Method for simultaneously detecting concentrations of 4 tetracycline drugs based on liquid chromatography-mass spectrometry

Abstract

The application provides a method for simultaneously detecting concentrations of four kinds of tetracycline drugs, i.e., Ivermectin, Minocycline, Tigecycline and Omadacycline, based on liquid chromatography-mass spectrometry, comprising: preparing a standard solution of the four kinds of tetracycline drugs; diluting the standard solution to prepare eight standard curve working solutions with different concentrations and four quality control working solutions with different concentrations; diluting the standard curve working solutions and the quality control working solutions with different concentrations into plasma matrix to obtain samples with different concentrations in the standard curve and quality control samples; using a mixture of terramycin and Tigecycline-d9 as an internal standard working solution, the linear range of the standard curve is 20 ng / mL-2500 ng / mL; and calculating the drug concentration in a clinical biological sample according to the standard curve. The method realizes simultaneous determination of the four kinds of tetracycline drugs, and has the advantages of simple operation, rapid and accurate analysis, high recovery rate and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of clinical pharmaceutical testing, specifically to a method for simultaneously detecting the concentrations of four tetracycline drugs based on liquid chromatography-mass spectrometry (LC-MS). Background Technology

[0002] Tetracyclines are a class of commonly used antibacterial drugs in clinical practice, exhibiting strong antibacterial activity against a variety of pathogens, including atypical pathogens such as mycoplasma, chlamydia, and Legionella. Chlortetracycline, oxytetracycline, and tetracycline are the first-generation members of the tetracycline family; the second generation, represented by minocycline and doxycycline, has become commonly used in clinical practice; tigecycline is a third-generation tetracycline, widely used in the treatment of severe infections; omalicycline and eracycline belong to the third generation and were both approved by the FDA for clinical treatment in 2018. Due to their low resistance rates and high safety profiles, they undoubtedly provide more options for anti-infective therapy.

[0003] Studies have shown that elevated minimum inhibitory concentrations (MICs) and insufficient dosage of tigecycline may be major causes of treatment failure. The widespread clinical use of off-label high-dose tigecycline dosing regimens also increases the risk of toxic reactions. Patent CN111537649A discloses a kit for detecting antibacterial drugs in serum using ultra-high performance liquid chromatography-tandem mass spectrometry. The antibacterial drugs include: sulbactam, imipenem, linezolid, meropenem, moxifloxacin, piperacillin, tigecycline, cefoperazone, vancomycin, and teicoplanin. Except for tigecycline, other tetracycline antibiotics have not yet been addressed.

[0004] Omacycline was the first aminomethyltetracycline to successfully enter clinical application. Omacycline not only covers common pathogens of community-acquired bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections (ABSSSI), but also exhibits good antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, and Klebsiella pneumoniae. A 2024 paper reported the development of a UPLC-MS / MS method for determining omacycline concentrations in the serum of pneumonia patients, which involved the detection of omacycline but did not mention the detection of other tetracycline antibiotics.

[0005] Eracycline, a third-generation tetracycline, is recommended as a fluorocycline in combination with other drugs for the treatment of bloodstream infections. Compared to tigecycline, eracycline has higher concentrations in lung tissue and a lower incidence of adverse events. Therefore, eracycline has advantages over tigecycline, but its clinical efficacy requires further research to confirm. Regarding the detection of eracycline blood concentration, patent document CN119023862A discloses a method for determining human plasma eracycline concentration using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), which only involves the detection of eracycline and does not mention the detection of other tetracycline antibiotics. Patent document CN120369844A discloses a method for detecting human serum eracycline concentration using HPLC-MS / MS, which also only involves the detection of eracycline and does not mention the detection of other tetracycline antibiotics.

[0006] Currently, there are no reports on the simultaneous detection of eracycline, minocycline, tigecycline, and omalicycline. Therefore, it is necessary to propose an analytical method that is simple to operate and highly accurate, capable of simultaneously detecting the concentrations of four tetracycline antibiotics. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting the concentration of four tetracycline drugs based on liquid chromatography-mass spectrometry (LC-MS) to solve the problem that the existing technology cannot simultaneously detect the concentration of four tetracycline antibiotics. This method has the advantages of being simple, rapid, accurate, having high recovery rate, and high sensitivity.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: The present invention provides a method for simultaneously detecting the plasma concentrations of four tetracycline drugs (eiracycline, minocycline, tigecycline, and omalicycline) based on liquid chromatography-mass spectrometry (LC-MS), comprising the following steps: preparing standard stock solutions of the four tetracycline drugs under yellow light and ice bath conditions; diluting the standard stock solutions to prepare eight standard curve working solutions and four quality control working solutions of different concentrations; diluting the standard curve working solutions and quality control working solutions of each concentration into the plasma matrix to obtain samples and quality control samples of each concentration in the standard curve; mixing the samples and quality control samples of each concentration in the standard curve with a pre-prepared internal standard working solution, vortexing, centrifuging, and then mixing the supernatant with a certain ratio of... After the diluent for the example is mixed, an appropriate amount of the solution is analyzed using a liquid chromatography-mass spectrometry (LC-MS) instrument. A standard curve is plotted with drug concentration as the X-axis and the ratio of drug peak area to internal standard peak area as the Y-axis. Additionally, the clinical test sample is mixed with a pre-prepared internal standard working solution, vortexed, centrifuged, and the supernatant is mixed with a certain proportion of diluent. An appropriate amount of the solution is then analyzed using LC-MS. The drug concentration in the clinical biological sample is calculated based on the standard curve. The processing of samples at various concentrations in the standard curve, quality control samples, and plasma samples in the clinical biological samples is performed under yellow light and ice bath conditions. The method is as follows: 50 µL of plasma sample is placed in a 1.5 mL centrifuge tube, and 200 µL of internal standard working solution is added. After vortexing, the mixture is centrifuged at 2–8 °C and 14000 rpm. The supernatant is added to the centrifuge tube at a ratio of 2:1 with the diluent, mixed thoroughly, centrifuged again, and the supernatant is added to the sample injection tube for analysis using LC-MS.

[0009] Optionally, in the above method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS), the concentration of the standard stock solutions of eiracycline, minocycline, tigecycline, and omalicycline is 1.00 mg / mL.

[0010] Optionally, in the above method for simultaneously detecting the blood concentrations of four tetracycline drugs—eracycline, minocycline, tigecycline, and omalicycline—in plasma using liquid chromatography-mass spectrometry (LC-MS), the internal standard working solution is a mixed internal standard working solution of oxytetracycline and tigecycline-d9 prepared in methanol, wherein the concentration of oxytetracycline in the internal standard working solution is 500 ng / mL and the concentration of tigecycline-d9 is 25 ng / mL.

[0011] Optionally, in the above method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS), the preparation of each concentration sample and quality control sample in the standard curve is carried out under yellow light and ice bath conditions. The concentrations of the standard curves for eiracycline, minocycline, tigecycline, and omalicycline are 20 ng / mL, 40 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, and 2500 ng / mL, respectively, and the concentrations of the quality control samples are 20 ng / mL, 60 ng / mL, 600 ng / mL, and 1500 ng / mL, respectively.

[0012] Optionally, in the above method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eparacycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS), when using a LC-MS analyzer for analysis: The liquid chromatography conditions are as follows: Mobile phase A is an aqueous solution of ammonium acetate with a concentration of 10 mM containing 0.3% formic acid; mobile phase B is an acetonitrile solution containing 0.3% formic acid; the needle washing solution is a mixed solution of acetonitrile, water, isopropanol, methanol, and formic acid.

[0013] The gradient elution program was as follows: 0.00–1.00 min, mobile phase B ratio 3%; 1.00–1.70 min, mobile phase B ratio 3–7%; 1.70–2.50 min, mobile phase B ratio 7–98%; 2.50–4.0 min, mobile phase B ratio 98%; 4.01–6.00 min, mobile phase B ratio 3%; elution flow rate was 0.2 mL / min, injection volume was 2–5 µL, and sample collection time was 6.00 min. The mass spectrometry conditions were as follows: the ion source was an electrospray ion source, the detection mode was positive ion mode, the scanning mode was multiple ion reaction monitoring (MRM), the drying gas temperature was 350℃, the drying gas flow rate was 5 L / min, the nebulizing gas pressure was 30 psi, the auxiliary gas temperature was 350℃, the auxiliary gas flow rate was 11 L / min, and the capillary voltage was 4000 V.

[0014] Optionally, in the above method for simultaneously detecting the blood concentrations of four tetracycline drugs (eiracycline, minocycline, tigecycline, and omalicycline) in plasma based on liquid chromatography-mass spectrometry (LC-MS), the chromatographic conditions are as follows: Agilent SB-C18 column, RRHD 1.8µm, 2.1 × 50mm; injector temperature 4~8℃; column temperature 40℃.

[0015] Optionally, in the above method for simultaneously detecting the blood concentrations of four tetracycline drugs—eracycline, minocycline, tigecycline, and omalicycline—in plasma using liquid chromatography-mass spectrometry (LC-MS), the injection washing solution contains 0.1% formic acid, and the volume ratio of acetonitrile, water, methanol, and isopropanol is 1:1:1:1.

[0016] Optionally, in the above method for simultaneously detecting the blood concentrations of four tetracycline drugs—eracycline, minocycline, tigecycline, and omalicycline—in plasma using liquid chromatography-mass spectrometry (LC-MS), the solvent used to prepare the standard stock solution is dimethyl sulfoxide, and the diluent is a 50% methanol aqueous solution containing 0.1% formic acid.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method of this invention enables the simultaneous determination of four tetracycline antibiotics, and has the advantages of simple operation, rapid and accurate analysis, high recovery rate, and high sensitivity. Specifically, multi-drug analysis can be completed using the same chromatographic mass spectrometry process. Clinicians only need to label the types of drugs used and quickly generate results, eliminating the need for frequent column and mobile phase changes and reducing labor costs. It supports pharmacokinetic studies in special populations, reduces batch-to-batch variation with a unified method, and provides stable and efficient technical support for individualized dosing and PK / PD studies, thereby improving the overall quality of TDM services and the accuracy of clinical diagnosis and treatment. The amount of plasma used for detection is small, only 50 µL, and the amount of blood drawn from patients is small, making it particularly suitable for the elderly, children, and critically ill patients with difficult blood collection. The pretreatment operation of the method of this invention is simple, and the process can be completed in one precipitation step, saving time and effort.

[0018] (2) This invention uses a mixed internal standard working solution. Oxytetracycline, a similar compound with a similar structure to the analyte, serves as an internal standard for elastocycline and minocycline; tigecycline-d9 is an isotopic internal standard, serving as an internal standard for both tigecycline and omalicycline. Since the mass spectrometry signal responses of tigecycline and omalicycline are not on the same order of magnitude as those of elastocycline and minocycline, and their ionization capabilities differ significantly, two internal standards are used. By selecting appropriate internal standards, there is no interference between them and the four analyte compounds, thus ensuring the accuracy of the detection results.

[0019] (3) The linear range of the standard curve of the present invention can cover the sample concentration, and pretreatment can be performed directly without dilution, thus eliminating the dilution step. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0021] Figure 1Chromatograms of four tetracycline drugs and internal standards provided in embodiments of the present invention; Figure 2 The chromatogram of a double-blank plasma sample provided in an embodiment of the present invention; Figure 3 Chromatogram of a single blank plasma sample (mixed internal standard) provided for an embodiment of the present invention; Figure 4 Chromatograms of the lower limit of quantitation provided in embodiments of the present invention; Figure 5 Chromatograms of ilacycline and internal standard provided in embodiments of the present invention; Figure 6 Chromatograms of minocycline and internal standard provided in embodiments of the present invention; Figure 7 Chromatograms of tigecycline and internal standard provided in embodiments of the present invention; Figure 8 Chromatograms of omalicycline and internal standard provided in embodiments of the present invention; Figure 9 This is the ilacycline standard curve provided in the embodiments of the present invention; Figure 10 The minocycline standard curve provided in the embodiments of the present invention; Figure 11 The omalicycline standard curve provided in this embodiment of the invention; Figure 12 The tigecycline standard curve provided in this embodiment of the invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0023] The present invention provides a method for simultaneously detecting the concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS), comprising the following steps: Prepare standard stock solutions of four tetracycline drugs under yellow light and ice bath conditions; The standard stock solution was diluted to prepare 8 standard curve working solutions and 4 quality control working solutions of different concentrations. The working solutions for each concentration of the standard curve and the working solutions for quality control were diluted into the plasma matrix to obtain the samples and quality control samples at each concentration in the standard curve; The samples at each concentration in the standard curve and the quality control samples were mixed with the pre-prepared internal standard working solution. After vortexing, the mixture was centrifuged, and the supernatant was mixed with a certain proportion of diluent (50% methanol (containing 0.1% formic acid)). An appropriate amount of the solution was then analyzed using liquid chromatography-mass spectrometry (LC-MS). A standard curve was plotted with drug concentration as the X-axis and the ratio of drug peak area to internal standard peak area as the Y-axis. The clinical sample to be tested was mixed with the pre-prepared internal standard working solution, vortexed, centrifuged, and the supernatant was mixed with a certain proportion of diluent. An appropriate amount of solution was taken for analysis using liquid chromatography-mass spectrometry (LC-MS), and the drug concentration in the clinical biological sample was calculated based on the standard curve.

[0024] The plasma used is human plasma, and all solution preparation and plasma pretreatment processes are carried out under yellow light and ice bath conditions.

[0025] Example 1. Experimental Materials (1) Reagents and consumables: Methanol, isopropanol, dimethyl sulfoxide, water and formic acid were all of chromatographic grade (Fisher); the chromatographic column was SB-C18, RRHD 1.8µm, 2.1 ×50mm.

[0026] (2) Drugs: Iracycline (MCE Company, batch number 232624); Tigecycline (MCE Company, batch number 119745); Minocycline (MCE Company, batch number 227983); Omacycline (MCE Company, batch number 94776); Oxytetracycline (MCE Company, batch number 213158); Tigecycline-d9 (MCE, batch number 320926).

[0027] (3) Instruments: 6465B Triple Quad LC / MS (Agilent) Ultivo; 1290 high performance liquid chromatograph (Agilent); multi-tube vortex mixer (TARGIN-TECH); Vortex (DLAB); centrifuge (Sigma).

[0028] (4) The samples were biological samples collected by the General Hospital of the Chinese People's Liberation Army.

[0029] (5) The diluent used in the preparation and sample processing method is 50% methanol (containing 0.1% formic acid).

[0030] 2. Liquid Chromatography Conditions Mobile phase A: 10 mM ammonium acetate aqueous solution (containing 0.3% formic acid); Mobile phase B: Acetonitrile solution (containing 0.3% formic acid); Needle washing solution: a mixed solution of acetonitrile / water / isopropanol / methanol / formic acid; The chromatographic column was SB-C18, RRHD 1.8µm, 2.1 × 50mm; sample acquisition time was 6.0 min, injector temperature was 8ºC; column temperature: 40ºC, injection volume: 5µL. Gradient elution was used, and the elution program is shown in Table 1. Table 1 3. Mass spectrometry conditions The ion source was an electrospray ion source, the detection mode was positive ion mode, the scanning mode was multiple ion reaction monitoring (MRM) scheme, the nebulizer gas was nitrogen, and the mass spectrometry conditions are shown in Table 2.

[0031] Table 2 Mass Spectrometry Conditions The ion pairs and mass spectrometry parameters of the analyte and internal standard are shown in Table 3: Table 3 Ion pair parameters 2. Solution preparation and sample processing methods (1) Preparation of stock solution of the analyte standard Tetracycline drugs generally suffer from stability issues. For example, minocycline requires moisture-proof storage, omalicycline standards require moisture-proof and light-protected storage, and eracycline stock solution is unstable in solution. To ensure stability, the entire preparation process is carried out under light, and all samples are prepared in an ice bath, thus guaranteeing stability.

[0032] The powders of eracycline, minocycline, tigecycline, and omalicycline are dissolved in a solution. Preferably, standard stock solutions of eracycline, minocycline, tigecycline, and omalicycline are prepared with an appropriate amount of dimethyl sulfoxide (DMSO) to a concentration of 1.00 mg / mL and stored at -80ºC for later use.

[0033] (2) Preparation of internal standard working solution Oxytetracycline and tigecycline-d9 were used as internal standards to avoid interference with the four analyte drugs, thus ensuring the accuracy of the test results. Appropriate amounts of oxytetracycline and tigecycline-d9 standards were dissolved and prepared into 1.00 mg / mL internal standard stock solutions, which were stored at -80°C. An appropriate amount of the internal standard stock solution was then used to prepare an internal standard working solution of suitable concentration using an organic solvent. The solution used to prepare the internal standard working solution was methanol, acetonitrile, or a mixture of methanol and acetonitrile. Preferably, methanol, a protein precipitant, was used to prepare the mixed internal standard working solution, with concentrations of oxytetracycline and tigecycline-d9 of 500 ng / mL and 25 ng / mL, respectively.

[0034] (3) Preparation of standard curve working solution and quality control working solution Take appropriate amounts of the stock solutions of eracycline, minocycline, tigecycline, and omalicycline, and add them separately to diluent to prepare standard curve working solutions of 8 concentrations. Then, take appropriate amounts of the stock solutions of the three drugs and add them separately to diluent to prepare quality control sample working solutions of 4 concentrations. The diluent is 50% methanol (containing 0.1% formic acid). The specific procedures are as follows: Preparation of working solutions for standard curves at different concentrations: Accurately pipette 13 µL each of iracycline, minocycline, tigecycline and omalicycline stock solutions, add 208 µL of 50% methanol solution (containing 0.1% formic acid) to prepare the upper limit of quantitation working solution, and then dilute sequentially from high to low concentration to obtain 8 working solutions for standard curves at different concentrations.

[0035] Preparation of working solutions for quality control samples at four different concentrations: Accurately pipette 12 µL each of the stock solutions of eracycline, minocycline, tigecycline and omalicycline, add 352 µL of 50% methanol solution (containing 0.1% formic acid) to prepare HQC working solutions, and then dilute them sequentially from high to low concentration to obtain working solutions for quality control samples at four different concentrations.

[0036] The working solution concentrations for the standard curve were 400, 800, 2000, 4000, 10000, 20000, 40000, and 50000 ng / mL; the working solution concentrations for the quality control samples were 400, 1200, 12000, and 30000 ng / mL.

[0037] (4) Preparation of standard curve samples and quality control samples Because plasma is a commonly used clinical specimen, it is easy to obtain and can comprehensively reflect the utilization of drugs in the human body; the working solution is diluted with plasma, and the dilution factor can be up to 20 times, and a series of standard curve concentrations are prepared in sequence.

[0038] Specifically, the eight standard curve working solutions and four quality control working solutions of different concentrations prepared in (3) were diluted in a certain proportion into the plasma matrix (containing K2EDTA anticoagulant). Specifically, 10.0 µL of each concentration mixed working solution in (3) was added to 190 µL of blank plasma matrix to obtain the standard curve samples and quality control samples of each concentration. The concentrations of eracycline, minocycline, tigecycline and omalicycline were 20 ng / mL, 40 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL and 2500 ng / mL, respectively, and the concentrations of the quality control samples were 20 ng / mL, 60 ng / mL, 600 ng / mL and 1500 ng / mL, respectively.

[0039] The linear range of the standard curve in this invention is reasonably set, ranging from 20 to 2500 ng / mL. Based on clinical pharmacokinetic studies, the peak concentrations of omalicycline, minocycline, eracycline, and tigecycline are 2116 ng / mL, 2100-5100 ng / mL, 2125 ng / mL, and 900 ng / mL, respectively. The linear range of this invention covers the sample concentrations, allowing for direct pretreatment with minimal need for dilution, thus eliminating the need for dilution steps.

[0040] (5) Pretreatment of plasma samples Plasma samples include plasma samples of various concentrations in the standard curve, quality control samples, and plasma samples from clinical biological samples, and their pretreatment methods are the same. The pretreatment method is as follows: Take 20.0~100µL of plasma sample, preferably 50.0µL, and centrifuge it in a 1.5mL centrifuge tube (EP tube) for 10 minutes. Then add 200µL of internal standard working solution, vortex for 1 minute, and centrifuge at 14000rpm (2~8°C) for 10 minutes. Take the supernatant and add it to the centrifuge tube at a ratio of 2:1 with the diluent (preferably take 200µL of supernatant and add it to 100µL of diluent). Mix well, centrifuge again (under the same conditions), and add the supernatant to the injection tube for analysis using a liquid chromatography-mass spectrometry (LC-MS / MS) instrument. The injection volume can be 2~5µL, preferably 5µL.

[0041] For double blank samples, use an equal volume of methanol instead of the internal standard working solution during processing.

[0042] (6) Sample detection: The drug concentration was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS). The mixed extract of eracycline, minocycline, tigecycline and omalicycline was injected into the HPLC system, and the chromatographic peaks of the four drugs and the internal standard were detected. The drug concentration in the clinical biological sample could then be calculated.

[0043] During testing, the concentrations of each standard curve were used ( c Using the x-axis as the horizontal axis and the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard as the vertical axis (y-axis), a regression equation is obtained, a standard curve is plotted, and the ratio is obtained through clinical sample testing to calculate the drug concentration.

[0044] 3. Standard Curve Chromatograms of four tetracycline antibiotics are shown below. Figure 1 The chromatogram for double-blank plasma detection is shown below. Figure 2 The relevant chromatograms of iracycline, tigecycline, minocycline, and omacycline are shown in [reference needed]. Figures 3-8 ; (1) Standard curve: The internal standard method was used, with drug concentration as the X-axis and the ratio of drug peak area to internal standard peak area as the Y-axis, using a weighted average of 1 / X.2 The least squares method was used for regression calculations, and a standard curve was plotted. The linear regression equations, linear ranges, and correlation coefficients for each drug are shown in Table 4 below. (Standard curves are shown below.) Figures 9-12 .

[0045] Table 4 5. Accuracy and Precision Accuracy and precision experiments were conducted in three batches over a period of at least two days. Quality control samples for accuracy and precision experiments were freshly prepared on the day of testing. Each accuracy and precision analysis batch contained quality control samples at four concentration levels: lower limit of quantitation (LLOQ), low concentration (LQC), medium concentration (MQC), and high concentration (HQC), with six samples at each concentration. The accuracy and precision of all four compounds met the requirements, as shown in Table 5.

[0046] Table 5 Table 6. Extraction Recovery Rate Recovery rates were calculated by comparing the response values ​​of the analytes and internal standards in the extracted and unextracted samples. As shown in Table 6, the extraction recovery precision for the four compounds at low, medium, and high concentrations was within 15.0%, and the extraction recovery precision across the three concentrations was also within 15.0%, meeting industry standards.

[0047] In summary, this invention provides a method for simultaneously detecting the plasma concentrations of eracycline, minocycline, tigecycline, and omalicycline using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Oxytetracycline and tigecycline-d9 are used as internal standards to increase the accuracy of the detection results. This method exhibits good linearity, high sensitivity, short analysis time, strong specificity, and good accuracy and precision.

[0048] The above embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or improve the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features, within the scope of the technology disclosed in the present invention; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS), characterized in that, Includes the following steps: Standard stock solutions of four tetracycline drugs were prepared under yellow light and ice bath conditions. The standard stock solution was diluted to prepare 8 standard curve working solutions and 4 quality control working solutions of different concentrations. The working solutions for each concentration of the standard curve and the working solutions for quality control were diluted into the plasma matrix to obtain the samples and quality control samples at each concentration in the standard curve; The samples and quality control samples of each concentration in the standard curve were mixed with the pre-prepared internal standard working solution, vortexed, centrifuged, and the supernatant was mixed with a certain proportion of diluent. An appropriate amount of solution was taken and analyzed by liquid chromatography-mass spectrometry. The standard curve was plotted with the drug concentration as the X-axis and the ratio of the drug peak area to the internal standard peak area as the Y-axis. as well as The clinical test sample was mixed with the pre-prepared internal standard working solution, vortexed, centrifuged, and the supernatant was mixed with a certain proportion of diluent. An appropriate amount of the solution was then analyzed using a liquid chromatography-mass spectrometry (LC-MS) system. The drug concentration in the clinical biological sample was then calculated based on the standard curve. The processing of plasma samples from each concentration in the standard curve, the quality control samples, and the clinical biological samples was carried out under yellow light and ice bath conditions, as follows: 50 µL of plasma sample was placed in a 1.5 mL centrifuge tube, and 200 µL of the internal standard working solution was added. After vortexing, the sample was centrifuged at 2-8℃ and 14000 rpm. The supernatant was added to the centrifuge tube at a ratio of 2:1 with the diluent, mixed thoroughly, and centrifuged again. The supernatant was then added to the sample injection tube and analyzed using a liquid chromatography-mass spectrometry (LC-MS) system.

2. The method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, The concentration of the standard stock solutions of eracycline, minocycline, tigecycline and omalicycline is 1.00 mg / mL.

3. The method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, The internal standard working solution is a mixed internal standard working solution of oxytetracycline and tigecycline-d9 prepared with methanol, wherein the concentration of oxytetracycline in the internal standard working solution is 500 ng / mL and the concentration of tigecycline-d9 is 25 ng / mL.

4. The method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, The standard curves were prepared under yellow light and ice bath conditions. The concentrations of the standard curves for eracycline, minocycline, tigecycline, and omalicycline were 20 ng / mL, 40 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, and 2500 ng / mL, respectively. The concentrations of the quality control samples were 20 ng / mL, 60 ng / mL, 600 ng / mL, and 1500 ng / mL, respectively.

5. The method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, When analyzing using a liquid chromatography-mass spectrometry (LC-MS) system: The liquid chromatography conditions are as follows: Mobile phase A is an aqueous solution of ammonium acetate with a concentration of 10 mM and containing 0.3% formic acid; mobile phase B is an acetonitrile solution containing 0.3% formic acid; the washing solution is a mixed solution of acetonitrile, water, isopropanol, methanol, and formic acid; the gradient elution program is 0.00~1.00 min, and the proportion of mobile phase B is 3%; 1.00-1.70 min, the proportion of mobile phase B is 3-7%; 1.70-2.50 min, the proportion of mobile phase B is 7-98%; 2.50~4.0 min, the proportion of mobile phase B is 98%; 4.01~6.00 min, the proportion of mobile phase B is 3%; the elution flow rate is 0.2 mL / min, the injection volume is 2~5 µL, and the sample collection time is 6.00 min; The mass spectrometry conditions were as follows: the ion source was an electrospray ion source, the detection mode was positive ion mode, the scanning mode was multiple ion reaction monitoring (MRM), the drying gas temperature was 350℃, the drying gas flow rate was 5 L / min, the nebulizing gas pressure was 30 psi, the auxiliary gas temperature was 350℃, the auxiliary gas flow rate was 11 L / min, and the capillary voltage was 4000 V.

6. The method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS) according to claim 5, characterized in that, The chromatographic column used in the liquid chromatography conditions was an Agilent SB-C18, RRHD 1.8µm, 2.1 × 50mm; the injector temperature was 4~8℃, and the column temperature was 40℃.

7. The method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS) according to claim 5, characterized in that, The injection washing solution contains 0.1% formic acid, and the volume ratio of acetonitrile, water, methanol and isopropanol is 1:1:1:

1.

8. The method for simultaneously detecting the plasma concentrations of four tetracycline drugs—eiracycline, minocycline, tigecycline, and omalicycline—based on liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, The solvent used to prepare the standard stock solution is dimethyl sulfoxide, and the diluent is a 50% methanol aqueous solution containing 0.1% formic acid.

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