CN113376271A - High-sensitivity detection and analysis method for chitosan oligosaccharide in biological sample based on LC-MS - Google Patents
High-sensitivity detection and analysis method for chitosan oligosaccharide in biological sample based on LC-MS Download PDFInfo
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Abstract
本发明涉及一种基于LC‑MS的生物样品中壳寡糖的高灵敏度检测分析方法，所述壳寡糖包括壳二糖、壳三糖、壳四糖和壳五糖，所述检测分析方法包括以下步骤：(a)取生物样品进行前处理，得到样品滤液，后进行LC‑MS分析，得到样品LC‑MS信号；(b)称取壳寡糖标准物并分别将其配成标准储备液，再将标准储备液用水逐级稀释，配制一系列浓度的标准溶液，之后与空白试剂混合进行LC‑MS分析，得到标准曲线；(c)根据步骤(b)得到的标准曲线对步骤(a)得到的样品LC‑MS信号进行分析，得到生物样品中壳寡糖的含量。与现有技术相比，本发明具有简单快速、检测限低、检测灵敏度高和重复性好的优点，用于生物样本中壳寡糖含量的测定，能够满足生物样本中目标物的检测要求。
The present invention relates to a high-sensitivity detection and analysis method of chitosan oligosaccharide in biological samples based on LC-MS. The method includes the following steps: (a) taking biological samples for pretreatment to obtain sample filtrate, and then performing LC-MS analysis to obtain sample LC-MS signals; (b) weighing chitosan oligosaccharide standard substances and making them into standard reserves respectively solution, then dilute the standard stock solution with water step by step to prepare standard solutions of a series of concentrations, and then mix with blank reagents for LC-MS analysis to obtain a standard curve; (c) according to the standard curve obtained in step (b), compare step ( a) The obtained sample LC-MS signal is analyzed to obtain the content of chitosan oligosaccharide in the biological sample. Compared with the prior art, the present invention has the advantages of simplicity and rapidity, low detection limit, high detection sensitivity and good repeatability, is used for the determination of chitosan oligosaccharide content in biological samples, and can meet the detection requirements of target substances in biological samples.
Description
Technical Field
The invention belongs to the field of food and biological detection, and particularly relates to a high-sensitivity detection and analysis method for chitosan oligosaccharide in a biological sample based on LC-MS.
Background
Chitosan Oligosaccharide (COS) is a degradation product of chitosan, and is an oligosaccharide formed by connecting 2-10N-acetylglucosamine through beta-1, 4 glycosidic bonds. The chitosan oligosaccharide is the only cationic saccharide in nature, is safe and nontoxic, has wide functional effects, and has the functions of reducing weight, reducing cholesterol, regulating immunity, resisting tumor, inhibiting putrefying bacteria, regulating gastrointestinal tract and the like. The chitosan oligosaccharide has small molecular weight, is easily soluble in water, can be absorbed by human bodies, animal bodies and plant bodies, and has good physiological activity.
The chitosan oligosaccharide has no ultraviolet absorption group and no chromophoric group, so that the quantitative detection of the chitosan oligosaccharide is difficult, and particularly, the chitosan oligosaccharide is easily interfered by biological source components in a biological sample, so that the trace detection is more difficult. The existing quantitative detection method for chitosan oligosaccharide mainly comprises high performance liquid chromatography, high performance anion exchange chromatography-amperometry, capillary electrophoresis, fluorescence photometry and the like.
The separation and detection technology of chitosan oligosaccharide is always one of the concerns of researchers, and although the analysis method of chitosan oligosaccharide is various, the improvement of the existing method to make it have better specificity and sensitivity is always a hotspot and difficult problem of research. In the liquid phase separation of the chitosan oligosaccharide, the chitosan oligosaccharide contains two functional groups of amino and hydroxyl, so that on one hand, the polarity of the chitosan oligosaccharide is strong, and the chitosan oligosaccharide is difficult to be well preserved on a reversed phase C18 column directly, and on the other hand, the ultraviolet absorption is weak, and the high-sensitivity response is difficult to be obtained on an ultraviolet detector. In order to improve the problems of chitosan oligosaccharide retention and detection, an amino column is usually used for separation, and a differential refraction method detector is used for detection, but the amino column has short service life, the differential refraction method has low sensitivity and is not suitable for applying gradient elution to separate samples, and the rapid and trace detection of chitosan oligosaccharide in a complex system is difficult to realize.
Patent CN109884209A discloses a method for detecting high performance liquid chromatography tandem mass spectrometry of chitosan oligosaccharide (degree of polymerization DP 2-7) in health products, which comprises the following steps: (1) preparing a standard solution and carrying out high performance liquid chromatography tandem mass spectrometry detection to obtain spectrograms of the mixed standard solutions with different concentrations; (2) respectively making a concentration-chromatographic peak area relation curve graph of the standard spectrogram obtained in the step (1) according to different polymerization degrees (DP 2-7) of the chitosan oligosaccharide; (3) pretreating a chitosan oligosaccharide sample and detecting by high performance liquid chromatography tandem mass spectrometry; (4) and (3) determining the content of the chitosan oligosaccharide in the sample, namely determining the polymerization degree of the chitosan oligosaccharide according to the retention time of the spectrogram of the sample obtained in the step (3), comparing the polymerization degree with the standard curve obtained in the step (2), and determining the content of the chitosan oligosaccharide by using an external standard method. The linear range of chitobiose detection in the method is 0.5-20ug/mL, the linear range of chitotriose is 0.5-20ug/mL, and the linear range of chitotetrasaccharide is 0.5-20 ug/mL; in the method, the detection quantitative range of chitobiose is 0.002-2.56ug/mL, the linear range of chitotriose is 0.02-5.12ug/mL, the linear range of chitotetrasaccharide is 0.2-25.6ug/mL, and the lower limit of the quantitative limit is lower than that of the method, which shows that the method has higher sensitivity. The method is only suitable for measuring the chitosan oligosaccharide in the health care product, can measure the chitosan oligosaccharide in a complex biological system, eliminates the interference of endogenous substances in a biological sample through sample pretreatment, and realizes trace detection in the complex biological system.
Disclosure of Invention
The invention aims to provide a detection and analysis method for chitosan oligosaccharide in a biological sample based on LC-MS, which can be used for measuring the content of chitosan oligosaccharide with different polymerization degrees and can also be used for measuring the content of chitosan oligosaccharide with a single polymerization degree.
The purpose of the invention is realized by the following technical scheme:
a high-sensitivity detection and analysis method for chitosan oligosaccharide in a biological sample based on LC-MS, wherein the chitosan oligosaccharide comprises chitobiose, chitotriose, chitotetraose and chitopentaose, and the molecular weight of the chitobiose is as follows: 341.1545, molecular weight of chitotriose 502.2243, molecular weight of chitotetrasaccharide 663.2931, molecular weight of chitopentasaccharide 824.3619, said detection and analysis method comprising the steps of:
(a) pretreating a biological sample to obtain sample filtrate, and then performing LC-MS analysis to obtain a sample LC-MS signal;
(b) weighing chitosan oligosaccharide standard substances, preparing standard stock solutions from the chitosan oligosaccharide standard substances, diluting the standard stock solutions with water step by step, preparing a series of standard solutions with concentration, mixing the standard solutions with a blank reagent, treating according to the operation of pretreatment of a biological sample, and then performing LC-MS analysis to obtain a standard curve;
(c) and (c) analyzing the sample LC-MS signal obtained in the step (a) according to the standard curve obtained in the step (b) to obtain the content of the chitosan oligosaccharide in the biological sample.
In the step (a) and the step (b), the condition parameters adopted by the LC-MS analysis are as follows:
the chromatographic conditions are as follows: the mobile phase A is water containing ammonia water, and the mobile phase B is acetonitrile containing ammonia water; gradient elution or equal proportion elution is adopted for elution;
the mass spectrum conditions are as follows: ionization (ESI) was performed using an electrospray ionization ion source, and scanning was performed in positive ion target selective ion scanning mode (t-SIM).
In the step (a) and the step (b), the condition parameters adopted by the LC-MS analysis are as follows:
the chromatographic conditions are as follows:
the chromatographic column adopts Waters ACQUITY UPLC Glycan BEH Amide with the model of 2.1 multiplied by 100mm and 1.7um, the column temperature is 45 ℃, the sample injection amount is 2uL, the flow rate is 0.2mL/min, the mobile phase A is water containing 0.1% (v/v) ammonia water, the mobile phase B is acetonitrile containing 0.1% (v/v) ammonia water, and when gradient elution is adopted, the elution gradient is as follows:
the mass spectrum conditions are as follows:
electrospray ionization ion source (ESI), positive ion scan mode; selective ion scanning mode (SIM) ((SIM))Specifically chitobiose M/z 341.1545[ M + H ]]+Chitotriose M/z 502.2243[ M + H ]]+Chitotetraose 663.1931[ M + H ]]+Chitopentasaccharide 824.3619[ M + H ]]+) Setting the selection scanning parameters as follows: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931-664.2931, chitopentasaccharide: 823.3619-825.3619; the resolution is 70000; the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the voltage of the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen gas.
In step (a), the biological sample comprises a liquid sample and a solid sample, the liquid sample comprises serum, plasma, blood, body fluid, homogenate and urine, and the solid sample comprises stool, liver, kidney, heart, lung, spleen, pancreas, brain, cerebellum and intestinal contents.
When the biological sample is a liquid sample, the sample pretreatment process specifically comprises the following steps: adding a methanol-acetonitrile solvent into a liquid sample, uniformly mixing by vortex, standing, centrifuging, taking supernate, filtering the supernate to obtain sample filtrate, and then loading the sample filtrate into a liquid phase small bottle with an inner insertion tube for LC-MS analysis.
The volume ratio of the liquid sample to the methanol-acetonitrile solvent is 1: 1-1: 5, preferably 1: 3, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solvent is 1: 3-3: 1, preferably 1: 1, the rotation speed of vortex is 2500-3200 rpm, preferably 3200rpm, the time of vortex is 30-90 s, preferably 30s, the standing temperature is-20 ℃, the standing time is 10-30 min, preferably 15min, the centrifugation temperature is 4 ℃, the rotation speed of centrifugation is 12000-15000 rpm, preferably 13000rpm, the time of centrifugation is 5-15 min, preferably 10min, and a 0.22um filter membrane is adopted for filtration.
When the biological sample is a solid sample, the sample pretreatment process specifically comprises the following steps: mixing a solid sample and water, grinding, sequentially carrying out primary vortex, primary standing, ultrasonic treatment, secondary standing and primary centrifugation to obtain a first supernatant, adding a methanol-acetonitrile solvent into the first supernatant, carrying out tertiary standing after secondary vortex mixing, carrying out secondary centrifugation to obtain a second supernatant, filtering the second supernatant to obtain a sample filtrate, and then loading the sample filtrate into a liquid phase small bottle with an inner insertion tube for LC-MS analysis.
The mass-to-volume ratio of the solid sample to the water is 100 mg: 300 uL-100 mg: 900uL, preferably 100 mg: 500uL, 2 grains of pre-cooled grinding beads (the grinding beads are placed in a refrigerator for pre-cooling at 4 ℃ in advance) are added during grinding, the grinding frequency is 30-90 Hz, preferably 60Hz, the grinding time is 90-180 s, preferably 120s, the rotating speed of the first vortex is 2500-3200 rpm, preferably 3200rpm, the time of the first vortex is 30-90 s, preferably 30s, the temperature of the first standing is-20 ℃, the time of the first standing is 10-30 min, preferably 10min, ultrasound is carried out in an ice water bath, the power of ultrasound is 300W, the time of ultrasound is 10-30 min, preferably 10min, the temperature of the second standing is-20 ℃, the time of the second standing is 10-30 min, preferably 10min, the temperature of the first centrifugation is 4 ℃, the rotating speed of the first centrifugation is 3000-6000 rpm, preferably 4000rpm, and the time of the first centrifugation is 5-15 min, preferably 10min, the volume ratio of the first supernatant to the methanol-acetonitrile solvent is 1: 1-1: 5, preferably 1: 3, in the methanol-acetonitrile solvent, the volume ratio of methanol to acetonitrile is 1: 3-3: 1, preferably 1: 1, the rotating speed of the second vortex is 2500-3200 rpm, preferably 3200rpm, the time of the second vortex is 30-90 s, preferably 30s, the temperature of the third standing is-20 ℃, the time of the third standing is 10-30 min, preferably 10min, the temperature of the second centrifugation is 4 ℃, the rotating speed of the second centrifugation is 12000-15000 rpm, preferably 13000rpm, the time of the second centrifugation is 5-15 min, preferably 10min, and a filter membrane of 0.22um is adopted for filtration.
In the step (b), when the chitosan oligosaccharide standard substance is chitosan, the concentration of the serial standard solutions of chitosan is 0.04-51.2ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is vortexed and mixed uniformly to obtain a standard serum/tissue sample, wherein the concentration of chitosan in the standard serum/tissue sample is 0.002-2.56ug/mL, and the concentration is the range of the concentration in the actual sample which can be detected by the method, and the following steps are the same;
when the chitosan oligosaccharide standard substance is chitotriose, the concentration of a series of standard solutions of the chitotriose is 0.4-102.4ug/mL, the series of standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is swirled and mixed uniformly to obtain a standard serum/tissue sample, wherein the concentration of the chitotriose in the standard serum/tissue sample is 0.02-5.12 ug/mL;
when the chitosan oligosaccharide standard substance is chitosan, the concentration of the serial standard solutions of chitosan tetrasaccharide is 4-512ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is swirled and mixed uniformly to obtain a standard serum/tissue sample, wherein the concentration of chitosan tetrasaccharide in the standard serum/tissue sample is 0.2-25.6 ug/mL;
when the chitosan oligosaccharide standard substance is chitosan pentasaccharide, the concentration of the serial standard solutions of the chitosan pentasaccharide is 20-1280ug/mL, when the chitosan oligosaccharide standard substance is the chitosan pentasaccharide, the concentration of the serial standard solutions of the chitosan pentasaccharide is 20-1280ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is swirled and mixed to obtain a labeled serum/tissue sample, wherein the concentration of the chitosan pentasaccharide in the labeled serum/tissue sample is 1-64 ug/mL.
In the step (b), the LC-MS analysis process is specifically as follows: mixing the standard solution and a blank reagent, adding a methanol-acetonitrile solvent after primary vortex mixing, standing after secondary vortex mixing, centrifuging to obtain a supernatant, filtering the supernatant to obtain a sample filtrate, and then loading the sample filtrate into a liquid phase small bottle with an inner insertion tube for LC-MS analysis.
The volume ratio of the standard solution, the blank reagent and the methanol-acetonitrile solvent is 5: 95: 300, the rotating speed of the first vortex is 2500-3200 rpm, preferably 3200rpm, the time of the first vortex is 30-90 s, preferably 30s, the rotating speed of the second vortex is 2500-3200 rpm, preferably 3200rpm, the time of the second vortex is 30s, the standing temperature is-20 ℃, the standing time is 15min, the centrifugation temperature is 4 ℃, the rotating speed of the centrifugation is 13000rpm, the centrifugation time is 10min, and a filter membrane of 0.22um is adopted for filtration. In the step (b), when the blank reagent is serum without chitosan oligosaccharide, the standard curve equation of chitobiose is that y is 10000000x +789150, and R2 is 0.9992;
when the blank reagent is serum without chitosan oligosaccharide, the standard curve equation of chitotriose is 3000000x-200043, and R2 is 0.9998;
when the blank reagent is the kidney homogenate supernatant without the chitosan oligosaccharide, the standard curve equation of the chitobiose is that y is 20000000x +859579, and R2 is 0.9989;
when the blank reagent is cecal content without chitosan oligosaccharide, the standard curve of the chitotetraose is y 1000000x-371077, and R2 0.9975;
when the blank reagent is cecal content without chitosan oligosaccharide, the standard curve of the chitopentasaccharide is 569254x-1000000, and R2 is 0.9991.
The invention adopts LC-MS technology, carries out quantification by external standard method, adopts the chromatogram and the mass spectrum to carry out mass spectrum signal, chromatogram signal and linear relation investigation, establishes qualitative and quantitative methods of chitobiose, chitotriose, chitotetraose and chitopentaose by using an ultra-performance liquid chromatogram-electrostatic ion orbitrap mass spectrometer, and optimizes instrument analysis method by using standard products of four compounds, including liquid chromatogram condition, mass spectrum detection condition and mass spectrum parameter optimization, thereby achieving the detection requirement of accurately, rapidly and sensitively measuring chitosan oligosaccharide in a complex system.
The principle of the invention is specifically as follows:
aiming at the defects that in the prior art, the detection sensitivity of chitosan oligosaccharide is low, the chromatographic column has long balance time, the chitosan oligosaccharide is easily interfered by an impurity peak in a complex system, retention time drift easily occurs, the removal effect of impurity during sample pretreatment is not ideal, so that the influence on the service life of the chromatographic column is large, a plurality of projects cannot be detected simultaneously and the like, the invention develops a high-sensitivity detection and analysis method for chitosan oligosaccharide in a complex biological sample by utilizing an ultra-high performance liquid chromatography-electrostatic ion orbital trap mass spectrum system, improves the problem of retention of chitosan oligosaccharide in chromatography and greatly improves the detection sensitivity.
The method utilizes the characteristics of high sensitivity and strong anti-interference capability of the ultra-high performance liquid chromatography-electrostatic ion orbit trap high-resolution mass spectrometry technology, accurately extracts the accurate mass number of the target compound by selecting an ion scanning mode, eliminates matrix interference to a certain extent, has a simple and quick sample pretreatment mode, and is suitable for processing samples on a large scale.
The method optimizes the sample pretreatment and chromatographic conditions, so that more impurities can be removed in the sample pretreatment, and the interference of macromolecular substances such as protein in the sample to be detected is effectively reduced by adopting the methanol-acetonitrile mixed organic solvent in the pretreatment process of the sample to be detected. The chromatographic conditions with better separation effect are selected to ensure that the chitobiose, the chitotriose, the chitotetrasaccharide and the chitopentasaccharide are effectively separated from the impurities, thereby ensuring the accuracy of detection data and obtaining good separation results.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. according to the invention, the standard substances of chitobiose, chitotriose and chitotetraose are utilized to perform instrument analysis method optimization including liquid chromatography condition optimization, mass spectrum detection condition optimization and mass spectrum parameter optimization, so that the mass spectrum can be accurately determined, and the quantitative sensitivity is optimal.
2. The invention adopts LC-MS technology and uses ultra-high performance liquid chromatography-electrostatic ion orbitrap mass spectrum combination instrument to establish qualitative and quantitative methods of chitobiose, chitotriose, chitotetraose and chitopentaose in the contents of serum, liver, kidney, heart, lung, spleen, pancreas, brain, cerebellum and intestinal tract.
3. The method is simple and easy to implement, greatly reduces the pretreatment time and cost, can simultaneously detect the contents of chitobiose, chitotriose, chitotetraose and chitopentaose in a complex system, and can also only detect the content of one of the substances.
The method has the advantages of simplicity, rapidness, low detection limit, high detection sensitivity and good repeatability, is used for measuring the content of the chitosan oligosaccharide in the biological sample, can meet the detection requirement of a target object in the biological sample, has high sensitivity and selectivity and strong anti-interference capability, and can be used as an effective tool for qualitative and quantitative analysis of the chitosan oligosaccharide.
Drawings
FIG. 1 is a chromatogram of chitobiose in a serum sample of Experimental example 1;
FIG. 2 is a graph showing the linear relationship of chitobiose in serum obtained from the standard solution in Experimental example 1;
FIG. 3 is a chromatogram of chitotriose in a serum sample of Experimental example 2;
FIG. 4 is a graph showing the linear relationship of chitotriose in serum obtained from a standard solution in Experimental example 2;
FIG. 5 is a chromatogram of chitobiose in a kidney homogenate sample of Experimental example 3;
FIG. 6 is a graph showing the linear relationship of chitobiose obtained from the standard solution in the kidney homogenate in Experimental example 3;
FIG. 7 is a chromatogram of chitotetraose in a sample of homogenized caecum contents from Experimental example 4;
FIG. 8 is a graph of the linearity of chitotetraose in the homogenate of the caecum contents obtained from the standard solution in Experimental example 4;
FIG. 9 is a chromatogram of chitopentaose from a sample of homogenate of caecum contents in Experimental example 5;
FIG. 10 is a graph of the linearity of chitopentaose in caecum content homogenate from the standard solution in Experimental example 5;
FIG. 11 is a chromatogram of standard chitobiose, chitotriose, chitotetrasaccharide and chitopentasaccharide in a blank reagent.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
A high-sensitivity detection and analysis method for chitosan oligosaccharide in a biological sample based on LC-MS, wherein the chitosan oligosaccharide comprises chitobiose, chitotriose, chitotetraose and chitopentaose, and the detection and analysis method comprises the following steps:
(a) pretreating a biological sample to obtain sample filtrate, and then performing LC-MS analysis to obtain a sample LC-MS signal;
(b) weighing chitosan oligosaccharide standard substances, preparing standard stock solutions from the chitosan oligosaccharide standard substances, diluting the standard stock solutions with water step by step, preparing a series of standard solutions with concentration, mixing the standard solutions with a blank reagent, treating according to the operation of pretreatment of a biological sample, and then performing LC-MS analysis to obtain a standard curve;
(c) and (c) analyzing the sample LC-MS signal obtained in the step (a) according to the standard curve obtained in the step (b) to obtain the content of the chitosan oligosaccharide in the biological sample.
In the step (a) and the step (b), the condition parameters adopted by the LC-MS analysis are as follows:
the chromatographic conditions are as follows: the mobile phase A is water containing ammonia water, and the mobile phase B is acetonitrile containing ammonia water; gradient elution or equal proportion elution is adopted for elution;
the mass spectrum conditions are as follows: an electrospray ionization ion source is adopted for ionization, and a positive ion target selection ion scanning mode is adopted for scanning.
In the step (a) and the step (b), the condition parameters adopted by the LC-MS analysis are as follows:
the chromatographic conditions are as follows:
the chromatographic column adopts Waters ACQUITY UPLC Glycan BEH Amide with the model of 2.1 multiplied by 100mm and 1.7um, the column temperature is 45 ℃, the sample injection amount is 2uL, the flow rate is 0.2mL/min, the mobile phase A is water containing 0.1% (v/v) ammonia water, the mobile phase B is acetonitrile containing 0.1% (v/v) ammonia water, and when gradient elution is adopted, the elution gradient is as follows:
the mass spectrum conditions are as follows:
using positive ion mode, target-selective ion scan, scan parameters are: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931-664.2931, chitopentasaccharide: 823.3619-825.3619; the resolution was 70000, the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the voltage of the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen gas.
In step (a), the biological sample comprises a liquid sample and a solid sample, the liquid sample comprises serum, plasma, blood, body fluid, homogenate, and urine, and the solid sample comprises stool, liver, kidney, heart, lung, spleen, pancreas, brain, cerebellum, and intestinal contents.
When the biological sample is a liquid sample, the sample pretreatment process specifically comprises the following steps: adding a methanol-acetonitrile solvent into a liquid sample, uniformly mixing by vortex, standing, centrifuging to obtain a supernatant, filtering the supernatant to obtain a sample filtrate, and then loading the sample filtrate into a liquid phase small bottle with an inner insertion tube for LC-MS analysis, wherein the volume ratio of the liquid sample to the methanol-acetonitrile solvent is 1: 1-1: 5, the volume ratio of the methanol to the acetonitrile in the methanol-acetonitrile solvent is 1: 3-3: 1, the rotation speed of the vortex is 2500-3200 rpm, the vortex time is 30-90 s, the standing temperature is-20 ℃, the standing time is 10-30 min, the centrifugation temperature is 4 ℃, the rotation speed of the centrifugation is 12000-15000 rpm, the centrifugation time is 5-15 min, and a filter membrane of 0.22um is adopted for filtration.
When the biological sample is a solid sample, the sample pretreatment process specifically comprises the following steps: firstly, mixing a solid sample and water, grinding, then sequentially carrying out primary vortex, primary standing, ultrasonic treatment, secondary standing and primary centrifugation to obtain a first supernatant, taking the first supernatant, adding a methanol-acetonitrile solvent, carrying out tertiary standing after secondary vortex mixing, then carrying out secondary centrifugation to obtain a second supernatant, taking the second supernatant, filtering to obtain a sample filtrate, and then loading the sample filtrate into a liquid phase small bottle with an inner insertion tube for LC-MS analysis, wherein the mass-to-volume ratio of the solid sample to the water is 100 mg: 300 uL-100 mg: 900uL, adding pre-cooling grinding beads during grinding, wherein the grinding frequency is 30-90 Hz, the grinding time is 90-180 s, the rotation speed of the first vortex is 2500-3200 rpm, the time of the first vortex is 30-90 s, the temperature of the first standing is-20 ℃, the time of the first standing is 10-30 min, ultrasound is carried out in an ice water bath, the power of ultrasound is 300W, the time of ultrasound is 10-30 min, the temperature of the second standing is-20 ℃, the time of the second standing is 10-30 min, the temperature of the first centrifugation is 4 ℃, the rotation speed of the first centrifugation is 3000-6000 rpm, the time of the first centrifugation is 5-15 min, the volume ratio of the first supernatant to the methanol-acetonitrile solvent is 1: 1-1: 5, the volume ratio of the methanol to the acetonitrile in the methanol-acetonitrile solvent is 1: 3-3: 1, and the rotation speed of the second vortex is 2500-3200 rpm, the time of the second vortex is 30-90 s, the temperature of the third standing is-20 ℃, the time of the third standing is 10-30 min, the temperature of the second centrifugation is 4 ℃, the rotating speed of the second centrifugation is 12000-15000 rpm, the time of the second centrifugation is 5-15 min, and a 0.22um filter membrane is adopted for filtration.
In the step (b), when the chitosan oligosaccharide standard substance is chitosan, the concentration of the serial standard solutions of chitosan is 0.04-51.2ug/mL, adding the serial standard solutions into blank serum or blank tissue homogenate supernatant, and performing vortex mixing to obtain a standard serum/tissue sample, wherein the concentration of chitosan in the standard serum/tissue sample is 0.002-2.56 ug/mL;
when the chitosan oligosaccharide standard substance is chitotriose, the concentration of a series of standard solutions of the chitotriose is 0.4-102.4ug/mL, the series of standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is swirled and mixed uniformly to obtain a standard serum/tissue sample, wherein the concentration of the chitotriose in the standard serum/tissue sample is 0.02-5.12 ug/mL;
when the chitosan oligosaccharide standard substance is chitosan, the concentration of the serial standard solutions of chitosan tetrasaccharide is 4-512ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is swirled and mixed uniformly to obtain a standard serum/tissue sample, wherein the concentration of chitosan tetrasaccharide in the standard serum/tissue sample is 0.2-25.6 ug/mL;
when the chitosan oligosaccharide standard substance is chitosan pentasaccharide, the concentration of the serial standard solutions of the chitosan pentasaccharide is 20-1280ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the serum/tissue sample is subjected to vortex mixing to obtain a standard serum/tissue sample, wherein the concentration of the chitosan pentasaccharide in the standard serum/tissue sample is 1-64 ug/mL.
Example 1
An LC-MS-based detection and analysis method for chitosan oligosaccharide in a biological sample, which is used for analyzing the content of chitosan in serum of rats after the gavage of chitosan, namely the biological sample is a serum sample. The method comprises the following specific steps:
1. administration blood sampling
8-week-old, null male Wistar rats (230 ± 10g) were acclimatized and fasted for 12h, then gavaged with chitobiose solution, 200uL of blood was taken from the caudal vein after 0.5h (n ═ 3, i.e., three rats sampled at this time point, the same below) and 2h (n ═ 3, one for one from the rats sampled at 0.5h, the same below), and serum was taken after standing overnight at 4 ℃.
2. Method for processing serum sample
The sample pretreatment method comprises the following steps: adding 300uL of methanol-acetonitrile (v/v is 1: 1) solvent into 100uL of serum sample, violently whirling for 30s at 3200rpm, mixing uniformly, standing for 15min at-20 ℃, centrifuging for 10min at 13000rpm at 4 ℃, taking 200uL of supernatant, filtering with a 0.22um filter membrane to obtain sample filtrate, putting the sample filtrate into a liquid phase vial with an inner cannula, and carrying out on-machine detection on the sample filtrate (detection parameters are described below), wherein the chromatogram of chitobiose in the serum sample of a rat 1 at 0.5h is shown in FIG. 1 (the chromatogram of the serum sample of the other two rats at 0.5h and the chromatogram of the serum sample of three rats at 2h are similar to those in FIG. 1 and are not shown in the other figures), and FIG. 1 can show that the chitobiose can obtain clear chromatographic signals with good peak shapes under a positive ion selective ion scanning mode.
3. Standard curve sample preparation method
Weighing 10mg of chitobiose (purchased from Shanghai-sourced leaf Biotech, Ltd.) and dissolving in 1mL of ultrapure water to obtain a chitobiose stock solution with the final concentration of 10 mg/mL; diluting with ultrapure water to obtain 0.1mg/mL chitobiose working solution, and storing in a refrigerator at 4 deg.C in dark place. Diluting the chitobiose working solution with ultrapure water to obtain a series of standard solutions, wherein the concentration of chitobiose is 0.04-51.2ug/mL (specifically 0.04ug/mL, 0.16ug/mL, 0.8ug/mL, 3.2ug/mL, 12.8ug/mL, 25.6ug/mL, 51.2ug/mL), adding 5uL of the standard solutions into 95uL of blank serum, respectively, adding chitobiose in serum to obtain a serum with a concentration of 0.002-2.56ug/mL (specifically 0.002ug/mL, 0.008ug/mL, 0.04ug/mL, 0.16ug/mL, 0.64ug/mL, 1.28ug/mL, 2.56ug/mL), mixing by vortexing at 3200rpm for 30s, adding 300uL of methanol-acetonitrile (1 v/1 rpm) to obtain a solution, mixing by vortexing at-3200 rpm for 30min, centrifuging at-15 min, centrifuging at 13000 deg.10 uL, after passing through a 0.22um filter, the filtrate was placed in a liquid phase vial with an internal cannula, and the filtrate was subjected to on-machine detection (detection parameters are described below), giving a chromatogram shown in a in FIG. 11.
4. The technical conditions are as follows:
chromatographic conditions are as follows: waters ACQUITY UPLC Glycan BEH Amide column, 2.1 × 100mm, 1.7 um; column temperature: 45 ℃; the sample injection amount is 2 uL; the flow rate is 0.2 mL/min; mobile phase A: water containing 0.1% (v/v) ammonia, B: acetonitrile containing 0.1% (v/v) ammonia; the elution gradient was:
mass spectrum conditions: electrospray ionization ion source (ESI), positive ion scan mode; selection of a Targeted Selective ion scanning mode (t-SIM) (specifically chitobiose M/z 341.1545[ M + H)]+Chitotriose M/z 502.2243[ M + H ]]+Chitotetraose 663.1931[ M + H ]]+Chitopentasaccharide 824.3619[ M + H ]]+) Setting the selection scanning parameters as follows: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931-664.2931, chitopentasaccharide: 823.3619-825.3619; the resolution is 70000; the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen.
5. LC-MS analysis was performed in positive ion mode selective ion scanning mode to determine the chitobiose content in serum, the ion for quantification of chitobiose being M/z 341.1545([ M + H) ("M + H")]+) (ion deviation < 6X 10-6)。
6. Measurement results
Standard curve equation for chitobiose in blank serum: y is 10000000x +789150, and R2 is 0.9992, as shown in fig. 2 (where the abscissa is the concentration of chitobiose in serum, the unit is ug/mL, and the ordinate is the peak area of chitobiose), and fig. 2 shows that the linear relationship of chitobiose in serum obtained by the quantitative analysis of the present method is good.
Specific results of the chitobiose content in the serum of the rat after the gavage with chitobiose are shown in table 1, wherein the detection results obtained by respectively taking blood from the same rat at two time points in the transverse behavior are the same as the following results.
TABLE 1 content of chitobiose in serum
Example 2
A detection and analysis method for chitosan oligosaccharide in a biological sample based on LC-MS is used for analyzing the content of chitotriose in the serum of a rat after the gavage of the chitotriose, namely the biological sample is a serum sample. The method comprises the following specific steps:
1. administration blood sampling
8-week-old null male Wistar rats (230 ± 10g) were acclimatized and fasted for 12h before gavage of chitotriose solution, 200uL of blood was taken from the caudal vein after 0.5h (n ═ 3) and 2h (n ═ 3), and serum was taken after standing overnight at 4 ℃.
2. Method for processing serum sample
The sample pretreatment method comprises the following steps: adding 300uL of methanol-acetonitrile (v/v is 1: 1) solvent into 100uL of serum sample, violently whirling for 30s at 3200rpm, mixing uniformly, standing for 15min at-20 ℃, centrifuging for 10min at 13000rpm at 4 ℃, taking 200uL of supernatant, filtering with a 0.22um filter membrane to obtain sample filtrate, putting the sample filtrate into a liquid phase vial with an inner cannula, and carrying out on-machine detection on the sample filtrate (detection parameters are described below), wherein a chromatogram of chitotriose in the serum sample of a rat 1 at 0.5h is shown in fig. 3 (the chromatogram of the serum sample of the other two rats at 0.5h and the chromatogram of the serum sample of three rats at 2h are similar to those in fig. 3 and are not shown additionally), and fig. 3 can show that the chitotriose can obtain clear chromatographic signals with good peak shapes under a positive ion selective ion scanning mode.
3. Standard curve sample preparation method
Weighing 10mg of chitotriose (purchased from Shanghai-sourced leaf Biotechnology Co., Ltd.) and dissolving the chitotriose in 1mL of ultrapure water to obtain a chitotriose stock solution with the final concentration of 10 mg/mL; diluting with ultrapure water to prepare 1mg/mL chitotriose working solution, and storing in a refrigerator at 4 ℃ in a dark place for later use. Diluting the chitotriose working solution with ultrapure water to obtain a series of standard solutions, wherein the concentration range of the chitotriose is 0.4-102.4ug/mL (specifically 0.4ug/mL, 1.6ug/mL, 6.4ug/mL, 12.8ug/mL, 25.6ug/mL, 51.2ug/mL, 102.4ug/mL), respectively adding 5uL of the standard solutions into 95uL of blank serum, wherein the concentration of the chitotriose in the serum is 0.02-5.12ug/mL (specifically 0.02ug/mL, 0.08ug/mL, 0.32ug/mL, 0.64ug/mL, 1.28ug/mL, 2.56ug/mL, 5.12ug/mL), mixing by vortexing at 3200rpm for 30s, adding 300uL of methanol-acetonitrile (v/v 1: 1) solvent, mixing vigorously at 3200rpm for 30min, vortexing at-15 min, centrifuging at 13000 ℃ for 13000 ℃ and centrifuging at 20 uL, after passing through a 0.22um filter, the filtrate was placed in a liquid phase vial with an internal cannula, and the filtrate was subjected to on-machine detection (detection parameters are described below), giving a chromatogram shown in b of FIG. 11.
4. The technical conditions are as follows:
chromatographic conditions are as follows: waters ACQUITY UPLC Glycan BEH Amide column, 2.1 × 100mm, 1.7 um; column temperature: 45 ℃; the sample injection amount is 2 uL; the flow rate is 0.2 mL/min; mobile phase A: water containing 0.1% (v/v) ammonia, B: acetonitrile containing 0.1% (v/v) ammonia; the elution gradient was:
mass spectrum conditions: electrospray ionization ion source (ESI), positive ion scan mode; selective ion scanning mode (SIM) (specifically chitobiose M/z 341.1545[ M + H ]]+Chitotriose M/z 502.2243[ M + H ]]+Chitotetraose 663.1931[ M + H ]]+) Chitopentasaccharide 824.3619[ M + H ]]+) Setting the selection scanning parameters as follows: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931-664.2931, chitopentasaccharide: 823.3619-825.3619; the resolution is 70000; the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen.
5. LC-MS analysis was performed in positive ion mode selective ion scanning mode and chitotriose content in serum was measured as M/z 502.2243([ M + H ] +) (ion deviation < 6 × 10-6) for quantitative analysis, and the chromatogram of chitotriose in serum is shown in fig. 3.
6. Measurement results
Standard curve equation for chitotriose in blank serum: y is 3000000x-200043, and R2 is 0.9998, as shown in fig. 4 (wherein, the abscissa is the concentration of chitotriose in serum, and the unit is ug/mL, and the ordinate is the peak area of chitotriose), fig. 4 shows that the linear relationship of chitotriose obtained by the quantitative analysis of the method in serum is good.
Specific results of chitotriose content in rat serum after gavage with chitotriose are shown in Table 2.
TABLE 2 content of chitotriose in serum
Example 3
An LC-MS-based detection and analysis method for chitosan oligosaccharide in a biological sample, which is used for analyzing the content of chitosan in the kidney after the chitosan is perfused into a rat, namely the biological sample is the kidney. The specific steps are as follows:
1. drug administration for collecting kidney
Male Wistar rats (230 ± 10g) aged 8 weeks were subjected to seven-day adaptive feeding, then, to gastric lavage of chitobiose solution, and the rats were sacrificed after 1h of neck amputation (n ═ 3), and then, the kidneys were dissected, the surface was washed with physiological saline to remove blood, and the water was blotted with filter paper.
2. Kidney sample processing method
Accurately weighing 100mg kidney sample in an EP tube, adding 2 grains of pre-cooled grinding beads (pre-cooling the grinding beads in a refrigerator at 4 ℃) and 500uL of pre-cooled normal saline, and grinding for 120s by a tissue grinder under 60 Hz. Then vortexed at 3200rpm for 30s, and allowed to stand at-20 ℃ for 10min, followed by sonication in an ice water bath at 300W for 10 min. Standing at-20 deg.C for 10min, and centrifuging at 4000rpm for 10 min. 100uL of the supernatant was taken, 300uL of methanol-acetonitrile (v/v ═ 1: 1) solvent was added, vortexed and mixed at 3200rpm for 30s, and then allowed to stand at-20 ℃ for 10min, and after taking out, it was centrifuged at 13000rpm for 10 min. 200uL of the supernatant was filtered through a 0.22um filter and placed in a liquid phase vial for detection.
3. Standard curve sample preparation method
10mg of chitobiose (purchased from Shanghai-derived leaf Biotech Co., Ltd.) was weighed and dissolved in 1mL of ultrapure water to obtain a chitobiose stock solution having a concentration of 10mg/mL, diluted with ultrapure water to 0.1mg/mL of chitobiose working solution, and stored in a refrigerator at 4 ℃ in a dark place for use. Diluting the chitobiose working solution with ultrapure water to obtain a series of standard solutions, wherein the chitobiose concentration ranges from 0.04-51.2ug/mL (specifically 0.04ug/mL, 0.16ug/mL, 0.8ug/mL, 3.2ug/mL, 12.8ug/mL, 25.6ug/mL, and 51.2ug/mL), adding 5uL of the standard solutions into the supernatant of the blank kidney tissue homogenate, respectively, the chitobiose concentration in the supernatant of the kidney tissue homogenate is from 0.002-2.56ug/mL (specifically 0.002ug/mL, 0.008ug/mL, 0.04ug/mL, 0.16ug/mL, 0.64ug/mL, 1.28ug/mL, and 2.56ug/mL), adding 300uL of methanol-acetonitrile (v/v: 1 rpm), mixing, vigorously at 3200rpm for 30min, vortexing at-15 min, and centrifuging at 13000 ℃ for 15min, taking 200uL of supernatant, filtering with 0.22um filter membrane, loading into liquid phase vial with inner cannula, performing on-machine detection (detection parameters are described below) on the filtrate, and obtaining chromatogram shown in fig. 11 a.
4. The technical conditions are as follows:
chromatographic conditions are as follows: waters ACQUITY UPLC Glycan BEH Amide column, 2.1 × 100mm, 1.7 um; column temperature: 45 ℃; the sample injection amount is 2 uL; the flow rate is 0.2 mL/min; mobile phase A: water containing 0.1% (v/v) ammonia, B: acetonitrile containing 0.1% (v/v) ammonia; the elution gradient was:
mass spectrum conditions: electrospray ionization ion source (ESI), positive ion scan mode; selective ion scanning mode (SIM) (specifically chitobiose M/z 341.1545[ M + H ]]+Chitotriose M/z 502.2243[ M + H ]]+Chitotetraose 663.1931[ M + H ]]+) Chitopentasaccharide 824.3619[ M + H ]]+) Setting the selection scanning parameters as follows: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931 to 664.2931, in which the first and second substrates are bonded together,shell pentasaccharide: 823.3619-825.3619; the resolution is 70000; the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen.
5. LC-MS analysis was performed in positive ion mode selective ion scanning mode to determine chitobiose content in kidney, and COS 2 was used as an ion for quantitative analysis of M/z 341.1555([ M + H) (]+) (ion deviation < 6X 10-6) The chromatogram of chitobiose in kidney homogenate is shown in fig. 5, and fig. 5 can show that chitobiose can obtain clear chromatographic signals with good peak shape in positive ion selective ion scanning mode.
6. Standard curve equation for chitobiose in blank kidney: y 20000000x +859579, R2 0.999 as shown in fig. 6 (wherein the abscissa is the concentration of chitobiose in kidney homogenate in ug/mL and the ordinate is the area of the chitobiose peak); FIG. 6 shows that chitobiose obtained by the quantitative analysis of the present method has a good linear relationship in kidney samples.
The concentration in the standard was the concentration of chitobiose in rat kidney homogenate prepared from 100mg kidney tissue (approximately 100uL in volume) homogenized with 500uL of physiological saline, thus the concentration in the kidney was 6 times the concentration in the kidney homogenate. The results of the chitobiose content in rat kidney after gavage with chitobiose are shown in Table 3.
TABLE 3 content of chitobiose in Kidney
Example 4
An LC-MS-based method for detecting and analyzing chitosan oligosaccharide in a biological sample, which is used for analyzing the content of chitosan in caecum contents of rats after chitosan gavage, and the biological sample is the caecum contents. The specific steps are as follows:
1. administration to obtain the cecal content
Male Wistar rats (230 ± 10g) at 8 weeks of age were gavaged with a chitotetraose solution after seven days of acclimation, sacrificed at 4h neck (n ═ 3), and the cecal contents were dissected out.
2. Method for processing cecal content sample
100mg of cecum content was accurately weighed into an EP tube, 2 beads (pre-cooled beads in a refrigerator at 4 ℃ C.) and 500uL of pre-cooled physiological saline were added, and the mixture was ground with a tissue grinder at 60Hz for 120 seconds. Then vortexed at 3200rpm for 30s, and allowed to stand at-20 ℃ for 10min, followed by sonication in an ice water bath at 300W for 10 min. Standing at-20 deg.C for 10min, and centrifuging at 4000rpm for 10 min. 100uL of the supernatant was taken, 300uL of methanol-acetonitrile (v/v ═ 1: 1) solvent was added, vortexed and mixed at 3200rpm for 30s, and then allowed to stand at-20 ℃ for 10min, and after taking out, it was centrifuged at 13000rpm for 10 min. 200uL of the supernatant was filtered through a 0.22um filter and placed in a liquid phase vial for detection.
3. Standard curve sample preparation method
Weighing 10mg of chitotetraose (purchased from Shanghai-derived leaf Biotechnology Co., Ltd.) and dissolving in 1mL of ultrapure water to obtain a chitotetraose stock solution with a concentration of 10mg/mL, diluting with ultrapure water to obtain a chitotetraose working solution with a concentration of 1mg/mL, and storing in a refrigerator at 4 ℃ in a dark place for later use. Diluting the chitotetraose working solution with ultrapure water to obtain a series of standard solutions, wherein the concentration range of the chitotetraose is 4-512ug/mL (specifically 4ug/mL, 16ug/mL, 32ug/mL, 64ug/mL, 128ug/mL, 256ug/mL, 512ug/mL), respectively adding 5uL of the standard solutions into 95uL of the supernatant of the cecum content homogenate, and mixing with 3200rpm by vortex for 30s to obtain labeled kidney samples (specifically 0.2ug/mL, 0.8ug/mL, 1.6ug/mL, 3.2ug/mL, 6.4ug/mL, 12.8ug/mL, 25.6ug/mL) with the concentration range of 0.2-25.6 ug/mL. Subsequently, 300. mu.L of methanol-acetonitrile (v/v. 1: 1) was added, vigorously vortexed at 3200rpm for 30 seconds and mixed, left to stand at-20 ℃ for 15min, then centrifuged at 13000rpm for 10min at 4 ℃, 200. mu.L of the supernatant was taken, filtered through a 0.22um filter and put into a liquid phase vial with an internal cannula, and the filtrate was subjected to on-machine detection (detection parameters are described below) to obtain a chromatogram shown in c in FIG. 11.
4. The technical conditions are as follows:
chromatographic conditions are as follows: waters ACQUITY UPLC Glycan BEH Amide column, 2.1 × 100mm, 1.7 um; column temperature: 45 ℃; the sample injection amount is 2 uL; the flow rate is 0.2 mL/min; mobile phase A: water containing 0.1% (v/v) ammonia, B: acetonitrile containing 0.1% (v/v) ammonia; the elution gradient was:
mass spectrum conditions: electrospray ionization ion source (ESI), positive ion scan mode; ion scanning mode (SIM) was selected (specifically chitobiose M/z 341.1545[ M + H ═ 341.1545]+Chitotriose M/z 502.2243[ M + H ]]+Chitotetraose M/z 663.1931[ M + H ]]+) Chitopentasaccharide M/z 824.3619[ M + H ]]+) Setting the selection scanning parameters as follows: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931-664.2931, chitopentasaccharide: 823.3619-825.3619; the resolution is 70000; the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen.
5. LC-MS analysis was performed in positive ion mode selective ion scanning mode to determine the content of chitotetraose in the caecum contents, the ion of chitotetraose used for quantitative analysis being M/z-663.2931 ([ M + H ])]+) (ion deviation < 6X 10-6) The chromatogram of chitotetraose in the caecum content is shown in fig. 7, and fig. 7 can show that the chitotetraose can obtain a clear chromatographic signal with a good peak shape in a positive ion selective ion scanning mode.
6. Standard curve equation for chitotetraose in blank cecal contents: y 1000000x 371077, R2 0.9975 as shown in fig. 8 (wherein the abscissa is the concentration of chitotetraose in the cecal content homogenate in ug/mL and the ordinate is the peak area of chitotetraose); FIG. 8 shows that chitotetraose quantitatively analyzed by this method has a good linear relationship in samples of the caecum contents.
The standard was chitotetraose in rat cecal knot content homogenate prepared from 100mg of cecal knot content (approximately 100uL in volume) plus 500uL of physiological saline, thus the concentration in cecal knot content was 6 times higher than that in cecal knot content homogenate. The specific results of chitotetraose content in the homogenate of rat cecal nodule contents after gavage with chitotetraose are shown in Table 4.
TABLE 4 content of chitotetraose in cecal contents
Example 5
An LC-MS-based detection and analysis method for chitosan oligosaccharide in a biological sample is used for analyzing the content of the chitosan pentasaccharide in caecum contents of rats after the chitosan pentasaccharide is perfused into the stomachs, and the biological sample is the caecum contents. The specific steps are as follows:
1. administration to obtain the cecal content
Male Wistar rats (230 ± 10g) at 8 weeks of age were gavaged with a solution of chitopentaose after seven days of acclimation, sacrificed at 5h neck (n ═ 3), and the cecal contents were dissected out.
2. Method for processing cecal content sample
Accurately weighing 100mg of cecum content into an EP tube, adding 2 precooled grinding beads and 500uL of precooled normal saline, and grinding for 120s by a tissue grinder at 60 Hz. Then vortexed at 3200rpm for 30s, and allowed to stand at-20 ℃ for 10min, followed by sonication in an ice water bath at 300W for 10 min. Standing at-20 deg.C for 10min, and centrifuging at 4000rpm for 10 min. 100uL of the supernatant was taken, 300uL of methanol-acetonitrile (v/v ═ 1: 1) solvent was added, vortexed and mixed at 3200rpm for 30s, and then allowed to stand at-20 ℃ for 10min, and after taking out, it was centrifuged at 13000rpm for 10 min. 200uL of the supernatant was filtered through a 0.22um filter and placed in a liquid phase vial for detection.
3. Standard curve sample preparation method
Weighing 10mg of chitopentaose (purchased from Shanghai-derived leaf Biotechnology Co., Ltd.) and dissolving in 1mL of ultrapure water to obtain a chitopentaose stock solution with a concentration of 10mg/mL, diluting with ultrapure water to obtain a 5mg/mL chitopentaose working solution, and storing in a refrigerator at 4 ℃ in a dark place for later use. Diluting the shell pentasaccharide working solution with ultrapure water to obtain a series of standard solutions, wherein the concentration range of the shell pentasaccharide is 20-1280ug/mL (specifically 20ug/mL, 40ug/mL, 80ug/mL, 160ug/mL, 320ug/mL, 640ug/mL, 1280ug/mL), adding 5uL of the standard solutions into 95uL of the cecum content homogenate supernatant, and mixing uniformly by swirling at 3200rpm for 30s to obtain labeled cecum content samples (specifically 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 16ug/mL, 32ug/mL, 64 ug/mL) with the concentration range of 1-64 ug/mL. Subsequently, 300. mu.L of methanol-acetonitrile (v/v. 1: 1) was added, vigorously vortexed at 3200rpm for 30 seconds and mixed, left to stand at-20 ℃ for 15min, then centrifuged at 13000rpm for 10min at 4 ℃, 200. mu.L of the supernatant was taken, filtered through a 0.22um filter and put into a liquid phase vial with an internal cannula, and the filtrate was subjected to on-machine detection (detection parameters are described below) to obtain a chromatogram shown in FIG. 11 d.
4. The technical conditions are as follows:
chromatographic conditions are as follows: waters ACQUITY UPLC Glycan BEH Amide column, 2.1 × 100mm, 1.7 um; column temperature: 45 ℃; the sample injection amount is 2 uL; the flow rate is 0.2 mL/min; mobile phase A: water containing 0.1% (v/v) ammonia, B: acetonitrile containing 0.1% (v/v) ammonia; the elution gradient was:
mass spectrum conditions: electrospray ionization ion source (ESI), positive ion scan mode; ion scanning mode (SIM) was selected (specifically chitobiose M/z 341.1545[ M + H ═ 341.1545]+Chitotriose M/z 502.2243[ M + H ]]+Chitotetraose M/z 663.1931[ M + H ]]+) Chitopentasaccharide M/z 824.3619[ M + H ]]+) Setting the selection scanning parameters as follows: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931-664.2931, chitopentasaccharide: 823.3619-825.3619; the resolution is 70000; the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen.
5. LC-MS analysis was performed in positive ion mode selective ion scanning mode to determine the content of chitopentasaccharide in the caecum contents, the ion of chitopentasaccharide used for quantitative analysis being M/z-824.3619 ([ M + H ])]+) (ion deviation < 6X 10-6) The chromatogram of chitopentaose in the caecum content is shown in FIG. 9, and FIG. 9 can illustrate that chitopentaose is in positive ionThe ion scanning mode is selected to obtain clear chromatographic signals with good peak shapes.
6. Standard curve equation for chitopentaose in blank cecal contents: y-569254 x-1000000, R2-0.9991, as shown in fig. 10 (where the abscissa is the concentration of chitopentaose in the cecum content homogenate in ug/mL and the ordinate is the peak area of chitopentaose); FIG. 10 shows that the chitopentaose quantitatively analyzed by this method has a good linear relationship in the samples of the caecum contents.
The standard yeast is the concentration of chitopentasaccharide in the homogenate of rat ceca content prepared from 100mg of ceca content (about 100uL in volume) plus 500uL of physiological saline, and thus the concentration in the ceca content is 6 times higher than that in the homogenate of ceca content. The specific results of the content of chitopentaose in the homogenate of the rat cecal content after gavage of chitopentaose are shown in Table 5.
TABLE 5 content of chitopentaose in cecal contents
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A high-sensitivity detection and analysis method for chitosan oligosaccharide in a biological sample based on LC-MS is characterized in that the chitosan oligosaccharide comprises chitobiose, chitotriose, chitotetraose and chitopentaose, and the detection and analysis method comprises the following steps:
(a) pretreating a biological sample to obtain sample filtrate, and then performing LC-MS analysis to obtain a sample LC-MS signal;
(b) weighing chitosan oligosaccharide standard substances, preparing standard stock solutions from the chitosan oligosaccharide standard substances, diluting the standard stock solutions with water step by step, preparing a series of standard solutions with concentration, mixing the standard solutions with a blank reagent, treating according to the operation of pretreatment of a biological sample, and then performing LC-MS analysis to obtain a standard curve;
(c) and (c) analyzing the sample LC-MS signal obtained in the step (a) according to the standard curve obtained in the step (b) to obtain the content of the chitosan oligosaccharide in the biological sample.
2. The method for detecting and analyzing chitosan oligosaccharide in biological samples based on LC-MS as claimed in claim 1, wherein the condition parameters used in LC-MS analysis in step (a) and step (b) are:
the chromatographic conditions are as follows: the mobile phase A is water containing ammonia water, and the mobile phase B is acetonitrile containing ammonia water; gradient elution or equal proportion elution is adopted for elution;
the mass spectrum conditions are as follows: an electrospray ionization ion source is adopted for ionization, and a positive ion target selection ion scanning mode is adopted for scanning.
3. The method for detecting and analyzing chitosan oligosaccharide in biological samples based on LC-MS as claimed in claim 2, wherein the condition parameters used in the LC-MS analysis in step (a) and step (b) are:
the chromatographic conditions are as follows:
the chromatographic column adopts Waters ACQUITY UPLC Glycan BEH Amide with the model of 2.1 multiplied by 100mm and 1.7um, the column temperature is 45 ℃, the sample injection amount is 2uL, the flow rate is 0.2mL/min, the mobile phase A is water containing 0.1% (v/v) ammonia water, the mobile phase B is acetonitrile containing 0.1% (v/v) ammonia water, and when gradient elution is adopted, the elution gradient is as follows:
the mass spectrum conditions are as follows:
using positive ion mode, target-selective ion scan, scan parameters are: chitobiose: 340.1545-342.1545, chitotriose: 501.2233-503.2233, chitotetraose: 662.2931-664.2931, chitopentasaccharide: 823.3619-825.3619; the resolution was 70000, the flow rate of the sheath gas was 40arb, the flow rate of the mixed gas was 10arb, the heating temperature of the mixed gas was 350 ℃, the temperature of the ion source was 320 ℃, the voltage of the positive ion spray was 3.5kV, the voltage of the S-lens RF was 50V, and both the ion source and the collision gas were high-purity nitrogen gas.
4. The method for high-sensitivity detection and analysis of chitosan oligosaccharide in biological sample based on LC-MS as claimed in claim 1, wherein in step (a), said biological sample comprises liquid sample and solid sample, said liquid sample comprises serum, plasma, blood, body fluid, tissue homogenate and urine, said solid sample comprises feces, liver, kidney, heart, lung, spleen, pancreas, brain, cerebellum and intestinal contents.
5. The LC-MS-based high-sensitivity detection and analysis method for chitosan oligosaccharide in biological samples as claimed in claim 4, wherein when the biological sample is a liquid sample, the sample pretreatment process specifically comprises: adding a methanol-acetonitrile solvent into a liquid sample, uniformly mixing by vortex, standing, centrifuging, taking supernate, filtering the supernate to obtain sample filtrate, and then loading the sample filtrate into a liquid phase small bottle with an inner insertion tube for LC-MS analysis.
6. The high-sensitivity detection and analysis method for chitosan oligosaccharide in a biological sample based on LC-MS as claimed in claim 5, wherein the volume ratio of the liquid sample to the methanol-acetonitrile solvent is 1: 1-1: 5, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solvent is 1: 3-3: 1, the rotation speed of vortex is 2500-3200 rpm, the time of vortex is 30-90 s, the temperature of standing is-20 ℃, the time of standing is 10-30 min, the temperature of centrifugation is 4 ℃, the rotation speed of centrifugation is 12000-15000 rpm, the time of centrifugation is 5-15 min, and a 0.22um filter membrane is adopted for filtration.
7. The LC-MS-based high-sensitivity detection and analysis method for chitosan oligosaccharide in biological samples as claimed in claim 4, wherein when the biological sample is a solid sample, the sample pretreatment process specifically comprises: mixing a solid sample and water, grinding, sequentially carrying out primary vortex, primary standing, ultrasonic treatment, secondary standing and primary centrifugation to obtain a first supernatant, adding a methanol-acetonitrile solvent into the first supernatant, carrying out tertiary standing after secondary vortex mixing, carrying out secondary centrifugation to obtain a second supernatant, filtering the second supernatant to obtain a sample filtrate, and then loading the sample filtrate into a liquid phase small bottle with an inner insertion tube for LC-MS analysis.
8. The method for high-sensitivity detection and analysis of chitosan oligosaccharide in biological samples based on LC-MS as claimed in claim 7, wherein the mass-to-volume ratio of the solid sample to water is 100 mg: 300 uL-100 mg: 900uL, adding pre-cooling grinding beads during grinding, wherein the grinding frequency is 30-90 Hz, the grinding time is 90-180 s, the rotation speed of the first vortex is 2500-3200 rpm, the time of the first vortex is 30-90 s, the temperature of the first standing is-20 ℃, the time of the first standing is 10-30 min, ultrasound is carried out in an ice water bath, the power of ultrasound is 300W, the time of ultrasound is 10-30 min, the temperature of the second standing is-20 ℃, the time of the second standing is 10-30 min, the temperature of the first centrifugation is 4 ℃, the rotation speed of the first centrifugation is 3000-6000 rpm, the time of the first centrifugation is 5-15 min, the volume ratio of the first supernatant to the methanol-acetonitrile solvent is 1: 1-1: 5, the volume ratio of the methanol to the acetonitrile in the methanol-acetonitrile solvent is 1: 3-3: 1, and the rotation speed of the second vortex is 2500-3200 rpm, the time of the second vortex is 30-90 s, the temperature of the third standing is-20 ℃, the time of the third standing is 10-30 min, the temperature of the second centrifugation is 4 ℃, the rotating speed of the second centrifugation is 12000-15000 rpm, the time of the second centrifugation is 5-15 min, and a 0.22um filter membrane is adopted for filtration.
9. The LC-MS-based high-sensitivity detection and analysis method for chitosan oligosaccharide in a biological sample, as claimed in claim 1, wherein in the step (b), when the chitosan oligosaccharide standard substance is chitosan, the concentration of the serial standard solutions of chitosan is 0.04-51.2ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is vortexed and mixed to obtain a labeled serum/tissue sample, wherein the concentration of chitosan in the labeled serum/tissue sample is 0.002-2.56 ug/mL;
when the chitosan oligosaccharide standard substance is chitotriose, the concentration of a series of standard solutions of the chitotriose is 0.4-102.4ug/mL, the series of standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is swirled and mixed uniformly to obtain a standard serum/tissue sample, wherein the concentration of the chitotriose in the standard serum/tissue sample is 0.02-5.12 ug/mL;
when the chitosan oligosaccharide standard substance is chitosan, the concentration of the serial standard solutions of chitosan tetrasaccharide is 4-512ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the mixture is swirled and mixed uniformly to obtain a standard serum/tissue sample, wherein the concentration of chitosan tetrasaccharide in the standard serum/tissue sample is 0.2-25.6 ug/mL;
when the chitosan oligosaccharide standard substance is chitosan pentasaccharide, the concentration of the serial standard solutions of the chitosan pentasaccharide is 20-1280ug/mL, the serial standard solutions are taken and added into blank serum or blank tissue homogenate supernatant, and the serum/tissue sample is subjected to vortex mixing to obtain a standard serum/tissue sample, wherein the concentration of the chitosan pentasaccharide in the standard serum/tissue sample is 1-64 ug/mL.
10. The method for high-sensitivity detection and analysis of chitosan oligosaccharide in biological sample according to claim 1, wherein in step (b), when the blank reagent is serum without chitosan oligosaccharide, the standard curve equation of chitobiose is (y) 10000000x +789150, R2＝0.9992；
When the blank reagent is serum without chitosan oligosaccharide, the standard curve equation of chitotriose is that y is 3000000x-200043, R2＝0.9998；
When the blank reagent is kidney homogenate supernatant without chitosan oligosaccharide, the standard curve equation of chitobiose is that y is 20000000x +859579, R2＝0.9989；
When the blank reagent is cecal content without chitosan oligosaccharide, the standard curve of the chitotetraose is that y is 1000000x-371077, R2＝0.9975；
When the blank reagent is cecal content without chitosan oligosaccharide, the standard curve of the chitopentasaccharide is 569254x-1000000, R2＝0.9991。
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