CN102183659A - Method for detecting carbamazepine - Google Patents

Abstract

The invention discloses a carbamazepine detection method, comprising the following steps: contacting a sample to be tested with an anti-carbamazepine specific antibody; content, wherein the anti-carbamazepine specific antibody is obtained by immunizing animals with the carbamazepine immunogen. The detection method of the present invention has high specificity, can accurately determine whether there is carbamazepine in the sample to be tested, and can also accurately determine the content of carbamazepine.

Description

A kind of carbamazepine detection method

technical field

The invention relates to a detection method, in particular to a detection method of carbamazepine.

Background technique

Carbamazepine (5 H -dibenzo[ b , f ]azepine-5-carboxamide, 5H-dibenzo[b,f]azepine-5-carboxamide), its structural formula is shown in formula (II).

Carbamazepine is a class of antiepileptic and mood stabilizing drugs, mainly used in the treatment of epilepsy, bipolar disorder and trigeminal neuralgia. Carbamazepine has many side effects, including: anaphylaxis that may be life-threatening; toxicity to the trigeminal nerve that may cause severe damage to the skin and internal organs. It is therefore very important to monitor the patient's blood drug levels during treatment.

Contents of the invention

The object of the present invention is to provide a method for detecting carbamazepine.

The technical scheme that the present invention takes is:

A method for detecting carbamazepine, comprising the following steps:

1) Contact the sample to be tested with the specific antibody against carbamazepine;

2) According to the combination of the substance in the sample to be tested and the antibody, determine the content of carbamazepine in the sample,

Wherein the anti-carbamazepine specific antibody is obtained by immunizing animals with a carbamazepine immunogen, and the structural formula of the carbamazepine immunogen is shown in (I):

In the formula, R is a linking group, and the carrier has immunogenicity.

Preferably, the carrier is an immunogenic protein.

R is -O-(CH ₂ ) _n -COO- , n is an integer between 1 and 20, and R is selected as -O-(CH ₂ ) ₄ -COO- .

Step 2) Use an enzyme immunoassay. The enzyme immunoassay is a homogeneous enzyme immunoassay or an enzyme-linked immunosorbent assay.

The sample to be tested is a physiological sample, preferably a blood sample.

The detection method of the present invention has high specificity, can accurately determine whether there is carbamazepine in the sample to be tested, and can also accurately determine the content of carbamazepine.

Description of drawings

Fig. 1 is carbamazepine ELISA detection response curve;

Figure 2 is the average phase enzyme immunoreaction curve of Kabamaze.

Detailed ways

A method for detecting carbamazepine, comprising the following steps:

1) Contact the sample to be tested with the specific antibody against carbamazepine;

2) According to the combination of the substance in the sample to be tested and the antibody, determine the content of carbamazepine in the sample,

Wherein the anti-carbamazepine specific antibody is obtained by immunizing animals with a carbamazepine immunogen, and the structural formula of the carbamazepine immunogen is shown in (I):

In the formula, R is a linking group, and the carrier has immunogenicity.

Preferably, the carrier is an immunogenic protein. Proteins are usually chosen as carriers, although other sufficiently large immunogenic substances can also be used as carriers. The most commonly used immunogenic carriers include serum albumin, hemocyanin (KLH) and thyroglobulin. The choice of carrier is the basic common sense of those skilled in the art.

R is -O-(CH ₂ ) _n -COO- , n is an integer between 1 and 20, and R is selected as -O-(CH ₂ ) ₄ -COO- .

Below in conjunction with embodiment, further illustrate the present invention.

The chemical structures of carbamazepine derivatives used in the following examples are shown in formula (III).

The synthetic route of this carbamazepine derivative is as follows:

Concrete synthetic steps are as follows:

Synthesis of compound 2:

1) Accurately weigh 2.5 g of potassium nitrosodisulfonate ((KSO ₃ ) ₂ NO, Fremy's salt, 9.32 mmol) and 1.8 g of Na ₂ HPO ₄ (12.7 mmol) into a beaker , add 95ml of double distilled water to dissolve, and adjust the pH to 7.22; weigh 0.55g of compound 1 (12.76 mmol) into 60ml of acetone solution;

2) Mix the above two solutions and stir vigorously to obtain a purple solution;

3) Add the above purple solution into the acetone solution, keep stirring for 10 minutes, filter, and place in the refrigerator overnight;

4) Concentrate the overnight solution with an argon rotary evaporator, extract with 500ml ether, and evaporate the organic phase solvent after extraction;

5) The evaporation residue was purified by flash column chromatography with a bonded silica gel column, the eluent was a 4:1 mixed hexane (Hexane) and tetraethylammonium chloride (Tetraethoxypropane, TEAC) solution, and finally ether ( Diethyl ether, Et ₂ O) recrystallized dark red crystalline powdery substance is iminobenzoquinone compound 2 (0.12g, 21%).

Synthesis of compound 3:

1) Accurately weigh 1.2 g of compound 2 (5.8 mmol) and dissolve it in 50 ml of CHCl ₃ solution, and place it in a separatory funnel;

2) Weigh 2.5 sodium dithionite (Na ₂ S ₂ O ₄ ) (14.3 mmol) and dissolve it in 20 ml of water to make a solution;

3) Add excess Na ₂ S ₂ O ₄ solution to the above separatory funnel, shake gently until the color of the organic solution layer changes from red to yellow, and let stand to separate layers;

4) The aqueous phase was extracted and separated with CHCl ₃ , and the obtained organic phase was absorbed and dried with Na ₂ SO ₄ , and the solvent was evaporated by rotary distillation.

5) Compound 3 (1.1 g, 92%) was obtained as pale yellow-green crystals by recrystallization from the residue extracted with CHCl ₃ .

Synthesis of compound 4:

1) Accurately weigh 1.1g of compound 3 into 10 ml CHCl ₃ solution, then add 1ml of triethylamine (Triethylamine, TEA);

2) Add 2 g tert-butylchlorodimethylsilane (TBDMSCl) (15.2 mmol) to the solution, stir at room temperature for 3 days, and then evaporate the solvent;

3) Add water to dissolve, extract and separate with chloroform (CHCl ₃ ), the obtained organic phase is dried with Na ₂ SO ₄ , and the solvent is evaporated by rotary distillation.

Through the above process, 1.6 g of crude compound 4 can be obtained.

Synthesis of compound 5:

1) Weigh 1.6 g of compound 4 into 10 ml of CHCl ₃ solution, then add 2 ml of TMSOCN (4.95 mmol), stir at room temperature for 2 days, and evaporate the solvent;

2) Add water to dissolve, extract with CHCl ₃ , wash the obtained organic phase with brine first, then add Na ₂ SO ₄ to dry, and finally evaporate the solvent by rotary distillation.

Through the above process, 1.6 g of yellow crude compound 5 was finally obtained.

Synthesis of compound 6:

1) Weigh 1.6g compound 5 (4.37 mmol) and 2 g tetrabutylammonium fluoride (TBAF) (15.2 mmol) into a beaker, add 20 ml tetrahydrofuran (Tetrahydrofuran, THF) to dissolve, stir at room temperature After 4 hours, the solvent was evaporated;

2) Add distilled water to dissolve, extract and separate with ethyl acrylate (EA), the obtained organic phase is dried with Na ₂ SO ₄ , and the solvent is evaporated by rotary distillation;

3) After the crude extract was purified by chromatographic column (EA/PE (ethyl acrylate/polyethylene) = 1:1), 1.0 g of compound 6 was obtained. (75% yield from compound 3 to compound 6)

Synthesis of compound 7:

1) Accurately weigh 1.0 g of compound 6 (4.0 mmol) into 30 ml of acrylonitrile (ACN), add 1.38 g of K ₂ CO ₃ (10.0 mmol) and 1.16 g of compound A (5 - bromo-n-valerate) (6.0 mmol), stirred overnight at room temperature;

2) The solution was concentrated by vacuum filtration, and then extracted and separated with ethyl acetate (Acetoacetate, EtOAc);

3) The obtained organic phase is added with Na ₂ SO ₄ for drying, and then vacuum-filtered;

4) The crude compound was purified by flash chromatography on a silica gel bonded column, and the eluent was EA and PA solution EA/PE (ethyl acrylate/polyethylene) mixed in a ratio of 1:3, and finally 1.1 g of white solid compound 7 was obtained , and the yield was 76%.

Similarly, by changing the amount of -CH ₂ - in compound A, analogs of compound 7 can be obtained.

Synthesis of carbamazepine derivatives:

1) Weigh 1.1 g of compound 7 (3.0 mmol) into 20 ml of tetrahydrofuran (Tetrahydrofuran, THF);

2) Then weigh 0.48 g of lithium hydroxide (LiOH·H ₂ O) (11.8 mmol) containing crystal water and dissolve it in 10 ml of distilled water;

3) Mix the above two solutions and stir at 50°C for 6 hours, TLC shows that the hydrolysis is complete;

4) Concentrate and acidify the mixed solution until the pH value of the water layer is equal to 3, and separate the solid and liquid by filtration;

5) The white solid was recrystallized from methanol (Methanol, MeOH) to obtain 230 mg of carbamazepine derivatives, with a yield of 48%.

Similarly, analogues as shown in formula (III) can be obtained, the only difference being the number of -CH ₂ -.

Carbamazepine derivatives were scanned by Bruker Avance III plus 400 MHz for nuclear magnetic resonance spectroscopy, and TMS was used as the internal standard. The results are as follows: ¹ H NMR (DMSO-d6, 400MHz): 12.04 (s, 1H), 7.37-7.45 (m, 3H), 7.29-7.35 (m, 2H), 5.53 (s, 1H), 3.98 (t, 2H, J = 6.4 Hz), 2.28 (t, 2H, J = 7.2 Hz), 1.63-1.75 (m, 4H). Characterized as a carbamazepine derivative represented by formula (Ⅲ).

The obtained derivatives were analyzed and identified by chromatography/mass spectrometry (LCMS). The instrument was Agilent's tandem quadrupole mass spectrometer LC/MSD1200 series, and the ion source was in positive or negative ionization mode. The column specification is: Welchrom XB-C18 (50×4.6 mm, 5 μm), the column temperature is 30°C, the flow rate is 1.5 mL/min, and the mobile phase is acetonitrile-water ratio of 5%-60%.

LCMS results showed: purity 99.2%; retention time 2.769 min; molecular weight 352; molecular ion: 353 (M+1).

Synthesis of BSA-Carbamazepine Derivative Immunogen

The immunogen is formed by linking BSA and carbamazepine through –O-(CH ₂ ) ₄ -COO- groups. The specific synthesis method is as follows:

1) Dissolve bovine serum albumin (Bovine Serum Albumin, BSA) (200 mg) in 50 ml 0.2 M, pH 8.5 phosphate buffer;

2) Add the following chemicals into a small beaker and stir to dissolve: 200 mg of synthetic carbamazepine derivatives, 3.5 ml of dimethylformamide (DMF), 3.5 ml of ethanol, 7.0 ml of 10mM potassium phosphate buffer at pH 5.0 Liquid, 200 mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50 mg N-hydroxysuccinimide (N-hydroxysuccinimide, Sulfo-NHS), these chemical The product was stirred and dissolved at room temperature for 30 min;

3) The dissolved solution was added dropwise to the BSA solution, and stirred overnight at 2-8°C to obtain the antigen; the synthesized antigen was purified by dialysis to obtain the carbamazepine immunogen.

Preparation of specific antibody against carbamazepine

Prepare antibodies by conventional methods, the general steps are as follows:

Dilute the synthetic carbamazepine immunogen to 1.0 mg/ml with PBS, then mix 1.0 ml of the antigen solution with Freund's complete adjuvant, and inject it into rabbits;

After 2 to 3 weeks, inject the rabbit with 1.0 ml of the same antigen solution and Freund's incomplete adjuvant once, and then once every four weeks, twice in total, and the antibody titer obtained is about 1:3000.

Carbamazepine ELISA test

Carbamazepine was tested by ELISA using the prepared antibody.

The test utilizes a competitive immunoassay to determine the amount of carbamazepine in a fluid sample.

The carbamazepine in the sample competes with the conjugated carbamazepine derivative (HRP-carbamazepine derivative enzyme conjugate) for binding to limited sites on the antibody coated on the enzyme-linked plate. If there is little or no carbamazepine in the liquid sample, the HRP enzyme-conjugated carbamazepine derivative will bind to the antibody in the plate. On the contrary, if the liquid sample contains a large amount or a certain amount of carbamazepine, the enzyme-carbamazepine derivative conjugate will reduce the binding to the antibody, thereby weakening the chromogenic signal. Therefore, the absorbance produced by the assay is inversely proportional to the amount of carbamazepine in the fluid sample. Its dose-response curve is shown in Figure 1.

Kamaxi mean phase enzyme immunoassay

The EMIT (Enzyme Multiplied Immunoassay Technique) test of carbamazepine was carried out by using the prepared antibody.

The test is a competitive reaction, and the antibody-bound carbamazepine and free carbamazepine in the reaction system do not need to be separated by a solid phase. The basic principle of the test is the combination of free carbamazepine in liquid samples and carbamazepine derivatives coupled to glucose-6-phosphate dehydrogenase (G6PDH) to specific antibodies site competition. The carbamazepine in the liquid sample competitively displaces the antibody-bound carbamazepine enzyme conjugate and releases it from the antibody binding site, thereby reactivating the enzyme. Therefore, the more carbamazepine content in the liquid sample, the more free carbamazepine derivative-G6PDH enzyme conjugate, so that a stronger signal can be obtained.

The dose-effect curve obtained by the homogeneous immunoassay is shown in Fig. 2 .

drug interference test

Select 30 commonly used compounds and drugs, adjust their concentration to 10.0 μg/ml, and carry out interference test determination. The test results are shown in the following table:

ID# Compound name Concentration equivalent to carbamazepine (μg/ml) 1 Acetylsalicylic acid < 0.1 2 Amobarbital < 0.1 3 Ampicillin < 0.1 4 Phenylethylamine < 0.1 5 caffeine < 0.1 6 Methamphetamine < 0.1 7 Chlorpromazine < 0.1 8 Chlordiazepoxide < 0.1 9 d-methamphetamine < 0.1 10 Fenoprofen < 0.1 11 Gemfibrozil < 0.1 12 Gentisic acid < 0.1 13 Hydrocodone < 0.1 14 ibuprofen < 0.1 15 Imipramine < 0.1 16 (L)-Ephedrine < 0.1 17 Lidocaine < 0.1 18 Naproxen < 0.1 19 Nicotinamide < 0.1 20 penicillin < 0.1 twenty one Phenylephrine < 0.1 twenty two Phenylpropanolamine < 0.1 twenty three Procainamide < 0.1 twenty four procaine < 0.1 25 quinidine < 0.1 26 Zaminic acid < 0.1 27 Ecgonine methyl ester < 0.1 28 Ecgonine < 0.1 29 stable < 0.1 30 Cotinine < 0.1

According to the method of carbamazepine ELISA test, the above-mentioned compounds were tested in multiple wells, and the results were all negative. It can be seen that the antibody of the present invention is an anti-carbamazepine specific antibody.

Claims (8)

1. carbamazepine detection method may further comprise the steps:

1) sample to be tested is contacted with anti-carbamazepine specific antibody;

2) according to the combine situation of the material in the sample to be tested with antibody, the content of carbamazepine in the judgement sample,

Wherein anti-carbamazepine specific antibody is obtained by carbamazepine immunogen immune animal, and the immunogenic structural formula of described carbamazepine is shown in (I):

In the formula, R is a linking group, and carrier has immunogenicity.

2. carbamazepine detection method according to claim 1 is characterized in that: carrier is for having immunogenic protein.

3. carbamazepine detection method according to claim 1 is characterized in that: R Wei – O-(CH ₂) _n-COO-, n are the integers between 1 to 20.

4. carbamazepine detection method according to claim 3 is characterized in that: R Wei – O-(CH ₂) ₄-COO-.

5. carbamazepine detection method according to claim 1 is characterized in that: step 2) the inspection-free determination method of use enzyme.

6. carbamazepine detection method according to claim 5 is characterized in that: the inspection-free determination method of enzyme is homogeneous EIA method or enzyme-linked immunosorbent assay method.

7. carbamazepine detection method according to claim 1 is characterized in that: sample to be tested is the physiology sample.

8. carbamazepine detection method according to claim 7 is characterized in that: the physiology sample is a blood sample.

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