution into the prepared standard working solution of escitalopram with each concentration, adding 300 mu L of acetonitrile, uniformly mixing and injecting, detecting according to the processing and detecting conditions of the embodiment 2, and drawing by using the peak area and the concentration of the quantitative ion chromatographic peak to obtain a standard curve, wherein the results show that the linear range and the quantitative limit of the escitalopram are as follows:

escitalopram

(1) Detection Line (LOD): 0.033 ng/mL.

(2) Limit of quantitation (LOQ): 0.1 ng/mL.

(3) Linear range: the linearity is good in the range of 25ng/L to 1500ng/L, and the correlation coefficient R2 is larger than 0.998.

Preparing standard intermediate solution containing escitalopram into high, medium and low 3 concentrations respectively, performing sample-adding recovery rate experiment and precision experiment, detecting under the same detection conditions, and repeatedly analyzing and determining for 3 batches, wherein the recovery rate and precision of escitalopram are shown in the following table 2:

TABLE 2

Adding quantity of scalar	5ng/mL	25ng/mL	100ng/mL
				Average recovery rate	100.4％	100.12％	102.71％
Precision RSD	2.92％	1.39％	1.99％

As can be seen from Table 2, the average recovery of escitalopram in the range of 3 addition levels of low, medium and high was 100.12% to 102.71%, and the relative standard deviation was 1.39% to 2.92%.

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

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.