A method and system for extracting drugs from bodily fluids

Abstract

The application discloses a method and system for extracting drugs from body fluid, comprising: preparing a eutectic solvent, taking a body fluid sample and placing it in a container, adding a buffer salt solution to adjust the pH of the sample solution, adding the eutectic solvent to the container containing the body fluid containing the drug, and uniformly dispersing the body fluid and the eutectic solvent; adding magnetic particles to the above and mixing; separating the magnetic particles in the above step using an external magnet, discarding the supernatant, and obtaining a magnetic mixture containing the target drug; adding a desorption agent to the magnetic mixture obtained in the above step to desorb the target drug. The method and system have obvious operation simplicity and time efficiency. This method does not require a centrifugal separation step, nor does it require complex preparation of magnetic DES, greatly simplifying the operation process and reducing sample processing time.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical analysis technology and relates to a method and system for extracting drugs from body fluids. Background Technology

[0002] In the field of pharmaceutical analysis, traditional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) methods have long dominated. While these methods are effective in certain situations, they typically rely on the use of large amounts of organic solvents. These solvents not only burden the environment but also pose safety risks such as flammability and toxicity during operation. Furthermore, these traditional methods are complex, time-consuming, and costly, especially when processing large volumes of samples or requiring rapid analysis, where their shortcomings are particularly evident.

[0003] With the promotion of green chemistry principles and increased environmental awareness, researchers are seeking more environmentally friendly, efficient, and economical drug extraction methods. Under this trend, eutectic solvents (DES) have become a favored new type of green solvent due to their low vapor pressure, high thermal stability, and excellent solubility. DES are liquid mixtures formed by simply mixing components such as amines, alcohols, or organic acids, through hydrogen bonding interactions. They are environmentally friendly, inexpensive, and easy to prepare. However, DES also has limitations in drug extraction from biological fluids, such as the need for cumbersome centrifugation steps after extraction to achieve phase separation. Although magnetic DES have also been developed, their synthesis typically involves multiple stages, including selecting suitable magnetic materials, preparation, and modification, which are both time-consuming and complex. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the first aspect of this application is to provide a method for extracting drugs from body fluids.

[0005] The second aspect of this application is a system for extracting drugs from bodily fluids.

[0006] The first aspect of this application is to provide a method for extracting a drug from body fluids, comprising:

[0007] (1) Preparation of eutectic solvent;

[0008] (2) Take a body fluid sample and place it in a container, add a buffer salt solution to adjust the pH of the sample solution, add the eutectic solvent to the container containing the body fluid containing the drug, and make the body fluid and the eutectic solvent evenly dispersed.

[0009] (3) Add magnetic particles to step (2) and mix;

[0010] (4) Use an external magnet to separate the magnetic particles from step (3), discard the supernatant, and obtain a magnetic mixture containing the target drug.

[0011] (5) Add a desorbent to the magnetic mixture obtained in step (4) to desorb and obtain the target drug.

[0012] In some embodiments, the drug is a hydrophobic drug.

[0013] In some embodiments, the hydrophobic drug includes antidepressants, anticancer drugs, antiviral drugs, antibacterial drugs, or anxiolytic drugs.

[0014] In some embodiments, the bodily fluid is blood, plasma, urine, saliva, cerebrospinal fluid, or tears.

[0015] As some embodiments, the magnetic particles are Fe3O4-based magnetic nanoparticles.

[0016] In some embodiments, the ratio of the magnetic particles to the body fluid is (5-50) mg: 1 mL.

[0017] In some embodiments, the buffer salt is a borax buffer salt, a phosphate buffer salt, or an acetate buffer salt.

[0018] In some embodiments, the eutectic solvent includes at least one alcohol and at least one salt, wherein the salt is choline chloride, zinc chloride, or ammonium bromide; the alcohol includes glycerol, ethylene glycol, or octanol; and the ratio of the salt to the alcohol is 1:3.

[0019] In some embodiments, the desorption solvent includes ethanol, acetonitrile, methanol, or acetone.

[0020] A second aspect of this application is to provide a system for extracting a drug from bodily fluids, characterized in that the system is used to perform the method described in the first aspect of this application.

[0021] Compared with the prior art, the beneficial effects of this application are as follows:

[0022] This invention proposes a method and system for extracting drugs from body fluids, offering significant advantages in ease of operation and time efficiency. This method eliminates the need for centrifugation and the complex preparation of magnetic DES, greatly simplifying the process and reducing sample processing time. Furthermore, this application uses an environmentally friendly eutectic solvent as the extraction medium, effectively reducing the use of toxic organic solvents, and the magnetic particles are reusable, thus demonstrating high environmental friendliness. These features make this invention superior to existing technologies in terms of sample processing efficiency and environmental friendliness, providing a safer and more efficient solution for drug extraction and analysis. Attached Figure Description

[0023] Figure 1 A flowchart of a method for extracting drugs from body fluids according to an embodiment of this application is shown;

[0024] Figure 2 The results of using the extraction method of Example 1 to detect antidepressants in urine are shown in a comparative graph.

[0025] Figure 3 The effect of DES volume on extraction recovery rate in the method of Example 2 is shown;

[0026] Figure 4 The effect of the amount of magnetic particles on the extraction recovery rate in the method of Example 3 is shown;

[0027] Figure 5 The effect of DES microextraction time on extraction recovery rate in the method of Example 4 is shown;

[0028] Figure 6 The effect of magnetic particle size on extraction recovery rate in the method of Example 5 is shown;

[0029] Figure 7 The effect of desorption time on extraction recovery rate in the method of Example 6 is shown;

[0030] Figure 8 The effect of the number of times the magnetic particles are reused on the extraction recovery rate is shown in the method of Example 7. Detailed Implementation

[0031] The present application will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0032] Example 1

[0033] A method for extracting drugs from body fluids. Figure 1 The flowchart of this method is shown, which specifically includes the following steps:

[0034] (1) Preparation of eutectic solvent (DES): Methyltrioctylammonium bromide and octanol are mixed in a molar ratio of 1:3, heated to 50°C and stirred until completely dissolved to form a uniform eutectic solvent.

[0035] (2) DES microextraction: Take 200 μL of filtered urine or blood sample and place it in a 1.5 mL centrifuge tube. Add 800 μL of phosphate buffer solution (50 mM) to adjust the pH to 10.0. Add 10 μL of prepared DES and vortex for 1 minute to fully disperse the DES in the sample.

[0036] (3) Addition of magnetic particles: Add 10 mg of magnetic particles, which are iron oxide-based magnetic nanoparticles, to the mixture after vortexing. Continue vortexing for 30 seconds to allow the magnetic particles to combine with DES and form magnetic DES.

[0037] (4) Magnetic separation: Use an external magnet to quickly separate the magnetic DES from the aqueous phase, discard the supernatant, and leave the magnetic DES containing the target analyte.

[0038] (5) Desorption: Add 100 μL of ethanol as a desorbent to a centrifuge tube containing magnetic DES, and vortex for 1 minute to desorb the target analyte from the magnetic particles. Collect the ethanol eluent containing the target analyte and use it directly for liquid chromatography-ultraviolet detection analysis.

[0039] (6) Detection: The collected eluent was directly used for liquid chromatography-ultraviolet (LC-UV) detection analysis. Specific chromatographic conditions were set as follows: an Agilent Zorbax SB-C18 column (150 × 4.6 mm id, 5 μm) was used. The mobile phase consisted of water and acetonitrile (ACN) in a 48:52 volume ratio containing 20 mM sodium phosphate buffer (pH 3.0). The flow rate was maintained at 1.0 mL / min throughout the chromatographic process. The detection wavelength was set to 230 nm, and the sample injection volume was 20 μL.

[0040] Optionally, the eutectic solvent in step (1) above is a mixture of at least one alcohol and at least one salt, which forms a stable liquid in a certain proportion, has the property of being immiscible with water and being able to effectively extract hydrophobic drugs from body fluids. Commonly used alcohols may include glycerol, ethylene glycol or octanol, while salts may include choline chloride, zinc chloride or ammonium bromide.

[0041] Optionally, the urine sample in step (2) above may contain hydrophobic drugs, including but not limited to antidepressants, anticancer drugs, antiviral drugs, antibacterial drugs, anxiolytic drugs or other hydrophobic drugs.

[0042] Optionally, the desorption solvent in step (5) above is ethanol, acetonitrile, methanol or acetone.

[0043] Optionally, the instrumental analysis described in step (6) above can be high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), ultraviolet-visible spectroscopy (UV-Vis) analysis, infrared spectroscopy (IR) analysis, or nuclear magnetic resonance (NMR) analysis, etc.

[0044] Example 2

[0045] The difference between this embodiment and Embodiment 1 is that the amount of DES used in step (2) is adjusted to 5, 10, 15, 20, 30, and 40 μL, respectively. The rest is the same as in Embodiment 1.

[0046] Example 3

[0047] The difference between this embodiment and Embodiment 1 is that the amount of magnetic particles used in step (3) is adjusted to 5 mg, 15 mg, 20 mg and 40 mg respectively. The rest is the same as in Embodiment 1.

[0048] Example 4

[0049] The difference between this embodiment and Embodiment 1 is that the vortex time in step (2) is adjusted to 0.5, 1, 2, 4, 6 and 10 minutes respectively. The rest is the same as Embodiment 1.

[0050] Example 5

[0051] The difference between this embodiment and Embodiment 1 is that the particle size distribution of the magnetic particles in step (3) is adjusted to 20 nm, 50 nm, and 2000 nm. The rest is the same as in Embodiment 1.

[0052] Example 6

[0053] The difference between this embodiment and Embodiment 1 is that the desorption time in step (5) is adjusted to 0.5, 1, 2, 4, 6 and 10 minutes respectively. The rest is the same as in Embodiment 1.

[0054] Example 7

[0055] The difference between this embodiment and Embodiment 1 is that the number of times the magnetic particles are reused in step (3) is used as a variable to examine the effect of reusing the magnetic particles 1 to 6 times on the extraction recovery rate. The rest is the same as in Embodiment 1.

[0056] This application applies the methods for extracting drugs from body fluids described in Examples 1 to 6 to the detection of antidepressants in urine. The aforementioned antidepressants include: fluoxetine (FLX), sertraline (SER), imipramine (IMP), and amitriptyline (AMP). The detection results are as follows: Figures 2 to 8 As shown.

[0057] Figure 2 A comparative graph showing the results of using the extraction method of Example 1 to detect antidepressants in urine is presented. Figure 2 As shown, curve a is the chromatogram of the untreated antidepressant standard solution (5 μg / mL), and curve b is the detection chromatogram of spiked urine (5 μg / mL) treated using the extraction method of Example 1 of this application. The chromatographic peaks of curve b match well with the chromatographic peaks of each analyte in the standard solution. Curve c is the detection chromatogram of spiked urine (5 μg / mL) extracted using magnetic particles without the addition of DES. Figure 2 As shown, the peak signal of the antidepressant in curve c is very weak. The above data indicates that the use of DES and magnetic particles in this embodiment for extracting antidepressants from urine demonstrates a synergistic effect. The entire extraction process is completed in approximately three minutes, and the recovery rate of the four antidepressants exceeds 80%.

[0058] The extraction method of this application has the advantages of being green and environmentally friendly, as follows: (1) Use of eutectic solvent (DES): This method uses a eutectic solvent composed of methyltrioctylammonium bromide and octanol in a 1:3 molar ratio. As a novel green solvent, the eutectic solvent has the characteristics of low toxicity and easy biodegradability, which is in line with the principles of green chemistry. (2) Magnetic particle DES microextraction method: Magnetic particles are used to assist in the phase separation of the eutectic solvent and the sample solution, which can avoid the cumbersome centrifugation steps. (3) Use of low-toxicity ethanol as a desorbent: In the extraction and desorption steps, low-toxicity ethanol is selected as the desorbent, which further reduces the toxicity and environmental risk of the method. (4) Fast and efficient extraction process: The entire extraction process takes about 3 minutes to complete, which has the characteristics of high efficiency and energy saving.

[0059] Figure 3 The effect of DES volume on extraction recovery in the method of Example 2 is shown. Figure 3 As shown, the amount of DES used in step (2) was adjusted accordingly. When the amount of DES was 5-40 μL, the overall extraction recovery rate of antidepressants in body fluids was relatively consistent. Based on the premise of reducing the amount of DES while ensuring the extraction recovery rate, 10 μL of DES was selected as the preferred embodiment.

[0060] Figure 4 The effect of the amount of magnetic particles used on the extraction recovery rate in the method of Example 3 is shown. For example... Figure 4As shown, the amount of magnetic particles used in step (3) was adjusted accordingly. When the amount of magnetic particles was 5-10 mg, the extraction recovery rate of the above four antidepressants in body fluids was all above 80%. The extraction recovery rate decreased slightly with the increase of the amount of magnetic particles, possibly because too many magnetic particles led to an increase in residual desorption solution. Therefore, based on the premise of reducing the amount of magnetic particles and ensuring the extraction recovery rate, using 10 mg of magnetic particles is the preferred embodiment.

[0061] Figure 5 The effect of DES microextraction time on extraction recovery is shown in the method of Example 4. For example... Figure 5 As shown, when the DES microextraction vortex time in step (2) was adjusted to 0.5, 1, 2, 4, 6, and 10 minutes respectively, the extraction recovery rates of the four antidepressants in body fluids were comparable. Figure 5 As shown, the extraction method of this application only requires 0.5 minutes of vortexing time to achieve full extraction of antidepressant drugs.

[0062] Figure 6 The effect of magnetic particle size on extraction recovery in the method of Example 5 is shown. Figure 6 As shown, the size of the magnetic particles in step (3) was adjusted accordingly. When the size of the magnetic particles was 20 nm, 500 nm and 2000 nm respectively, the extraction recovery rate of the above four antidepressants in body fluid was comparable, all greater than 80%.

[0063] Figure 7 The effect of desorption time on extraction recovery is shown in the method of Example 6. For example... Figure 7 As shown, the desorption time in step (5) was adjusted to 0.5, 1, 2, 4, 6 and 10 minutes respectively. Within the range of investigation, the desorption time had little effect on the extraction recovery rate. The shortest desorption vortex time of only 0.5 minutes was required to achieve sufficient desorption.

[0064] Figure 8 The effect of the number of times the magnetic particles were reused on the extraction recovery rate in the method of Example 7 is shown. Figure 8 As shown, the magnetic particles in step (3) are reused, and the magnetic particles are cleaned with ethanol three times between each reuse. Figure 8 The results showed that there was no significant difference in the extraction recovery rate obtained by reusing the same magnetic particles six times, indicating that the magnetic particles have excellent reusability.

[0065] Experimental Example 1

[0066] This application further investigated the quantitative analytical performance of antidepressants. The results are shown in Tables 1 and 2. Good linear calibration curves were obtained for the four antidepressants in the concentration range of 0.01–10.00 μg / mL, with correlation coefficients exceeding 0.999. The limits of quantitation (LOQs) ranged from 0.068 to 0.092 μg / mL. The relative recoveries (93.8–111.6%) and precision (RSD ≤ 6.0%) at low, medium, and high concentrations were all at high levels.

[0067] Table 1. Linear range, regression data, limit of detection, and limit of quantitation for four antidepressants.

[0068]

[0069] Table 2. Relative recoveries and relative standard deviations (RSDs) of the developed method at three different spiking levels (0.2, 2.0, and 5.0 μg / mL) within and between days (n=3).

[0070]

[0071] Experimental Example 2

[0072] This experimental example is the same as Example 1. To evaluate the feasibility of the proposed method for analyzing antidepressants in real samples, a single-blind experiment was conducted on spiked urine and plasma samples: three urine and plasma samples were spiked with different concentrations of the drug (2.5, 6.8, and 9.0 μg / mL) by a person who did not disclose the spiking level to the operator until the final determined level was revealed. As shown in Table 3, the detected concentrations were in good agreement with the spiked concentrations, with a relative error not exceeding ±8.8%. These results demonstrate the applicability and reliability of this method for analyzing antidepressants in real urine and plasma samples.

[0073] Table 3. Results of single-blind experiments on urine and plasma measurements.

[0074]

[0075] The second aspect of this application also provides a system for extracting drugs from body fluids, which is used to perform the method for extracting drugs from body fluids according to the first aspect of this application. This system enables rapid, environmentally friendly extraction of drugs from body fluids, is simple to operate, and possesses high efficiency and accuracy in drug extraction.

[0076] The above embodiments are merely preferred embodiments of this application and should not be construed as limiting the scope of protection of this application. Any non-substantial changes and substitutions made by those skilled in the art based on this application shall fall within the scope of protection claimed by this application.

Claims

1. A method for extracting a drug from body fluids, characterized in that, include: (1) Preparation of eutectic solvent; The preparation process of the eutectic solvent includes: mixing methyltrioctylammonium bromide and octanol in a molar ratio of 1:3, heating and stirring until dissolved to form the eutectic solvent; (2) Take a body fluid sample and place it in a container, add buffer salt solution to adjust the pH of the sample solution, add the eutectic solvent to the container containing the body fluid containing the drug, and disperse the body fluid and the eutectic solvent evenly; wherein, The body fluid sample is blood or urine; The drug is fluoxetine, sertraline, imipamine, or amitriptyline; The buffer salt solution is a phosphate buffer salt; (3) Add magnetic particles to step (2) and mix; The magnetic particles are Fe3O4-based magnetic nanoparticles, and the ratio of the magnetic particles to the body fluid is (5-50) mg: 1 mL. (4) Use an external magnet to separate the magnetic particles from step (3), discard the supernatant, and obtain a magnetic mixture containing the target drug; (5) Add a desorbent to the magnetic mixture obtained in step (4) to desorb and obtain the target drug; the desorbent is selected from ethanol, acetonitrile, methanol or acetone.

2. A system for extracting drugs from body fluids, characterized in that, The system is used to perform the method as described in claim 1.

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