A high-precision, interference-resistant endotoxin analysis method and kit

Abstract

The present invention discloses a method and kit for analyzing endotoxins in complex matrix samples based on liquid chromatography-mass spectrometry. 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid or 3-hydroxyoctadecanoic acid characteristic of endotoxins are released by KOH methanol solution for quantitative analysis of various types of endotoxin content; the hydrolyzate is derivatized with isoquinoline-1-carboxylic acid hydrazine to significantly improve the ionization efficiency of the hydrolyzate; using optimized liquid chromatography and mass spectrometry parameters, high stability, high sensitivity and broad spectrum detection of endotoxins from different bacterial sources are achieved. The method has strong anti-interference ability, is applicable to complex matrices, and solves the problem of high false negative rate caused by matrix interference in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a high-precision, interference-resistant endotoxin analysis method and a matching kit based on liquid chromatography-mass spectrometry technology. Background Art

[0002] Endotoxin is a component of the cell wall of Gram-negative bacteria, also known as lipopolysaccharide, and is present in the cell wall of Gram-negative bacteria. Endotoxin is composed of three parts: O-antigen, core polysaccharide, and lipid A. The O-antigen is the outermost part, composed of dozens of identical oligosaccharide units, and is not necessary for the integrity of the outer membrane. The core polysaccharide is composed of heptyl core polysaccharide, galactose, 2-keto-3-deoxyoctanoic acid, etc., and is present in all Gram-negative bacteria. It can undergo non-stoichiometric substitution with other sugars and is variable. Lipid A is the active center and toxicity center of lipopolysaccharide. Lipid A is composed of D-glucosamine disaccharide units connected by β-1,6-glycosidic bonds. The hydroxyl and amino groups connected to the sugar can be replaced by hydroxy fatty acids or fatty acyl fatty acids. The main difference is the number of carbon atoms in the substituted fatty acid.

[0003] Endotoxins are recognized by Toll-like receptor 4 (TLR4) in the host's innate immune system, mediating activation of MAPK and NF-κB signaling pathways and inducing the secretion of inflammatory mediators. Endotoxins also participate in endotoxin tolerance mechanisms, which regulate excessive inflammatory responses. Excessive endotoxins can also cause severe pathophysiological reactions, manifesting as fever, hypotension, shock, organ failure, and even death. However, endotoxins are thermally and chemically stable, requiring heating at 250°C for 30 minutes to destroy their biotoxicity. This leads to the potential for excessive endotoxins in the production of pharmaceuticals, foods, and livestock products, posing a health risk to humans and livestock. Therefore, it is necessary to develop a method for analyzing endotoxins in complex matrices.

[0004] An early method for determining endotoxins was the rabbit fever test, which involves injecting a test sample into rabbits and observing changes in body temperature. However, due to large individual differences, long cycles, and cumbersome procedures, it is not suitable for batch testing. Currently, the main method for determining endotoxins is the Limulus amebocyte lysate (LAS) method. The 2020 edition of the Pharmacopoeia of the People's Republic of China stipulates the use of LAS for the determination of endotoxins in medical devices and pharmaceuticals. However, it is not suitable for endotoxin determination in samples with complex matrices, such as food, feed, blood, and tissue. This is because the LAS method can react nonspecifically with β-glucan in the sample, resulting in a high false positive rate. The use of synthetic recombinant Factor C has high specificity and is not limited by the shortage of LAS. However, the standardization of the recombinant Factor C method requires rigorous validation. For example, the linear range of the standard curve is narrow, and multiple repeated tests are required to ensure reliability. In addition, the sample dilution factor needs to be adjusted according to the specific product, which increases the complexity of the experimental design. For samples with complex matrices, additional verification of interfering factors (such as pH value and component interference) is still required, and the operation process is cumbersome. Jean-Paul et al. (Comparative Example 1) reported (https: / / pmc.ncbi.nlm.nih.gov / articles / PMC4479340) that 3-hydroxytetradecanoic acid was used in endotoxin quantification using GC-MS and LC-MS, eliminating interference from matrices such as β-glucan. However, 3-hydroxytetradecanoic acid alone cannot be used to determine all types of endotoxins. For example, Helicobacter pylori and Francisella endotoxins do not contain 3-hydroxytetradecanoic acid, but rather one 3-hydroxyhexadecanoic acid and two 3-hydroxyoctadecanoic acid residues, resulting in false negative results. Summary of the Invention

[0005] In response to the technical difficulties and shortcomings of endotoxin detection, the purpose of the present disclosure is to propose a high-precision, interference-resistant endotoxin analysis method and kit.

[0006] Based on the above objectives, the present disclosure provides an analysis method for endotoxins, comprising:

[0007] (1) directly treating the sample to be tested with an alkaline solution to obtain 3-hydroxy fatty acids in the endotoxin, wherein the 3-hydroxy fatty acids are one or more of 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid and 3-hydroxyoctadecanoic acid, and the hydrolyzate contains KOH and methanol;

[0008] (2) using isoquinoline-1-carboxylic acid hydrazine to carry out a derivatization reaction on the 3-hydroxy fatty acid to obtain derivative products of different 3-hydroxy fatty acids, thereby enhancing the ionization efficiency of the hydrolyzed product and improving the sensitivity of mass spectrometry detection;

[0009] (3) Detecting the derivative product by liquid chromatography-mass spectrometry, wherein the mass spectrometry is a triple quadrupole mass spectrometry, the ionization mode is an electrospray positive ion mode, and the data acquisition mode is a multiple ion scanning mode; and determining the content of endotoxin by analyzing the quantitative results of 3-hydroxy fatty acids in the test sample.

[0010] Based on the same inventive concept, the embodiments of the present disclosure also provide a high-precision, interference-resistant endotoxin analysis method and kit, wherein the detection kit is applied to the aforementioned endotoxin analysis method; wherein the detection kit includes the methanol solution for KOH; EDC, HOAt and isoquinoline-1-carboxylic acid hydrazine solution for derivatization reaction; 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid and 3-hydroxyoctadecanoic acid standards.

[0011] As can be seen from the above, the present disclosure provides a high-precision, anti-interference endotoxin analysis method and kit, which releases 3-hydroxy fatty acids that can represent endotoxins through KOH methanol solution, and indirectly realizes the quantitative analysis of endotoxins by quantitative analysis of 3-hydroxy fatty acids. This scheme detects multiple 3-hydroxy fatty acids simultaneously, realizes a comprehensive analysis of endotoxins from different sources, and the scheme is not interfered with by β-glucan, thereby increasing the quantitative accuracy of endotoxins. Through the reaction of isoquinoline-1-carboxylic acid hydrazine with 3-hydroxy fatty acids, 3-hydroxy fatty acids are converted from negative ion mode detection with low ionization efficiency to positive ion mode detection with high ionization efficiency, significantly improving the detection sensitivity of 3-hydroxy fatty acids, and the detection limit of different 3-hydroxy fatty acids is as low as 6~15 pg / ml, and the quantitative limit is as low as 20~50 pg / ml. At the same time, the derivatization reaction of 3-hydroxy fatty acids with isoquinoline-1-carboxylic acid hydrazine is simple to operate, and the reaction conditions are mild. The derivatization reaction can be completed within 1 hour at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the present disclosure or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0013] Figure 1 Schematic diagram of the endotoxin detection principle involved in the present disclosure;

[0014] Figure 2 Comparison of mass spectrometric signal responses before and after the reaction of 3-hydroxytetradecanoic acid with isoquinoline-1-carboxylic acid hydrazine;

[0015] Figure 3 For the separation of 2-hydroxy fatty acids and 3-hydroxy fatty acids;

[0016] Figure 4 is the conversion coefficient of E. coli in the solvent. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0018] The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should have the usual meanings understood by persons of ordinary skill in the art to which the present disclosure pertains. The "first," "second," and similar terms used in the embodiments of the present disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different components. The terms "comprising" or "including" may be open, semi-enclosed, or closed.

[0019] The abbreviations used in the examples of this disclosure have their conventional meanings in the chemical and biological arts.The chemical structures and formulas set forth herein are constructed according to standardized valence rules known in the chemical arts.

[0020] The embodiment of the present disclosure uses 3-hydroxy fatty acids in the endotoxin structure as the characteristic structure of endotoxin. 3-hydroxy fatty acids can be released by alkaline hydrolysis. Liquid chromatography-mass spectrometry is used to quantitatively analyze the type and content of 3-hydroxy fatty acids, thereby achieving quantitative analysis of endotoxins and solving the interference problem of β-glucan. The technical principle and route of the present disclosure are shown in FIG. Figure 1 .

[0021] The inventors of the present disclosure have discovered that the carbon chain lengths of 3-hydroxy fatty acids in the endotoxin structures of different Gram-negative bacteria are different, but the carbon chain lengths and numbers of 3-hydroxy fatty acids in the endotoxins of Gram-negative bacteria of the same species are fixed.

[0022] In a first aspect, the detection method comprises:

[0023] (1) The sample to be tested is subjected to alkaline hydrolysis to obtain 3-hydroxy fatty acids, wherein the alkaline hydrolysis solution comprises KOH and methanol. It should be noted that the sample to be tested can be food, medicine, serum, plasma, plant and animal tissues, etc., and this disclosure is not limited to this.

[0024] In some embodiments, the alkaline hydrolysis solution is a 2-6% KOH methanol solution. The present disclosure has attempted to use a 2-6% NaOH aqueous solution and a 2-6% NaOH methanol solution for hydrolysis. The results confirmed that the efficiency of NaOH in hydrolyzing endotoxins was low and did not meet the quantitative accuracy requirements of the detection method. Hydrolysis was performed using a 2-6% KOH methanol solution, and the hydrolysis efficiency of 3-hydroxy fatty acids in endotoxins reached 98% as verified by high-performance liquid chromatography. It should be noted that the % described above for KOH concentration is mass %.

[0025] For example, the concentration of 2-6% KOH methanol solution can be 2%, 3%, 4%, 4.5%, 5%, or 6%.

[0026] In some embodiments, the alkaline hydrolysis treatment conditions include: a sample to alkaline solution ratio of 1:10 to 1:20, a hydrolysis time of 2 to 12 hours, and a hydrolysis temperature of 30 to 80° C. It should be noted that the sample to alkaline solution ratio is a mass ratio.

[0027] Exemplarily, the ratio of the sample to the alkaline solution can be 1:10, 1:15, or 1:20.

[0028] For example, the sample hydrolysis time can be 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours.

[0029] For example, the sample hydrolysis temperature can be 30°C, 40°C, 50°C, 60°C, 70°C, or 80°C.

[0030] In some embodiments, the alkaline hydrolysis product comprises one or more of 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, and 3-hydroxyoctadecanoic acid. It should be noted that if no 3-hydroxy fatty acid is detected, the sample can be determined to be free of endotoxins.

[0031] Exemplarily, the alkaline hydrolysis product derived from E. coli endotoxin is 3-hydroxytetradecanoic acid.

[0032] (2) The 3-hydroxy fatty acid produced by hydrolysis is subjected to a derivatization reaction to obtain a derivative product.

[0033] The inventors of this document have found that the direct use of liquid chromatography-mass spectrometry to determine the signal response of 3-hydroxy fatty acids in the hydrolyzate is low, which is very different from the detection sensitivity of existing Limulus amebocyte lysate reagents and cannot meet the requirements for the analysis of endotoxins in actual samples. The reason for the analysis is that 3-hydroxy fatty acids contain carboxyl groups with strong electronegativity, which results in 3-hydroxy fatty acids only being able to be subjected to mass spectrometry analysis in negative ion mode, and the detection sensitivity is low. In order to solve this technical bottleneck, the present invention uses isoquinoline-1-carboxylic acid hydrazine to carry out a derivatization reaction with 3-hydroxy fatty acids. Since isoquinoline-1-carboxylic acid hydrazine carries three nitrogen atoms that are easily positively charged, it is very easy to be ionized under a positive ion electrospray ion source, thereby achieving an improvement in the sensitivity of endotoxin detection. Figure 2 Comparison of mass spectrometric signal responses before and after the reaction of 3-hydroxytetradecanoic acid with isoquinoline-1-carboxylic acid hydrazine at the same concentration.

[0034] In some embodiments, the 3-hydroxy fatty acid and isoquinoline-1-carboxylic acid hydrazine derivatization reaction solution is an acetonitrile solution containing 250-1250 mmol / L EDC, 30-90 mmol / L HOAt, and 20-200 mmol / L isoquinoline-1-carboxylic acid hydrazine. Using this derivatization solution, various 3-hydroxy fatty acids in the sample can be efficiently derivatized, with a derivatization efficiency exceeding 95%.

[0035] For example, the EDC concentration in the derivatization solution may be 250 mmol / L, 500 mmol / L, 700 mmol / L, 1000 mmol / L, or 1250 mmol / L.

[0036] For example, the HOAt concentration in the derivatization solution may be 30 mmol / L, 60 mmol / L, or 90 mmol / L.

[0037] For example, the concentration of isoquinoline-1-carboxylic acid hydrazine in the derivatization solution may be 20 mmol / L, 50 mmol / L, 100 mmol / L, 150 mmol / L, or 200 mmol / L.

[0038] In some embodiments, the derivatization reaction of 3-hydroxy fatty acids with isoquinoline-1-carboxylic acid hydrazine occurs at a temperature of 4-50°C and a reaction time of 15-90 minutes. Reaction temperature is positively correlated with reaction rate; higher temperatures shorten reaction times, while lower temperatures require longer reaction times. Within the reaction time and temperature ranges described herein, using the derivatization solution described above, the derivatization efficiency of 3-hydroxy fatty acids exceeds 95%, and no byproducts are produced.

[0039] For example, the reaction temperature can be 4°C, 20°C, 37°C, or 50°C.

[0040] For example, the reaction time can be 15 minutes, 30 minutes, 60 minutes, or 90 minutes.

[0041] (3) Using liquid chromatography-mass spectrometry, the derivative products of the 3-hydroxy fatty acid are detected to obtain quantitative results of endotoxin.

[0042] In some embodiments, after the derivatization reaction is completed, the volume is fixed with acetonitrile and then liquid chromatography-mass spectrometry detection is directly performed.

[0043] Optionally, after the derivatization reaction, the sample is evaporated to dryness using a low-temperature vacuum rotary evaporator, and the dried sample is redissolved in 0.1-1 ml of 30-100% methanol or acetonitrile for liquid chromatography-mass spectrometry detection.

[0044] During actual sample testing, the alkaline hydrolysis product may also contain 2-hydroxy fatty acids, which have the same molecular weight as 3-hydroxy fatty acids and produce the same daughter ions. If the effective separation of 2-hydroxy fatty acids and 3-hydroxy fatty acids cannot be achieved, it will have a negative impact on the endotoxin quantitative results. This document publicly describes the liquid phase conditions for separating 3-hydroxy fatty acids, including a C8 chromatographic column with an inner diameter of 2.1 mm, a column length of 10 cm, a column temperature of 40°C, and a flow rate of 0.3 ml / min. Mobile phase A is water containing 0.1% formic acid, and phase B is acetonitrile containing 0.1% formic acid. Under these conditions, the separation of 2-hydroxy fatty acids and 3-hydroxy fatty acids can be achieved, eliminating interference ( Figure 3 ).

[0045] Optionally, the length of the C8 column can be 15 cm, and the column temperature can be between 35°C and 50°C.

[0046] Furthermore, the inventors of the present disclosure optimized parameters of the liquid chromatography-mass spectrometry instrument, such as collision voltage and ion pair information, to improve the sensitivity and versatility of endotoxin detection. Specific parameters are shown in Table 1.

[0047] Table 1 Some detection parameter ranges of liquid chromatography-mass spectrometry

[0048]

[0049] Optionally, the methods described in this disclosure may be used 13 C or 2 The isotope internal standard of 3-hydroxy fatty acid with H is used to realize the internal standard method quantification. Correspondingly, the detection parameters of 3-hydroxy fatty acid with isotope labeling need to be increased, and the corresponding parent ion mass-to-charge ratio increases. 13 C or 2 H marks the number of elements, and other parameters remain unchanged.

[0050] For example, 10 13When C-labeled 3-hydroxydecanoic acid is used as the internal standard, the mass-to-charge ratio of the corresponding parent ion can be set to 368.0-368.4.

[0051] The liquid chromatography-mass spectrometry method described in this disclosure directly measures the content of 3-hydroxy fatty acids. Therefore, it is necessary to determine the conversion coefficient between 3-hydroxy fatty acids and endotoxins to obtain the endotoxin content in the sample. When using Limulus amebocyte lysate (LA-lysate) to quantitatively analyze endotoxins, although the toxicity and pyrogenicity of different bacterial endotoxins vary, the Chinese Pharmacopoeia, the United States Pharmacopoeia, the European Pharmacopoeia, and the Japanese Pharmacopoeia all stipulate that Escherichia coli endotoxins are used as the reference standard. Therefore, this disclosure also uses Escherichia coli endotoxins to determine the conversion coefficient between 3-hydroxy fatty acids and endotoxins.

[0052] In some embodiments, the E. coli endotoxin reference substance of different concentrations was treated according to the endotoxin hydrolysis conditions described in the present disclosure, and the content of 3-hydroxytetradecanoic acid was determined by liquid chromatography-mass spectrometry after derivatization with isoquinoline-1-carboxylic acid hydrazine. The known endotoxin concentration was used as the horizontal axis, and the peak area of ​​3-hydroxytetradecanoic acid was measured as the vertical axis. A linear regression method between 3-hydroxytetradecanoic acid and endotoxin concentration was established ( Figure 4 ), which is used to calculate the endotoxin content in the sample to be tested.

[0053] Alternatively, according to the structure of Escherichia coli endotoxin, 1 mole of endotoxin contains 4 moles of 3-hydroxytetradecanoic acid, and the molar concentration of endotoxin in the sample can be calculated using the measured molar concentration of 3-hydroxytetradecanoic acid.

[0054] Secondly, embodiments of the present disclosure also provide a detection kit for high-precision, interference-resistant endotoxin analysis, which is applicable to the aforementioned method for detecting endotoxin content; wherein the detection kit includes the aforementioned KOH methanol solution, isoquinoline-1-carboxylic acid hydrazine, and a 3-hydroxy fatty acid standard. Using the detection kit provided by embodiments of the present disclosure, endotoxin testing can be performed directly on the sample to be tested, eliminating the need to purchase separate detection reagents, thereby saving detection time and improving detection efficiency.

[0055] The technical solution of the embodiment of the present disclosure has at least the following advantages: (1) KOH is directly used to hydrolyze the sample to be tested to produce 3-hydroxy fatty acids, which reduces the loss during the endotoxin extraction process and improves the quantitative accuracy; (2) the sensitivity of mass spectrometry detection is improved by derivatizing 3-hydroxy fatty acids with isoquinoline-1-carboxylic acid hydrazine; (3) the liquid phase and mass spectrometry acquisition parameters are optimized to improve the quantitative accuracy and precision of endotoxins in unknown samples. (4) The present disclosure measures the characteristic 3-hydroxy fatty acids in endotoxins, which are not contained in the sample matrix and are particularly not interfered with by β-glucan. In summary, the endotoxin method of the present disclosure is flexible to use, simple to operate, has strong anti-interference ability, high sensitivity, and high quantitative accuracy.

[0056] In order to make the technical solution of the present disclosure clearer and easier to understand, the method for detecting lipopolysaccharide content provided by the present disclosure is described in detail below with reference to specific examples.

[0057] Example 1 Analysis of endotoxin content in fermented feed

[0058] 1) Accurately weigh 1 mg of solid standards of 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, and 3-hydroxyoctadecanoic acid and dissolve them in the corresponding volume of methanol to obtain a 4 nmol / ml stock solution of the standards.

[0059] 2) Using the standard stock solution, prepare a standard gradient solution of 0.004 nmol / ml, 0.040 nmol / ml, 0.100 nmol / ml, 0.200 nmol / ml, and 0.400 nmol / ml for drawing a quantitative standard curve;

[0060] 3) Accurately weigh 200 mg of feed sample and add 2 mL of a 4% methanolic KOH hydrolysis solution. Mix thoroughly and hydrolyze at 50°C for 6 h. After hydrolysis, add 2 M hydrochloric acid in methanol to neutralize the solution. Centrifuge at 14,000 rpm for 10 min, remove the supernatant, and vacuum dry. Redissolve the dried sample in 500 μl of acetonitrile and centrifuge at 14,000 rpm for 10 min. Remove the supernatant and set aside.

[0061] 4) Mix 100 μl of the serially diluted standard solution and sample extract with an equal volume of derivatization reagent (500 mmol / L LEDC, 15 mmol / L HOAt, and 40 mmol / L isoquinoline-1-carboxylic acid hydrazine). Derivatize at room temperature for 1 hour. Terminate the reaction with 1% formic acid in water, and analyze the supernatant using liquid chromatography-mass spectrometry.

[0062] LC / MS conditions were as follows: Quantification of hydroxy fatty acid derivatives was performed using a Waters UPLC high-performance liquid chromatograph coupled to a Sciex 6500 triple quadrupole-linear ion trap mass spectrometer. Column: C8 1.8 µm (2.1 × 100 mm); Mobile phase A: Water with 0.1% formic acid; Mobile phase B: Acetonitrile with 0.1% formic acid; Gradient settings: as shown in Table 2.

[0063] Table 2 Elution gradient

[0064]

[0065] Triple quadrupole mass spectrometer conditions: Data acquisition was performed in MRM scan mode. Ion source: ESI; backflush: 30 psi; ion source voltage: 5500 V; heating temperature: 500°C; pyrolysis gas: Medium; ion source gas: 50 kPa; inlet voltage: 10 V; collision cell output voltage: 13 V. Ion pair information is shown in Table 3.

[0066] Table 3 Ion pair information

[0067]

[0068] 5) According to the liquid chromatography results, the linear equations and linear relationships of the standard curves of the five compounds and the determination results of different 3-hydroxy fatty acids are shown in Table 4.

[0069] Table 4 Linear equation of the standard curve and 3-hydroxy fatty acid content in the samples

[0070]

[0071] Judging from the above test results, the feed contains not only 3-hydroxytetradecanoic acid, but also a variety of other types of 3-hydroxy fatty acids. It also confirms that the endotoxin content determined by only 3-hydroxytetradecanoic acid is low and limited.

[0072] Example 2 Analysis of endotoxin content in serum

[0073] Dissolve 1 mg of E. coli endotoxin powder in 1 mL of aqueous solution to obtain a 1 mg / mL solution. Use this stock solution to prepare gradient solutions of 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 180 ng / mL. Accurately measure 100 μl of the E. coli endotoxin aqueous gradient solution and add 2 mL of a 2% methanolic KOH solution to the total volume. Mix thoroughly and incubate in a 40°C water bath for 10 hours. After hydrolysis is complete, add 2 M hydrochloric acid in methanol to neutralize the solution. Centrifuge at 14,000 rpm for 10 minutes, and remove the supernatant by vacuum drying.

[0074] 2) Accurately measure 100 μl of serum and add 900 μl of 2% KOH methanol hydrolysis solution. Mix thoroughly and incubate in a 40°C water bath for 10 hours. After hydrolysis, add 2 M hydrochloric acid in water to neutralize the solution. Centrifuge at 14,000 rpm for 10 minutes and remove the supernatant by vacuum drying.

[0075] 3) After spin-drying, 100 μl of a derivatizing agent (400 mmol / L EDC, 20 mmol / L HOAt, and 50 mmol / L isoquinoline-1-carboxylic acid hydrazine) was added to each of the spin-dried endotoxin control and serum samples for derivatization at 4°C for 90 minutes. The derivatization reaction was terminated by adding 1% aqueous formic acid. The supernatant was analyzed by liquid chromatography-mass spectrometry.

[0076] LC / MS conditions were as follows: Quantification of hydroxy fatty acid derivatives was performed using a Waters UPLC high-performance liquid chromatograph coupled to a Sciex 6500 triple quadrupole-linear ion trap mass spectrometer. Column: C8 1.8 µm (2.1 × 100 mm); Mobile phase A: Water with 0.1% formic acid; Mobile phase B: Acetonitrile with 0.1% formic acid; Gradient settings: as shown in Table 5.

[0077] Table 5 Elution gradient

[0078]

[0079] Triple quadrupole mass spectrometry conditions: Data acquisition was performed in MRM scan mode. Ion source: ESI; backflush: 30 psi; ion source voltage: 5500 V; heating temperature: 500°C; pyrolysis gas: Medium; ion source gas: 50 kPa; inlet voltage: 10 V; collision cell output voltage: 13 V. Ion pair information is shown in Table 6.

[0080] Table 6 Ion pair information

[0081]

[0082] 5) The results of the conversion coefficient determination of E. coli endotoxin and 3-hydroxytetradecanoic acid are shown in Table 7.

[0083] Table 7 Escherichia coli endotoxins and 3-hydroxy fatty acid content in samples

[0084]

[0085] According to the test results, the known concentration of Escherichia coli endotoxin was linearly related to the peak area of ​​3-hydroxytetradecanoic acid, with a correlation coefficient of 0.9951 and a linear equation of y=38.655 x + 3259.1. Using this linear equation, the endotoxin content in the serum was determined to be 131.18-733.17 ng / mL.

[0086] Comparative Example 1

[0087] 1) To 20 µl of plasma, hydrolyze the sample by adding 75 µl of 150 mM sodium chloride and 300 µl of 8 mol / L hydrochloric acid at 90°C for 4 hours. 3-Hydroxytetradecanoic acid was then extracted with 600 µl of distilled water and 5 ml of hexane. After evaporating the hexane in vacuo, the fatty acid was dissolved in 100 µl of a 40% A / 60% B mobile phase mixture (mobile phase A: 5 mM ammonium acetate, pH 5.0; mobile phase B: acetonitrile / 5 mM ammonium acetate (pH 7.3), 96.7% / 3.3% by volume).

[0088] 3-Hydroxytetradecanoic acid was quantified using an Agilent Infinity 1200 binary HPLC system equipped with a Poroshell 120 EC C18 column (100 × 4.6 mm, 2.7 µm). The column temperature was set at 30°C, and the injection volume was 10 µl. A 7-minute mobile phase gradient was established as follows: 80% phase B at a flow rate of 1 ml / min from 0 to 0.5 min; then, the proportion of phase B was linearly increased to 100% over 1 min, while the flow rate was reduced to 0.5 ml / min. These conditions were maintained for 1 min; the flow rate was then increased back to 1 ml / min over 2.5 min; and finally, the column was equilibrated with 80% phase B at a flow rate of 1 ml / min for 2.5 min. Mass spectrometry was performed on an Agilent QQQ 6460 triple quadrupole mass spectrometer equipped with a JetStream electrospray ionization source (negative ion mode, gas temperature 300°C, gas flow rate 10 L / min, nebulizer pressure 20 psi, sheath gas temperature 200°C, sheath gas flow rate 11 L / min, capillary voltage 3500 V), and nitrogen collision gas. Selected reaction monitoring (SRM) mode was used for quantitative detection of specific ions using the following parameters: 3-hydroxytetradecanoic acid, precursor ion 243.2 Da, product ion 59 Da, fragmentor voltage 93 V, and collision energy 9 eV.

[0089] Compared with Example 1, Comparative Example 1 discloses the use of 8M HCl at 90°C for 4 h to hydrolyze endotoxins to produce 3-hydroxytetradecanoic acid, which has a high hydrolysis temperature. Example 1 uses KOH to achieve the hydrolysis of 3-hydroxy fatty acids at room temperature, with a hydrolysis efficiency of 98%. Only 3-hydroxytetradecanoic acid is used for endotoxin analysis. Example 1 uses 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, and 3-hydroxyoctadecanoic acid, a total of six unique 3-hydroxy fatty acids derived from Gram-negative bacterial endotoxins, which can measure a more comprehensive range of endotoxins.

[0090] In Comparative Example 1, the hydrolyzate was extracted and purified and then directly used for LC-MS analysis. The detection limit of 3-hydroxytetradecanoic acid was 12 ng / ml, which was much lower than the detection sensitivity of 0.3 EU / ml (equivalent to 6 pg / ml) of the Limulus amebocyte lysate. In Example 2, the 3-hydroxy fatty acids produced by hydrolysis were derivatized with isoquinoline-1-carboxylic acid hydrazine, which greatly improved the ionization efficiency and sensitivity of 3-hydroxy fatty acid compounds. For example, the sensitivity of 3-hydroxytetradecanoic acid after derivatization was increased by 187.9 times ( Figure 2 ), the detection sensitivity reached 10 pg / ml, which is comparable to that of the Limulus amebocyte lysate.

Claims

1. A high-precision and interference-resistant endotoxin analysis method, characterized in that: include: (1) Using a methanol solution containing 2% to 6% KOH to directly treat the endotoxin in the sample to be tested, hydrolyzing it to produce 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid and 3-hydroxyoctadecanoic acid, the ratio of sample to alkaline solution is 1:10 to 1:20, the hydrolysis time is 2 to 12 hours, and the hydrolysis temperature is 37 to 80°C; (2) chemically derivatizing the hydrolyzate with isoquinoline-1-carboxylic acid hydrazine, wherein the derivatization reaction solution is an acetonitrile solution containing 250-1250 mmol / L EDC, 30-90 mmol / L HOAt, and 20-200 mmol / L isoquinoline-1-carboxylic acid hydrazine, the reaction temperature is 4-50° C., and the reaction time is 15-90 minutes; (3) Liquid chromatography-mass spectrometry was used to determine the type and content of the derivatized 3-hydroxy fatty acids to achieve accurate quantitative analysis of endotoxins.

2. The analysis method according to claim 1, characterized in that A liquid chromatography-triple quadrupole mass spectrometer was used for analysis in multiple ion scanning mode under positive ion mode. The detection range of the parent ion of 3-hydroxydecanoic acid was set to 358.0-358.4; the detection range of the parent ion of 3-hydroxydodecanoic acid was set to 386.1-386.5; the detection range of the parent ion of 3-hydroxytetradecanoic acid was set to 414.1-414.5; the detection range of the parent ion of 3-hydroxyhexadecanoic acid was set to 442.1-442.5; and the detection range of the parent ion of 3-hydroxyoctadecanoic acid was set to 470.2-470.

6. The product ions generated by the collision of the above five parent ions ranged from 170.0-170.2 and 188.0-188.2, respectively. The collision energy for generating the product ions of 170.0-170.2 was 30-50 eV, and the collision energy for generating the product ions of 188.0-188.2 was 20-40 eV.

3. A high-precision and interference-resistant endotoxin detection kit, characterized in that: The detection kit is applied to the endotoxin analysis method according to claim 1; wherein the detection kit comprises the KOH methanol solution for hydrolysis, EDC, HOAt and isoquinoline-1-carboxylic acid hydrazine solution for derivatization reaction, and 3-hydroxy fatty acid standard.

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