Serum propofol separation and extraction method and content determination method
By using separation and extraction methods and the color development reaction of molecular probes in serum samples, the problem of low detection efficiency of propofol in the prior art was solved, and a rapid and accurate determination of propofol concentration was achieved.
Patent Information
- Application Number
- CN202411323922.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2025-03-11
AI Technical Summary
The detection method of propofol in the prior art is inefficient and requires multiple hours to consume, making it difficult to achieve rapid detection.
A serum propofol separation and extraction method is used to remove proteins from serum samples, mix them with organic solvents using ultrasound, centrifuge to obtain an organic phase containing propofol, and develop color through the reaction of molecular probes and alkali solution, and measure the ultraviolet absorbance value to determine the propofol concentration.
The rapid and accurate detection of propofol in serum is achieved, the detection efficiency is improved, and the impact on the ultraviolet absorbance value is reduced by optimizing organic solvents and protein precipitants.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of propofol detection, and in particular to a serum propofol separation and extraction method and a content determination method. Background Art
[0002] In the prior art, the commonly used methods for analyzing the drug concentration of propofol in vivo include HPLC, LC-MS, GC-MS, etc. Although these methods can accurately detect the concentration of propofol, they take several hours and are too inefficient. In order to achieve the enrichment, separation and rapid detection of propofol, the physical and chemical properties of propofol are understood through literature research, and a separation and enrichment method for propofol is developed to achieve efficient extraction from serum samples; and based on the special phenol structure of propofol, the optical absorption / fluorescence emission molecular probe of the above propofol is designed and synthesized, and a corresponding detection method is developed to obtain the corresponding linear range, detection limit and anti-interference ability and other information. Summary of the invention
[0003] To address the deficiencies in the prior art, the present invention provides a serum propofol separation and extraction method and a content determination method, which can separate and extract propofol in serum and accurately and quickly detect the propofol concentration.
[0004] In order to achieve the purpose of the present invention, the following scheme is proposed: A method for separating and extracting serum propofol comprises the following steps: S1. removing proteins from serum samples to obtain protein-free supernatant; S2. Ultrasonic mixing of the supernatant and the organic solvent is performed, and the mixture is centrifuged to obtain an organic phase containing propofol; the organic solvent is selected from one or more of methyl tert-butyl ether, dichloromethane, and n-hexane.
[0005] A method for measuring propofol content comprises the following steps: S1. removing proteins from serum samples to obtain protein-free supernatant; S2, ultrasonically mixing the supernatant and the organic solvent, and centrifuging to obtain an organic phase containing propofol; the organic solvent is selected from one or more of methyl tert-butyl ether, dichloromethane, and n-hexane; S3. Mix the organic phase with the molecular probe and alkaline solution to develop color, and measure the absorbance value.
[0006] Furthermore, in step S1, a 50% trichloroacetic acid aqueous solution is selected as a protein precipitant to remove protein from serum.
[0007] Furthermore, in step S2, the organic solvent is selected from n-hexane.
[0008] Furthermore, in step S2, the volume ratio of the supernatant to the organic solvent is 1:1.
[0009] Furthermore, in step S3, the volume ratio of the organic phase to the molecular probe is 1:1.
[0010] Furthermore, in step S3, the molecular probe is an ethanol solution of 2,6-dichloroquinone-4-chloroimide, and the concentration of the molecular probe is 20 mg / L.
[0011] Furthermore, in step S3, the alkaline solution is an ethanol solution of NaOH.
[0012] The method for determining the content of propofol specifically includes the following steps: ultrasonically mixing 700 μL of serum sample with 50 μL of 50% trichloroacetic acid, centrifuging to obtain a deproteinized supernatant; ultrasonically mixing 600 μL of the supernatant with 600 μL of n-hexane, centrifuging to obtain an organic phase containing propofol; mixing 500 μL of the organic phase with 500 μL of an ethanol solution of 2,6-dichloroquinone-4-chloroimide and 50 μL of an ethanol solution of 0.25 M NaOH for color development, and measuring the ultraviolet absorbance value; substituting the absorbance value at 600 nm into the baseline standard of the day to obtain the propofol concentration in the serum.
[0013] The beneficial effects of the present invention are: 1. Six organic solvents were used to extract propofol from water samples with different concentrations. It was found that methyl tert-butyl ether, dichloromethane and n-hexane had good linearity and were suitable as propofol extractants.
[0014] 2. Further experiments found that if an aqueous solution of trichloroacetic acid is selected as a protein precipitant to remove protein from serum, then methyl tert-butyl ether and dichloromethane as extractants will extract trichloroacetic acid and affect the amount of alkali added, so n-hexane is preferred as the extractant for propofol.
[0015] 3. Since organic solvents can extract small protein molecules in serum and affect the stability of ultraviolet absorbance values, this scheme selects 50% trichloroacetic acid aqueous solution as a protein precipitant to remove proteins from serum, thereby reducing the impact on ultraviolet absorbance values; 50% trichloroacetic acid as a protein precipitant has the advantage of good linearity, while NaOH / methanol solution as a protein precipitant leads to reduced detection sensitivity, and 10% trichloroacetic acid as a protein precipitant leads to protein residues and poor linearity.
[0016] 4. Although the signal value of propofol aqueous solution is good, the UV / fluorescence determination of drugs in actual blood samples is greatly interfered by solvents and has high requirements on equipment. Therefore, the present invention selects 2,6-dichloroquinone-4-chloroimine molecular probe for the detection of propofol. The molecular probe reacts with propofol and color development occurs when an alkaline solution is added.
[0017] 5. Since n-hexane and methanol are not completely miscible, the reaction between propofol in the n-hexane-extracted serum and the molecular probe will cause stratification. Therefore, the solvent methanol of the molecular probe was replaced with ethanol, and the NaOH / methanol solution was also replaced with NaOH / ethanol solution. The experiment found that when detecting propofol in water, the molecular probe with ethanol solvent still maintained good detection performance for propofol and had good linearity; when detecting propofol in serum, the detection of propofol by the molecular probe with ethanol solvent was basically consistent with that of the probe with methanol solvent. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The UV measurement graph of propofol aqueous solution with different concentrations between 200-300nm is shown; Figure 2 The fitting of different concentrations of propofol aqueous solution at 210nm is shown Figure 1 ; Figure 3 The fitting of propofol aqueous solution with different concentrations at 270 nm is shown. Figure 2 ; Figure 4 The fitting of 210nm after fluorescence measurement of propofol aqueous solution with different concentrations is shown. Figure 1 ; Figure 5 The fitting of 210nm after fluorescence measurement of propofol aqueous solution with different concentrations is shown. Figure 2 ; Figure 6 The UV measurement graph of propofol aqueous solution with different concentrations between 400-800nm is shown; Figure 7 The fitting graphs of propofol aqueous solutions with different concentrations at 572 nm are shown; Figure 8 The diagram shows the color development of the molecular probe of propofol aqueous solution with different concentrations; Fig. 9 A is the UV determination diagram of dichloromethane extraction of propofol aqueous solution with different concentrations, B is the fitting diagram, and C is the color development diagram; Fig.10 A is the UV determination diagram of methyl tert-butyl ether extracting different concentrations of propofol aqueous solution, B is the fitting diagram, and C is the color development diagram; Fig.11 A is the UV determination diagram of ethyl acetate extraction of propofol aqueous solution with different concentrations, B is the fitting diagram, and C is the color development diagram; Fig.12 A is the UV determination diagram of n-hexane extraction of propofol aqueous solution with different concentrations, B is the fitting diagram, and C is the color development diagram; Fig.13A is the UV determination diagram of toluene extraction of propofol aqueous solution with different concentrations, B is the fitting diagram, and C is the color development diagram; Fig.14 A is the UV determination diagram of petroleum ether extracting different concentrations of propofol aqueous solution, B is the fitting diagram, and C is the color development diagram; Fig.15 A flow chart for the separation and extraction of propofol from deproteinized serum is shown; Fig.16 A is the UV determination diagram of dichloromethane extraction of deproteinized serum solutions of different concentrations of propofol, B is the fitting diagram, and C is the color development diagram; Fig.17 A is the UV determination diagram of dichloromethane extraction of deproteinized serum solutions of different concentrations of propofol, B is the fitting diagram, and C is the color development diagram; Fig.18 A is the UV determination diagram of methyl tert-butyl ether extraction of deproteinized serum solutions of different concentrations of propofol, B is the fitting diagram, and C is the color development diagram; Fig.19 A flow chart for separation and extraction of propofol in serum is shown; Fig. 20 A is the UV measurement graph of serum with different propofol concentrations treated with NaOH methanol solution, and B is the fitting graph; Fig.21 A is the UV measurement graph of serum with different propofol concentrations treated with 10% trichloroacetic acid, and B is the fitting graph; Fig. 22 A is the UV measurement diagram of 50% trichloroacetic acid serum with different concentrations of propofol, and B is the fitting diagram; Fig.23 A is the UV determination diagram of propofol in water sample after the probe solvent was improved, and B is the fitting diagram; Fig.24 A is the UV determination diagram of propofol in serum sample after the probe solvent was improved, and B is the fitting diagram; Fig.25 A is the UV measurement of propofol at different concentrations after serum incubation, and B is the fitting Figure 1 , C is the fitting Figure 2 ; Fig.26 A is the UV measurement graph of different concentrations of propofol in serum, B is the baseline standard graph of the day, and C is the UV measurement graph of propofol detection in serum of two patients. DETAILED DESCRIPTION
[0019] 1.1 UV / fluorescence determination of propofol aqueous solution In the clinical use of propofol, the blood concentration of the patient is about 0.5-1.0 mg / L when the patient loses consciousness. During the entire clinical anesthesia process, the general use concentration of propofol is 2-6 mg / L, so the blood concentration range of propofol in clinical detection is at least 0.5-6 mg / L. First, the ultraviolet / fluorescence measurement of propofol aqueous solution is carried out, the characteristic peak position of propofol is marked, and the fitting of the peak corresponding to the specific wavelength is calculated to judge the detectable signal value of the propofol aqueous solution.
[0020] pass Figure 1-Figure 5 It can be clearly seen that the signal value of propofol aqueous solution is better, but because the UV / fluorescence determination of drugs in actual blood samples is greatly interfered by the solvent and has high requirements for equipment, it is necessary to combine colorimetric molecular probes for related detection of propofol.
[0021] 1.2 Chromogenic molecular probes By observing the structure of propofol, it is a compound with no substituent in the para position of the phenolic hydroxyl group. This position can be detected using Gibbs reagent: the methanol solution of 2,6-dichloroquinone-4-chloroimine reacts with propofol and an alkaline solution is added to produce color. The reaction equation is as follows: A NaOH aqueous solution with pH = 13 was prepared, and the color-developing molecular probe was mixed with different concentrations of propofol aqueous solutions (0.4, 1, 1.6, 2, 6, 10 mg / L) at a ratio of 1:1 (500 μL), and then 50 μL of NaOH aqueous solution was added to each solution to develop color. UV measurement was performed, and the results were as follows: Figure 6-Figure 8 As shown, the absorbance fitting of the molecular colorimetric probe has excellent linearity in the propofol concentration range of 0.2-5 mg / L. Therefore, the molecular probe can be used as a detection reagent for propofol for subsequent optimization and powerful verification of propofol enrichment.
[0022] 1.3 Separation, enrichment and detection of propofol Propofol has a certain degree of hydrophobicity. In clinical anesthesia, propofol can highly bind to proteins to generate signals and thus exert its anesthetic effect. It cannot directly react with molecular probes to develop color, and the various amino acids contained in serum will react with molecular probes to produce false positive reactions. Therefore, the effective separation and enrichment of propofol in serum is a prerequisite for the successful detection of propofol blood concentration in serum.
[0023] 1.3.1 Separation and extraction of propofol in water Six water-immiscible organic solvents were selected for extraction and color development of the propofol aqueous solution: methyl tert-butyl ether, dichloromethane, ethyl acetate, n-hexane, petroleum ether, and toluene.
[0024] Take 600 μL of propofol of different concentrations (0.4, 1, 1.6, 2, 6, 10 mg / L) and mix them with 600 μL of different organic solvents by ultrasonic mixing. After centrifugation, take 500 μL of the organic phase, mix it with 500 μL of molecular probe and 50 μL of NaOH aqueous solution for color development, and measure the ultraviolet absorbance value. The results are as follows: Figure 9-14 As shown in Table 1, for the extraction of propofol in water samples, methyl tert-butyl ether, dichloromethane and n-hexane were finally selected as good extractants for the next step of operation optimization.
[0025] Table 1 Extraction results of propofol in water by six organic solvents 1.3.2 Separation and extraction of propofol from deproteinized serum Since organic solvents can extract small protein molecules in serum and thus affect the stability of ultraviolet absorbance values, a 50% trichloroacetic acid aqueous solution was prepared as a protein precipitant to remove serum protein. The alkaline solution added during the color development process was replaced with a 0.25M NaOH methanol solution to remove the signals of amino acid small molecules and molecular probe color development in order to facilitate complete color development. Methyl tert-butyl ether, dichloromethane, and n-hexane were selected as extractants to extract and color the propofol-deproteinized serum solution. The specific operation process is as follows: Fig.15 shown.
[0026] Take 4 mL of serum from 2 tubes, add 400 μL of 50% trichloroacetic acid aqueous solution to each tube, mix by ultrasound, centrifuge at 10,000 rpm, and take the supernatant as deproteinized serum. Deproteinized serum solutions with different concentrations of propofol (0.4, 1, 1.6, 2, 6, 10 mg / L) were prepared with deproteinized serum. 600 μL of deproteinized serum solutions with different concentrations of propofol were ultrasonically mixed with 600 μL of different organic solvents (methyl tert-butyl ether, dichloromethane, n-hexane), and after centrifugation, 500 μL of the organic phase was mixed with 500 μL of molecular probe and 50 μL of 0.25 M NaOH methanol solution for color development, and the ultraviolet absorbance value was measured. The results are as follows: Figure 16-18 As shown in Table 2, n-hexane was finally selected as the extractant for serum samples to separate and enrich propofol, and the subsequent operation process was optimized.
[0027] Table 2 Extraction results of propofol in deproteinized serum by three organic solvents 1.3.3 Separation and extraction of propofol from serum 0.25M NaOH methanol solution, 10% trichloroacetic acid aqueous solution, and 50% trichloroacetic acid aqueous solution were prepared as protein precipitants to remove serum protein. Hexane was selected as the extractant to extract and color the propofol-deproteinized serum solution. The specific operation process is as follows: Fig.19 shown.
[0028] To simulate the situation where real serum samples contain propofol, serum solutions with different propofol concentrations (0.4, 1, 1.6, 2, 6, and 10 mg / L) were directly prepared with serum and divided into three groups. 50 μL of 0.25 M NaOH methanol solution, 10% trichloroacetic acid aqueous solution, and 50% trichloroacetic acid aqueous solution were added to remove proteins, respectively. 600 μL of serum solutions with different propofol concentrations were mixed with 600 μL of n-hexane by ultrasonication. After centrifugation, 500 μL of the organic phase was mixed with 500 μL of the molecular probe and 50 μL of 0.25 M NaOH methanol solution for color development. The ultraviolet absorbance was measured. The results are shown in the figure. Figure 20-22 As shown in Table 3, 50% trichloroacetic acid was finally selected as the protein precipitant for serum samples, and the subsequent operation process was optimized.
[0029] Table 3 Detection results of propofol in serum by three protein precipitants 1.3.4 Solvent optimization of chromogenic probes The propofol in the serum extracted by n-hexane reacted with the molecular probe and stratified. This was caused by the incomplete miscibility of n-hexane and methanol. After the solvent mixing test, ethanol and n-hexane were completely miscible. Therefore, the solvent methanol of the molecular probe 2,6-dichloroquinone-4-chloroimide was replaced with ethanol, and the 0.25M NaOH / methanol solution in the final alkali addition operation was also replaced with 0.25M NaOH / ethanol solution. The solvent replacement method was verified. In order to verify the usability of the improved probe, propofol in water was first detected. Fig.24 It can be seen that the molecular probe changed to ethanol solvent still maintains good detection performance for propofol and has good linearity, so the detection of propofol in serum was further verified. The detection of propofol in serum samples after the probe solvent was improved was basically the same as that of the methanol probe, with only the characteristic peak shifted to the right by 7nm, which was caused by the solvent effect.
[0030] 1.3.5 Environmental optimization of serum samples In order to present the most realistic clinical serum samples containing propofol and demonstrate the detection range of the molecular probe, serum samples with different concentrations of propofol (0.4, 0.8, 1, 1.6, 2, 8, 12, 20, 40, 100, 200 mg / L) were incubated at 37°C, and then a 50% trichloroacetic acid aqueous solution was added to remove the protein. 600 μL of serum solutions with different concentrations of propofol were ultrasonically mixed with 600 μL of n-hexane. After centrifugation, 500 μL of the organic phase was mixed with 500 μL of the molecular probe (ethanol solution) and 50 μL of 0.25 M NaOH ethanol solution for color development, and the ultraviolet absorbance value was measured. The results are shown in the figure. Fig.25 As shown, at a molecular probe concentration of 20 mg / L, a good linear fit was achieved in the range of 0.2-20 mg / L, and the requirements of a larger concentration range (0.2-100 mg / L) were met through nonlinear fitting. In general, for the detection of clinical serum propofol, the current probe usage and operating procedures have met the detection range of 0.5-6 mg / L through separation and extraction detection, and have good linearity.
[0031] 1.4 Verification of real samples from clinical patients In order to verify the applicability of molecular probes and operation procedures in real clinical patient serum and establish a real and effective clinical rapid detection method, we cooperated with local tertiary hospitals to obtain serum samples of two patients during anesthesia. First, the propofol sample of simulated serum was used to make the baseline standard of the day. 700μL of clinical serum samples of two patients were taken and mixed with 50μL of 50% trichloroacetic acid by ultrasound at 10,000 revolutions per minute for 3 minutes. After centrifugation, 600μL of protein-free serum was taken and mixed with 600μL of n-hexane by ultrasound at 10,000 revolutions per minute for 3 minutes. After centrifugation, 500μL of organic phase was taken and mixed with 500μL of molecular probe (ethanol solution) and 50μL of 0.25M NaOH ethanol solution for color development, and the ultraviolet absorbance value was measured. The absorbance value at 600nm was substituted into the baseline standard of the day to obtain the propofol concentration in the patient's serum.
[0032] The results are as follows Fig.26 As shown, by making the baseline standard for the day, the linear equation is y=0.01505+0.0178x, the corresponding absorbance of propofol detection in the serum sample of patient 1 is 0.02786, and the corresponding absorbance of propofol detection in the serum sample of patient 2 is 0.02898. By substituting the linear regression equation of the baseline standard for the day, the propofol concentration in the serum of patient 1 is approximately 0.72 mg / L, and the propofol concentration in the serum of patient 2 is approximately 0.78 mg / L.
[0033] The above method is based on the ultraviolet instrument in the laboratory. When the actual platform is built in the clinic, the volume of serum and the volume of various drugs used can be adjusted accordingly. In addition, the current operating procedure can complete the clinical propofol concentration detection without enrichment. If you want to improve the detection signal of low-concentration propofol, you can increase the mixing ratio of serum and n-hexane to enrich propofol.
[0034] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.
Claims
1. A method for separating and extracting serum propofol, characterized in that: The following steps are involved: S1. removing proteins from serum samples to obtain protein-free supernatant; S2. Ultrasonic mixing of the supernatant and the organic solvent is performed, and the mixture is centrifuged to obtain an organic phase containing propofol; the organic solvent is selected from one or more of methyl tert-butyl ether, dichloromethane, and n-hexane.
2. A method for determining propofol content, characterized in that: The following steps are involved: Using the serum propofol separation and extraction method as claimed in claim 1; S3. Mix the organic phase with the molecular probe and alkaline solution to develop color, and measure the absorbance value.
3. The method for measuring propofol content according to claim 2, characterized in that: In step S1, a 50% trichloroacetic acid aqueous solution is selected as a protein precipitant to remove protein from serum.
4. The method for measuring propofol content according to claim 2, characterized in that: In step S2, the organic solvent is selected from n-hexane.
5. The method for measuring propofol content according to claim 2, characterized in that: In step S2, the volume ratio of the supernatant to the organic solvent is 1:
1.
6. The method for measuring propofol content according to claim 2, characterized in that: In step S3, the volume ratio of the organic phase to the molecular probe is 1:
1.
7. The method for measuring propofol content according to claim 2, characterized in that: In step S3, the molecular probe is an ethanol solution of 2,6-dichloroquinone-4-chloroimide.
8. The method for measuring propofol content according to claim 2, characterized in that: In step S3, the molecular probe concentration is 20 mg / L.
9. The method for measuring propofol content according to claim 2, characterized in that: In step S3, the alkaline solution is an ethanol solution of NaOH.
10. The method for measuring propofol content according to claim 2, characterized in that: The following steps are involved: S1. Ultrasonic mix 700 μL of serum sample with 50 μL of 50% trichloroacetic acid, centrifuge to obtain the protein-free supernatant; S2, taking 600 μL of the supernatant and mixing it with 600 μL of n-hexane by ultrasonication, and centrifuging to obtain an organic phase containing propofol; S3. Take 500 μL of the organic phase, mix it with 500 μL of 2,6-dichloroquinone-4-chloroimide ethanol solution and 50 μL of 0.25M NaOH ethanol solution for color development, and measure the ultraviolet absorbance value; substituting the absorbance value at 600 nm into the baseline standard of the day to obtain the propofol concentration in the serum.