Simultaneous determination of 12 antibiotics in serum by HPLC with fluorescence detection

Abstract

The application discloses a detection kit for simultaneously determining the concentrations of 12 kinds of antibiotic drugs in trace serum and application, the 12 kinds of antibiotics including vancomycin, meropenem, imipenem, cilastatin, trimethoprim, linezolid, voriconazole, piperacillin, cefoperazone, sulfamethoxazole, cefepime and ceftazidime; the detection kit comprises pretreatment reagents and a liquid chromatography mobile phase; the pretreatment reagents comprise a protein precipitant and purified water; the protein precipitant comprises 50v / v% of methanol and 50v / v% of acetonitrile; the liquid chromatography mobile phase comprises phase A and phase B, the phase A comprises 0.02% of formic acid and 99.98% of water, and the phase B comprises 100% of acetonitrile. The method of the application only needs 10 muL of human serum sample, and can complete the detection within 4 min, can quantitatively determine 12 kinds of antibiotics with significant structural differences, and is simple, rapid, sensitive, accurate and specific.

Description

Technical Field

[0001] This invention relates to the field of biomedical analysis technology, specifically to the subfield of therapeutic drug monitoring (TDM) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection, and particularly to a detection kit and its application for simultaneously determining the concentration of 12 antibiotics in trace amounts of serum. Background Technology

[0002] Currently, commonly used traditional methods for drug concentration detection include liquid chromatography and immunoassay. Although these methods offer certain advantages in terms of ease of operation, lowering the operational threshold for basic detection scenarios, they still reveal significant limitations in practical applications: on the one hand, due to limitations in detection principles or reagent specificity, they are susceptible to interference and cross-reactions, affecting the reliability of detection results; on the other hand, these methods lack the ability to differentiate drug molecular forms, failing to distinguish between the parent drug and metabolites, and have poor adaptability in scenarios involving simultaneous detection of multiple drugs, making it difficult to meet the current higher requirements for detection accuracy, analytical dimensions, and efficiency.

[0003] In recent years, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has effectively overcome the shortcomings of traditional methods due to its excellent high sensitivity, high specificity, and ability to analyze multiple components simultaneously. It has gradually become the recognized "gold standard" in the field of therapeutic drug monitoring, providing technical support for concentration monitoring in scenarios involving the combined use of multiple drugs.

[0004] However, current research on LC-MS / MS in the field of antibiotic detection still has significant shortcomings: most studies focus on the detection of single or a few antibiotics, making it difficult to cover the common clinical need for multi-antibiotic combination therapy; although some studies have reported LC-MS / MS methods that can simultaneously detect multiple antibiotics, such as analytical protocols that can simultaneously detect 9, 10, 12, 18, and 19 antibiotics, these existing methods still have three key deficiencies, making it difficult to fully adapt to the needs of actual clinical applications: 1. Low detection efficiency: The processing time for a single sample is relatively long, usually at least 6 minutes, which cannot meet the needs of rapid drug concentration detection results in clinical diagnosis and treatment, and may affect the timely adjustment of treatment plans.

[0005] 2. Large sample requirement: A single test requires more than 100 μL of serum sample. However, blood samples from special populations such as anemic patients, elderly patients, and newborns are difficult to collect and the total amount is limited. Therefore, this method is not very applicable to these populations.

[0006] 3. Insufficient detection coverage: Currently, there is no mature LC-MS / MS method for simultaneously measuring the concentrations of 12 antibiotics in serum, including vancomycin, linezolid, voriconazole, sulfamethoxazole, trimethoprim, piperacillin, cefoperazone, ceftazidime, cefbirol, meropenem, imipenem, and cilastatin. There are still significant gaps in the relevant technical field, which cannot provide effective technical support for monitoring the concentrations of these antibiotics in combination. Summary of the Invention

[0007] The purpose of this invention is to provide a detection kit and its application for simultaneously determining the concentration of 12 antibiotics in trace amounts of serum. It is a simple, rapid, sensitive and specific LC-MS / MS method for simultaneously determining the concentration of 12 antibiotics in serum, including vancomycin, linezolid, voriconazole, sulfamethoxazole, trimethoprim, piperacillin, cefoperazone, ceftazidime, cefbilor, meropenem, imipenem and cilastatin.

[0008] To address the aforementioned technical problems, this invention provides a detection kit for simultaneously determining the concentrations of 12 antibiotics in trace amounts of serum. The 12 antibiotics include: vancomycin, meropenem, imipenem, cilastatin, trimethoprim, linezolid, voriconazole, piperacillin, cefoperazone, sulfamethoxazole, cefbirol, and ceftazidime; the detection kit includes pretreatment reagents and a liquid chromatography mobile phase. The pretreatment reagents include a protein precipitant and purified water; the protein precipitant includes 50 v / v% methanol and 50 v / v% acetonitrile. The mobile phase of the liquid chromatography comprises: phase A and phase B, wherein phase A comprises 0.02% formic acid and 99.98% water, and phase B comprises 100% acetonitrile.

[0009] Based on a general technical concept, the present invention provides an application of the aforementioned detection kit in the simultaneous determination of the concentrations of 12 antibiotics in trace amounts of serum, the application method comprising: S1. Mix serum and protein precipitant at a mass ratio of 1:3, centrifuge to collect the supernatant, and dilute with purified water to obtain the treated serum sample. S2. The processed serum sample is analyzed in an LC-MS / MS system to obtain the concentrations of 12 antibiotics in the serum.

[0010] In the above application, further, the centrifugation in S1 specifically refers to centrifuging at 13000 rpm for 5 minutes at 4°C.

[0011] In the above application, further, in S1, the volume ratio of supernatant to purified water is 1:19.

[0012] Furthermore, in the above application, the liquid chromatography conditions in the LC-MS / MS system of S2 are as follows: Column: ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm); Mobile phase: Phase A is 0.02% formic acid and 99.98% water, and Phase B is 100% acetonitrile; Flow rate: 0.35 mL / min; Injection volume: 5 μL; Column temperature: 40℃; Elution program: 0 min~2.5 min: Phase A 97%→50%, Phase B 3%→50%; 2.51 min~3.30 min: Phase A 10%, Phase B 90%; 3.31 min~4.00 min: Phase A 97%, Phase B 3% → Equilibrium.

[0013] In the above application, further, in the LC-MS / MS system of S2, the mass spectrometer adopts ESI+ mode, the ion source temperature is 150℃, the desolventizing temperature is 500℃, the flow rate of desolventizing gas N2 is 800 L / h, and the flow rate of cone-shaped N2 is 50 L / h.

[0014] Furthermore, in the above application, the MRM parameters in the mass spectrometer are as follows:

[0015] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention provides a method for simultaneously determining the concentrations of 12 antibiotics in trace amounts of serum. The high efficiency of simultaneous multi-component detection significantly accelerates the therapeutic drug monitoring process. This invention overcomes the limitation of traditional LC-MS / MS detection methods, which can only detect a few antibiotics at a time. By specifically optimizing the core parameters of chromatography and mass spectrometry, it achieves simultaneous quantitative detection of 12 antibiotics with significant structural differences (including vancomycin, meropenem, etc.). The single-needle sample run time is only 4 minutes. Compared with the traditional scheme that requires multiple methods and multiple detections, it greatly shortens the detection cycle and solves the industry pain point of cumbersome and time-consuming therapeutic drug monitoring (TDM) process. It can quickly provide feedback on the concentration data of multiple antibiotics in the patient's serum, providing timely support for the precise adjustment of the dosing regimen in clinical practice and significantly improving the efficiency of TDM work.

[0016] (2) This invention provides a method for simultaneously determining the concentrations of 12 antibiotics in trace amounts of serum, covering the universality of multiple classes of antibiotics and broadening the scope of application for therapeutic drug monitoring. The 12 antibiotics detected by this invention cover multiple clinically commonly used categories such as β-lactams, azoles, and glycopeptides. The chemical properties (polarity, hydrophobicity, structural stability, etc.) of compounds in each category differ significantly (e.g., the physicochemical properties of vancomycin (a glycopeptide) and meropenem (a β-lactam) differ greatly). Through systematic optimization of chromatographic columns, mobile phases, and mass spectrometry parameters, this method successfully meets the detection needs of different classes of antibiotics, solving the problem of narrow applicability of single methods in existing technologies and the need to establish separate detection methods for different classes. This greatly broadens the application scenarios of TDM and can meet the actual needs of clinical combination drug use and multi-target monitoring, improving the practicality and promotion value of the method.

[0017] (3) This invention provides a method for simultaneously determining the concentrations of 12 antibiotics in a trace amount of serum. This method uses trace sample testing, improving patient compliance and the breadth of monitoring. This invention requires only 10 μL of human serum sample to simultaneously quantify 12 antibiotics, significantly reducing sample volume compared to the 50 μL or more required by traditional TDM methods. This miniaturized design is particularly suitable for special populations with difficult blood collection, such as children, the elderly, and critically ill patients, effectively reducing the physical burden of blood collection on patients and significantly improving patient compliance with TDM. Simultaneously, the need for trace samples expands the application scenarios of TDM (such as primary healthcare institutions and rapid emergency monitoring), solving the problem of limited monitoring coverage caused by high sample volume requirements in existing methods, and promoting the widespread application of TDM technology.

[0018] (4) This invention provides a method for simultaneously determining the concentrations of 12 antibiotics in trace serum samples. Addressing the technical contradictions of low recovery rates (only about 30%) of glycopeptide antibiotics (such as vancomycin) in pure acetonitrile and poor precipitation effects in pure methanol (due to numerous sample impurities and insufficient stability), this invention innovatively screens a mixed precipitant system of methanol:acetonitrile = 1:1 (v / v). This scheme not only increases the vancomycin recovery rate to over 60%, but also achieves satisfactory recoveries for all 12 analytes, while ensuring clear supernatant and minimal impurity residues after sample processing. This solves the industry problem of balancing recovery rate and precipitation effect during pretreatment. Furthermore, this pretreatment process is well-suited for 10 μL trace samples, effectively reducing sample loss and improving the sensitivity and reliability of the method, thus ensuring accurate quantification of trace antibiotics in trace samples.

[0019] (5) This invention provides a method for simultaneously determining the concentrations of 12 antibiotics in trace serum. By optimizing the selection of an ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column and matching it with an optimized scheme using 0.02% formic acid aqueous solution - 100% acetonitrile solution as the mobile phase, 95% high aqueous phase as the gradient starting ratio, and a flow rate of 0.35 mL / min, this invention successfully solves the technical problems of inconsistent retention capacity, poor peak shape, and severe matrix interference of antibiotics with different polarities on the chromatographic column. The high aqueous phase starting gradient design ensures that highly polar compounds obtain sufficient retention time, avoiding peak broadening or overlap caused by early elution; the combination of slow gradient elution and a specific chromatographic column effectively separates matrix impurities from target analytes, reduces matrix effects, and improves the selectivity and sensitivity of the method; at the same time, the optimized flow rate of 0.35 mL / min compresses the single-needle run time to 4 min while ensuring separation effect, achieving a perfect balance between separation efficiency and detection speed.

[0020] (6) This invention provides a method for simultaneously determining the concentrations of 12 antibiotics in trace serum. Addressing the issues of large differences in the responses of the 12 antibiotics and the potential for detector saturation and poor linearity in some high-response, high-concentration compounds (such as sulfamethoxazole, trimethoprim, and piperacillin), this invention effectively avoids signal saturation by precisely optimizing the collision energy of these compounds, ensuring good linearity for all 12 analytes. This optimization scheme solves the technical pain points of inaccurate quantification and narrow linear range caused by response differences in multi-component simultaneous detection, ensuring the accuracy of antibiotic detection results across different concentration ranges, meeting the core requirements of TDM for accurate drug concentration quantification, and providing reliable data support for clinical dosage adjustment. Attached Figure Description

[0021] 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.

[0022] Figure 1 The chromatograms are of 12 antibacterial agents obtained by the method of the present invention in Experiment 1 of the present invention.

[0023] Figure 2 The results are from the detection conducted in Experiment 3 of this invention using Group 1 and Group 2 as the mobile phase, respectively.

[0024] Figure 3 This is a comparison of the standard curves of piperacillin and sulfamethoxazole under different mass spectrometry parameters in Experiment 4 of this invention.

[0025] Figure 4The results of Deming regression analysis were performed on the concentrations of 12 antibiotics in fingertip blood and venous blood in Experiment 5 of this invention. Detailed Implementation

[0026] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0027] All materials, reagents, and instruments used in the following examples were commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Vancomycin (100% purity), meropenem (86.8% purity), imipenem (93.2% purity), cilastatin (94.9% purity), and trimethoprim (99.8% purity) were purchased from the China National Institutes for Food and Drug Control (Beijing, China). Linezolid (99.61% purity), voriconazole (98.43% purity), piperacillin sodium (96.15% purity), cefoperazone sodium (98.32% purity), and sulfamethoxazole (99.78% purity) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cefbirol (92.0% purity) was purchased from Shenyang Sanjiu Pharmaceutical Co., Ltd. (Shenyang, China). Ceftazidime (99.9% purity) was purchased from CFW Laboratories, Inc. (Walnut, USA). Acetonitrile and methanol (HPLC grade) were purchased from Merck, Inc. (Darmstadt, Germany). Formic acid (HPLC grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO, HPLC grade) was supplied by Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ultrapure water was prepared using a GenePure XCAD plus system purchased from Thermo Fisher Scientific, Inc. (Thermo Fisher Scientific, Germany).

[0028] To validate the method, blank serum used to assess matrix effects, specificity, matrix stability, and to prepare calibration curve samples and quality control samples (QCs) was obtained from anonymous patients after obtaining approval from the institutional review board.

[0029] Example 1 A test kit for simultaneously determining the concentrations of 12 antibiotics in trace amounts of serum, including a protein precipitant.

[0030] The protein precipitant is a mixed solvent of methanol and acetonitrile (50:50, v / v).

[0031] Example 2 One embodiment of the detection kit is used for the simultaneous detection of the concentrations of 12 antibiotics in trace amounts of serum, and the method of application includes: (1) Serum sample pretreatment: 1.1 Transfer 10.0 μL of serum sample and 30.0 μL of protein precipitant to an Eppendorf tube and vortex for 5.0 minutes; 1.2 Then, centrifuge the sample at 13,000 rpm for 5 minutes at 4°C; 1.3 Next, add 380 μL of purified water to 20.0 μL of supernatant. Shake thoroughly for 5.0 minutes; 1.4. Inject the analytical solution into the liquid chromatography-tandem mass spectrometry system for analysis.

[0032] (2) LC-MS / MS system analysis.

[0033] 2.1 Liquid chromatography conditions.

[0034] Column: ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm).

[0035] Mobile phase: Phase A (0.02% formic acid and 99.98% water), Phase B (100% acetonitrile).

[0036] Flow rate: 0.35 mL / min; injection volume: 5 μL; column temperature: 40℃.

[0037] Elution program: 0 min~2.5 min: Phase A 97%→50%, Phase B 3%→50%; 2.51 min~3.30 min: Phase A 10%, Phase B 90%; 3.31 min~4.00 min: Phase A 97%, Phase B 3% → Equilibrium.

[0038] 2.2 After 0.3 minutes, the switching valve is switched to mass spectrometry (MS). After 3.0 minutes, the switching valve is switched back to waste liquid. Mass spectrometry conditions: (ESI+ mode, MRM parameters are shown in Table 1) Capillary voltage: 2.5 kV, ion source temperature: 150℃; desolvation temperature: 500℃.

[0039] Nitrogen gas was used as both the desolventizing gas and the conical gas. The flow rate of the desolventizing gas (N2) was 800 L / h, and the flow rate of the conical gas (N2) was 50 L / h.

[0040] Table 1: Mass spectrometry parameters of 12 antibiotics

[0041] In Table 1, the linear ranges of the 12 compounds are inconsistent, and their responses in LC-MS / MS are also inconsistent. For compounds with high response and high concentration, such as sulfamethoxazole, trimethoprim, and piperacillin, the response in the instrument is prone to saturation, which prevents the upper limit of quantitation from reaching the target sample response and results in poor linearity. Therefore, we optimized the collision energy of compounds such as sulfamethoxazole, trimethoprim, and piperacillin to avoid detector saturation and improve the linearity of the method.

[0042] (3) Quantitative analysis.

[0043] 3.1. Plot the standard curve: The calibration curve was generated using the least squares regression method, with 1 / x 2 As a weighting factor for peak area.

[0044] Prepare eight concentration gradient standard solutions of vancomycin (0.400, 0.800, 2.00, 4.00, 12.0, 24.0, 72.0, and 80.0 μg / mL) within the concentration range of 0.400–80.0 μg / mL, and inject them into the LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area of ​​standard" on the y-axis.

[0045] Prepare eight concentration gradient standard solutions of linezolid (0.100, 0.200, 0.500, 1.00, 3.00, 6.00, 18.0, and 20.0 μg / mL) within the concentration range of 0.100–20.0 μg / mL, and inject them into the LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0046] Prepare eight standard solutions of voriconazole standard at concentrations ranging from 0.100 to 20.0 μg / mL (0.100, 0.200, 0.500, 1.00, 3.00, 6.00, 18.0, and 20.0 μg / mL), and inject them into an LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0047] Eight standard solutions of sulfamethoxazole were prepared at concentrations ranging from 1.20 to 240 μg / mL (1.20, 2.40, 6.00, 12.0, 36.0, 72.0, 216, and 240 μg / mL), and injected into an LC-MS / MS system for detection. A calibration curve was plotted with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0048] Prepare eight concentration gradient standard solutions of trimethoprim (0.100, 0.200, 0.500, 1.00, 3.00, 6.00, 18.0, and 20.0 μg / mL) within the concentration range of 0.100–20.0 μg / mL, and inject them into the LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0049] Prepare eight standard solutions of piperacillin standard at concentrations ranging from 2.50 to 500 μg / mL (2.50, 5.00, 12.5, 25.0, 75.0, 150, 450, and 500 μg / mL), and inject them into an LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0050] Prepare eight standard solutions of cefoperazone standard at concentrations ranging from 2.50 to 500 μg / mL (2.50, 5.00, 12.5, 25.0, 75.0, 150, 450, and 500 μg / mL), and inject them into an LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0051] Prepare eight concentration gradient standard solutions of ceftazidime (0.100, 0.200, 0.500, 1.00, 3.00, 6.00, 18.0, and 20.0 μg / mL) within the concentration range of 1.00–200 μg / mL, and inject them into the LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0052] Prepare eight concentration gradient standard solutions of ceftobiprole (0.300, 0.600, 1.50, 3.00, 9.00, 18.0, 54.0, and 60.0 μg / mL) within the concentration range of 0.300–60.0 μg / mL, and inject them into the LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0053] Prepare eight standard solutions of meropenem standard at concentrations ranging from 0.300 to 60.0 μg / mL (0.300, 0.600, 1.50, 3.00, 9.00, 18.0, 54.0, and 60.0 μg / mL), and inject them into an LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0054] Prepare eight concentration gradient standard solutions of imipenem (0.300, 0.600, 1.50, 3.00, 9.00, 18.0, 54.0, and 60.0 μg / mL) within the concentration range of 0.300–60.0 μg / mL, and inject them into the LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0055] Prepare eight standard solutions of cystatin standard at concentrations ranging from 0.300 to 60.0 μg / mL (0.300, 0.600, 1.50, 3.00, 9.00, 18.0, 54.0, and 60.0 μg / mL), and inject them into an LC-MS / MS system for detection. Plot a calibration curve with "standard concentration" on the x-axis and "characteristic ion peak area" on the y-axis.

[0056] The linear range, regression equation, correlation coefficient, and lower limit of quantitation for 12 antibacterial drugs are listed in Table 2.

[0057] Table 2: Linear range, regression equation, correlation coefficient and lower limit of quantification of 12 antibacterial drugs.

[0058]

[0059] The results in Table 2 show that the 12 antibacterial drugs exhibit good linearity, with r² > 0.99. The limit of quantitation (LLOQ) for each analyte was determined as the lowest point of the calibration curve, and the signal-to-noise ratio (S / N) for each LLOQ was greater than 10.

[0060] 3.2 Concentration Calculation: Substitute the "target peak area" in the sample into the calibration curve to calculate the actual concentration of antibiotic in the sample.

[0061] Experiment 1: To examine the precision, accuracy, matrix effect, and recovery rate of the method in Example 2.

[0062] Four human serum samples of different concentrations were prepared and tested according to the method in Example 1. Precision, accuracy, matrix effect, and recovery were statistically analyzed, and the results are listed in Table 3.

[0063] Table 3: Precision, accuracy, matrix effect and recovery rate of quantification of 12 antibiotics in human serum.

[0064]

[0065] The results in Table 3 show that at the lower limit of quantitation (LLOQ), the coefficient of variation (CV) for all compounds ranged from 0.8% to 19.3%; at other concentration levels, the CV ranged from 0.6% to 12.0%. The deviation of accuracy (DEV) for all compounds ranged from -12.4% to 13.2%. Therefore, this method meets the requirements of the relevant guidelines for bioanalytical method validation. The average extraction recoveries for all compounds ranged from 60.4% to 113.9%, while the matrix effect ranged from 86.1% to 107.5%. All CVs were below 15.0%. These results indicate that pretreatment of plasma samples by protein precipitation can achieve stable extraction efficiency without significant interference from the serum matrix.

[0066] Figure 1 The figures show chromatograms of 12 antibacterial agents obtained using the method of this invention. In the figures, A represents a blank human serum sample; B represents a blank serum sample with the limit of quantitation (LLOQ) added; C represents a blank serum sample with the limit of quantitation (ULOQ) added; and D represents a typical clinical serum sample. The figures show that the peak areas of the 12 antibiotics studied, at their respective retention times, are all within the range of 0.0% to 8.9% of the peak area of ​​the corresponding channel in the LLOQ sample. Furthermore, analysis of ULOQ samples containing only one of the 12 antibiotics showed that the interfering peak areas between these antibiotics were all less than 15.0% of the peak area of ​​the corresponding analyte in the LLOQ. Therefore, the selectivity and specificity of this analytical method meet acceptable standards.

[0067] Experiment 2: Investigate the effect of different precipitants on the recovery rate.

[0068] Six precipitant systems were set up, each with a volume of 150 μL.

[0069] Group 1: Pure acetonitrile; Group 2: Pure methanol; Group 3: Methanol:acetonitrile = 3:7 (v / v); Group 4: Methanol:acetonitrile = 5:5 (v / v); Group 5: Methanol: Acetonitrile = 7:3 (v / v).

[0070] The method in Example 2 was used to detect healthy human serum without the target analyte (with no interfering peaks verified by LC-MS / MS). Table 4 shows the statistical results of the recovery rate of healthy human serum analyzed using groups 1 to 5 as precipitants.

[0071] Table 4: Statistical results of recovery rates for different precipitants.

[0072]

[0073] The results in Table 4 show that when pure acetonitrile was used as the precipitant, the recoveries of vancomycin and imipenem were low, only around 30%. Methanol, as the precipitant, achieved higher extraction recoveries, but the precipitation effect was poor, resulting in contaminated samples and affecting the stability of the method. Using a methanol-acetonitrile (1:1, v / v) mixed solvent as the protein precipitant, satisfactory extraction recoveries were obtained for all 12 target compounds (recoveries of vancomycin and imipenem exceeded 60%, while recoveries of the remaining compounds ranged from 80% to 110%).

[0074] Experiment 3: Investigating the effect of liquid chromatography parameters on analytes.

[0075] Since the properties of each analyte are different, their retention capacity and behavior on the chromatographic column are inconsistent. The effects of different elution procedures on the analytes were investigated.

[0076] Group 1: Column: ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm). Flow rate: 0.35 mL / min; Injection volume: 5 μL; Column temperature: 40℃.

[0077] Mobile phase: Phase A (0.1% formic acid and 99.9% water), Phase B (0.1% formic acid and 99.9% acetonitrile).

[0078] Elution program: 0 min~2.5 min: Phase A 97%→50%, Phase B 3%→50%; 2.51 min~3.30 min: Phase A 10%, Phase B 90%; 3.31 min~4.00 min: Phase A 97%, Phase B 3% → Equilibrium.

[0079] Group 2: Column: ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm). Flow rate: 0.35 mL / min; Injection volume: 5 μL; Column temperature: 40℃.

[0080] Mobile phase: Phase A (0.02% formic acid and 99.98% water), Phase B (100% acetonitrile).

[0081] Elution program: 0 min~2.5 min: Phase A 97%→50%, Phase B 3%→50%; 2.51 min~3.30 min: Phase A 10%, Phase B 90%; 3.31 min~4.00 min: Phase A 97%, Phase B 3% → Equilibrium.

[0082] Figure 2 The results are shown for detection using Group 1 and Group 2 as mobile phases, respectively. As can be seen from the figure, when using 0.1% formic acid water + 0.1% formic acid acetonitrile as the mobile phase, the response values ​​of ceftazidime (A1) and cefoperazone (B1) (2.478e+003 and 1.295e+003, respectively) are significantly lower than those using 0.02% formic acid water + 100% acetonitrile as the mobile phase (A2: 1.751e+004, B2: 3.441e+005), indicating that the latter can improve the detection sensitivity of these two compounds.

[0083] From C1 and C2 in the figure, we can see that under the two mobile phase conditions, the correlation coefficient (r) of trimethoprim is... 2 The correlation coefficients for group 2 (trimethoprim and 0.97877 and 0.991599 respectively) were greater than 0.98 and closer to 1, indicating that using a mobile phase of 0.02% formic acid water + 100% acetonitrile can ensure good linearity and meet the requirements for quantitative analysis.

[0084] In addition, by optimizing the initial ratio of the mobile phase, gradient, flow rate and other parameters, this invention ensures that highly polar compounds have a certain retention capacity on the chromatographic column, obtain good peak shape and sensitivity, and at the same time reduces the separation of matrix effects and matrix interference, improving the selectivity and sensitivity of the method. With a flow rate of 0.35 mL / min, the single-needle sample run time is only 4 min.

[0085] Experiment 4: Investigating the effect of mass spectrometry parameters on the linearity of the method.

[0086] The linear ranges of the 12 compounds were inconsistent, as were their responses in LC-MS / MS. For compounds with high responses and high concentrations, such as piperacillin and sulfamethoxazole, the response in the instrument was prone to saturation, preventing the upper limit of quantitation from being reached and resulting in poor linearity. Therefore, we adjusted the collision energy in the mass spectrometer for each compound.

[0087] For piperacillin: according to the mass spectrometry parameters in Table 1 of Example 1, the linearity of the standard curves was investigated for A1: cone voltage (cone=38) + collision energy (Collision=20) and A2: cone voltage (cone=34) + collision energy (Collision=20).

[0088] Figure 3 A1 and A2 in the figure represent the standard curves of piperacillin with two different mass spectrometry parameters. A1 shows that while piperacillin achieves the highest response, high-concentration samples exhibit response saturation, resulting in poor linearity of the standard curve. In A1, r... 2 =0.879750, deviating from the ideal value). Adjusting the piperacillin parameters to cone=34+Collision=20 (a non-optimal combination) resolved the response saturation problem in high-concentration samples, and the linearity of the standard curve was significantly improved (r in A2). 2 =0.998579, close to 1) For sulfamethoxazole: according to the mass spectrometry parameters in Example 1, the linearity of the standard curves was investigated for B1: cone voltage (cone=32) + collision energy (Collision=18) and B2: cone voltage (cone=50) + collision energy (Collision=18).

[0089] Figure 3 B1 and B2 in the figure represent the standard curves of sulfamethoxazole with two different mass spectrometry parameters. From B1, it can be seen that when using the optimal cone voltage (cone=32) + collision energy (Collision=18), the problem of "high response but high concentration saturation" also occurs, and the linearity of the standard curve is poor (r in B1). 2 =0.937004). Adjusting the sulfamethoxazole parameters to cone=50+Collision=18 (a non-optimal combination) also eliminated high concentration saturation, and the standard curve showed good linearity (r in B2). 2 =0.997145).

[0090] Therefore, we optimized the collision energies of compounds such as piperacillin and sulfamethoxazole to avoid detector saturation and achieve good linearity in our method.

[0091] Experiment 5: To examine the detection effects of venous blood sampling and fingertip blood sampling.

[0092] A total of 379 paired blood samples (venous and capillary blood samples) were collected from patients treated with vancomycin (60 cases), linezolid (42 cases), voriconazole (43 cases), trimethoprim / sulfamethoxazole (37 cases), piperacillin (36 cases), cefoperazone (30 cases), ceftazidime (25 cases), cefbirol (21 cases), meropenem (45 cases), and imipenem-cilastatin (40 cases).

[0093] Figure 4 Deming regression analysis was performed on the concentrations of 12 antibiotics in finger-prick and venous blood. The antibiotics included vancomycin (VAN), linezolid (LIZ), voriconazole (VRO), sulfamethoxazole (SMZ), trimethoprim (TMP), piperacillin (PIP), cefoperazone (CEF), ceftazidime (CTD), cefbirol (CBL), meropenem (MEP), imipenem (IMP), and cilastatin (CST).

[0094] The figure shows a strong linear correlation between the concentrations of each antibiotic in finger-prick blood and venous blood, with regression slopes ranging from 0.910 to 1.03, and both the slope and intercept within the 95% confidence interval. Therefore, from a statistical perspective, the concentrations of these 12 antibiotics in venous blood and finger-prick blood samples can be considered equivalent.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A detection kit for simultaneously determining the concentrations of 12 antibiotics in trace amounts of serum, characterized in that, The 12 antibiotics include: vancomycin, meropenem, imipenem, cilastatin, trimethoprim, linezolid, voriconazole, piperacillin, cefoperazone, sulfamethoxazole, cefbirol, and ceftazidime; the detection kit includes pretreatment reagents and a liquid chromatography mobile phase; The pretreatment reagents include a protein precipitant and purified water; the protein precipitant is a mixed solvent comprising 50 v / v% methanol and 50 v / v% acetonitrile. The mobile phase of the liquid chromatography comprises: phase A and phase B, wherein phase A comprises 0.02% formic acid and 99.98% water, and phase B comprises 0.1% formic acid and 99.9% acetonitrile.

2. The application of the detection kit according to claim 1 in the simultaneous determination of the concentrations of 12 antibiotics in trace amounts of serum, characterized in that, The application method includes: S1. Mix serum and protein precipitant at a mass ratio of 1:3, centrifuge to collect the supernatant, and dilute with purified water to obtain the treated serum sample. S2. The processed serum sample is analyzed in an LC-MS / MS system to obtain the concentrations of 12 antibiotics in the serum.

3. The application according to claim 2, characterized in that, The centrifugation in S1 specifically refers to centrifuging at 13,000 rpm for 5 minutes at 4°C.

4. The application according to claim 2, characterized in that, In S1, the volume ratio of supernatant to purified water is 1:

19.

5. The application according to claim 2, characterized in that, The liquid chromatography conditions in the LC-MS / MS system of S2 are as follows: Column: ACQUITY UPLC BEH C18 (2.1mm × 50mm, 1.7μm); Mobile phase: Phase A consists of 0.02 v / v formic acid and 99.98 v / v water, and Phase B consists of 100 v / v acetonitrile; Flow rate: 0.35 mL / min; Injection volume: 5 μL; Column temperature: 40℃; Washing procedure: 0 min~2.5 min: Phase A 97%→50%, Phase B 3%→50%; 2.51 min~3.30 min: Phase A 10%, Phase B 90%; 3.31 min~4.00 min: Phase A 97%, Phase B 3% → Equilibrium.

6. The application according to claim 2, characterized in that, The mass spectrometer in the LC-MS / MS system of S2 uses ESI. + The mode was set with an ion source temperature of 150℃, a desolventizing temperature of 500℃, a desolventizing gas N2 flow rate of 800 L / h, and a conical N2 flow rate of 50 L / h.

7. The application according to claim 6, characterized in that, The MRM parameters in the mass spectrometer are:

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