CN115078612B - An analytical method for detecting chemicals based on modified Cr-MOF - Google Patents

Abstract

The present invention relates to an analytical method for detecting chemicals based on modified Cr-MOF, which utilizes dispersed solid phase extraction technology, adopts modified Cr-MOF solid phase extraction to exclude proteins in biological samples and enrich risperidone, quetiapine, aripiprazole and its metabolites 9-hydroxyrisperidone, N-dealkylated quetiapine, dehydrogenated aripiprazole, and performs high performance liquid chromatography detection. The method grafts ethylenediamine on the basis of MIL-101 (Cr) to form a modified Cr-MOF, improves the enrichment efficiency of risperidone, quetiapine, aripiprazole and its metabolites, and excludes proteins in the biological sample system to save the step of protein precipitation. The present invention is simple to operate, saves time and cost of pre-treatment, has good extraction effect, and the raw materials used are cheap and easy to obtain, and the application environment is friendly, and the market prospect is broad. The materials used in the present invention are expected to become special materials for biological sample extraction.

Description

An analytical method for detecting chemicals based on modified Cr-MOF

Technical Field

The invention belongs to the field of chemical detection and relates to an analytical method for detecting chemicals based on modified Cr-MOF.

Background Art

According to current data from the World Health Organization, mental illness (i.e., schizophrenia or bipolar disorder, manifested as hallucinations, delusions, or paranoia) affects approximately 83 million people worldwide. As a result, a series of antipsychotic drugs have emerged to provide effective treatment for various symptoms of mental illness. Antipsychotic drugs can have many side effects on the human body, which vary depending on the type of drug, the dosage taken, or the individual differences of the patient, but most have common side effects. Therefore, blood drug concentration monitoring is particularly important in psychiatry. Effective clinical management can be carried out for patients receiving psychotropic drugs. Avoid medical complications, poisoning, unresponsiveness or non-compliance. Therefore, it is necessary to strengthen the detection of psychotropic drugs in blood drug concentration monitoring.

Blood drug concentration monitoring refers to the analysis of the concentration of the drug to be measured in the blood under the guidance of pharmacokinetic principles, in order to evaluate the efficacy or determine the dosing regimen, so as to individualize the dosing regimen, improve the level of drug treatment, and achieve clinical safety, effectiveness, and rational medication. Since serum contains a large amount of protein and the concentration of psychotropic drugs in the blood is generally low, and patients often use other drugs together, this may cause certain interference to the analysis. Therefore, the pretreatment method must have high selectivity and high sensitivity to accurately perform qualitative and quantitative analysis on the subsequent analysis. In the prior art, the common analysis and detection of psychotropic drugs is pretreatment technology combined with high performance liquid chromatography. The pretreatment methods for determining psychotropic drugs in serum mainly include liquid-liquid extraction (LLE), protein precipitation (PP), and solid phase extraction (SPE). At present, the commonly used liquid-liquid extraction method has the problems of excessive use of organic reagents and low extraction rate; protein precipitation has the defects of low protein precipitation efficiency, easy clogging of high performance liquid chromatography, and interference analysis of other drugs; solid phase extraction has the defects of long extraction time, cumbersome steps (need to remove protein before extraction), easy clogging of extraction column, and special equipment.

Summary of the invention

The purpose of the present invention is to overcome the defects of the prior art and design an analytical method for detecting chemicals based on modified Cr-MOF, using dispersed solid phase extraction (DSPE) technology and modified Cr-MOF as a solid phase extraction material. The method has low toxicity, less use of organic reagents, and high protein exclusion rate. It can exclude proteins in serum samples in one step and enrich three psychotropic drugs and their metabolites at the same time.

Dispersed solid phase extraction (DSPE) is a new sample pretreatment technology developed based on nanomaterials. In recent years, many researchers have introduced MOFs materials into dispersed solid phase extraction as adsorbents. MOFs are a type of porous coordination polymer self-assembled by metal ions and organic ligands with coordination bonds. They have the characteristics of large specific surface area, variable and adjustable structure, and stable chemical properties.

In order to achieve the above object, the present invention provides an analytical method for detecting chemicals based on modified Cr-MOF: the method comprises removing proteins and enriching the serum sample to be tested by dispersed solid phase extraction technology, and then performing ultraviolet detection to obtain the concentration of the relevant drug, which specifically comprises the following steps:

1) Take the modified Cr-MOF in a centrifuge tube, add buffer and serum to the centrifuge tube, vortex to make the modified Cr-MOF evenly dispersed in the sample, centrifuge at 80000-10000r/min for 4-6min, and pour out the supernatant; the ratio of buffer to serum is 4:1;

2) Add the elution solution to the precipitate in step 1), vortex, centrifuge at 8000r/min for 5min after sufficient elution, pour the supernatant into a new centrifuge tube, blow dry with nitrogen, re-dissolve with the re-solution, disperse evenly, and subject the obtained reaction system to high performance liquid chromatography detection.

The preparation method of the modified Cr-MOF in step 1) comprises the following steps:

S1, dissolving chromium nitrate nonahydrate and terephthalic acid in ultrapure water, adding a 40% hydrofluoric acid aqueous solution by mass, and stirring to fully dissolve the chromium nitrate nonahydrate, terephthalic acid, ultrapure water and hydrofluoric acid aqueous solution in a mass ratio of (80-90):(30-40):1:(10-20);

S2, reacting the mixture obtained in step 1) in a solvent thermal reactor at 210-230° C. for 7-9 hours, and washing the obtained mixture with DMF and hot ethanol;

S3, soaking the sample in step 2) in anhydrous ethanol for 18-22 hours, washing and drying after the reaction is completed, and then placing the sample in a 30mmol/L ammonium fluoride solution at 58-62°C for 9-11 hours, washing with warm deionized water, and vacuum drying at 90-110°C overnight to obtain purified Cr-MOF;

S4. Dissolve the purified Cr-MOF in anhydrous ethanol, continue to add ethylenediamine, move it into a reactor for reaction, wash it with ethanol several times after the reaction, and vacuum dry it at 90-11°C for 10-14h to obtain modified Cr-MOF.

In step 1), the buffer solution is an acidic aqueous solution.

The pH value of the buffer solution is 3-5.

The eluting solution in step 2) is a methanol solution, a methanol solution with a volume ratio of 2% acetic acid, acetonitrile or acetone.

The reconstitution solution described in step 2) is an acetonitrile aqueous solution;

The volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:4.

The detection conditions of the high performance liquid chromatography in step 2) are: using waters ACQUity UPLC C18 50mm×2.1mm, 1.7um as the chromatographic column; the detector is a diode array UV detector, and the measurement wavelength is 250nm; the injection volume of the automatic sampler is 20uL; the mobile phase A is: 975ml ultrapure water + 25ml 2M ammonium acetate solution + 1ml formic acid + 1ml triethylamine, and the mobile phase B is acetonitrile.

The psychotropic drugs are risperidone, quetiapine, N-desalkyl quetiapine, dehydroaripiprazole, aripiprazole and its metabolite 9-hydroxyrisperidone.

The reaction conditions in step S4 are as follows: 85-95° C. for 10-14 h.

The mass ratio of the purified Cr-MOF to the ethylenediamine in step S4 is 50:1-2.

The present invention has the following advantages:

The modified Cr-MOF material has good adsorption efficiency for three psychotropic drugs and their metabolites. It can enrich the drugs and exclude proteins in serum samples in one step, saving the protein removal step. The modified Cr-MOF material is used as a dispersed solid phase extraction material and a high performance liquid chromatography-ultraviolet detector is used to establish an analysis method for three psychotropic drugs and their metabolites in serum samples. The method is simple to operate, saves pretreatment time and cost, has good extraction effect, and is green and environmentally friendly. The raw materials used in the present invention are cheap and easy to obtain, the application environment is friendly, and the market prospects are broad. The material of the present invention is expected to become a special material for biological sample extraction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a transmission electron micrograph of the Cr-MOF material MIL-101 and the modified Cr-MOF in Example 1.

Figure 2 Infrared images of Cr-MOF material MIL-101 and modified Cr-MOF in Example 1.

FIG3 is a UV chromatogram of 6 psychotropic drugs spiked in Example 2.

FIG. 4 shows the effect of the amount of adsorbent on the extraction effect in Example 4.

FIG. 5 shows the effect of buffer pH on the extraction effect in Example 5.

FIG. 6 shows the effect of extraction time on extraction effect in Example 6.

FIG. 7 shows the effect of the type of eluent on the extraction effect in Example 7.

DETAILED DESCRIPTION

The present invention will be further described in the following examples, but the present invention is not limited thereto.

Example 1

Preparation of modified Cr-MOF:

First, 800 mg of chromium nitrate nonahydrate and 332 mg of terephthalic acid were dissolved in 9.5 ml of ultrapure water, and 0.1 ml of hydrofluoric acid (40 w/w%) was added dropwise, and the mixture was stirred and dissolved thoroughly; the obtained mixture was reacted in a solvent thermal reactor at 220° C. for 8 h, and after the reaction, the obtained mixture was washed with DMF and hot ethanol;

The washed material was soaked in anhydrous ethanol at 100°C for 20 h. After the reaction was completed, it was washed and dried. Then, the material was placed in a 30 mmol/L ammonium fluoride solution at 60°C for 10 h. After washing with warm deionized water, it was vacuum dried at 100°C overnight to obtain purified Cr-MOF (MIL-101).

Weigh 0.3 g of purified MIL-101, dissolve it in 30 ml of anhydrous ethanol, add 0.18 ml of ethylenediamine, transfer it into a reactor and react at 90 ° C for 12 h. After the reaction, wash it with ethanol several times and vacuum dry it at 100 ° C for 12 h to obtain the modified Cr-MOF.

As shown in Figures 1 and 2, Figure 1 is a transmission electron microscope image of the unmodified and modified Cr-MOF obtained, and Figure 2 is an infrared image of the unmodified and modified Cr-MOF.

Example 2

In this example, the modified Cr-MOF prepared in Example 1 was used for dispersed solid phase extraction.

The modified Cr-MOF was used for dispersed solid phase extraction and combined with high performance liquid chromatography to analyze three psychotropic drugs and their metabolites in biological samples. The specific steps are as follows:

Take 9 mg of modified Cr-MOF in a 1.5 ml centrifuge tube, add 800 ml of buffer (PH=3) and 200 ml of serum (the concentration of the six psychotropic drugs in the total system is 200 ng/ml) into the centrifuge tube, vortex for 5 minutes to make the modified Cr-MOF evenly dispersed in the sample, centrifuge at 8000 r/min for 5 minutes, pour out the supernatant, and set aside the precipitate.

Add 1 ml of 2% ammonia methanol solution to the above precipitate, vortex for 8 minutes, and centrifuge at 8000 r/min for 5 minutes after thorough washing. Pour the supernatant into a new centrifuge tube, blow dry with nitrogen, and then redissolve and disperse evenly with 1 ml of ultrapure water: acetonitrile (80:20). The obtained reaction system is subjected to high performance liquid chromatography detection. The high performance liquid chromatography conditions are as follows:

HPLC detection conditions: Waters ACQUity UPLC C18 (50mm×2.1mm, 1.7um) was selected as the chromatographic column; the detector was a diode array UV detector, and the detection wavelength was 250nm; the injection volume of the automatic sampler was 10ul. Mobile phase A (975ml ultrapure water + 25 2M ammonium acetate solution + 1ml formic acid + 1ml triethylamine), mobile phase B was acetonitrile, and the elution procedure was as follows:

The results detected in this embodiment are shown in the following table:

Example 3

Testing the exclusion effect of modified Cr-MOF on proteins:

A 1 mg/mL bovine serum albumin solution was prepared to simulate the serum environment. 9 mg of modified Cr-MOF was weighed into a 1.5 mL centrifuge tube, 1 mL of bovine serum albumin solution was added, vortexed for 5 min, centrifuged at 8000 r/min for 5 min, and the supernatant was placed in a new centrifuge tube.

The protein in the supernatant was determined by the Folin-phenol method. 0.25 mL of the solution in the supernatant after enrichment and centrifugation was placed in a test tube, and 0.25 mL of ultrapure water was added to dilute it by half. Then 2.5 mL of Folin-phenol reagent A was added, mixed, and allowed to stand at room temperature for 10 min. Then 0.25 mL of Folin-phenol reagent B was added, mixed immediately, and allowed to stand at room temperature for 30 min. The absorbance value at a wavelength of 500 nm was measured, and the protein concentration in the sample system after enrichment was calculated by comparing with the standard curve.

The protein removal rate of modified Cr-MOF was calculated using the following formula:

BSA rejection (%)=C ₀ /C _f ×100

C ₀ - concentration of supernatant after SPE, C _{f -} initial concentration

The protein exclusion rate of the modified Cr-MOF was measured to be 92.93%, indicating that the modified Cr-MOF in the present invention has a good protein exclusion effect. While enriching the drug, the protein in the serum sample system is excluded from the material, eliminating the step of organic solvent protein precipitation in the traditional solid phase extraction method.

Example 4

Effect of adsorbent dosage on drug recovery rate:

This example investigates the effect of the amount of adsorbent on the drug recovery rate. 3, 5, 7, 9, 11, 13, and 15 mg of modified Cr-MOF were weighed in 1 mL of biological sample, and the concentration of six psychotropic substances in the sample was 200 ng/ml. As shown in Figure 4, when the amount of modified Cr-MOF increased from 1 mg to 9 mg, the recovery rates of risperidone, 9-hydroxyrisperidone, N-dealkylated quetiapine, and aripiprazole increased with the increase in the amount of adsorbent, indicating that as the amount of modified Cr-MOF increased, the number of adsorption sites increased and the adsorption amount increased; but when the amount of adsorbent was greater than 9 mg, the amount of modified Cr-MOF was then increased, and the extraction recovery rates of risperidone, 9-hydroxyrisperidone, N-dealkylated quetiapine, and aripiprazole all decreased, the recovery rate of dehydroaripiprazole increased, and the recovery rate of quetiapine remained basically unchanged. Considering various factors, the optimal amount of the extractant in the present invention is 9 mg.

Example 5

Effect of buffer pH on drug recovery:

This example studies the effect of buffer pH on the activity of the surface adsorption sites of the adsorbent. The extraction recovery results under the conditions of buffer pH of 3, 5, 7, 9, and 11 were investigated respectively. From the results in Figure 5, it can be seen that the recovery rates of the six psychotropic drugs basically decrease gradually from pH 3 to 11; because under acidic conditions, the modified Cr-MOF is protonated and electrostatically interacts with the alkaline drugs; therefore, the optimal pH of the extraction buffer is 3.

Example 6

Effect of extraction time on drug recovery

This example studies the effect of extraction time on drug recovery. The extraction time affects the adsorption equilibrium between the drug to be tested and the modified MOF material, and affects the drug extraction recovery. The present invention investigates the recovery rates of six psychotropic drugs when the extraction times are 1, 2, 5, 8, and 11 min, respectively. As shown in FIG6 , when the extraction time increases from 1 min to 5 min, the recovery rates of all drugs increase. After further extending the extraction time, except for a slight increase in 9-hydroxyrisperidone, the recovery rates of the remaining drugs decrease, indicating that the adsorption has reached equilibrium. Therefore, the optimal extraction time is 5 min.

Example 7

Study on the effect of different types of eluent on drug recovery

This example studies the effect of different elution solvents on the elution efficiency of the drug to be tested adsorbed on the modified Cr-MOF. This example studies the effect of three pure organic solvents, methanol, acetonitrile and acetone, on the extraction recovery rate. From the results of Figure 7, it can be seen that methanol has the best elution efficiency for the three psychotropic drugs and their metabolites, so methanol is the best elution solvent.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (9)

1. An analytical method for detecting chemicals based on modified Cr-MOF, which is characterized in that: the method comprises the steps of removing protein from a serum sample to be detected by a dispersion solid phase extraction technology, enriching, and then carrying out ultraviolet detection to obtain the concentration of related medicines, wherein the protein exclusion rate reaches 92.93 percent, and the method specifically comprises the following steps of:

1) Adding the modified Cr-MOF into a centrifuge tube, adding buffer solution and serum into the centrifuge tube, swirling to uniformly disperse the modified Cr-MOF into a sample, centrifuging for 4-6min at 80000-10000r/min, and pouring out supernatant; wherein the ratio of the buffer solution to the serum is 4:1, a step of;

2) Adding an eluting solution into the precipitate in the step 1), swirling, fully eluting, centrifuging for 5min at 8000r/min, pouring the supernatant into a new centrifuge tube, blowing nitrogen to dry, re-dissolving by using a re-dissolving solution, uniformly dispersing, and detecting the prepared reaction system by using high performance liquid chromatography;

The preparation method of the modified Cr-MOF in the step 1) comprises the following steps:

S1, dissolving chromium nitrate nonahydrate and terephthalic acid in ultrapure water, dropwise adding 40% hydrofluoric acid aqueous solution by mass percent, and fully stirring and dissolving, wherein the mass ratio of the chromium nitrate nonahydrate to the terephthalic acid to the ultrapure water to the hydrofluoric acid aqueous solution is (80-90): (30-40): 1 (10-20);

S2, reacting the mixture prepared in the step S1 in a solvothermal reaction kettle at 210-230 ℃ for 7-9 hours, and washing the prepared mixture with DMF and hot ethanol;

S3, soaking the sample in the step S2 in absolute ethyl alcohol for 18-22 hours, washing and drying after the reaction is finished, putting the sample into an ammonium fluoride solution for reacting for 9-11 hours at 58-62 ℃, washing with warm deionized water, and drying overnight at 90-110 ℃ in vacuum to obtain the purified Cr-MOF;

s4, dissolving the purified Cr-MOF in absolute ethyl alcohol, continuously adding ethylenediamine, transferring into a reaction kettle for reaction, washing with ethanol for multiple times after the reaction is finished, and vacuum drying at 90-110 ℃ for 10-14h to obtain the modified Cr-MOF.

2. The method for analysis of modified Cr-MOF-based detection chemicals according to claim 1, wherein: in step 1), the buffer solution is an acidic aqueous solution.

3. The method for analysis of modified Cr-MOF-based detection chemicals according to claim 1, wherein: the pH value of the buffer solution is 3-5.

4. The method for analysis of modified Cr-MOF assay chemistry according to claim 1, wherein: the eluting solution in the step 2) is methanol solution, methanol solution of ammonia water with the volume ratio of 2%, acetonitrile or acetone.

5. The method for analysis of modified Cr-MOF assay chemistry according to claim 1, wherein: the complex solution in the step 2) is acetonitrile water solution;

The volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:4.

6. The method for analysis of modified Cr-MOF assay chemistry according to claim 1, wherein: the detection conditions of the high performance liquid chromatography in the step 2) are as follows: a column of waters ACQUity UPLC C18 mm.times.2.1 mm,1.7um was used; the detector is a diode array ultraviolet detector, and the measuring wavelength is 250nm; sample injection amount of the automatic sample injector: 20uL; the mobile phase A is: 975ml of ultrapure water +25ml of 2M ammonium acetate solution +1ml of formic acid +1ml of triethylamine, mobile phase B being acetonitrile.

7. The method for analysis of modified Cr-MOF assay chemistry according to claim 1, wherein: the chemicals comprise psychotropic drugs and metabolites thereof, wherein the psychotropic drugs are risperidone, quetiapine, N-dealkylquetiapine, dehydroaripiprazole and aripiprazole, and the metabolites are 9-hydroxyrisperidone.

8. The method for analysis of modified Cr-MOF assay chemistry according to claim 1, wherein: in step S4, the reaction is shifted the reaction conditions in the kettle are as follows: reacting for 10-14h at 85-95 ℃.

9. The method for analysis of modified Cr-MOF assay chemistry according to claim 1, wherein: the mass ratio of the purified Cr-MOF to the ethylenediamine in the step S4 is 50:1-2.