阅读说明：本技术 一种超高效液相色谱串联质谱技术检测血清中抗菌药物的方法 (Method for detecting antibacterial drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology ) 是由 成晓亮 李美娟 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种超高效液相色谱串联质谱技术检测血清中抗菌药物的方法,所述的抗菌药物包括：舒巴坦(SBT)、亚胺培南(IPN)、利奈唑胺(LNZ)、美洛培南(MPN)、莫西沙星(MXC)、哌拉西林(PRC)、替加环素(TGC)、头孢哌酮(CFZ)、万古霉素(VCM)、替考拉宁(TCL)；采用超高效液相色谱串联质谱法检测预处理血清中上述抗菌药物的含量,利用质谱同位素内标法定量,以标准品与内标物的浓度比为X轴,标准品与内标物峰面积比为Y轴,建立校准曲线,计算出血清中目标药物的浓度；本发明方法前处理过程简单且灵敏度高、特异性强,5min之内完成抗菌药物的分离和检测,为临床上抗菌药物的治疗浓度监测提供一种可靠的检测方法。(The invention discloses a method for detecting antibacterial drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the antibacterial drugs comprise: sulbactam (SBT), Imipenem (IPN), Linezolid (LNZ), Meropenem (MPN), Moxifloxacin (MXC), Piperacillin (PRC), Tigecycline (TGC), Cefoperazone (CFZ), Vancomycin (VCM), Teicoplanin (TCL); detecting the content of the antibacterial drug in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, quantifying by utilizing a mass spectrometry isotope internal standard method, establishing a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the concentration of the target drug in the serum; the method has simple pretreatment process, high sensitivity and strong specificity, completes the separation and detection of the antibacterial agent within 5min, and provides a reliable detection method for the clinical monitoring of the treatment concentration of the antibacterial agent.)

1. A method for detecting antibacterial drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology is characterized in that the antibacterial drugs comprise SBT, IPN, LNZ, MPN, MXC, PRC, TGC, CFZ, VCM and TCL;

detecting the contents of SBT, IPN, LNZ, MPN, MXC, PRC, TGC, CFZ, VCM and TCL in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry method, quantifying by utilizing a mass spectrum isotope internal standard method, establishing a calibration curve by taking the concentration ratio of a standard substance and an internal standard substance as an X axis and the peak area ratio of the standard substance and the internal standard substance as a Y axis, and calculating the concentration of a target drug in the preprocessed serum;

(1) specific chromatographic conditions are as follows:

mobile phase A: water containing 0.01 to 0.2 percent of formic acid;

mobile phase B: acetonitrile;

a chromatographic column: ACQUITY UPLC CSH C18 (2.1X 50mm,1.7 μm);

the gradient elution mode is adopted, and is shown in the table 1;

the flow rate is 0.2-0.5 mL/min, the collection time of each sample is 5.0min, the column temperature is 30-50 ℃, and the sample injection volume is 0.2-10 mu L;

TABLE 1 mobile phase gradient elution parameters

(2) Specific mass spectrum conditions: in an electrospray ionization detection mode, a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 3.0kV (ESI +) and 2.5kV (ESI-); source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard substance and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 2;

TABLE 2 Mass spectrometric parameters

2. The method for detecting the antibacterial drugs in the serum according to the ultra performance liquid chromatography-tandem mass spectrometry technology of claim 1, wherein the serum is human or animal serum.

3. The method for detecting the antibacterial drugs in the serum according to the ultra performance liquid chromatography-tandem mass spectrometry technology of claim 1, wherein the pretreated serum is prepared according to the following method: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing an internal standard into the centrifuge tube, and oscillating at high speed for 5 min; 14000r/min, centrifuging at 4 ℃ for 5min, taking 60 mu L of supernatant to a plastic lining tube, and injecting samples.

4. The method for detecting the antibacterial drugs in the serum according to the ultra performance liquid chromatography-tandem mass spectrometry technology of claim 1, wherein the standard substance is prepared according to the following steps:

respectively preparing the antibacterial drugs into standard substance mother solutions with the concentrations respectively as follows: SBT 100mg/mL, LNZ 20mg/mL, MPN5mg/mL, MXC 10mg/mL, PRC 10mg/mL, TGC10mg/mL, VCM 50mg/mL, TCL 50mg/mL, IPN2mg/mL and CFZ 50mg/mL, then respectively transferring SBT 20 mu L, LNZ 10 mu L, MPN 40 mu L, MXC 20 mu L, PRC 20 mu L, TGC 8 mu L, VCM 16 mu L, TCL 40 mu L, IPN 400 mu L, CFZ200 mu L, adding 226 mu L methanol solution, and fully mixing to obtain 1mL mixed standard stock solution;

adding 10 μ L of the mixed standard stock solution into 190 μ L of blank serum matrix as a first high-value concentration point (S7); taking the first high-value concentration point (S7) and diluting the first high-value concentration point with an equal volume of blank serum matrix to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S7) with 3 times volume of blank serum substrate to obtain a third high-value concentration point (S5); diluting the second high concentration point (S6) with 9 times volume of blank serum matrix to obtain a fourth high concentration point (S4); diluting the third high concentration point (S5) with 9 times volume of blank serum matrix to obtain a fifth high concentration point (S3); diluting the fourth high concentration point (S4) with blank serum matrix of 4 times volume to obtain a sixth high concentration point (S2); the fifth high concentration point (S3) was diluted with 4 volumes of blank serum substrate to obtain the seventh high concentration point (S1).

5. The method for detecting the antibacterial drugs in the serum according to the ultra performance liquid chromatography tandem mass spectrometry technology of claim 3, wherein the protein precipitant containing the internal standard is prepared by the following method: respectively preparing isotope internal standard mother liquor with the concentration of 1mg/mL from the antibacterial drugs by using methanol; preparing the isotope internal standard mother liquor into mixed internal standard solution containing 150000ng/mL SBT-d5, 5000ng/mL LNZ-d3, 5000ng/mL MPN-d6, 5000ng/mL MXC-13CD3, 5000ng/mL PRC-d5 and 10000ng/mL TGC-d9 by using methanol solution; and adding 19.8mL of protein precipitant into 200 mu L of the mixed internal standard solution, and uniformly mixing to obtain the protein precipitant containing the internal standard.

6. The method for detecting the antibacterial drugs in the serum according to the ultra performance liquid chromatography-tandem mass spectrometry technology of claim 5, wherein the protein precipitator is a mixed solution of methanol, acetonitrile and isopropanol in a volume ratio of 1:1: 2.

7. The method for detecting antibacterial drugs in serum according to the ultra performance liquid chromatography-tandem mass spectrometry technology of claim 4, wherein the blank serum matrix is blank serum without target drugs.

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting antibacterial drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology.

Background

Bacterial infections are an important source of morbidity and mortality, and the use of antibiotic therapy is an important aspect of inpatient management, particularly in Intensive Care Units (ICU). However, it has been demonstrated that antibiotic treatment based on standard dosing regimens often fails to achieve effective therapeutic blood levels in patients, which greatly affects the therapeutic efficacy and may result in patients with multiple bacterial resistance. Because of the inter-individual variability in pharmacokinetics among different patients, simply increasing the dosage of a drug may cause dangerous toxic side effects. Strict in vivo monitoring of drug concentrations is therefore required, particularly for drugs with narrow therapeutic concentrations.

Sulbactam (SBT) is a beta-lactamase inhibitor, and has an inhibiting effect on beta-lactamase produced by gram-positive and gram-negative bacteria (except copper green pseudomonas). Cefoperazone (CFZ) is a third-generation cephalosporin with a wide antimicrobial spectrum and is particularly effective against gram-negative bacilli. The combination of sulbactam and cefoperazone has synergistic antibacterial activity. The cefoperazone/sulbactam is widely applied to serious infection patients such as ICU (acute coronary syndrome) and the like as one of antibiotics with low drug resistance rate to a few of widely-resistant acinetobacter baumannii.

Piperacillin (PRC) is broad-spectrum semisynthetic acylurea penicillin, has the characteristics of wide antibacterial spectrum and strong antibacterial action, particularly has strong antibacterial action on pseudomonas aeruginosa, is widely applied at home and abroad at present, and is considered as the most valuable penicillin. Piperacillin is mainly used for clinically treating diseases such as pneumonia, septicemia, respiratory tract infection, biliary tract infection, urinary system infection, subacute endocarditis, suppurative meningitis and the like caused by pseudomonas aeruginosa and other sensitive gram-negative bacilli.

Tigecycline (TGC) is a new type of glycyltetracycline antibiotics developed and marketed in recent years, and has a bactericidal effect by inhibiting the synthesis of bacterial proteins by preventing amino acid tRNA from entering ribosome A site. Tigecycline has broad-spectrum antibacterial activity, and bacteria sensitive to tigecycline comprise methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus, penicillin-resistant streptococcus pneumoniae, acinetobacter baumannii, gram-negative bacteria producing extended-spectrum beta-lactamase and the like.

Imipenem (IPN) is a carbapenem antibiotic, has the characteristics of wide antibacterial spectrum, strong antibacterial activity and high stability to beta-lactamase, is mainly used for treating multiple drug resistant gram negative bacillus infection, mixed infection of severe aerobes and anaerobes and severe infection with undetermined pathogen, and is a first-line medicament for treating severe infection.

Meropenem (MPN) is an artificial semi-synthetic carbapenem antibiotic, has an ultra-broad spectrum of antibacterial activity, shows extremely strong activity to aerobic and anaerobic bacteria which are common in clinic, and is clinically used for treating respiratory, urinary tract, surgery, obstetrics and gynecology, skin and soft tissue infection and bacterial meningitis due to strong tissue penetrating power and wide distribution of meropenem.

Linezolid (LNZ) is the first novel clinical oxazolidinone antibacterial drug, has a unique action mechanism, has no cross-resistance with other drugs, has no obvious influence on pharmacokinetics by age and sex, and has a good antibacterial effect on most gram-positive bacteria including multidrug-resistant enterococcus, staphylococcus, streptococcus pneumoniae and the like.

Moxifloxacin (MXC) belongs to a fourth-generation novel fluoroquinolone broad-spectrum antibacterial drug, and has the bactericidal effect by inhibiting the activity of a subunit A of DNA helicase and topoisomerase IV of bacteria, and blocking the replication of DNA. The moxifloxacin has good antibacterial activity on gram-positive bacteria, gram-negative bacteria, anaerobic bacteria and atypical pathogens (mycoplasma, chlamydia and legionella), and has the advantages of strong tissue penetration, high bioavailability, long action time, less adverse reaction and the like. Moxifloxacin is a commonly used clinical antibacterial drug and is mainly applied to acute attack of chronic bronchitis, chronic obstructive pulmonary disease, community-acquired pneumonia and skin and soft tissue infection.

Vancomycin (VCM) is a glycopeptide antibacterial drug, has strong bactericidal activity, rarely induces and generates drug resistance, has obvious curative effect on infection caused by gram-positive bacteria, methicillin-resistant staphylococcus aureus and the like, and is a common drug for clinically treating severe infection.

Teicoplanin (TCL) is a bactericidal novel glycopeptide antibiotic produced by actinoplanes fermentation, has a mechanism of action similar to vancomycin and other glycopeptide antibiotics, is one of important antibiotics used for treating multiple drug-resistant bacterial infections after vancomycin, and is clinically used for treating various serious gram-positive bacterial infections, particularly methicillin-resistant staphylococcus infections. Is suitable for treating severe staphylococcal infection (especially methicillin resistant staphylococci), such as sepsis, septic endocarditis, acute osteomyelitis and acute attack of chronic osteomyelitis, septic arthritis, neutropenia complicated infection of patients, etc.

At present, methods for measuring antibacterial drugs in human serum or plasma in domestic and foreign literature reports mainly comprise an HPLC-UV method and an LC-MS/MS method, but have the defects of low sensitivity, long analysis time of a single sample, large sample consumption and the like, and some methods even adopt an external standard method for quantification, have matrix interference and poor accuracy. Chinese patents (CN 110361481A and CN 110361482A) respectively disclose a kit for monitoring the concentration of vancomycin in blood and a detection method thereof and a kit for monitoring the concentration of linezolid in blood and a detection method thereof, the two inventions are the same, a multidimensional online solid phase extraction liquid chromatography analysis technology is used for measuring vancomycin and linezolid in blood, the sample amount used in the method is 4mL, an external standard method is used for quantification, and the analysis time of a single sample is as long as 17 min; the method for applying and monitoring Chinese medicaments in 2013, 4, 10, 2 and titled as 'measuring the concentration of moxifloxacin in blood plasma by HPLC-MS/MS method' adopts levofloxacin as an internal standard to detect the concentration of moxifloxacin in blood plasma of a patient, and the method is not applicable to patients taking levofloxacin; in an article entitled "LC-MS/MS method for simultaneously determining the concentrations of voriconazole and tigecycline in human plasma" at volume 24 of journal 35 of Chinese clinical pharmacology, other drugs are also used as internal standards for quantitative analysis, and the universal applicability and the accuracy are lacked. The isotope internal standard and the object to be detected have basically the same or consistent physical and chemical properties, chromatographic behavior and response characteristics, and are the most accurate quantification method at present. Moreover, clinical samples are extremely precious, so the dosage of pretreatment samples is as small as possible, and the instrument is seriously polluted due to too large sample injection amount, and the maintenance cost of the instrument is increased.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting antibacterial drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology.

The antibacterial drug comprises: sulbactam (Sulbactam, SBT), Imipenem (ipicenm, IPN), Linezolid (Linezolid, LNZ), Meropenem (Meropenem, MPN), Moxifloxacin (Moxifloxacin, MXC), piperacillin (Pipracil, PRC), Tigecycline (Tigecycline, TGC), Cefoperazone (CFZ), Vancomycin (Vancomycin, VCM), Teicoplanin (Teicoplanin, TCL).

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a method for detecting antibacterial drugs in serum by ultra performance liquid chromatography tandem mass spectrometry technology comprises SBT, IPN, LNZ, MPN, MXC, PRC, TGC, CFZ, VCM and TCL;

detecting the content of the antibacterial drugs in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, quantifying by utilizing an isotope internal standard method, establishing a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the concentration of the 10 antibacterial drugs in the serum;

(1) specific chromatographic conditions are as follows:

mobile phase A: water containing 0.01 to 0.2 percent of formic acid;

mobile phase B: acetonitrile;

a chromatographic column: ACQUITYUPLC CSH C18 (2.1X 50mm,1.7 μm);

the gradient elution mode is adopted, and is shown in the table 1;

the flow rate is 0.2-0.5 mL/min, the collection time of each sample is 5.0min, the column temperature is 30-50 ℃, and the sample injection volume is 0.2-10 mu L;

TABLE 1 mobile phase gradient elution parameters

(2) Specific mass spectrum conditions: in an electrospray ionization detection mode, a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 3.0kV (ESI +) and 2.5kV (ESI-); source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard substance and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 2;

TABLE 2 Mass spectrometric parameters

Wherein the serum is human or animal serum.

Wherein the pretreated serum is prepared by the following method: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing an internal standard into the centrifuge tube, and oscillating at high speed for 5 min; 14000r/min, centrifuging at 4 ℃ for 5min, taking 60 mu L of supernatant to a plastic lining tube, and injecting samples.

Wherein, the protein precipitant containing the internal standard is prepared by the following method: respectively preparing isotope internal standard mother liquor with the concentration of 1mg/mL from the antibacterial drugs by using methanol; preparing the isotope internal standard mother liquor into mixed internal standard solution containing 150000ng/mL SBT-d5, 5000ng/mL LNZ-d3, 5000ng/mL MPN-d6, 5000ng/mL MXC-13CD3, 5000ng/mL PRC-d5 and 10000ng/mL TGC-d9 by using methanol solution; and taking 200 mu L of mixed internal standard solution, and adding 19.8mL of protein precipitator to obtain the protein precipitator containing the internal standard.

Wherein the protein precipitator is a mixed solvent of methanol, acetonitrile and isopropanol.

The standard substance is prepared according to the following steps: respectively preparing the antibacterial drugs into standard substance mother solutions with the concentrations respectively as follows: SBT 100mg/mL, LNZ 20mg/mL, MPN5mg/mL, MXC 10mg/mL, PRC 10mg/mL, TGC10mg/mL, VCM 50mg/mL, TCL 50mg/mL, IPN2mg/mL and CFZ 50mg/mL, then SBT 20 μ L, LNZ 10 μ L, MPN 40 μ L, MXC 20 μ L, PRC 20 μ L, TGC 8 μ L, VCM 16 μ L, TCL 40 μ L, IPN 400 μ L, CFZ200 μ L respectively, 226 μ L methanol solution is added, and the mixture is fully mixed to obtain 1mL mixed standard stock solution.

The invention prepares the mixed standard stock solution into seven calibrator solutions with different concentration points by using a blank serum matrix, and the preparation process is as follows: adding 10 μ L of the mixed standard stock solution into 190 μ L of blank serum matrix as a first high-value concentration point (S7);

taking the first high-value concentration point (S7) and diluting the first high-value concentration point with an equal volume of blank serum matrix to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S7) with 3 times volume of blank serum substrate to obtain a third high-value concentration point (S5); diluting the second high concentration point (S6) with 9 times volume of blank serum matrix to obtain a fourth high concentration point (S4); diluting the third high concentration point (S5) with 9 times volume of blank serum matrix to obtain a fifth high concentration point (S3); diluting the fourth high concentration point (S4) with blank serum matrix of 4 times volume to obtain a sixth high concentration point (S2); diluting the fifth high concentration point (S3) with blank serum matrix 4 times the volume to obtain a seventh high concentration point (S1), the specific process is as shown in Table 3 below;

TABLE 3 Standard Curve preparation and concentration (unit: ng/mL)

Wherein the blank serum matrix is blank serum without target drugs;

taking 50 mu L of each concentration point of seven calibration sample samples with different concentrations, putting the 50 mu L of each concentration point in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard into the centrifuge tube, and oscillating at a high speed for 5 min; centrifuging at 14000r/min at 4 deg.C for 5min, collecting supernatant 60 μ L, and introducing into plastic liner tube with sample amount of 1 μ L.

Has the advantages that: the method has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, can finish the separation and detection of 10 antibacterial drugs within 5min, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical antibacterial drugs, and provides a reliable detection method for monitoring the treatment concentration of clinical antibacterial drugs.

Drawings

FIG. 1 is an ion flow chart of antibacterial drug standard extraction;

FIG. 2 is an ion flow diagram of the extraction of antibacterial agents from serum.

Detailed Description

For the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.