Method for measuring concentration of cefditoren in K2EDTA human plasma by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)
By employing acetonitrile precipitation and optimized LC-MS/MS methods, the challenge of detecting cefotaxime concentration in K2EDTA-anticoagulated human plasma was solved, achieving efficient and accurate concentration determination suitable for high-throughput sample analysis.
Patent Information
- Application Number
- CN202511297429.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-11
- Publication Date
- 2025-11-28
AI Technical Summary
The existing technology lacks an LC-MS/MS method for determining the concentration of ceftoran in K2EDTA-anticoagulated human plasma, and cefotetan and ceftoran have significant differences in drug characteristics, making direct application impossible.
Samples were treated by acetonitrile precipitation and detected by liquid chromatography-tandem mass spectrometry. A mobile phase of 0.05-0.3 wt% formic acid aqueous solution and acetonitrile was used, with acetonitrile accounting for 30-50% of the mobile phase. The flow rate was 0.7-0.9 mL/min. The elution program and ionization parameters were optimized by combining positive ion mode and multiple reaction monitoring mass spectrometry.
It significantly improves the separation and detection response of ceftriaxone, reduces matrix interference, improves detection accuracy and efficiency, and ensures the accuracy and precision of quantification, making it suitable for high-throughput sample analysis.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of chromatographic detection, in particular to a method for determining the concentration of cefotetan in K2EDTA human plasma by LC-MS / MS. BACKGROUND
[0002] Cefotetan is a semi-synthetic third-generation cephalosporin antibiotic, which is an oral aminothiazole cephalosporin and is used in the clinic for the treatment of infectious diseases caused by sensitive bacteria. In order to ensure the safety and effectiveness of its clinical medication, it is crucial to accurately monitor the blood drug concentration in the human body. At present, LC-MS / MS (liquid chromatography tandem mass spectrometry) has become a commonly used method for drug concentration detection due to its high sensitivity and high specificity. However, there is no special patent technology disclosed for the LC-MS / MS determination method of cefotetan concentration in K2EDTA anticoagulated human plasma.
[0003] The literature Shi, M., Yin, L., Cai, L. et al. LC-MS / MS Method for the Quantitation of Cefotetan in Human Plasma and Its Application to Pharmacokinetic Study. Chem. Res. Chin. Univ. 30, 900-904 (2014) establishes a LC-MS / MS determination method for cefotetan in human plasma, which uses acetonitrile to precipitate protein to treat the sample, and the chromatographic column is Zorbax XDBC8 column, and the mobile phase is acetonitrile / 1% formic acid solution. However, cefotetan and cefotetan are different drugs with different structural characteristics, and K2EDTA anticoagulated plasma is not used. SUMMARY
[0004] The present application provides a method for determining the concentration of cefotetan in K2EDTA human plasma by LC-MS / MS, comprising the following steps: preparing standard curve samples containing cefotetan-d3, quality control samples, test samples containing cefotetan, blank samples, zero concentration samples and upper limit of quantification samples without internal standard; using liquid chromatography tandem mass spectrometry to detect the standard curve samples containing cefotetan-d3, quality control samples, test samples containing cefotetan, blank samples, zero concentration samples and upper limit of quantification samples without internal standard; the mobile phase of the liquid chromatography: 0.05-0.3wt% formic acid aqueous solution and acetonitrile, and the volume ratio of acetonitrile in the mobile phase is 30-50%.
[0005] The gradient of the concentration of the standard curve sample is: 10.0 ng / mL (lower limit of quantification, LLOQ), 20.0 ng / mL, 50.0 ng / mL, 200 ng / mL, 400 ng / mL, 800 ng / mL, 1600 ng / mL and 2000 ng / mL (upper limit of quantification, ULOQ).
[0006] The gradient of the concentration of the quality control sample is: lower limit of quantification (LLOQ QC, 10.0 ng / mL), low concentration (LQC, 30.0 ng / mL), medium concentration (GMQC, 150 ng / mL MQC, 600 ng / mL), high concentration (HQC, 1500 ng / mL).
[0007] The flow rate of the mobile phase is 0.7-0.9 mL / min.
[0008] The present application can significantly improve the separation degree and detection response value of Cefditoren by limiting the elution program of liquid chromatography (aqueous solution containing 0.05-0.3 wt% formic acid and acetonitrile, the volume ratio of acetonitrile in the mobile phase is 30-50%, and the flow rate of the mobile phase is 0.7-0.9 mL / min), to ensure the symmetry and sharpness of the peak type, and reduce the matrix interference. As an ion pair reagent, formic acid can enhance the positive charge density of the amino group in the Cefditoren molecule through protonation (-COOH→-COO-+H + ), and promote its ionization efficiency in the electrospray ion source; when the proportion of acetonitrile is 30-50%, it can not only ensure the moderate retention of the target on the octadecylsilane bonded phase chromatographic column (avoiding the overlap of the matrix interference substance and the early peak), but also make the target desorb quickly through its strong elution capacity, reducing the peak broadening; the flow rate of 0.7-0.9 mL / min matches the pore size of the chromatographic column , balancing the mass transfer efficiency and column pressure of the mobile phase, further maintaining the sharpness of the peak type.
[0009] The test sample is pretreated, and the pretreatment includes: precipitation with acetonitrile.
[0010] The present application selects acetonitrile precipitation method to significantly improve the extraction recovery rate of Cefditoren, reduce the matrix effect, and ensure the detection accuracy. As a strong polar organic solvent, acetonitrile can effectively remove most of the matrix protein interference by destroying the hydrogen bond network of proteins in plasma, making the proteins denature and precipitate quickly; in addition, the carboxyl group in the molecular structure of Cefditoren has good compatibility with acetonitrile, and has high solubility in acetonitrile, which can reduce the adsorption loss of the target in the precipitation process, thereby improving the recovery rate; at the same time, acetonitrile is compatible with the subsequent mobile phase (containing acetonitrile), avoiding the solvent effect between the sample solution and the mobile phase, further reducing the matrix interference.
[0011] The volume ratio of the acetonitrile in the mobile phase is 35-45%.
[0012] The elution time of the liquid chromatography is less than 2 minutes.
[0013] Optionally, the elution time of the liquid chromatography is 1.5 minutes.
[0014] The elution time of the present application is less than 2 minutes, which can significantly improve the detection efficiency, is suitable for high-throughput sample analysis, and reduces peak broadening and matrix residue. Under the optimized mobile phase ratio and flow rate, the hydrophobic interaction between cefditoren and octadecylsilane bonded phase is weak, and the "retention-elution" process can be completed in a short time; at the same time, the elution time is less than 2 minutes, which avoids the diffusion of the target in the chromatographic column and reduces the risk of peak broadening; in addition, fast elution can reduce the adsorption of strongly retained matrix components in the plasma in the column, reduce column pollution, prolong the service life of the chromatographic column, and ensure the stability of long-term detection.
[0015] The stationary phase of the chromatographic column used in the liquid chromatography is octadecylsilane bonded phase, wherein the pore size of the stationary phase matrix is 100-300 angstrom.
[0016] Optionally, the particle size of the bonded phase is 2.5-5 microns.
[0017] Optionally, the type of the chromatographic column is 5 microns C18(2) 4.6 mm x 150 mm.
[0018] The ionization mode of the mass spectrometer is: electrospray ion source, positive ion mode, multiple reaction monitoring.
[0019] The ion source parameters of the mass spectrometer include the following parameters: collision gas 9.00 psi, curtain gas 20.00 psi, first ion source gas 30.00 psi, second ion source gas 60.00 psi, ion source spray voltage 5500.00 V, ion source temperature 550.00 DEG C.
[0020] The reaction ion parameters of cefditoren in the mass spectrometer are: the monitored ion pair is 507.2 / 241.2, the de-clustering voltage is 160.00 V, the inlet voltage is 10.00 V, the outlet voltage is 10.00 V, the collision energy is 26.00 eV, and the residence time is 300.00 msec.
[0021] The reaction ion parameters of cefditoren-d3 in the mass spectrometer are: the monitored ion pair is 510.2 / 244.2, the de-clustering voltage is 160.00 V, the inlet voltage is 10.00 V, the outlet voltage is 10.00 V, the collision energy is 26.00 eV, and the residence time is 300.00 msec.
[0022] The present application can maximize ionization efficiency and detection specificity of cefditoren and internal standard through specific mass spectrometry parameters, ensuring quantitative accuracy and precision. Cefditoren molecule contains amino thiazole structure, which is easy to combine protons to form [M+H] + molecular ion (m / z 507.2) has similar ionization behavior with [M+H] + (m / z 510.2) of internal standard cefditoren-d3, ensuring quantitative consistency; under multiple reaction monitoring mode, selecting characteristic fragment ions (241.2, 244.2) can effectively exclude the influence of isomers or interferents in the matrix; ion source parameters (such as spray voltage 5500V, temperature 550℃) can promote droplet desolvation and ion evaporation, improving ionization efficiency; the combination of collision gas 9.00psi and collision energy 26eV can make the quasi-molecular ion fragment into characteristic fragments stably, ensuring the reproducibility of detection response.
[0023] Advantages
[0024] 1. The present application can significantly improve the separation degree and detection response value of cefditoren by limiting the elution program of liquid chromatography, reducing matrix interference, and thus improving the detection accuracy.
[0025] 2. The present application can improve the recovery rate and reduce matrix interference by performing precipitation pretreatment on the test sample with acetonitrile.
[0026] 3. The elution time of the present application is less than 2min, which can significantly improve the detection efficiency, is suitable for high-throughput sample analysis, and at the same time reduces peak broadening and matrix residue.
[0027] 4. The present application can maximize ionization efficiency and detection specificity of cefditoren and internal standard through specific mass spectrometry parameters, ensuring quantitative accuracy and precision.
[0028] 5. The present application establishes a standardized LC-MS / MS determination method for cefditoren in K2EDTA anticoagulated human plasma. BRIEF DESCRIPTION OF DRAWINGS
[0029] Figure 1 The conditions of liquid chromatography of Example 1.
[0030] Figure 2 The conditions of mass spectrometry of Example 1 (Q1: quadrupole mass analyzer 1; Q3: quadrupole mass analyzer 3).
[0031] Figure 3 The precision, accuracy and recovery rate test results of Example 1.
[0032] Figure 4 The selectivity test results of Example 1.
[0033] Figure 5 Results of Example 1 robustness test.
[0034] Figure 6 Sub-ion scan chromatogram of cefditoren of Example 1.
[0035] Figure 7 Sub-ion scan chromatogram of cefditoren-d3 of Example 1.
[0036] Figure 8 Standard curve obtained from standard curve samples of Example 1.
[0037] Figure 9 Liquid chromatogram of test sample of Example 1.
[0038] Figure 10 Liquid chromatogram of test sample of Comparative Example 1.
[0039] Figure 11 Liquid chromatogram of test sample of Comparative Example 2.
[0040] Figure 12 Liquid chromatogram of test sample of Comparative Example 3.
[0041] Figure 13 Liquid chromatogram of test sample of Comparative Example 4. DETAILED DESCRIPTION
[0042] Example 1
[0043] A method for determining the concentration of cefditoren in K2EDTA human plasma by LC-MS / MS is as follows: prepare standard curve samples containing cefditoren-d3, quality control samples containing cefditoren-d3, test samples containing cefditoren, blank samples, zero concentration samples, and upper limit of quantification samples without internal standard; detect the standard curve samples containing cefditoren-d3, quality control samples, test samples containing cefditoren, blank samples, zero concentration samples, and upper limit of quantification samples without internal standard using liquid chromatography tandem mass spectrometry.
[0044] The concentration gradient of the standard curve samples (both solvents are 50wt% methanol aqueous solution) is: 10.0ng / mL (lower limit of quantification, LLOQ), 20.0ng / mL, 50.0ng / mL, 200ng / mL, 400ng / mL, 800ng / mL, 1600ng / mL, and 2000ng / mL (upper limit of quantification, ULOQ).
[0045] The concentration gradient of the quality control sample (solvent is 50wt% methanol aqueous solution) is: lower limit of quantification (LLOQ QC, 10.0 ng / mL), low concentration (LQC, 30.0 ng / mL), medium concentration (GMQC, 150 ng / mL and MQC, 600 ng / mL), high concentration (HQC, 1500 ng / mL).
[0046] The preparation of the blank sample: 50 μL of blank matrix is mixed with 25 μL of methanol.
[0047] The preparation of the zero concentration sample: 50 μL of blank matrix is mixed with 25 μL of internal standard solution.
[0048] The preparation of the upper limit of quantification sample without internal standard: 50 μL of high concentration quality control sample is mixed with 25 μL of methanol.
[0049] The preparation of the internal standard solution: cefditoren-d3 standard and methanol are taken to prepare an internal standard solution of 300 ng / mL; the blank matrix is blank human plasma provided by Zibo City Traditional Chinese Medicine Hospital with K2EDTA as an anticoagulant.
[0050] The specific test is the following steps: 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 upper limit of quantification sample without internal standard are prepared in a 2.2 mL 96-well polypropylene plate under room temperature (25℃) and white light conditions; 300 μL of acetonitrile is added to each of the above samples, which is mixed uniformly and then precipitated; after precipitation, 4000ppm centrifugation is performed for 5 min, and 200 μL of supernatant is taken and moved to another 96-well polypropylene plate, which is detected using a liquid chromatograph mass spectrometer.
[0051] Chromatogram acquisition and chromatographic peak integration are completed by AB Sciex Analyst software (version 1.7.2). The standard curve is obtained by regression using Watson LIMS system (version 7.6.1), and the chromatographic response ratio of the analyte to the internal standard is used as the vertical coordinate. The linear regression is performed on the concentration (x) of the analyte in the plasma and the response ratio (y) by weighted (W=1 / x 2 ) least squares method, and the obtained regression equation (y=ax+b) is the standard curve. The drug concentration of the test sample is calculated by the standard curve equation obtained by fitting. As shown in Figure 8 , a=0.00151; b=-0.00106; R 2 =0.9989.
[0052] The conditions of the liquid chromatograph are shown in Figure 1 , and the mass spectrometry conditions are shown in Figure 2 , wherein the cefditoren daughter ion scanning spectrum is shown in Figure 6The cefltofen-d3 sub-ion scanogram is shown as follows: Figure 7
[0053] Comparative Example 1
[0054] The specific implementation is the same as that of Example 1; the difference is that 300 μL of methanol is added to each of the above samples in Comparative Example 1, and after mixing evenly, precipitation is performed.
[0055] Comparative Example 2
[0056] The specific implementation is the same as that of Example 1; the difference is that the elution procedure in Comparative Example 2 is as follows: the mobile phase is a water solution containing 0.1 wt% formic acid and methanol, 0 min: the volume ratio of methanol in the mobile phase is 60%; 0-0.20 min: the volume ratio of methanol in the mobile phase is 60.0%-90.0%; 0.20-0.90 min: the volume ratio of methanol in the mobile phase is 90%, 0.90-0.91 min: the volume ratio of methanol in the mobile phase is 90%-60%.
[0057] Comparative Example 3
[0058] The specific implementation is the same as that of Example 1; the difference is that the elution procedure in Comparative Example 3 is isocratic elution: the mobile phase is a water solution containing 0.1 wt% formic acid and methanol, and the volume ratio of the methanol in the mobile phase is 30%.
[0059] Comparative Example 4
[0060] The specific implementation is the same as that of Example 1; the difference is that the elution procedure in Comparative Example 4 is isocratic elution: the mobile phase is a water solution containing 0.1 wt% formic acid and acetonitrile, and the volume ratio of the acetonitrile in the mobile phase is 60%.
[0061] Performance test method and data
[0062] I. Methodology validation of the test method of Example 1
[0063] 1. Precision and accuracy
[0064] Intra-batch precision and accuracy
[0065] The intra-batch precision and accuracy were evaluated using quality control samples (LLOQ QC, LQC, GMQC, MQC and HQC), each with 6 replicates at each concentration.
[0066] The precision was investigated by calculating the coefficient of variation (% CV) of each concentration level of the quality control sample, and the accuracy was investigated by calculating the deviation (Diff %) of the average measured concentration of each concentration level of the quality control sample from its theoretical concentration.
[0067] Acceptance criteria:
[0068] The deviation between the mean measured concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQ QC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQ QC, the coefficient of variation should not exceed 20.0%).
[0069] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples must have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQ QC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level must meet the above standards.
[0070] Inter-batch precision and accuracy analysis
[0071] Inter-batch precision and accuracy were assessed by examining at least three independent validation analytical batches (intra-batch precision and accuracy analysis batches, completed within at least two days) using freshly prepared quality control samples with a blank matrix.
[0072] The quality control samples used to calculate inter-batch precision and accuracy were derived from the quality control samples (LLOQ QC, 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.
[0073] Acceptance criteria:
[0074] 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 LLOQ QC, the deviation should be within ±20.0%).
[0075] 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 LLOQ QC shall not exceed 20.0%).
[0076] If an analytical batch fails to meet the acceptance criteria, three additional accuracy and precision analytical batches are tested to validate the methodology.
[0077] like Figure 3 As shown, Example 1 exhibits excellent precision and accuracy.
[0078] 2. Extraction recovery rate
[0079] 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 blank sample extract 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.
[0080] 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).
[0081] 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.
[0082] 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.
[0083] 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%.
[0084] 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.
[0085] 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.
[0086] 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%.
[0087] like Figure 3 As shown, Example 1 exhibits excellent extraction and recovery rates.
[0088] Selective
[0089] Matrix selectivity (endogenous interference)
[0090] 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.
[0091] 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).
[0092] 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.
[0093] 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.
[0094] 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) with 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.
[0095] Interference of the analyte to the internal standard
[0096] 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.
[0097] 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.
[0098] Interference of internal standard with analyte
[0099] 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.
[0100] 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 samples of the standard curve that meet the acceptance criteria in the same analytical batch.
[0101] Selectivity of analysis batch
[0102] 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.
[0103] 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.
[0104] like Figure 4 As shown, Example 1 exhibits excellent selectivity.
[0105] Durability
[0106] The precision was assessed by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, with samples injected by different analysts (Analyst A and Analyst B) or on different instruments (LC-MS / MS, two sets of instruments of the same model with identical parameters). The accuracy was assessed by calculating the deviation (Diff%) between the mean measured concentration of the quality control samples at each concentration level and their theoretical concentration.
[0107] Acceptance criteria:
[0108] The deviation between the mean measured concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQ QC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQ QC, the coefficient of variation should not exceed 20.0%).
[0109] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples must have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQ QC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level must meet the above standards.
[0110] The mass spectrometer model is TripleQuad 5500+; the chromatographic 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).
[0111] Figure 5 The durability test results for Example 1 are as follows, and they meet the requirements.
[0112] II. Analyze the liquid chromatograms of Example 1 and Comparative Examples 1-4, as follows: Figures 9-10 As shown, compared to the preferred embodiment 1 ( Figure 9 In Comparative Example 1, the methanol precipitation signal was significantly reduced. Figure 10 );like Figure 9 , 11 As shown in -13, compared to the preferred embodiment 1 ( Figure 9 The elution method in Comparative Example 2 showed poor peak shape and severe tailing. Figure 11 ); During isocratic elution in Comparative Example 3, the response value was too low, and the retention time was too late. Figure 12 Comparative Example 4: The mobile phase was changed, but the flow rate was too slow, resulting in a later retention time. Figure 13 ).
Claims
1. A method for determining the concentration of ceftoran in human plasma containing K2EDTA using LC-MS / MS, characterized in that, Includes the following steps: Prepare standard curve samples containing ceftriaxone-d3, quality control samples, test samples containing ceftriaxone, blank samples, zero-concentration samples, and samples with upper limit of quantitation (UPQ) without internal standard; analyze these samples using liquid chromatography-tandem mass spectrometry (LC-MS / MS); the mobile phase of the LC-MS consists of an aqueous solution containing 0.05-0.3 wt% formic acid and acetonitrile, wherein the volume percentage of acetonitrile in the mobile phase is 30-50%.
2. The method for determining the concentration of ceftriaxone in human plasma with K2EDTA by LC-MS / MS according to claim 1, characterized in that, The flow rate of the mobile phase is 0.7-0.9 mL / min.
3. The method for determining the concentration of ceftriaxone in human plasma with K2EDTA by LC-MS / MS according to claim 2, characterized in that, The test sample underwent pretreatment, which included precipitation using acetonitrile.
4. The method for determining the concentration of ceftriaxone in human plasma with K2EDTA by LC-MS / MS according to claim 3, characterized in that, The acetonitrile accounts for 35-45% of the volume of the mobile phase.
5. The method for determining the concentration of ceftriaxone in human plasma with K2EDTA by LC-MS / MS according to claim 1 or 4, characterized in that, The elution time for the liquid chromatography is <2 min.
6. The method for determining the concentration of ceftoran in human plasma with K2EDTA by LC-MS / MS according to claim 5, characterized in that, The liquid chromatography column used has an octadecylsilane-bonded stationary phase, wherein the pore size of the stationary phase matrix is [missing information].
7. The method for determining the concentration of cefotaxime in human plasma with K2EDTA by LC-MS / MS according to claim 1 or 6, 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 ceftriaxone in human plasma with K2EDTA by LC-MS / MS according to claim 7, characterized in that, The ion source parameters of the mass spectrometer include the following parameters: Collision gas 9.00psi, curtain gas 20.00psi, first ion source gas 30.00psi, second ion source gas 60.00psi, ion source spray voltage 5500.00V, ion source temperature 550.00℃.
9. The method for determining the concentration of ceftoran in human plasma with K2EDTA by LC-MS / MS according to claim 8, characterized in that, The reaction ion parameters of ceftriaxone in the mass spectrometer were as follows: monitored ion pair 507.2 / 241.2, declustering voltage 160.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 26.00eV, and residence time 300.00msec.
10. The method for determining the concentration of ceftriaxone in human plasma with K2EDTA by LC-MS / MS according to claim 9, characterized in that, The reaction ion parameters of cefotaxime-d3 in the mass spectrometer were as follows: monitored ion pair 510.2 / 244.2, declustering voltage 160.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 26.00eV, and residence time 300.00msec.