Method for determining concentration of cefixime in K2EDTA human plasma by liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of chromatographic detection, in particular to a method for determining the concentration of cefixime in K2EDTA human plasma through liquid chromatography-tandem mass spectrometry. Comprising the following steps: preparing a standard curve solution containing cefixime-13C-15N2 by using K2EDTA (Ethylene Diamine Tetraacetic Acid) human plasma as a matrix, preparing a test solution containing cefixime, and precipitating the solutions by using acetonitrile; determining the concentration of the cefixime test solution after precipitation by using an internal standard method; a liquid chromatography tandem mass spectrometer is used for determination, a mobile phase of liquid chromatography is composed of a mobile phase A and a mobile phase B, the mobile phase A is an aqueous solution containing 10 mM ammonium acetate, and the mobile phase B is acetonitrile. The invention provides an efficient and accurate cefixime concentration detection method by optimizing chromatographic conditions and mass spectrum parameters, so that the accuracy and reliability of data are ensured, and the requirements of biological sample analysis in clinical tests are met.

Description

Technical Field

[0001] This invention relates to the field of chromatographic detection technology, specifically to a method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Background Technology

[0002] Cefixime, a third-generation oral cephalosporin antibiotic, is widely used to treat infectious diseases of the respiratory and urinary systems caused by susceptible bacteria. Because its clinical efficacy is closely related to plasma drug concentration, accurate and sensitive detection methods are needed to monitor plasma cefixime concentrations, providing data support for pharmacokinetic studies and clinical medication guidance. Currently, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) has become the mainstream technique for quantitative analysis of cefixime in biological matrices due to its combination of high separation efficiency and high detection sensitivity. Currently, the mobile phase for LC-MS / MS detection of cefixime often uses an aqueous solution containing formic acid / ammonium acetate and an organic phase combination of acetonitrile / methanol. However, its robustness and accuracy still need improvement to meet the stringent requirements of biological sample analysis in clinical trials.

[0003] Chinese invention patent application CN111521706A discloses a method for rapid detection of cefixime concentration in plasma. In the liquid chromatography part, mobile phase A (0.1% formic acid + 5mM ammonium acetate aqueous solution) and mobile phase B (0.5% formic acid acetonitrile) are eluted at a ratio of 50:50. However, the elution time is relatively long, reaching 2.4 min, and the detection accuracy and precision are not investigated. Summary of the Invention

[0004] This invention provides a method for determining the concentration of cefixime in K2EDTA-treated human plasma using liquid chromatography-tandem mass spectrometry, comprising the following steps: preparing a cefixime-treated plasma matrix using K2EDTA-treated human plasma as a substrate. 13 C- 15 Prepare a standard curve solution of N2 and a test solution containing cefixime. Precipitate all solutions with acetonitrile. Determine the concentration of cefixime in the test solution after precipitation using the internal standard method.

[0005] The determination was performed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) instrument. The mobile phase of the liquid chromatography consisted of mobile phase A and mobile phase B. Mobile phase A was an aqueous solution containing 10 mM ammonium acetate, and mobile phase B was acetonitrile.

[0006] This invention uses an aqueous solution containing 10 mM ammonium acetate as mobile phase A and acetonitrile as mobile phase B, significantly improving the precision and accuracy of detection. No formic acid or other modifiers are added. Cefixime, as a cephalosporin compound, contains polar functional groups in its molecular structure. 10 mM ammonium acetate provides a stable buffer environment for the mobile phase, maintaining the ionized state of cefixime in solution and preventing structural changes due to pH fluctuations. Acetonitrile, with its strong elution ability, interacts with the hydrophobic structure of cefixime, promoting rapid elution of the target analyte from the chromatographic column. This mobile phase combination effectively solves the problem of insufficient mobile phase robustness in existing technologies. Combined with internal standard determination, it significantly improves the precision and accuracy of detection. Compared with mobile phase systems containing formic acid, the deviation and coefficient of variation of quality control samples are controlled within a better range.

[0007] The elution process of the mobile phase includes: a flow rate of 0.8-1.2 mL / min and a volume content of acetonitrile in the mobile phase of 30-60%.

[0008] Optionally, the elution procedure of the mobile phase includes: a flow rate of 1 mL / min and a volume content of 40% acetonitrile in the mobile phase.

[0009] A mobile phase flow rate of 0.8-1.2 mL / min and an acetonitrile volume content of 30-60% create a highly efficient elution system, reducing detection time to within 2 minutes. The molecular weight and polarity of cefixime are well-suited to this flow rate and organic phase ratio, ensuring thorough separation of the target analyte from plasma matrix impurities while avoiding peak broadening or low detection efficiency caused by excessively slow elution. Compared to the existing 2.4-minute elution time, this method significantly shortens the analysis cycle. Furthermore, precise control of elution intensity ensures symmetrical peak shape and stable response, meeting the high-throughput detection requirements of biological samples in clinical trials.

[0010] The chromatographic column is designed with octyl-bonded silica gel as the stationary phase, a packing particle size of 4-6 micrometers, a column length of 100-150 mm, and an inner diameter of 4-6 mm, which is highly compatible with the hydrophobic-hydrophilic balance of cefixime. The hydrophobic groups of the octyl-bonded silica gel can specifically interact with the nonpolar structure in the cefixime molecule, achieving effective separation of the target analyte from interfering impurities. The 4-6 micrometer packing particle size and the reasonable combination of column length and inner diameter ensure separation efficiency while reducing column pressure, improving mobile phase mass transfer efficiency, and reducing peak diffusion. These column parameters ensure stable retention time and high peak resolution for cefixime, providing high-purity target components for subsequent mass spectrometry detection and laying the foundation for accurate detection.

[0011] The mass spectrometry acquisition time is <2 min.

[0012] Optionally, the mass spectrometry acquisition time is 1-1.5 min.

[0013] Optionally, the mass spectrometry acquisition time is 1.4 min.

[0014] The concentration gradients of the standard curve solutions were 20.0 ng / mL, 40.0 ng / mL, 100 ng / mL, 250 ng / mL, 1000 ng / mL, 2000 ng / mL, 4000 ng / mL, and 5000 ng / mL.

[0015] The chromatographic column used in the liquid chromatography-tandem mass spectrometer has all of the following characteristics: the stationary phase is octyl-bonded silica gel, the particle size of the packing material is 4-6 micrometers, the column length is 100-150 mm, and the inner diameter is 4-6 mm.

[0016] The standard curve solution uses a 40-60% acetonitrile aqueous solution as a dilution solution.

[0017] Optionally, the standard curve solution may be diluted with a 50% aqueous acetonitrile solution.

[0018] The ionization mode of the mass spectrometer is: electrospray ionization source, positive ion mode, multiple reaction monitoring.

[0019] The ion source parameters of the mass spectrometer include: collision gas 8 psi, curtain gas 20.0 psi, first ion source gas 50.0 psi, second ion source gas 60.0 psi, ion source spray voltage 5500 V, and ion source temperature 550 °C.

[0020] The cefixime reaction ion parameters in the mass spectrometer include: monitored ion pair 454.0 / 285.0, declustering voltage 100.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 22.00eV, and residence time 200.00msec.

[0021] The mass spectrometer showed cefixime- 13 C- 15 The N2 reaction ionic parameters are as follows: monitored ion pair 457.1 / 288.2, declustering voltage 100.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 22.00eV, and residence time 200.00msec.

[0022] Beneficial effects 1. This invention uses an aqueous solution containing 10 mM ammonium acetate as mobile phase A and acetonitrile as mobile phase B, which significantly improves the precision and accuracy of detection.

[0023] 2. Setting the mobile phase flow rate to 0.8-1.2 mL / min and controlling the acetonitrile volume content to 30-60% can form a highly efficient elution system, shortening the detection time to within 2 minutes.

[0024] 3. The chromatographic column is designed with octyl-bonded silica gel as the stationary phase, a packing particle size of 4-6 micrometers, a column length of 100-150 mm, and an inner diameter of 4-6 mm. This design is highly compatible with the hydrophobic-hydrophilic balance of cefixime, enabling effective separation of the target analyte from interfering impurities and improving the selectivity of the target analyte.

[0025] 4. By setting up standard curve solutions with specific gradient concentrations, this invention can reduce matrix effects and further improve the overall precision, recovery rate, and selectivity of the detection.

[0026] 5. By setting specific chromatographic parameters, this invention achieves intra-batch quality control CV ≤ 6%, inter-batch quality control ≤ 7%, and an overall analyte recovery rate of up to 83.0%, demonstrating excellent robustness and selectivity. Attached Figure Description

[0027] Figure 1 The conditions for liquid chromatography in Example 1 are as follows.

[0028] Figure 2 The conditions for mass spectrometry in Example 1 are (Q1: quadrupole mass analyzer 1; Q3: quadrupole mass analyzer 3).

[0029] Figure 3 The results of precision, accuracy and recovery tests for Example 1 are shown.

[0030] Figure 4 The results are from the selective test in Example 1.

[0031] Figure 5 The results are from the durability test of Example 1.

[0032] Figure 6 Example 1: Cefixime ion scanning spectrum.

[0033] Figure 7 Example 1 Cefixime - 13 C- 15 N2 daughter ion scanning spectrum.

[0034] Figure 8 Example 1: Standard curve obtained from the standard curve sample.

[0035] Figure 9 The results of precision, accuracy, and recovery tests are for Comparative Example 1. Detailed Implementation

[0036] Example 1 A method for determining the concentration of cefixime in K2EDTA-treated human plasma using liquid chromatography-tandem mass spectrometry includes the following steps: preparing a cefixime-treated plasma matrix using K2EDTA-treated human plasma as a substrate. 13 C- 15 Prepare a standard curve solution of N2 and a test solution containing cefixime. Precipitate all solutions with acetonitrile. Determine the concentration of cefixime in the test solution after precipitation using the internal standard method.

[0037] The standard curve solution contained cefixime- 13 C- 15 The concentration gradient of N2 was 20.0 ng / mL (lower limit of quantitation, LLOQ), 40.0 ng / mL, 100 ng / mL, 250 ng / mL, 1000 ng / mL, 2000 ng / mL, 4000 ng / mL and 5000 ng / mL (upper limit of quantitation, ULOQ); the standard curve solution was diluted with 50% acetonitrile aqueous solution.

[0038] The quality control sample concentration gradient is as follows: lower limit of quantitation (LLOQQC, 20.0 ng / mL), low concentration (LQC, 60.0 ng / mL), medium concentration (GMQC, 300 ng / mL, MQC, 1500 ng / mL), and high concentration (HQC, 3750 ng / mL).

[0039] The dilution control sample (DQC) was 10000 ng / mL. This DQC was then diluted with blank plasma (dilution factor: 5) to prepare a test solution containing cefixime with a concentration of 2000 ng / mL.

[0040] The testing process also includes the preparation of quality control solutions, blank samples (50 μL blank matrix mixed with 25 μL methanol), zero-concentration samples (50 μL blank matrix mixed with 25 μL internal standard solution), and samples without internal standard at the upper limit of quantitation (50 μL cefixime- 13 C- 15 The quality control solution with the highest N2 concentration was mixed with 25 μL of methanol.

[0041] Preparation of the internal standard solution: Take cefixime - 13 C- 15 Prepare an internal standard solution of 300 ng / mL using N2 standard and methanol.

[0042] The blank matrix was blank human plasma provided by Zibo Municipal Hospital of Traditional Chinese Medicine, with K2EDTA as the anticoagulant.

[0043] The specific testing steps are as follows: In a 2.2 mL 96-well polypropylene plate, at room temperature (25℃) and under white light, prepare 75 μL of standard curve sample, 75 μL of quality control sample, 75 μL of test sample, 75 μL of blank sample, zero concentration sample, and sample without internal standard for the upper limit of quantitation; add 300 μL of acetonitrile to each of the above samples, mix well and precipitate, centrifuge the above sample plate at 4℃ and 3220g for about 5 minutes; transfer 150 µL of supernatant to another clean 96-well polypropylene plate at room temperature, add 150 µL of purified water, vortex mix at room temperature for about 3 minutes, and place it into an autosampler.

[0044] Chromatographic acquisition and peak integration were performed using ABSciex Analyst software (version 1.7.2). The standard curve was obtained using regression analysis with the Watson LIMS system (version 7.6.1), with the chromatographic response ratio of the analyte to the internal standard as the ordinate, and weighted averages (W=1 / x) calculated. 2 The least squares method uses linear regression between the concentration (x) of the analyte in plasma and the response ratio (y). The resulting regression equation (y=ax+b) is the standard curve. The drug concentration of the sample is calculated from the fitted standard curve equation. Figure 8 As shown, a = 0.000583; b = 0.000899; R 2 =0.9968.

[0045] The conditions for the liquid chromatography are as follows: Figure 1 As shown, the mass spectrometry conditions are as follows: Figure 2 As shown, the ion scanning spectrum of cefixime is as follows: Figure 6 As shown, cefixime - 13 C- 15 N2 ion scanning spectrum as follows Figure 7 As shown.

[0046] Comparative Example 1 The specific implementation method is the same as in Example 1; the difference is that the volume content of acetonitrile in the mobile phase of Comparative Example 1 is 20% (0-2 min); the mobile phase A is an aqueous solution of 10 mM ammonium acetate containing 0.1 wt% formic acid.

[0047] Performance testing methods and data 1. The test methods of Example 1 and Comparative Example 1 were validated methodologically (for Comparative Example 1, only the precision and accuracy were validated). 1. Precision and accuracy Intra-batch precision and accuracy Intra-batch precision and accuracy were assessed using quality control samples (LLOQQC, LQC, GMQC, MQC, and HQC), with six replicates for each concentration of quality control sample.

[0048] Precision was assessed by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and accuracy was assessed by calculating the deviation (Diff%) between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level.

[0049] Acceptance criteria: The deviation between the measured mean concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQQC, the coefficient of variation should not exceed 20.0%).

[0050] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples should have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQQC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level should meet the above standards.

[0051] Inter-batch precision and accuracy analysis Inter-batch precision and accuracy were assessed by examining at least three independent validation analysis batches (intra-batch precision and accuracy analysis batches, completed within at least two days) using freshly prepared quality control samples with a blank matrix.

[0052] The quality control samples used to calculate inter-batch precision and accuracy were derived from the quality control samples (LLOQQC, LQC, GMQC, MQC, and HQC) prepared to examine intra-batch precision and accuracy, with six replicates for each concentration level quality control sample per validation analysis batch.

[0053] Acceptance criteria: The deviation of the overall mean concentration of each quality control sample at each concentration level from its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%).

[0054] The overall coefficient of variation for the measured concentration of each quality control sample at each concentration level shall not exceed 15.0% (the coefficient of variation for LLOQQC shall not exceed 20.0%).

[0055] If an analytical batch fails to meet the acceptance criteria, three additional accuracy and precision analytical batches are tested to validate the methodology.

[0056] like Figure 3 As shown, Example 1 exhibits excellent precision and accuracy, such as Figure 9 The precision and accuracy of Comparative Example 1 shown are significantly lower than those of Example 1.

[0057] 2. Extraction recovery rate Blank matrix from the same batch (or source) as the routine quality control samples (extracted samples, test samples) was used as blank samples. After extraction, the analyte and internal standard were added to the extract of the blank samples to prepare low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC). Six replicates of each concentration were used as reference samples.

[0058] Test samples are routine quality control samples or samples prepared using the same process, including low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC) (6 replicates for each concentration).

[0059] Analyte extraction recovery rate calculation: The peak area of ​​the analyte in each concentration of routine quality control sample (test sample) is divided by the average peak area of ​​the analyte in the reference sample of the same concentration.

[0060] Internal standard extraction recovery rate calculation: The peak area of ​​the internal standard in each routine quality control sample (test sample) is divided by the average peak area of ​​the internal standard in the reference sample.

[0061] Acceptance criteria: The overall coefficient of variation for analyte extraction does not exceed 15.0%; the coefficient of variation for internal standard extraction recovery does not exceed 15.0%.

[0062] If the independent extraction recovery of the analyte or internal standard does not meet the acceptance criteria, the extraction recovery of the analytical method can be evaluated by the extraction recovery after internal standard correction.

[0063] Extraction recovery rate calculation for internal standard correction: The ratio of the peak area of ​​the analyte to its internal standard in each concentration routine quality control sample (test sample) divided by the mean ratio of the peak area of ​​the analyte to its internal standard in the reference sample of the same concentration.

[0064] Acceptance criteria for extraction recovery after internal standard correction: The overall coefficient of variation of extraction recovery after internal standard correction shall not exceed 15.0%.

[0065] like Figure 3 As shown, Example 1 exhibits excellent extraction and recovery rates.

[0066] Selective Matrix selectivity (endogenous interference) Matrix selectivity was evaluated by examining blank biological matrices from at least six different individuals, a high-lipid matrix from one individual, and a hemolyzed matrix from one individual, with measurements of blank samples without internal standards and LLOQ-level samples, respectively.

[0067] High-lipid matrix: via commercially available or simulated hyperlipidemic plasma (1 ± 0.1 mg low-density lipoprotein (LDL) and 3 ± 0.3 mg triglycerides added to 1 mL of blank plasma).

[0068] Hemolysing matrix: Whole blood was frozen at -80°C for at least 30 minutes, thawed, vortexed for at least 1 minute, and then mixed with conventional blank matrix (1:49, v:v) to prepare hemolysing blank matrix.

[0069] Acceptance criteria: The response value of interfering components in the blank matrix at the analyte retention time shall not exceed 20.0% of the analyte response value of the LLOQ sample prepared with the same individual blank matrix; the response value at the internal standard retention time shall not exceed 5.0% of the internal standard response value of the LLOQ sample prepared with the same individual blank matrix.

[0070] If a blank matrix sample from a certain source (batch) does not meet the acceptance criteria, the same method will be used to evaluate the interference of three additional blank matrices from different sources (batches) on the analyte and internal standard. If a high-lipid matrix or hemolyzed matrix does not meet the acceptance criteria, the same method will be used to evaluate one additional high-lipid matrix or hemolyzed matrix from a different source.

[0071] Interference of the analyte to the internal standard Three samples containing only a single analyte and without an internal standard were prepared, processed, and analyzed in parallel to determine the upper limit of quantitation concentration.

[0072] Acceptance criteria: The average peak area of ​​the internal standard at the retention time of a sample containing only a single analyte should not exceed 5.0% of the average peak area of ​​the internal standard in the samples meeting the lower limit of quantitation of the standard curve in the same analytical batch.

[0073] Interference of internal standard with analyte Three samples containing only a single internal standard and without the analyte were prepared, processed, and analyzed in parallel. The concentration of the internal standard was the actual concentration used.

[0074] Acceptance criteria: The average peak area at the retention time of the analyte should not exceed 20.0% of the average peak area of ​​the analyte in the standard curve samples that meet the acceptance criteria in the same analytical batch.

[0075] Selectivity of analysis batch The selectivity of the analytical batch was evaluated using the first blank matrix sample and the first blank quality control sample of the analytical batch.

[0076] Acceptance criteria: The peak area of ​​the analyte in the detection channel of both blank samples does not exceed 20.0% of the average peak area of ​​the analyte in the samples with the limit of quantitation of the effective standard curve; the peak area of ​​the internal standard in the detection channel of the first blank matrix sample does not exceed 5.0% of the average peak area of ​​the internal standard in the samples with the limit of quantitation of the effective standard curve in the same analytical batch.

[0077] like Figure 4 As shown, Example 1 exhibits excellent selectivity.

[0078] Durability The samples were injected by different analysts (Analyst A and Analyst B) or at different concentrations (high, medium, low concentrations and LLOQ). The precision was assessed by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and the accuracy was assessed by calculating the deviation (Diff%) between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level.

[0079] Acceptance criteria: The deviation between the measured mean concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQQC, the coefficient of variation should not exceed 20.0%).

[0080] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples should have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQQC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level should meet the above standards.

[0081] The mass spectrometer model is TripleQuad5500+; the chromatography system includes a liquid phase pump (LC-30AD), a controller (CBM-20A), a degasser (DGU-20A5R(C), a column oven (CTO-20A), and an injection system (SIL-30ACMP).

[0082] Figure 5 The durability test results for Example 1 are as follows, and they meet the requirements.

Claims

1. A method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: Preparation of cefixime-containing formulations using K2EDTA-treated human plasma as a matrix. 13 C- 15 A standard curve solution of N2 was prepared, and a test solution containing cefixime was prepared. All solutions were precipitated with acetonitrile. The concentration of cefixime in the test solution after precipitation was determined using the internal standard method. The determination was performed using liquid chromatography-tandem mass spectrometry. The mobile phase of the liquid chromatography consisted of mobile phase A and mobile phase B. Mobile phase A was an aqueous solution containing 10 mM ammonium acetate, and mobile phase B was acetonitrile.

2. The method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The elution process of the mobile phase includes: a flow rate of 0.8-1.2 mL / min and a volume content of acetonitrile in the mobile phase of 30-60%.

3. The method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that, The mass spectrometer acquisition time is <2 min.

4. The method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The concentration gradients of the standard curve solutions were 20.0 ng / mL, 40.0 ng / mL, 100 ng / mL, 250 ng / mL, 1000 ng / mL, 2000 ng / mL, 4000 ng / mL, and 5000 ng / mL.

5. The method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that, The chromatographic column used in the liquid chromatography-tandem mass spectrometry instrument includes all of the following characteristics: The stationary phase is octyl-bonded silica gel, the filler particles have a diameter of 4-6 micrometers, the column length is 100-150 mm, and the inner diameter is 4-6 mm.

6. The method for determining the concentration of cefixime in human plasma using K2EDTA by liquid chromatography-tandem mass spectrometry according to claim 5, characterized in that, The standard curve solution uses a 40-60% acetonitrile aqueous solution as a dilution solution.

7. The method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The ionization mode of the mass spectrometer is: electrospray ionization source, positive ion mode, multiple reaction monitoring.

8. The method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The ion source parameters of the mass spectrometer include: collision gas 8 psi, curtain gas 20.0 psi, first ion source gas 50.0 psi, second ion source gas 60.0 psi, ion source spray voltage 5500 V, and ion source temperature 550 °C.

9. The method for determining the concentration of cefixime in human plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The cefixime reaction ion parameters in the mass spectrometer include: monitored ion pair 454.0 / 285.0, declustering voltage 100.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 22.00eV, and residence time 200.00msec.

10. The method for determining the concentration of cefixime in human plasma using K2EDTA by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The mass spectrometer showed cefixime- 13 C- 15 The N2 reaction ionic parameters are as follows: monitored ion pair 457.1 / 288.2, declustering voltage 100.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 22.00eV, and residence time 200.00msec.

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