CN112986452B - Method for determining tandospirone concentration in human plasma - Google Patents
Method for determining tandospirone concentration in human plasma Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to the technical field of medicine detection, and discloses a method for determining the concentration of tandospirone in human plasma. The invention overcomes the defect of accurate quantitative detection of lower concentration level and can meet the requirement of content determination in organisms with smaller dosage.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for determining the concentration of tandospirone in human plasma.
Background
The main action parts of tandospirone are concentrated in the hippocampus, amygdala and other brain marginal systems of the emotional center and the raphe nucleus of the nerve projecting 5-HT energy. By stimulating 5-HT1A autoreceptor, 5-HT nervine projected from the central suture to hippocampus is regulated, and 5-HT effect of behavior suppression system is inhibited to exert anxiolytic effect. Since the binding site of tandospirone is relatively concentrated, an anxiolytic effect with higher selectivity can be exerted. Has good therapeutic effect on symptoms such as dysphoria with symptoms of nerve disorder, essential hypertension, peptic ulcer, insomnia, etc. Especially, hypertension, peptic ulcer, insomnia and the like are often only treated for symptoms, the treatment course is long, the curative effect is not obvious, and the anxiolytic can relieve the psychological factors causing the diseases and improve the curative effect by introducing the anxiolytic into the treatment of the diseases (called psychosomatic symptoms in Japan), so that the effect is remarkable.
At present, high performance liquid chromatography is mainly used, and the lower detection limit cannot meet the content measurement in organisms at a small dosage. CN200810135089.7 discloses a method for detecting different configurations of tandospirone citrate by adopting high performance liquid chromatography to realize the control of the quality of the preparation, which cannot realize the measurement of small dosage in organisms; the literature (Huanghongyong, pharmaceutical guidance, volume 32, No. 6 of 2013, No. 32, 707-710) discloses a human body bioequivalence experiment of two tandospirone citrate preparations, a high performance liquid chromatography method is adopted to detect the content of tandospirone citrate in human plasma, the linearity is good within the range of 10-1000 mug/L, the lowest detection limit is 10 mug/L, accurate quantitative detection of a lower level cannot be realized, and the limitation exists in the actual application process.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for measuring the concentration of tandospirone in human plasma, which adopts an LC-MS/MS method to measure the concentration of tandospirone in human plasma, overcomes the defect of accurate quantitative detection of a lower concentration level, can meet the content measurement requirement in organisms with smaller dosage, has the challenge that the lower limit of the quantification is only 3 pg/mL, has signal-to-noise ratio meeting the standard requirement in the detection process, has no interference of impurity peaks, and has high content accuracy of measured medicines.
In order to solve the technical problems, the invention adopts the following technical scheme:
in one aspect, the present invention provides a method for determining the concentration of tandospirone in human plasma, comprising the steps of: detecting tandospirone in a preprocessed human plasma sample by adopting a high performance liquid chromatography-tandem mass spectrometry technology, separating the tandospirone from impurities by utilizing high performance liquid chromatography, quantifying by utilizing an isotope internal standard method, taking the concentration ratio of a standard substance in a standard solution and an internal standard substance in an internal standard working solution as an X axis, and obtaining the standard substance and the internal standard substance in the standard solutionAnd taking the peak area ratio of the internal standard substance in the working solution as the Y axis, establishing a standard curve regression equation, performing linear least square regression calculation by using the peak area ratio of the object to be measured and the internal standard substance as the theoretical concentration ratio of the standard substance and the internal standard substance in the standard curve regression equation, and calculating the actual measurement concentration of tandospirone in the human plasma sample by using the obtained standard curve regression equation. According to the expression of the invention, the standard substance is a tandospirone standard substance; the internal standard substance is tandospirone-d 8 (ii) a The object to be detected is tandospirone in a human plasma sample to be detected.
Further, the chromatographic conditions of the high performance liquid tandem mass spectrometry technology are as follows:
a chromatographic column: phenomenex Gemini 3 mu m C6-Phenyl 110A
Mobile phase A: 5mM ammonium acetate; mobile phase B: acetonitrile;
flow rate: 0.500mL/min to 0.700 mL/min;
washing the needle washing liquid: 10 s; sample introduction amount: 10 mu L of the solution; column temperature: 35 ℃;
needle washing liquid: 50% methanol; washing the needle washing liquid: 10 s; sample introduction amount: 7 mu L of the solution; column temperature: 35 ℃; stopping time: 5.50 min.
The mobile phase A and the mobile phase B adopted by the invention have important significance for improving response, cannot pollute a mass spectrometer, and cannot influence the service life of the mass spectrometer.
Further, the chromatographic conditions of the high performance liquid tandem mass spectrometry technology further comprise a mobile phase proportion which is set as follows:
within 0.00-3.00 min, the volume ratio of the mobile phase A to the mobile phase B is 70: 30, the flow rate is 0.500 mL/min;
within 3.00-3.10 min, the volume ratio of the mobile phase A to the mobile phase B is 20: 80, the flow rate is 0.500 mL/min;
within 3.10-3.11 min, the volume ratio of the mobile phase A to the mobile phase B is 5: 95 at a flow rate of 0.500 mL/min;
within 3.11-4.50 min, the volume ratio of the mobile phase A to the mobile phase B is 5: 95 at a flow rate of 0.700 mL/min;
and within 4.50-4.51 min, the volume ratio of the mobile phase A to the mobile phase B is 20: 80, the flow rate is 0.700 mL/min;
within 4.51-5.50 min, the volume ratio of the mobile phase A to the mobile phase B is 70: 30 at a flow rate of 0.500 mL/min.
Further, the chromatographic conditions of the high performance liquid tandem mass spectrometry technology further include mass spectrometry conditions, specifically:
ESI source is adopted, ionization mode: electrospray ionization positive ion mode (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM);
the parameters for tandospirone are set as follows: q1 Mass: 384.000Da, Q3 Mass: 122.300Da, Dwell: 100.00msec, DP: 100, EP: 10, CE: 39, CXP: 10, Polarity: positive;
tandospirone-d 8The parameter settings are as follows: q1 Mass: 392.200Da, Q3 Mass: 122.000Da, Dwell: 100.00msec, DP: 100, EP: 10, CE: 39, CXP: 10, Polarity: positive;
the mass spectrum switching valve is set as follows: 1) Total time: 1.0 min, Position: b; 2) Total time: 3.5 min, Position: a;
the ion source parameters were set as follows: IS: 3500.00, respectively; gas 1: 60.00; gas 2: 80.00; CAD: 9.00; and (4) CUR: 35.00; temp (° C): 600.00.
further, after the detection is carried out by using the high performance liquid tandem mass spectrometry technology, the retention time of the standard substance tandospirone is 2.56 min, and the internal standard substance tandospirone-d 8The retention time is 2.51 min, the peak appears, the peak shape is good, and no impurity peak in human plasma interferes the determination of the compound, which shows that the method has stronger specificity.
Further, the standard curve regression equation is as follows:
the regression equation of the standard curve of the measured tandospirone in human plasma is as follows: y =0.00185X-4.44e-005, R is 0.9991; the linear range of tandospirone is 3.000-6000 pg/mL, and the lowest quantitative lower limit of the blood concentration of tandospirone is 3.000 pg/mL.
Further, the pretreated human plasma sample is prepared by the following method: and (3) taking human plasma in a sample hole, adding an internal standard working solution, adding a precipitator, performing vortex and centrifugation, and performing sample injection LC-MS/MS system detection.
Further, the pretreated human plasma sample is prepared by the following method: taking human plasma in a sample hole, adding 50 mu L of internal standard working solution, adding 300 mu L of precipitator, sealing plates, vortexing for 3min, centrifuging, 2450g, and carrying out 4 ℃ for 10 min; and (5) detecting by a 10-mu-L sample injection LC-MS/MS system.
Further, the standard solution formulation process is as follows: weighing tandospirone in a volumetric flask, and dissolving with methanol to prepare a standard reference substance stock solution; diluting the standard reference substance stock solution with 50% acetonitrile aqueous solution to prepare a standard series of tandospirone working solution; and (3) taking standard series of tandospirone working solutions, respectively diluting the standard series of tandospirone working solutions into blank plasma, and preparing the standard solutions of tandospirone with series concentrations.
And further, before detection of the standard solution, adding an internal standard working solution for dilution, adding a precipitator, performing vortex, centrifuging, and performing sample injection LC-MS/MS system detection.
Further, the internal standard working solution is prepared by the following steps: weighing tandospirone-d 8Dissolving in a volumetric flask by methanol to prepare an internal standard reference substance stock solution; diluting the stock solution of the internal standard reference substance with 50% acetonitrile aqueous solution to prepare tandospirone-d 8An internal standard working solution.
Further, the concentration of the standard reference substance stock solution is 1 mg/mL-1. Further, the precipitating agent is acetonitrile.
Further, the concentration range of the standard series tandospirone working solution is 0.06-120 ng/mL; the concentration range of the tandospirone working solution in the standard curve working solution is preferably 120, 102, 60, 12, 2.4, 0.6, 0.12 and 0.06 ng/mL.
Further, in the standard curve plasma sample, the concentration range of tandospirone is 3-6000 pg/mL, and preferably 3, 6, 30, 120, 600, 3000, 5100 and 6000 pg/mL.
Further, tandospirone-d 8The concentration was 3 ng/mL.
In another aspect, the invention is implemented by the following scheme: a method for determining the concentration of tandospirone in human plasma, comprising the steps of:
step 1) preparing a standard solution and an internal standard working solution;
step 2) detecting the standard solution by adopting a high performance liquid tandem mass spectrometry technology, separating the standard substance and the internal standard substance by utilizing high performance liquid chromatography, and establishing a standard curve regression equation by taking the concentration ratio of the standard substance in the standard solution and the internal standard substance in the internal standard working solution as an X axis and the peak area ratio of the standard substance in the standard solution and the internal standard substance in the internal standard working solution as a Y axis;
step 3) preparing a pretreated human plasma sample;
and 4) detecting tandospirone in the preprocessed human plasma sample by adopting a high performance liquid chromatography-tandem mass spectrometry technology, separating the tandospirone from impurities by utilizing high performance liquid chromatography, performing linear least square regression calculation by comparing peak areas of the object to be detected and the internal standard substance with theoretical concentration ratios of the standard substance and the internal standard substance in a standard curve regression equation, and calculating the actually measured concentration of the tandospirone in the human plasma sample by using the obtained standard curve regression equation.
The invention selects a chromatographic column: phenomenex Gemini 3 mu m C6-Phenyl 110A; the method is used for separating non-polar protein compounds, and is suitable for determining that the response value of low concentration of tandospirone in plasma is higher, the peak shape is better, the tandospirone is symmetrical and not trailing, and the peak-off time is not too far ahead or too far behind. Compared with the octadecylsilane chemically bonded silica gel column adopted in the prior art, the chromatographic column can be easily eluted, and the experimental time is shortened.
Considering the influence of the flow rate on the drug peak-out time and the influence of the peak-out effect caused by the overlarge sample injection amount, the flow rate is selected to be 0.500 mL/min-0.700 mL/min, and the sample injection amount is 10 mu L, so that the drug peak-out effect and the response value can be ensured, the drug can be completely eluted, the pollution to a chromatographic column is avoided, and the recovery rate and the sensitivity of mass spectrometry detection can be improved.
The invention adopts a gradient elution mode to detect tandospirone in plasma, the elution proportion of a mobile phase is changed at different time, and the problems of plasma matrix effect and the like are comprehensively considered, so that the peak shape of a drug is symmetrical and perfect, the drug can be sufficiently eluted, the recovery rate is high, and the interference is small.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for determining the concentration of tandospirone in human plasma, which adopts a liquid chromatography-mass spectrometry combined method for detection, combines the separation capability of a liquid chromatograph for effectively separating different compounds and the strong component identification capability of a mass spectrometer, can effectively separate and identify the tandospirone in the plasma, and carries out quantitative detection. Compared with the existing liquid phase detection method, the detection lower limit of the method can meet the content determination requirement in organisms with smaller dosage, the signal-to-noise ratio in the detection process meets the standard requirement, the interference of impurity peaks is avoided, and the content accuracy of the determined medicine is high.
Under the chromatographic conditions adopted in the experiment, the retention time of tandospirone is about 2.56 min, and tandospirone-d 8The retention time is about 2.51 min, the peak shape is good, the measurement is not interfered by the miscellaneous peak, and the base line is stable; the method has high specificity, can accurately determine the concentration of tandospirone in blood plasma, has high sensitivity, and has the minimum limit of blood plasma quantification of 3.000 pg/mL; the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of tandospirone. The linear range of the plasma standard curve of the tandospirone in the method is as follows: 3.000-6000 pg/mL, and the precision RSD in and among batches is less than 9.1%. The method has good reproducibility and high accuracy, is not interfered by a substrate, a hyperlipemia substrate and a hemolysis substrate, and has no interference phenomenon of the blood plasma of different crowds to the method.
The method can meet the pharmacokinetic requirement of human bodies and can be applied to clinical pharmacokinetic research.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a standard graph of tandospirone in human plasma by LC-MS/MS;
FIG. 2 is a tonogram of tandospirone in human plasma by LC-MS/MS;
FIG. 3LC-MS/MS method for detecting tandospirone-in human plasmad 8Liquid mass diagram of (1);
FIG. 4 shows a scanning mass spectrum of tandospirone positive ion multi-reaction detection;
FIG. 5 tandospirone-d 8And (3) detecting and scanning a mass spectrogram by positive ion multi-reaction.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
1. Main instrument
API 6500 Plus type triple quadrupole liquid chromatograph-mass spectrometer equipped with electrospray ionization source and analysis (Version 1.6.3) data processing system, equipped with Agilent1260 autosampler, liquid chromatograph, column oven (Agilent, USA)
METTLER TOLEDO XPR2/A type millionth-one-day (METTLER, Switzerland)
METTLER TOLEDO AB135-S type one hundred thousand balance (METTLER, Switzerland)
One ten thousandth of a balance of Sartorius model BS224S (beijing sidoris).
2. Chromatographic conditions
A chromatographic column: phenomenex Gemini 3 mu m C6-Phenyl 110A; mobile phase A: 5mM ammonium acetate; mobile phase B: acetonitrile; flow rate: 0.500mL/min to 0.700 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: 35 ℃; needle washing liquid: 50% methanol; washing the needle washing liquid: 10 s; sample introduction amount: 7 mu L of the solution; column temperature: 35 ℃; stopping time: 5.50 min.
The flow phase ratio is set as follows:
within 0.00-3.00 min, the volume ratio of the mobile phase A to the mobile phase B is 70: 30, the flow rate is 0.500 mL/min;
within 3.00-3.10 min, the volume ratio of the mobile phase A to the mobile phase B is 20: 80, the flow rate is 0.500 mL/min;
within 3.10-3.11 min, the volume ratio of the mobile phase A to the mobile phase B is 5: 95 at a flow rate of 0.500 mL/min;
within 3.11-4.50 min, the volume ratio of the mobile phase A to the mobile phase B is 5: 95 at a flow rate of 0.700 mL/min;
and within 4.50-4.51 min, the volume ratio of the mobile phase A to the mobile phase B is 20: 80, the flow rate is 0.700 mL/min;
within 4.51-5.50 min, the volume ratio of the mobile phase A to the mobile phase B is 70: 30 at a flow rate of 0.500 mL/min.
3. Conditions of Mass Spectrometry
ESI source is adopted, ionization mode: electrospray ionization positive ion mode (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM);
the parameters for tandospirone are set as follows: q1 Mass: 384.000Da, Q3 Mass: 122.300Da, Dwell: 100.00msec, DP: 100, EP: 10, CE: 39, CXP: 10, Polarity: positive;
tandospirone-d 8The parameter settings are as follows: q1 Mass: 392.200Da, Q3 Mass: 122.000Da, Dwell: 100.00msec, DP: 100, EP: 10, CE: 39, CXP: 10, Polarity: positive;
the mass spectrum switching valve is set as follows: 1) Total time: 1.0 min, Position: b; 2) Total time: 3.5 min, Position: a;
the ion source parameters were set as follows: IS: 3500.00, respectively; gas 1: 60.00; gas 2: 80.00; CAD: 9.00; and (4) CUR: 35.00; temp (° C): 600.00.
4. anticoagulant: heparin sodium
5. Internal standard: tandospirone-d 8
6. Data processing
Chromatographic retention time and chromatographic peak area were collected and processed by Analyst (Version1.6.3) and quantified. And (3) performing linear least square regression calculation according to the ratio of the peak area of the analyte to the peak area of the internal standard to the theoretical concentration of the analyte in the standard curve to the concentration of the internal standard, and calculating the actually measured concentration of the analyte in the sample according to the obtained regression equation.
7. The pretreatment method before the detection of the sample comprises the following steps:
taking whole blood, adding anticoagulant heparin sodium, treating by a protein precipitation method to prepare blank plasma, refrigerating for sample dilution or preparation, and unfreezing before use.
Adding 50 mu L of internal standard working solution before using a 100 mu L standard curve plasma sample, adding 300 mu L of acetonitrile, performing vortex for 3min, centrifuging, 2450g, 4 ℃, and 10min for sample introduction.
Adding 50 mu L of internal standard working solution before 100 mu L SST is used, adding 300 mu L acetonitrile, rotating for 3min, centrifuging, 2450g, 4 ℃, 10min, and waiting for sample injection.
Adding 50 mu L of internal standard working solution before using a 100 mu L quality control plasma sample, adding 300 mu L of acetonitrile, rotating for 3min, centrifuging, 2450g, 4 ℃, 10min, and waiting for sample injection.
Adding 50 mu L of 50% acetonitrile solution before using 100 mu L of blank plasma, adding 300 mu L of acetonitrile, rotating for 3min, centrifuging, 2450g, 4 ℃, 10min, and waiting for sample injection. Recording as follows: double blank or Carryover samples.
Or adding 50 mu L of internal standard working solution before 100 mu L of blank plasma is used, adding 300 mu L of acetonitrile, rotating for 3min, centrifuging, 2450g, 4 ℃, 10min, and waiting for sample injection. Recording as follows: blank sample.
Adding 50 mu L of 50% acetonitrile solution before using 100 mu L of ultrapure water, adding 300 mu L of acetonitrile, rotating for 3min, centrifuging, 2450g, 4 ℃, 10min, and waiting for sample injection. Recording as follows: reagent and Materials blank samples.
Remarking: double blank for Double blank, carryover for residual investigation, blank for single blank with internal standard only, Reagent and Materials blank for Reagent consumables acceptance.
Example 1
1. Solution preparation
Mobile phase a (5 mM aqueous ammonium acetate): 770.8mg of ammonium acetate was weighed out and dissolved in 2L of ultrapure water, degassed by ultrasound, and mixed well.
Mobile phase B (acetonitrile): 2L of acetonitrile was measured into a brown solvent bottle.
Diluted solution (50% acetonitrile in water): 100mL of acetonitrile and 100mL of ultrapure water were transferred to an appropriate solvent bottle and mixed well.
Needle washing solution preparation (50% methanol aqueous solution): 500mL of methanol and 500mL of ultrapure water were weighed out and transferred to an appropriate solvent bottle, followed by mixing.
2. Preparing a standard solution:
preparation of standard reference substance stock solution
Weighing a certain amount of tandospirone reference substance, recording the weight, placing the weighed tandospirone reference substance into self-made cup-shaped aluminum foil paper, placing the aluminum foil paper into a 20mL brown wide-mouth glass bottle, calculating the actual weight of an analyte according to the actual weighing value and the tandospirone content, adding a proper amount of methanol, finally preparing the tandospirone with the concentration of 1.000mg/mL, and shaking up. Storing at-20 deg.C.
Table 1: preparation of standard curve working solution
Table 2: preparation of Standard Curve plasma samples
3. Preparation of internal standard solution
Internal standard stock solution preparation
1039 muL of methanol is added to dissolve the reference substance with the specification of 1.04mg, and the reference substance is shaken up to obtain the internal standard stock solution with the concentration of 1.000mg/mL, and the internal standard stock solution is stored at the temperature of minus 20 ℃.
Internal standard working solution preparation
Tandospirone-taking-d 8 Adding 990 μ L of 50% acetonitrile water into an IS Stock 10 μ L-1.5 mL centrifuge tube, and uniformly mixing to obtain IS Spike1 (10 μ g/mL);
and taking 30 mu L of IS Spike1 (10 mu g/mL) to a 100mL volumetric flask, adding 50% acetonitrile water to the volume to be calibrated, and uniformly mixing to obtain IS Spike (3 ng/mL).
4. Preparation of quality control standard solution
Preparing a quality control reference substance stock solution:
weighing a certain amount of tandospirone reference substance, recording the weight, placing the weighed tandospirone reference substance into self-made cup-shaped aluminum foil paper, placing the aluminum foil paper into a 20mL brown wide-mouth glass bottle, calculating the actual weight of an analyte according to the actual weighing value and the tandospirone content, adding a proper amount of methanol, finally preparing the tandospirone with the concentration of 1.000mg/mL, and shaking up. Storing at-20 deg.C.
Table 3: preparation of quality control working solution
Table 4: preparation of quality control plasma sample
The following quality control L/M/H QC concentration is consistent with the precision and the accuracy L/M/HOQ QC concentration, and the preparation method is the same as the above.
5. Methodology validation
5.1 Linear Range and lower quantitative limits
Taking 10 muL of each standard series working solution, diluting the standard series working solutions into blank plasma to enable the total volume to be 0.2 mL, wherein the concentration value of tandospirone in a prepared standard curve plasma sample is as follows: 3.000 pg/mL.
Taking 100 mu L of blank plasma to Double blank, blank and carryover sample holes, taking 100 mu L of ultrapure water to RB sample holes, and taking 100 mu L of each verification sample to the corresponding sample hole.
Taking 50 mu L of IS Spike diluent (50% acetonitrile) to RB, Double blank and carryover sample wells; and taking 50 mu L of IS Spike solution to the corresponding sample well.
And taking 300 mu L of acetonitrile precipitant to the corresponding sample hole.
Plate sealed, vortex for 3 min.
Centrifugation at 4 ℃ and 2450g for 10 min.
10 μ L of sample was introduced into the chromatography system for analysis.
And (4) conclusion: the linear curve of the obtained standard curve sample is shown in figure 1, wherein the regression equation of the standard curve of tandospirone in human plasma measured by an LC-MS/MS method is y =0.00185X-4.44e-005, and R is 0.9991; an average of 7 determinations, SD of R was 0.0005., CV was 0.1%; slope SD of.0.00056, CV of 32.8%; the linear range of the tandospirone is 3.000-6000 pg/mL, the lowest quantitative lower limit of the blood concentration of the tandospirone is 3.000pg/mL, and the linear relation in the linear range is good.
5.2 precision and accuracy
Four quality control plasma samples with different concentrations were taken and tested, respectively, for HOQ QC (4800 pg/mL), MOQ QC (200.0 pg/mL), LOQ QC (8.000 pg/mL), and LLOQ (3.000 pg/mL). As a review of accuracy and precision within and between batches. Each concentration was prepared in 6 replicates and tested at least three times. The mean values were used to assess batch accuracy and precision.
Table 5: LC-MS/MS method for determining precision and accuracy of tandospirone in and among batches in plasma
Injecting: this point is unresponsive.
The results show that: LLOQ concentration level Standard plasma samples should be measured within the range of 80.0% to 120.0% of the standard value, and within-batch to inter-batch precision (RSD) should be no greater than 20%. The measured values of the low, medium and high concentration level standard plasma samples should be within the range of 85.0% -115.0% of the standard values, and the batch-to-batch precision (RSD) is less than 15.0%. The method has good precision and accuracy in the detection of tandospirone in plasma.
5.3 System applicability:
taking a pretreated standard plasma sample with the concentration of 3.000pg/mL, wherein the concentration of the standard plasma sample is the lower limit of quantitation (LLOQ); 10 μ L of sample injection detection is repeatedly analyzed for 5 times;
and respectively recording chromatographic peak area values and retention time of the object to be detected and the internal standard, and calculating the chromatographic peak area ratio of the object to be detected and the internal standard and the variation coefficient of the chromatographic peak retention time.
Table 6: system applicability of LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) method for determining tandospirone in plasma
And (4) conclusion: the signal to noise ratio (S/N is not less than 5) of the substance to be detected, and the variation coefficient of the ratio of the area of the substance to be detected to the area of the internal standard chromatographic peak obtained by 5 times of repeated analysis is less than 15%; the variation coefficient of the retention time of the sample to be tested and the internal standard chromatographic peak obtained by 5 times of repeated analysis is less than 5%. The retention time variation coefficient of the tested substance tandospirone is 0.3-0.6%, the retention time variation coefficient of the internal standard is 0.1-0.7%, and the variation coefficient of the area ratio is 4.0-6.4%; the method is applicable to the detection of tandospirone in heparin sodium plasma.
5.4 Selectivity
Selectivity refers to the ability to distinguish interference in a biological matrix when an analyte is measured by chromatographic methods. Before the method is validated, at least 6 different batches of blank substrates need to be screened for method validation.
Taking 100 muL of blank plasma from different sources of 6 Chinese people, adding no internal standard, and sampling 10 muL into a chromatographic system for analysis.
Taking 100 mu L of 6 blank plasma from different sources, preparing a standard plasma sample with tandospirone concentration as a quantitative lower limit concentration level, wherein the concentration of the standard plasma sample is 3.000pg/mL, and after pretreatment, injecting 10 mu L of the sample into a chromatographic system for analysis;
the results were recorded for each blank sample and for the standard plasma sample for the lower limit concentration level of quantitation, respectively.
Table 7: comparison table for selective investigation of blank healthy human plasma from six different sources on analytes and internal standards
And (4) conclusion: the peak area of the chromatographic peak at the retention time of the analyte in at least 6 blank matrix samples from different sources is lower than 20.0% of the peak area of the to-be-measured substance at the corresponding blank matrix LLOQ concentration from different sources, and the peak area of the internal standard at the retention time of the internal standard is lower than 5.0% of the peak area of the internal standard at the corresponding blank matrix LLOQ concentration from different sources. The measured value of the LLOQ concentration prepared by 6 blank matrixes from different sources is 80.0-120.0% of the theoretical value. The method provided by the invention aims at the interference range of the analyte in the blank matrix samples from different sources to be 0%, and the internal standard interference response value is 0%. Therefore, blank plasma of different human bodies does not interfere with the detection result of the tandospirone, the method can be used for detecting the tandospirone in heparin sodium plasma of different human bodies, and the plasma of different people has no influence on the detection method.
5.5 recovery
5.5.1 extraction recovery of analytes
Analyte recovery sample
The concentrations are respectively: LOQ QC (8.000 pg/mL), MOQ QC (200.0 pg/mL), HOQ QC (4800 pg/mL) tandospirone quality control plasma samples, and 6 parts are prepared in parallel;
recovery reference sample:
the extraction process is the same as the extraction operation for the extraction recovery sample, and the concentration of the extraction recovery sample is the same as the concentration of the initial yield reference sample, but the analyte and internal standard solution do not participate in the extraction process.
5.5.2 extraction recovery of internal standard
Extraction recovery sample of internal standard
6 parts of blank plasma samples are prepared in parallel, internal standard working solution is added, after precipitation, supernatant solution with proper volume is taken, and standard solution with concentration equivalent to that of the medium-concentration quality control sample is added.
Recovery of internal standard reference sample:
the extraction procedure was the same as for the internal standard extraction recovery samples, but both the analyte and internal standard solutions were added after precipitation.
And (4) conclusion: 1) the extraction recovery rates of tandospirone in the high, medium and low concentration ranges are respectively 95.4, 97.3 and 96.4, and the corresponding coefficient of variation CV values are 0.7, 5.1 and 3.8; the total recovery was 96.4. and the total recovery variation CV value was 3.6.
4) Internal standard tandospirone-d 8The extraction recovery rate of (2) was 103.0, and the CV value of recovery was.5.5.
5.6 matrix Effect
1) Matrix effects refer to the inhibition or enhancement of the ionization of an analyte by a component present in a biological matrix. Taking blank human plasma of 6 different batches respectively, preparing one part for each batch, and adding a standard solution and an internal standard solution with the same concentration as the low-concentration and high-concentration quality control samples after pretreatment for analysis.
The analysis was carried out in 6 replicates of pure solutions corresponding to the concentrations of the low-concentration and high-concentration quality control samples, in the absence of matrix.
2) Preparation of hemolysis matrix: and taking 100 muL of blank whole blood, ultrasonically damaging blood cells, taking 40 muL of blank whole blood, adding 1960 muL of normal blank plasma, and uniformly mixing to obtain 2% of hemolyzed plasma subjected to ultrasonic damage, wherein the hemolyzed plasma is regarded as severe hemolysis.
Two samples of the analyte at two concentration levels (LOQ QC, HOQ QC) were prepared 6 times using simulated hemolyzed plasma, processed and then injected into a chromatographic system for analysis.
3) The simulated hyperlipidemia plasma samples were used for evaluation (5% middle and long chain fat emulsion injection hyperlipidemia plasma samples were prepared), 6 portions of each low and high concentration level (LOQ QC, HOQ QC) sample of the analyte were prepared from the simulated hyperlipidemia plasma samples, and after processing, the samples were injected into a chromatographic system for analysis.
And (4) conclusion: coefficient of variation CV% of matrix factor normalized by internal standard: 1.3-1.8%; mean deviation in accuracy of the lysomatrix effect range: 1.3-6.8%; mean accuracy deviation range for the hyperlipidemia matrix effect: 2.7-8.1%; the method of the invention has no obvious matrix effect, and has no obvious hemolysis and hyperlipemia matrix effect.
5.7 stability
And (3) carrying out stability investigation on human plasma quality control samples with low and high concentrations (LOQ QC and HOQ QC) of the object to be detected under the following conditions:
1) repeated freeze thawing at-70 deg.C for 5 times.
2) The mixture is placed at room temperature for 22h to be stable.
3) 4 days at-20 ℃ and 66 days at-70 ℃.
4) Whole blood stability: the change of the ratio of 0h peak area to 2h peak area of an analyte with low concentration level and high concentration level (LOQ QC and HOQ QC) in whole blood under ice-water bath is respectively considered, after the corresponding investigation time point is placed, a whole blood sample is centrifugally separated into plasma, an internal standard is added for processing according to the corresponding plasma sample processing operation method, and each concentration level is parallel to 6 parts.
The sample after stability investigation and the sample prepared at present are detected simultaneously, and the comparison result of each concentration is as follows:
table 8: standing at room temperature for 22h for stabilization-short term stability results
Table 9: 4d stability at-20 ℃ long term stability results
Table 10: 66d stabilization at-70 ℃ long term stability results
Table 11: -70 ℃ repeated freeze-thaw 5 times stability results
Table 12: stability results of whole blood
The CV value of the concentration of each drug in the plasma detected by the method of the invention under the investigation condition is less than 5%, which indicates that the method of the invention has stronger stability to tandospirone in whole blood and plasma under the investigation condition.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for determining the concentration of tandospirone in human plasma, comprising the steps of: detecting tandospirone in a preprocessed human plasma sample by adopting a high performance liquid tandem mass spectrometry technology, separating the tandospirone from impurities by utilizing high performance liquid chromatography, quantifying by utilizing an isotope internal standard method, taking the concentration ratio of a standard substance in a standard solution and an internal standard substance in an internal standard working solution as an X axis, taking the peak area ratio of the standard substance in the standard solution and the internal standard substance in the internal standard working solution as a Y axis, establishing a standard curve regression equation, performing linear least square regression calculation by taking the peak area ratio of a substance to be detected and the internal standard substance as the theoretical concentration ratio of the standard substance and the internal standard substance in the standard curve regression equation, and calculating the actually measured concentration of the tandospirone in the human plasma sample by using the obtained standard curve regression equation;
the internal standardThe preparation process of the solution is as follows: weighing tandospirone-d 8Dissolving in a volumetric flask by methanol to prepare an internal standard reference substance stock solution; diluting the stock solution of the internal standard reference substance with 50% acetonitrile aqueous solution to prepare tandospirone-d 8An internal standard working solution;
the chromatographic conditions of the high performance liquid tandem mass spectrometry technology are as follows:
a chromatographic column: phenomenex Gemini 3 mu m C6-Phenyl 110A
Mobile phase A: 5mM ammonium acetate; mobile phase B: acetonitrile;
flow rate: 0.500mL/min to 0.700 mL/min;
needle washing liquid: 50% methanol; washing the needle washing liquid: 10 s; sample introduction amount: 7 mu L of the solution; column temperature: 35 ℃; stopping time: 5.50 min;
the chromatographic conditions of the high performance liquid tandem mass spectrometry technique further comprise a mobile phase proportion which is set as follows:
within 0.00-3.00 min, the volume ratio of the mobile phase A to the mobile phase B is 70: 30, the flow rate is 0.500 mL/min;
within 3.00-3.10 min, the volume ratio of the mobile phase A to the mobile phase B is 20: 80, the flow rate is 0.500 mL/min;
within 3.10-3.11 min, the volume ratio of the mobile phase A to the mobile phase B is 5: 95 at a flow rate of 0.500 mL/min;
within 3.11-4.50 min, the volume ratio of the mobile phase A to the mobile phase B is 5: 95 at a flow rate of 0.700 mL/min;
and within 4.50-4.51 min, the volume ratio of the mobile phase A to the mobile phase B is 20: 80, the flow rate is 0.700 mL/min;
within 4.51-5.50 min, the volume ratio of the mobile phase A to the mobile phase B is 70: 30, the flow rate is 0.500 mL/min;
the standard curve regression equation is as follows:
the regression equation of the standard curve of the measured tandospirone in human plasma is as follows: y =0.00185X-4.44e-005, correlation coefficient R is 0.9991; the linear range of tandospirone is 3.000-6000 pg/mL, and the lowest quantitative lower limit of the blood concentration of tandospirone is 3.000 pg/mL;
the chromatographic conditions of the high performance liquid tandem mass spectrometry technology further comprise mass spectrometry conditions, and specifically comprise the following steps:
ESI source is adopted, ionization mode: electrospray ionization positive ion mode; the scanning mode is as follows: monitoring multiple reactions;
the parameters for tandospirone are set as follows: q1 Mass: 384.000Da, Q3 Mass: 122.300Da, Dwell: 100.00msec, DP: 100, EP: 10, CE: 39, CXP: 10, Polarity: positive;
tandospirone-d 8The parameter settings are as follows: q1 Mass: 392.200Da, Q3 Mass: 122.000Da, Dwell: 100.00msec, DP: 100, EP: 10, CE: 39, CXP: 10, Polarity: positive;
the mass spectrum switching valve is set as follows: 1) Total time: 1.0 min, Position: b; 2) Total time: 3.5 min, Position: a;
the ion source parameters were set as follows: IS: 3500.00, respectively; gas 1: 60.00; gas 2: 80.00; CAD: 9.00; and (4) CUR: 35.00; temp (° C): 600.00.
2. the method for determining tandospirone concentration in human plasma according to claim 1, characterized in that the pretreated human plasma sample is prepared as follows: and (3) taking human plasma in a sample hole, adding an internal standard working solution, adding a precipitator, performing vortex and centrifugation, and performing sample injection LC-MS/MS system detection.
3. The method for determining tandospirone concentration in human plasma according to claim 1, characterized in that the standard solution is formulated as follows: weighing tandospirone in a volumetric flask, and dissolving with methanol to prepare a standard reference substance stock solution; diluting the standard reference substance stock solution with 50% acetonitrile solution to prepare a standard series of tandospirone working solutions; taking standard series of tandospirone working solutions, respectively diluting the standard series of tandospirone working solutions into blank plasma, and preparing standard solutions of tandospirone with series concentrations;
and before detection, the standard solution is diluted by adding an internal standard working solution, added with a precipitator, vortexed, centrifuged and detected by a sample injection LC-MS/MS system.
4. The method for determining the concentration of tandospirone in human plasma according to claim 3, wherein the concentration range of the standard series of tandospirone working solutions is 0.06-120 ng/mL;
in the standard solution of tandospirone with the series of concentrations, the concentration range of the tandospirone is 3.000-6000 pg/mL;
tandospirone-d 8The concentration was 3 ng/mL.
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| EP3035046A1 (en) * | 2014-12-19 | 2016-06-22 | Evonik Degussa GmbH | Method for determining a dialkyl disulfide |
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| EP3035046A1 (en) * | 2014-12-19 | 2016-06-22 | Evonik Degussa GmbH | Method for determining a dialkyl disulfide |
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