A rapid visual detection method for rocuronium bromide based on water-soluble pyrene derivatives

Abstract

The present invention relates to the field of rocuronium bromide detection, and discloses a rapid detection method based on pyrene derivatives and sugammadex sodium. Specifically, a method for detecting rocuronium bromide is disclosed, using pyrene derivatives and sugammadex sodium of the structure shown in formula (I) as probes. The pyrene derivatives have good water solubility and can achieve high sensitivity and specific detection of rocuronium bromide. The detection method provided by the present invention has high detection efficiency and sensitivity, and has the characteristics of visualization, and is easy to use.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a rapid visual detection method of rocuronium bromide based on a water-soluble pyrene derivative. Background Art

[0002] Rocuronium bromide is a non-depolarizing neuromuscular relaxant with a quaternary ammonium salt structure and a rigid hydrophobic sterol skeleton. Compared with other muscle relaxants, rocuronium bromide has the advantages of rapid onset, fewer metabolites, no obvious adverse cardiovascular reactions, and no accumulation, and is now widely used in clinical anesthesia technology.

[0003] However, the duration of action and dosage effects of rocuronium are different and unpredictable in different patients. This, to a certain extent, leads to the easy occurrence of excessive use of rocuronium during surgery, resulting in a high rate of residual muscle relaxation caused by rocuronium after the removal of the tracheal tube, about 45%. Residual muscle relaxation can cause patients to have insufficient respiratory control and incomplete recovery after the trachea is removed, posing a life-threatening risk. For decades, doctors have been using nerve stimulation methods to roughly judge the degree of residual neuromuscular blockade, and are unable to accurately grasp the specific concentration of rocuronium. Therefore, rapid and accurate monitoring of the content of rocuronium is of great significance for controlling the patient's status and conducting pharmacokinetic studies. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a rapid visual detection method for rocuronium bromide based on water-soluble pyrene derivatives, which overcomes the defects of low detection efficiency and high cost in the existing methods for detecting rocuronium bromide.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] First, the present invention provides the use of a pyrene derivative and sugammadex sodium represented by formula (I) in the detection of rocuronium bromide:

[0007]

[0008] The pyrene derivative with the structure shown in the above formula (I) has good water solubility and can detect rocuronium bromide with high sensitivity and specificity, showing obvious specificity and anti-interference ability in the detection.

[0009] The conformation and aggregation state of the pyrene derivative of the structure shown in formula (I) of the present invention in a sugammadex sodium solution show different states to external stimuli, thereby causing changes in its optical properties, such as the on- or quenching of fluorescence.

[0010] Therefore, when the above-mentioned pyrene derivatives and sugammadex sodium are used for the detection of rocuronium bromide, the effect of fluorescence response can be achieved, that is, the fluorescence of the pyrene derivative is quenched, and the quenching effect is visible to the naked eye under ultraviolet light.

[0011] Moreover, the detection limit of the above-mentioned pyrene derivatives and sugammadex sodium for rocuronium bromide is as low as 0.3 μmol / L, and the visualization effect in the solution is obvious.

[0012] The reason why the pyrene derivatives and sugammadex sodium provided by the present invention have excellent rocuronium bromide detection performance may be based on the following principle: the pyrene derivatives of the present invention and sugammadex sodium can form inclusion complexes in the solution through multiple non-covalent interactions, and rocuronium bromide can replace the pyrene derivatives from sugammadex sodium, resulting in a change in the probe fluorescence signal.

[0013] The present invention has no particular limitation on the source of the pyrene derivatives of the structure represented by the above formula (I), which may be commercially available or prepared by methods well known to those skilled in the art.

[0014] In order to obtain a pyrene derivative of the structure shown in formula (I) with higher quality and yield, the present invention provides a preferred preparation method for preparing the pyrene derivative of the structure shown in formula (I) as described below, specifically:

[0015] Second, the present invention provides a method for preparing the above-mentioned pyrene derivative, comprising the following steps:

[0016] (1) in the presence of a first solvent, subjecting the compound represented by formula (II) to a first contact reaction with phosphorus tribromide to obtain a compound represented by formula (III);

[0017] (2) In the presence of a second solvent, the compound represented by formula (III) is subjected to a second contact reaction with trimethylamine to obtain a compound represented by formula (I).

[0018]

[0019]

[0020] The present invention has no particular limitation on the source of the compound represented by formula (II), and the compound can be obtained, for example, from commercial sources or synthesized using methods provided by prior art.

[0021] Preferably, the conditions of the first contact reaction include: stirring at room temperature for 2-3 hours.

[0022] Preferably, the conditions of the second contact reaction include: stirring the reaction at 35-45° C. for 24-28 hours.

[0023] Preferably, after the first contact reaction, the method further comprises: sequentially subjecting the obtained reaction mixture to a solvent removal treatment and a first precipitation treatment to obtain a compound represented by formula (III).

[0024] Preferably, the first precipitation treatment specifically includes:

[0025] The crude solid obtained after solvent removal was purified by silica gel column chromatography.

[0026] The eluent of the silica gel chromatography column method is preferably ether / petroleum ether;

[0027] The volume ratio of diethyl ether to petroleum ether is preferably 10:90 to 5:95.

[0028] Preferably, after the second contact reaction, the method further comprises: removing the solvent from the obtained reaction mixture to obtain the compound represented by formula (I).

[0029] The first solvent and the second solvent in step (1) and step (2) are independently preferably any one or more of dichloromethane and tetrahydrofuran.

[0030] Third, the present invention provides a reagent, test paper or kit for detecting rocuronium bromide, comprising the above-mentioned pyrene derivative or the pyrene derivative prepared by the above-mentioned preparation method and sugammadex sodium.

[0031] Fourth, the present invention provides a rapid visual detection method based on water-soluble pyrene derivatives. Specifically, the present invention provides a method for detecting rocuronium bromide, using the above-mentioned pyrene derivatives or the pyrene derivatives prepared by the above-mentioned preparation method and sugammadex sodium as probes.

[0032] In the present invention, the concentration of the probe is preferably 4-6 μmol / L for pyrene derivatives and 8-12 μmol / L for sugammadex sodium. The concentration of the pyrene derivative is more preferably 5 μmol / L, and the concentration of sugammadex sodium is more preferably 10 μmol / L.

[0033] The specific detection method of the present invention is preferably as follows: in a 10mmol / L PBS buffer with a pH of 7.4, the above-mentioned probe concentration is 5μmol / L of a pyrene derivative, and 10μmol / L of sodium gluconate is used to perform a spectral test of rocuronium bromide. During the detection, the excitation wavelength is 350nm, and the slit width of the excitation light and the emission light is 1.0nm. The fluorescence intensity at an emission wavelength of 396nm is selected to calculate the fluorescence quenching efficiency of the probe, and a detection standard curve is established with the concentration of rocuronium bromide as the horizontal coordinate and the fluorescence quenching efficiency of the probe as the vertical coordinate. In a 10mmol / L PBS buffer with a pH of 7.4, the above-mentioned probe concentration is 5μmol / L of a pyrene derivative, and 10μmol / L of sodium gluconate is used to perform a visual detection of rocuronium bromide, and the probe undergoes fluorescence quenching that can be identified by the naked eye under ultraviolet light.

[0034] Compared with the prior art, the present invention provides a rapid detection method based on water-soluble pyrene derivatives, using pyrene derivatives of the structure shown in formula (I) and sugammadex sodium as probes. The above pyrene derivatives have good water solubility and can achieve high sensitivity and specificity in detecting rocuronium bromide. The detection method provided by the present invention has high detection efficiency and sensitivity, and has the characteristics of visualization and is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the fluorescence quenching efficiency (λ) of the probe (5 μmol / L pyrene derivative, 10 μmol / L sugammadex sodium) after adding different concentrations of rocuronium bromide to PBS buffer (10 mmol / L pH = 7.4). ex =350nm,λ em =396nm), Figure 1 The middle inset is a photograph taken under 365nm ultraviolet light before and after the addition of rocuronium;

[0036] Figure 2 is the fluorescence quenching degree (λ) of the probe (5 μmol / L pyrene derivative, 10 μmol / L sugammadex sodium) in PBS buffer (10 mmol / L pH = 7.40) and different substances (10 μmol / L) ex =350nm,λ em =396nm), (uric acid (UA), citric acid (CA), ascorbic acid (AA), glucose (GLC), Mg 2+ , Ca 2+ and HCO 3 － ). DETAILED DESCRIPTION

[0037] To further illustrate the present invention, the rapid visual detection method of rocuronium bromide based on water-soluble pyrene derivatives provided by the present invention is described in detail below in conjunction with examples.

[0038] Preparation of spare materials:

[0039] Preparation of buffer solution: Weigh disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride solids in proportion, and prepare 500 mL of 10 mmol / L PBS buffer solution with distilled water. Store it in a refrigerator at 4°C for later use.

[0040] Preparation of pyrene derivative mother solution: Weigh the solid represented by formula (I), prepare a mother solution with a concentration of 5 mmol / L with distilled water, and divide it into solutions of equal volume in small bottles for later use. When testing the spectrum, dilute it with the prepared PBS buffer solution (10 mmol / L, pH=7.4) to a certain concentration for testing.

[0041] Preparation of sugammadex sodium mother solution: Weigh sugammadex sodium solid, prepare a mother solution with a concentration of 10mmol / L with distilled water, and divide it into solutions of equal volume in small bottles for later use. When testing the spectrum, dilute it with the prepared PBS buffer solution (10mmol / L, pH=7.4) to a certain concentration for testing.

[0042] Preparation of the test substance and the interfering substance: Prepare rocuronium bromide with distilled water to a 1 mmol / L stock solution for later use. Prepare other interfering substances with distilled water to a 10 mmol / L stock solution. Store these solutions in a 4°C refrigerator for later use.

[0043] Preparation of actual samples: Rocuronium bromide injection Rocuronium bromide injection was purchased from Merck and diluted 100 times in PBS buffer for experiments; fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., and the serum was centrifuged at 4000g for 15 minutes in a 3000MWCO ultrafiltration tube, and the ultrafiltrate obtained was used for experiments.

[0044] Example 1

[0045] Preparation of the compound represented by formula (I)

[0046] At 0°C, phosphorus tribromide (1.98 mmol) dissolved in dichloromethane (20 mL) was added dropwise to 1-pyrenebutanol (0.50 g, 1.80 mmol) dissolved in anhydrous dichloromethane (30 mL) within 30 min. The mixture was then stirred at room temperature for 2 h. After the reaction was completed, it was washed with water several times and the organic phase was evaporated to dryness. The obtained solid was then purified by silica gel chromatography and eluted with ether / petroleum ether (5:95, v / v) to obtain the compound described in (III). The compound described in (III) (0.10 g, 0.30 mmol) was added to a solution of 6 mL trimethylamine-ethanol solution (33%, w / w) and 6 mL THF and stirred at 40°C for 24 h. After the reaction was completed, the reaction mixture was concentrated and then added to a large amount of THF to dry to obtain the compound shown in (I) (hereinafter referred to as the pyrene derivative).

[0047] Its characteristics are as follows: 1 H NMR(500MHz,DMSO-d6)δ8.40(d,J=9.5Hz,1H),8.31-8.24(m,4H),8.16(m,2H),8.08(t,J=7 .8Hz,1H),8.01(d,J=8.0Hz,1H),3.45-3.40(m,4H),3.09(s,9H),1.90(m,2H),1.80(m,2H). 13 C NMR(125MHz,DMSO-d6)δ136.6(s),131.4(s),130.9(s),129.9(s),128.5(s),127.94(s),127.92(s),127.80(s),127.08(s),126.6 8(s),125.51(s),125.47(s),125.34(s),124.74(s),124.61(s),123.88(s),65.79(s),52.82(s),32.57(s),28.43(s),22.34(s).

[0048] The probe dissolves well in water and can be dissolved by adding water.

[0049] Example 2

[0050] Fluorescence spectrum test :

[0051] Take 1 μL of pyrene derivative mother solution, 1 μL of sugammadex sodium mother solution and 998 μL of 10 mmol / L PBS buffer and add them to a 1 mL sample pool. After mixing evenly, measure the fluorescence spectrum of the probe buffer and record the fluorescence intensity at a wavelength of 396 nm as I 0Subsequently, a certain concentration gradient of rocuronium bromide was gradually added to the sample pool, and the corresponding fluorescence spectrum was measured after uniform mixing, and the fluorescence intensity at 396 nm was recorded as I.

[0052] The results are as follows Figure 1 As shown (that is, Figure 1 It indicates the degree of fluorescence quenching of the above-mentioned pyrene derivatives at 396 nm in PBS (10 mmol / L, pH = 7.4) buffer with the concentration of rocuronium bromide. QI = [(I 0 -I) / I 0 ]×100%,λ ex =350nm, the slit width of the excitation light and emission light is 1.0nm.)

[0053] The experimental results show that the pyrene derivatives are excited at an excitation wavelength of 350nm and emit an emission peak at 396nm. With the addition of rocuronium bromide, the fluorescence emission intensity of the pyrene derivatives gradually decreases. When the concentration of rocuronium bromide is added to 10μmol / L, the quenching degree of the pyrene derivatives reaches nearly 50%. According to the detection limit calculation method, the detection limit of the probe for rocuronium bromide is 0.3μmol / L.

[0054] Figure 1 The middle illustration is a photo taken under 365nm ultraviolet light before and after the addition of rocuronium bromide. It can be seen that after the addition of rocuronium bromide, the corresponding solution fluorescence is quenched.

[0055] Example 3

[0056] Optional Studies:

[0057] Common interfering substances in the detection of rocuronium bromide were selected, including uric acid (UA), citric acid (CA), ascorbic acid (AA), glucose (GLC), Mg 2+ , Ca 2+ and HCO 3 － In the test, the concentration of the aforementioned pyrene derivative was 5 μmol / L, the concentration of sugammadex sodium was 10 μmol / L, and the concentration of all interfering substances was 10 μmol / L. The fluorescence spectrum was tested under the same test conditions. The degree of fluorescence quenching of the probe at 396 nm before and after the addition of the interfering substance was used as a parameter to measure the degree of influence of the aforementioned probe on the analyte.

[0058] The results are as follows Figure 2 As shown (that is, Figure 2Figure 3 shows the fluorescence quenching degree of the above probes in PBS (10 mmol / L, pH = 7.4) buffer solution with rocuronium bromide and other interfering substances).

[0059] As can be seen from the figure, except for rocuronium bromide, the QI of all other substances is ≈ 0, while the QI of rocuronium bromide is ≈ 50%, which is much higher than other compounds. This result shows that the aforementioned probe has excellent selectivity for rocuronium bromide.

[0060] Example 4

[0061] Application of actual samples :

[0062] In order to verify the feasibility of this detection method in actual samples, rocuronium bromide injection was selected Used for determination of spike recovery.

[0063] After adding 2.0μmol / L, 5.0μmol / L and 8.0μmol / L rocuronium bromide, the spike recoveries were 99.50%, 101.60% and 100.12% (RSD<5%), respectively. The results showed that the method has good accuracy and can be used in rocuronium bromide injection. Application.

[0064] Example 5

[0065] Fetal bovine serum was selected to explore the detection of rocuronium under physiological conditions. The detection method and indicators were similar to the above-mentioned detection steps.

[0066] After adding 2.0 μmol / L, 5.0 μmol / L and 8.0 μmol / L rocuronium, the spike recoveries were 93.03%, 103.33% and 107.56% (RSD < 3%), respectively, indicating that the aforementioned probe can be used for the quantitative detection of rocuronium under physiological conditions to a certain extent.

[0067] It can be seen from the above results that the pyrene derivatives of the structure represented by formula (I) of the present invention have good water solubility and can detect rocuronium bromide with high sensitivity and specificity.

[0068] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of the pyrene derivative represented by formula (I) and sugammadex sodium in the detection of rocuronium bromide; 2. A method for detecting rocuronium bromide, characterized in that, The pyrene derivative represented by formula (I) and sugammadex sodium are used as probes; 3. The method according to claim 2, characterized in that The concentration of the pyrene derivative represented by formula (I) is 4-6 μmol / L, and the concentration of sugammadex sodium is 8-12 μmol / L.

4. A reagent, test paper or kit for detecting rocuronium bromide, characterized in that: It includes a pyrene derivative represented by formula (I) and sugammadex sodium; 5. The use according to claim 1, or the method according to any one of claims 2 to 3, characterized in that: The pyrene derivative represented by the formula (I) is prepared according to the following method: (1) in the presence of a first solvent, subjecting the compound represented by formula (II) to a first contact reaction with phosphorus tribromide to obtain a compound represented by formula (III); (2) in the presence of a second solvent, subjecting the compound represented by formula (III) to a second contact reaction with trimethylamine to obtain a compound represented by formula (I); 6. The use or method according to claim 5, characterized in that: The conditions of the first contact reaction include: stirring at room temperature for 2-3 hours, The conditions of the second contact reaction include: stirring the reaction at 35-45° C. for 24-28 hours.

7. The use or method according to claim 5, characterized in that: After the first contact reaction, the method further comprises: sequentially performing a solvent removal treatment and a first precipitation treatment on the obtained reaction mixture to obtain a compound represented by formula (III); After the second contact reaction, the method further comprises: removing the solvent from the obtained reaction mixture to obtain the compound represented by formula (I).

8. The use or method according to claim 7, characterized in that: The first precipitation treatment specifically includes: The crude solid obtained after solvent removal was purified by silica gel column chromatography.

9. The use or method according to claim 8, characterized in that: The eluent of the silica gel column chromatography method is ether / petroleum ether; The volume ratio of ether to petroleum ether is 10:90 to 5:

95.

10. The use or method according to claim 5, characterized in that: The first solvent and the second solvent are independently selected from any one or more of dichloromethane and tetrahydrofuran.

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