A detection method for therapeutic drugs extracted from serum based on ultrafiltration

Abstract

The present invention relates to a detection method for extracting therapeutic drugs in serum based on ultrafiltration. The method comprises the following steps: S1, after mixing a sample, an internal standard solution and a stabilizer, ultrafiltration is performed; the stabilizer contains an aqueous solution of hydrochloric acid and glycine; S2, the filtrate after ultrafiltration is subjected to liquid chromatography-tandem mass spectrometry detection to obtain the content of therapeutic drugs in a variety of serums; the therapeutic drugs in the serum include at least one of voriconazole, vancomycin, tazobactam, meropenem, imipenem, teicoplanin, linezolid, amikacin and moxifloxacin. The detection method of the present invention can quickly and accurately detect 9 kinds of therapeutic drugs in serum at the same time, the pretreatment is simple and easy to operate, time-saving and labor-saving, efficient and accurate, one test, one injection detection of 9 kinds of therapeutic drugs, greatly improves the efficiency and accuracy of medical examinations, is convenient for doctors and patients, and provides an effective and feasible basis for monitoring the content of therapeutic drugs in the human body.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and relates to a detection method for extracting therapeutic drugs in serum based on ultrafiltration. Background Art

[0002] Bacterial infection is an important source of morbidity and mortality, and the use of antibiotics has become an important aspect of hospitalized patient management, especially in the intensive care unit (ICU). However, it has been shown that the blood drug concentration of patients treated with antibiotics based on standard dosing regimens often cannot reach effective therapeutic concentrations, which greatly affects the therapeutic effect and may cause patients to develop multi-bacterial resistance. Because there are individual differences in drug pharmacokinetics among different patients, simply increasing the drug dose may cause dangerous toxic side effects. Therefore, strict in vivo drug concentration monitoring is required, especially for drugs with narrow therapeutic concentrations.

[0003] Imipenem (IPM) is a carbapenem antibiotic characterized by a broad antibacterial spectrum, strong antibacterial activity and high stability to β-lactamase. It is mainly used to treat multidrug-resistant Gram-negative bacillus infections, severe mixed infections of aerobic and anaerobic bacteria, and severe infections with unidentified pathogens. It is the first-line drug for the treatment of severe infections.

[0004] Meropenem (MEM) is an artificial semi-synthetic carbapenem antibiotic with an ultra-broad-spectrum antibacterial activity, showing strong activity against common aerobic and anaerobic bacteria in clinical practice. Due to its strong tissue penetration and wide distribution, meropenem is clinically used to treat respiratory, urinary tract, surgical, obstetrics and gynecology, skin and soft tissue infections and bacterial meningitis.

[0005] Linezolid (LZD) is the first new oxazolidinone antibiotic used in clinical practice. The drug has a unique mechanism of action, no cross-resistance with other drugs, and no significant effect of age and gender on its pharmacokinetics. It has a good antibacterial effect on most Gram-positive bacteria, including multidrug-resistant Enterococci, Staphylococci and Streptococcus pneumoniae.

[0006] Moxifloxacin (MXF) is a fourth-generation new fluoroquinolone broad-spectrum antibacterial drug. It inhibits the activity of bacterial DNA helicase A subunit and topoisomerase IV, blocks DNA replication, and exerts a bactericidal effect. Moxifloxacin has good antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, anaerobic bacteria, and atypical pathogens (mycoplasma, Chlamydia, Legionella), and has the advantages of strong tissue penetration, high bioavailability, long duration of action, and few adverse reactions. Moxifloxacin is a commonly used antibacterial drug in clinical practice, mainly used for acute exacerbations of chronic bronchitis, chronic obstructive pulmonary disease, community-acquired pneumonia, and skin and soft tissue infections.

[0007] Vancomycin (VA) is a glycopeptide antibacterial drug with strong bactericidal activity and rarely induces drug resistance. It is effective against infections caused by Gram-positive bacteria and methicillin-resistant Staphylococcus aureus, and is a common drug for the clinical treatment of severe infections.

[0008] Teicoplanin (TEC) is a new bactericidal glycopeptide antibiotic produced by fermentation of Actinomyces spp. Its mechanism of action is similar to that of vancomycin and other glycopeptide antibiotics. It is one of the important antibiotics used to treat multidrug-resistant bacterial infections after vancomycin. It is clinically used to treat various serious Gram-positive bacterial infections, especially methicillin-resistant Staphylococcus aureus infections. It is suitable for the treatment of severe Staphylococcus aureus (especially methicillin-resistant Staphylococcus aureus) infections, such as sepsis, septic endocarditis, acute osteomyelitis and acute exacerbation of chronic osteomyelitis, septic arthritis, and concurrent infections in patients with neutropenia.

[0009] Voriconazole (VOR) Voriconazole is a new triazole antifungal drug and one of the common drugs for the treatment of invasive fungal infections. However, due to its potential drug interactions and genetic polymorphism of the metabolic enzyme cytochrome P450 (CYP) 2C19, its pharmacokinetics vary greatly between individuals, which may cause serious adverse drug reactions (ADRs), and some ADRs are even irreversible. Studies have shown that the blood concentration of voriconazole is closely related to clinical efficacy and ADR. Therefore, it is recommended to monitor the blood concentration of patients using voriconazole.

[0010] Tazobactam (TAZ) Tazobactam is a β-lactamase inhibitor with antibacterial activity against many Gram-positive and Gram-negative aerobic and anaerobic bacteria. It is widely used in sepsis patients with lung, skin and soft tissue, abdominal cavity, urinary tract infections, etc. However, when patients use piperacillin / tazobactam during CRRT, its pharmacokinetics may be greatly affected. Monitoring the blood concentration of tazobactam during CRRT can help clinicians choose a more reasonable way of administration.

[0011] Amikacin (AK) Amikacin is a broad-spectrum aminoglycoside antibiotic, which is made by modifying the structure of kanamycin. It has antibacterial activity against many Gram-negative bacteria and some Gram-positive bacteria. It is suitable for the treatment of various infections. It is commonly used in clinical practice for respiratory tract infections, bone and joint infections, sepsis (including neonatal sepsis), central nervous system infections, postoperative infections and recurrent urinary tract infections. Because its improper use may cause a certain degree of ototoxicity and renal toxicity, it should be used under the condition of blood drug concentration monitoring.

[0012] Therapeutic Drug Monitoring (TDM) is a clinical pharmacology practice, and its significance is mainly reflected in the following aspects: 1. Individualized drug administration: Different patients may have significant differences in metabolism and response to the same drug. TDM can monitor individual drug concentrations, thereby adjusting the drug administration plan and achieving personalized treatment. 2. Improving efficacy: By monitoring the drug concentration in the blood, it can ensure that the drug is within the therapeutic window, achieving both therapeutic effects and poor efficacy caused by concentrations below the therapeutic level. 3. Preventing toxicity: Excessive drug concentrations may cause toxic reactions. Through TDM, the treatment plan can be adjusted in time to avoid or reduce adverse drug reactions. 4. Drug interactions: Patients may use multiple drugs at the same time, and there may be interactions between these drugs. TDM helps to discover these interactions and manage them by adjusting the dosage. 5. Improving patient compliance: Through TDM, it can be found whether the patient takes the drug according to the doctor's instructions, thereby improving the patient's treatment compliance. 6. Legal and regulatory requirements: In some cases, such as drug overdose or poisoning, TDM can provide legal evidence and also help regulators supervise drug use. 7. Economic benefits: Although TDM itself incurs certain costs, by accurately adjusting drug dosages, it can reduce medical expenses caused by drug overdose or treatment failure, thereby bringing long-term economic benefits. 8. Scientific research and education: TDM data are of great value for new drug development, research on drug metabolism and effects, and medical education. 9. Medication safety for special populations: For special populations (such as pregnant women, children, the elderly or patients with renal insufficiency), TDM can help determine the appropriate drug dosage to ensure safety and effectiveness. Through TDM, the medical team can better manage patients' drug treatment, optimize treatment outcomes, and improve patients' quality of life.

[0013] The accuracy of therapeutic drug monitoring (TDM) depends largely on the sample pretreatment steps. The purpose of pretreatment is to extract and purify drugs from complex biological matrices for accurate concentration determination. The following are some of the pretreatment methods commonly used in TDM: 1. Protein precipitation: This is a simple and commonly used pretreatment method. By adding organic solvents (such as methanol and acetonitrile), proteins in plasma or serum are precipitated, and then the precipitated proteins are removed by centrifugation, leaving the drug molecules in the supernatant for analysis. However, the pretreatment purification of protein precipitation is not thorough enough, which is easy to contaminate the chromatographic column and mass spectrometer, and the matrix effect will be obvious. 2. Solid phase extraction (SPE): An extraction method using solid phase adsorbents. By selecting a specific type of adsorbent, the target drug can be specifically adsorbed and then eluted with an appropriate solvent. However, the sample pretreatment is complicated and the preparation cost is high. Moreover, 9 different types of drugs require the selection of several SPE solid phase cartridges, and at least two pretreatments are required to treat these 9 therapeutic drugs. The pretreatment time is long and the throughput is low. 3. Ultrafiltration: Ultrafiltration membranes are used to separate macromolecular substances (such as proteins) from small molecule drugs. This is suitable for the pretreatment of drugs with smaller molecular weights, but the cost of ultrafiltration membranes is relatively high.

[0014] With the rapid development of modern mass spectrometry technology, chromatography-tandem mass spectrometry has become the main method for therapeutic drug monitoring due to its high sensitivity, strong specificity, and ability to rapidly test a large number of samples. Existing patents or methods developed based on LC-MS / MS technology for therapeutic drug monitoring have their own advantages, but still have the following disadvantages. 1. Low throughput, few detection targets. 2. Incomplete pretreatment and purification can easily contaminate the chromatographic column and mass spectrometer; 3. Complex sample pretreatment and high preparation cost.

[0015] Therefore, there is an urgent need for a method based on LC-MS / MS technology to effectively detect therapeutic drugs in serum. Summary of the invention

[0016] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for detecting therapeutic drugs in serum extracted by ultrafiltration. The present invention makes the therapeutic drugs (especially imipenem and meropenem) in the sample free and stable by adding a specific stabilizer, and extracts the therapeutic drugs from the serum sample by ultrafiltration. The method is simple and fast, and the pretreatment of 9 therapeutic drugs can be basically completed within 30 minutes. After adding isotope internal standard dilution, the matrix effect and interference are reduced to ensure the accuracy and stability of quantification. Various substances with different properties can be extracted by ultrafiltration, which is convenient and fast, and can also save costs. The cost of each therapeutic drug is lower; and the ultrafiltration method can choose tubular and plate types. The ultrafiltration cost of the plate type is more than 10 times cheaper than that of the tubular type, which is close to the cost of the protein precipitation method. When the plate type ultrafiltration is selected, the cost is lower and cheaper.

[0017] By using a reverse phase chromatography system and selecting a water-resistant reverse phase C18 chromatographic column as the chromatographic column for therapeutic drugs, chromatographic retention of nine therapeutic drugs can be ensured without adding an ion pair reagent to the mobile phase. Using methanol, 0.75% formic acid and 10mmol / L ammonium acetate in water as the mobile phase, gradient elution is used to complete the chromatographic separation of nine therapeutic drugs within 5 minutes, ensuring that each therapeutic drug has a good signal. The short chromatographic separation time can greatly improve the detection flux of therapeutic drugs and improve the detection efficiency. It can be used for the rapid extraction of a large number of samples. The method provided by the present invention has the advantages of multiple detection types, easy operation, short detection time, high detection flux, high sensitivity, strong specificity, and wide detection range for monitoring therapeutic drugs in human serum.

[0018] The purpose of the present invention can be achieved by the following scheme:

[0019] In a first aspect, the present invention provides a method for detecting therapeutic drugs in serum extracted by ultrafiltration, comprising the following steps:

[0020] S1, after mixing the sample, internal standard solution and stabilizer, ultrafiltration is performed;

[0021] The stabilizer is an aqueous solution containing hydrochloric acid and glycine;

[0022] S2, subjecting the filtrate after ultrafiltration to liquid chromatography-tandem mass spectrometry detection to obtain the content of therapeutic drugs in multiple serums;

[0023] The therapeutic drugs in the serum include at least one of voriconazole, vancomycin, tazobactam, meropenem, imipenem, teicoplanin, linezolid, amikacin, and moxifloxacin.

[0024] As an embodiment of the present invention, in step S1, the concentration of hydrochloric acid in the stabilizer is 0.01-1M, and the concentration of glycine is 0.1-10 mg / mL.

[0025] As an embodiment of the present invention, in step S1, the internal standard includes voriconazole-IS, vancomycin-IS, tazobactam-IS, meropenem-IS, imipenem-IS, teicoplanin-IS, linezolid-IS, amikacin-IS, and moxifloxacin-IS.

[0026] Further, in the internal standard solution, the concentrations of the internal standards are: 0.5-2 μg / mL voriconazole-IS, 1-10 μg / mL vancomycin-IS, 1-10 μg / mL tazobactam-IS, 1-10 μg / mL meropenem-IS, 1-10 μg / mL imipenem-IS, 1-10 μg / mL teicoplanin-IS, 0.5-2 μg / mL linezolid-IS, 1-10 μg / mL amikacin-IS, 0.25-1 μg / mL moxifloxacin-IS;

[0027] The internal standard solution also includes a stabilizer; wherein the concentration of hydrochloric acid is 0.01-1M, and the concentration of glycine is 0.1-10 mg / mL.

[0028] As an embodiment of the present invention, in step S1, the volume ratio of the sample, stabilizer and internal standard solution is 4-6:2-3:0.8-1.2. The mixing is performed by vortex oscillation. The present invention adjusts the pH value of the serum sample and adds a stabilizer to free and stabilize the therapeutic drug in the sample.

[0029] As an embodiment of the present invention, in step S1, the ultrafiltration method comprises transferring the mixed solution to an ultrafiltration plate under positive pressure or centrifugation. The mixed solution is a mixed solution of the sample, the internal standard solution and the stabilizer.

[0030] Furthermore, the specification of the ultrafiltration plate is 3KD, 350μL.

[0031] Furthermore, the centrifugal speed is 12000-15000 r / min, and the time is 15-30 min; the positive pressure is 0.1-0.4 MPa, and the time is 15-30 minutes.

[0032] The preferred ultrafiltration device of the present invention is an ultrafiltration plate. The ultrafiltration plate is cheap, has a fast pre-treatment speed for large batches of samples, has a good purification effect, and is very suitable for promotion and application.

[0033] In some embodiments, 100 μL of sample is added to a single well of a 96-well ultrafiltration plate (3KD, 350 μL), 20 μL of internal standard solution is added, 50 μL of stabilizer is added, and the mixture is vortexed and mixed for 5 minutes; a 96-well receiving plate is placed under the ultrafiltration plate, corresponding to the well positions one by one, and the assembled ultrafiltration plate and receiving plate are placed on a positive pressure device, filtered at 0.2 MPa for 20 minutes, the 96-well receiving plate is removed, and the plate is directly detected by liquid chromatography-tandem mass spectrometry.

[0034] The liquid chromatography-tandem mass spectrometer used in the liquid chromatography-tandem mass spectrometry of the present invention is the RZ-500 series produced by Shanghai Ruikang Biotechnology Co., Ltd.

[0035] As an embodiment of the present invention, in step S2, the parameters of the liquid chromatography-tandem mass spectrometry include:

[0036] Chromatographic conditions:

[0037] Mobile phase A: aqueous solution containing formic acid and ammonium acetate;

[0038] Mobile phase B: methanol solution containing formic acid and ammonium acetate;

[0039] Chromatographic column model: C18 30×50mm 3μm;

[0040] Column temperature: 40°C;

[0041] Injection volume: 2 μL;

[0042] The gradient elution method was adopted, and the flow rate and gradient were as follows:

[0043] Time (minutes) Flow rate (mL / min) A％ B％ 0 0.4 100 0 1 0.4 75 25 2.5 0.4 5 95 3 0.4 5 95 4 0.4 100 0 5 0.4 100 0

[0044] Mass spectrometry conditions:

[0045] Electrospray ion source (ESI), positive ion MRM scanning analysis, ion source parameters: spray voltage 3000V, transfer tube temperature 280℃, ion source temperature 350℃, sheath gas 50Arb, auxiliary gas 15Arb; Q1 / Q3 ion channels were selected as:

[0046]

[0047] In some embodiments, step S2 specifically includes the following steps: transferring the solution in the lower receiving plate to a 96-well injection plate, and performing detection on a liquid chromatography-tandem mass spectrometer.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The present invention makes the therapeutic drugs (especially imipenem and meropenem) in the sample free and stable by adding a specific stabilizer, and uses ultrafiltration to intercept macromolecules such as proteins and pigments in serum, so that the therapeutic drugs are extracted and purified, and multiple therapeutic drugs are separated by chromatographic columns, and quantitative detection is performed using multiple reaction monitoring (MRM) mode on a liquid chromatography-tandem mass spectrometry system. This method can quickly and accurately detect 9 therapeutic drugs in serum at the same time, the pretreatment is simple and easy to operate, time-saving and labor-saving, efficient and accurate, and 9 therapeutic drugs can be detected in one test and one injection, which greatly improves the efficiency and accuracy of medical examinations, is convenient for doctors and patients, and provides an effective and feasible basis for monitoring the content of therapeutic drugs in the human body.

[0050] 2. The ultrafiltration method used in the present invention can quickly extract 9 therapeutic drugs of different properties, with a wide range of extraction types and high efficiency, and can complete the extraction and purification within 30 minutes. The ultrafiltration plate is used as the ultrafiltration material to reduce costs and realize automated operation. Compared with the traditional protein precipitation method, the sample purification is cleaner and more thorough, which can greatly reduce the loss of the chromatographic column and mass spectrometer during sample analysis and detection and improve the sensitivity of detection.

[0051] 3. The pretreatment of the present invention does not dilute the sample substantially, and can detect serum samples with lower concentrations of therapeutic drugs. The isotope internal standard method is used for quantification, which can greatly eliminate the matrix effect and achieve accurate quantification. The ultra-high performance liquid chromatography-tandem mass spectrometry method is used to detect 9 therapeutic drugs, and the method simultaneously detects the peak time and ion pair of the target. The specificity is high, which can greatly avoid the interference of cross reactions, thereby improving accuracy.

[0052] 4. The detection method of the present invention has high throughput. With one pretreatment and one injection, nine therapeutic drugs can be detected at the same time. The types of therapeutic drugs that can be detected are very comprehensive. In addition, the instrument analysis time is short, and the detection and analysis can be completed within 5 minutes. At the same time, it is of great significance for the detection of a large number of samples. In addition, the detection method of the present invention has good detection repeatability, good specificity, high sensitivity, simple sample processing, and high degree of automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0054] Figure 1 It is the total ion current diagram of 9 therapeutic drugs;

[0055] Figure 2 The chromatogram is the lower limit of quantitation (0.5 μg / mL) of amikacin (AK);

[0056] Figure 3 This is the chromatogram of the lower limit of quantification (0.2 μg / mL) of imipenem (IPM)

[0057] Figure 4 This is the chromatogram of the lower limit of quantification (0.2 μg / mL) of vancomycin (VA);

[0058] Figure 5 This is the chromatogram of the lower limit of quantification of tazobactam (0.2 μg / mL);

[0059] Figure 6 This is the chromatogram of the lower limit of quantification (0.5 μg / mL) of meropenem (MEM);

[0060] Figure 7The chromatogram is the lower limit of quantitation (0.005 μg / mL) of moxifloxacin (MXF);

[0061] Figure 8 This is the chromatogram of the lower limit of quantification (0.05 μg / mL) of linezolid (LZD);

[0062] Fig. 9 The chromatogram is the lower limit of quantification (0.5 μg / mL) of teicoplanin (TEC);

[0063] Fig.10 This is the chromatogram of the lower limit of quantification (0.05 μg / mL) of voriconazole (VOR). DETAILED DESCRIPTION

[0064] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0065] The detection method comprises the following steps: adding 100 μL of sample into a single well of a 96-well ultrafiltration plate (3KD, 350 μL), adding 20 μL of internal standard solution, adding 50 μL of stabilizer, and vortexing and mixing for 5 minutes; placing a 96-well receiving plate under the ultrafiltration plate, corresponding to the well positions one by one, placing the assembled ultrafiltration plate and receiving plate on a positive pressure device, filtering at 0.2 MPa for 20 minutes, removing the 96-well receiving plate, and directly detecting on a liquid chromatography-tandem mass spectrometer.

[0066] Preparation of reagents:

[0067] Stabilizer: aqueous solution containing hydrochloric acid and glycine (0.1 M hydrochloric acid 1 mg / mL glycine);

[0068] Internal standard solution: 1 μg / mL voriconazole-IS, 5 μg / mL vancomycin-IS, 5 μg / mL tazobactam-IS, 5 μg / mL meropenem-IS, 5 μg / mL imipenem-IS, 5 μg / mL teicoplanin-IS, 1 μg / mL linezolid-IS, 5 μg / mL amikacin-IS, 0.5 μg / mL moxifloxacin-IS in aqueous solution of hydrochloric acid (0.1 M) and glycine (1 mg / mL).

[0069] Standard stock solution: Accurately weigh 10 mg of 9 therapeutic drug standards (voriconazole, vancomycin, tazobactam, meropenem, imipenem, teicoplanin, linezolid, amikacin, and moxifloxacin) into 9 brown reagent bottles, then use a pipette to pipette 10 mL of glycine hydrochloride aqueous solution into each brown reagent bottle, ultrasonicate for 5 min, and vortex mix to obtain 9 therapeutic drug stock solutions. The concentration of the 9 therapeutic drug stock solutions is 1 mg / mL.

[0070] The liquid chromatography-tandem mass spectrometer was the RZ-500 series from Shanghai Ruikang Biotechnology Co., Ltd.

[0071] Chromatographic conditions:

[0072] Mobile phase A: aqueous solution containing formic acid and ammonium acetate

[0073] Mobile phase B: methanol solution containing formic acid and ammonium acetate

[0074] Chromatographic column model: C18 30×50mm 3μm

[0075] Column temperature: 40°C

[0076] Injection volume: 2 μL

[0077] The gradient elution method is adopted, and the flow rate and gradient are shown in Table 1 (HPLC separation flow rate and gradient)

[0078] Table 1 Liquid phase elution gradient

[0079]

[0080]

[0081] Mass spectrometry conditions:

[0082] Electrospray ion source (ESI), positive ion MRM scanning analysis, ion source parameters: spray voltage 3000V, transfer tube temperature 280°C, ion source temperature 350°C, sheath gas 50Arb, auxiliary gas 15Arb; Q1 / Q3 ion channels were selected as (Table 2):

[0083] Table 2 Parent ion and daughter ion parameters

[0084]

[0085] Example 1 Screening of stabilizers

[0086] Among the 9 antibiotics tested by the present invention, two carbapenem antibiotics, imipenem and meropenem, are particularly unstable and easy to be explained due to their drug characteristics. Therefore, it is necessary to screen out a suitable stabilizer to ensure the relative stability of the imipenem and meropenem to be tested during the detection process.

[0087] 1. Solution preparation

[0088] The configuration of the stabilizer in this embodiment is shown in Table 3:

[0089] Table 3

[0090] Group Components and content Stabilizer solution 1 Contains 1mg / mL ascorbic acid, 0.1% formic acid Stabilizer solution 2 Contains 1mg / mL ascorbic acid, 1mol / L hydrochloric acid Stabilizer solution 3 Contains 1mg / mL glycine, 0.1mol / L hydrochloric acid

[0091] 2. Test steps

[0092] The stability test was only conducted on imipenem and meropenem, two substances with relatively poor stability.

[0093] The samples were prepared to contain 4 μg / mL and 40 μg / mL of meropenem and imipenem, respectively, as low and high groups of experimental samples. Take the above-packed low-concentration group experimental samples and high-concentration group experimental samples, add appropriate amounts of internal standards, and then add stabilizers (the stabilizers are stabilizer solution 1, stabilizer solution 2, and stabilizer solution 3, respectively), vortex and oscillate to mix the samples evenly, let stand, transfer all samples to ultrafiltration tubes, centrifuge at high speed, remove the ultrafiltration tubes, transfer the extract from the lower layer of centrifugation to the sample injection plate, and load the samples on a liquid chromatography-tandem mass spectrometer for detection.

[0094] The test structure of the low concentration group experimental samples is shown in Table 4 and Table 5:

[0095] Table 4

[0096]

[0097] Table 5

[0098]

[0099] The results of mass spectrometer detection experiments of the high concentration group are shown in Tables 6 and 7 below:

[0100] Table 6

[0101]

[0102] Table 7

[0103]

[0104] Experimental conclusion:

[0105] By comparing the above experimental results, comprehensively considering the accuracy and CV value, stabilizer solution 3 (an aqueous solution containing 1 mg / mL glycine and 0.1 mol / L hydrochloric acid) was selected as a stabilizer to make imipenem and meropenem relatively more stable in the sample.

[0106] Example 2 Optimization of stabilizer component content

[0107] 1. Solution preparation

[0108] The configuration of the stabilizer in this embodiment is shown in Table 8:

[0109] Table 8

[0110] Group Components and content Stabilizer solution 3 Contains 1 mg / mL glycine, 0.1 mol / L hydrochloric acid Stabilizer solution 4 Contains 0.1 mg / mL glycine, 0.01 mol / L hydrochloric acid Stabilizer solution 5 Contains 10 mg / mL glycine, 1 mol / L hydrochloric acid

[0111] 2. Test steps

[0112] The stability test was only conducted on imipenem and meropenem, two substances with relatively poor stability.

[0113] The samples were prepared to contain 4 μg / mL and 40 μg / mL of meropenem and imipenem, respectively, as low and high groups of experimental samples. Take the above-packed low-concentration group experimental samples and high-concentration group experimental samples, add appropriate amounts of internal standards, and then add stabilizers (the stabilizers are stabilizer solution 3, stabilizer solution 4, and stabilizer solution 5, respectively), vortex and oscillate to mix the samples evenly, let stand, transfer all samples to ultrafiltration tubes, centrifuge at high speed, remove the ultrafiltration tubes, transfer the extract from the lower layer of centrifugation to the sample injection plate, and load the samples on a liquid chromatography-tandem mass spectrometer for detection.

[0114] The test structure of the experimental samples in the low concentration group is shown in Tables 9 and 10.

[0115] Table 9

[0116]

[0117] Table 10

[0118]

[0119] The results of mass spectrometer detection experiments of the high concentration group are shown in Tables 11 and 12 below:

[0120] Table 11

[0121]

[0122] Table 12

[0123]

[0124] Experimental conclusion:

[0125] By comparing the above experimental results, comprehensively considering the accuracy, CV value, and material cost, an aqueous solution containing 1 mg / mL glycine and 0.1 mol / L hydrochloric acid was selected as the stabilizer.

[0126] Example 3 Linear range and sensitivity

[0127] In the experiment, blank serum samples without therapeutic drugs were used, and different concentrations of each therapeutic drug were added to the blank serum to prepare 6 therapeutic drug spiked sample series (calibrator series) with different concentrations, where the concentrations of each therapeutic drug were:

[0128] Voriconazole: 0.05μg / mL, 0.25μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL

[0129] Vancomycin: 0.2μg / mL, 1μg / mL, 2μg / mL, 4μg / mL, 20μg / mL, 40μg / mL

[0130] Tazobactam: 0.2μg / mL, 1μg / mL, 2μg / mL, 4μg / mL, 20μg / mL, 40μg / mL

[0131] Meropenem: 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL

[0132] Imipenem: 0.2μg / mL, 1μg / mL, 2μg / mL, 4μg / mL, 20μg / mL, 40μg / mL

[0133] Teicoplanin: 0.5μg / mL, 2.5μg / mL, 5μg / mL, 10μg / mL, 50μg / mL, 100μg / mL

[0134] Linezolid: 0.05μg / mL, 0.25μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL

[0135] Amikacin: 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL

[0136] Moxifloxacin: 0.005μg / mL, 0.025μg / mL, 0.05μg / mL, 0.1μg / mL, 0.5μg / mL, 1μg / mL.

[0137] The prepared samples were treated experimentally according to the aforementioned sample pretreatment method and then tested on a mass spectrometer. The test data were subjected to linear fitting by the mass spectrometer software to obtain the linear range, standard curve equation, linear correlation coefficient and detection limit, as shown in Table 13, respectively.

[0138] Table 13 Linear range and equation of each therapeutic drug

[0139]

[0140] Table 13 shows that the curves of each treatment drug are well correlated. (R>0.995). Figure 1 It can be seen that this method can separate and detect 9 kinds of antibiotics intact within 5 minutes, with high analysis efficiency. Figures 2 to 10 It can be seen that the peak shapes of the nine antibiotics were good at the quantitative limit concentration, and the signal-to-noise ratios were all greater than 10, which fully met the requirements for analytical detection.

[0141] Example 4 Precision and Accuracy

[0142] In the experiment, blank serum (homemade) samples without therapeutic drugs were used, and low, medium and high concentrations of standard solutions of therapeutic drugs were added to the blank serum (see Table 14 for the theoretical addition amounts), and 6 parallel samples were prepared for each concentration. After experimental treatment according to the aforementioned sample pretreatment steps, the samples were tested on a mass spectrometer, and the data obtained are shown in Table 14.

[0143] Table 14: Blank serum spike recovery and precision

[0144]

[0145] As shown in Table 14, the precision and accuracy of sample detection are good. (CV<15%, DIFF<15%)

[0146] Example 5 Detection of clinical samples

[0147] Ten serum samples were selected and subjected to experimental treatment according to the aforementioned sample pretreatment steps and then tested on a mass spectrometer. The data obtained are shown in Table 15.

[0148] Table 15 Clinical sample test results

[0149]

[0150] The results of each therapeutic drug in serum were within the reference range.

[0151] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for detecting therapeutic drugs in serum extracted by ultrafiltration, characterized in that: The following steps are involved: S1, after mixing the sample, internal standard solution and stabilizer, ultrafiltration is performed; The stabilizer is an aqueous solution containing hydrochloric acid and glycine; S2, subjecting the filtrate after ultrafiltration to liquid chromatography-tandem mass spectrometry to obtain the content of therapeutic drugs in multiple serums; The therapeutic drugs in the serum include at least one of voriconazole, vancomycin, tazobactam, meropenem, imipenem, teicoplanin, linezolid, amikacin, and moxifloxacin.

2. The detection method according to claim 1, characterized in that: The concentration of hydrochloric acid in the stabilizer is 0.01-1M, and the concentration of glycine is 0.1-10 mg / mL.

3. The detection method according to claim 1, characterized in that: In step S1, the internal standard includes voriconazole-IS, vancomycin-IS, tazobactam-IS, meropenem-IS, imipenem-IS, teicoplanin-IS, linezolid-IS, amikacin-IS, and moxifloxacin-IS.

4. The detection method according to claim 3, characterized in that: In the internal standard solution, the concentrations of the internal standards are: 0.5-2 μg / mL voriconazole-IS, 1-10 μg / mL vancomycin-IS, 1-10 μg / mL tazobactam-IS, 1-10 μg / mL meropenem-IS, 1-10 μg / mL imipenem-IS, 1-10 μg / mL teicoplanin-IS, 0.5-2 μg / mL linezolid-IS, 1-10 μg / mL amikacin-IS, 0.25-1 μg / mL moxifloxacin-IS; The internal standard solution also includes a stabilizer; wherein the concentration of hydrochloric acid is 0.01-1M, and the concentration of glycine is 0.1-10 mg / mL.

5. The detection method according to claim 1, characterized in that: In step S1, the volume ratio of the sample, stabilizer and internal standard solution is 4-6:2-3:0.8-1.

2.

6. The detection method according to claim 1, characterized in that: In step S1, the ultrafiltration method includes transferring the mixed solution to an ultrafiltration plate for positive pressure or centrifugation.

7. The detection method according to claim 6, characterized in that: The specification of the ultrafiltration plate is 3KD, 350 μL.

8. The detection method according to claim 6, characterized in that: The centrifugal speed is 12000-15000 r / min, and the time is 15-30 min; or, the positive pressure is 0.1-0.4 MPa, and the time is 15-30 minutes.

9. The detection method according to claim 1, characterized in that: In the liquid chromatography-tandem mass spectrometry detection, the chromatographic conditions are: Mobile phase A: aqueous solution containing formic acid and ammonium acetate; Mobile phase B: methanol solution containing formic acid and ammonium acetate; Chromatographic column model: C18 30×50mm 3μm; Column temperature: 40°C; Injection volume: 2 μL; The gradient elution method was adopted, and the flow rate and gradient were as follows:

10. The detection method according to claim 1, characterized in that: In the liquid chromatography-tandem mass spectrometry detection, the mass spectrometry conditions are: Electrospray ionization source ESI, positive ion MRM scanning analysis, ion source parameters: spray voltage 3000V, transfer tube temperature 280℃, ion source temperature 350℃, sheath gas 50Arb, auxiliary gas 15Arb; Q1 / Q3 ion channels are selected as:

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