CN113092615A - Method for detecting antiepileptic drug and metabolite thereof in hair by liquid chromatography-mass spectrometry - Google Patents
Method for detecting antiepileptic drug and metabolite thereof in hair by liquid chromatography-mass spectrometry Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
Abstract
The invention belongs to the technical field of detection of medicines and metabolites thereof, and particularly relates to a method for detecting antiepileptic medicines and metabolites thereof in hair by liquid chromatography-mass spectrometry. The invention provides a system for judging whether the dosage of the antiepileptic drug is excessive or insufficient by detecting or inputting the content of the antiepileptic drug or the metabolite thereof in hair. The detection method comprises the following steps: (1) pretreating a hair sample to obtain a solution to be detected; (2) and detecting the concentration of the antiepileptic drug in the hair by adopting a liquid chromatography-mass spectrometry combined method. The detection method is simple, convenient and sensitive, can reflect the long-term drug use condition of a subject by detecting the anti-epileptic drug in the hair, has low requirements on simple and convenient material acquisition and storage of the hair sample, and is suitable for large-scale clinical popularization.
Description
Technical Field
The invention belongs to the technical field of detection of medicines and metabolites thereof, and particularly relates to a method for detecting antiepileptic medicines and metabolites thereof in hair by liquid chromatography-mass spectrometry.
Background
Epilepsy, a common chronic neurological disorder, is treated primarily by antiepileptic drugs. The sufficient treatment course is particularly important for controlling epileptic seizure when reasonably taking the medicine, at present, the concentration of plasma/serum antiepileptic medicine (or metabolite thereof) of a patient is mainly detected by venous blood sampling, but the venous blood sampling is invasive, only the current in-vivo medicine concentration level is reflected, and the patient with blood sampling difficulty accompanying special diseases, children, pregnant women, old people and other special populations need to regularly go to a hospital to draw blood to detect the medicine concentration. Therefore, there is a need in the art to develop a simpler and sensitive method for detecting the concentration of antiepileptic drugs (or metabolites thereof) to meet the detection requirement of patients in situations where blood collection is inconvenient.
The hair can be collected at home, the sampling process is non-invasive and painless, the hair can be transported and stored at normal temperature, the hair grows slowly, and the hair from the scalp to different sections of the far end can reflect the medication conditions of patients in different periods. In the prior art, the literature "liquid chromatography-tandem mass spectrometry method for measuring 11 opioid alkaloids in hair, pharmaceutical science, 2011, 46 (12): 1501-1506' provides a technical scheme for detecting 11 opioid alkaloids such as heroin, morphine, monoacetylmorphine and the like in hair by liquid chromatography-mass spectrometry.
However, the composition contained in the hair is very complex, and the content of antiepileptic drugs (or their metabolites) in the hair of a person taking them is extremely low. If the method for detecting the hair sample by the liquid chromatography-mass spectrometry is used for detecting the antiepileptic drug (or the metabolite thereof), how to effectively separate the antiepileptic drug (or the metabolite thereof) from other interference components is a very difficult problem.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting an anti-epileptic drug and a metabolite thereof in hair by liquid chromatography-mass spectrometry, which aims to: provides a simple and sensitive detection method for detecting the anti-epileptic drugs in the hair, thereby reflecting the long-term drug administration condition of the subject.
An anti-epileptic drug usage monitoring system comprising computer equipment for executing a computer program comprising the steps of:
a. detecting or infusing the content of an antiepileptic drug or a metabolite thereof in hair, wherein the antiepileptic drug comprises at least one of levetiracetam, lamotrigine or oxcarbazepine, and the metabolite of the antiepileptic drug is selected from 10-hydroxy-carbamazepine;
b. when the content of levetiracetam exceeds 2.5 mu g/mg, the content of lamotrigine exceeds 1 mu g/mg, the content of oxcarbazepine exceeds 0.05 mu g/mg or the content of 10-hydroxy-carbamazepine exceeds 1.75 mu g/mg, the overdose administration is prompted;
c. when the content of levetiracetam is lower than 0.5 mu g/mg, the content of lamotrigine is lower than 0.125 mu g/mg, the content of oxcarbazepine is lower than 0.002 mu g/mg or the content of 10-hydroxy-carbamazepine is lower than 0.15 mu g/mg, the medicine is indicated to be supplemented.
The invention also provides a method for detecting the anti-epileptic drugs and the metabolites thereof in the hair by liquid chromatography-mass spectrometry, which comprises the following steps:
(1) pretreating a hair sample to obtain a solution to be detected;
(2) detecting the concentrations of the antiepileptic drugs and the metabolites thereof in the hair by adopting a liquid chromatography-mass spectrometry combined method; the chromatographic conditions are as follows: the mobile phase A is 0.1-1 wt.% of formic acid and 5-10mmol/L of ammonium acetate aqueous solution, and the mobile phase B is acetonitrile-methanol-formic acid solution with the volume ratio of (300- & ltSUB & gt 700) & ltSUB & gt to (300- & ltSUB & gt 700) & ltSUB & gt 1 & ltSUB & gt; the chromatographic column is a C18 column.
Preferably, in the step (1), the pretreatment specifically includes the following steps:
(1.1) cleaning and airing a hair sample;
(1.2) grinding the hair sample into powder and incubating;
and (1.3) adding the incubated supernatant into acetonitrile solution containing the antiepileptic drug isotope internal standard or metabolite isotope internal standard thereof to be detected, shaking and centrifuging, and adding the centrifuged supernatant of the organic layer into the mobile phase A to obtain the solution to be detected.
Preferably, in the step (1.1), the cleaning process comprises 2-4 times of washing with ultrapure water and 1-2 times of acetone cleaning, and preferably comprises 3 times of washing with ultrapure water and 1 time of acetone cleaning.
Preferably, in step (1.2), the incubation is performed in methanol, preferably the ratio of the amount of the powder to the amount of methanol is 10-30mg:1ml, preferably 20mg:1 ml; and/or, the incubation condition is incubation for 1-12h at 40-65 ℃, preferably incubation for 4h at 60 ℃.
Preferably, in the step (1.3), the concentration of the antiepileptic isotope internal standard or metabolite isotope internal standard in the acetonitrile solution is 30-40ng/ml, preferably 20 ng/ml; and/or the dosage ratio of the supernatant after incubation to the acetonitrile solution is 20ul:80-160ul, preferably 20ul:80 ul; and/or the dosage ratio of the organic layer supernatant to the mobile phase A is 40ul to 160ul, and preferably 40ul to 160 ul.
Preferably, the chromatographic conditions further comprise: mobile phase a is 0.1 wt.% aqueous solution of formic acid and 5mmol/L ammonium acetate, and mobile phase B is acetonitrile-methanol-formic acid solution in a volume ratio of 500:500: 1; and/or, the chromatographic column is Kinetex analytical column 5 μm C18100A, 100X 2.1mm or ACQUITY UPLC BEH C1850 mm X2.1 mm X1.7 μm; and/or the flow rate is 0.3-0.45mL/min, preferably 0.4 mL/min; and/or the sample amount is 5-10 μ L, preferably 7.5 μ L; and/or the column temperature is 35-40 ℃, preferably 40 ℃;
and/or, the gradient elution procedure is:
and/or the mass spectrum conditions of the mass spectrum are as follows: the ion source is ESI, the scanning type is MRM, and the mass spectrum detection mode is a positive ion mode.
Preferably, the concentrations of the antiepileptic drug and its metabolites are obtained by a standard curve method.
Preferably, the concentration standard working solution of the standard curve method is prepared by the following method:
preparing a reference substance of the antiepileptic drug and the metabolite thereof into a solution, and diluting the solution into a series of concentrations;
(II) taking a hair sample of a normal person who does not take the antiepileptic drug, and pretreating according to the same method as the step (1) to prepare a blank biological matrix sample;
(III) mixing the reference substance solutions with the series of concentrations in the step (I) with the blank biological matrix sample obtained in the step (II) respectively to prepare a drug-containing biological matrix sample, adding acetonitrile containing an isotope internal standard of the substance to be detected, separating supernatant, and mixing the supernatant with the mobile phase A to obtain a working solution containing the reference substance with the series of concentrations;
the working solution was used to obtain a standard curve.
Preferably, in the step (i), the solvent used for preparing and diluting the solution is at least one of water, DMSO or acetonitrile; and/or, in the step (III), the control solution and the blank biological matrix sample are mixed according to the volume ratio of 1 (5-25), and the volume ratio is preferably 1: 19; and/or, the drug-containing biological matrix sample is mixed with the acetonitrile according to the volume ratio of 1 (4-8), and the volume ratio is preferably 1: 4; and/or mixing the supernatant with the mobile phase A according to a volume ratio of 1 (4-9), wherein the volume ratio is preferably 1: 4.
Preferably, the antiepileptic drug comprises at least one of levetiracetam, lamotrigine or oxcarbazepine, and the metabolite of the antiepileptic drug is selected from 10-hydroxy-carbamazepine.
The "isotope internal standard" used in the present invention and the detected substance (antiepileptic drug or metabolite thereof) are the same substance, but several hydrogen atoms therein are replaced by deuterium, or 12C is replaced by 13C, and its physicochemical properties are basically unchanged, for example: "levetiracetam-d 6" is where 6 hydrogens of levetiracetam are replaced by deuterium, "licarbazepine-d 4" is where 4 hydrogens of licarbazepine are replaced by deuterium, lamotrigine-13C-d 3 is lamotrigine where 12C is replaced by 13C, and where 3 hydrogens are replaced by deuterium.
After the detection method is adopted, the antiepileptic drug and the metabolite thereof can be effectively extracted from the hair sample, and the antiepileptic drug and the metabolite thereof are effectively separated in the liquid chromatogram, so that the simple, convenient and sensitive detection of the antiepileptic drug and the metabolite thereof is realized, and the long-term medication condition, the medication compliance and the drug accumulation condition of a subject are reflected. The hair sample is simple in material taking and low in storage requirement, and is suitable for large-scale clinical popularization.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a chromatogram obtained in example 1;
FIG. 2 is the mass spectrometric detection of ion pairs obtained in example 1;
FIG. 3 is a standard curve obtained in test example 3;
FIG. 4 shows the results of the concentration measurements of levetiracetam, lamotrigine and oxcarbazepine metabolites in the test sample of test example 3.
Detailed Description
Example 1 detection of the levetiracetam, lamotrigine and oxcarbazepine metabolite content in Hair
(1) Establishment of antiepileptic drug standard curve
a. Preparation of control solutions: respectively weighing 0.2mg of levetiracetam and lamotrigine, 0.5mg of oxcarbazepine metabolite (10-hydroxy-carbamazepine) standard reference substance, dissolving levetiracetam in 1ml of water, dissolving lamotrigine in 1ml of DMSO, dissolving oxcarbazepine metabolite 10-hydroxy-carbamazepine in 1ml of DMSO, adding 7ml of diluent (20% of acetonitrile and 80% of water), mixing to obtain 20ug/ml of levetiracetam and lamotrigine, and mixing 50ug/ml of oxcarbazepine metabolite 10-hydroxy-carbamazepine mixed I-grade working solution reference substance solution;
b. preparation of standard working solutions of a series of concentrations: taking the reference substance solution in the step a, adding a diluent (20% acetonitrile and 80% water) to prepare a reference substance solution with a series of concentrations, adding a blank hair incubation liquid (namely a blank biological matrix) according to a ratio of 1:19 to prepare a biological matrix containing the medicine, wherein the specific preparation concentration and the specific preparation process are as follows:
TABLE 1 configured concentration and course of drug-containing biomatrix
Preparing an internal standard working solution: respectively weighing 1mg of levetiracetam-d 6, lamotrigine-13C-d 3, licarbazepine-d 4, levetiracetam-d 6 dissolved in 1ml of water, lamotrigine-13C-d 3 dissolved in 1ml of DMSO, licarbazepine-d 4 dissolved in 1ml of DMSO, mixing the three internal standards, adding acetonitrile for diluting to the concentration of each internal standard of 20ng/ml, and preparing the following steps:
TABLE 2 internal standard working fluid configuration
Adding internal standard working solution into the medicated biological matrix at a volume ratio of 1:5, mixing, centrifuging at 4 deg.C and 13000rpm for 5min, collecting supernatant, adding mobile phase A at a volume ratio of 1:4, and mixing;
c. respectively sucking a series of concentration standard working solutions, injecting the working solutions into an LC-MS/MS instrument, and measuring peak areas to obtain a standard curve;
the chromatographic conditions were as follows:
a chromatographic column: ACQUITY UPLC BEH C18; taking 0.1% formic acid 5mmol/L ammonium acetate water as a mobile phase A, and acetonitrile: methanol: formic acid 500:500:1 is mobile phase B, and the gradient elution procedure is:
TABLE 3 elution gradient
The strong needle washing liquid is formic acid: methanol: acetonitrile is 1:100:100, weak needle washing liquid is 20% acetonitrile, water and 0.5% formic acid, and plunger rod washing liquid is 20% acetonitrile, water;
the chromatographic peak time is as follows: levetiracetam RT ═ 0.99min, levetiracetam-d 6 RT ═ 0.98min, lamotrigine RT ═ 1.31min, lamotrigine-13C-d 3 RT ═ 1.29min, oxcarbazepine metabolite 10-hydroxy-carbamazepine RT ═ 1.42min, licarbazepine-d 4 RT ═ 1.41min (see fig. 1).
Mass spectrum conditions: the ion source is ESI, the scanning type is MRM, and the mass spectrum detection mode is a positive ion mode;
the detected ion pairs are respectively: levetiracetam m/z 171.0 → 126.1, lamotrigine m/z 255.9 → 211.1, oxcarbazepine metabolite 10-hydroxy-carbamazepine m/z 255.0 → 237.2, levetiracetam-d 6m/z 177 → 131.1, lamotrigine-13C-d 3 m/z 259.0 → 214.0, licarbazepine-d 4 m/z 259.0 → 241.0 (see fig. 2).
(2) And (3) determining the content of the antiepileptic drug in the sample to be tested:
d. preparation of test solution
Clipping the hair of the occiput of a subject by using scissors to stick to the scalp, reserving 3ml of the near end, putting the hair into an aluminum foil paper bag, temporarily storing at normal temperature, washing the hair sample by using ultrapure water for 2-4(3) times and acetone for 1-2(1) times, then drying the hair, grinding the hair sample into grey powder by using a hair grinding instrument, adding methanol into the hair to prepare 20mg/ml solution, incubating for 4 hours at 60 ℃, adding 20ul of the supernatant incubation liquid, adding 80ul of acetonitrile containing isotope internal standards (levetiracetam-d 6, lamotrigine-13C-d 3 and licarbazepine-d which are respectively 20ng/ml), carrying out vortex oscillation for 1min, then carrying out centrifugation (the centrifugation conditions T is 4 ℃, the rotation speed 13000rpm and the time is 5min), taking 40ul of the supernatant of the organic layer, adding 160ul of the mobile phase A for reconstruction, and carrying out vortex mixing to obtain the hair shampoo;
e. determination of antiepileptic drugs
And (3) taking 7.5 mu L of the test solution, injecting the test solution into an LC-MS/MS instrument for detection, detecting under the same condition in the step d, monitoring whether the operation process is polluted or not by using a blank sample, and obtaining the content of each antiepileptic drug in the hair according to the standard curve in the step (1).
The advantageous effects of the present invention are described below by way of test examples.
Test example 1 chromatographic condition screening test
1. The test method comprises the following steps:
a. preparing mobile phases with different concentration ratios: mobile phase a1 was an aqueous solution of 0.1% formic acid and 5mmol/L ammonium acetate, mobile phase a2 was an aqueous solution of 1% formic acid and 5mmol/L ammonium acetate, mobile phase A3 was an aqueous solution of 1% formic acid and 10mmol/L ammonium acetate, mobile phase B1 was a solution of acetonitrile-methanol-formic acid in a volume ratio of 700:300:1, mobile phase B2 was a solution of acetonitrile-methanol-formic acid in a volume ratio of 500:500:1, and B3 was a solution of acetonitrile-methanol-formic acid in a volume ratio of 300:700: 1.
b. Pretreatment of a hair sample: cutting the occipital hair of a tested person by scissors attached to the scalp, reserving 3ml of the proximal end, placing the hair in an aluminum foil paper bag for temporary storage at normal temperature, taking a hair sample, washing the hair sample for 3 times by using ultrapure water and washing the hair for 1 time by using acetone, drying the hair sample, grinding the hair sample into grey powder by using a hair grinding instrument, adding methanol into the hair to prepare 20mg/ml solution, incubating the hair sample for 4 hours at 60 ℃, taking 40ul of supernatant incubation liquid, adding 160ul of acetonitrile containing isotope internal standards of the drug to be tested (levetiracetam-d 6, lamotrigine-13C-d 3 and licarbazepine-d which are respectively 20ng/ml), carrying out vortex oscillation for 1min, then carrying out centrifugation (the centrifugation condition T is 4 ℃, the rotation speed is 13000rpm and the time is 5min), taking 3 parts of 40ul of supernatant liquid, respectively adding 160ul of mobile phase A1, carrying out vortex mixing to obtain No. 1 to-be-tested liquid, adding 160ul mobile phase A2 to obtain No. 2 to obtain No..
c. Sample injection detection under different conditions: respectively sucking 7.5 μ L of No. 1-3 solution to be detected, and injecting into LC-MS/MS instrument with acid UPLC BEH C18 chromatographic column under the following different conditions.
Condition 1: elution of the corresponding mobile phase a and mobile phase B1 (acetonitrile-methanol-formic acid solution at 700:300:1 by volume) in a gradient for 2 min;
condition 2: elution of the corresponding mobile phase a and mobile phase B2 (acetonitrile-methanol-formic acid solution at a volume ratio of 500:500: 1) in a gradient for 2 min;
condition 3: elution of the corresponding mobile phase a and mobile phase B3 (acetonitrile-methanol-formic acid solution at a volume ratio of 300:700: 1) in a gradient for 2 min;
d. and observing, recording and comparing the peak time, the peak shape, the responsivity and other differences.
The other unexplained test conditions were the same as in example 1.
2. And (3) test results:
the peak of the No. 1 liquid to be detected (mobile phase A1) under the condition 2 (mobile phase B2) is obviously superior to other solutions and conditions, the responsivity is highest, the peak shape is most beautiful, and no tailing or impurity peak interference exists.
Table 4 preliminary experiment (responsivity-peak area) for finding optimum conditions of mobile phase ratio
The experimental example shows that the flow matching ratio provided by the invention can effectively improve the response values of levetiracetam, lamotrigine and oxcarbazepine metabolites, and has good shape and no impurity peak interference.
Test example 2 Hair pretreatment Condition exploration
1. The test method comprises the following steps:
a. the hair of the back occipital part of a subject (taking levetiracetam 50bid, lamotrigine 1000bid and oxcarbazepine 450bid for 1 year of antiepileptic treatment, regularly applying the medicine and not adjusting the dosage) is cut by scissors, a proximal end 3ml hair sample is left, washed by ultrapure water for 3 times and acetone for 1 time and then dried.
b. The cleaned hair sample was cut to 1-2mm with scissors. One half of the hair is ground into grey white powder by using a hair grinder, the grey white powder is divided into 4 parts, the grey white powder is weighed and then is respectively filled into No. 1-4 EP tubes, and a proper amount of methanol is added to prepare a solution of 20 mg/ml. The other half of the hair sample without grinding is divided into 4 parts, weighed and then subpackaged into No. 5-8 EP tubes, and a proper amount of methanol is added to prepare a 20mg/ml solution.
c. The hair in different numbered EP tubes was incubated under the corresponding conditions according to table 1-1: 1/2/5/6 sample at 60 deg.C for 1h,3/4/7/8 sample at 60 deg.C for 12 h;
sample No. 2/4/6/8 was pretreated according to method B, as per table 5: taking 20ul of supernatant fluid, adding 80ul of acetonitrile containing isotope internal standards (20 ng/ml each of levetiracetam-d 6, lamotrigine-13C-d 3 and licarbazepine-d) of drugs to be detected, carrying out vortex oscillation for 1min, then centrifuging (the centrifugation condition T is 4 ℃, the rotating speed is 13000rpm, and the time is 5min), taking 40ul of supernatant fluid of an organic layer, adding 160ul of mobile phase A phase for reconstruction, carrying out vortex mixing, and taking 7.5 mu L of supernatant fluid to inject into an LC-MS/MS instrument for detection;
table 5 milling, incubation and pretreatment conditions the subject sample numbers of the pre-experiment were investigated
e. The response values (peak areas) of levetiracetam, lamotrigine and oxcarbazepine metabolites in each sample were recorded and compared.
2. And (3) test results:
the responsiveness of the three antiepileptic drugs in the ground hair sample is obviously higher than that of the hair sample without grinding, the responsiveness of the sample incubated for 12h is higher than that of the sample incubated for 1h, and the responsiveness of the pretreatment method B is higher than that of the method A.
TABLE 6 grinding, incubation and pretreatment conditions pre-experiments (responsivity-peak area)
According to the test example, the pretreatment condition of the hair sample provided by the invention can effectively improve the response values of levetiracetam, lamotrigine and oxcarbazepine metabolites, so that the sensitivity and the accuracy of the detection method are improved.
Test example 3 Hair incubation time Condition exploration
1. The test method comprises the following steps:
a. the hair of the back occipital part of a subject (taking levetiracetam 50bid, lamotrigine 1000bid and oxcarbazepine 450bid for 1 year of antiepileptic treatment, regularly applying the medicine and not adjusting the dosage) is cut by scissors, a proximal end 3ml hair sample is left, washed by ultrapure water for 3 times and acetone for 1 time and then dried.
b. The cleaned hair sample is cut to 1-2mm by scissors, ground into grey powder by a hair grinder, divided into 6 parts, weighed and then respectively loaded into No. 1-6 EP tubes, and added with a proper amount of methanol to prepare a 20mg/ml solution.
c. Samples No. 1-6, incubated at 60 ℃ for 1, 2, 4, 6, 8, 12 hours, respectively;
d. pretreating a sample: taking 20ul of supernatant fluid, adding 80ul of acetonitrile containing isotope internal standards (20 ng/ml each of levetiracetam-d 6, lamotrigine-13C-d 3 and licarbazepine-d) of drugs to be detected, carrying out vortex oscillation for 1min, then centrifuging (the centrifugation condition T is 4 ℃, the rotating speed is 13000rpm, and the time is 5min), taking 40ul of supernatant fluid of an organic layer, adding 160ul of mobile phase A phase for reconstruction, carrying out vortex mixing, and taking 7.5 mu L of supernatant fluid to inject into an LC-MS/MS instrument for detection;
e. the response values (peak areas) of levetiracetam, lamotrigine and oxcarbazepine metabolites in each sample were recorded and compared.
2. And (3) test results:
the three antiepileptic drugs in the sample had the highest responsiveness after incubation for 4 h.
TABLE 7 incubation test Pre-experiment (responsivity-Peak area)
According to the experimental example, the incubation condition of the hair sample provided by the invention can effectively improve the response values of the levetiracetam, lamotrigine and oxcarbazepine metabolites, so that the sensitivity and the accuracy of the detection method are improved.
Experimental example 4 verification of precision, accuracy and linearity
1. The test method comprises the following steps:
a. taking 190ul of blank hair incubation liquid, respectively adding 10ul of quantitative lower limit working solution or quality control working solution, and preparing quantitative lower limit drug-containing hair incubation liquid with levetiracetam and lamotrigine of 2ng/ml and oxcarbazepine metabolite concentration of 5ng/ml, and quality control series drug-containing hair incubation liquid with levetiracetam and lamotrigine of 5, 50, 800ng/ml and oxcarbazepine metabolite concentration of 12.5, 125, 2000 ng/ml;
b. taking 20ul of medicated hair incubation liquid, adding 80ul of acetonitrile containing 20ng/ml of isotope internal standards of drugs to be tested (levetiracetam-d 6, lamotrigine-13C-d 3 and licarbazepine-d 4 respectively), carrying out vortex oscillation for 1min, centrifuging (T is 4 ℃, the rotating speed is 13000rpm, and the time is 5min), taking 40ul of organic layer supernatant, adding 160ul of mobile phase A for reconstruction, carrying out vortex mixing, and taking 7.5 mu L of the mixture to be injected into an LC-MS/MS instrument for detection;
c. and measuring 3 batches of 6 repeated samples of each concentration, calculating the actually measured concentration according to the following standard curve of each batch, and calculating the precision and the accuracy between days and in days by using single-factor variance analysis in Excel software.
2. And (3) test results:
2.1 three batch precision and accuracy analysis
The daily and daytime RSD of the LLOQ samples of the levetiracetam, lamotrigine and oxcarbazepine metabolites and the low, medium and high concentration quality control samples are less than 20 percent, the accuracy is within 87-96 percent, and the standard requirements are met.
TABLE 8 precision and accuracy analysis of levetiracetam, lamotrigine and oxcarbazepine metabolites in hair
(three batches of 6 replicate samples per concentration n ═ n)
2.2 Standard Curve linearity verification (see FIG. 3)
Standard curve for levetiracetam concentration in hair: y 0.00843x-0.00162(r 0.9986);
standard curve for lamotrigine concentration in hair: y 0.00199x +0.000832(r 0.9980);
standard curve for oxcarbazepine metabolite 10-hydroxy-carbamazepine concentration in hair: y is 0.015x-0.0014(r is 0.9970).
2.3 test of samples from subjects
The method can be used for sample injection once, and can simultaneously detect the concentrations of levetiracetam, lamotrigine and oxcarbazepine metabolites in hair, wherein the detection concentration range of levetiracetam and lamotrigine is 2-1000 ng/ml, the detection concentration range of oxcarbazepine metabolites is 5-2500 ng/ml, fig. 4 shows the hair detection results of 1 subject taking the three drugs simultaneously, the patient takes 1g bid of levetiracetam, 200mg bid of lamotrigine and oxcarbazepine 300mg earlier and 600mg later for 1 year, the measured levetiracetam concentration in hair is 668ng/ml, the concentration of lamotrigine is 3240ng/ml, and the concentration of oxcarbazepine metabolite 10-hydroxy-carbamazepine is 1060ng/ml (see fig. 4).
As can be seen from the above examples and test examples, the method of the present invention has the advantages of high precision and accuracy, combined detection requirements, and good linearity of the standard curve, so that the present invention realizes the simple and sensitive detection of the content of the antiepileptic drug and the metabolite thereof in the hair of the patient by using the hair sample. Because the hair sample is easy to obtain and has low storage requirement, the invention is very suitable for large-scale clinical popularization.
Claims (10)
1. The utility model provides an antiepileptic medicine uses monitoring system which characterized in that: comprising a computer device for executing a computer program comprising the steps of:
a. detecting or infusing the content of an antiepileptic drug or a metabolite thereof in hair, wherein the antiepileptic drug comprises at least one of levetiracetam, lamotrigine or oxcarbazepine, and the metabolite of the antiepileptic drug is selected from 10-hydroxy-carbamazepine;
b. when the content of levetiracetam exceeds 2.5 mu g/mg, the content of lamotrigine exceeds 1 mu g/mg, the content of oxcarbazepine exceeds 0.05 mu g/mg or the content of 10-hydroxy-carbamazepine exceeds 1.75 mu g/mg, the overdose administration is prompted;
c. when the content of levetiracetam is lower than 0.5 mu g/mg, the content of lamotrigine is lower than 0.125 mu g/mg, the content of oxcarbazepine is lower than 0.002 mu g/mg or the content of 10-hydroxy-carbamazepine is lower than 0.15 mu g/mg, the medicine is indicated to be supplemented.
2. A method for testing anti-epileptic drugs and metabolites thereof in hair by using the system of claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) pretreating a hair sample to obtain a solution to be detected;
(2) detecting the concentrations of the antiepileptic drugs and the metabolites thereof in the hair by adopting a liquid chromatography-mass spectrometry combined method; the chromatographic conditions are as follows: the mobile phase A is 0.1-1 wt.% of formic acid and 5-10mmol/L of ammonium acetate aqueous solution, and the mobile phase B is acetonitrile-methanol-formic acid solution with the volume ratio of (300- & ltSUB & gt 700) & ltSUB & gt to (300- & ltSUB & gt 700) & ltSUB & gt 1 & ltSUB & gt; the chromatographic column is a C18 column.
3. The method of claim 2, wherein: in the step (1), the pretreatment specifically comprises the following steps:
(1.1) cleaning and airing a hair sample;
(1.2) grinding the hair sample into powder and incubating;
and (1.3) adding the incubated supernatant into acetonitrile solution containing the antiepileptic drug isotope internal standard or metabolite isotope internal standard thereof to be detected, shaking and centrifuging, and adding the centrifuged supernatant of the organic layer into the mobile phase A to obtain the solution to be detected.
4. A method according to claim 3, characterized by: in the step (1.1), the cleaning process comprises 2-4 times of washing with ultrapure water and 1-2 times of cleaning with acetone, preferably 3 times of washing with ultrapure water and 1 time of cleaning with acetone.
5. A method according to claim 3, characterized by: in step (1.2), the incubation is performed in methanol, preferably the ratio of the amount of the powder to the amount of methanol is 10-30mg:1ml, preferably 20mg:1 ml; and/or, the incubation condition is incubation for 1-12h at 40-65 ℃, preferably incubation for 4h at 60 ℃.
6. A method according to claim 3, characterized by: in the step (1.3), the concentration of the antiepileptic drug isotope internal standard or metabolite isotope internal standard thereof in the acetonitrile solution is 30-40ng/ml, preferably 20 ng/ml; and/or the dosage ratio of the supernatant after incubation to the acetonitrile solution is 20ul:80-160ul, preferably 20ul:80 ul; and/or the dosage ratio of the organic layer supernatant to the mobile phase A is 40ul to 160ul, and preferably 40ul to 160 ul.
7. The method of claim 2, wherein: the chromatographic conditions further comprise: mobile phase a is 0.1 wt.% aqueous solution of formic acid and 5mmol/L ammonium acetate, and mobile phase B is acetonitrile-methanol-formic acid solution in a volume ratio of 500:500: 1; and/or, the chromatographic column is Kinetex analytical column 5 μm C18100A, 100X 2.1mm or ACQUITY UPLC BEH C1850 mm X2.1 mm X1.7 μm; and/or the flow rate is 0.3-0.45mL/min, preferably 0.4 mL/min; and/or the sample amount is 5-10 μ L, preferably 7.5 μ L; and/or the column temperature is 35-40 ℃, preferably 40 ℃;
and/or, the gradient elution procedure is:
and/or the mass spectrum conditions of the mass spectrum are as follows: the ion source is ESI, the scanning type is MRM, and the mass spectrum detection mode is a positive ion mode.
8. The method of claim 2, wherein: the concentrations of the antiepileptic drug and the metabolite thereof are obtained by a standard curve method.
9. The method of claim 8, wherein: the concentration standard working solution of the standard curve method is prepared by the following method:
preparing a reference substance of the antiepileptic drug and the metabolite thereof into a solution, and diluting the solution into a series of concentrations;
(II) taking a hair sample of a normal person who does not take the antiepileptic drug, and pretreating according to the same method as the step (1) to prepare a blank biological matrix sample;
(III) mixing the reference substance solutions with the series of concentrations in the step (I) with the blank biological matrix sample obtained in the step (II) respectively to prepare a drug-containing biological matrix sample, adding acetonitrile containing an isotope internal standard of the substance to be detected, separating supernatant, and mixing the supernatant with the mobile phase A to obtain a working solution containing the reference substance with the series of concentrations;
the working solution was used to obtain a standard curve.
10. The method of claim 9, wherein: in the step (I), the solvent adopted for preparing and diluting the solution is at least one of water, DMSO or acetonitrile; and/or, in the step (III), the control solution and the blank biological matrix sample are mixed according to the volume ratio of 1 (5-25), and the volume ratio is preferably 1: 19; and/or, the drug-containing biological matrix sample is mixed with the acetonitrile according to the volume ratio of 1 (4-8), and the volume ratio is preferably 1: 4; and/or mixing the supernatant with the mobile phase A according to a volume ratio of 1 (4-9), wherein the volume ratio is preferably 1: 4.
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