WO2020220618A1

WO2020220618A1 - One-step quechers pretreatment device

Info

Publication number: WO2020220618A1
Application number: PCT/CN2019/113800
Authority: WO
Inventors: 刘洋; 何小维; 王羽; 黄强; 张斌; 扶雄
Original assignee: 华南理工大学
Priority date: 2019-04-30
Filing date: 2019-10-28
Publication date: 2020-11-05
Also published as: CN110031578A

Abstract

A one-step QuEChERS pretreatment device. The device comprises an outer sleeve (1) and an inner sleeve (2) inserted into the outer sleeve (1). The inner wall of an open end of the outer sleeve (1) is provided with an outer tube thread (1-1). The inner sleeve (2) has an outer wall provided with an inner tube thread (2-1) that matches the outer tube thread (1-1), a lower end provided with a pushable piston (3), and a middle portion provided with a filter screen (4). An inner tube hole (2-2) is provided in the tube wall between the piston (3) and the filter screen (4). The piston (3) is sealedly fitted with the inner wall of the inner sleeve (2). The device can implement effective extraction of drugs in the blood of biological samples and the purification of blood matrix so as to obtain analysis samples in one step. The analysis samples can be directly analyzed with instruments. The device can complete operations of a QuEChERS method in the sleeves so as to save consumables and save space, is an efficient and environmentally friendly pretreatment analysis device, and can be widely applied to forensic poisons and sample pretreatment in forensic clinical analysis.

Description

One-step pre-processing device for QuEChERS

Technical field

The invention relates to the field of pretreatment devices for the analysis of amphetamine drugs, in particular to a QuEChERS one-step pretreatment device.

Background technique

As the most routine biological test material in clinic, blood can accurately and timely reflect the drug concentration in the body and the status of each drug in the target organ. The blood test material contains a large amount of organic matter, including proteins (hemoglobin, plasma protein, enzymes and protein hormones) , Non-protein nitrogen-containing compounds (nitrogen-containing substances in the blood except protein, mainly urea, uric acid, creatine, creatinine, amino acids, ammonia, peptides, bilirubin), sugars and other organic matter and vitamins, lipids (including Organic substances such as steroid hormones often interfere with the analysis of target substances in instrumental analysis. At present, QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) is a very efficient processing method for the processing of complex matrices.

QuEChERS is essentially the derivation and further development of solid phase extraction technology and matrix solid phase dispersion technology. The basic principle of this method is to extract the homogenized sample with acetonitrile (or acidified acetonitrile), then use extraction salt to salt out and stratify, use matrix dispersion extraction mechanism, use PSA (ethylenediamine-N-propylsilane) or Other adsorbents such as C18 (octadecyl silane) are combined with most of the interfering substances (organic acids, fatty acids, carbohydrates, etc.) in the matrix and removed by centrifugation to achieve the purpose of purification.

Compared with the traditional pre-processing analysis method, the QuEChERS method has many advantages: (1) The internal standard method is used for correction, which has good reproducibility, high accuracy and precision. The use of internal standard methods can effectively eliminate differences between commodities and reduce analysis errors between batches. (2) Efficient, fast, and high analysis throughput. Batch processing of 10 to 20 samples only takes 30 to 40 minutes to complete the relevant pre-processing process. (3) The solvent consumption is small and the use of chlorinated organic solvents is avoided, the pollution is small, and it conforms to the "green chemistry" environmental protection concept. (4) The space required for the experiment is small, and the process is only completed in the Teflon centrifuge tube and the solvent is added, and then immediately sealed, reducing the damage to the staff. (5) The method is easy to operate and the analysis cost is low. The reagents, utensils and devices used in this method are all basic configuration items of the laboratory, which are cheap and easy to obtain.

Regarding the very popular QuEChERS technology, although it has been continuously applied in the pretreatment of analysis and detection in 2003, the literature reports at home and abroad have been used in pesticide analysis, drug analysis, mycotoxin analysis, and polycyclic aromatic hydrocarbons. Analysis and other aspects have been widely used, but are rarely involved in the field of toxicology analysis. For QuECh ERS for drug analysis, there are only a few reports on the QuEChERS method used. Luca et al. used QuECh ERS to analyze benzene in blood. Liquid quality analysis of diazapine drugs, the lowest detection limit is 0.5ng/mL, the limit of quantification is 0.05～2ng/mL[Anzillotti L, Odoardi S, Strano-Rossi S. Cleaning up blood samples using a modified "QuEChERS" procedure for the determination of drugs of abuse and benzodiazepines by UPLC-MSMS(☆)[J].Forensic Science International, 2014, 243: 99-106.]. Matsuta et al. used a modified Qu ECh ERS to analyze 13 toxicants such as amphetamine in the blood, and the recovery rate could reach 59% to 93% at a concentration of 0.5ug/mL [Matsuta S, Nakanishi K, Miki A, et al. Development of a simple one-pot extraction method for various drugs and metabolites of forensic interest in blood by modifying the QuEChERS method[J].Forensic Science International, 2013, 232(1-3): 40-45.]. However, all of the above methods are used to improve and optimize the reagents in the QuECh ERS method. Many transfer and pipetting processes are still inevitable during the processing process. Many consumables are required, and obvious system errors will affect the experimental results. For the QuEChERS processing device, there is an invention that uses a centrifuge tube for extraction, purification and separation. Patent CN 103698195 B "QuEChERS Method Pack for Efficient Homogeneous Extraction of Pesticides and Veterinary Drug Residues" used fruits and vegetables, meat, aquatic products, food, wine, tea , The extraction tube in the method package of the tobacco invention, the syringe filter membrane filter, and the salting package tool used can only make the extracted liquid squeezed out of the lower layer after the test sample is extracted. The nozzle will be blocked, the separation of the matrix and the extraction liquid will be poor, and subsequent transfer and pipetting will cause large errors.

Summary of the invention

technical problem

The solution to the problem

Technical solutions

The present invention provides a QuEChERS one-step pre-processing device, which can complete the extraction, separation, purification and other experimental processes in a sleeve, avoiding complicated liquid extraction and pipetting processes; at the same time, it can effectively reduce the production of sample transfer Loss, reduce system error, save consumables, purify complex matrix blood, single sample processing can be completed within 10 minutes; micro-solid phase extraction, separation and purification process can also be performed.

The invention uses mixed organic reagents with high polarity to extract amphetamine drugs, and at the same time improves the extraction method, which greatly increases the extraction efficiency, better removes macromolecular compounds such as phospholipids and proteins in the blood, and reduces the influence of interferences on target compounds , So that the recovery rate is greatly increased. The addition of the purification package allows the complex matrix in the blood to be significantly purified, and the amount of sample consumed is small. The device of the present invention has the characteristics of simplicity, high efficiency, low cost, environmental friendliness, etc., and can use solid phase extraction to meet the pretreatment process of the extraction and analysis of drugs in a variety of samples. The sleeve device of the present invention can be used in conjunction with an analysis and measurement instrument. Realize efficient, fast and automated drug analysis.

The purpose of the present invention is achieved through the following technical solutions.

A QuEChERS one-step pretreatment device, comprising an outer sleeve 1 and an inner sleeve 2 inserted into the outer sleeve 1; the inner wall of the open end of the outer sleeve 1 is provided with an outer pipe thread 1-1;

The outer wall of the inner sleeve 2 is provided with an inner pipe thread 2-1 that matches the outer pipe thread 1-1, a pushable piston 3 is provided at the lower end, and a filter screen 4 is provided in the middle; between the piston 3 and the filter screen 4 The wall of the tube is provided with an inner tube small hole 2-2; the piston 3 is in a sealing fit with the inner wall of the inner sleeve 2.

Preferably, the upper end of the inner sleeve is provided with an organic filter membrane 4-1. After the sample is roughly filtered through the filter, the organic filter membrane 4-1 can be used for secondary filtration and purification.

The pore diameter of the inner tube can be different according to the state of the sample and the size of the purifying agent selected for different drugs of the processed sample, and is preferably 20 to 200 um.

Preferably, the outer pipe thread and the inner pipe thread are made of rubber sealing material. Due to the twisting of the thread of the inner and outer tube, the liquid compressed by the air pressure from the outer cavity can enter the inner tube cavity.

Preferably, the outer sleeve and the inner sleeve have better sealing performance.

Preferably, the volume ratio of the outer sleeve to the inner sleeve is 3:2 to 3:1.

Preferably, the piston is made of a rubber material with high sealing properties.

Preferably, the pore size of the filter is 10um-100um, more preferably 20um-60um.

Preferably, an extraction reagent solution and an extraction reagent pack are placed in the cavity of the outer sleeve.

Preferably, the extraction reagent solution includes acetonitrile; the extraction reagent includes grinding beads, anhydrous magnesium sulfate and sodium borate.

Preferably, a purification bag is placed in the inner sleeve to achieve sample purification in the inner sleeve cavity.

Further preferably, the extraction reagent package, extraction reagent solution, and purification package are packaged with light-proof tin foil.

Preferably, the purification includes PSA and C18.

Preferably, the bottom of the piston is larger than the middle.

The device can be used for the pretreatment of liquid samples, preferably blood; it can also be used for the analysis of drugs, pesticide residues, toxins, etc.

The above device is used to realize a one-step pretreatment of QuEChERS suitable for drug analysis in blood. It is mainly divided into three parts, namely, sample loading, extraction, separation, purification and filtration process, which specifically includes the following steps:

1) Sample loading and extraction process

Add the sample to the outer sleeve cavity, open the pre-packed extraction reagent solution, add it to the outer sleeve cavity, and add the grinding beads in the extraction reagent pack at the same time, shake it manually for 5-10 seconds, and then add the extraction reagent pack In the anhydrous magnesium sulfate and sodium borate, insert the inner sleeve into the outer sleeve. The inner sleeve has inner pipe threads and the outer sleeve has matching outer pipe threads. Screw the inner sleeve to a certain scale and load the whole The process is completed. At this time, the bottom of the piston and the liquid are kept at a certain distance. In order to prevent the liquid from splashing into the purification bag during the vortex vibration, after screwing the inner sleeve to a certain scale, vortex at 2500r/min for 30s and then stand for 1min. After stratification, the supernatant and waste liquid are obtained, and the extraction process is completed;

2) Separation process

After the extraction is completed, continue to screw the inner sleeve to a certain scale so that the bottom of the piston contacts the bottom of the outer sleeve and stops rotating. At this time, the supernatant is squeezed up, the liquid level is higher than the purifying agent, and the air in the outer sleeve cavity is compressed. The internal pressure is greater than atmospheric pressure. Because there are many small holes on the wall of the inner casing pipe where the purifying agent is installed, the supernatant liquid extracted is under the action of the liquid surface pressure difference and the atmospheric pressure difference. The supernatant enters the purification agent layer through the small holes in the inner tube, completes the separation process, and enters the next step of purification package purification;

3) Purification and filtration process

The purified supernatant rises in the inner sleeve cavity. After all the supernatant enters the inner sleeve 2 cavity, vortex for 1 min. However, the liquid level at this time is below the filter membrane, due to the liquid surface pressure The force of difference and atmospheric pressure difference is too small to allow the purified liquid to pass through the filter membrane with smaller holes. Therefore, the sleeve can be turned over, so that while the filter screen 4 is freely filtered due to gravity, the inner sleeve continues to rotate, and the piston pushes the purified supernatant to move downwards. When the piston passes over the small hole of the inner tube, a In a closed space, when the inner sleeve continues to be screwed down, the piston pushes the liquid forward, and the pressure in the closed space is greater. Therefore, the thrust and internal pressure generated during the process of continuing to screw the inner sleeve make the purified The liquid can pass through the filter with relatively large resistance. When the purified and coarsely filtered liquid passes through the organic filter membrane, the entire extraction process is completed, and the purified sample is obtained for direct instrument detection.

Compared with the prior art, the present invention has the following advantages and effects:

1. The device of the present invention can successfully implement sample loading and extraction of drugs in blood of biological samples, separation of extract and matrix, and purification of samples such as blood matrix. The analysis sample can be obtained in one step, which can be directly analyzed by the instrument.

2. The present invention uses the QuEChERS one-step device and its reagent package to effectively extract drugs in complex sample matrices such as blood, such as amphetamine drugs, and use the material package to purify complex sample matrices such as blood, and finally perform sample detection and analysis. The processing of a single sample can be completed within 10 minutes, which reduces the complicated process of liquid extraction and pipetting. At the same time, it can effectively reduce the loss in sample transfer and reduce system errors.

3. The method is simple to operate for the analysis method of toxic drugs in blood samples. The QuEChERS method can be completed in the cannula device of the present invention, saving consumables and saving space. It is an efficient and environmentally friendly pre-processing analysis device. It is widely used in forensic poisons and sample pre-processing for forensic clinical analysis.

The beneficial effects of the invention

Beneficial effect

Brief description of the drawings

Description of the drawings

Figure 1 is an overall schematic diagram of the QuECHERS method of the present invention to extract the casing.

Fig. 2 is a schematic diagram of a part of the sample and extraction liquid added in the present invention.

Fig. 3 is a partial schematic diagram of the assembly sleeve for adding sample and extraction liquid according to the present invention.

Figure 4 is a partial schematic diagram of the extraction layering of the present invention.

Figure 5 is a partial schematic diagram of the pressure difference generated in the present invention.

Fig. 6 is a schematic diagram of the process in which the extract of the present invention passes through the small holes of the inner tube, enters the inner tube, and is purified by the purifying agent.

Fig. 7 is a schematic diagram of the supernatant liquid passing through the filter mesh of the present invention.

Figure 8 is a schematic diagram of the supernatant of the present invention entering the organic filter membrane after being filtered.

Invention embodiment

Embodiments of the invention

The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings, but the implementation of the present invention is not limited thereto.

The structure diagram of a QuEChERS one-step pretreatment device of the present invention is shown in Figure 1, and includes an outer sleeve 1 and an inner sleeve 2 inserted into the outer sleeve 1. The outer pipe thread 1 is provided on the inner wall of the open end of the outer sleeve 1 -1; The outer wall of the inner sleeve 2 is provided with an inner pipe thread 2-1 that matches with the outer pipe thread 1-1, the lower end is provided with a pushable piston 3, and the middle is provided with a filter 4; the piston 3 and the filter The tube wall between the nets 4 is provided with an inner tube small hole 2-2; the piston 3 is in a sealing fit with the inner wall of the inner sleeve 2; the upper end of the inner sleeve is provided with an organic filter membrane 4-1.

Example 1

As shown in Figure 2, the QuEChERS method package for the high-efficiency homogeneous extraction of amphetamines and other drugs of the present invention includes: an outer tube containing an extraction reagent solution (acetonitrile mixed reagent), an extraction reagent package 6 (grinding beads, anhydrous magnesium sulfate, Salting out package such as sodium borate); the inner sleeve is placed with PSA, C18 and other fillers. In addition, the detailed structure of the inner sleeve is shown in Figure 2. One end is equipped with a porous outer wall, the middle is equipped with a filter, and the other end is equipped with an organic filter membrane.

Example 2

As shown in Figure 2, the QuEChERS method package for the high-efficiency homogeneous extraction of amphetamines and other drugs of the present invention includes: an outer tube containing an extraction reagent solution (acetonitrile mixed reagent), an extraction reagent package (grinding beads, anhydrous magnesium sulfate, boric acid) Sodium salting out package); the inner sleeve is placed with PSA, C18 and other fillers. In addition, the detailed structure of the inner sleeve is shown in Figure 2. One end has a porous outer wall, the middle has a filter screen, and the other end contains an organic filter membrane. The outer packaging of the extraction reagent solution and the extraction reagent pack 6 is a light-proof and moisture-proof tin foil bag.

The specification of the outer sleeve is 5ml, the extraction reagent solution (1mL acetonitrile mixed reagent), the extraction reagent pack 6 includes 4-6 grinding beads, 150mg anhydrous magnesium sulfate, 30mg sodium borate and other salting out bags; 20mg PSA is placed in the inner sleeve , 10mg C18 and other fillers, in addition to the 4mL inside, the outer wall of the inner sleeve is threaded, and there are small holes, the lower end has a piston, contains a filter, and the other end contains an organic filter membrane.

Example 3

As shown in Figure 1, the QuEChERS method package for the efficient homogeneous extraction of amphetamines and other drugs of the present invention includes: an outer tube, which contains an extraction reagent solution (acetonitrile mixed reagent), an extraction reagent package (grinding beads, anhydrous magnesium sulfate, boric acid) Sodium salting-out package); the inner sleeve is placed with PSA, C18 and other fillers. In addition, the detailed structure of the inner sleeve is shown in Figure 2. One end has a porous outer wall, a filter membrane in the middle, and an organic filter membrane at the other end. The specification of the outer casing 1 is 15ml, the extraction reagent solution (1mL acetonitrile mixed reagent), the extraction reagent package 6 includes 4-6 grinding beads, 150mg anhydrous magnesium sulfate, 30mg sodium borate and other salting out packages; 20mgPSA, 10mg C18 and other fillers, the inside of the sleeve is 10mL, the outer wall of the inner sleeve is threaded, and there are small holes, the lower end has a piston, contains a filter, and the other end contains an organic filter membrane.

Example 4

As shown in Figure 1, the QuEChERS method package for the high-efficiency homogeneous extraction of amphetamines and other drugs of the present invention includes: an outer tube containing an extraction reagent solution (acetonitrile mixed reagent), an extraction reagent package (grinding beads, anhydrous magnesium sulfate, sodium borate) The inner sleeve is equipped with PSA, C18 and other fillers. In addition, the detailed structure of the inner sleeve is shown in Figure 2, with a porous outer wall at one end, a filter membrane in the middle, and an organic filter membrane at the other end. The specification of the outer sleeve is 50ml, the extraction reagent solution (1mL acetonitrile mixed reagent), the extraction reagent package 6 includes 4-6 grinding beads, 150mg anhydrous magnesium sulfate, 30mg sodium borate and other salting out packages; the inner sleeve includes 20mg PSA, Packing such as 10mgC18, in addition, the inside is 40mL, the outer wall of the inner sleeve is threaded, and there are small holes, the lower end is equipped with a piston, which contains a filter, and the other end contains an organic filter membrane.

Example 5

As shown in Fig. 1, the steps of a QuEChERS method package for the efficient homogeneous extraction of amphetamines and other drugs using the present invention are as follows:

1) Sample loading and extraction process

As shown in Figure 1, unscrew the QuECHERS extraction sleeve, add a certain amount of sample into the outer sleeve 1 cavity, open the pre-packed extraction reagent solution, add the outer sleeve 1 cavity, and add the extraction reagent at the same time Shake the grinding beads in the bag manually for 5-10 seconds, and then add the anhydrous magnesium sulfate, sodium borate and other reagents in the extraction reagent bag, as shown in Figure 2. Insert the inner sleeve 2 into the outer sleeve 1, the inner sleeve has an inner sleeve thread 2-1, and the outer sleeve has a matched outer pipe thread 1-1, screw the inner sleeve to a certain scale, as shown in Figure 3. Shown. The entire sample loading process is completed. Note that the bottom of the piston 3 and the liquid are kept at a certain distance at this time, and the bottom of the piston is relatively large, in order to prevent the liquid from splashing into the purification bag 5 during the vortex vibration. After screwing the inner sleeve to a certain scale, vortex at 2500r/min for 30s and then let it stand for 1min. The liquid stratification is shown in Figure 4, the supernatant and waste liquid, the extraction process is completed.

2) Separation process

After the extraction is completed, continue to screw the inner sleeve 2 to a certain scale, so that the bottom of the piston 3 contacts the bottom of the outer sleeve 1, and stops rotating, as shown in FIG. 5. At this time, the supernatant is squeezed up, the liquid level is higher than the purification bag, and the air in the cavity of the outer sleeve 1 is compressed, and the internal pressure is greater than the atmospheric pressure. Since the inner casing 2 is equipped with a purification bag, there are multiple small holes on the wall, as shown in Figure 5-1. Therefore, the extracted supernatant liquid is under the action of the liquid surface pressure difference and the atmospheric pressure difference. The supernatant liquid enters the purification agent layer through the small holes 2-2 of the inner tube, completes the separation process, and enters the next step of purification package purification.

3) Purification and filtration process

The purified supernatant rises in the cavity of the inner sleeve 2 as shown in Figs. 6 and 7. After all the supernatant liquid enters the cavity of the inner sleeve 2, vortex for 1 min, but the liquid level at this time is below the filter 4. Because the force of the liquid surface pressure difference and the atmospheric pressure difference is too small, the purified liquid cannot pass through the filter 4 with smaller holes. Therefore, the sleeve can be turned over, so that while the filter screen 4 is freely filtered due to gravity, the inner sleeve 2 is continuously rotated, so that the piston 3 pushes the purified supernatant to move downward. When the piston 3 passes over the small hole 2-2 of the inner tube, a closed space is formed. When the inner sleeve 2 continues to be screwed down, the more the piston 3 pushes the liquid forward, and the greater the pressure in the enclosed space. Therefore, the thrust and internal pressure generated during the process of continuing to screw the inner sleeve 2 make the purged The liquid can successively pass through the filter 4 and the organic filter membrane 4-1 with relatively large resistance (see Figure 8). When the purified liquid passes through the liquid of the filter membrane 4-1, the entire extraction process is completed, and the purified The sample is directly tested by the instrument.

The samples are whole blood, plasma, and serum. The device can also be used for analysis and processing of drugs in biological samples such as urine and saliva, and the device of the invention can also be used for pre-processing analysis.

The drugs are drugs of abuse, such as the listed amphetamine drugs. The device can also be used to analyze other types of drugs in complex matrix samples, such as pesticide residues, mycotoxins, and other analytical tests. The device of the invention can also be used for Pre-processing analysis.

The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Modifications and improvements can be made to the technical methods of the present invention, including the specifications of the sleeves used, the package of reagents, and the reagents contained in the package. All should fall within the protection scope determined by the claims of the present invention.

Claims

A QuEChERS one-step pretreatment device, which is characterized in that it comprises an outer sleeve (1) and an inner sleeve (2) inserted into the outer sleeve (1); the inner wall of the open end of the outer sleeve (1) is provided with an outer pipe thread (1-1); The outer wall of the inner sleeve (2) is provided with an inner pipe thread (2-1) that matches the outer pipe thread (1-1), the lower end is provided with a pushable piston (3), and the middle is provided There is a filter screen (4); the pipe wall between the piston (3) and the filter screen (4) is provided with an inner tube hole (2-2); the inner wall of the piston (3) and the inner sleeve (2) Sealed fit.
The QuEChERS one-step pretreatment device according to claim 1, wherein the upper end of the inner sleeve is provided with an organic filter membrane (4-1).
The QuEChERS one-step pretreatment device according to claim 1, wherein the outer pipe thread and the inner pipe thread are made of rubber sealing material.
The QuEChERS one-step pretreatment device according to claim 1, wherein the volume of the inner sleeve is smaller than the volume of the outer sleeve.
The QuEChERS one-step pre-processing device according to claim 1, wherein the piston is made of rubber with high sealing properties.
The QuEChERS one-step pretreatment device according to claim 1, wherein the pore size of the filter is 10um-100um.
The QuEChERS one-step pretreatment device according to claim 1, wherein an extraction reagent solution and an extraction reagent pack are placed in the cavity of the outer sleeve; a purification pack is placed in the inner sleeve.
The QuEChERS one-step pretreatment device according to claim 7, wherein the extraction reagent solution comprises acetonitrile; and the extraction reagent comprises grinding beads, anhydrous magnesium sulfate and sodium borate.
The QuEChERS one-step pretreatment device according to claim 7, wherein the purification includes PSA and C18.
The QuEChERS one-step pretreatment device according to claim 1, wherein the bottom of the piston is larger than the middle.