Detection method of drug metabolism molecule ceftiofur sodium

Abstract

The invention discloses a method for detecting drug metabolism molecule ceftiofur sodium, and belongs to the technical field of drug detection. Comprising the following steps: pre-treating a sample, spotting the sample on an MALDI target plate, co-crystallizing the sample and a matrix, and detecting the ceftiofur sodium by adopting matrix-assisted laser desorption / ionization mass spectrometry and imaging analysis, thereby obtaining the ceftiofur sodium, the matrix comprises [alpha]-cyano-4-hydroxycinnamic acid. According to the method, the matrix-assisted laser desorption / ionization mass spectrometry and imaging analysis are adopted for detecting the ceftiofur sodium in the sample for the first time, and the result shows that the alpha-cyano-4-hydroxycinnamic acid shows low background interference, high sensitivity and relatively good reproducibility when being used as the matrix in a positive ion mode. Meanwhile, the established method is further applied to detection of actual samples, and the result is satisfactory.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting drug metabolite molecule ceftiofur sodium. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Ceftiofur sodium is a third-generation cephalosporin antibiotic with a broad spectrum of antibacterial activity, and has strong antibacterial effects on both Gram-positive and Gram-negative bacteria. It mainly exerts its bactericidal effect by blocking the synthesis of bacterial cell walls.

[0004] The main detection methods for ceftiofur sodium are high performance liquid chromatography (HPLC), etc. HPLC method has obvious advantages in the detection of ceftiofur sodium, especially in terms of accuracy, sensitivity and cost-effectiveness, but it also has some limitations, such as sensitivity to the environment and the requirement for sample pretreatment. The sample pretreatment process is complicated and time-consuming.

[0005] Matrix-assisted laser desorption / ionization mass spectrometry and imaging analysis (MALDI-MS&MSI) is a soft ionization technique that mixes the analyte with the matrix, which is usually a small molecule aromatic compound that can absorb laser energy. The matrix and analyte are mixed and coated on a metal target plate. After the solvent evaporates, a co-crystal is formed. The energy of the laser is absorbed by the matrix, causing the sample molecules to desorb and ionize from the surface of the target plate. The ionized sample molecules are accelerated and enter the flight tube, separated according to the mass-to-charge ratio (m / z) in the field-free drift region, and finally reach the detector. The detector records the flight time of the ion to determine the m / z value of the ion; MALDI-MSI (mass spectrometry imaging) is an extension of MALDI-MS that allows two-dimensional imaging of the distribution of molecules in tissue samples. The image presentation of molecular distribution is achieved by rastering the tissue sample with a highly collimated laser and collecting individual mass spectra in a serial manner. This technique can be used to analyze the distribution of drugs and their metabolites in tissues.

[0006] The advantages of MALDI-MS and MALDI-MSI include high sensitivity, high throughput, simple sample preparation, non-destructive analysis of samples, and the ability to analyze macroscopic biological samples. These technologies are widely used in the identification of biomarkers, disease mechanism research, and drug distribution analysis. However, there is no report on the detection of ceftiofur sodium using MALDI-MS&MSI technology. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a method for detecting the drug metabolite molecule ceftiofur sodium. The detection method provided by the present invention can quickly and accurately analyze and detect the content of ceftiofur sodium in a sample, providing strong support for drug quality control and clinical application.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] The first aspect of the present invention provides a method for detecting a drug metabolite molecule, ceftiofur sodium, comprising:

[0010] After the sample is pre-treated, it is spotted on a MALDI target plate, and after the sample and the matrix are co-crystallized, matrix-assisted laser desorption / ionization mass spectrometry and imaging analysis are used to detect ceftiofur sodium, and the obtained product is obtained;

[0011] The matrix comprises α-cyano-4-hydroxycinnamic acid.

[0012] In some embodiments of the present invention, the ceftiofur sodium is from wastewater, serum, urine or biological tissue.

[0013] In some embodiments of the present invention, the sample is wastewater, and the pre-treatment is filtration.

[0014] Preferably, the pretreatment comprises: filtering the wastewater and storing it at 0-5° C., spotting the matrix and the wastewater on a MALDI target plate, and analyzing the wastewater after co-crystallization with the matrix.

[0015] Preferably, the filter membrane is 0.2-0.3 μm; and the matrix is ​​α-cyano-4-hydroxycinnamic acid.

[0016] In some embodiments of the present invention, the sample contains serum or urine, and the pre-treatment is to remove proteins.

[0017] Preferably, the pretreatment comprises: precipitating high-abundance proteins in the sample, vortexing, collecting the supernatant after precipitation, spotting the matrix and the sample on a MALDI target plate, and analyzing the sample after co-crystallization with the matrix.

[0018] Preferably, acetonitrile is added to the sample to precipitate high-abundance proteins; and the matrix is ​​α-cyano-4-hydroxycinnamic acid.

[0019] In some embodiments of the present invention, when the sample is a biological tissue, the pretreatment is to freeze and fix the biological tissue and then slice it, spray the matrix on the surface of the biological tissue, dry it, and then analyze it.

[0020] Preferably, the solvent of the matrix is ​​50% acetonitrile, the solute is α-cyano-4-hydroxycinnamic acid, and the concentration is 8-12 mg / mL, preferably 10 mg / mL.

[0021] In some embodiments of the present invention, the MALDI-MS test uses a RapifleXTM MALDI instrument with a laser wavelength of 355 nm and a positive ion mode.

[0022] In some embodiments of the present invention, mass spectrometry data is acquired by capturing 1000 lasers at a laser frequency of 500 Hz, with a scan range of 300-800 m / z.

[0023] In some embodiments of the present invention, ion source voltage 1 and ion source voltage 2 operate at 20 and 19.55 kV, respectively;

[0024] LasAtten value is 15%;

[0025] Lens and PIE are set to 11.383 and 2.632 respectively;

[0026] Reflector voltage 1, reflector voltage 2 and reflector voltage 3 are at 22.5, 1.55 and 9.25 kV respectively.

[0027] In some embodiments of the present invention, the delayed extraction time is 100 ns;

[0028] Beam scanning range: 30×30μm 2 , result field: 10 4 ×10 4 μm 2 .

[0029] In some embodiments of the present invention, for kidney tissue samples, the mode is M5, and the resulting area size is 200×200 μm 2 ;

[0030] Laser intensity: 15%.

[0031] The beneficial effects of the present invention are:

[0032] The present invention uses matrix-assisted laser desorption / ionization mass spectrometry and imaging analysis for the first time to detect ceftiofur sodium in samples. The results show that the use of positive ion mode and α-cyano-4-hydroxycinnamic acid as a matrix shows low background interference, high sensitivity and good reproducibility. At the same time, the established method was further applied to the detection of actual samples, and the results were satisfactory. The detection method provided by the present invention is simple, practical and easy to promote. MALDI-MS&MSI technology has shown its unique advantages in the detection of ceftiofur sodium, especially in rapid and accurate analysis and drug resistance detection. With the development of technology, this detection method will continue to provide strong support for drug quality control and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 The mass spectra of 100 μg / L ceftiofur sodium in Example 1 of the present invention in positive ion (a) and negative ion (b) modes;

[0035] Figure 2 The mass spectra of ceftiofur sodium detected by laser desorption ionization assisted by different matrices including CHCA (a), DHB (b), SA (c), NTA (d), HNTP (e) and NPG (f) in Example 1 of the present invention;

[0036] Figure 3 In Example 1 of the present invention, MALDI-MS mass spectrometry analysis of 100 μg / L ceftiofur sodium was performed using a CHCA matrix containing different concentrations of NaCl in the positive ion mode; wherein, from 1 to 6, the concentration of NaCl in the CHCA matrix is ​​0, 10, 25, 100, 250 and 500 mM, respectively;

[0037] Figure 4 The MALDI-MS mass spectra (a) and the working curve (b) of ceftiofur sodium at concentrations of 0.5, 5, 25, 50, and 75 μg / L in Example 1 of the present invention are respectively shown;

[0038] Figure 5 The chromatogram of the determination of ceftiofur sodium by the HPLC-MS / MS method in Example 1 of the present invention;

[0039] Figure 6 The mass spectrometry images are obtained by applying different concentrations of standard solutions of ceftiofur sodium (5, 10, 25, 50, 100 μg / L) to the surface of rat kidney tissue sections in Example 1 of the present invention;

[0040] Figure 7 This is a MADLI-MSI image of a rat kidney tissue section after exposure to ceftiofur sodium in Example 1 of the present invention. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0042] Example 1

[0043] 1. Experimental methods

[0044] (1) Experimental reagents

[0045] The required experimental drugs and materials are shown in Table 1 below:

[0046] Table 1 List of experimental drugs

[0047]

[0048] (2) Experimental instruments

[0049] The required experimental equipment is shown in Table 2 below:

[0050] Table 2 List of experimental equipment

[0051]

[0052]

[0053] (3) MALDI instrument experimental conditions and HPLC-MS / MS conditions

[0054] MALDI instrument experimental conditions:

[0055] The MALDI analysis experiment was analyzed using Bruker's RapifleXTM MALDI model, with a laser wavelength of 355nm and positive ion mode. At a laser frequency of 500Hz, mass spectrometry data was acquired by capturing 1000 lasers, and the original mass spectra in the range of m / z300-800 were collected. The sample spatial resolution of mass spectrometry imaging was set to 100μm. Ion source voltage 1 and ion source voltage 2 operated at 20 and 19.55kV, respectively. The LasAtten value was 15%. Lens and PIE were set to 11.383 and 2.632, respectively. Reflector voltage 1, reflector voltage 2, and reflector voltage 3 operated at 22.5, 1.55, and 9.25kV, respectively. The delayed extraction time was 100ns. Beam scanning range: 30×30μm 2 , result field: 10 4 ×10 4 For kidney tissue samples, the mode is M5, and the resulting area size is 200×200 μm 2 . Laser intensity: 15%.

[0056] The acquired MALDI mass spectrometry data were processed and analyzed using Flex analysis software (4.0). The mass spectrometry imaging data were analyzed using SCiLS2018b software, and the mass spectrometry imaging results were processed by normalizing the total ion counts.

[0057] HPLC-MS / MS instrument experimental conditions:

[0058] HPLC-MS / MS detection: Thermo U-3000 (Dionex, CA, USA) HPLC system, Sciex QTrap 5500 mass spectrometry system produced by AB Sciex, USA.

[0059] Electrospray ionization source was used in positive ion multiple reaction monitoring mode. The parent ion was m / z 523.9 and the product ion was 241.2 m / z. The chromatographic column was Thermofield C18 (2.1 μm, 100mm×2.1mm) to separate the target analytes. The flow rate was 0.35mL / min and the injection volume was 5.0μL. Mobile phase A: 1% formic acid in water, mobile phase B: methanol. The initial mobile phase condition was 20% B, which was maintained for 4 minutes, and then the methanol content was increased to 65% and maintained for 4 minutes. Subsequently, the mobile phase B component was reduced to the initial condition (20% B) for 2 minutes.

[0060] (4) Sample pretreatment

[0061] Wastewater sample: Wastewater used to clean the sodium-aluminum barrel of ceftiofur was collected, filtered through a 0.22 μm filter membrane, and stored in a 4°C refrigerator. 1 μL of the matrix solution was accurately transferred with a pipette and spotted on a MALDI target plate (Ground 384). Then, 1 μL of the wastewater sample was accurately transferred with a pipette and spotted on a MALDI target plate. After the sample and the matrix were co-crystallized, they were placed in a MALDI mass spectrometer for analysis.

[0062] Serum sample pretreatment: Add 1 mL of acetonitrile to 1 mL of human serum sample for high-abundance protein precipitation, vortex for 1 min, collect the supernatant after precipitation, and set aside for later use. Use a pipette to accurately transfer 1 μL of matrix solution and spot it on the MALDI target plate (Ground 384), then use a pipette to accurately transfer 1 μL of serum supernatant and spot it on the MALDI target plate. After the sample and matrix are co-crystallized, place it in the MALDI mass spectrometer for analysis.

[0063] Urine sample pretreatment: Add 1 mL of acetonitrile to 1 mL of urine sample to precipitate high-abundance proteins, vortex for 1 min, collect the supernatant after precipitation, and set aside for later use. Use a pipette to accurately transfer 1 μL of matrix solution and spot it on the MALDI target plate (Ground 384), then use a pipette to accurately transfer 1 μL of serum supernatant and spot it on the MALDI target plate. After the sample and matrix are co-crystallized, place it in the MALDI mass spectrometer for analysis.

[0064] Ceftiofur sodium exposure experiment in rats: Rats were injected with 50 mg / kg of ceftiofur sodium. Animal experiments and treatments were in accordance with the regulations of the Animal Care and Use Committee of Shandong Analysis and Testing Center.

[0065] Biological tissue sample imaging method: Rat kidney tissue was frozen in a -80℃ refrigerator. Before slicing, it was transferred to the sample holder in the cryo-microtome, and Tissue-Tek OCT composite solution (a water-soluble mixture of polyethylene glycol and polyvinyl alcohol, the ratio of the two was 1:1) was poured on the biological tissue for fixation. Thermo Cryostar NX50 cryostat was used to prepare 10μm thick biological tissue sections at -30℃, and the sections were placed on ITO conductive glass slides. The matrix spraying was carried out using HTXtm-sprayer (Carrboro, USA), and the matrix was sprayed on the surface of the biological tissue. During spraying, the nozzle was kept at a distance of 15-25cm from the tissue surface. The matrix solvent was 50% acetonitrile; the matrix concentration was CHCA 10mg / mL; the carrier gas was N2; the gas pressure was 10psi; the nozzle temperature was 80℃; the number of spraying times was 8 times; the speed was 800mm / min; the track spacing was 3mm; the matrix solution flow rate was 0.125mL / min. All samples on the ITO conductive glass slides need to be dried in a vacuum dryer for 15 minutes before mass spectrometry imaging to completely cover the co-crystallization on the surface of the biological tissue for MALDI mass spectrometry imaging analysis.

[0066] 2. Results and Discussion

[0067] (1) Optimization of positive and negative ion modes

[0068] Ceftiofur sodium was dissolved in water to obtain a 100 ppb aqueous solution of ceftiofur sodium, which was used as a standard solution for detection. The standard solution was subjected to MALDI-MS analysis using the above experimental conditions. Figure 1 It can be seen that ceftiofur sodium has a strong mass spectrometric response in the positive ion mode, and there is no corresponding mass spectrometric peak in the negative ion mode. Therefore, it can be seen that ceftiofur sodium is suitable for detection in the positive ion model.

[0069] (2) Comparison of different matrices

[0070] The above experimental conditions were used to perform MALDI-MS analysis on a standard solution (100 ppb ceftiofur sodium in water). Different organic matrices were compared, including α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), 4-nitroaniline (NTA), 5-nitro-3-(trifluoromethyl)pyridin-2-ol (HNTP), and 2-nitrophloroglucinol (NPG). Figure 2 It can be seen that when CHCA is used as the matrix, the detected response of ceftiofur sodium is the highest, so the experiment selected CHCA as the matrix for the next experiment of ceftiofur sodium.

[0071] (3) Salt tolerance test

[0072] Molecular analysis in actual samples often encounters the influence of coexisting high concentrations of salt, which can seriously reduce the ionization efficiency and suppress the analyte signal. In order to evaluate the salt tolerance of the prepared matrix in the positive ion mode, NaCl solution with a concentration range of 10-500 mM was used as the solvent for ceftiofur sodium to simulate the real sample environment. Figure 3 It can be seen that after adding 500 mM NaCl to the ceftiofur sodium solution, the peak intensity of the analyte dropped to 85.6% of the initial intensity. The signal became relatively stable, indicating that the method has strong salt tolerance.

[0073] (4) Reproducibility study

[0074] Signal reproducibility is a common problem in MALDI-MS. In positive ion mode, the signal reproducibility between points and samples was evaluated by analyzing 100 μg / L aqueous solution of ceftiofur sodium. The mass spectrometry results are shown in Table 3. In positive ion mode, the signal reproducibility between samples and between points was 7.23 and 9.65%, respectively. The above results show that the method has good reproducibility.

[0075] The reproducibility test is shown in Table 3 below:

[0076] Table 3 Point-to-point and sample-to-sample relative standard deviations of ceftiofur sodium

[0077] Target Mass-to-charge ratio Point-to-Point (RSD) Sample to sample (RSD) Ceftiofur Sodium 546.55 7.23％ 9.65％

[0078] 3. Methods and applications

[0079] (1) MALDI-MS determination of ceftiofur sodium

[0080] Ceftiofur sodium was detected by MALDI-MS method, and the concentration points of 0.1, 5, 25, 50, and 75 μg / L were prepared to establish a working curve. Figure 4 As shown, the experiment found that ceftiofur sodium had good linearity in the range of 0.5-75 μg / L, and the detection limit of the method was 0.025 μg / L. The linear correlation coefficient (R2) was 0.9922, indicating that the method had good linearity, the precision of the method was 8.32%, and the recovery rate was between 87.4-107.6%.

[0081] The method was further applied to the detection of real samples such as wastewater from the cleaning of sodium ceftiofur barrels, human serum samples, and urine (Table 4). The recoveries of the spiked wastewater, human serum samples, and urine samples of ceftiofur barrels were studied at 1, 10, and 50 μg / L. The spiked recoveries of wastewater from the cleaning of sodium ceftiofur barrels, human serum samples, and urine were 80.0% to 107.0%, 93.0% to 106.6%, and 97.4% to 111.0%, respectively. The results showed that the experimentally established MALDI-MS method can successfully determine ceftiofur sodium in wastewater from the cleaning of sodium ceftiofur barrels, human serum samples, and urine.

[0082] Table 4 MALDI-MS determination results of ceftiofur sodium in actual samples.

[0083]

[0084] ND: Not detected.

[0085] In order to evaluate the reliability of the established analytical method, the MALDI-MS analysis results of the wastewater sample from the washing of sodium ceftriaxone aluminum barrels were compared with the HPLC-MS / MS analysis results. As shown in Table 4, the MALDI-MS method can detect 26.1 μg / L sodium ceftriaxone in the wastewater. Figure 5 As shown, the HPLC-MS / MS analysis method determined the sodium thiothioate concentration to be 25.8 μg / L at a retention time of 6.36 min. The analysis results of the two methods were highly consistent, indicating that the MALDI-MS method can successfully determine sodium thiothioate in actual samples.

[0086] (2) MALDI-MSI determination of ceftiofur sodium

[0087] In order to further verify the applicability of MALDI-MSI, rats were selected as model samples, and a certain amount of ceftiofur sodium was exposed to the rats, and then mass spectrometry imaging analysis of their kidneys was performed. Different concentrations of ceftiofur sodium standard solutions (5, 10, 25, 50, 100 μg / L) were dripped on the surface of rat kidney tissue sections, and the mass spectrometry imaging was shown in the figure below. Figure 5 As shown. The experiment found that the rat kidney slices exposed to ceftiofur sodium showed different degrees of molecular distribution. The internal organs are the site of metabolism and also become the main site of drug molecule aggregation. The results show that MALDI-MSI can reliably visualize the distribution of ceftiofur sodium in animal tissues.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the drug metabolite molecule ceftiofur sodium, characterized in that: include: After the sample is pre-treated, it is spotted on a MALDI target plate, and after the sample and the matrix are co-crystallized, matrix-assisted laser desorption / ionization mass spectrometry and imaging analysis are used to detect ceftiofur sodium, and the obtained product is obtained; The matrix comprises α-cyano-4-hydroxycinnamic acid.

2. The detection method according to claim 1, characterized in that The ceftiofur sodium comes from waste water, blood serum, urine or biological tissue.

3. The detection method according to claim 2, characterized in that The sample is wastewater, and the pre-treatment is filtration; Preferably, the pretreatment comprises: filtering the wastewater and storing it at 0-5°C, spotting the matrix and the wastewater on a MALDI target plate, and analyzing the wastewater after co-crystallization with the matrix; Preferably, the filter membrane is 0.2-0.3 μm; and the matrix is ​​α-cyano-4-hydroxycinnamic acid.

4. The detection method according to claim 2, characterized in that The sample contains serum or urine, and the pretreatment is to remove protein; Preferably, the pretreatment comprises: precipitating high-abundance proteins in the sample, vortexing, collecting the supernatant after precipitation, spotting the matrix and the sample on a MALDI target plate, and analyzing the sample after co-crystallization with the matrix; Preferably, acetonitrile is added to the sample to precipitate high-abundance proteins; and the matrix is ​​α-cyano-4-hydroxycinnamic acid.

5. The detection method according to claim 2, characterized in that: When the sample is a biological tissue, the pretreatment is to freeze and fix the biological tissue and then slice it, spray the matrix on the surface of the biological tissue, dry it, and then analyze it; Preferably, the solvent of the matrix is ​​50% acetonitrile, the solute is α-cyano-4-hydroxycinnamic acid, and the concentration is 8-12 mg / mL, preferably 10 mg / mL.

6. The detection method according to claim 1, characterized in that MALDI-MS tests were performed using a RapifleXTM MALDI instrument with a laser wavelength of 355 nm in positive ion mode.

7. The detection method according to claim 1, characterized in that Mass spectrometry data were acquired by capturing 1000 laser pulses at a laser frequency of 500 Hz, with a scan range of 300-800 m / z.

8. The detection method according to claim 1, characterized in that The ion source voltage 1 and ion source voltage 2 were operated at 20 and 19.55 kV, respectively; LasAtten value is 15%; Lens and PIE are set to 11.383 and 2.632 respectively; Reflector voltage 1, reflector voltage 2 and reflector voltage 3 are at 22.5, 1.55 and 9.25 kV respectively.

9. The detection method according to claim 1, characterized in that: The delay extraction time is 100ns; Beam scanning range: 30×30μm 2 , result field: 10 4 ×10 4 μm 2 .

10. The detection method according to claim 1, characterized in that: For kidney tissue samples, the mode was M5 and the resulting area size was 200 × 200 μm. 2 ; Laser intensity: 15%.

Images