Method for detecting the concentration of cefiderocol in serum

Abstract

The present invention belongs to the technical field of analysis and detection, and specifically relates to a method for detecting the concentration of cefiderocol in serum. In the present invention, liquid chromatography tandem mass spectrometry is used to quantitatively analyze the concentration of cefiderocol in serum. The detection method of the present invention can quickly and accurately detect the concentration of cefiderocol in serum, realize cefiderocol therapeutic drug monitoring (TDM), and realize individualized precision treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting the concentration of cefiderocol in serum. Background Art

[0002] Cefiderocol, chemical formula: C 30 H 34 ClN7O 10 S2, a novel antimicrobial agent, consists of a modified cephalosporin structure with an added catechol group that can chelate iron ions, allowing cefiderocol to penetrate the cell membrane of Gram-negative bacteria (including multidrug-resistant strains) through iron transport channels. Using this transport mechanism, cefiderocol avoids bacterial defense mechanisms such as efflux pumps and ultimately binds to penicillin-binding protein (PBP3). In addition, cefiderocol has a new side chain, and modifications of the side chain C-3 and C-7 make cefiderocol highly stable to various β-lactamases including carbapenemases. Infections caused by carbapenem-resistant enterobacteriaces (CRE) and non-fermenting Gram-negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia are currently a major global health problem, resulting in a significant increase in hospital morbidity and mortality, and are a serious health threat.

[0003] Among the new β-lactams recently licensed for the treatment of difficult-to-treat (DTR) Gram-negative infections, cefiderocol is a promising option given its valuable in vitro activity against CRE, DTR Pseudomonas aeruginosa, carbapenem-resistant Acinetobacter baumannii, and Stenotrophomonas maltophilia. However, limited population-specific drug-specific pharmacokinetic (PK) and drug-specific pharmacokinetic / pharmacodynamic (PK / PD) parameter data are available for cefiderocol in sick patients, especially critically ill patients. This is particularly important because in this population, there are several comorbidities and altered physiological states that can modify the PK and PK / PD characteristics of most β-lactam antibiotics used in clinical practice. Therefore, the establishment of an accurate and rapid method for the determination of cefiderocol drug concentrations in serum is highly necessary and desirable.

[0004] To this end, the present invention is proposed. Summary of the invention

[0005] The main purpose of the invention is to provide a method for detecting the concentration of cefiderocol in serum, and the detection method can quickly and accurately detect the concentration of cefiderocol in serum.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions.

[0007] The present invention provides a method for detecting the concentration of cefiderocol in serum, which comprises the following steps:

[0008] Adding blank serum to the standard solution of the series concentrations to obtain the standard curve working solution; and adding the standard solution of low, medium and high concentrations to the blank serum to obtain three level quality control working solutions;

[0009] An internal standard solution and a precipitant are added to the serum to be tested, the standard curve working solution, and the quality control working solution, respectively, and the mixture is vortexed and centrifuged. The obtained supernatant is subjected to liquid chromatography tandem mass spectrometry quantitative analysis, and a standard curve is drawn with the concentration of cefiderocol in the sample as the abscissa and the ratio of the peak area of ​​cefiderocol to the peak area of ​​the internal standard as the ordinate, and the cefiderocol concentration corresponding to the ratio of the measured peak area of ​​cefiderocol to the peak area of ​​the internal standard is read on the standard curve;

[0010] Wherein, the standard solution is a cefiderocol solution, and the internal standard solution is a solution containing cefiderocol-D8;

[0011] The mass spectrometry conditions in the liquid chromatography tandem mass spectrometry analysis include:

[0012] The ion spray voltage was 5500 V, the temperature was 550 °C, the nebulizer gas was 60 psi, the auxiliary heater gas was 60 psi, the curtain gas was 30 psi, and the collision gas was 9 psi;

[0013] Liquid phase conditions include:

[0014] The chromatographic column was a Waters ACQUITY UPLC C8 column with an inner diameter of 2.1 mm, a column length of 50 mm, and a particle size of 1.7 μm. The column temperature was 40 °C, the injection volume was 5 μl, the injector temperature was 15 °C, and the flow rate was 0.4 ml / min;

[0015] Different volumes of mobile phase A and mobile phase B were mixed for gradient elution.

[0016] In some embodiments of the present invention, the series of concentrations includes at least 10 concentrations, and the concentration range of cefiderocol in the standard curve working solution is 0.01 μg / mL~100 μg / mL, that is, the linear range of the standard curve is 0.01 μg / mL~100 μg / mL.

[0017] In some embodiments of the present invention, the concentration of cefiderocol in the standard curve working solution includes 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL.

[0018] In some embodiments of the present invention, the concentration of cefiderocol in the quality control working solution includes 1 μg / mL, 10 μg / mL, and 50 μg / mL.

[0019] In some embodiments of the present invention, the concentration of the internal standard solution is 100 μg / mL.

[0020] In some embodiments of the present invention, the volume ratio of the standard curve working solution, the quality control working solution or the test serum, the internal standard solution and the precipitant is 100:10:300.

[0021] In some embodiments of the invention, the precipitation agent comprises methanol.

[0022] In some embodiments of the present invention, the liquid phase conditions further include: mobile phase A is a 0.1% formic acid in water solution, and mobile phase B is a 0.1% formic acid in methanol solution.

[0023] In some embodiments of the present invention, the elution procedure comprises:

[0024] From 0 to 0.5 min, the volume content of mobile phase A was maintained at 92%, and the volume content of mobile phase B was maintained at 8%;

[0025] From 0.5 min to 0.6 min, the volume content of mobile phase A was adjusted from 92% to 2%, and the volume content of mobile phase B was adjusted from 8% to 98%;

[0026] From 0.6 min to 2 min, the volume content of mobile phase A was maintained at 2%, and the volume content of mobile phase B was maintained at 98%;

[0027] From 2 min to 2.1 min, the volume content of mobile phase A was adjusted from 2% to 92%, and the volume content of mobile phase B was adjusted from 98% to 8%;

[0028] From 2.1 min to 3 min, the volume content of mobile phase A was maintained at 92%, and the volume content of mobile phase B was maintained at 8%.

[0029] Compared with the prior art, the present invention achieves the following technical effects:

[0030] The present invention utilizes the high separation performance of liquid chromatography and the high selectivity and high sensitivity of mass spectrometry to establish a liquid chromatography-tandem mass spectrometry quantitative analysis method, which can quickly, simply and accurately detect the concentration level of cefiderocol in serum, realize cefiderocol therapeutic drug monitoring (TDM), and achieve individualized precision treatment.

[0031] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0033] Figure 1 This is a schematic diagram of the calibration curve of Cefiderocol;

[0034] Figure 2 It is a schematic diagram of the lowest point of the standard curve STD1 (STD: Standard, i.e., the standard curve working solution), the substance peak of the analyte Cefiderocol and the internal standard peak of Cefiderocol-D8;

[0035] Figure 3 The highest point of the standard curve STD10 is the substance peak of the analyte Cefiderocol and the internal standard peak of Cefiderocol-D8;

[0036] Figure 4 The peak of the blank sample followed by the highest point of the standard curve is the substance peak of cefiderocol and the internal standard peak of cefiderocol-D8;

[0037] Figure 5 The chromatograms of Cefiderocol and Cefiderocol-D8 of the quality control (QCL) samples with 100% acetonitrile as the precipitant;

[0038] Figure 6 The chromatograms of cefiderocol and cefiderocol-D8 of the QCL samples were 100% methanol as the precipitant, mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in methanol, the chromatographic column was ACQUITY UPLC C8 column (2.1 mm × 50 mm, 1.7 μm, Waters), and under the chromatographic conditions shown in Table 1 ;

[0039] Figure 7The chromatograms of cefiderocol and cefiderocol-D8 of the QCL samples with a Kinetex F5 column (3 mm × 100 mm, 2.6 μm, Phenomenex);

[0040] Figure 8 The chromatograms of cefiderocol and cefiderocol-D8 of the QCL samples with the Ultimate XB-C18 column (4.6 mm × 50 mm, 5 μm, Welch);

[0041] Fig. 9 The chromatograms of cefiderocol and cefiderocol-D8 of the QCL samples using the Boltimate C18 column (2.1 mm × 50 mm, 2.7 μm, Yuexu Technology);

[0042] Fig.10 The chromatograms of cefiderocol and cefiderocol-D8 of the QCL samples on the Accucore™ aQ C18 column (2.1 mm × 50 mm, 2.6 μm, ThermoScientific™);

[0043] Fig.11 Chromatograms of Cefiderocol and Cefiderocol-D8 for QCL samples with the liquid phase time set to 4.5 min / sample;

[0044] Fig.12 Chromatograms of Cefiderocol and Cefiderocol-D8 of QCL samples separated by isocratic elution with 8% methanol in water;

[0045] Fig.13 Chromatograms of Cefiderocol and Cefiderocol-D8 of QCL samples separated by liquid phase elution with 12% methanol in water;

[0046] Fig.14 Chromatograms of Cefiderocol and Cefiderocol-D8 of QCL samples separated by liquid phase elution with 15% methanol in water;

[0047] Fig.15 The chromatograms of cefiderocol and cefiderocol-D8 of Sample 5 with 0.1% formic acid water as mobile phase A and 0.1% formic acid methanol as mobile phase B;

[0048] Fig.16 The chromatograms of Cefiderocol and Cefiderocol-D8 of Sample 5 with 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B;

[0049] Fig.17 This is the sample stability test chart. DETAILED DESCRIPTION

[0050] The exemplary embodiments of the present invention will be described in more detail below with reference to specific embodiments. It should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0051] In a series of critically ill patients treated with cefiderocol for pneumonia and / or bloodstream infection caused by carbapenem-resistant Acinetobacter baumannii, the present invention found that the microbial eradication rate showed a proportional increase trend when the pharmacokinetic / pharmacodynamic target achievement shifted from suboptimal to quasi-optimal and optimal, indicating that cefiderocol real-time therapeutic drug monitoring (TDM) has potential application value in challenging clinical scenarios.

[0052] Therapeutic drug monitoring (TDM) refers to the measurement of active drug in a biological matrix at defined times to control whether its concentration is within the therapeutic range for optimal therapeutic efficacy and tolerability. This practice is very useful to guide therapeutic adjustments of drugs (especially those with a narrow therapeutic index) in cases of polypharmacy (where the risk of drug interactions is expected to be high) and in special pathophysiological conditions (such as hepatic or renal insufficiency, pregnancy, patients in intensive care units, etc.). In addition, it can even be used to assess patient compliance and prevent the development of resistance by optimizing drug exposure. TDM of antibiotics represents an integrated practice in multiple settings: in particular, this strategy has been applied to limit the toxicity of specific classes of antimicrobial agents, such as oxazolidinones and fluoroquinolones. In addition, the current global increase in antimicrobial resistance has led to a progressive increase in the minimum inhibitory concentration (MIC) of several drugs against many bacterial strains, making the achievement of adequate drug concentrations particularly critical for therapeutic success. Moreover, antimicrobial drugs are the most commonly taken drugs in critically ill patients, who present complex physiological characteristics and huge intra- and inter-individual pharmacokinetic variability. For these reasons, comparison of TDM results with drug-specific pharmacokinetic / pharmacodynamic (PK / PD) parameters based on the drug's killing kinetics (time- versus concentration-dependent killing) and bacterial susceptibility greatly increases the probability of treatment success. This evidence has expanded the use of TDM to many classes of antibiotic agents that do not have a narrow therapeutic index, such as the β-lactams, and has contributed to the wider application of this practice, avoiding toxicity and maximizing therapeutic efficacy.

[0053] Currently, the gold standard for TDM is considered to be liquid chromatography, which is usually combined with tandem mass spectrometry (LC-MS / MS). Liquid chromatography-mass spectrometry is a combined detection method that uses liquid chromatography as the separation system and mass spectrometry as the detection system. The sample is separated from the mobile phase in the mass spectrometry part and ionized. The mass analyzer of the mass spectrometer separates the ion fragments according to the mass-to-charge ratio, and the mass spectrum is obtained by the detector. Liquid chromatography-mass spectrometry detection has high selectivity and high sensitivity, and is increasingly widely used in clinical applications. It can detect a variety of markers that cannot be detected by traditional means.

[0054] Therefore, the present invention establishes a detection and analysis method for cefiderocol in serum based on liquid chromatography tandem mass spectrometry technology. After the serum sample is pretreated by protein precipitation, the accurate detection of cefiderocol serum level is achieved through a liquid chromatography tandem mass spectrometry platform.

[0055] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0056] 1. Preparation of mobile phase solution

[0057] 1. Prepare mobile phase A.

[0058] Add 1 mL of formic acid to 1000 mL of ultrapure water (taking the preparation of 1 L of mobile phase A as an example, the amount of each component added must be reduced proportionally when reducing the amount). Use ultrasonic vibration for 5 minutes to expel the gas and then use it. Store at 20℃~25℃ and the shelf life is one week.

[0059] 2. Prepare mobile phase B.

[0060] Add 1 mL of formic acid to 1000 mL of methanol (take the preparation of 1 L of mobile phase B as an example, reduce the amount of each component added proportionally when reducing the amount), use ultrasonic vibration for 5 minutes to expel the gas and then use it. Store at 20℃~25℃ and the shelf life is one month.

[0061] 2. Preparation of working fluid

[0062] Cefiderocol and Cefiderocol-D8 were purchased from Tianjin Alta Technology Co., Ltd.

[0063] 1. Prepare the internal standard solution.

[0064] Cefiderocol-D8 was dissolved in dimethyl sulfoxide to prepare an internal standard solution with a concentration of 100 μg / mL.

[0065] 2. Prepare a series of standard solutions (S1-S10).

[0066] S10: 100 μL Cefiderocol (1 mg / mL), final concentration 1000 μg / mL.

[0067] S9: 50 μL S10 + 50 μL 50% methanol, the final concentration is 500 μg / mL.

[0068] S8: 60 μL S9 + 90 μL 50% methanol, the final concentration is 200 μg / mL.

[0069] S7: 50 μL S8 + 50 μL 50% methanol, the final concentration is 100 μg / mL.

[0070] S6: 50 μL S7 + 50 μL 50% methanol, the final concentration is 50 μg / mL.

[0071] S5: 50 μL S6 + 200 μL 50% methanol, the final concentration is 10 μg / mL.

[0072] S4: 50 μL S5 + 50 μL 50% methanol, the final concentration is 5 μg / mL.

[0073] S3: 50 μL S4 + 200 μL 50% methanol, the final concentration is 1 μg / mL.

[0074] S2: 50 μL S3 + 50 μL 50% methanol, the final concentration is 0.5 μg / mL.

[0075] S1: 50 μL S2 + 200 μL 50% methanol, the final concentration is 0.1 μg / mL.

[0076] 3. Prepare the standard curve working solution.

[0077] 90 μL of blank serum sample was added to 10 μL of standard solution (S1-S10) to obtain 100 μL of standard curve working solution (STD1-STD10). The concentrations of cefiderocol in the standard curve working solution were 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL.

[0078] 4. Prepare quality control working solution.

[0079] 90 μL of blank serum sample was added to 10 μL (S5 / S7 / S9) solution to obtain 100 μL quality control (QCL / QCM / QCH) working solution. The concentration of cefiderocol in the quality control working solution was 1 μg / mL, 10 μg / mL, and 50 μg / mL, respectively.

[0080] In the embodiment of the present invention, the linearity r of the standard curve should be greater than 0.99, and the accuracy of the low, medium and high quality control samples should be within ±15% of the theoretical value.

[0081] 3. Sample Pretreatment

[0082] Take a 2 mL centrifuge tube, add 100 μL of standard curve working solution, 10 μL of internal standard solution (concentration of 100 μg / mL), and 300 μL of 100% methanol in sequence, vortex for 10 min, and centrifuge at 13000 rpm for 10 min; take 150 μL of supernatant (1) and transfer it to a 96-well plate for testing.

[0083] Take a 2 mL centrifuge tube, add 100 μL of quality control working solution, 10 μL of internal standard solution (concentration of 100 μg / mL), and 300 μL of 100% methanol in sequence, vortex for 10 min, and centrifuge at 13000 rpm for 10 min; take 150 μL of supernatant (2) and transfer it to a 96-well plate for testing.

[0084] Take a 2 mL centrifuge tube, add 100 μL of the serum sample to be tested, 10 μL of the internal standard solution (concentration of 100 μg / mL), and 300 μL of 100% methanol in sequence, vortex for 10 min, and centrifuge at 13000 rpm for 10 min; take 150 μL of the supernatant (3) and transfer it to a 96-well plate for testing.

[0085] It is worth mentioning that the precipitant in the present invention is 100% methanol after repeated comparisons. Compared with the traditional use of acetonitrile as a precipitant, the use of methanol as a precipitant in the present invention is more sensitive. In addition, the present invention also conducted a comparative verification. If 100% acetonitrile is used as a precipitant for precipitation, the response intensity is reduced. For details, see Figure 5 Therefore, 100% methanol in the present invention has a better effect, see Figure 6 .

[0086] 4. Liquid chromatography tandem mass spectrometry method conditions

[0087] 1. The liquid chromatography conditions are shown in Table 1.

[0088] Table 1 Summary of HPLC conditions

[0089]

[0090] 2. The mass spectrometry conditions are shown in Table 2.

[0091] Table 2 Summary of mass spectrometry conditions

[0092]

[0093] 3. The ion pair parameters are shown in Table 3.

[0094] Table 3 Summary of ion pair parameters

[0095]

[0096] * is the quantitative ion pair

[0097] It is worth mentioning that, in order to achieve the purpose of more sensitive detection and faster detection speed, the chromatographic column used in the present invention is ACQUITY UPLC C8 column (2.1mm×50mm, 1.7μm, Waters) and gradient elution is performed under the chromatographic conditions shown in Table 1. At the same time, comparative tests are also carried out in the present invention to effectively illustrate the technical effects of the present invention.

[0098] When the chromatographic column was ACQUITY UPLC C8 column (2.1 mm × 50 mm, 1.7 μm, Waters) and the chromatographic conditions shown in Table 1, the samples were subjected to the same pretreatment and it was found that: when mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile, the results were as follows: Fig.16 ,contrast Fig.15 It can be seen that the characteristic peak is slightly delayed, which affects the quantification, and the response intensity is low. Therefore, in the present invention, mobile phase A is 0.1% formic acid water, and mobile phase B is 0.1% formic acid methanol, which has a better effect. Fig.15 .

[0099] Under the above-determined mobile phase and the chromatographic conditions shown in Table 1 (except the chromatographic column), after the samples were subjected to the same pretreatment, it was found that: if the chromatographic column was Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex), Figure 7 It can be seen from the figure that the characteristic peak is delayed, which affects the quantification and the response intensity is low. If the chromatographic column is Ultimate XB-C18column (4.6mm×50mm, 5μm, Welch), Figure 8 It can be seen from the figure that the characteristic peak has a poor shape and there are interference peaks around the characteristic peaks, which are very close to each other, affecting the quantification and making it impossible to quantify accurately. If the chromatographic column is a Boltimate C18 column (2.1mm×50mm, 2.7μm, Yuexu Technology), Fig. 9 It can be seen that the response intensity is Figure 6 Low; if the chromatographic column is Accucore™ aQ C18 column (2.1mm×50mm, 2.6μm, Thermo Scientific™), Fig.10 It can be seen that the response strength is still relatively Figure 6 Therefore, the chromatographic column in the present invention is ACQUITY UPLC C8 column (2.1 mm × 50 mm, 1.7 μm, Waters) for better effect, see Figure 6 .

[0100] Under the above-determined mobile phase and chromatographic column conditions, isocratic elution separation was first attempted, with 8% methanol-water solution, 12% methanol-water solution, and 15% methanol-water solution being used for liquid phase separation. Figure 12 to Figure 14 , it can be seen that with the increase of the organic phase ratio, the peak time gradually moves forward, but the characteristic peak shape is poor and there are interference peaks, so gradient separation is attempted; when the liquid phase time is set to 4.5 min / sample (Table 4), see Fig.11 It can be found that the characteristic peak shape is poor and the response intensity is low; in order to improve the separation efficiency, a 3-min liquid phase time elution gradient is set (Table 1), and it is found that the peak shape is better and the response intensity is higher. Therefore, the liquid phase time is set to 3 min / sample for better results, and compared with the existing 4 min elution time or even 5.5 min elution time, the present invention can achieve more efficient detection.

[0101] Table 4 4.5min mobile phase gradient (poor peak shape, low response)

[0102]

[0103] 5. Instrumental analysis steps

[0104] 1. Turn on the computer power, enter the Windows operating system, check whether the instrument is in good condition, log in to the Analyst software, and enter the workstation main interface.

[0105] 2. Double-click the Hardware configuration button, click LCMS, and then click Activate profile on the right to activate the instrument. The LCMS module is displayed as a green check mark, indicating that the connection is successful.

[0106] 3. Double-click Build Acquistion Batch in the Acquire column, select the established method, and edit the sample sequence (sample number, sample tray position, injection volume, etc.) in the sequence editing menu bar.

[0107] 4. Click Equilibrate in the menu bar, select the previously established LC / MS method to be run, and set the equilibrium time to 10 to 20 minutes.

[0108] 5. Enter the Submit interface on the right side of the Batch Editor, select the sample sequence to be analyzed and click the Submit button to submit.

[0109] 6. After the equilibrium time is over, the instrument status in the lower right corner of the main interface is displayed as Ready. Click Start sample in the menu bar to start sample analysis and data acquisition.

[0110] 7. Double-click the Open Date File option in the Explore option on the left to view the chromatogram image during the detection process at any time.

[0111] 8. After sample analysis and collection, use Mμltiquant to create a quantitative list and perform quantitative analysis.

[0112] 6. Standby steps

[0113] 1. Click the Standby button in the menu bar, and you can see the flow rate slowly decreases to zero. After the system pressure drops to 0, double-click Hardware configuration, click LCMS in the pop-up dialog box, and then click Deactivateprofile on the right to deactivate the liquid chromatography-mass spectrometry. The LCMS module is displayed as a cross, and you can disconnect.

[0114] 2. Exit the Analyst software and shut down the computer.

[0115] VII. Performance Verification Results of the Method

[0116] 1. Linear

[0117] A series of serial dilutions were performed on the standard solution of known concentration (the concentrations were 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.1 μg / mL, 0.05 μg / mL, and 0.01 μg / mL), and an equal amount of internal standard solution was added. The calibration points were fitted by the least square method to form a calibration curve. The linearity requirement was r>0.99, and the deviation between the value of each concentration point and the theoretical value was less than 15%. The deviation between the detected value and the theoretical value at the limit of Quantification (LoQ) point was less than 20%. The linear range obtained from the experiment was 0.01~100 μg / mL, LoQ=0.01 μg / mL, and the cefiderocol standard curve r=0.99951. The linearity was good, and the quantification at the LoQ point was accurate. Figure 1 and Figure 2 As shown, it is sufficient to show that the detection sensitivity and linearity of the present invention are good. Compared with the detection limit of 0.02 μg / mL in the existing linear range, it is more accurate and has higher sensitivity, which can meet the needs of clinical detection and scientific research.

[0118] 2. Precision and carryover

[0119] The prepared low, medium and high quality control working solutions were tested three times for three consecutive days. The intra-day precision coefficients of variation of cefiderocol were calculated to be 4.42%, 1.93% and 0.93%, all less than 10%, and the inter-day precisions were 5.22%, 4.15% and 4.57%, all less than 10%, respectively, proving that the precision was good (see Table 5 for details). The blank sample was measured immediately after the highest point of the calibration curve. The signal response of the blank sample was less than 0.1% of the highest point of the calibration curve. See Table 5 for details. Figure 3 to Figure 4 , proving that the detection method of the present invention has no significant carryover contamination.

[0120] Table 5 Detection precision of Cefiderocol

[0121]

[0122] 3. Accuracy and Matrix Effects

[0123] Spiked recovery rate: Take at least 5 samples from the population from different sources, select an equal amount of serum from each sample and mix them, then select 2 equal amounts from the mixed serum, add low / high value standard solutions of known concentrations, respectively, carry out the same pretreatment and sampling analysis process, and perform three parallel measurements. The spiked recovery rate % = (measured sample concentration after spiked-measured matrix sample concentration) / theoretical spiked concentration * 100% is used to calculate, and the data in Table 6 are obtained. The spiked recovery rate of cefiderocol is 102.2%~105.5%, indicating that the detection method of the present invention has good accuracy.

[0124] Matrix effect: Take at least 5 samples from different sources, extract according to the sample pretreatment method except that no internal standard solution is added, add the corresponding concentration of internal standard solution and different concentrations of standard solution to the extracted matrix sample, and prepare low and high levels of matrix extraction spiked samples (A), and inject and analyze. Prepare the corresponding low and high concentration levels of standard solutions with the same injection reagent, inject and analyze, and obtain the average signal intensity of each concentration point in the pure solution (B). Calculate the area ratio of the analyte to the internal standard peak in each group of samples. The matrix effect of cefiderocol is 107.5% and 109.9% respectively by calculating the formula matrix effect = (area ratio of sample A / area ratio of sample B) x 100%, as shown in Table 6, which meets the experimental requirements.

[0125] Recovery rate of spiked samples after extraction: Take at least 5 samples from different sources, select an equal amount of serum from each sample and mix them, then select 2 equal amounts from the mixed serum, and perform the same pretreatment process (but without adding standard and internal standard solution). After centrifugal precipitation and taking the supernatant, add low / high value standards of known concentrations respectively, and measure three times in parallel. The formula of spiked recovery rate % = (measured sample concentration after spiked sample concentration - measured matrix sample concentration) / theoretical spiked concentration * 100% is used for calculation, and the data in Table 6 are obtained. The recovery rate of cefiderocol after extraction is 94.3%~97.2%, indicating that the accuracy of the detection method of the present invention is good.

[0126] Table 6 Recovery rate and matrix effect of Cefiderocol

[0127]

[0128] 4. Stability

[0129] Take at least 5 samples from different sources, select an equal amount of serum from each sample and mix them, then select 6 equal parts from the mixed serum, add the median standard (STD5), and place them at -20℃, 4℃, and 20℃ for 20 hours and at 4℃, 20℃ for 2 hours, and 20℃ for 0 hours, respectively, and then perform the same pretreatment and injection process. The relative concentration (%) is calculated using the formula: measured sample concentration after placement / theoretical sample concentration*100%, and the result is Fig.17 The data shown shows that the relative concentration is 87.3% after 2 hours at 20°C, 24.1% after 20 hours at 20°C, 81.7% after 20 hours at 4°C, and 95.2% after 20 hours at -20°C. This result shows that blood should be tested as soon as possible after collection, and samples stored at 4°C should be less than 20 hours. Samples stored for more than 20 hours should be stored at -20°C.

[0130] The equipment and process scales described herein are intended to simplify the description of the invention. Applications, modifications and variations of the invention will be apparent to those skilled in the art.

[0131] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for detecting cefiderocol concentration in serum, characterized in that, The following steps are involved: Adding blank serum to the standard solution of the series concentrations to obtain the standard curve working solution; and adding the standard solution of low, medium and high concentrations to the blank serum to obtain three level quality control working solutions; An internal standard solution and a precipitant are added to the serum to be tested, the standard curve working solution, and the quality control working solution, respectively, and the mixture is vortexed and centrifuged. The obtained supernatant is subjected to liquid chromatography tandem mass spectrometry quantitative analysis, and a standard curve is drawn with the concentration of cefiderocol in the sample as the abscissa and the ratio of the peak area of ​​cefiderocol to the peak area of ​​the internal standard as the ordinate, and the cefiderocol concentration corresponding to the ratio of the measured peak area of ​​cefiderocol to the peak area of ​​the internal standard is read on the standard curve; Wherein, the standard solution is a cefiderocol solution, the internal standard solution is a solution containing cefiderocol-D8; the precipitant is methanol; the volume ratio of the standard curve working solution, the quality control working solution or the test serum, the internal standard solution and the precipitant is 100:10:300, and the concentration of the internal standard solution is 100 μg / mL; the cefiderocol concentration in the quality control working solution includes 1 μg / mL, 10 μg / mL, and 50 μg / mL; the concentration range of cefiderocol in the standard curve working solution is 0.01 μg / mL~100 μg / mL, and the cefiderocol concentration in the standard curve working solution includes 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL; The mass spectrometry conditions in the liquid chromatography tandem mass spectrometry analysis include: The ion spray voltage was 5500 V, the temperature was 550 °C, the nebulizer gas was 60 psi, the auxiliary heating gas was 60 psi, the curtain gas was 30 psi, and the collision gas was 9 psi; the declustering voltages of cefiderocol and cefiderocol-D8 were 108 V and 114 V, respectively; the ion reactions used for quantitative analysis were cefiderocol: m / z: 752.2→285.1, cefiderocol-D8: m / z: 760.1→293.1; the collision voltage of cefiderocol: m / z: 752.2→285.1 was 22 V, and the collision voltage of cefiderocol-D8: m / z: 760.1→293.1 was 29 V; Liquid phase conditions include: The chromatographic column was a Waters ACQUITY UPLC C8 column with an inner diameter of 2.1 mm, a column length of 50 mm, and a particle size of 1.7 μm. The column temperature was 40 °C, the injection volume was 5 μl, the injector temperature was 15 °C, and the flow rate was 0.4 ml / min; Different volumes of mobile phase A and mobile phase B were mixed for gradient elution; The elution procedure includes: From 0 to 0.5 min, the volume content of mobile phase A was maintained at 92%, and the volume content of mobile phase B was maintained at 8%; From 0.5 min to 0.6 min, the volume content of mobile phase A was adjusted from 92% to 2%, and the volume content of mobile phase B was adjusted from 8% to 98%; From 0.6 min to 2 min, the volume content of mobile phase A was maintained at 2%, and the volume content of mobile phase B was maintained at 98%; From 2 min to 2.1 min, the volume content of mobile phase A was adjusted from 2% to 92%, and the volume content of mobile phase B was adjusted from 98% to 8%; From 2.1 min to 3 min, the volume content of mobile phase A was maintained at 92%, and the volume content of mobile phase B was maintained at 8%.

2. The method for detecting cefiderocol concentration in serum as claimed in claim 1, wherein The liquid phase conditions also include: Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in methanol.

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