A method for detecting the content of hydrophobic drugs in body fluids

Abstract

The present invention discloses a method for detecting the content of hydrophobic drugs in body fluids, which method relates to a hydrophobic lipophilic fiber-supported liquid phase extraction device in a syringe, combined with flow injection mass spectrometry (FIA-MS) for rapid detection of drugs in body fluids. A hydrophobic lipophilic fiber attached with an oily solvent such as n-octanol is placed between a syringe needle and a syringe to construct a simple and easy-to-use device for extracting hydrophobic drugs in body fluid samples. The sample solution is drawn into the syringe by the syringe plunger rod, and the hydrophobic drug can be quickly extracted onto the hydrophobic lipophilic fiber, and then the desorption solvent is drawn for desorption, and the desorption liquid is analyzed and detected using FIA-MS. This method can quickly analyze hydrophobic drugs in complex body fluid samples, enhance the ability to resist matrix interference, and improve detection sensitivity. In addition, the present invention expands the extraction technology in the syringe, and is expected to develop into an automated pre-treatment device to achieve fully automated analysis.

Description

Technical Field

[0001] The invention belongs to the field of drug detection and relates to a method for detecting the content of hydrophobic drugs in body fluids. Background Art

[0002] Drug therapy is the main treatment for many diseases. The detection of drug concentration in body fluids is of great significance for determining safe and effective drug dosages. Therefore, it is particularly important to establish a rapid detection method for hydrophobic drugs.

[0003] The complex biological matrix is ​​a huge challenge for the detection of antidepressants, and sample pretreatment is required to minimize matrix interference caused by endogenous compounds (proteins, lipids, etc.). At present, the existing sample pretreatment methods for hydrophobic drug detection methods mainly include solid phase extraction (SPE) and liquid liquid extraction (LLE). Solid phase extraction has good reproducibility and can be automated, and is generally more popular. However, before loading the sample, the extraction column must undergo activation, equilibrium, and cleaning steps, making sample pretreatment complicated, time-consuming, and labor-intensive. The liquid-liquid extraction method uses the different solubility of the target in the two-phase solution to achieve the purpose of effective separation, but the liquid-liquid extraction requires more solvents, and the extraction process requires continuous oscillation and centrifugation, which is complicated to operate. In addition, when used for body fluid samples containing proteins, liquid-liquid extraction is prone to emulsification, resulting in poor reproducibility. Therefore, it is of great significance to develop a simple and easy pretreatment method for the detection of hydrophobic drugs in body fluids.

[0004] Supported liquid extraction (SLE) uses diatomaceous earth treated by a special process as an adsorbent to quickly adsorb water in the sample matrix and form a liquid film on the surface of the adsorbent, thereby separating the target compound from the aqueous phase and achieving enrichment and purification of the target compound. This method uses a corresponding extraction device and only requires two steps, namely, loading and eluting, to extract the target compound from the aqueous phase. Compared with sample pretreatment methods such as LLE and SPE, SLE has higher sample extraction capacity and efficiency, and has the advantages of less matrix interference and less sample volume required. It is widely used in the pretreatment of human biological samples. Its disadvantage is that it requires steps such as drying and re-dissolution, which seriously affects the speed and throughput of pretreatment and is not conducive to rapid and high-throughput analysis. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a method for detecting the content of hydrophobic drugs in body fluids, which method has the characteristics of rapidity, accuracy, good specificity and high sensitivity.

[0006] One of the purposes of the present invention is achieved by the following technical solution:

[0007] A method for detecting drug content in body fluids, comprising the following steps:

[0008] (1) loading the internal hydrophobic and oleophilic fiber filler into an extraction device, and adding an extractant onto the internal hydrophobic and oleophilic fiber filler;

[0009] (2) adding the body fluid to be detected onto the internal hydrophobic and oleophilic fiber filler to mix the extractant with the body fluid to be detected; then adding a desorption solvent into the extraction device to obtain a desorption liquid containing the drug to be analyzed;

[0010] (3) Detecting the desorption liquid containing the hydrophobic drug to be analyzed.

[0011] Furthermore, the internal hydrophobic and oleophilic fiber filler is any fiber with hydrophobic and oleophilic properties, such as natural fiber (kapok fiber, feather, etc.) or synthetic fiber (polypropylene fiber, polyurethane fiber or alkyl vinyl polymer fiber, etc.).

[0012] Furthermore, the extractant is any organic solvent that is immiscible with water (n-octanol, n-hexane, etc.), a deep eutectic solvent, an ionic liquid, etc.

[0013] Furthermore, the desorption solvent is acetonitrile, methanol, anhydrous ethanol, 80% acetonitrile, 80% methanol or 80% ethanol.

[0014] Furthermore, the desorption solvent also includes formic acid, and the volume content of the formic acid is 0-1.0%.

[0015] Furthermore, in the step (2), the number of extractions and the number of desorptions are one or more.

[0016] Furthermore, the drug is selected from antidepressants (one or more of imipramine, sertraline, fluoxetine, and amitriptyline), glucocorticoid drugs (one or more of prednisone, dexamethasone, and hydrocortisone), and sartan drugs (one or more of candesartan and valsartan).

[0017] Furthermore, the detection method in step (3) is flow injection-mass spectrometry.

[0018] Furthermore, the body fluid is plasma, urine or cerebrospinal fluid.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a method for detecting the content of hydrophobic drugs in body fluids, which relates to a hydrophobic lipophilic fiber-supported liquid phase extraction device in a syringe, which is combined with FIA-MS for rapid detection of hydrophobic drugs in body fluids. A hydrophobic lipophilic fiber attached with an oily solvent such as n-octanol is placed between a syringe needle and a syringe to construct a simple and easy-to-use device for extracting hydrophobic substances. The sample solution is drawn into the syringe by the syringe plunger rod, and the hydrophobic drug can be quickly extracted onto the hydrophobic lipophilic fiber, and then a desorption solvent is added for desorption, and the desorbed liquid is analyzed and detected by FIA-MS. This method can quickly analyze hydrophobic drugs in complex body fluid samples, enhance the ability to resist matrix interference, and improve the detection sensitivity. In addition, the present invention expands the extraction technology in the syringe, and is expected to be developed into an automated pre-treatment device to achieve fully automated analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the detection process of an embodiment of the present invention;

[0022] Figure 2 Graph showing the test results of Examples 2-6 of the present invention;

[0023] Figure 3 This is a test result diagram of Example 7 of the present invention;

[0024] Figure 4 This is a comparison chart of the effects of using fiber-supported liquid phase extraction-flow injection-mass spectrometry on the detection results in plasma in Example 8 of the present invention;

[0025] Figure 5 This is a comparison chart of the effects of supported liquid extraction-flow injection-mass spectrometry on the detection results in urine in Example 8 of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0027] Example 1

[0028] A method for detecting the content of a hydrophobic antidepressant drug in a body fluid comprises the following steps: Figure 1 shown.

[0029] (1) Extraction and desorption:

[0030] (1.1) Preparation of standard solutions: Prepare stock solutions of imipramine (IMP), sertraline (SER), fluoxetine (FLX), amitriptyline (AMT), d3-sertraline (d3-SER) and d6-fluoxetine (d6-FLX): Accurately weigh 10.0 mg of standard powder into a 10 mL beaker, dissolve it with acetonitrile-water solution (1:1, v / v), transfer it to a 10.0 mL volumetric flask, wash the beaker three times with acetonitrile-water solution (1:1, v / v), transfer the washing solution to the volumetric flask, add acetonitrile-water solution (1:1, v / v) to the volumetric flask to the mark, shake well to obtain the standard stock solution (1.0 mg / mL). Transfer the prepared stock solution to a brown screw-capped bottle and store it in a -20°C refrigerator for later use.

[0031] (1.2) Plasma and urine samples of healthy volunteers were collected from the hospital of our school. All the providers had not taken the four antidepressants selected for this study, and they had signed informed consent before the collection. Immediately after the sample was collected, it was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant was collected and stored in a -20°C refrigerator for later use. When used, the sample was thawed at room temperature, a certain amount of antidepressant standard was added, vortexed for 1 minute, and then diluted to the required multiple with potassium dihydrogen phosphate buffer (25mM, pH10.0). Plasma was diluted 5 times and urine was diluted 2 times.

[0032] (1.3) Weigh 10.0 mg of internal hydrophobic and oleophilic fiber filler - natural kapok fiber and load it into the space between the needle and the syringe of a disposable plastic syringe (5.0 mL), and draw 10.0 μL of n-octanol onto the kapok fiber. Extraction: draw 1.0 mL of the pretreated body fluid sample into the above device, push and pull 3 times to allow n-octanol to fully adsorb the antidepressant. Desorption: draw 100 μL of acetonitrile solution (containing 0.1% formic acid) into the syringe, push and pull 3 times to allow the antidepressant to be fully desorbed from the kapok fiber, and collect the desorbed liquid in a 2.0 mL centrifuge tube for subsequent FIA-MS detection.

[0033] (2) Detection: The MS system uses a Waters triple quadrupole tandem mass spectrometer (TQD), equipped with an online degasser, quaternary gradient pump, column oven, autosampler, electrospray ionization source, etc. In this experiment, sample information was collected in multiple reaction monitoring mode under positive ion scanning (ESI+). The cone voltage was 3.0 kV, the ion source temperature was set to 150 °C, the desolvation temperature was 500 °C, the desolvation gas flow rate was 1000 L / h, and the cone gas flow rate was 45 L / h. Each analyte was manually and automatically (IntelliStart) tuned the cone voltage, collision energy (Table 2.1) and automatically optimized the dwell time (Dwell time) using MassLynx 3.1 software.

[0034] Table 1 Mass spectrometry acquisition parameters for antidepressant drugs

[0035]

[0036] Note: * indicates quantitative ion.

[0037] Example 2

[0038] The difference between this embodiment and embodiment 1 is that the amount of kapok fiber in step (1.3) of embodiment 1 is adjusted to 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg and 5 mg respectively, and the rest is the same as embodiment 1.

[0039] Test results such as Figure 2 As shown in Figure A, when 1.0 to 2.0 mg of kapok fiber was used as the support material for liquid phase microextraction, the recovery rates of the four antidepressants gradually increased, and when the amount of kapok fiber was from 2.5 to 5.0 mg, the recovery rate gradually decreased to a stable state. This may be because when there is less kapok fiber, there is less n-octanol attached, so as the amount of kapok fiber increases, the recovery rate gradually increases; when the amount of kapok fiber is more, the resistance of the syringe may be too large, resulting in excessive loss of n-octanol, and the recovery rate will decrease instead.

[0040] Example 3

[0041] The difference between this embodiment and embodiment 1 is that the amount of n-octanol in step (1.3) of embodiment 1 is increased from 0 to 10 μL. The rest is the same as embodiment 1.

[0042] Test results such as Figure 2 As shown in Figure B, when the amount of n-octanol increased from 0 to 10 μL, the signal intensity of each antidepressant continued to increase; and when the amount was further increased (10-20 μL), the recovery rate remained unchanged. This may be because when the amount of n-octanol was small, the amount of antidepressants extracted was correspondingly small; when the amount of n-octanol was too large, the kapok fiber was completely infiltrated and reached saturation.

[0043] Example 4

[0044] The difference between this embodiment and embodiment 1 is that the pH of the sample in step (1.3) of embodiment 1 is adjusted to 4, 6, 7, 8, 9, 10, and 12 respectively. The rest is the same as embodiment 1.

[0045] Since alkaline antidepressants are easily dissociated in aqueous solution, this experiment inhibited their dissociation by adding pH control buffer, thereby enhancing the extraction efficiency. This study used a 25mM phosphate buffer system as the pH control buffer and investigated the effect of the pH of the sample solution on the extraction efficiency in the range of pH 4.0-12.0. The results are shown in Figure 2. Figure 2 As shown in Figure C, at pH 4.0-10.0, the recovery rate gradually increased and reached a plateau, and then gradually decreased at pH 10.0-12.0.

[0046] Example 5

[0047] The difference between this embodiment and embodiment 1 is that the desorption solvent in step (1.3) of embodiment 1 is adjusted to acetonitrile, methanol, anhydrous ethanol, 80% acetonitrile, 80% methanol, 80% ethanol, and the rest is the same as embodiment 1.

[0048] The results are as follows Figure 2 As shown in D, the desorption solvents are acetonitrile, methanol, anhydrous ethanol, 80% acetonitrile, 80% methanol, and 80% ethanol, and the four antidepressants all have high recovery rates.

[0049] Example 6

[0050] The difference between this embodiment and embodiment 1 is that the content of formic acid in the desorption solvent in step (1.3) of embodiment 1 is adjusted to 0%, 0.1%, 0.2%, 0.5% and 1.0% respectively, and the rest is the same as embodiment 1.

[0051] The results are as follows Figure 2 As shown in Figure E, when the formic acid content was between 0% and 1.0%, the recoveries of the four antidepressants were at a relatively high level, all above 40%.

[0052] Example 7

[0053] The difference between this embodiment and embodiment 1 is that the extraction times and desorption times in step (1.3) of embodiment 1 are adjusted to 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, and 15 times respectively. The rest is the same as embodiment 1.

[0054] The results are as follows Figure 3 As shown, when the extraction times were 2-15 times, the recovery rates of the four antidepressants were above 40%, and when the desorption times were 1-10 times, the recovery rates of the four antidepressants were above 45%.

[0055] Example 8

[0056] A method for detecting the content of hydrophobic sartan drugs in body fluids. The difference between this embodiment and embodiment 1 is that: the stock solutions of imipramine (IMP), sertraline (SER), fluoxetine (FLX), amitriptyline (AMT), d3-sertraline (d3-SER) and d6-fluoxetine (d6-FLX) in step (1.1) of embodiment 1 are adjusted to candesartan and valsartan stock solutions, the kapok fiber in step (1.3) is adjusted to polypropylene fiber, the n-octanol in step (1.3) is adjusted to a hydrophobic deep co-solvent (prepared by mixing decanoic acid and methyltrioctylammonium bromide in a ratio of 2:1), the mass spectrometry detection parameters in step (2) are adjusted to the following Table 2, and the rest are the same as in embodiment 1.

[0057] Table 2 Mass spectrometry acquisition parameters for sartan drugs

[0058]

[0059] Note: * indicates quantitative ion.

[0060] The test results showed that after the blank plasma was spiked with 100 ng / mL, the recoveries of candesartan and valsartan stock solution were 78% and 82%, respectively; after the blank urine was spiked with 100 ng / mL, the recoveries of candesartan and valsartan stock solution were 85% and 88%, respectively, both of which were relatively good.

[0061] Example 9

[0062] The influence of supported liquid extraction-flow injection-mass spectrometry on the detection results

[0063] To further explore the effect of in syringe-SLE-FIA-MS on detection sensitivity, this study compared the mass spectrometry signal intensities obtained by FIA-MS analysis of antidepressants with those obtained by in syringe-SLE-FIA-MS analysis in the positive ion MRM mode.

[0064] like Figure 4 and Figure 5As shown in the figure, when spiked plasma and urine were directly analyzed by FIA-MS (black line), the signal was weak; however, when SLE-FIA-MS (grey line) was used for analysis using kapok fiber, the signal intensity was greatly improved. The higher signal intensity obtained after insyringe-SLE-FIA-MS analysis may have the following two reasons: (1) most of the impurities coexisting in the biological sample were removed in the SLE step, which is conducive to improving the ionization efficiency of the ESI source for the analyte; (2) the analyte can be effectively enriched on the kapok fiber, resulting in an increase in the analyte concentration. The above results show that rapid pretreatment with SLE before FIA-MS analysis can significantly improve the detection sensitivity.

[0065] Example 10

[0066] Methodological validation

[0067] 10.1 Matrix Effects

[0068] To study the effect of coexisting matrices in plasma and urine samples on analyte detection, the signals of antidepressants in plasma, urine and standard solutions were investigated. In actual plasma samples, the mass spectrometry signals were significantly reduced compared with those in standard solutions, indicating that coexisting interferents in plasma and urine may affect the electrospray ionization efficiency. The matrix effect was calculated by comparing the mass spectrometry signals of blank plasma and blank urine samples with the same spiked amount (1 μg / mL) and the standard samples.

[0069] Table 3

[0070]

[0071] The results are shown in Table 3. The matrix effects of imipramine, fluoxetine, sertraline and amitriptyline in plasma and urine are in the range of 15.6-27.2% and 50.1-80.5%, respectively. The results show that different sample matrices interfere with the detection of antidepressants. The matrix effect was calculated by internal standard normalization method, and the matrix effect of sertraline in plasma and urine was 82.9-89.5%, 95.7-100.9%, and the matrix effect of fluoxetine in plasma and urine was 85.6-90.8%, 101.0-103.4%, which met the requirements of detection. Therefore, an isotope-labeled internal standard is required to correct the signal fluctuations caused by the matrix effect.

[0072] 10.2 Standard Curve and Limit of Quantitation

[0073] Under the optimized SLE conditions, the concentration of each analyte was set as the independent variable, d6-FLX was used as the internal standard for AMT and IMP, and the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard was set as the dependent variable to establish the standard curve.

[0074] Table 4

[0075]

[0076] The results are shown in Table 4. Each target analyte has a good linear relationship within the linear range of 1.0-1000 ng / mL.

[0077] Table 5

[0078] Detection AMT IMP SER FLX Antidepressant detection in plasma 8 4 25 80 Antidepressant detection in plasma with SLE 0.1 0.05 1 2 Antidepressant detection in urine 0.2 0.4 5 8 Antidepressant detection in urine with SLE 0.04 0.02 0.2 0.4

[0079] The signal-to-noise ratio method was used to determine the quantification limits of antidepressants themselves and the quantification limits after treatment with the SLE method in plasma and urine. The results are shown in Table 5. After treatment with the SLE method, the quantification limits of antidepressants in plasma increased by 25-80 times, and the quantification limits in urine increased by 5-25 times, significantly improving the detection sensitivity.

[0080] 10.3 Precision and Accuracy

[0081] Relative recovery was used to evaluate the accuracy of the detection and analysis method, and RSD was used to evaluate the intra-day precision and inter-day precision of the detection and analysis method.

[0082] Table 6

[0083]

[0084] The results are shown in Table 6. The relative recoveries of three different concentrations of antidepressants, low (2.0 ng / mL), medium (20.0 ng / mL), and high (500.0 ng / mL), were between 84.7-103.5% and 87.6-112.3%, and the RSDs of intra-day precision and inter-day precision were between 5.6-12.4% and 1.0-12.0%, respectively, indicating that the established method meets the analytical requirements.

[0085] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. An application of a method for detecting drugs in body fluids in detecting the content of hydrophobic drugs, characterized in that: The process of detecting the content of hydrophobic drugs based on the method of detecting drugs in body fluids includes: (1) Loading the internal hydrophobic and oleophilic fiber filler into the extraction device, and adding the extractant onto the internal hydrophobic and oleophilic fiber filler; (2) adding the body fluid to be detected onto the internal hydrophobic and oleophilic fiber filler by suction, so that the extractant and the body fluid to be detected are mixed; then adding a desorption solvent into the extraction device to obtain a desorption liquid containing the drug to be analyzed; (3) testing the desorption liquid containing the drug to be analyzed; The drugs to be analyzed are imipramine, sertraline, fluoxetine, amitriptyline, candesartan and valsartan.

2. The use according to claim 1, characterized in that The internal hydrophobic and oleophilic fiber filler is natural fiber or synthetic fiber; The natural fiber is kapok fiber or feather; The synthetic fiber is polypropylene fiber, polyurethane fiber or alkyl vinyl polymer fiber.

3. The use according to claim 1, characterized in that The extractant is an organic solvent, a deep eutectic solvent or an ionic liquid that is immiscible with water.

4. The use according to claim 3, characterized in that The organic solvent is n-octanol or n-hexane.

5. The use according to claim 1, characterized in that The desorption solvent is acetonitrile, methanol, ethanol or acetone.

6. The use according to claim 5, characterized in that The desorption solvent also includes formic acid, and the volume content of the formic acid is 0-1.0%.

7. The use according to claim 1, characterized in that The number of extractions and the number of desorptions in the step (2) are one or more.

8. The use according to claim 1, characterized in that The detection method in step (3) is flow injection-mass spectrometry.

9. The use according to claim 1, characterized in that The body fluid is plasma, urine or cerebrospinal fluid.

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