Handheld solid sample in-situ extraction device and application thereof in mass spectrometric detection

Abstract

The invention discloses a handheld solid sample in-situ extraction device and application in mass spectrometric detection.The device is an off-line in-situ extraction device and comprises a handheld extraction system A, a liquid supply system for providing a solvent for the handheld extraction system A and a negative pressure system for driving an extracting solution to flow in the handheld extraction system A; or the device is an online in-situ extraction device and comprises a handheld extraction system B and a liquid supply system for providing a solvent for the handheld extraction system B. According to the handheld solid sample in-situ extraction device provided by the invention, chemical information of a target sample can be rapidly obtained in an off-line extraction or on-line analysis form in cooperation with various mass spectrometry technologies, so that the flexibility and the application mode of the in-situ extraction detection device are enhanced, and pollution caused by approaching mass spectrometry is reduced.

Description

Technical Field

[0001] The invention belongs to the mechanical field and relates to a sample chemical component extraction device, in particular to a handheld solid sample in-situ extraction device and an application thereof in mass spectrometry detection. Background Art

[0002] Mass spectrometry (MS) has excellent sensitivity, specificity, analysis speed and molecular coverage, and is widely used in food and drug safety, environmental monitoring, forensic identification, clinical analysis and other fields. Although mass spectrometry technology has powerful analytical capabilities, traditional mass spectrometry analysis usually requires coupling with instruments such as gas chromatography (GC) or liquid chromatography (LC), which takes a long time to analyze and requires high sample pretreatment, and cannot meet the requirements of in-situ analysis. In-situ mass spectrometry avoids complex sample processing and can quickly and efficiently sample and detect samples.

[0003] Ambient ionization techniques can analyze samples with complex chemical compositions under natural conditions. They require few sample pretreatment steps, bringing the obvious advantages of simplicity, speed and versatility. Ambient ionization techniques also combine direct sampling and ionization of tiny areas on solid samples, thus achieving controllable in-situ sampling and ionization, including desorption electrospray ionization (DESI), liquid microjunction-surface sampling (LMJ-SSP), and liquid extraction surface analysis (LESA). However, the cleanliness levels of the sampling end and the mass spectrometry end of the atmospheric pressure ionization technology are completely different: due to the complexity of the detected samples, the sampling end faces a relatively harsh working environment, while the mass spectrometry end is a vacuum environment with very high cleanliness requirements. The above atmospheric pressure ionization technology usually fits the sampling end and the mass spectrometry end together or places them very close to each other. The analysis of complex samples can easily cause mass spectrometry contamination, errors and instrument loss. On the other hand, the close coupling between the sampling end and the mass spectrometry end loses the portability of the sampling end, and the resulting steric hindrance limits the size and distance of the sample, which limits its application in clinical sampling, field sampling, etc. while lacking flexibility.

[0004] In order to realize in-situ and portable extraction of solid samples and reduce the contamination of the samples to the mass spectrometer, the present invention is specially proposed. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a handheld solid sample in-situ extraction device and its application in mass spectrometry detection.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A handheld solid sample in-situ extraction device, which is an offline in-situ extraction device, wherein the offline refers to the detection and analysis of the extraction liquid after the extraction is completed. The offline in-situ extraction device comprises a handheld extraction system A, a liquid supply system for providing a solvent to the handheld extraction system A, and a negative pressure system for driving the extraction liquid to flow in the handheld extraction system A;

[0008] The handheld extraction system A comprises: a handheld housing A1, a sampling pipe A2 penetrating the handheld housing A1, a three-way pipe fitting A3 and a three-way pipe fitting A4 arranged along the sampling pipe A2 and sleeved outside the sampling pipe A2 and fixed inside the handheld housing A1, and a liquid collecting pipe A5 for collecting liquid in the sampling pipe A2; one end of the sampling pipe A2 is a sampling end, and the other end is a collecting end, and both the sampling end and the collecting end extend out of the handheld housing A1; the three-way pipe fitting A3 and the three-way pipe fitting A4 are respectively arranged at the sampling end and the collecting end of the sampling pipe A2; the liquid collecting pipe A5 is arranged at the collecting end of the sampling pipe A2;

[0009] The main pipe of the three-way pipe fitting A3 is sleeved on the sampling pipe A2 near the sampling end, and the branch pipe is provided with an adapted plug A33, on which the pipe A8 for conveying the solvent in the liquid supply system A9 to the three-way pipe fitting A3 is tightly provided; the main pipe near the collecting end is provided with an adapted plug A32, on which the plug A32 is tightly provided with a fixed sleeve for fixing the sampling pipe A2, and the fixed sleeve is tightly sleeved outside the sampling pipe A2; the main pipe near the sampling end is provided with an adapted plug A31, on which the plug A31 is tightly provided with a nested pipe A6 sleeved outside the sampling pipe A2, and a cavity is reserved between the inner wall of the nested pipe A6 and the outer wall of the sampling pipe A2 for the solvent conveyed to the three-way pipe fitting A3 to flow out to the sampling end of the sampling pipe A2;

[0010] The main pipe of the three-way pipe fitting A4 is sleeved on the sampling pipe A2 near the collecting end, and the branch pipe is provided with an adapted plug A43, on which the pipe A10 for extracting the gas in the three-way pipe fitting A4 to the negative pressure system A11 is tightly provided; the end near the sampling end of the main pipe is provided with an adapted plug A41, on which the plug A41 is tightly provided with a fixed sleeve for fixing the sampling pipe A2, and the fixed sleeve is tightly sleeved outside the sampling pipe A2; the end near the collecting end of the main pipe is provided with an adapted plug A42, on which the plug A42 is tightly provided with a sleeve The nested tube A7 outside the sampling pipe A2 extends out of the handheld housing A1 and is tightly inserted into the liquid collecting pipe A5. A cavity is left between the inner wall of the nested tube A7 and the outer wall of the sampling pipe A2 for the negative pressure system to draw the gas in the liquid collecting pipe A5 through the three-way pipe fitting A4 to form a negative pressure environment in the liquid collecting pipe A5. A wall-attached connection is provided between the end of the sampling pipe A2 and the inner wall of the liquid collecting pipe A5 to facilitate the extraction liquid in the sampling pipe A2 to flow into the liquid collecting pipe A5 along the inner wall of the liquid collecting pipe A5 driven by the air pressure difference.

[0011] Preferably, the liquid supply system A9 comprises a syringe and a syringe pump for controlling the syringe to inject the solvent.

[0012] Preferably, the sampling pipe A2 is a quartz capillary.

[0013] Preferably, the nested tubes A6, A7 are ETFE tubes.

[0014] Preferably, the fixed sleeve is a PEEK tube.

[0015] A handheld solid sample in-situ extraction device, which is an online in-situ extraction device, wherein the online means that the extraction liquid is directly transported to a mass spectrometry system for analysis and detection while the extraction is being performed, and the online in-situ extraction device comprises a handheld extraction system B and a liquid supply system that provides a solvent for the handheld extraction system B;

[0016] The handheld extraction system B comprises: a handheld housing B1, a sampling pipe B2 running through the handheld housing B1, a three-way pipe fitting B3 and a two-way pipe fitting B4 arranged along the sampling pipe B2 and sleeved outside the sampling pipe B2 and fixed inside the handheld housing B1; one end of the sampling pipe B2 is a sampling end, and the other end is a mass spectrometer connection end, and both the sampling end and the mass spectrometer connection end extend out of the handheld housing B1; the three-way pipe fitting B3 is arranged at the sampling end near the sampling end of the sampling pipe B2, and the two-way pipe fitting B4 is arranged at the mass spectrometer connection end of the sampling pipe B2;

[0017] The main pipe of the three-way pipe fitting B3 is sleeved on the sampling pipe B2 near the sampling end, and the branch pipe is provided with an adaptive plug B33, on which the pipe B6 for conveying the solvent in the liquid supply system B7 to the three-way pipe fitting B3 is tightly provided; the end of the main pipe near the mass spectrometer connection is provided with an adaptive plug B32, on which the plug B32 is tightly provided with a fixed sleeve for fixing the sampling pipe B2, and the fixed sleeve is tightly sleeved outside the sampling pipe B2; the end of the main pipe near the sampling end is provided with an adaptive plug B31, on which the plug B31 is tightly provided with a nested pipe B5 sleeved outside the sampling pipe B2, and a cavity is reserved between the inner wall of the nested pipe B5 and the outer wall of the sampling pipe B2 for the solvent conveyed to the three-way pipe fitting B3 to flow out to the sampling end of the sampling pipe B2;

[0018] The two-way pipe fitting B4 is sleeved on the sampling pipeline B2 near the mass spectrometer connection end. The two-way pipe fitting B4 is provided with matching plugs B41 and B42 near the sampling end and the mass spectrometer connection end respectively. The plugs B41 and B42 are respectively tightly provided with fixed sleeves for fixing the sampling pipeline B2. The fixed sleeves are tightly sleeved on the outside of the sampling pipeline B2, which is driven by the pressure difference formed by the negative pressure in the mass spectrometer system to transport the extract to the mass spectrometer system B8.

[0019] Preferably, the liquid supply system comprises a syringe and a syringe pump for controlling the syringe to inject the solvent.

[0020] Preferably, the sampling pipe B2 is a quartz capillary.

[0021] Preferably, the nested tube B5 is an ETFE tube.

[0022] Preferably, the fixed sleeve is a PEEK tube.

[0023] Beneficial effects:

[0024] 1. The present invention provides a detection device for in-situ analysis of the surface of a solid sample, which is divided into an offline in-situ extraction device and an online in-situ extraction device. The chemical information of the target sample can be quickly obtained by offline extraction or online analysis in combination with a variety of mass spectrometry techniques.

[0025] 2. The present invention realizes two different detection modes by means of two handheld device structures, which enhances the flexibility and application of the in-situ extraction detection device and reduces the contamination caused by proximity to the mass spectrometer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is an off-line in-situ extraction device and structure diagram;

[0027] Figure 2 It is an online in-situ extraction device and structure diagram;

[0028] Figure 3The representative mass spectrum (A) of the buspirone standard sampled on the glass slide and the spectrum (B) of the blank matrix 2,5-dihydroxybenzoic acid are used for the offline in situ extraction device combined with MALDI-TOF MS;

[0029] Figure 4 The offline in situ extraction device combined with MALDI-TOF MS is used to detect representative mass spectra in rat brain slices, and the lipid signals with mass-to-charge ratios ranging from 750 to 850 and small molecule metabolite signals ranging from 100 to 180 are magnified and displayed;

[0030] Figure 5 The offline in-situ extraction device combined with MALDI-TOF MS is used to detect various chemical components in a variety of Chinese herbal medicine pieces, including representative mass spectra of extracts from honeysuckle, chrysanthemum, rose, and coptis root pieces; A is a representative mass spectra of extracts from honeysuckle; B is a representative mass spectra of extracts from chrysanthemum; C is a representative mass spectra of extracts from rose; D is a representative mass spectra of extracts from coptis root;

[0031] Figure 6 The representative mass spectra of the off-line in-situ extraction device combined with MALDI-TOF MS for detecting safflower and dyed chrysanthemum, wherein A is the representative mass spectra of the standard reference substance of hydroxysafflor yellow A; B is the representative mass spectra of the standard medicinal material extract of safflower; C is the representative mass spectra of the extract of analyte 1; D is the representative mass spectra of the extract of analyte 2;

[0032] Figure 7 The online in situ extraction device combined with HESI-Q Exactive MS is used to detect the representative mass spectrum of rat brain slices, and the lipid signals with a mass-to-charge ratio range of 720-850 and the small molecule metabolite signals with a mass-to-charge ratio range of 120-200 are magnified.

[0033] Figure 8 The online in-situ extraction device combined with HESI-Q Exactive MS is used to detect the representative mass spectra of safflower and dyed chrysanthemum; A is the ion signal of analyte 1 in the mass-to-charge ratio range of 405-430; B is the ion signal of analyte 1 in the mass-to-charge ratio range of 606-618; C is the ion signal of analyte 2 in the mass-to-charge ratio range of 405-430; D is the ion signal of analyte 2 in the mass-to-charge ratio range of 606-618;

[0034] Fig. 9The online in-situ extraction device is combined with HESI-Q Exactive MS to detect the representative mass spectra of hydroxysafflor yellow A standard sample points on a glass slide and hydroxysafflor yellow A in the Chinese herbal medicine safflower; wherein A is a representative mass spectrum of hydroxysafflor yellow A standard sample points detected in full scan mode; B is a representative mass spectrum of hydroxysafflor yellow A standard sample points detected in ion fragmentation mode; C is a representative mass spectrum of safflower pieces detected in full scan mode; D is a representative mass spectrum of hydroxysafflor yellow A in safflower pieces detected in ion fragmentation mode; E is the inferred ion fragmentation mode of hydroxysafflor yellow A;

[0035] Among them: A is an offline handheld extraction system, A1 is a handheld housing, A2 is a sampling pipeline, A3 is a three-way pipe fitting, A4 is a three-way pipe fitting, A5 is a liquid collecting tube, A6 is a nested tube, A7 is a nested tube, A8 is a liquid supply pipeline, A9 is a liquid supply system, A10 is a negative pressure pipeline, A11 is a negative pressure system, A31, A32, A33, A41, A42, and A43 are plugs; B is an online handheld extraction system, B1 is a handheld housing, B2 is a sampling pipeline, B3 is a three-way pipe fitting, B4 is a two-way pipe fitting, B5 is a nested tube, B6 is a liquid supply pipeline, B7 is a liquid supply system, B8 is a mass spectrometry system, and B31, B32, B33, B41, and B42 are plugs. DETAILED DESCRIPTION

[0036] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0037] Example 1: Offline in-situ extraction device

[0038] The method for setting up and using the handheld solid sample in-situ extraction device for offline in-situ extraction provided by the present invention is: assembling a handheld extraction system A, installing a liquid supply system for providing solvent to the handheld extraction system A, installing a negative pressure system for driving the extraction liquid to flow in the handheld extraction system A, using the handheld solid sample in-situ extraction device to realize offline in-situ extraction, and detecting and analyzing the extraction liquid after the extraction is completed.

[0039] The specific operation and usage are as follows:

[0040] (1) Assemble the handheld extraction system A: Figure 1As shown, it includes a handheld housing A1, a sampling pipe A2 running through the handheld housing A1, a three-way pipe fitting A3 and a three-way pipe fitting A4 arranged along the sampling pipe A2 and sleeved outside the sampling pipe A2 and fixed inside the handheld housing A1, and a liquid collecting pipe A5 for collecting liquid in the sampling pipe A2. One end of the sampling pipe A2 is a sampling end, and the other end is a collecting end, both of which extend out of the handheld housing A1; the three-way pipe fitting A3 and the three-way pipe fitting A4 are respectively arranged at the sampling end and the collecting end of the sampling pipe A2; the liquid collecting pipe A5 is arranged at the collecting end of the sampling pipe A2. The main pipe of the three-way pipe fitting A3 is sleeved on the sampling pipe A2 near the sampling end, and the branch pipe is provided with an adaptive plug A33, on which the pipe A8 for conveying the solvent in the liquid supply system A9 to the three-way pipe fitting A3 is tightly arranged; the near collecting end of the main pipe is provided with an adaptive plug A32, on which the plug A32 is tightly provided with a fixed sleeve for fixing the sampling pipe A2, and the fixed sleeve is tightly sleeved outside the sampling pipe A2; the near sampling end of the main pipe is provided with an adaptive plug A31, on which the plug A31 is tightly provided with a nested tube A6 sleeved outside the sampling pipe A2, and a cavity is reserved between the inner wall of the nested tube A6 and the outer wall of the sampling pipe A2 for the solvent conveyed to the three-way pipe fitting A3 to flow out to the sampling end of the sampling pipe A2. The main pipe of the three-way pipe fitting A4 is sleeved on the sampling pipe A2 near the collecting end, and the branch pipe is provided with an adapted plug A43, on which the pipe A10 for extracting the gas in the three-way pipe fitting A4 to the negative pressure system A11 is tightly provided; the end near the sampling end of the main pipe is provided with an adapted plug A41, on which the plug A41 is tightly provided with a fixed sleeve for fixing the sampling pipe A2, and the fixed sleeve is tightly sleeved outside the sampling pipe A2; the end near the collecting end of the main pipe is provided with an adapted plug A42, on which the plug A42 is tightly provided with a sleeve for the sampling pipe A2; The nested tube A7 outside the sampling pipe A2 extends out of the handheld housing A1 and is tightly inserted into the liquid collecting pipe A5. A cavity is left between the inner wall of the nested tube A7 and the outer wall of the sampling pipe A2 for the negative pressure system to draw the gas in the liquid collecting pipe A5 through the three-way pipe fitting A4 to form a negative pressure environment in the liquid collecting pipe A5. A wall-attached connection is made between the end of the sampling pipe A2 and the inner wall of the liquid collecting pipe A5 to facilitate the extraction liquid in the sampling pipe A2 to flow into the liquid collecting pipe A5 along the inner wall of the liquid collecting pipe A5 driven by the air pressure difference.

[0041] The handheld housing A1 is made by computer software modeling combined with 3D printing technology, including the forward and rear parts. The rear part is approximately flat with a wall thickness of 2.5mm, and is provided with grooves and screw holes for fixing the three-way pipe fittings A3 and A4. The forward part is approximately a hollow semi-cylinder with a wall thickness of 2.5mm. There is a conical structure near the sampling end, and there are holes at the tip of the cone for the sampling pipe A2 and the nested pipe A6 to pass through. The forward and rear parts can be fixed with screws through the screw holes. An extended part is set on the left side of the handheld housing to accommodate the branch pipes of the three-way pipe fittings A3 and A4. The above screw holes are all 3mm, and the screws are fixed with M3 screws and nuts. The sampling pipe A2 is a quartz capillary with an outer diameter of 362μm, an inner diameter of 150μm, and a length of about 160mm.

[0042] During assembly, first, the three-way pipe fittings A3 and A4 are embedded in the grooves of the back part and fixed with screws. The sampling pipeline A2 is passed through the main pipes of the three-way pipe fittings A3 and A4, and the matching plug A32 is installed at the near collecting end of the main pipe of the three-way pipe fitting A3. The plug A32 is tightly provided with a fixed sleeve for fixing the sampling pipeline A2. The fixed sleeve is tightly sleeved outside the sampling pipeline A2. The fixed sleeve is selected to have an outer diameter of 1 / 16 inch, an inner diameter of 405 μm, and a length of about 25 mm. PEEK tube. Since the inner diameter of the fixed sleeve of 405 μm is slightly larger than the outer diameter of the sampling pipeline A2 of 362 μm, the hand-tight plug A32 causes the fixed sleeve to produce a slight elastic deformation, thereby sealing and fixing the sampling pipeline A2. The near sampling end of the main pipe of the three-way pipe fitting A3 is provided with a matching plug A31, and the plug A31 is tightly provided with a nested tube A6 sleeved outside the sampling pipeline A2. The nested tube A6 is an ETFE tube with an outer diameter of 1 / 16 inch, an inner diameter of 1000μm, and a length of about 40mm. Since the inner diameter of the nested tube A6 of 1000μm is much larger than the outer diameter of the sampling pipe A2 of 362μm, a cavity is left between the inner wall of the nested tube A6 and the outer wall of the sampling pipe A2 for the solvent delivered to the three-way pipe fitting A3 to flow out to the sampling end of the sampling pipe A2. The plug A32 will seal and fix the sampling pipe A2 only when it is tightened, and the relative position of the sampling pipe A2 and the nested tube A6 can be adjusted when it is loosened. The sampling pipe A2 and the nested tube A6 can have two positional relationships: when the sampling pipe A2 does not pass through the nested tube A6 and is about 1-2 mm away from the sampling end of the nested tube A6, the nested tube A6 can protect the sampling pipe A2 and form a solvent reflux from the nested tube A6 to the sampling pipe A2 in the nested tube A6, but when the negative pressure and flow rate are unstable or mismatched, there will be a situation of droplet interruption and solvent overflow, resulting in extraction failure or contamination of the sample; when the sampling pipe A2 passes through the outside of the nested tube A6 and is about 1-2 mm away from the sampling end of the nested tube A6, it is only necessary to let the negative pressure supply system provide sufficient negative pressure, so that the solvent can flow out of the nested tube A6 and contact the analyte, and then enter the sampling pipe A2 under the drive of the air pressure difference, which reduces the matching requirements of the liquid flow rate and the negative pressure and simplifies the process of adjusting the flow rate and the negative pressure. The sampling pipe A2 and the nested tube A6 are passed through the hole of the cone tip near the sampling end of the forward part of the handheld housing A1, and the nested tube A6 is adjusted to protrude about 2 to 3 mm from the hole of the cone tip. A PEEK tube with an outer diameter of 1 / 16 inch, an inner diameter of 500 μm and a length of about 30 cm is used as pipeline A8. One end of pipeline A8 is connected to the liquid supply system A9, and the other end is tightly fixed to the branch pipe of the three-way pipe fitting A3 through an adapted plug A33.

[0043] An adaptable plug A41 is installed near the sampling end of the main pipe of the three-way pipe fitting A4. The plug A41 is tightly provided with a fixed sleeve for fixing the sampling pipe A2, and the fixed sleeve is tightly sleeved outside the sampling pipe A2. The fixed sleeve is selected to be a PEEK tube with an outer diameter of 1 / 16 inch, an inner diameter of 405 μm, and a length of about 25 mm. Since the inner diameter of the fixed sleeve 405 μm is slightly larger than the outer diameter of the sampling pipe A2 362 μm, the hand-tight plug A41 causes the fixed sleeve to produce a slight elastic deformation, thereby sealing and fixing the sampling pipe A2. An adaptable plug A42 is provided near the collecting end of the main pipe of the three-way pipe fitting A4. The plug A42 is tightly provided with a nested tube A7 sleeved outside the sampling pipe A2. The nested tube A7 extends out of the handheld housing A1 and is tightly inserted into the liquid collecting pipe A5. The nested tube A7 is a PEEK tube with an outer diameter of 1 / 16 inch, an inner diameter of 1000 μm, and a length of about 35 mm. Since the inner diameter of the nested tube A7 of 1000 μm is much larger than the outer diameter of the sampling pipe A2 of 362 μm, a cavity is left between the inner wall of the nested tube A7 and the outer wall of the sampling pipe A2 for the negative pressure system to extract the gas in the liquid collecting pipe A5 through the three-way pipe fitting A4 and form a negative pressure environment in the liquid collecting pipe A5. The end of the sampling pipe A2 and the inner wall of the liquid collecting pipe A5 are connected to the wall to facilitate the extraction liquid in the sampling pipe A2 to flow into the liquid collecting pipe A5 along the inner wall of the liquid collecting pipe A5 driven by the pressure difference. The characteristic of the sampling pipe A2 will be sealed and fixed only when the plug A41 is tightened, and the length of the sampling pipe A2 extending out of the nested tube A7 is adjusted to about 20 to 25 mm by loosening the plug A41. An ETFE tube with an outer diameter of 1 / 16 inch, an inner diameter of 1000 μm and a length of about 30 cm is used as the pipeline A10. One end of the pipeline A10 is connected to the negative pressure supply system A11, and the other end is tightly fixed to the branch pipe of the three-way pipe fitting A4 through an adapted plug A43.

[0044] Before sampling, take an EP tube with a hole punched in the bottle cap in advance, with a hole diameter of about 1.6 mm as the liquid collecting tube A5, and insert the nested tube A7 tightly into the liquid collecting tube A5. Since the hole diameter of about 1.6 mm is close to the outer diameter of the nested tube A7 of 1 / 16 inch, the liquid collecting tube A5 and the nested tube A7 can be tightly connected and sealed. Adjust the position of the liquid collecting tube A5 so that the end of the sampling pipeline A2 and the inner wall of the liquid collecting tube A5 form a wall-attached connection mode, which is convenient for the extract in the sampling pipeline A2 to flow into the liquid collecting tube A5 along the inner wall of the liquid collecting tube A5 under the drive of the air pressure difference, and the nested tube A7 extends into the liquid collecting tube A5 by about 15 to 20 mm, thereby preventing the extract from entering the negative pressure system. Similarly, the liquid collecting tube A5 can also be replaced with a sampling vial equipped with a bottle cap and a gasket. When the nested tube A7 is tightly inserted into the liquid collecting tube A5, the gasket of the sampling vial can still ensure the sealing effect after puncture, thereby eliminating the punching process.

[0045] (2) Installing a liquid supply system for providing solvent to the handheld extraction system A: The liquid supply system A9 includes a syringe and a syringe pump for controlling the injection of solvent by the syringe, and a fixing component for connecting the syringe to the pipeline A8.

[0046] (3) Installing a negative pressure system for driving the extraction liquid to flow in the handheld extraction system A: The negative pressure system A11 includes a small vacuum pump and a regulating component for controlling the output voltage of the vacuum pump, and a fixing component for connecting the small vacuum pump to the pipeline A10.

[0047] (4) Use a handheld solid sample in-situ extraction device to achieve offline in-situ extraction: first use a syringe to draw a suitable amount of solvent, and fix the syringe on the syringe pump; then push the syringe until the nested tube A6 is filled with solvent and there are no bubbles; then prepare the analyte, and bring the sampling end of the handheld extraction system A close to the analyte until the sampling pipe A2 is about to contact the analyte; then turn on the negative pressure supply system, and run the syringe pump to push the syringe to the front end of the probe to form a liquid junction, and start sampling when the liquid junction is formed; when a sufficient amount of extract is collected in the liquid collection pipe A5, lift the handheld extraction system A to end sampling. At this time, the liquid junction is still forming, and clean solvent flows into and washes the sampling pipe A2 to prevent the residue of the extract; after washing is completed, turn off the syringe pump, and then turn off the negative pressure supply system, and the offline in-situ extraction work is completed. In addition, the handheld extraction system A can also achieve negative pressure concentration of a small amount of extract with a lower boiling point solvent. The specific operation is: after washing is completed, turn off the syringe pump and invert the handheld extraction system A. It can be seen that the extract flows to the bottom of the tube and gradually evaporates and concentrates under the action of negative pressure.

[0048] (5) Detection and analysis of the extract: After the offline in-situ extraction is completed, the extract can be detected and analyzed. The extract can be detected by a variety of analytical instruments, including liquid chromatography-mass spectrometry (LC-MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), etc. Among them, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has the characteristics of high detection flux, high sensitivity, wide application range, mainly single-charged molecular ion peaks, and is suitable for the analysis of multi-component samples. In addition, MALDI-MS has no strict requirements on sample processing and is simple to operate. Therefore, the present invention uses MALDI-MS to detect the extract.

[0049] Taking matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis as an example, it is necessary to spot and add matrix on an indium-tin oxide (ITO) coated glass slide. Matrix components such as aminoacridine (9-AA) are negative ion detection matrices, and 2,5-dihydroxybenzoic acid (DHB) are positive ion detection matrices. Spotting experiments are divided into two loading methods combined with matrix addition: matrix re-dissolution spotting method and matrix spray method. The matrix re-dissolution spotting method uses 10 microliters of prepared matrix solution to directly dissolve the evaporated sample extract; the matrix spray method uses 10 microliters of pure solvent to dissolve, and then the matrix is ​​evenly deposited on the ITO glass slide through a matrix spray instrument. Taking the commercial MALDI mass spectrometer UltrafleXtreme MALDI TOF / TOF MS as an example, the ITO glass slide is placed on the target plate, the target plate is inserted into the target, and the software is opened to determine the chemical components in the extract according to the general operation method of the MALDI mass spectrometer. After the aforementioned extract treatment process, a good signal can be obtained.

[0050] Example 2: Online in-situ extraction device

[0051] The method for constructing and using the handheld solid sample in-situ extraction device for online in-situ extraction provided by the present invention is: assembling a handheld extraction system B, installing a liquid supply system for providing solvent to the handheld extraction system B, and using the handheld solid sample in-situ extraction device to realize online in-situ extraction and detection and analysis.

[0052] The specific operation and usage are as follows:

[0053] (1) Assemble the handheld extraction system B: Figure 2 As shown, it includes a handheld shell B1, a sampling pipe B2 running through the handheld shell B1, a three-way pipe fitting B3 and a two-way pipe fitting B4 arranged along the sampling pipe B2 and sleeved on the outside of the sampling pipe B2 and fixed in the handheld shell B1; one end of the sampling pipe B2 is the sampling end, and the other end is the mass spectrometer connection end, and the sampling end and the mass spectrometer connection end both extend out of the handheld shell B1; the three-way pipe fitting B3 is arranged at the sampling end near the sampling end of the sampling pipe B2, and the two-way pipe fitting B4 is arranged at the mass spectrometer connection end near the sampling pipe B2.

[0054] The main pipe of the three-way pipe fitting B3 is sleeved on the sampling pipeline B2 near the sampling end, and the branch pipe is provided with an adaptive plug B33, on which the pipe B6 for conveying the solvent in the liquid supply system B7 to the three-way pipe fitting B3 is tightly arranged; the main pipe near the mass spectrometer connection end is provided with an adaptive plug B32, on which the plug B32 is tightly provided with a fixed sleeve for fixing the sampling pipeline B2, and the fixed sleeve is tightly sleeved outside the sampling pipeline B2; the main pipe near the sampling end is provided with an adaptive plug B31, on which the plug B31 is tightly provided with a nested tube B5 sleeved outside the sampling pipeline B2, and a cavity is reserved between the inner wall of the nested tube B5 and the outer wall of the sampling pipeline B2 for the solvent conveyed to the three-way pipe fitting B3 to flow out to the sampling end of the sampling pipeline B2.

[0055] The two-way pipe fitting B4 is sleeved on the sampling pipeline B2 near the mass spectrometer connection end. The two-way pipe fitting B4 is provided with matching plugs B41 and B42 at the near sampling end and the near mass spectrometer connection end respectively. The plugs B41 and B42 are tightly provided with fixed sleeves for fixing the sampling pipeline B2 respectively. The fixed sleeves are tightly sleeved on the outside of the sampling pipeline B2 which transports the extract to the mass spectrometer system B8 under the drive of the air pressure difference formed by the negative pressure in the mass spectrometer system.

[0056] The handheld housing B1 is made by computer software modeling combined with 3D printing technology, including the forward and rear parts. The rear part is approximately flat with a wall thickness of 2.5mm, and is provided with grooves for fixing the three-way pipe fitting B3 and the two-way pipe fitting B4, and is provided with screw holes for fixing the three-way pipe fitting B3 and the two-way pipe fitting B4. The forward part is approximately a hollow semi-cylinder with a wall thickness of 2.5mm. It is slightly narrowed near the mass spectrometer connection end, and has a conical structure near the sampling end. There are holes at the tip of the cone for the sampling pipe B2 and the nested tube B5. The forward and rear parts can be fixed with screws through the through screw holes. An extended part is provided on the left side of the handheld housing to accommodate the branch pipe of the three-way pipe fitting B3. The above screw holes are all 3mm, and the screws are fixed with M3 screws and nuts.

[0057] During assembly, first embed the three-way pipe fitting B3 and the two-way pipe fitting B4 into the groove of the back part, and fix the three-way pipe fitting B3 and the two-way pipe fitting B4 with screws. Take a quartz capillary with an outer diameter of 362μm, an inner diameter of 150μm, and a length of about 75cm as the sampling pipeline B2, and pass the sampling end of the sampling pipeline B2 through the main pipes of the three-way pipe fitting B3 and the two-way pipe fitting B4. An adaptable plug B32 is provided at the near-mass spectrometer connection end of the main pipe of the three-way pipe fitting B3, and a fixed sleeve for fixing the sampling pipeline B2 is tightly provided on the plug B32, and the fixed sleeve is tightly sleeved outside the sampling pipeline B2. A PEEK tube with an outer diameter of 1 / 16 inch, an inner diameter of 405 μm, and a length of about 25 mm is selected as a fixed sleeve. Since the inner diameter of the fixed sleeve is slightly larger than the outer diameter of the sampling pipe B2, 362 μm, the fixed sleeve is slightly elastically deformed by the hand-tight plug B32, thereby sealing and fixing the sampling pipe B2. A matching plug B31 is provided at the near sampling end of the main pipe of the three-way pipe fitting B3, and a nested tube B5 is tightly provided on the plug B31 and is sleeved outside the sampling pipe B2. An ETFE tube with an outer diameter of 1 / 16 inch, an inner diameter of 1000 μm, and a length of about 40 mm is selected as the nested tube B5. Since the inner diameter of the nested tube B5, 1000 μm, is much larger than the outer diameter of the sampling pipe B2, 362 μm, a cavity is left between the inner wall of the nested tube B5 and the outer wall of the sampling pipe B2 for the solvent transported to the three-way pipe fitting B3 to flow out to the sampling end of the sampling pipe B2. By utilizing the characteristic that the sampling pipe B2 will be sealed and fixed only when the plug B32 is tightened, the relative position of the sampling pipe B2 and the nested tube B5 can be adjusted when loosening. The sampling pipe B2 and the nested tube B5 can have two positional relationships: when the sampling pipe B2 does not pass through the nested tube B5 and is about 1-2mm away from the sampling end of the nested tube B5, the nested tube B5 can protect the sampling pipe B2 and form a reflux of the solvent from the nested tube B5 to the sampling pipe B2 in the nested tube B5, but when the negative pressure and the flow rate are unstable or mismatched, there will be a situation of liquid droplet interruption and solvent overflow, resulting in extraction failure or contamination of the sample; when the sampling pipe B2 passes through the outside of the nested tube B5 by about 1-2mm, it is only necessary to let the negative pressure supply system provide sufficient negative pressure, so that the solvent can flow out of the nested tube B5 and contact the analyte, and then enter the sampling pipe B2 driven by the air pressure difference, which reduces the matching requirements of the liquid flow velocity and the negative pressure and simplifies the process of adjusting the flow rate and the negative pressure. Pass the sampling pipe B2 and the nested pipe B5 through the hole at the tip of the cone near the sampling end of the forward part, and adjust the nested pipe B5 to protrude about 2 to 3 mm from the hole at the tip of the cone. Take a PEEK tube with an outer diameter of 1 / 16 inch, an inner diameter of 500 μm, and a length of about 30 cm as pipe B6. One end of pipe B6 is connected to the liquid supply system B7, and the other end is tightly fixed to the branch pipe of the three-way pipe fitting B3 through an adapted plug B33.

[0058] Pass the sampling pipe B2 through the main pipe of the two-way pipe fitting B4, and install matching plugs B41 and B42 at the near sampling end and near the mass spectrometer connection end of the main pipe. The plugs B41 and B42 are respectively tightly provided with fixed sleeves for fixing the sampling pipe B2. The fixed sleeves are tightly sleeved on the outside of the sampling pipe B2 in the mass spectrometer system B8 under the drive of the air pressure difference formed by the negative pressure in the mass spectrometer system. A PEEK tube with an outer diameter of 1 / 16 inch and an inner diameter of 405 μm is selected as the fixed sleeve. The fixed sleeve tightly fixed on the plug B41 near the sampling end is about 25 mm long. Since the inner diameter of the fixed sleeve 405 μm is slightly larger than the outer diameter of the sampling pipe B2 362 μm, the fixed sleeve is slightly elastically deformed by the hand-tight plug B41, thereby sealing and fixing the sampling pipe B2. The fixed sleeve tightly fixed on the plug B42 near the mass spectrometer connection end extends out of the handheld housing B1, but its length does not exceed the distance from the sampling pipe B2 extending from the plug B42 to the mass spectrometer connection end. The two-way pipe fitting B4 is used to make a handheld structure that is easy to grasp and to fix and protect the sampling pipe B2.

[0059] During the sampling process, driven by the pressure difference formed by the negative pressure in the mass spectrometry system, the extract in the sampling pipeline B2 is transported to the mass spectrometry system B8. The mass spectrometry system B8 is a commercial mass spectrometer equipped with a heated electrospray ionization (H-ESI) ion source and a QExactive combined quadrupole Orbitrap mass spectrometer, and includes a heated electrospray ionization (H-ESI) ion source and a QExactive combined quadrupole Orbitrap mass spectrometer, and a fixed component connecting the heated electrospray ionization (H-ESI) ion source and the sampling pipeline B2.

[0060] (2) Installing a liquid supply system for providing solvent to the handheld extraction system B: The liquid supply system B7 includes a syringe and a syringe pump for controlling the syringe to inject solvent, and a fixing component for connecting the syringe to the pipeline B6.

[0061] (3) Use a handheld solid sample in-situ extraction device to achieve online in-situ extraction and detection and analysis: First, use a syringe to draw a sufficient amount of solvent and fix the syringe on the syringe pump; then push the syringe until the nested tube B5 is filled with solvent and there are no bubbles; take the commercial heated electrospray ionization (H-ESI) ion source and QExactive combined quadrupole Orbitrap mass spectrometer as an example, operate the software interface of the mass spectrometer B8, switch the mass spectrometer status from "standby" to "on", at this time the HESI ion source starts to run and the Venturi effect generates negative pressure inside the ion source, run the syringe pump to push the syringe to the front end of the probe to form a liquid junction. Observe whether the mass spectrometer signal is stable, and start collecting signals when the total ion current is stable. Prepare the analyte, bring the sampling end of the handheld extraction system B close to the analyte until the sampling pipe B2 is about to touch the analyte, at which time the liquid junction contacts the analyte and sampling begins; when enough signals are collected, lift the handheld extraction system B to end sampling, at which time the liquid junction is still forming, and clean solvent flows into and washes the sampling pipe B2 to prevent the residue of the extract; after washing is completed, turn off the injection pump, and after the mass spectrometer signal gradually disappears, switch the mass spectrometer state to "standby" state, and the online in-situ extraction work is completed.

[0062] Example 3: Application example of off-line in-situ extraction device

[0063] (1) Offline in situ extraction device combined with MALDI-TOF MS for detection of buspirone standards spotted on glass slides

[0064] In order to investigate the detection capability and accuracy of the offline in-situ extraction device for small molecule drug components, in this example, the offline in-situ extraction device was combined with MALDI-TOF MS to detect the buspirone standard sampled on a glass slide. Buspirone belongs to the azaspirocyclic decanedione class of compounds, which has an anti-anxiety effect. Due to its good ionization efficiency, it is often used to evaluate the detection performance of mass spectrometry analysis.

[0065] First, prepare the buspirone sample: take the buspirone standard (Mw 385.2472) and prepare a solution of 1 pg / ml. Use a pipette to spot the buspirone standard solution on a glass slide with a sample volume of 1 microliter. After evaporation, set aside.

[0066] Subsequently, sampling was performed using an offline in-situ extraction device: according to the in-situ extraction device provided in Example 1, and in accordance with the operation of Example 1-(4), the injection parameters were as follows: methanol:water = 8:2 (v / v) was used as the extraction solvent, the injection pump flow rate was 30 μL / min, the extract collection volume was 100 μL, 50 μL of solvent was used for washing, and the spotting and extraction were repeated 6 times; a pre-punched 1.5 ml EP tube was selected as the liquid collection tube A5, and the handheld liquid microjunction surface extraction probe device was aligned with the evaporated buspirone standard sample point on the glass slide for offline in-situ extraction.

[0067] The extract was then tested and analyzed according to the operation of Example 1-(5): Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for analysis, 2,5-dihydroxybenzoic acid (DHB) was used as the detection matrix in the positive ion mode, and the matrix was deposited by the matrix spray method. The specific operation was that the extract was evaporated to dryness under a nitrogen blow instrument for concentration, 10 microliters of pure solvent was re-dissolved, and the sample was spotted on an indium-tin oxide (ITO) coated glass slide with a sample volume of 1 μL. The matrix was then evenly deposited on the ITO glass slide by a matrix spray instrument, and the operation was carried out according to the instructions for use of the MALDI-MS instrument, and detection was carried out in the positive ion mode. Representative MALDI-MS mass spectra are shown in Figure 3 , representative spectrum from the detection of buspirone standard sample Figure 3 Buspirone [M+H] can be seen at m / z 386.2 on A + Addition peaks and isotope peak clusters. And representative spectrum in the background matrix Figure 3 In B there is no signal at m / z 386.2.

[0068] (2) Offline in situ extraction device combined with MALDI-TOF MS for detection of endogenous small molecule metabolites and lipids in rat brain slices

[0069] In order to investigate the detection capability of the offline in situ extraction device for endogenous small molecule metabolites and lipids in biological tissues, in this example, the offline in situ extraction device was combined with MALDI-TOF MS to detect sagittal slices of rat brain.

[0070] First, prepare the sagittal slices of the rat brain: take the rat brain stored in a -80°C refrigerator, cut the rat brain along the sagittal plane and slice it on a freezing microtome, the slice thickness is 10 μm, and the slices are transferred to glass slides by melting mounting. The sagittal slices of the rat brain are ready.

[0071] Subsequently, sampling was performed using an offline in-situ extraction device: according to the in-situ extraction device provided in Example 1, and in accordance with the operation of Example 1-(4), the injection parameters were as follows: 0.1% trifluoroacetic acid in methanol: water = 7.5:2.5 (v / v) was used as the extraction solvent, the injection pump flow rate was 30 μL / min, the extract collection volume was 100 μL, 50 μL of solvent was used for washing, and the extraction was repeated 5 times; a pre-punched 1.5 ml EP tube was selected as the liquid collection tube A5, and the handheld liquid microstructure surface extraction probe device was aligned with the sagittal section of the rat brain on the glass slide for offline in situ extraction.

[0072] The extract was then tested and analyzed according to the operation of Example 1-(5): Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for analysis, 2,5-dihydroxybenzoic acid (DHB) was used as the detection matrix in the positive ion mode, and the matrix was deposited by the matrix spray method. The specific operation was that the extract was evaporated to dryness under a nitrogen blow instrument for concentration, 10 microliters of pure solvent was re-dissolved, and the sample was spotted on an indium-tin oxide (ITO) coated glass slide with a sample volume of 1 μL. The matrix was then evenly deposited on the ITO glass slide by a matrix spray instrument, and the operation was carried out according to the instructions for use of the MALDI-MS instrument, and detection was carried out in the positive ion mode. Representative MALDI-MS mass spectra are shown in Figure 4 From the spectrum, we can see lipid signals distributed in the mass-to-charge ratio range of 750-850, including glycerophospholipid lipids (GP): phosphatidylcholine (PC), phosphatidylethanolamine (PE), etc., sphingolipid lipids (SP): sphingomyelin (SM) and other lipids; we can also see small molecule metabolite signals distributed in the mass-to-charge ratio range of 100-180, including γ-aminobutyric acid (GABA), glutamate (Glu), acetylcholine (Ach), etc.

[0073] (3) Offline in-situ extraction device combined with MALDI-TOF MS was used to detect various chemical components in a variety of Chinese herbal medicines, including honeysuckle, chrysanthemum, rose, and coptis root.

[0074] In order to investigate the detection capability of the offline in-situ extraction device for various chemical components in Chinese herbal medicines, in this embodiment, an offline in-situ extraction device combined with MALDI-TOF MS was used to detect various chemical components in Chinese herbal medicines such as honeysuckle, chrysanthemum, rose, and coptis.

[0075] First, prepare the Chinese herbal medicine slices: take the honeysuckle, chrysanthemum, rose, and coptis slices purchased from the Chinese medicine pharmacy and weigh about 0.5 mg of the slices on a scale. Place the 0.5 mg slices in a 35mm x 12mm culture dish to spread them evenly. The Chinese herbal medicine slices of honeysuckle, chrysanthemum, rose, and coptis are ready.

[0076] Subsequently, sampling was performed using an offline in-situ extraction device: according to the in-situ extraction device provided in Example 1, and in accordance with the operation of Example 1-(4), the injection parameters were as follows: methanol: water = 8:2 (v / v) was used as the extraction solvent, the injection pump flow rate was 30 μL / min, the extract collection volume was 100 μL, and the washing solvent was 50 μL. The extraction was repeated 5 times for each piece of Chinese medicine. Among them, the alkaloid content in the Coptis chinensis pieces was high and had a strong ionization efficiency. The extract collection volume was increased to 50 μL, and the washing volume was increased to 300 μL to completely wash the residue in the sampling pipe A2; a pre-punched 1.5 ml EP tube was selected as the liquid collection tube A5, and the handheld liquid micro-junction surface extraction probe device was aimed at the Chinese medicine piece to be tested for offline in-situ extraction.

[0077] The extract was then tested and analyzed according to the operation of Example 1-(5): Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for analysis, 2,5-dihydroxybenzoic acid (DHB) was used as the detection matrix in the positive ion mode, and the matrix was deposited by the matrix spray method. The specific operation was that the extract was evaporated to dryness under a nitrogen blow instrument for concentration, 10 microliters of pure solvent was re-dissolved, and the sample was spotted on an indium-tin oxide (ITO) coated glass slide with a sample volume of 1 μL. The matrix was then evenly deposited on the ITO glass slide by a matrix spray instrument, and the operation was carried out according to the instructions for use of the MALDI-MS instrument, and detection was carried out in the positive ion mode. Representative MALDI-MS mass spectra are shown in Figure 5 From the atlas, we can see the characteristic chemical components of the following types of Chinese herbal medicines:

[0078] (a) Representative spectrum of extracts from honeysuckle slices Figure 5 A variety of characteristic chemical components can be detected in A, including various flavonoids and flavonoid glycosides, such as rutin, in the form of [M+H] + , mass-to-charge ratio is m / z 611.1; kaempferol-rutinoside, additive form is [M+H] + , mass-to-charge ratio is m / z 595.1; luteolin-glucoside, additive form is [M+H] + , mass-to-charge ratio is m / z 499.1, etc.

[0079] (b) Representative spectrum of extracts from chrysanthemum slices Figure 5 A variety of characteristic chemical components can be detected in B, including various flavonoids and flavonoid glycosides, such as acaciatin, in the form of [M+H] + , mass-to-charge ratio is m / z 285.1; quercetin, additive form is [M+H] + , mass-to-charge ratio is m / z 465.2; luteolin-glucoside, additive form is [M+H] + , mass-to-charge ratio is m / z 499.1, etc.

[0080] (c) Representative spectrum of rose extracts Figure 5 A variety of characteristic chemical components can be detected in C, including various organic acids and phenolic acid compounds, such as gallic acid, in the form of [M+H] + , mass-to-charge ratio is m / z 171.1; the proanthocyanidin addition form is [M+H] + , mass-to-charge ratio is m / z 579.1, etc.

[0081] (d) Representative spectrum of extracts from Coptis chinensis slices Figure 5 A variety of characteristic chemical components can be detected in D, including a variety of alkaloid compounds, such as berberine, in the form of [M+H] + , mass-to-charge ratio is m / z 336.1; berberine, additive form is [M+H] + , mass-to-charge ratio is m / z 322.1; coptisine, additive form is [M+H] + , mass-to-charge ratio is m / z 321.1; corydaline, additive form is [M+H] + , mass-to-charge ratio is m / z 342.2, etc.

[0082] (4) Offline in situ extraction device combined with MALDI-TOF MS for detection and identification of safflower and dyed chrysanthemum based on chemical composition

[0083] In order to investigate the application capability of the offline in-situ extraction device in identifying the authenticity of Chinese herbal medicine pieces, in this example, the offline in-situ extraction device was combined with MALDI-TOF MS to detect and identify safflower and dyed chrysanthemum according to chemical composition. Dyed chrysanthemum has been reported as an adulterant of safflower.

[0084] First, prepare the Chinese herbal medicine slices: take the safflower slices purchased from the Chinese medicine pharmacy and the chrysanthemum slices dyed with the food coloring sunset yellow solution, grind them in an electric powder grinder in advance so that the two slices cannot be directly identified in appearance, and weigh about 0.5 mg of the slices on the balance, place the 0.5 mg slices in a 35mm x 12mm petri dish to spread evenly, and the Chinese herbal medicine slices safflower and dyed chrysanthemum are ready. Then randomly shuffle the petri dishes and mark the two types of Chinese herbal medicine slices to be tested as test 1 and test 2.

[0085] Subsequently, sampling was performed using an offline in-situ extraction device: according to the in-situ extraction device provided in Example 1, and in accordance with the operation of Example 1-(4), the injection parameters were as follows: methanol: water = 8:2 (v / v) was used as the extraction solvent, the injection pump flow rate was 30 μL / min, the extract collection volume was 100 μL, 50 μL of solvent was used for washing, and each piece of Chinese medicine was extracted 5 times; a pre-punched 1.5 ml EP tube was selected as the liquid collection tube A5, and the handheld liquid micro-junction surface extraction probe device was aimed at the Chinese medicine piece to be tested for offline in-situ extraction.

[0086] At the same time, 0.5 mg of safflower slices were weighed on a balance, and methanol: water = 8:2 (v / v) was used as the extraction solvent. 500 μL of the extraction solvent was added for ultrasonic extraction for 30 minutes as the standard medicinal material extract. Subsequently, methanol: water = 8:2 (v / v) was used as the solvent to prepare a standard solution containing 0.5 μg / mL of hydroxysafflor yellow A (HSYA) standard.

[0087] The extract was then tested and analyzed according to the operation of Example 1-(5): matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for analysis, aminoacridine (9-AA) was used as the detection matrix in negative ion mode, and the matrix was deposited by matrix spray method. Specifically, the extract was evaporated to dryness under a nitrogen blow instrument for concentration, 10 microliters of pure solvent was re-dissolved, and the sample was spotted on an indium-tin oxide (ITO) coated glass slide with a sample volume of 1 μL. At the same time, the standard medicinal material extract and the standard of hydroxysafflor yellow A (HSYA) were spotted on the same ITO coated glass slide for comparison with a sample volume of 1 μL. The matrix was then uniformly deposited on the ITO glass slide by a matrix spray instrument, and the operation was carried out according to the instructions for use of the MALDI-MS instrument, and detection was carried out in negative ion mode. Representative MALDI-MS mass spectra are shown in Figure 6 From the atlas, we can see the characteristic chemical components of the following types of Chinese herbal medicines:

[0088] (a) Representative spectra of standard herbal extracts Figure 6 Representative spectra of standard reference substances of A and hydroxysafflor yellow A (HSYA) Figure 6 The characteristic component hydroxysafflor yellow A can be detected in both B and the additive form is [MH] - , mass-to-charge ratio is m / z 611.1.

[0089] (b) Representative spectrum of analyte 1 Figure 6 Hydroxysafflor yellow A, a characteristic component of safflower, can be detected in C in the additive form of [MH] - , the signal with mass-to-charge ratio of m / z 611.1, sunset yellow pigment could not be detected from analyte 1. According to the existing literature, the characteristic peak of sunset yellow pigment contains the sum of [M-2Na+H]- , the signal with mass-to-charge ratio of m / z 407.3 and the sum form is [M-Na] - , a signal with a mass-to-charge ratio of m / z 429.3. Based on the above chemical information, it can be seen that analyte 1 may be safflower slices.

[0090] (c) Representative spectrum of analyte 2 Figure 6 Hydroxysafflor yellow A, a characteristic component of safflower, could not be detected in D, and the characteristic peak of sunset yellow pigment could be detected from analyte 2, including the addition form of [M-2Na+H] - , the signal with a mass-to-charge ratio of m / z 407.3 and the summed form is [M-Na] - , with a mass-to-charge ratio of m / z 429.3. Based on the above chemical information, it can be seen that analyte 2 may be dyed chrysanthemum slices, that is, safflower adulteration.

[0091] Example 4: Application example of online in-situ extraction device

[0092] (1) Online in situ extraction device combined with HESI-Q Exactive MS for detection of endogenous small molecule metabolites and lipids in rat brain slices

[0093] In order to investigate the detection capability of the online in situ extraction device for endogenous small molecule metabolites and lipids in biological tissues, in this example, the online in situ extraction device was combined with HESI-Q Exactive MS to detect sagittal sections of rat brain.

[0094] First, prepare the sagittal slices of the rat brain: take the rat brain stored in a -80° refrigerator, cut the rat brain along the sagittal plane and slice it on a freezing microtome, the slice thickness is 10 μm, and the slices are transferred to glass slides by melt mounting. The sagittal slices of the rat brain are ready.

[0095] Subsequently, an online in-situ extraction device was used for sampling and detection and analysis: according to the in-situ extraction device provided in Example 2 and the operation in Example 2-(3), the injection parameters were as follows: 0.1% trifluoroacetic acid in methanol: water = 7.5: 2.5 (v / v) was used as the extraction solvent, and the injection pump flow rate was 10 μL / min; the parameters of the heated electrospray ionization (H-ESI) ion source were set as follows: sheath gas pressure 15 psi, ion source voltage 4000V, ion transfer tube temperature 320°C, ion source needle heating temperature 300°C, and detection mode was positive ion mode; the mass spectrometer state was switched from "on" to "on" and the injection pump was operated to push the syringe to the front end of the probe to form a liquid junction. After the mass spectrometer signal was stable, the handheld liquid microjunction surface extraction probe device was aligned with the tissue section, and the mass spectrometer was set to start sampling; the sampling parameters were as follows: 30 μL of the extract was collected and detected cumulatively, 50 μL of solvent was used for washing, and the extraction was repeated 5 times, and then the detection data of each time was saved and analyzed. The representative mass spectrogram is shown in Figure 7 From the spectrum, we can see lipid signals distributed in the mass-to-charge ratio range of 700-850, including lipids such as glycerophospholipids (GP), phosphatidylcholine (PC), and phosphatidylethanolamine (PE); we can also see small molecule metabolite signals distributed in the mass-to-charge ratio range of 100-180, including tyrosine, and the sum form is [M+H] + , mass-to-charge ratio is m / z 180.0658; phenylalanine, additive form is [M+H] + , mass-to-charge ratio is m / z164.0706; DOPA, the addition form is [M+H] + , mass-to-charge ratio is m / z 196.0632, etc.

[0096] (2) Online in situ extraction device combined with HESI-Q Exactive MS for detection and identification of safflower and dyed chrysanthemum based on chemical composition

[0097] In order to investigate the application capability of the online in-situ extraction device in identifying the authenticity of Chinese herbal medicine slices, in this example, the online in-situ extraction device was combined with HESI-Q Exactive MS to detect and identify safflower and dyed chrysanthemum according to chemical composition. Dyed chrysanthemum has been reported as an adulterant of safflower.

[0098] First, prepare the Chinese herbal medicine slices: take the safflower slices purchased from the Chinese medicine pharmacy and the chrysanthemum slices dyed with the food coloring sunset yellow solution, grind them in an electric powder grinder in advance so that the two slices cannot be directly identified in appearance, and weigh about 0.5 mg of the slices on the balance, place the 0.5 mg slices in a 35mm x 12mm petri dish to spread evenly, and the Chinese herbal medicine slices safflower and dyed chrysanthemum are ready. Then randomly shuffle the petri dishes and mark the two types of Chinese herbal medicine slices to be tested as test 1 and test 2.

[0099] Subsequently, an online in-situ extraction device was used for sampling and detection and analysis: according to the in-situ extraction device provided in Example 2, and according to the operation in Example 2-(3), the injection parameters were as follows: 0.1% trifluoroacetic acid in methanol: water = 7.5: 2.5 (v / v) was used as the extraction solvent, and the injection pump flow rate was 10 μL / min; the parameters of the heated electrospray ionization (H-ESI) ion source were set as follows: sheath gas pressure 15 psi, ion source voltage 3500 V, ion transfer tube temperature 320°C, ion source needle heating temperature 300°C, and detection mode was negative ion mode; the mass spectrometer state was switched from "on" to "on" and the injection pump was operated to push the syringe to the front end of the probe to form a liquid junction. After the mass spectrometer signal was stable, the handheld liquid microjunction surface extraction probe device was aligned with the sample to be tested, and the mass spectrometer was set to start sampling; the sampling parameters were as follows: 30 μL of the extract was collected and detected cumulatively, 50 μL of solvent was used for washing, and the extraction was repeated 5 times, and then the detection data of each time was saved and analyzed. The representative mass spectrum is shown in Figure 8 , the characteristic chemical components of the following types of Chinese herbal medicines:

[0100] (a) Representative spectrum of analyte 1 Figure 8 Hydroxysafflor yellow A, a characteristic component of safflower, can be detected in A and 8B in the additive form of [MH] - , the signal with mass-to-charge ratio of m / z 611.1586, sunset yellow pigment could not be detected from analyte 1. According to the existing literature, the characteristic peak of sunset yellow pigment contains the sum of [M-2Na+H] - , the signal with mass-to-charge ratio of m / z 407.3 and the sum form is [M-Na] - , a signal with a mass-to-charge ratio of m / z 429.3. Based on the above chemical information, it can be seen that analyte 1 may be safflower slices.

[0101] (b) Representative spectrum of analyte 2 Figure 8 C. Hydroxysafflor yellow A, a characteristic component of safflower, could not be detected in 8D, and the characteristic peak of sunset yellow pigment could be detected from analyte 2, including the addition form of [M-2Na+H] - , the signal with a mass-to-charge ratio of m / z 407.3 and the summed form is [M-Na] - , with a mass-to-charge ratio of m / z 429.3. Based on the above chemical information, it can be seen that analyte 2 may be dyed chrysanthemum slices, that is, safflower adulteration.

[0102] (3) The online in-situ extraction device combined with the HESI-Q Exactive MS was used to detect the MS and MS / MS results of the hydroxysafflor yellow A standard sample on the glass slide and the hydroxysafflor yellow A in the Chinese herbal medicine safflower.

[0103] In order to investigate the ability of the online in-situ extraction device to detect the characteristic chemical components of Chinese herbal medicines and obtain secondary mass spectrometry information, in this example, an online in-situ extraction device was combined with HESI-Q Exactive MS to detect the MS and MS / MS results of hydroxysafflor yellow A standard on a glass slide and hydroxysafflor yellow A in the Chinese herbal medicine safflower.

[0104] First, prepare the Chinese medicine slices: take the safflower slices purchased from the Chinese medicine pharmacy, weigh about 0.5 mg of the slices on the balance, and place the 0.5 mg slices in a 35mm x 12mm petri dish to spread evenly. After the safflower Chinese medicine slice samples are prepared, methanol: water = 8: 2 (v / v) is used as a solvent to prepare a standard solution containing 0.5 μg / mL of hydroxysafflor yellow A (HSYA) standard. The prepared hydroxysafflor yellow A (HSYA) standard solution is spotted on a glass slide for comparison, with a sample volume of 1 μL as the standard sample point of hydroxysafflor yellow A (HSYA).

[0105] Subsequently, an online in-situ extraction device was used for sampling and detection and analysis: according to the in-situ extraction device provided in Example 2 and the operation in Example 2-(3), the injection parameters were as follows: 0.1% trifluoroacetic acid in methanol: water = 7.5: 2.5 (v / v) was used as the extraction solvent, and the injection pump flow rate was 10 μL / min; the parameters of the heated electrospray ionization (H-ESI) ion source were as follows: sheath gas pressure was 15 psi, ion source voltage was 3500 V, ion transfer tube temperature was 320°C, ion source needle heating temperature was 300°C, and the detection mode was negative ion mode; the mass spectrometer state was switched from "on" to "on" and the injection pump was operated to push the syringe to the front end of the probe to form a liquid junction. After the mass spectrometer signal was stable, the handheld liquid micro-junction surface extraction probe device was aligned with the sample to be tested, and the mass spectrometer was set to start sampling; when the target compound signal was observed (in this embodiment, it was the signal of hydroxysafflor yellow A (HSYA) at m / z 611.1586), the mass spectrometer acquisition mode was adjusted from "Full" to "Non-condensing". Scan” is in “MS / MS” mode, and the preset m / z 611.1 parent ion detection window is used to collect the secondary ion fragments of the target. The sampling parameters are as follows: 50 μL of extract is collected and tested cumulatively, 50 μL of solvent is used for washing, and the extraction is repeated 5 times. The detection data of each time is then saved and analyzed. The representative mass spectra are shown in Fig. 9 , the characteristic chemical composition and secondary ion fragment information of the following samples:

[0106] (a) Characteristic spectrum of the HSYA standard sample point Fig. 9 A high-response HSYA signal can be detected in A, and the summed form is [MH] -, mass-to-charge ratio was m / z 611.1586, and the spectrum was obtained in MS / MS scanning mode. Fig. 9 In B, characteristic secondary ion fragments of HSYA can be detected, including m / z 325.07, m / z 473.11, and m / z 491.12.

[0107] (b) Characteristic spectrum from safflower slices Fig. 9 C can detect the characteristic component HSYA signal, the sum form is [MH] - , mass-to-charge ratio was m / z 611.1586, and the spectrum was obtained in MS / MS scanning mode. Fig. 9 The characteristic secondary ion fragments of HSYA can be detected in D, including m / z 325.07, m / z 473.11 and m / z 491.12.

[0108] Combining the existing literature and the structural information of HSYA, it can be determined that the ion fragmentation pathway of HSYA is as follows: Fig. 9 E.

[0109] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A handheld solid sample in-situ extraction device, which is an offline in-situ extraction device, wherein the offline means that the extraction liquid is tested and analyzed after the extraction is completed, and is characterized by: The off-line in-situ extraction device comprises a handheld extraction system A, a liquid supply system for providing a solvent to the handheld extraction system A, and a negative pressure system for driving the extraction liquid to flow in the handheld extraction system A; The handheld extraction system A comprises: a handheld housing A1, a sampling pipe A2 penetrating the handheld housing A1, a three-way pipe fitting A3 and a three-way pipe fitting A4 arranged along the sampling pipe A2 and sleeved outside the sampling pipe A2 and fixed inside the handheld housing A1, and a liquid collecting pipe A5 for collecting liquid in the sampling pipe A2; one end of the sampling pipe A2 is a sampling end, and the other end is a collecting end, and both the sampling end and the collecting end extend out of the handheld housing A1; the three-way pipe fitting A3 and the three-way pipe fitting A4 are respectively arranged at the sampling end and the collecting end of the sampling pipe A2; the liquid collecting pipe A5 is arranged at the collecting end of the sampling pipe A2; The main pipe of the three-way pipe fitting A3 is sleeved on the sampling pipe A2 near the sampling end, and the branch pipe is provided with an adapted plug A33, on which the pipe A8 for conveying the solvent in the liquid supply system A9 to the three-way pipe fitting A3 is tightly provided; the main pipe near the collecting end is provided with an adapted plug A32, on which the plug A32 is tightly provided with a fixed sleeve for fixing the sampling pipe A2, and the fixed sleeve is tightly sleeved outside the sampling pipe A2; the main pipe near the sampling end is provided with an adapted plug A31, on which the plug A31 is tightly provided with a nested pipe A6 sleeved outside the sampling pipe A2, and a cavity is reserved between the inner wall of the nested pipe A6 and the outer wall of the sampling pipe A2 for the solvent conveyed to the three-way pipe fitting A3 to flow out to the sampling end of the sampling pipe A2; The main pipe of the three-way pipe fitting A4 is sleeved on the sampling pipe A2 near the collecting end, and the branch pipe is provided with an adapted plug A43, on which the pipe A10 for extracting the gas in the three-way pipe fitting A4 to the negative pressure system A11 is tightly provided; the end near the sampling end of the main pipe is provided with an adapted plug A41, on which the plug A41 is tightly provided with a fixed sleeve for fixing the sampling pipe A2, and the fixed sleeve is tightly sleeved outside the sampling pipe A2; the end near the collecting end of the main pipe is provided with an adapted plug A42, on which the plug A42 is tightly provided with a sleeve The nested tube A7 outside the sampling pipe A2 extends out of the handheld housing A1 and is tightly inserted into the liquid collecting pipe A5. A cavity is left between the inner wall of the nested tube A7 and the outer wall of the sampling pipe A2 for the negative pressure system to draw the gas in the liquid collecting pipe A5 through the three-way pipe fitting A4 to form a negative pressure environment in the liquid collecting pipe A5. A wall-attached connection is provided between the end of the sampling pipe A2 and the inner wall of the liquid collecting pipe A5 to facilitate the extraction liquid in the sampling pipe A2 to flow into the liquid collecting pipe A5 along the inner wall of the liquid collecting pipe A5 driven by the air pressure difference.

2. The in-situ extraction device according to claim 1, characterized in that: The liquid supply system comprises a syringe and a syringe pump for controlling the syringe to inject solvent.

3. The in-situ extraction device according to claim 1, characterized in that: The sampling pipe A2 is a quartz capillary.

4. The in-situ extraction device according to claim 1, characterized in that: The nested tubes A6 and A7 are ETFE tubes.

5. The in-situ extraction device according to claim 1, characterized in that: The fixed sleeve is a PEEK tube.

6. A handheld solid sample in-situ extraction device, which is an online in-situ extraction device, wherein the extraction liquid is directly transported to the mass spectrometry system for analysis and detection while the extraction is being carried out, and is characterized in that: The online in-situ extraction device comprises a handheld extraction system B and a liquid supply system for providing solvent to the handheld extraction system B; The handheld extraction system B comprises: a handheld housing B1, a sampling pipe B2 running through the handheld housing B1, a three-way pipe fitting B3 and a two-way pipe fitting B4 arranged along the sampling pipe B2 and sleeved outside the sampling pipe B2 and fixed inside the handheld housing B1; one end of the sampling pipe B2 is a sampling end, and the other end is a mass spectrometer connection end, and both the sampling end and the mass spectrometer connection end extend out of the handheld housing B1; the three-way pipe fitting B3 is arranged at the sampling end near the sampling end of the sampling pipe B2, and the two-way pipe fitting B4 is arranged at the mass spectrometer connection end of the sampling pipe B2; The main pipe of the three-way pipe fitting B3 is sleeved on the sampling pipe B2 near the sampling end, and the branch pipe is provided with an adaptive plug B33, on which the pipe B6 for conveying the solvent in the liquid supply system B7 to the three-way pipe fitting B3 is tightly provided; the end of the main pipe near the mass spectrometer connection is provided with an adaptive plug B32, on which the plug B32 is tightly provided with a fixed sleeve for fixing the sampling pipe B2, and the fixed sleeve is tightly sleeved outside the sampling pipe B2; the end of the main pipe near the sampling end is provided with an adaptive plug B31, on which the plug B31 is tightly provided with a nested pipe B5 sleeved outside the sampling pipe B2, and a cavity is reserved between the inner wall of the nested pipe B5 and the outer wall of the sampling pipe B2 for the solvent conveyed to the three-way pipe fitting B3 to flow out to the sampling end of the sampling pipe B2; The two-way pipe fitting B4 is sleeved on the sampling pipeline B2 near the mass spectrometer connection end. The two-way pipe fitting B4 is provided with matching plugs B41 and B42 near the sampling end and the mass spectrometer connection end respectively. The plugs B41 and B42 are respectively tightly provided with fixed sleeves for fixing the sampling pipeline B2. The fixed sleeves are tightly sleeved on the outside of the sampling pipeline B2, which is driven by the pressure difference formed by the negative pressure in the mass spectrometer system to transport the extract to the mass spectrometer system B8.

7. The in-situ extraction device according to claim 6, characterized in that: The liquid supply system comprises a syringe and a syringe pump for controlling the syringe to inject solvent.

8. The in-situ extraction device according to claim 6, characterized in that: The sampling pipe B2 is a quartz capillary.

9. The in-situ extraction device according to claim 6, characterized in that: The nested tube B5 is an ETFE tube.

10. The in-situ extraction device according to claim 6, characterized in that: The fixed sleeve is a PEEK tube.

Images