High performance liquid chromatography-tandem mass spectrometry detection method for BAB159 residues in soil

Abstract

The invention relates to the technical field of environmental sample detection, in particular to a high performance liquid chromatography-tandem mass spectrometry detection method for BAB159 residues in soil. Comprising the following steps: (1) mixing a soil sample with methanol, carrying out ultrasonic extraction, and then centrifuging to take supernate; (2) weighing a BAB159 standard substance, and adding acetonitrile to prepare a BAB159 standard stock solution; diluting to prepare a standard working solution with gradient concentration; (3) determining the matrix standard working solution with gradient concentration in a high performance liquid chromatography-mass spectrometer, and establishing a standard working curve by using an external standard method; and adding the standard working solution with the gradient concentration in the step (2) into the supernate in the step (1), and injecting into a high performance liquid chromatography-mass spectrometer for determination. The method is simple, efficient, high in sensitivity and good in accuracy, BAB159 in soil can be rapidly detected, and residues are prevented from harming aquatic organisms and the natural environment.

Description

Technical Field

[0001] This invention relates to the field of environmental sample detection technology, and in particular to a high-performance liquid chromatography-tandem mass spectrometry method for detecting BAB159 residues in soil. Background Technology

[0002] Antibiotic resistance has become a major threat to global public health. Currently, the development of antimicrobial compounds and the rational use of existing drugs remain the main strategies for effectively treating bacterial infections of animal origin. In previous laboratory studies, a lead compound, BAB159, with optimal activity was screened using a total synthetic method. Figure 1 BAB159 belongs to the benzoylaniline derivative class and possesses excellent antibacterial activity. It exhibits good antibacterial activity against the standard strain of Staphylococcus aureus (ATCC 25923) and methicillin-resistant Staphylococcus aureus (MRSA T144), with a minimum inhibitory concentration (MIC) as low as 0.015 μg / mL. Furthermore, BAB159 shows good activity against five major clinical bacterial groups, including Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens, and Streptococcus, outperforming many currently used clinical antibiotics (CN202311361694.7). BAB159 also demonstrates good antibacterial effects in vivo, significantly improving the survival rate of MRSA T144-infected giant wax moths and mice. In the giant wax moth infection model, it even outperforms the vancomycin positive control group at the same dose. In a mouse systemic infection model, BAB159 significantly reduces the bacterial load in the heart, liver, spleen, lungs, and kidneys of mice. BAB159 exhibits excellent antibacterial activity both in vivo and in vitro. Furthermore, toxicological studies have shown that BAB159 has low acute toxicity and no mutagenicity, reproductive toxicity, or genotoxicity, making it a promising novel antibacterial drug for clinical application (CN202311372798.8).

[0003] The promotion and application of new drugs should be based on a clear understanding of their biological activity, the exploration of their behavior, residue dynamics and ecotoxicity in the environment, and environmental safety evaluation and risk assessment (Tolls J. Sorption of veterinary pharmaceuticals in soils: a review. Environ Sci Technol. 2001, 35(17): 3397-406.). Current research shows that most veterinary antimicrobial drugs are not absorbed or metabolized by the body after entering the animal's body, and about 30%-90% of the original drug will be excreted into the environment through animal feces and urine (Gros M, Mas-Pla J, Boy-Roura M, et al. Veterinary pharmaceuticals and antibiotics in manure and slurry and their fate in amended agricultural soils: Findings from an experimental field site (Baix Empordà, NE Catalonia). Sci Total Environ, 2019, 654: 1337-1349.). After being excreted, antimicrobial drugs can enter the soil through feces or compost. Currently, there are few reports on the residual analysis methods of BAB159 in the ecological environment, its adsorption-desorption mechanism in soil, leaching behavior and influencing factors, and its toxicity to non-target organisms in the environment. To ensure the safe promotion and use of BAB159, in-depth research should be conducted as soon as possible on these unclear issues.

[0004] Therefore, studying the exposure level of BAB159 in soil is crucial. Currently, commonly used detection methods include potentiometric titration, high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Potentiometric titration has poor specificity and is easily affected by human operation; HPLC is greatly affected by the sample matrix and prone to false positives; GC-MS requires derivatization before measurement, which is cumbersome and hinders its widespread use; LC-MS / MS, with its high sensitivity, accuracy, and strong anti-interference ability, is the most suitable analytical method for detecting BAB159 residues in complex matrices. Currently, there is a lack of methods for detecting BAB159 in soil, both domestically and internationally. This invention aims to establish a high-performance liquid chromatography-tandem mass spectrometry method for detecting BAB159 in soil, providing technical support for the detection of BAB159 exposure levels in soil. Summary of the Invention

[0005] The purpose of this invention is to provide a high-performance liquid chromatography-tandem mass spectrometry method for detecting BAB159 residues in soil.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a high-performance liquid chromatography-tandem mass spectrometry method for detecting BAB159 residues in soil, comprising the following steps: (1) Sample pretreatment: Soil sample was mixed with methanol and extracted by ultrasonication, and then the supernatant was collected by centrifugation; (2) Preparation of standard solutions: Weigh out BAB159 standard, add an appropriate amount of dimethyl sulfoxide (DMSO) and dissolve it by sonication, then prepare BAB159 standard stock solution with acetonitrile; and prepare standard working solutions with gradient concentrations by dilution. (3) Determine the matrix standard working solutions of gradient concentration in a high performance liquid chromatography-mass spectrometry instrument and establish a standard working curve using the external standard method; after adding the standard working solutions of gradient concentration in step (2) to the supernatant of step (1), inject the solution into the high performance liquid chromatography-mass spectrometry instrument for determination; Chromatographic conditions: Column temperature: 38~42℃; Injection volume: 0.8~1.2 μL; Flow rate: 0.3~0.5 mL / min; Mobile phase: A is 0.1% formic acid water, B is acetonitrile; Isocratic elution: 30%A:70%B; Mass spectrometry conditions: Electrospray ionization source, negative ionization mode, spray voltage 4400~4600 V, ion source temperature 280~320 ℃, multiple reaction monitoring mode.

[0007] Preferably, the chromatographic column in step (3) has a size of 2.1 × 50 mm and a diameter of 2.6 μm.

[0008] Preferably, the type of chromatographic column in step (3) is a Kinetex C18 chromatographic column.

[0009] Preferably, the parameter settings for the mass spectrometry in step (3) are shown in Table 1: Table 1 Mass Spectrometry Parameters of BAB159 .

[0010] Preferably, the concentrations of the standard working solutions with gradient concentrations in step (2) are set to 5, 10, 50, 100, and 200 μg / mL, respectively.

[0011] Preferably, the concentration of the BAB159 standard stock solution in step (2) is 0.8~1.2 mg / mL.

[0012] Preferably, the ratio of soil sample to methanol in step (1) is 2g:8~12mL.

[0013] Preferably, the ultrasonic extraction in step (1) has a power of 300~500W and a time of 25~35 min.

[0014] Preferably, the centrifugation speed in step (1) is 9000~11000 r / min and the time is 8~12 min.

[0015] Preferably, the soil sample in step (1) is one or more of loam, sandy loam, silty loam or sand.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention establishes a high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for determining BAB159 residues in soil. Soil samples were extracted with methanol by ultrasonication, centrifuged at high speed, and the supernatant was filtered before LC-MS / MS analysis, with quantification using the external standard method. A Kinetex C18 column was used as the stationary phase, and the mobile phase was 0.1% formic acid aqueous solution and acetonitrile (V:V = 30:70). BAB159 showed good linearity in the concentration range of 5-200 ng / mL, with a correlation coefficient greater than 0.9928. The limit of detection (LOD) and limit of quantitation (LOQ) of this method were 0.5 ng / mL and 1 ng / mL, respectively. In blank soil samples with drug spiking concentrations of 5, 50, and 200 ng / mL, the average recovery rate of the BAB159 standard working solution was 65.97%–105.59%, and the intra-day and inter-day coefficient of variation was ≤8.24%. This method is simple, efficient, highly sensitive, and accurate, enabling rapid detection of BAB159 in soil and avoiding residual harm to aquatic organisms and the natural environment. Attached Figure Description

[0017] Figure 1 The structure of compound BAB159 is shown.

[0018] Figure 2 The recovery rate of BAB159 under different extractants.

[0019] Figure 3 This is the standard curve for BAB159 in different soil types. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] 1. Materials and Methods

[0023] 1.1 Instruments and Reagents

[0024] 1.1.1 Instruments: LC-20AD liquid chromatograph (Shimadzu Corporation, Japan), LCMS-8045 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan), small benchtop centrifuge (Sigma, Germany), electronic analytical balance (Sartorious, Germany), Milli-Q ultrapure water system (Millipore, USA).

[0025] 1.1.2 BAB159 active pharmaceutical ingredient (Wuhan Huisheng Biotechnology Co., Ltd.); methanol and acetonitrile were of chromatographic grade (Merk, Germany); and the test water was ultrapure water.

[0026] 1.2 Methods

[0027] 1.2.1 Sample Collection

[0028] Loam soil was collected from the edge of a paddy field at Huazhong Agricultural University in Wuhan. Sandy loam soil was collected from the edge of a farmland in Anyang, Henan. Silty loam soil was collected from the campus of China Agricultural University. Sandy soil was collected from Baiwang Mountain in Beijing. For each sample, three subsamples were collected from the top 0–20 cm of the soil layer using a multi-point sampling method. These subsamples were mixed together to obtain composite samples, which were then air-dried. One portion was sieved through a 2 mm sieve for soil experiments, and the other portion was sieved through a 1 mm sieve for determining the basic physicochemical properties of the soil.

[0029] 1.2.2 Instrument Conditions Chromatographic conditions: Kinetex C18 column (2.1 × 50 mm, 2.6 μm), column temperature: 40℃; injection volume: 1 μL; flow rate: 0.4 mL / min. Mobile phase: A is 0.1% formic acid in water, B is acetonitrile. Isocratic elution: 30% A: 70% B. Mass spectrometry conditions: Electrospray ionization source, negative ionization mode (ESI-), spray voltage 4500 V, ion source temperature 300℃, multiple reaction monitoring mode (MRM). Other mass spectrometry parameters are shown in Table 1.

[0030] 1.2.3 Preparation of Standard Solutions: Prepare BAB159 standard stock solution by accurately weighing 5.00 mg of BAB159 standard into a 5.00 mL volumetric flask. Add 1.00 mL of dimethyl sulfoxide (DMSO) and sonicate to dissolve. Dilute to the mark and shake well. Dilute with acetonitrile to prepare a 1 mg / mL BAB159 standard stock solution. Store at -20°C for later use. The standard stock solution is stable for up to three months. The standard mixed working solution should be prepared fresh for each use, based on the stock solution.

[0031] 1.2.4 Sample Pretreatment: Weigh approximately 2.00 g of soil sample into a 50 mL PTFE centrifuge tube, add 10 mL of methanol, vortex to mix, and then extract ultrasonically for 30 min. Centrifuge at 10000 r / min for 10 min. Filter the supernatant through a 0.22 μm organic syringe filter and perform quantification according to the instrument's operating conditions using the external standard method. If the detection level of BAB159 in the soil sample exceeds the linear range of the standard curve, it needs to be appropriately diluted with acetonitrile and re-measured.

[0032] 1.2.5 Construction of the matrix standard curve: Blank soil samples were taken and pretreated according to the method described in 1.2.4. An appropriate amount of BAB159 standard working solution was added to the treated samples to make the drug concentration in the samples 5, 10, 50, 100, and 200 ng / mL, with three replicates for each concentration. The samples were filtered through a 0.22 μm filter membrane for LC-MS / MS analysis. A standard curve was plotted with drug concentration (C) as the abscissa and peak area (A) as the ordinate, and the regression equation and correlation coefficient (r) were obtained.

[0033] 1.2.6 Determination of Limits of Detection and Limits of Quantification: Blank soil sample extracts were prepared according to the sample pretreatment method in 1.2.4. Mixed standard working solutions were added to prepare a series of low-concentration samples for LC-MS / MS analysis. Each concentration was measured three times, and the signal intensity S and noise intensity N were recorded for each measurement. The average values ​​of S and N were used to determine the limits of detection (LOD). The lowest concentration of the sample when S / N = 3 was taken as the limit of detection (LOD), and the lowest concentration of the sample when S / N = 10 was taken as the limit of quantification (LOQ).

[0034] 1.2.7 Addition Recovery and Precision Determination: Appropriate amounts of standard mixed working solution were added to four blank soil samples, with drug concentrations of 5, 50, and 200 ng / mL, and five replicates were made for each concentration. The samples were treated according to the sample pretreatment method and then analyzed by LC-MS / MS. The recovery rate and intra-day coefficient of variation were calculated, and the inter-day coefficient of variation was calculated after three consecutive days of detection.

[0035] 2 Results and Discussion

[0036] 2.1 Optimization of chromatographic conditions

[0037] 2.1.1 Optimization of chromatographic columns

[0038] The effects of two chromatographic columns—Waters BEH C18 (2.1 × 100 mm, 1.7 μm) and Kinetex C18 (2.1 × 50 mm, 2.6 μm)—on the analyte peaks were investigated. The analyte eluted on both columns. Although the former had higher sensitivity than the latter, the latter produced a better peak shape; therefore, the Kinetex C18 column was used for separation in this experiment. To avoid system residue affecting the results, a blank sample wash system should be added after high-concentration injections to prevent contamination.

[0039] 2.1.2 Optimization of the mobile phase

[0040] The chromatographic behavior of BAB159 quantitative ion pairs was investigated using a 100 ng / mL BAB159 standard solution as the mobile phase in water-acetonitrile, water-methanol, 0.1% formic acid-methanol, and 0.1% formic acid-acetonitrile solutions. When water-acetonitrile was used as the mobile phase, the chromatographic peak broadened and showed tailing. The response was slower than acetonitrile when using water-methanol as the mobile phase. Formic acid was added during detection to reduce tailing peaks; after adding formic acid, the chromatographic peaks became sharp and symmetrical. Ultimately, 0.1% formic acid-acetonitrile was chosen as the mobile phase. No interfering peaks appeared in the blank soil extract at the aforementioned retention times.

[0041] 2.2 Selection of Extractant

[0042] The structure of BAB159 is similar to that of the molluscicide niclosamide. Extraction solvents for niclosamide in soil include chloroform, acetonitrile, methanol, and ethyl acetate. Based on the compatibility and speed of liquid chromatography-mass spectrometry (LC-MS), this invention selected and compared four extraction solvents in the sample processing steps. Spiking recovery tests were conducted using BAB159 standard solutions on blank soil matrix samples. The spiked recoveries for chloroform extraction were 84.24%–86.3%, for acetonitrile extraction 70.37%–73.23%, for methanol extraction 80.43%–88.43%, and for ethyl acetate extraction 75.45%–78.69%. Comparatively, chloroform and methanol extraction yielded better spiked recoveries; however, chloroform as an extractant affected the peak shape of the drug. Therefore, methanol was ultimately chosen as the extractant. (See [link to relevant documentation]). Figure 2 .

[0043] 2.3 Determination of standard curve, limit of detection, and limit of quantitation

[0044] A standard curve was obtained by plotting the peak area of ​​BAB159 against the initial mass concentration. BAB159 showed good linearity in the concentration range of 5–200 ng / mL, with a correlation coefficient greater than 0.9928, as shown in Table 2. Figure 3As shown in the figure. The lowest concentration with a signal-to-noise ratio of 3 was taken as the limit of detection, and the lowest concentration with a signal-to-noise ratio of 10 was taken as the limit of quantitation. The limits of detection for the four soil samples were found to be 0.5 ng / mL and the limits of quantitation were 1 ng / mL.

[0045] Table 2 Regression equations and correlation coefficients of BAB159 in different soil types

[0046] 2.4 Adding recovery rate and precision

[0047] Appropriate amounts of standard stock solution were added to blank soil samples to achieve BAB159 concentrations of 5, 50, and 200 ng / mL, and recovery and precision tests were conducted. The results showed that in loam, the average recoveries of BAB159 were 74.99±1.84%, 71.53±0.24%, and 89.55±0.40%, with intra-day and inter-day coefficients of variation ≤8.24%. In sandy soil, the average recoveries were 71.97±2.97%, 86.15±1.58%, and 91.56±1.25%, with intra-day and inter-day coefficients of variation ≤7.14%. In silty loam, the average recoveries were 65.97±1.75%, 105.59±0.47%, and 95.48±2.75%, with intra-day and inter-day coefficients of variation ≤6.55%. In sandy loam soil, the average recoveries of BAB159 were 81.18±2.39%, 87.02±4.68%, and 101.24±1.26%, respectively, with intra-day and inter-day coefficients of variation ≤6.14%, indicating that the method is stable and reliable.

[0048] Table 3 Recovery rate and coefficient of variation of BAB159 in different soil types

[0049] 3. Conclusion

[0050] This invention establishes a method for detecting BAB159 in soil, employing extraction with the organic solvent methanol, followed by membrane filtration and analysis. This method exhibits high specificity, sensitivity, reproducibility, low cost, and meets the required recovery and precision. It can be used to detect BAB159 content in four types of soil, providing a methodological reference for future research on the environmental behavior of BAB159.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A high-performance liquid chromatography-tandem mass spectrometry method for detecting BAB159 residues in soil, characterized in that, Includes the following steps: (1) Sample pretreatment: Soil sample was mixed with methanol and extracted by ultrasonication, and then the supernatant was collected by centrifugation; (2) Preparation of standard solutions: Weigh BAB159 standard and add acetonitrile to prepare BAB159 standard stock solution; and prepare standard working solutions with gradient concentrations by dilution; (3) Determine the matrix standard working solutions of gradient concentration in a high performance liquid chromatography-mass spectrometry instrument and establish a standard working curve using the external standard method; after adding the standard working solutions of gradient concentration in step (2) to the supernatant of step (1), inject the solution into the high performance liquid chromatography-mass spectrometry instrument for determination; Chromatographic conditions: Column temperature: 38~42℃; Injection volume: 0.8~1.2μL; Flow rate: 0.3~0.5 mL / min; Mobile phase: A is 0.1% formic acid in water, B is acetonitrile; isocratic elution: 30% A: 70% B; mass spectrometry conditions: electrospray ionization source, negative ionization mode, spray voltage 4400~4600V, ion source temperature 280~320℃, multiple reaction monitoring mode.

2. The detection method according to claim 1, characterized in that, The chromatographic column in step (3) has a size of 2.1×50mm and a diameter of 2.6 μm.

3. The detection method according to claim 1, characterized in that, The type of chromatographic column in step (3) is a Kinetex C18 column.

4. The detection method according to claim 1, characterized in that, The parameter settings for the mass spectrometry described in step (3) are shown in Table 1: Table 1 5. The detection method according to claim 1, characterized in that, The concentrations of the standard working solutions with gradient concentrations in step (2) are set to 5, 10, 50, 100, and 200 μg / mL, respectively.

6. The detection method according to claim 1, characterized in that, The concentration of the BAB159 standard stock solution in step (2) is 0.8~1.2 mg / mL.

7. The detection method according to claim 1, characterized in that, The ratio of soil sample to methanol in step (1) is 2g:8~12ml.

8. The detection method according to claim 1, characterized in that, The ultrasonic extraction in step (1) uses a power of 300~500W and a time of 25~35min.

9. The detection method according to claim 1, characterized in that, In step (1), the centrifugation speed is 9000~11000 r / min and the time is 8~12 min.

10. The detection method according to claim 1, characterized in that, The soil sample in step (1) is one or more of the following: loam, sandy loam, silty loam, or sand.

Images