CN113884592A

CN113884592A - Method for detecting concentration of carbamazepine drug in dry blood spots

Info

Publication number: CN113884592A
Application number: CN202111139577.7A
Authority: CN
Inventors: 韩颖强; 吴思敏; 李苗苗; 顾婷; 卞超; 张延林
Original assignee: Joinn Laboratories Suzhou Co Ltd
Current assignee: Joinn Laboratories Suzhou Co Ltd
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2022-01-04

Abstract

The invention discloses a method for detecting the concentration of carbamazepine medicine in dry blood spots. The method comprises the following specific steps: taking about 30 mu L of whole blood, dripping the whole blood on a blank DBS card, airing the whole blood at room temperature, punching a hole by using a 6-mm circular puncher, sampling, adding 50.0 mu L of internal standard working solution, centrifuging for 2 min and carrying out ultrasound for 5min for primary extraction, adding 200 mu L of acetonitrile, uniformly mixing, carrying out ultrasound for 10min, centrifuging for 10min at 16000g, carrying out extraction again, precipitating protein, taking supernate, adding isometric ultrapure water, uniformly mixing, and carrying out LC-MS/MS analysis. The detection result has high accuracy, good sensitivity and no obvious concentration dependence. Meanwhile, the invention reduces the requirements on blood sampling personnel, blood sampling equipment, blood sampling environment, blood sampling amount, sample transportation and storage environment. The compound premix is applied to animal tests before clinic, can improve animal welfare and save test cost; the medical device is applied to clinic, is matched with an internet hospital, can save public medical resources, saves time and traffic cost of patients, and reduces hospital infection risks.

Description

Method for detecting concentration of carbamazepine drug in dry blood spots

Technical Field

The invention belongs to the technical field of blood concentration detection, and particularly relates to a method for detecting the concentration of carbamazepine in dry blood spots in vitro.

Background

Carbamazepine (Carbamazepine) is a psychotropic drug which is effective in the treatment of epilepsy, trigeminal neuralgia, glossopharyngeal neuralgia and manic-depressive disorder. The long-term administration can effectively inhibit the attack and the deterioration of the disease, but clinical researches find that the difference between metabolic individuals in vivo is large, the effective blood concentration range is narrow, the toxic and side effects are obvious and closely related to the blood concentration. Therefore, the blood concentration of the carbamazepine should be particularly concerned during the medicine taking period, and the medicine taking scheme is timely adjusted to avoid the reduction of the medicine effect or the generation of toxic and side effects.

At present, the carbamazepine blood concentration detection mostly adopts traditional detection matrixes such as blood plasma, blood serum and whole blood. The traditional detection matrix needs to be sent to a professional organization, collected and separated by professionals, and transported to a detection center through a cold chain for detection. Because the detection period is too long, the detection frequency is high, the blood collection amount is large, the work and life of a patient are seriously influenced, the psychological and physical burden of the patient is increased, and the timely monitoring of the carbamazepine blood concentration is difficult to realize. Especially when facing epidemic situation and some natural disasters, there is great potential safety hazard in patient's the hospital that goes to detect, and is difficult to realize timely, many times detect.

The pretreatment methods of carbamazepine biological samples reported in the literature include a protein precipitation method, a liquid-liquid extraction method, a solid-phase extraction method, a ligand binding method and the like. Protein precipitation is the simplest method, but the required sample amount is high, and the components of the sample solution after treatment are complex, so that the matrix effect and the lower limit of quantification of the compound are influenced.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention implements a method for detecting the concentration of carbamazepine medicine in dry blood spots.

The invention relates to a method for detecting the concentration of carbamazepine medicine in dry blood spots, which comprises the following steps:

step one, preparing DBS standard samples and quality control samples:

preparing a calibration standard sample and a quality control whole blood sample with the concentration of 0.0500-2000 ng/mL, vertically dropwise adding the calibration standard sample and the quality control whole blood sample at the central position of a sampling hole of a DBS card, enabling blood spots after sample application to be circular and the colors of the front side and the back side to be consistent, fully airing in a room-temperature ventilation environment to obtain a dry blood spot sample, and sealing and storing;

step two, DBS sample collection:

and taking about 10-50 mu L of whole blood sample, vertically and dropwise adding the whole blood sample to the central position of a sampling hole of the DBS card to ensure that the blood spot is round after sample loading and the colors of the front surface and the back surface are consistent, placing the sample in a room-temperature ventilation environment for 2-24 h, fully airing, and transferring the sample to room temperature, and sealing and storing at 2-8 ℃ or below-60 ℃.

Step three, sampling DBS samples:

and (3) adopting a 3-6-mm circular puncher to perform one or more punching sampling on the DBS sample and transferring the DBS sample into a clean EP tube. Before and after sampling, a puncher needs to punch holes at least twice on the blank part of clean filter paper or DBS card so as to avoid cross contamination among samples.

Step four, DBS sample treatment:

adding 20.0-100 mu L of oxcarbazepine internal standard working solution (the solvent is an aqueous solution with the methanol content of 0-50%) into an EP tube containing a dried blood spot sample, centrifuging for 1-10 min under the condition of about 2000-16000 g, carrying out ultrasonic treatment for 1-10 min, adding 100-500 mu L of acetonitrile, and mixing uniformly. And (3) performing ultrasonic treatment again for 1-20 min, centrifuging for 5-15 min at about 3200-16000 g, transferring the supernatant, adding ultrapure water with the same volume to dilute, and performing vortex mixing for analysis.

Step five, quantitative determination of DBS samples:

and (4) simultaneously processing a DBS calibration standard sample and a quality control sample according to the third step and the fourth step along with an unknown DBS sample, and analyzing the processed sample in a liquid mass spectrometer. And (3) adopting an internal standard method, respectively taking the concentration of a calibration standard sample and the area ratio of the carbamazepine and the oxcarbazepine in the calibration standard sample as horizontal and vertical coordinates, performing linear fitting by using a least square method to draw a standard curve, and substituting the area ratio of the carbamazepine and the oxcarbazepine in the unknown sample into calculation to obtain the concentration of the carbamazepine in the unknown sample.

The liquid phase method comprises the following steps:

a chromatographic column: poroshell 120SB-C18(2.7 μm, 3.0 x 100mm)

Mobile phase: the mobile phase is a mixed solution of the phase A and the phase B. Phase A is 5% acetonitrile water containing 0.1% formic acid; phase B was 95% acetonitrile in water containing 0.1% formic acid.

Elution procedure: gradient elution was performed at a flow rate of 0.6 mL/min. Gradient (for example phase B) is: 0.00-0.20 min, 35% -35%; 35 to 85 percent of the total amount of the mixture for 0.20 to 1.20 min; 1.20-2.00 min,85% -85%; 2.00-2.10 min,85% -35%; 2.10-3.00 min, 35% -35%.

The mass spectrometry method is as follows:

the ion source is an electrospray ion source (ESI source); the ionization mode is positive ion mode: the ion source voltage is 5000-5500V; the temperature of the ion source is 450-600 ℃; spraying mist, heating gas, air curtain gas and collision gas are all nitrogen gas; the pressure of the spraying air is 30-80 psi, the pressure of the heating air is 30-80 psi, and the pressure of the air curtain is 20-50 psi; the pressure of the collision gas is 6-12 psi; the scanning mode is multi-reaction monitoring; the ion pairs used for quantitative analysis of carbamazepine and the internal standard oxcarbazepine were: 237.1>194.2, 253.1>180.1, and the collision voltages are 27eV and 41eV, respectively.

The invention has the following beneficial effects:

compared with the traditional method, the DBS sample collection process is safer and more convenient, the limb end blood collection can be used for replacing vein blood collection, the blood collection amount is less, and the method is more friendly to the tested individuals. And no professional and specific environment are needed during sampling, and accurate quantification is not needed. The invention can be applied to preclinical pharmacokinetics tests and drug safety evaluation, can improve animal welfare and save experiment cost; meanwhile, the invention can also be applied to clinical blood concentration detection, and the patient can sample at home, thereby saving public medical resources, reducing epidemic exposure and propagation risks, and saving time and traffic expenditure of the patient.

DBS sample storage and transportation can be directly carried out at room temperature or low temperature (2-8 ℃), sample transportation and storage cost can be greatly saved, and sample stability risk caused by temperature runaway is reduced. The patient can directly carry out sample delivery through common logistics or a cold chain;

meanwhile, the pretreatment process of the DBS sample is simpler, the sample does not need to be accurately quantified by using a precise liquid transfer device, a relatively clean treated sample can be obtained by using a simple protein precipitation method with a very small sample amount, and a better quantification lower limit, high accuracy and high reproducibility result without obvious matrix effect and concentration dependence is obtained;

to sum up: the invention can be applied to preclinical pharmacokinetics tests and drug safety evaluation, and can save the experiment cost while improving the animal welfare. The invention can also be applied to clinical blood concentration detection, reduces epidemic risk caused by unnecessary going out of a patient, saves public medical resources, greatly shortens the blood concentration detection period of the patient, helps the patient to adjust the medication scheme in time, and avoids disease relapse and deterioration caused by drug effect reduction and toxic and side effects caused by blood concentration increase.

Drawings

FIG. 1 is a typical map of a standard curve for carbamazepine according to an embodiment of the present invention;

FIG. 2 is a typical spectrum of carbamazepine (upper) and oxcarbazepine (lower) in the lower limit of quantitation sample (LLOQ, carbamazepine concentration 0.500ng/mL) in the examples of the present invention;

FIG. 3 is a typical spectrum of carbamazepine (top) and oxcarbazepine (bottom) in the double Blank sample (Blank) in an example of the present invention;

FIG. 4 is a typical map of carbamazepine (top) and oxcarbazepine (bottom) in a Blank sample (Carryover Blank) after running a quantitative upper limit sample in an example of the present invention;

FIG. 5 is a graph relating plasma samples to DBS sample concentrations following intragastric administration of 2mg/kg carbamazepine to SD rats, wherein the different shape symbols represent different animal numbers;

FIG. 6 is a graph comparing plasma samples with DBS samples after 2mg/kg carbamazepine administration by gavage in SD rats, wherein ". smallcircle" represents plasma samples and "Δ" represents DBS samples.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The proper amount of the compound is determined by the ordinary technicians in the field according to the national technical specifications and the actual production conditions. The starting materials described in the present invention are all commercially available unless otherwise specified.

Examples

1. Drugs and reagents

The standard substance of the test substance, namely the carbamazepine (the batch number is 100142-201706, the content is 100.0 percent) and the standard substance, namely the oxcarbazepine (the batch number is 100657-201102, the content is 99.8 percent) are purchased from China food and drug testing research institute; DBS card Whatman^TM903Protein Saver Card ((batch: 10534612) from GE healthcare Ltd.; ultrapure water prepared from Millipop ultrapure water apparatus, methanol (chromatographically), acetonitrile (chromatographically), isopropanol (chromatographically) and formic acid (chromatographically) from Thermo Fisher Scientific Inc., Sprague Dawley rats from SPF animal houses, Inc., New drug research center of Showa, Suzhou.), and blank SD rat whole blood (anticoagulant K)₂EDTA) was collected by showa (suzhou) new drug research center, ltd.

2. Apparatus and conditions

The LC-MS consists of an Shimadzu LC-30A liquid phase system and an AB Sciex TripleQuadTM 6500 triple quadrupole tandem mass spectrum; an Eppendorf 5910R is adopted as a refrigerated centrifuge; the ultrasonic oscillator is SB-800DT from Ningbo Xinzhi Biotechnology GmbH.

Liquid phase conditions:

the column was prepared from Agilent Poroshell 120SB-C18(2.7 μm, 3.0 x 100 mm); the mobile phase A is 5% acetonitrile water containing 0.1% formic acid; the mobile phase B is 95% acetonitrile water containing 0.1% formic acid; the gradient elution procedure is shown in table 1 below:

TABLE 1 gradient elution procedure

Time (min)	Flow rate (mL/min)	％A	％B
				0.00	0.60	65	35
0.20	0.60	65	35
				1.20	0.60	15	85
2.00	0.60	15	85
				2.10	0.60	65	35
3.00	0.60	65	35

Mass spectrum conditions:

scanning was performed using TripleQuad 6500 mass spectrometry with ESI source, using positive ion MRM mode. The source and compound parameters are shown in tables 2 and 3 below:

table 2: source parameters:

table 3: compound parameters:

3. solution and sample preparation

Methanol is used as a solvent to prepare standard yeast with the concentration of carbamazepine of 1.00mg/mL and a quality control stock solution, and the standard yeast and the quality control stock solution are stored at the temperature of 2-8 ℃. Taking 50% methanol water as a solvent, diluting the stock solution of the carbamazepine standard curve into 10.0, 20.0, 40.0, 100, 200, 1000, 1800 and 2000ng/mL standard curve working solutions of carbamazepine, and diluting the stock solution of the carbamazepine quality control into 10.0, 30.0, 400 and 1600ng/mL standard curve working solutions of carbamazepine. With SD rat whole blood (K)₂EDTA anticoagulation) 20-fold dilution working solution with the preparation concentrations of 0.500, 1.00, 2.00, 5.00, 10.0, 50.0, 90.0 and 100ng/mL carbamazepine standardCurve whole blood samples and carbamazepine quality control whole blood samples with concentrations of 0.500, 1.50, 20.0 and 80.0ng/mL, respectively. Methanol is used as a solvent to prepare oxcarbazepine internal standard stock solution with the concentration of 1.00mg/mL, and the oxcarbazepine internal standard stock solution is stored at the temperature of 2-8 ℃. And diluting the oxcarbazepine internal standard stock solution into 2.00ng/mL oxcarbazepine internal standard working solution by using 50% methanol water as a solvent.

4. DBS sample preparation

A30.0 μ L standard curve and quality control whole blood sample are taken and vertically dropped on the specified position of the DBS card. In the dropping process, the contact between the gun head and the DBS card is avoided so as to avoid uneven blood spot distribution or damage of the DBS card. Meanwhile, the blood spot should be as regular and round as possible. And standing the card for not less than 2 hours in a room-temperature ventilation environment, and transferring the card to a refrigerator at the temperature of 2-8 ℃ for sealed storage after the blood spots are completely dried.

5. DBS sample treatment

The air dried DBS samples were sampled and transferred to a clean EP tube using a 6-mm diameter circular punch. Adding 50.0 μ L internal standard working solution into an EP tube containing a dry blood spot sample, centrifuging for 2 min under the condition of about 3200g, performing ultrasonic treatment for 5min, adding 200 μ L acetonitrile, and mixing. And performing ultrasonic treatment again for at least 10min, centrifuging at 2-8 ℃ for 10min at about 16000g, transferring the supernatant, adding ultrapure water with the same volume for dilution, performing vortex mixing, and performing sample injection by LC-MS/MS for 5 mu L analysis.

6. Methodology validation

6.1 Linear

The standard curve should contain 8 non-zero concentration points, and at least 6 non-zero concentration points and 75% of the standard curve points (including LLOQ and ULOQ) should meet the following requirements: except for the accuracy deviation (relative error, Bias%) of LLOQ sample within + -20.0%, the Bias% of other samples should be within + -15.0%, and the square value (determination coefficient) r of the correlation coefficient²≥0.98。

As a result: the standard curve is subjected to least square linear fitting, the linearity of the compound is good at 0.500-100 ng/mL (a typical standard curve graph is shown in figure 1), and r is²Are all greater than 0.98. Each calibration point of the six analysis batches meets the accuracy requirement, and the accuracy deviation between the calibration points is as follows: -5.35% -4.60%.

6.2 precision and accuracy

Using a standard curve, 4 concentration levels (LLOQ, QCL, QCM and QCH) and 5 samples of each concentration level to form 1 analysis batch, and running at least 3 analysis batches to evaluate accuracy and precision. The within-and between-lot accuracy deviation for each concentration level should be between ± 15%, LLOQ should be between ± 20%; the precision within and between batches should not exceed 15% and the LLOQ should not exceed 20%.

As a result: deviation in accuracy within batch: -5.95% -8.25%; deviation in accuracy between batches: -3.09% -5.46%; internal precision: 2.51 to 11.06 percent; batch precision: 5.62 to 8.64 percent.

6.3 sensitivity

Sensitivity requires that the signal-to-noise ratio of the lowest non-zero concentration point (LLOQ) of the standard curve is not less than 5, the accuracy deviation between batches is within plus or minus 20%, and the precision between batches is not more than 20%.

As a result: the LLOQ of the invention is 0.500ng/mL, and the response is shown in figure 2, and the calculated signal-to-noise ratio is 27. The maximum value of the intra-batch accuracy deviation was-5.88%, the maximum value of the intra-batch precision was 9.86%, the inter-batch accuracy deviation was-0.35%, and the inter-batch precision was 9.03%.

6.4 extraction recovery

And comparing the DBS samples with the LQC, MQC and HQC concentration levels with the sample with the LQC, MQC and HQC concentration levels prepared by adding standards after extracting the blank DBS sample, wherein the variation coefficient of the extraction recovery rate of the compound at each concentration point is less than 15%.

As a result: the average extraction recovery rate of carbamazepine LQC, MQC and HQC concentration is 106.1%, and the variation coefficient is 11.2%.

6.5 matrix Effect

Selecting at least six blank matrixes of different individuals, calculating the concentration level of LQC and HQC of each matrix, and the ratio of the peak area in the presence of the matrix to the peak area without the corresponding concentration of the matrix, namely the matrix factor, wherein the variation coefficient of the matrix factor after internal standard normalization is not more than 15%

As a result: the LQC matrix factor is 1.29 after internal standard normalization, and the coefficient of variation is 4.15%; the HQC matrix factor after internal standard normalization is 1.04, and the coefficient of variation is 3.40%.

6.6 Selectivity

Taking blank matrixes from at least six different sources, wherein the response of endogenous components is lower than 20% of the lower limit of the quantification of the target analyte (carbamazepine) and the response of an internal standard (oxcarbazepine) is 5%;

as a result: interference peaks (a representative spectrum is shown in figure 3) of the target analyte carbamazepine and the internal standard oxcarbazepine are not found in the blank matrix, and the method has good selectivity;

6.7 residues

The residue in the blank after high concentration sample should not exceed 20% of the LLOQ response and not exceed 5% of the internal standard response.

As a result: as shown in fig. 4, no residual peak was observed.

6.8 Effect of sample size

Preparing high and low concentration whole blood samples, loading 10.0 μ L, 20.0 μ L, 30.0 μ L, 40.0 μ L and 50.0 μ L respectively, and testing the accuracy by loading 30.0 μ L of a freshly prepared standard curve, wherein the accuracy deviation of each concentration point is within +/-15.0%.

The results show that: the accuracy deviation of high and low concentration dry blood spot samples with different sample loading amounts (10-50 mu L) is within +/-15.0%.

6.9 Effect of erythrocyte volume ratio

The whole blood of blank rats with HCT values of 0.30, 0.35, 0.40, 0.45 and 0.50 respectively is obtained after centrifugation and partial plasma addition or partial plasma removal. Blank rat whole blood with different HCT values is used as a matrix to prepare high-concentration and low-concentration level quality control samples, whole blood with a red blood cell volume ratio of 0.48 is used as DBS standard curve and quality control samples, and the accuracy of the samples to be tested is 85.0-115%.

As a result: when the volume ratio of the red blood cells is between 0.35 and 0.50, the accuracy of the quality control sample at high and low concentration levels meets the requirement.

6.10 Effect of sampling position

Preparing high-concentration and low-concentration level dry blood spot samples, respectively sampling at the central position and the periphery of the dry blood spot, and performing quantitative analysis by taking the central position of the freshly prepared DBS sample as a standard curve, wherein the accuracy deviation of the sample to be examined is within +/-15%, and the precision is not more than 15%.

The results show that: the accuracy deviation of the DBS samples with different sampling positions and high and low concentration levels is 0.95 percent and 4.13 percent respectively; the precision was 3.96% and 2.04%, respectively.

6.11 stability

Preparing low-concentration and high-concentration horizontal dry blood spot samples, respectively storing the samples at room temperature, 2-8 ℃ and below-60 ℃ for a certain time in a sealed manner, and then quantitatively analyzing the samples by taking the freshly prepared DBS samples as a standard curve, wherein the average accuracy deviation of the samples to be examined is within +/-15%.

The results show that: under the room temperature sealing condition, the carbamazepine DBS sample has good stability within 20 days, and the accuracy deviation of the low and high concentration levels is-6.67 percent and-11.50 percent respectively. (ii) a

Under the sealing condition of 2-8 ℃, the carbamazepine DBS sample has good stability within 55 days, and the accuracy deviation of low and high concentration horizontal levels is-11.33 percent and-6.56 percent respectively;

under the sealing condition below 60 ℃ below zero, the carbamazepine DBS sample has good stability within 55 days, and the accuracy deviation of low and high concentration horizontal levels is respectively-6.00% and-4.75%.

6.12 concentration differences between the SD rat dried blood spot sample and the SD rat plasma sample

Three female and male Sprague Dawley rats were selected, and 2mg/kg carbamazepine was administered by gavage, and jugular vein bleeds were performed at approximately 0.3mL each at 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 6h and 12h before administration (0h), after administration. About 90. mu.L of the suspension was dropped on a DBS card (3 portions in parallel, about 30. mu.L each) and dried at room temperature, and then the dried suspension was transferred to a refrigerator at 2 to 8 ℃ for storage under sealed conditions. Rest whole blood is put into the container K₂And (3) centrifuging the blood collection tube of the EDTA anticoagulant at the rotating speed of 2000g at the temperature of 2-8 ℃ within 1h, taking an upper layer plasma sample in a clean EP tube, and storing the upper layer plasma sample in an ultralow temperature refrigerator at the temperature of-60 ℃.

And (3) performing quantitative analysis on the plasma sample and the dry blood spot sample by adopting a system-verified LC-MS/MS method. The jugular vein is measured by taking the plasma sample concentration of the jugular vein as the abscissaThe DBS sample concentration is used as an ordinate, a plasma sample and dry blood spot sample concentration correlation distribution graph (figure 5) is drawn, and linear fitting is carried out to obtain a curve y which is 0.86757x +13.3878, r²0.9887. The resulting sample data were statistically analyzed using WinNonlin and plotted against the time of drug production. As can be seen from FIG. 6, the curve trends of the DBS samples and the plasma samples are completely consistent, and the Cmax, Tmax and T of the DBS samples and the plasma samples are calculated by using a non-compartmental model (NCA) method_1/2And calculating the deviation of the two, which is respectively: 1.45%, -0.01%, 9.29%. It can be known that there is no obvious difference between the blood concentration measured by the dry blood spot method and the plasma sample detection method.

The invention develops a method for detecting the concentration of carbamazepine medicine in dry blood spots. The method has the advantages of simple operation, small blood sampling amount, good test individual friendliness, high measurement result accuracy, good reproducibility and good correlation with the measurement result of the plasma sample. Is suitable for the research of clinical pharmacokinetics and the evaluation of drug safety.

Meanwhile, the method is safe and convenient in sampling process, does not need specific personnel, environment and equipment, and further does not need quantitative operation. The DBS sample of the fingertip blood can be collected by a patient, and is mailed to a detection center through conventional logistics, and after a detection result is obtained, the medication scheme can be adjusted timely and efficiently under the remote guidance of a doctor. Not only can effectively control the attack and the deterioration of the disease condition, but also can reduce the occurrence of adverse reactions or other toxic and side effects.

The parts of the invention not described in detail can be realized by adopting the prior art, and are not described in detail herein.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

Claims

1. A method for detecting the concentration of carbamazepine drug in dry blood spots, which is characterized by comprising the following steps:

step one, preparing DBS standard samples and quality control samples:

preparing a calibration standard sample and a quality control whole blood sample with known concentrations, vertically dropwise adding the calibration standard sample and the quality control whole blood sample at the central position of a sampling hole of a DBS card, enabling blood spots to be circular after sample loading and the colors of the front surface and the back surface to be consistent, fully airing in a room-temperature ventilation environment to obtain the DBS standard sample and the quality control sample, and sealing and storing;

step two, DBS sample collection:

taking a proper amount of whole blood sample, vertically and dropwise adding the whole blood sample to the center of a sampling hole of a DBS card, enabling the blood spots to be round after sample loading and the colors of the front surface and the back surface to be consistent, fully airing in a room-temperature ventilation environment to obtain a dry blood spot sample, and sealing and storing;

step three, sampling DBS samples:

adopting a circular puncher to perform one or more punching sampling on the DBS sample and transferring the DBS sample into a clean EP tube; before and after sampling, a puncher is needed to punch holes at least twice on the blank part of clean filter paper or DBS card so as to avoid cross contamination among samples;

step four, DBS sample treatment:

adding 20.0-100 mu L of internal standard working solution into an EP tube containing a dry blood spot sample, centrifuging for 1-10 min under the condition of 2000-16000 g, carrying out ultrasonic treatment for 1-10 min, adding 100-500 mu L of acetonitrile, and mixing uniformly; performing ultrasonic treatment for 1-20 min again, centrifuging for 5-15 min at 3200-16000 g, transferring supernatant, adding ultrapure water with the same volume for dilution, and performing vortex mixing for analysis;

step five, quantitative determination of DBS samples:

the DBS calibration standard sample and the quality control sample are processed simultaneously according to the third step and the fourth step along with the unknown DBS sample, and the processed sample is analyzed by a liquid mass spectrometer; and (3) adopting an internal standard method, respectively taking the concentration of a calibration standard sample and the area ratio of the carbamazepine and the oxcarbazepine in the calibration standard sample as horizontal and vertical coordinates, performing linear fitting by using a least square method to draw a standard curve, and substituting the area ratio of the carbamazepine and the oxcarbazepine in the unknown sample into calculation to obtain the concentration of the carbamazepine in the unknown sample.

2. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: in the first step, the concentration range of the known concentration calibration standard sample is 0.0500-2000 ng/mL.

3. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: in the second step, the whole blood source individual is rodent selected from but not limited to rat, mouse and rabbit or non-rodent mammal selected from but not limited to dog, monkey and human, and the whole blood loading amount is 10-50 μ L.

4. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: and the air drying time in the first step and the second step is 2-24 h.

5. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: the storage environment in the first step and the second step can be selected to be stored at room temperature, 2-8 ℃ or-60 ℃ in a sealing way according to different required storage time.

6. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: the diameter of the puncher in the third step is 3-6-mm.

7. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: in the fourth step, the internal standard working solution is a methanol/water solution (the content of methanol is 0-50%) containing oxcarbazepine.

8. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: the liquid phase method in the step five is as follows:

a chromatographic column: poroshell 120SB-C18, size 2.7 μm, 3.0 x 100 mm;

mobile phase: the mobile phase is a mixed solution of phase A and phase B; phase A is 5% acetonitrile water containing 0.1% formic acid; phase B is 95% acetonitrile water containing 0.1% formic acid;

elution procedure: gradient elution was performed at a flow rate of 0.6 mL/min; gradient (for example phase B) is: 0.00-0.20 min, 35% -35%; 0.20-1.20 min, 35% -85%; 1.20-2.00 min,85% -85%; 2.00-2.10 min,85% -35%; 2.10-3.00 min, 35% -35%.

9. The method of claim 1, wherein the concentration of carbamazepine drug in dry blood spots is measured by: the mass spectrometry method in the step five is as follows:

the ion source is an electrospray ion source; the ionization mode is positive ion mode: the ion source voltage is 5000-5500V; the temperature of the ion source is 450-600 ℃; spraying mist, heating gas, air curtain gas and collision gas are all nitrogen gas; the pressure of the spraying air is 30-80 psi, the pressure of the heating air is 30-80 psi, and the pressure of the air curtain is 20-50 psi; the pressure of the collision gas is 6-12 psi; the scanning mode is multi-reaction monitoring; the ion pairs used for quantitative analysis of carbamazepine and the internal standard oxcarbazepine were: 237.1>194.2, 253.1>180.1, and the collision voltages are 27eV and 41eV, respectively.