TWI495876B - Method for detecting endotoxin in a sample - Google Patents

Description

Method for analyzing endotoxin in a sample

The present invention relates to methods for analyzing samples, and more particularly to a method for analyzing endotoxin in a sample.

Endotoxin is a component of the cell wall of Gram-negative bacteria, also known as lipopolysaccharide. Endotoxins are toxic to the host and are only released when the bacteria die or dissolve the cells by artificial means, which is why they are called endotoxin. The toxic component of endotoxin is mainly lipid A (lipid A). Endotoxin is located at the outermost layer of the cell wall. The endotoxin of various bacteria is about the same, causing fever, microcirculatory disturbance, endotoxin shock, and disseminated intravascular coagulation.

The human body is extremely sensitive to bacterial endotoxin. Very small amounts (1-5ng/1Kg body weight) of endotoxin can cause an increase in body temperature, and the fever reaction will gradually subside after about 4 hours. In the case of natural infection, Gram-negative bacteria continue to grow and multiply, accompanied by successive deaths of Gram-negative bacteria and release of endotoxin, so the fever reaction will continue until the pathogens in the body are completely eliminated. On the other hand, endotoxemia can occur when a large number of Gram-negative pathogens in the bloodstream of a patient or human body die and a large amount of endotoxin released is introduced into the blood. A large amount of endotoxin acts on macrophages, neutrophils, endothelial cells, platelets, and the complement system and coagulation system, causing dysfunction and microcirculation Obstruction, its clinical manifestations of microcirculatory failure, hypotension, hypoxia, acidosis, etc., thus leading to patient shock, this pathological response is called endotoxin shock. It can be seen from the above that endotoxin can cause many diseases in human body, so detecting the content of endotoxin in human body is an important medical and clinical information.

Biomolecules can be detected by a number of different methods, wherein mass spectrometry can detect a wide range of molecular weights, which can detect small molecules such as peptides, sugars, fatty acids, etc., and can also detect, for example, proteins, Large molecules such as carbohydrates can be used to detect endotoxin using a mass spectrometer. For example, plasma desorption mass spectrometry (PDMS), matrix-assisted laser desorption free (MALDI) mass spectrometry, gas chromatography mass spectrometry (GC-MS), fast atomic impact (Fast) Atom bombardment, FAB) mass spectrometer to detect endotoxin. However, the above detection method requires a complicated and cumbersome purification and extraction step to purify the endotoxin before the test, and then the purified sample can be sent for measurement. Therefore, it is costly and requires lengthy steps such as purification and extraction. Therefore, how to detect endotoxin in a sample in a simple, rapid, direct and effective manner is an important subject in the technical field.

The present invention provides a method for analyzing endotoxin in a sample, comprising providing a sample comprising endotoxin; adding chitosan magnetic nanoparticles to the sample, and the chitosan magnetic nanoparticle is specific Sexually binds to the endotoxin and forms a complex; brings the external magnet close to the first portion of the sample, causing the complex in the sample to concentrate in the first portion; removing the second portion of the sample, which is different from the first portion; Endotoxin in the first part Perform a mass spectrometry step.

The above and other objects, features, and advantages of the invention will be apparent from

20‧‧‧Chitosan magnetic nanoparticles

22‧‧‧Magnetic core

24‧‧‧ chitosan

30‧‧‧Endotoxin

32‧‧‧The polysaccharide part of endotoxin

34‧‧‧Endotoxin in addition to the polysaccharide part

40‧‧‧Compound

HB‧‧‧ hydrogen bond

1 is a flow chart showing a method for analyzing endotoxin in a sample according to an embodiment of the present invention; and FIG. 2 is a schematic view showing a chitosan magnetic nano particle according to an embodiment of the present invention; A schematic diagram of a complex formed by chitosan magnetic nanoparticles and endotoxin formed in the embodiment of the invention; FIG. 4 is a UV spectrum analysis diagram of chitosan magnetic nanoparticles having a CuFeO ₂ core; The figure is a transmission electron microscopic analysis of chitosan magnetic nanoparticles with CuFeO ₂ core; the sixth figure is the Fourier transform infrared of chitosan and chitosan magnetic nanoparticles with CuFeO ₂ core. spectra; FIG. 7 Fe ₃ O ₄ having a system of several UV nuclear magnetic nanoparticles of chitosan FIG spectroscopy; FIG. 8 system having a transmissive core of Fe ₃ O ₄ magnetic nanoparticles of chitosan of Electron microscopic analysis; Fig. 9 is a Fourier transform infrared spectrum of chitosan and chitosan magnetic nanoparticles with Fe ₃ O ₄ nuclei; Fig. 10 is a Fourier transform infrared spectrum of endotoxin and complex Figure 11 is a Fourier transform infrared of endotoxin and complex Spectrum; FIG. 12 chitosan based solution having magnetic nanoparticles within the tube.

It will be appreciated that the following description provides many different embodiments or examples for implementing the invention. The specific elements and arrangements described below are intended to provide a brief description of the invention. Of course, these are by way of example only and not as a limitation of the invention. Moreover, repeated numbers or labels may be used in different embodiments. These repetitions are merely for the purpose of simplicity and clarity of the invention and are not to be construed as a limitation of the various embodiments and/or structures discussed. Furthermore, when a first material layer is on or above a second material layer, the first material layer is in direct contact with the second material layer. Alternatively, it is also possible to have one or more layers of other materials interposed, in which case there may be no direct contact between the first material layer and the second material layer.

It should be understood that the terms "first", "second", "third", etc. may be used herein to describe various elements, components, regions, layers, and/or portions, such elements, components, and regions. The layers, and/or parts are not to be limited by the terms, and the terms are used to distinguish different elements, components, regions, layers, and/or parts. Therefore, a first element, component, region, layer, and/or portion discussed below may be referred to as a second element, component, region, layer, and/or without departing from the teachings of the present invention. section.

Unless otherwise defined, all terms used herein (including technology And scientific terms have the same meaning as commonly understood by those of ordinary skill in the art. It will be understood that these terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with the relevant art and the context or context of the present disclosure, and should not be in an idealized or overly formal manner. Interpretation, unless specifically defined herein.

Here, the terms "about" and "about" are usually expressed within 20% of a given value or range, preferably within 10%, and more preferably within 5%. The quantity given here is an approximate number indicating that the terms "about" and "about" may be implied without specific explanation.

The method for analyzing endotoxin provided by the present invention utilizes chitosan magnetic nanoparticles to specifically bind to endotoxin and form a complex, and aggregates the complex using an external magnet, and then The toxin was subjected to mass spectrometry. Referring to Figure 1, method 100 is an endotoxin analysis method according to some embodiments of the invention, comprising step 102. Step 102 provides the same, this sample includes endotoxin. This endotoxin, also known as lipopolysaccharide, is a component of the cell wall of Gram-negative bacteria. In one embodiment, the sample is urine. In another embodiment, the sample is blood, RO water, dialysate, ultrapure dialysate or hemodiafiltration supplement or any other suitable sample.

In step 104, chitosan magnetic nanoparticle is added to the sample, and the chitosan magnetic nanoparticle can specifically bind to the endotoxin and form a complex. Referring to FIG. 2, the chitosan magnetic nanoparticle 20 has a magnetic core 22 and a chitosan 24, wherein the chitosan 24 can form a chemical bond with the magnetic core 22 and a single layer is applied to the surface of the magnetic core 22. on. Among them The sugar 24 is preferably bonded to the magnetic core 22 by a covalent bond.

The chitosan magnetic nanoparticle 20 is prepared by mixing and heating one or more compounds containing magnetic atoms to form a magnetic core 22. The compound containing a magnetic atom includes Fe ₂ (SO ₄ ) ₃ . 9H ₂ O, CuSO ₄ . 3H ₂ O, FeCl ₂ . 4H ₂ O, FeCl ₃ . 6H ₂ O or any other suitable compound. The temperature of this heating step is from about 90 ° C to about 180 ° C. The heating time is from about 3 hours to about 24 hours. Next, the obtained magnetic core 22 is mixed with chitosan 24 and heated to produce chitosan magnetic nanoparticles 20, wherein the heating temperature is from about 60 ° C to about 150 ° C. The heating time is from about 2 hours to about 5 hours.

In certain embodiments, the magnetic core 22 of the chitosan magnetic nanoparticles 20 is a CuFeO ₂ core. The chitosan magnetic nanoparticle 20 having a CuFeO ₂ core has a particle diameter of 5 nm to 7 nm.

In certain embodiments, the magnetic core 22 of the chitosan magnetic nanoparticles 20 is a Fe ₃ O ₄ core. The chitosan magnetic nanoparticle 20 having a Fe ₃ O ₄ core has a particle diameter of 2 nm to 10 nm.

Referring to Fig. 3, endotoxin 30 includes a polysaccharide component 32 and a moiety 34 other than the polysaccharide moiety 32. The chitosan magnetic nanoparticle 20 can specifically bind to the polysaccharide moiety 32 of the endotoxin and form a complex 40 with the endotoxin 30. The chitosan magnetic nanoparticle 20 and the polysaccharide moiety 32 of the endotoxin are non-covalently bonded, for example, a hydrogen bond, an electrostatic force, a hydrophobic force, or a vanadium bond. Van der Waal force. In one embodiment, as shown in FIG. 3, the chitosan magnetic nanoparticle 20 and the polysaccharide portion 32 of the endotoxin are mainly bound by a hydrogen bond HB. It should be understood that despite the third The figure shows only one chitosan magnetic nanoparticle 20 specifically binds to one endotoxin 30 and forms a complex 40, and a chitosan magnetic nanoparticle 20 can also be combined with multiple endotoxin 30 at the same time. And a complex 40 is formed. Similarly, the plurality of chitosan magnetic nanoparticles 20 can also be specifically combined with one endotoxin 30 to form a complex 40, or a plurality of chitosan magnetic nanoparticles 20 can also be simultaneously Endotoxin 30 specifically binds and forms a complex 40.

Next, in step 106, the external magnet is brought close to the first portion of the sample. Since the complex includes a magnetic core that is attracted by an external magnet, the complex is attracted to the first portion of the sample by the external magnet and collects there. The additional magnet can be a permanent magnet, an electromagnet, a superconducting magnet, or any other type of magnet. The permanent magnet can be a natural magnet (magnetite), an artificial magnet (aluminum alloy) or any other suitable permanent magnet that retains its magnetic properties for a long period of time. The non-permanent magnet may be an electromagnet that generates a magnetic force with an electric current or any other suitable non-permanent magnet. Alternatively, the outer magnet may be in the shape of a horseshoe magnet, a round magnet, a rod magnet, a ring magnet, a combination of the above, or any other suitable shape. The shape of the additional magnet is preferably adapted to the shape of the sample container to facilitate effective attraction of the complex to the first portion of the sample. The portion other than the first portion of the sample is the second portion, which is relatively far from the outer magnet relative to the first portion. After the outer magnet is near the first portion of the sample, the first portion has a first concentration and the second portion has a second concentration. Since the external magnet is close to the first portion of the sample, the complex concentration in the sample is attracted to it, so the first concentration is greater than the second concentration.

Next, step 108 removes the second portion of the sample, which is different from the first portion. Because most of the complexes containing endotoxin are gathered together. In the first part of this section, therefore, removing the second portion of the sample will only remove a small portion of the endotoxin from the original sample, while retaining most of the endotoxin in the remaining first portion. And removing the second part also means removing impurities other than endotoxin in the sample at the same time. Therefore, the removal step will increase the endotoxin concentration in the sample while also achieving the purification effect. And the increase in endotoxin concentration in the sample can increase the detection limit of subsequent analysis steps. In one embodiment, the detection limit of the endotoxin analysis method for performing the removal step is 67 to 250 times the detection limit of the endotoxin analysis method without the removal step.

Next, step 110 performs a mass spectrometry step on the endotoxin in the first portion of the sample. Plasma desorption mass spectrometry (PDMS), matrix-assisted laser desorption free (MALDI) mass spectrometry, gas chromatography mass spectrometry (GC-MS), fast atom bombardment (Fast atom bombardment) , FAB) mass spectrometer or any other suitable mass spectrometer to detect endotoxin in the sample. In one embodiment, the mass spectrometry step is a matrix assisted laser desorption free (MALDI) mass spectrometry step. At this point, it may be further included after the second portion is removed at step 108, the first portion is mixed with a matrix material, wherein the matrix material is used for matrix assisted laser desorption free (MALDI) mass spectrometry. The matrix material may be 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA), a combination of the above or any other suitable Matrix material. Since the endotoxin concentration in the sample is increased after the second portion is removed in step 108, and the purification effect is also achieved, the method of the present invention will increase the detection limit of the endotoxin analysis method. In one embodiment, the detection limit of the method of the invention is from 120 to 450 [mu]g endotoxin/mL sample.

The method for analyzing endotoxin provided by the present invention uses a simple shift In addition to the steps to replace the complicated and cumbersome purification or extraction steps. Thus, in one embodiment, there is no additional purification or extraction step prior to placing the external magnet near the first portion of the sample at step 106. In another embodiment, there is no additional purification or extraction step after the second portion is removed at step 108. In yet another embodiment, there is no additional purification or extraction step prior to step 106 of bringing the external magnet close to the first portion of the sample and after removing the second portion at step 108. Since the method of the present invention uses a simple removal step in combination with chitosan magnetic nanoparticles, the complicated and cumbersome purification, extraction and sample processing steps can be omitted, so that the method of the present invention is simpler, faster, direct and effective.

In summary, the method for analyzing endotoxin provided by the present invention utilizes chitosan magnetic nanoparticles to specifically bind to endotoxin and form a complex compound, which can be easily combined with the use of an external magnet and a simple analysis step. Fast, direct and efficient analysis, and can greatly increase the detection limit of analytical methods. Therefore, the method for analyzing endotoxin of the present invention can be applied to clinical medicine and patient diagnosis and treatment, and can quickly and effectively detect the endotoxin concentration in the human body. In addition, the analysis method of the endotoxin of the present invention is also very suitable for applications such as medical research.

[Comparative example]

Endotoxin was added to the urine (taken from the 21-year-old volunteer) and six samples with different endotoxin concentrations were set at 120 μg endotoxin/mL sample, 600 μg endotoxin/mL sample, and 750 μg endotoxin/mL. Sample, 1.5 mg endotoxin/mL sample, 2.25 mg endotoxin/mL sample, 3.0 mg endotoxin/mL sample. Take 5 μL of each of the 6 samples with different endotoxin concentrations to the sample. The plate was then individually instilled with 5 μL of 9-aminoacridine hydrochloride (9-AA) onto the sample which had been dropped onto the sample carrier, and then the sample was air-dried under ambient conditions. The matrix material in the negative ion mode was then used for matrix-assisted laser desorption free (MALDI) mass spectrometry to detect endotoxin in each sample. From the experimental results, the detection limit was 3.0 mg endotoxin/mL sample.

[Preparation Example 1]

0.01 mol Fe ₂ (SO ₄ ) ₃ . 9H ₂ O (purchased from Riedel de-Haen, India) and 0.01 mol CuSO ₄ . 3H ₂ O (available from Riedel de-Haen, India) was dissolved in 40 mL of water containing 2 mL of glycerol and a mixed solution was formed. 0.2 mol of NaOH was added to the mixed solution and stirred for 2 hours. Next, 4-isopropyl-benzaldehyde was added as a reducing agent, followed by heating to 180 ° C and reacting at this temperature for 24 hours. The reaction was then naturally cooled to room temperature and the resulting CuFeO ₂ core was collected by centrifugation. The obtained CuFeO ₂ core (0.2 g) was mixed with 0.2 g of chitosan for 5 hours to prepare chitosan magnetic nanoparticles having a CuFeO ₂ core. The chitosan magnetic nanoparticles having the CuFeO ₂ core were separated and purified by an external magnetic field.

The prepared chitosan magnetic nanoparticles having a CuFeO ₂ core were analyzed by UV spectroscopy. As shown in Fig. 4, the maximum absorption peak was 220 nm. In addition, an external magnet is also used to confirm the magnetic properties of the chitosan magnetic nanoparticles having a CuFeO ₂ core. As shown in Fig. 12, the external magnet located on the right side of the test tube will have a CuFeO ₂ core in the solution in the test tube. The polysaccharide magnetic nanoparticles (black) are attracted and collected to the inner wall of the right side of the test tube. The portion other than the inner wall on the right side of the test tube has almost no chitosan magnetic nanoparticles having a CuFeO ₂ core, so that the portion other than the right inner wall of the test tube seen by the naked eye is a transparent solution. Further, as shown in Fig. 5, chitosan magnetic nanoparticles having a CuFeO ₂ core were analyzed by transmission electron microscopy (TEM). By analyzing the results of a transmission electron microscope, the particle size of the chitosan magnetic nanoparticles having a CuFeO ₂ core can be analyzed. The analysis results show that about 90% of the chitosan magnetics having a CuFeO ₂ core The average particle diameter of the nanoparticles is about 5 nm, and about 10% of the chitosan magnetic nanoparticles having a CuFeO ₂ core have an average particle diameter of about 7 nm and an average particle diameter of 6 nm. In addition, chitosan and chitosan magnetic nanoparticles with CuFeO ₂ core were analyzed by a fourier transform infrared spectrometer. As shown in Fig. 6, the Fourier transform infrared spectrum of chitosan to 3450cm ^-1, 1660cm ^-1, 1250cm ^-1 has a peak, which in turn corresponds to OH stretching (strech), C = O stretching and CO stretching characteristic peaks. Having the Fourier CuFeO ₂ chitosan core of the magnetic nanoparticles transform infrared spectra were 3500cm ^-1, 1700cm ^-1, 1100cm ^-1 has a peak.

[Preparation Example 2]

Will be 0.6268g FeCl ₂ . 4H ₂ O and 1.7312g FeCl ₃ . 6H ₂ O was dissolved in 25 mL of water, followed by 40 mL of aqueous ammonia. This solution was then heated to 90. The Fe ₃ O ₄ core was obtained by reacting at ° C for 3 hours. 0.1 g of the obtained Fe ₃ O ₄ core was added to 30 mL of an acetic acid solution containing 0.3 g of chitosan, heated to 60 ° C and reacted at this temperature for 2 hours to obtain a dibutyl group having a Fe ₃ O ₄ core. Glycan magnetic nanoparticles.

The prepared chitosan magnetic nanoparticles having a Fe ₃ O ₄ core were analyzed by UV spectroscopy, and as shown in Fig. 7, the maximum absorption peak was 595 nm. Further, as shown in Fig. 8, chitosan magnetic nanoparticles having a Fe ₃ O ₄ core were analyzed by transmission electron microscopy (TEM). The particle size of the chitosan magnetic nanoparticles having a Fe ₃ O ₄ core can be analyzed by the analysis results of a transmission electron microscope, and the analysis results show that about 60% of the particles having the Fe ₃ O ₄ core The butadiene magnetic nanoparticles have an average particle diameter of about 5 nm, and about 40% of the chitosan magnetic nanoparticles having a Fe ₃ O ₄ core have an average particle diameter of about 10 nm, and a small portion has Fe. _The chitosan magnetic nanoparticles of the ₃ O ₄ core have an average particle diameter of about 2 nm or about 8 nm and an average particle diameter of 6 nm. In addition, chitin and a chitosan magnetic nanoparticle having a Fe ₃ O ₄ core are analyzed by a fourier transform infrared spectrometer, as shown in Fig. 9, a Fourier transform of chitosan infrared Spectroscopy at ^{3450cm -1, 3000cm -1, 1660cm -1} , 1250cm -1 has a peak, which in turn corresponds to OH stretching (strech), CH stretching, C = O stretching and CO stretching characteristic peaks. Fourier having chitosan Fe ₃ O ₄ magnetic nanoparticles of the nuclear transform infrared spectroscopy to ^{3400cm -1, 2850cm -1, 1625cm -1} , 1000cm -1 has a peak.

[Example 1]

Endotoxin was added to 1 mL of urine and six samples with different endotoxin concentrations were set at 120 μg endotoxin/mL sample, 600 μg endotoxin/mL sample, 750 μg endotoxin/mL sample, 1.5 mg endotoxin/ mL sample, 2.25 mg endotoxin/mL sample, 3.0 mg endotoxin/mL sample. Next, 1 mL of a magnetic nanoparticle solution containing chitosan magnetic nanoparticles having a CuFeO ₂ core was added to the sample and mixed for 30 minutes to form a chitin-containing magnetic nanoparticle having a CuFeO ₂ core and an endotoxin. The concentration of the magnetic nanoparticle solution was 0.5 g of chitosan magnetic nanoparticles having a CuFeO ₂ core/50 mL sample. The complex formed by the chitosan magnetic nanoparticles with CuFeO ₂ core and endotoxin is then attracted and collected to the first portion of the sample using an external magnet and the second portion is removed.

Next, the first portion containing the complex is mixed with 10 μL of 9-aminoacridine (9-AA) to form a mixed solution, and then 5 μL of the mixed solution is dropped onto the sample carrier, followed by the environment. The sample was air dried under the conditions. The matrix material in the negative ion mode was then used for matrix-assisted laser desorption free (MALDI) mass spectrometry to detect endotoxin in each sample. From the experimental results, the detection limit was 120 μg endotoxin/mL sample. Compared with the analytical method in which the comparative examples of the chitosan magnetic nanoparticles of the present invention are not used, the detection limit (120 μg endotoxin/mL sample) of the chitosan magnetic nanoparticles having a CuFeO ₂ core is used in the present invention. The detection limit of the comparative example (3.0 mg endotoxin/mL sample) was 250 times.

The endotoxin and the above complex were separately analyzed by Fourier transform infrared spectrometer. As shown in Fig. 10, compared with the Fourier transform infrared spectrum of endotoxin, the Fourier transform infrared spectrum of the complex compound was at OH characteristic peak of 3600 cm ^-1 . It is wider and slightly shifted at a C=O characteristic peak at 1660 cm ^-1 , which clearly shows that the chitosan magnetic nanoparticles having a CuFeO ₂ core in the complex are hydrogen bonded to the endotoxin.

[Example 2]

Endotoxin was added to 1 mL of urine and six samples with different endotoxin concentrations were set at 120 μg endotoxin/mL sample, 600 μg endotoxin/mL sample, 750 μg endotoxin/mL sample, 1.5 mg endotoxin/ mL sample, 2.25 mg endotoxin/mL sample, 3.0 mg endotoxin/mL sample. Next, 1 mL of a magnetic nanoparticle solution containing chitosan magnetic nanoparticles having a Fe ₃ O ₄ core was added to the sample and mixed for 30 minutes to form a chitosan magnetic nanoparticle having a Fe ₃ O ₄ core and The endotoxin forms a complex, and the concentration of the magnetic nanoparticle solution is 1 chitosan magnetic nanoparticle with a Fe ₃ O ₄ g core/50 mL sample. The complex formed by the chitosan magnetic nanoparticles with Fe ₃ O ₄ core and endotoxin is then attracted and concentrated to the first portion of the sample using an external magnet and the second portion is removed.

Next, the first portion containing the complex is mixed with 10 μL of 9-aminoacridine (9-AA) to form a mixed solution, and then 5 μL of the mixed solution is dropped onto the sample carrier, followed by the environment. The sample was air dried under the conditions. The matrix material in the negative ion mode was then used for matrix-assisted laser desorption free (MALDI) mass spectrometry to detect endotoxin in each sample. From the experimental results, the detection limit was 450 μg endotoxin/mL sample. Compared with the analytical method in which the comparative examples of the chitosan magnetic nanoparticles of the present invention are not used, the present invention uses the detection limit of chitosan magnetic nanoparticles having a Fe ₃ O ₄ core (450 μg endotoxin/mL sample). ) is 67 times the detection limit of the comparative example (3.0 mg endotoxin/mL sample).

The endotoxin and the above complex were separately analyzed by Fourier transform infrared spectrometer. As shown in Fig. 11, compared with the Fourier transform infrared spectrum of endotoxin, the Fourier transform infrared spectrum of the complex was at OH characteristic peak of 3600 cm ^-1 . It is wider and slightly shifted at a C=O characteristic peak at 1660 cm ^-1 , which clearly shows that the chitosan magnetic nanoparticles having a Fe ₃ O ₄ core in the complex are hydrogen bonded to the endotoxin.

Although the embodiments of the present invention and its advantages are disclosed above, it should be understood that those skilled in the art can make modifications, substitutions, and refinements without departing from the spirit and scope of the invention. In addition, the scope of the present invention is not limited to the processes, machines, manufacture, compositions, devices, methods, and steps in the specific embodiments described in the specification. Any one of ordinary skill in the art can. The processes, machines, fabrications, compositions, devices, methods, and procedures that are presently or in the future are understood to be used in accordance with the present invention as long as they can perform substantially the same function or achieve substantially the same results in the embodiments described herein. Accordingly, the scope of the invention includes the above-described processes, machines, manufactures, compositions, devices, methods, and steps. In addition, the scope of each of the claims constitutes an individual embodiment, and the scope of the invention also includes the combination of the scope of the application and the embodiments.

20‧‧‧Chitosan magnetic nanoparticles

22‧‧‧Magnetic core

24‧‧‧ chitosan

30‧‧‧Endotoxin

32‧‧‧The polysaccharide part of endotoxin

34‧‧‧Endotoxin in addition to the polysaccharide part

40‧‧‧Compound

HB‧‧‧ hydrogen bond

Claims (10)

A method for analyzing endotoxin in a sample, comprising: providing the same sample, the sample comprising an endotoxin; adding a chitosan magnetic nanoparticle to the sample, the chitosan magnetic nano The rice particles are specifically bound to the endotoxin and form a complex, wherein the chitosan magnetic nanoparticle consists essentially of a magnetic core and a chitosan coated on the surface of the magnetic core, and The magnetic core is a CuFeO ₂ core; an external magnet is brought close to the first portion of the sample to cause the complex in the sample to collect in the first portion; and the second portion of the sample is removed, the second portion is The first portion is different, the first portion has a first concentration, the second portion has a second concentration, and the first concentration is greater than the second concentration; a mass spectrometry step is performed on the endotoxin in the first portion. A method for analyzing endotoxin in a sample as described in claim 1 wherein the sample is urine. The method for analyzing endotoxin in a sample according to claim 1, wherein the chitosan magnetic nanoparticle having a CuFeO ₂ core has a particle diameter of 5 nm to 7 nm. A method for analyzing endotoxin in a sample as described in claim 1, wherein the chitosan magnetic nanoparticles are specifically bonded to the endotoxin by hydrogen bonding. The method for analyzing endotoxin in a sample according to claim 1, wherein the mass spectrometry step is a matrix-assisted laser desorption free (MALDI) mass spectrometry step. The method for analyzing endotoxin in a sample as described in claim 5 of the patent scope, Further comprising, after removing the second portion, mixing the first portion with a matrix material, wherein the matrix material is for matrix assisted laser desorption free (MALDI) mass spectrometry. A method for analyzing endotoxin in a sample as described in claim 6 wherein the matrix material is 2,5-dihydroxybenzoic acid (DHB) or 9-aminoacridine hydrochloride. (9-aminoacridine, 9-AA) or a combination of the above. A method for analyzing endotoxin in a sample as described in claim 1 wherein the detection limit of the method is 120 to 450 μg endotoxin/mL sample. A method of analyzing endotoxin in a sample as described in claim 1 wherein there is no additional purification or extraction step prior to bringing the external magnet close to the first portion of the sample. A method of analyzing endotoxin in a sample as described in claim 1 wherein there is no additional purification or extraction step after removal of the second portion.