A method for simultaneous determination of multiple antibiotic intermediates in beta-lactam antibiotic fermentation wastewater

Abstract

The application belongs to the technical field of wastewater detection, and discloses a method for simultaneously determining a plurality of antibiotic intermediates in beta-lactam antibiotic fermentation wastewater, which comprises the following steps: (1) adjusting the pH of a sample to prepare antibiotic fermentation wastewater into a test sample solution; (2) detecting the test sample solution by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the detection results of each component; wherein the chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase comprises a weak polarity phase and a strong polarity phase, the weak polarity phase is selected from methanol or acetonitrile, and the strong polarity phase is selected from formic acid-ammonium formate solution or acetic acid-ammonium acetate solution, wherein the acid content of the strong polarity phase is 0.02% to 0.2% by volume, and the ammonium salt concentration is 0.01M to 0.05M. The application has high accuracy, high sensitivity and strong specificity, can quickly and accurately detect the total amount of a plurality of antibiotic intermediates in antibiotic fermentation wastewater, provides a detection method basis for water quality evaluation of antibiotic fermentation wastewater discharge, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of wastewater detection technology, specifically relating to a method for the simultaneous determination of multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater. Background Technology

[0002] my country is the world's largest producer of β-lactam antibiotic raw materials, generating a large amount of pharmaceutical wastewater annually. This wastewater contains various β-lactam antibiotic intermediates, which also possess a certain degree of antibacterial activity. The main active ingredients include penicillin G (GK), 6-aminopenicillanic acid (6-APA), cephalosporin C (CPC), deacetyl-cephalosporin C (D-CPC), deacetoxy-cephalosporin C (DO-CPC), 7-aminocephalosporanic acid (7-ACA), and 7-aminodeacetoxycephalosporanic acid (7-ADCA). Among these, 7-ACA is a common intermediate for the vast majority of cephalosporins, 6-APA is a common intermediate for most penicillin derivatives (cillins), and GK and CPC are two important natural antibiotic and raw material intermediates. Compared with other antibiotics, penicillins and cephalosporins have a broad antibacterial spectrum, strong antibacterial activity, high efficacy, and few side effects, occupying an important position in the anti-infective drug market. The large quantities of pharmaceutical wastewater discharged into the environment each year, if not properly treated, could potentially lead to antibiotic resistance in environmental bacteria. Therefore, establishing detection and analytical methods for such substances is crucial for assessing their potential antibiotic resistance risk to bacteria and other microorganisms in the ecological environment.

[0003] In the field of wastewater treatment, the detection method for antibiotics typically employs liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS). Examples include "DB65 / T 4370-2021 Determination of Erythromycin and Penicillin in Water by Liquid Chromatography-Triple Quadrupole Mass Spectrometry," "T / PIAC 00001—2021 Method for the Determination of Penicillin in Antibiotic-Containing Fertilizer Residue, Organic Fertilizer Substrate, Crops, and Environmental Media," and "T / PIAC 00002—2021 Method for the Determination of Cephalosporins in Antibiotic-Containing Fertilizer Residue, Organic Fertilizer Substrate, Crops, and Environmental Media." These three standard methods offer simple sample pretreatment, high sensitivity and accuracy for determining antibiotic content in water, allowing for rapid and accurate determination. However, they only detect a limited number of antibiotics, and these types vary.

[0004] Other literature reports methods for detecting antibiotic intermediates, such as "Rapid Detection of Penicillin Intermediates and Two Penicillin Drugs in Milk by High Performance Liquid Chromatography." This literature employs liquid chromatography, but the pretreatment steps require repeated extraction with organic reagents, followed by further defatting, rotary evaporation, and solid-phase extraction purification of the extract. This is not only cumbersome but also increases the cost of detection. Furthermore, the sensitivity of the UV detector is lower than that of a triple quadrupole mass spectrometer. Similarly, papers such as "Study on Degradation Pathways of β-Lactam Antibiotics and Intermediates," "A Method for Detecting Related Substances of Ceftiofur Intermediate," and "An Analytical Method and Application for Cefixime Intermediates and Related Impurities" use liquid chromatography with a UV detector, which has limited sensitivity and cannot meet the current needs of environmental risk assessment.

[0005] Currently, there are few reports on methods for the simultaneous determination of multiple β-lactam antibiotic intermediates in microbial fermentation pharmaceutical wastewater, which makes it impossible to guarantee the environmental safety impact during the discharge of antibiotic fermentation wastewater. Summary of the Invention

[0006] In view of this, in order to solve the problems existing in the prior art, the present invention provides a rapid, accurate and highly sensitive detection method for simultaneously determining multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater.

[0007] To achieve the above objectives, the present invention aims to provide a method for the simultaneous determination of multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater, employing the following technical solution:

[0008] A method for simultaneous determination of multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater includes the following steps:

[0009] (1) Prepare a test solution from antibiotic fermentation wastewater for later use;

[0010] (2) The test solution was analyzed by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the qualitative or quantitative results of each component;

[0011] The chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase includes a weakly polar phase and a strongly polar phase, wherein the weakly polar phase is selected from methanol or acetonitrile, and the strongly polar phase is selected from formic acid-ammonium formate or acetic acid-ammonium acetate solution, wherein the acid content is 0.02% to 0.2% by volume, and the ammonium salt concentration is 0.01M to 0.05M.

[0012] In this invention, the "antibiotic fermentation wastewater" refers to the wastewater generated during the fermentation, washing, and extraction processes in the fermentation production of β-lactam antibiotic intermediates.

[0013] It is worth noting that antibiotic intermediates, compared to active pharmaceutical ingredients (APIs), are less prevalent and less concentrated, thus attracting less attention. However, exposure to the environment poses a similar risk of environmental resistance as APIs, warranting further investigation. Consequently, there are few reports on the simultaneous determination of multiple β-lactam antibiotic intermediates in wastewater treatment. In the food and pharmaceutical industries, liquid chromatography-ultraviolet (LC-UV) detection is commonly used for β-lactam antibiotic intermediates. However, this method has limitations in sensitivity, and different components require separate detection at different wavelengths, resulting in lower detection efficiency. For samples with complex matrices, high-level sample pretreatment is generally required. Simply extracting with an extractant and directly injecting the sample into the LC analyzer leads to chromatographic peaks that are severely affected by impurities, easily producing false positives. Solid-phase extraction (SPE) is necessary for purification, which increases sample pretreatment costs and reduces detection efficiency.

[0014] This invention aims to establish a rapid, accurate, and sensitive method for detecting multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater. It employs highly specific and sensitive liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS / MS), and through optimized mobile phase selection and gradient elution conditions, enables the elution of 7-ACA, which previously failed to elute, when the mobile phase is an ammonium salt. Simultaneously, the response intensity of 7-ADCA is significantly improved. Furthermore, reducing the organic phase ratio in the initial gradient elution conditions effectively improves the retention times of 6-APA and 7-ADCA, resolving the peak overlap problem in existing technologies and enabling the simultaneous determination of multiple antibiotic intermediates in antibiotic fermentation wastewater. In particular, the optimized scheme requires only pH adjustment, centrifugation, and filtration for sample pretreatment of the antibiotic fermentation wastewater, eliminating the need for solid-phase extraction and repeated solvent extraction. This method is simple, rapid, and yields accurate and highly sensitive results, demonstrating significant application potential.

[0015] Furthermore, the antibiotic intermediate includes one or more of GK, 6-APA, CPC, D-CPC, DO-CPC, 7-ACA, and 7-ADCA.

[0016] Furthermore, step (1) specifically includes:

[0017] (1.1) Adjust the pH of the antibiotic fermentation wastewater to 6-7;

[0018] (1.2) Centrifuge the wastewater after pH adjustment, take the supernatant, and filter it through a microporous membrane to obtain the final product.

[0019] Furthermore, in step (1.1), formic acid, acetic acid, or ammonia is used to adjust the pH; in step (1.2), the microporous filter membrane is 0.22 μm or 0.45 μm.

[0020] Furthermore, in step (2), the particle size of the chromatographic packing is 1.7 μm; the mobile phase is methanol as a weakly polar phase and 0.1% formic acid-0.02M ammonium formate aqueous solution as a strongly polar phase.

[0021] Furthermore, the chromatographic conditions also include a gradient elution program as follows:

[0022] The initial stage lasts for 1.0–5.0 min, and the volume ratio of methanol is 5%–20%.

[0023] The duration of the intermediate stage is 2.0–10.0 min, and the volume ratio of methanol is 40%–90%.

[0024] The final stage lasts 2.0–5.0 min, with a methanol volume ratio of 5%–20%; and,

[0025] The flow rate was 0.2–0.4 mL / min, the column temperature was 25–40 °C, and the injection volume was 1–15 μL.

[0026] Furthermore, the gradient elution procedure is as follows:

[0027] 0.00-4.00 min, 5% methanol; 4.01-7.00 min, 40%-60% methanol; 7.01-10.00 min, 5% methanol; and the flow rate was 0.25 mL / min, the column temperature was 35℃, and the injection volume was 2 μL.

[0028] Furthermore, in step (2), the mass spectrometry conditions include:

[0029] The ionization method is electrospray positive ion mode; the ion source temperature is 150-200℃; the desolvent gas temperature is 300-600℃; the desolvent gas flow rate is 400-1000L / Hr; the spray voltage is 2.8-4.0kV; the cone voltage is 10-40V; the collision voltage is 5-40V; and the scanning mode is multiple reaction monitoring mode.

[0030] Furthermore, the mass spectrometry conditions are as follows: ion source temperature 150℃; desolvation gas temperature 450℃; desolvation gas flow rate 900 L / Hr; spray voltage 4.0 kV; and multiple reaction monitoring conditions are shown in Table 1.

[0031] Table 17 Intermediate Multiple Reaction Monitoring Conditions for Antibiotics

[0032]

[0033]

[0034] .

[0035] Furthermore, in step (2), the content of the target component is calculated using the external standard method.

[0036] Compared with existing technologies, this invention reduces the requirements for sample pretreatment conditions and simplifies the operation process by employing liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS). Through optimized chromatographic conditions, it successfully separates antibiotic intermediates with similar molecular structures from β-lactam antibiotic fermentation wastewater, achieving simultaneous qualitative and quantitative detection of multiple antibiotic intermediates in antibiotic fermentation wastewater. Furthermore, this invention effectively improves detection sensitivity. The limits of detection for GK, 6-APA, CPC, D-CPC, DO-CPC, 7-ACA, and 7-ADCA are 0.6 μg / L, 0.7 μg / L, 1.6 μg / L, 1.1 μg / L, 0.7 μg / L, 0.9 μg / L, and 0.6 μg / L, respectively. The recoveries of all seven antibiotics at a spiked concentration of 0.05 mg / L are between 90% and 110.0%. The method established in this invention is highly accurate, sensitive, and specific, enabling rapid and effective detection of the total amount of antibiotic intermediates in antibiotic fermentation wastewater. This provides a methodological basis for evaluating the water quality of antibiotic fermentation wastewater discharge. In particular, in the preferred embodiment, the pretreatment method for antibiotic fermentation wastewater only requires pH adjustment, centrifugation, and filtration, eliminating the need for solid-phase extraction and repeated solvent extraction. This method is simple, rapid, and inexpensive, demonstrating high application potential. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0038] Figure 1 This is a total ion chromatogram of the standard solutions of various antibiotic intermediates in Example 1 of the present invention. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0041] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0042] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0043] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0044] This invention discloses a method for the simultaneous determination of multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater, belonging to the field of wastewater detection technology. The invention employs liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) and includes the following steps: (1) adjusting the sample pH to prepare a test solution from the antibiotic fermentation wastewater; (2) detecting the test solution using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) to obtain the detection results of each component; wherein the chromatographic conditions are: a C18 column; the mobile phase includes a weakly polar phase and a strongly polar phase, wherein the weakly polar phase is selected from methanol or acetonitrile, and the strongly polar phase is selected from formic acid-ammonium formate or acetic acid-ammonium acetate solution, wherein the acid content of the strongly polar phase is 0.02%–0.2% by volume, and the ammonium salt concentration is 0.01M–0.05M. This invention has high accuracy, high sensitivity, and strong specificity, and can rapidly and accurately detect the total amount of multiple antibiotic intermediates in antibiotic fermentation wastewater, providing a detection method basis for water quality evaluation of antibiotic fermentation wastewater discharge, and has good application prospects.

[0045] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0046] In the following examples and experimental cases, all reagents and materials used were commercially available.

[0047] Example 1

[0048] (1) Preparation of the test solution:

[0049] Take an appropriate amount of wastewater, adjust the pH to 6-7 with ammonia or formic acid, centrifuge at 4000 r / min for 10 min, take the supernatant, filter the supernatant through a 0.22 μm microporous membrane to obtain the test solution.

[0050] (2) Plotting the standard curve:

[0051] A mixed standard stock solution with a concentration of 1000 mg / L was prepared by using water to prepare GK, 6-APA, CPC, D-CPC, and DO-CPC. A standard stock solution with a concentration of 200 mg / L was prepared by using 0.02 M ammonium acetate solution to prepare a mixed standard stock solution with a concentration of 1.0 μg / L, 5.0 μg / L, 25.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 500.0 μg / L. The mixed standard working solutions were then analyzed by liquid chromatography-triple quadrupole mass spectrometry. The peak areas and corresponding standard working solution concentrations were measured to plot a standard curve, and the regression equation and correlation coefficient were calculated.

[0052] (3) Detection and analysis: The mixed standard working solution and the test sample were injected into the liquid chromatograph and detected and analyzed by liquid chromatography-triple quadrupole mass spectrometry. Then, the external standard method was used for quantitative analysis.

[0053] In steps (2) and (3) above, the instrument used for liquid chromatography-triple quadrupole method is an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer.

[0054] The chromatographic conditions were as follows: Waters-C18 column (2.1×100mm, 1.7μm); mobile phase: methanol and 0.1% formic acid-0.02M ammonium formate; gradient elution: 0.00-4.00 min, 5% methanol; 4.01-7.00 min, 40%-60% methanol; 7.01-10.00 min, 5% methanol; flow rate: 0.25 mL / min; column temperature: 35℃; injection volume: 10 μL.

[0055] Mass spectrometry conditions: electrospray positive ion mode; ion source temperature: 150℃; desolvent gas temperature: 450℃; desolvent gas flow rate: 900L / Hr; spray voltage: 4.0kV; specific parameters for the detected compounds are shown in Table 1.

[0056] The total ion chromatograms of the seven antibiotic standard solutions obtained in this embodiment are as follows: Figure 1 As shown.

[0057] To further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following methodological verification further clarifies the properties and application performance of the method for simultaneous determination of multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater disclosed in the present invention. However, this should not be construed as a limitation of the present invention. The method properties obtained by other determination experiments conducted by those skilled in the art based on the above-described invention and the applications based on the above properties are also considered to fall within the protection scope of the present invention.

[0058] Experiment Example 1: Linear Examination

[0059] The standard curve equations and linear correlation coefficients of GK, 6-APA, CPC, D-CPC, DO-CPC, 7-ACA and 7-ADCA were calculated according to the method in step (2) of Example 1, as shown in Table 2:

[0060] Table 27 Standard Curve Equations and Linear Correlation Coefficients for Antibiotic Intermediates

[0061] GK Y = 526.92X + 1250.70 0.9998 6-APA Y = 163.54X - 138.06 0.9996 CPC Y = 132.96X - 229.01 0.9994 D-CPC Y = 93.95X - 90.70 0.9995 DO-CPC Y = 69.38X - 3.461 0.9997 7-ACA Y = 210.18X - 526.81 0.9984 7-ADCA Y = 69.6987X - 5869.12 0.9955

[0062] As can be seen from the data in Table 2, the linearity of all seven antibiotic intermediates was good.

[0063] Experimental Example 2: Limit of Detection, Spiked Recovery and Precision

[0064] Using a blank assay where the target analyte was not detected, at least seven parallel determinations were performed on samples with concentrations or contents 3 to 5 times the estimated method detection limit, following all steps of sample analysis, and the detection limit was calculated. The spiked recovery rate at a sample spike concentration of 0.05 mg / L was investigated, and six parallel determinations were performed. The results are shown in Table 3.

[0065] Table 37: Limits of Detection, Spike Recovery, and Precision of Antibiotic Intermediates

[0066]

[0067] As shown in Table 3, the sensitivity of the seven antibiotic intermediates can reach about 1.0 μg / L, and the spiked recovery rates are all between 90% and 110.0%, indicating that the detection method has high accuracy and sensitivity.

[0068] Therefore, this invention utilizes liquid chromatography-triple quadrupole mass spectrometry to simultaneously determine multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater. This method is highly accurate, sensitive, and specific, enabling rapid and effective detection of the total amount of multiple antibiotic intermediates in antibiotic fermentation wastewater. It provides a methodological basis for evaluating the water quality of antibiotic fermentation wastewater discharge and has excellent application prospects.

[0069] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneous determination of multiple antibiotic intermediates in β-lactam antibiotic fermentation wastewater, characterized in that, Includes the following steps: (1) Prepare a test solution from the antibiotic fermentation wastewater for later use; (2) The test solution was analyzed by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the qualitative or quantitative results of each component; The chromatographic conditions were as follows: the chromatographic column was a C18 column; the mobile phase included a weakly polar phase and a strongly polar phase, wherein the weakly polar phase was selected from methanol or acetonitrile, and the strongly polar phase was selected from formic acid-ammonium formate or acetic acid-ammonium acetate solution, wherein the acid content was 0.02% to 0.2% by volume and the ammonium salt concentration was 0.01 M to 0.05 M; Step (1) specifically includes: (1.1) Adjust the pH of the antibiotic fermentation wastewater to 6-7; (1.2) Centrifuge the pH-adjusted wastewater, collect the supernatant, and filter it through a microporous membrane to obtain the final product; In step (2), the particle size of the chromatographic packing is 1.7 μm; the mobile phase is methanol as a weakly polar phase and 0.1% formic acid-0.02M ammonium formate aqueous solution as a strongly polar phase. Chromatographic conditions also include a gradient elution program: The initial stage lasted 1.0–5.0 min, and the volume ratio of methanol was 5%–20%. The intermediate stage lasts 2.0–10.0 min, and the volume ratio of methanol is 40%–90%. The final stage lasted 2.0–5.0 min, with a methanol volume ratio of 5%–20%; and, The flow rate was 0.2–0.4 mL / min, the column temperature was 25–40 ℃, and the injection volume was 1–15 μL. The gradient elution procedure is as follows: 0.00-4.00 min, 5% methanol; 4.01-7.00 min, 40%-60% methanol; 7.01-10.00 min, 5% methanol; and the flow rate was 0.25 mL / min, the column temperature was 35 ℃, and the injection volume was 2 μL.

2. The determination method according to claim 1, characterized in that, The antibiotic intermediates include one or more of penicillin G, 6-aminopenicillanic acid, cephalosporin C, deacetyl-cephalosporin C, deacetoxy-cephalosporin C, 7-aminocephalosporanic acid, and 7-aminodeacetoxycephalosporanic acid.

3. The determination method according to claim 1, characterized in that, In step (1.1), formic acid, acetic acid or ammonia is used to adjust the pH; in step (1.2), the microporous filter membrane is 0.22 μm or 0.45 μm.

4. The determination method according to claim 1, characterized in that, In step (2), the mass spectrometry conditions include: The ionization method is electrospray positive ion mode; the ion source temperature is 150–200 ℃; the desolvent gas temperature is 300–600 ℃; the desolvent gas flow rate is 400–1000 L / Hr; the spray voltage is 2.8–4.0 kV; the cone voltage is 10–40 V; the collision voltage is 5–40 V; and the scanning mode is multiple reaction monitoring mode.

5. The determination method according to claim 4, characterized in that, The mass spectrometry conditions were as follows: ion source temperature 150℃; desolvation gas temperature 450℃; desolvation gas flow rate 900 L / Hr; spray voltage 4.0 kV. Multiple reaction monitoring (MRM) conditions are shown in Table 1. Table 1. Multiple reaction monitoring conditions for seven antibiotic intermediates 。 6. The determination method according to claim 1, characterized in that, In step (2), the external standard method is used to calculate the content of the target component.

Images