Preparation method and application of ionic liquid MALDI matrix

Abstract

The invention discloses a preparation method and application of a novel ionic liquid MALDI matrix, wherein the ionic liquid matrix comprises an α-cyano-4-hydroxycinnamic acid-p-phenylenediamine complex. The ionic liquid matrix is ​​suitable for the detection of small molecules, has high detection sensitivity, low background noise, and has important application value in the fields of biology, medicine, food, etc.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and in particular to a preparation method and application of a novel ionic liquid MALDI matrix, and more particularly to an ionic liquid matrix, a kit, a method for detecting antibiotics in an analyte, and a kit for detecting antibiotics in an analyte. Background Art

[0002] Matrix-assisted laser desorption ionization (MALDI) is a soft ionization mass spectrometry technology that appeared in the 1980s. It can quickly analyze high molecular weight substances. There is no fragmentation or only a small amount of fragmentation during mass spectrometry detection. It has high detection precision and sensitivity, and has shown broad application prospects in many fields such as biology, food, and medicine. The mass spectrometry imaging technology based on MALDI can simultaneously meet the needs of spatial imaging and the detection of unknown substances, and has great application potential. Its working principle is: a laser of a certain intensity is used to irradiate the co-crystallized film formed by the sample and the matrix. The matrix absorbs energy from the laser, resulting in energy accumulation and rapid heat generation, thereby sublimating the matrix crystals, causing the matrix and analyte to expand and enter the gas phase. The detection is carried out according to the different mass-to-charge ratios (m / z) and the molecular weight of the sample is obtained. The matrix is ​​the key to detection using MALDI, and the research and development and application of the matrix are the development focus of this technology for wider application. At present, the common MALDI matrices are mainly organic small molecule matrices, such as α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA) and sinapinic acid (SA). In addition, inorganic matrices such as graphene and carbon nanotubes are also commonly used in MALDI due to their superior specific surface area and clean mass spectrometry background. However, organic small molecule matrices have many fragments in the low molecular weight region and poor uniformity when used. Inorganic matrices are expensive and complex to synthesize, which restricts the further development of these two types of matrices. Therefore, the concept of ionic liquid matrix is ​​introduced to increase the ionization efficiency while taking advantage of the advantages of liquid to improve uniformity and reduce the occurrence of "hot spot effect". Although the existing ionic liquids have shown outstanding advantages in the detection of large molecules, their application in small molecules is relatively rare.

[0003] Therefore, ionic liquid matrices for small molecules need to be studied. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to prepare an ionic liquid matrix suitable for small molecule detection.

[0005] It should be noted that the present invention is completed based on the following work of the inventors:

[0006] The inventors found that if the ionic liquid is synthesized based on the acid-base combination reaction of organic acid and organic base, the synthesis operation is economical and simple, there are no by-products, and the product is easy to purify. Furthermore, the inventors first screened organic acids suitable for synthesizing ionic liquids, including α-cyano-4-hydroxycinnamic acid (CHCA), DHB, 9-AA and 2,4,6-trihydroxyacetophenone (THAP), and selected CHCA with high sensitivity and relatively small background noise as the organic acid in the ionic liquid. Then, 16 kinds of organic bases that may be effective were synthesized and screened for ionic liquids. The detection sensitivity and uniformity of these 16 matrices for quinolones and sulfonamides were compared, and it was found that p-phenylenediamine was the most suitable organic base for making ionic liquids. Therefore, CHCA-based ionic liquids were synthesized for MALDI (matrix-assisted laser desorption ionization) analysis. The ionic liquid prepared by the embodiment of the present invention can not only be used as a matrix in MALDI for the purification and enrichment of small molecule veterinary drugs in samples, especially quinolones and sulfonamides, but also has important application value in the fields of biology, medicine, food, etc.

[0007] Therefore, according to the first aspect of the present invention, the present invention provides an ionic liquid matrix. According to an embodiment of the present invention, the ionic liquid matrix includes α-cyano-4-hydroxycinnamic acid-p-phenylenediamine complex (CHCA-p-phenylenediamine complex).

[0008] The inventors surprisingly found that the ionic liquid matrix of the embodiment of the present invention is suitable for the detection of small molecules, has high ionization efficiency, good uniformity, high detection sensitivity, and low background noise, and has important application value in the fields of biology, medicine, food, etc.

[0009] According to an embodiment of the present invention, in the CHCA-p-phenylenediamine composite, the molar ratio of CHCA to p-phenylenediamine is 1:0.5-3.

[0010] Further, according to the second aspect of the present invention, the present invention provides the use of an ionic liquid matrix, which is used as a reaction matrix in matrix-assisted laser desorption ionization. Thus, using the ionic liquid matrix as a detection matrix, the detection sensitivity is high and the background noise is small.

[0011] According to the third aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the aforementioned ionic liquid matrix. Thus, the kit is suitable for the detection of small molecules, has high detection sensitivity, low background noise, and has important application value in the fields of biology, medicine, food, etc. It should be noted that the kit has all the technical features and technical effects of the aforementioned ionic liquid matrix, which will not be repeated here.

[0012] According to the fourth aspect of the present invention, the present invention provides a method for detecting antibiotics in a test object. According to an embodiment of the present invention, the method comprises: extracting the test object to obtain an extract; mixing the extract with the aforementioned ionic liquid matrix to obtain a mixture; performing mass spectrometry imaging detection on the mixture to perform qualitative and / or quantitative detection of the antibiotics. Therefore, the method is suitable for the detection of antibiotics, with high detection sensitivity and low background noise. It should be noted that the method has all the technical features and technical effects of the aforementioned ionic liquid matrix, which will not be repeated here.

[0013] According to an embodiment of the present invention, the mixture is placed on a stainless steel plate for mass spectrometry imaging detection.

[0014] According to an embodiment of the present invention, the mass spectrometry imaging detection is MALDI-MSI mass spectrometry imaging detection.

[0015] According to an embodiment of the present invention, the detection conditions of the mass spectrometry imaging detection are: mass spectrometry detector: IT-TOF MS mass spectrometry detector; detection mode: positive ion mode.

[0016] According to an embodiment of the present invention, the imaging range of the mass spectrometry imaging detection is 50×50 microns.

[0017] According to an embodiment of the present invention, the imaging conditions of the mass spectrometry imaging detection are: sample voltage: 3.5 kilovolts; detector voltage: 1.7 kilovolts; laser intensity is 20.

[0018] According to an embodiment of the present invention, the antibiotics are quinolone antibiotics and sulfonamide antibiotics.

[0019] According to an embodiment of the present invention, in the CHCA-p-phenylenediamine complex, the molar ratio of CHCA to p-phenylenediamine is 1:0.5-3.

[0020] According to a fifth aspect of the present invention, the present invention provides a kit for detecting antibiotics in an object to be tested. According to an embodiment of the present invention, the kit includes the reagents, standard substances, auxiliary materials or a combination of at least one of the above-mentioned methods for detecting antibiotics in an object to be tested. Thus, the kit is suitable for the detection of antibiotics, with high sensitivity and low background noise. It should be noted that the method has all the technical features and technical effects of the above-mentioned kit for detecting antibiotics in an object to be tested, which will not be repeated here one by one.

[0021] According to an embodiment of the present invention, the antibiotics are quinolone antibiotics and sulfonamide antibiotics.

[0022] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0024] Figure 1 The results show that the CHCA, CHCA / Na and CHCA-p-phenylenediamine novel ionic liquids according to one embodiment of the present invention have a range of 1750-1200 cm -1 Schematic diagram of infrared spectrum of the range, where A is CHCA, B is CHCA / Na, and C is CHCA-p-phenylenediamine;

[0025] Figure 2 A molar ratio optimization diagram of the CHCA-p-phenylenediamine novel ionic liquid based on the detection of enrofloxacin according to one embodiment of the present invention and a schematic diagram of the detection effect using a CHCA matrix and a CHCA-p-phenylenediamine novel ionic liquid matrix are shown, wherein A is CHCA (left) and B is CHCA-p-phenylenediamine;

[0026] Figure 3 A schematic diagram showing the sensitivity comparison between the new CHCA-p-phenylenediamine ionic liquid and CHCA in detecting five quinolones and two sulfonamides according to one embodiment of the present invention;

[0027] Figure 4 A schematic diagram showing the homogeneity comparison between the new CHCA-p-phenylenediamine ionic liquid and CHCA in detecting four quinolones and two sulfonamides according to one embodiment of the present invention is shown;

[0028] Figure 5 A schematic diagram of a linear curve of the detection of enrofloxacin by the novel CHCA-p-phenylenediamine ionic liquid according to an embodiment of the present invention is shown, wherein A is a stainless steel target plate and B is fish slices. DETAILED DESCRIPTION

[0029] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0031] (I) Ionic liquid matrix and preparation method thereof

[0032] According to a first aspect of the present invention, the present invention provides an ionic liquid matrix. According to an embodiment of the present invention, the ionic liquid matrix includes a CHCA-p-phenylenediamine complex.

[0033] The inventors surprisingly found that the ionic liquid matrix of the embodiment of the present invention is suitable for the detection of small molecules, has high ionization efficiency, good uniformity, high detection sensitivity, and low background noise, and has important application value in the fields of biology, medicine, food, etc.

[0034] According to an embodiment of the present invention, in the CHCA-p-phenylenediamine complex, the molar ratio of CHCA to p-phenylenediamine is 1: 0.5-3. According to some preferred embodiments of the present invention, in the CHCA-p-phenylenediamine complex, the molar ratio of CHCA to p-phenylenediamine is 1: 1. Thus, the response intensity is higher when the molar ratio is 1: 1, and the uniformity is good in the imaging mode.

[0035] In order to facilitate the understanding of the aforementioned ionic liquid matrix, a method for preparing an ionic liquid matrix is ​​provided herein. According to an embodiment of the present invention, the method comprises: CHCA is dissolved in methanol by ultrasonic dissolution, and then p-phenylenediamine is added to the CHCA methanol solution, and vortexed for 10 minutes to obtain the ionic liquid matrix. This method does not require complex instruments and synthesis processes, is economical and simple to operate, and can not only realize MALDI analysis of antibiotics, especially quinolones and sulfonamides, but also provide new detection ideas for the fields of biology, medicine, food, etc.

[0036] According to an embodiment of the present invention, the vortex is a Vortex-Genie2 vortex mixer with a rotation frequency of 2000 rpm and a temperature of 25°C.

[0037] According to an embodiment of the present invention, the molar ratio of CHCA to p-phenylenediamine is 1:0.5-3, preferably, the molar ratio is 1:1. Thus, the response intensity is higher when the molar ratio is 0.5:1, and the uniformity is good in the imaging mode.

[0038] (II) Application of ionic liquid matrix

[0039] Further, according to the second aspect of the present invention, the present invention provides the use of an ionic liquid matrix, which is used as a reaction matrix in matrix-assisted laser desorption ionization. Thus, using the ionic liquid matrix as a detection matrix, the detection sensitivity is high and the background noise is small.

[0040] According to the third aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the aforementioned ionic liquid matrix. Thus, the kit is suitable for the detection of small molecules, has high detection sensitivity, low background noise, and has important application value in the fields of biology, medicine, food, etc. It should be noted that the kit has all the technical features and technical effects of the aforementioned ionic liquid matrix, which will not be repeated here.

[0041] According to the fourth aspect of the present invention, the present invention provides a method for detecting antibiotics in a test object. According to an embodiment of the present invention, the method comprises: extracting the test object to obtain an extract; mixing the extract with the aforementioned ionic liquid matrix to obtain a mixture; performing mass spectrometry imaging detection on the mixture to perform qualitative and / or quantitative detection of the antibiotics. Therefore, the method is suitable for the detection of antibiotics, with high detection sensitivity and low background noise. It should be noted that the method has all the technical features and technical effects of the aforementioned ionic liquid matrix, which will not be repeated here.

[0042] According to an embodiment of the present invention, the mixture is placed on a stainless steel plate for mass spectrometry imaging detection, thereby using the stainless steel plate as a support for the ionic liquid matrix to effectively avoid interference from external matrices.

[0043] According to an embodiment of the present invention, the mass spectrometry imaging detection is MALDI-MSI mass spectrometry imaging detection. Therefore, MALDI-MSI mass spectrometry imaging can simultaneously perform imaging and mass spectrometry detection on a specific area, effectively solving the problem of spatial distribution detection of small molecule veterinary drugs quinolones and sulfonamides.

[0044] According to an embodiment of the present invention, the detection conditions of the mass spectrometry imaging detection are: mass spectrometry detector: IT-TOF MS mass spectrometry detector; detection mode: positive ion mode. The inventors found that quinolones and sulfonamides are easy to obtain protons, and in the above positive ion mode, the response intensity is high and the ionization background is clean.

[0045] According to an embodiment of the present invention, the imaging range of the mass spectrometry imaging detection is 50×50 microns, which is just enough to cover the area of ​​each spotting circle in the stainless steel plate, saving detection time and reagents while ensuring that the spotting droplets are fully detected.

[0046] According to an embodiment of the present invention, the imaging conditions of the mass spectrometry imaging detection are: sample voltage: 3.5 kV; detector voltage: 1.7 kV; laser intensity is 20. Therefore, under this detection condition, the detected analyte appears in the form of molecular ions, with a clean background and low noise.

[0047] According to the embodiment of the present invention, the antibiotics are quinolone antibiotics and sulfonamide antibiotics. These two types of antibiotics are the first choice in veterinary drugs, have broad-spectrum therapeutic effects, and are easy to remain in meat. The detection of the above two types of antibiotics is conducive to ensuring the safety of food.

[0048] According to an embodiment of the present invention, in the CHCA-p-phenylenediamine composite, the molar ratio of CHCA to p-phenylenediamine is 1:0.5-3.

[0049] According to a fifth aspect of the present invention, the present invention provides a kit for detecting antibiotics in an object to be tested. According to an embodiment of the present invention, the kit includes the reagents, standard substances, auxiliary materials or a combination of at least one of the above-mentioned methods for detecting antibiotics in an object to be tested. Thus, the kit is suitable for the detection of antibiotics, with high sensitivity and low background noise. It should be noted that the method has all the technical features and technical effects of the above-mentioned kit for detecting antibiotics in an object to be tested, which will not be repeated here one by one.

[0050] According to an embodiment of the present invention, the antibiotics are quinolone antibiotics and sulfonamide antibiotics.

[0051] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0052] Below in conjunction with embodiment, the scheme of the present invention will be explained.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unrepresented specific techniques or conditions in the embodiment, according to the technology or conditions described in the document in this area or according to the product specification sheet.Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained commercially, for example, can be purchased from Sigma company.

[0053] Instruments and Materials

[0054] Shimadzu Scope / iMScope TRIO instrument was used for testing (Shimadzu Corporation, Japan);

[0055] KQ5200V ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0056] Milli-Q Reference ultrapure water machine (Merck Millipore, Germany);

[0057] LEICA CM 1950 (LEICA, Germany);

[0058] Vortex-Genie2 vortex mixer (Scientific Industries, USA);

[0059] SP12Z023 infrared detection instrument (Thermo Scientific)

[0060] Methanol HPLC grade (FISHER)

[0061] Enrofloxacin, lomefloxacin, pefloxacin, gatifloxacin, levofloxacin (Sigma-aldrich); CHCA matrix substance for MALDI-MS, ≥99.0% (HPLC) (Sigma-aldrich), p-phenylenediamine (chemically pure), all purchased from Sinopharm Group);

[0062] Sulfadimethoxazole, sulfamethoxazole (Sigma-aldrich) and sodium hydroxide solution were purchased from Sigma-aldrich.

[0063] Example 1

[0064] According to the method of the embodiment of the present invention, a new ionic liquid was synthesized using CHCA and p-phenylenediamine, and the feasibility and ionizability of the ionic liquid as a MALDI matrix were tested, as follows:

[0065] 1. Preparation of new ionic liquids

[0066] (1) Select CHCA with the best detection effect from CHCA, DHB, DAN and THAP;

[0067] (2) Weigh 10 mg of CHCA solid matrix, add it to 1 ml of methanol, and dissolve it by ultrasonication to obtain a CHCA methanol solution;

[0068] (3) Weigh an equimolar amount of p-phenylenediamine and add it to the CHCA methanol solution;

[0069] (4) Vortex for 10 minutes to obtain ionic liquid.

[0070] 2. Ionizability detection of ionic liquids

[0071] Infrared detection of ionic liquids shows the following results: Figure 1As shown, the infrared spectrum of CHCA-p-phenylenediamine ionic liquid is in the range of 1750-1200 cm -1 A broad double peak appears at the ions, which is speculated to be caused by hydrogen bonding. The disappearance of the carboxyl peak (1669 cm-1) and the appearance of the carboxylate peak (1580 cm-1) indicate that CHCA reacted with p-phenylenediamine, but its peak value is still higher than CHCA / Na, indicating that it still has ionization ability, which is consistent with the characteristics of ionic liquids.

[0072] Example 2

[0073] Enrofloxacin was detected using the CHCA-p-phenylenediamine ionic liquid prepared in Example 1, as follows:

[0074] 1. Analysis of Enrofloxacin Standard Products

[0075] (1) Dissolve 100 mg of enrofloxacin standard in methanol to prepare a 100 μg / mL enrofloxacin methanol solution.

[0076] (2) Take 20 μL of 100 μg / mL enrofloxacin methanol solution, add 20 μL of CHCA-p-phenylenediamine ionic liquid, and vortex for 30 seconds to mix.

[0077] (3) Take 1 μl of the mixed solution and spot it on a stainless steel target plate, and place it into a mass spectrometry imaging instrument.

[0078] The results are as follows Figure 2 As shown, this matrix exhibits higher sensitivity and uniformity in detecting enrofloxacin, as well as better point-to-point repeatability, and is a MALDI matrix worthy of recommendation.

[0079] 2. Explore the quantitative detection capability of enrofloxacin:

[0080] (1) Prepare 20 μL of methanol solution of enrofloxacin with different concentrations (5-500 μg / mL), mix with 20 μL of CHCA-p-phenylenediamine ionic liquid, vortex for 30 seconds, and then take 1 μL of it and spot it on a stainless steel target plate for detection.

[0081] (2) Prepare 20 μL of methanol solution of enrofloxacin at different concentrations (10-1000 μg / mL), mix with 20 μL of CHCA-p-phenylenediamine ionic liquid, vortex for 30 seconds, and then take 0.5 μL of it and spot it on grass carp slices for detection. Figure 5 As shown, the linear coefficients of the matrix for detecting enrofloxacin were all greater than 0.99, indicating that the ionic liquid matrix was feasible for the quantitative detection of enrofloxacin.

[0082] Example 3

[0083] The CHCA-p-phenylenediamine ionic liquid prepared in Example 1 was used to evaluate the universality of the detection of 5 quinolones and 2 sulfonamide veterinary drugs, as follows:

[0084] 100 μg / mL methanol solutions of enrofloxacin, levofloxacin, gatifloxacin, pefloxacin, lomefloxacin, sulfadimethazole and sulfamethoxazole were prepared respectively, 20 μL of the above solutions were taken, mixed with 20 μL of CHCA-p-phenylenediamine ionic liquid and CHCA methanol solution respectively, and vortexed for 30 seconds, and then 1 μL of them was spotted on a stainless steel target plate for detection and comparison of the results.

[0085] The experimental results are as follows Figure 3 and Figure 4 As shown in the figure, the detection of enrofloxacin, levofloxacin, gatifloxacin, pefloxacin, lomefloxacin, sulfadimethoxazole and sulfamethoxazole methanol using CHCA-p-phenylenediamine ionic liquid matrix showed higher sensitivity and uniformity than that using CHCA matrix.

[0086] In summary, the ionic liquid matrix of the embodiment of the present invention is suitable for the detection of small molecules, has high detection sensitivity and low background noise, and has important application value in the fields of biology, medicine, food, etc.

[0087] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0088] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for detecting antibiotics in an analyte, characterized in that: include: Extracting the analyte to obtain an extract; mixing the extract with an ionic liquid matrix to obtain a mixture, wherein the ionic liquid matrix comprises an α-cyano-4-hydroxycinnamic acid-p-phenylenediamine complex; and The mixture is subjected to MALDI-MSI mass spectrometry imaging detection to perform qualitative and / or quantitative detection of the antibiotics. The mixture is placed on a stainless steel plate for mass spectrometry imaging detection. Wherein, the detection conditions of the mass spectrometry imaging detection are: Mass spectrometer detector: IT-TOF MS mass spectrometer detector; Detection mode: positive ion mode; Wherein, the imaging range of the mass spectrometry imaging detection is 50×50 microns; Wherein, the imaging conditions of the mass spectrometry imaging detection are: Sample voltage: 3.5 kV; Detector voltage: 1.7 kV; The laser intensity is 20, Wherein, the antibiotics are quinolone antibiotics and sulfonamide antibiotics, Wherein, in the α-cyano-4-hydroxycinnamic acid-p-phenylenediamine complex, the molar ratio of α-cyano-4-hydroxycinnamic acid to p-phenylenediamine is 1:0.5-3.

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