CN111458430A - Liquid chromatography tandem mass spectrometry quantitative detection method for concentration of drug-resistant bacteria infection resisting drug - Google Patents
Liquid chromatography tandem mass spectrometry quantitative detection method for concentration of drug-resistant bacteria infection resisting drug Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
A liquid chromatography tandem mass spectrometry quantitative detection method for drug resistance bacteria infection drug concentration comprises the steps of using a standard product as an tedizolid standard product, using an isotope internal standard tedizolid-d 3 as an internal standard, drawing a standard curve by using the concentration ratio of a standard working solution to an internal standard working solution as an abscissa and the area ratio of a standard product peak area to an internal standard peak area as an ordinate, carrying out L C-MS/MS analysis on a processed biological sample, carrying out quantification through the standard curve, calculating the content of the tedizolid in a sample to be detected, and using a tedizolid chromatogram as shown in figure 3.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a liquid chromatography-tandem mass spectrometry quantitative detection method for drug resistance bacteria infection drug concentration.
Background
Drug-resistant bacteria refer to pathogenic bacteria having drug resistance. Overuse of antibiotics has prompted many drug resistant strains, and bacterial resistance is considered a serious threat to human health. For example, staphylococcus aureus is a gram-positive coccoid bacterium, is a common colonizing bacterium of human skin and nasal cavity, is one of the most prevalent pathogens of nosocomial and community infections, and can cause diseases such as suppuration, otitis media, sepsis, enteritis, sinusitis, osteomyelitis, pneumonia and even septicemia after infection. However, with the widespread use of antibiotics, there are many resistant varieties (resistant strains) in staphylococcus aureus, such as methicillin-resistant staphylococcus aureus (MRSA), which is one of them and is known as a "superbacteria" that is highly resistant to methicillin. The occurrence of drug-resistant bacteria increases the difficulty of curing infectious diseases, and infection caused by the drug-resistant bacteria is greatly related to the increase of morbidity and mortality, so that medicines which have broad-spectrum antibacterial action on most of clinically common gram-positive cocci and have no cross drug resistance with other antibacterial medicines are developed.
The tedizolid is an oxazolidinone compound, is regarded as a clinical substitute of linezolid, and is a second-generation product. The tedizolid shows good antibacterial action on methicillin-sensitive or drug-resistant staphylococcus aureus, vancomycin-resistant enterococcus and penicillin-sensitive or drug-resistant streptococcus pneumoniae, is an ideal medicine for treating Acute Bacterial Skin and Skin Structure Infection (ABSSSI), and has good clinical application prospect. (ABSSSI is a clinically common and fatal disease with an increasing incidence of gram-positive bacteria (primarily Staphylococcus aureus) and methicillin-resistant bacteria are the leading causes of infection).
However, the concentration of the antibacterial drug must be kept within an effective and safe dosage range to achieve good therapeutic effects, and the excessive blood concentration may cause serious side effects of the body, including impairment of liver and kidney functions and bone marrow suppression; if the blood concentration is too low, there is no antibacterial effect. In addition, because the individual infected with the drug has functional differences such as age, weight, gastrointestinal tract and the like, and is influenced by a plurality of factors such as genetic factors, environmental factors, drug interaction and the like, the drug metabolism and elimination processes in the patient body are greatly different, so that the antibacterial drug is usually at a narrow and variable effective blood concentration level, and the dosage of the antibacterial drug is adjusted according to the constitution and the illness state of the patient and the monitored blood concentration when the antibacterial drug is taken. Therefore, the monitoring of the blood concentration of the antibacterial agent has important guiding significance for the treatment of drug-resistant bacteria infected patients, and can assist doctors to optimize the administration dosage in a very narrow treatment window, thereby ensuring the effective antibacterial dosage and avoiding the occurrence of serious side effects caused by overhigh dosage.
Disclosure of Invention
The invention aims to provide a high-quality and effective liquid chromatography tandem mass spectrometry quantitative detection method for concentration of a drug resistant bacterial infection resistant drug, aiming at the defects of narrow therapeutic window of the drug resistant bacterial infection resistant drug, large individual tolerance capability and large in vivo elimination difference, so that the dose controllability of the drug resistant bacterial infection resistant drug is enhanced, the therapeutic effect of the drug resistant bacterial infection resistant drug is ensured, and the dose individuation and clinical accurate treatment of the drug resistant bacterial infection resistant drug are realized.
The L C-MS/MS analysis is short for liquid chromatography-tandem mass spectrometry (L iquid chromatography-tandem mass spectrometry, L C-MS/MS).
In order to achieve the purpose, the invention provides the following technical scheme:
a liquid chromatography tandem mass spectrometry quantitative detection method for drug resistance bacteria infection drug concentration comprises the steps of taking a standard substance in a standard solution as a tedizolid standard substance, taking an internal standard substance in an internal standard solution as an isotope internal standard tedizolid-d 3, drawing a standard curve by taking the concentration ratio of a standard working solution to an internal standard working solution as a horizontal coordinate and the area ratio of a standard substance peak area to an internal standard substance peak area as a vertical coordinate, carrying out L C-MS/MS analysis on a processed biological sample, carrying out quantification through the standard curve, and calculating the content of the tedizolid in a sample to be detected.
Preparing a standard solution, namely precisely weighing 5mg of a drug-resistant bacteria infection resisting tedizolid standard substance by using a balance, dissolving, diluting and fixing the volume to 10m L by using a chromatographic pure methanol solution to obtain a stock solution A with the concentration of 500 mu g/m L;
preparing an internal standard solution, namely precisely weighing the isotope internal standard tedizolid-d 31mg by using a balance, dissolving, diluting and fixing the volume to 10m L by using a chromatographic pure methanol solution to obtain a stock solution B with the concentration of 100 mu g/m L;
and (3) biological sample treatment, namely unfreezing a plasma sample at room temperature, adding 10 mu l of internal standard isotope internal standard tedizolid-d 3 to 1.5m L centrifuge tube, sucking 100 mu l of the plasma sample, adding 0.6m L chromatographic pure methanol, carrying out vortex mixing oscillation for 5min, centrifuging for 15min, taking 200 mu l of supernatant, drying by blowing with nitrogen, redissolving with 100 mu l of chromatographic pure acetonitrile, carrying out vortex mixing oscillation for 1min, centrifuging for 5min, taking 90 mu l of supernatant, and carrying out L C-MS/MS analysis.
The standard working solution is a tedizolid solution standard working solution which is prepared by diluting the stock solution A into 0.48825 mu g/m L, 3.90625 mu g/m L, 15.625 mu g/m L, 31.25 mu g/m L, 62.5 mu g/m L, 125.0 mu g/m L, 250.0 mu g/m L and 500.0 mu g/m L, and the diluent is chromatographically pure methanol;
the internal standard working solution is Tedizolid-d 3 internal standard working solution diluted to 20.0 mu g/m L by the stock solution B, and the diluent is chromatographic pure methanol.
The blood collection tube for collecting the plasma sample is a green cap blood collection tube containing heparin sodium.
In the L C-MS/MS analysis, the following chromatographic conditions were used:
the chromatographic column comprises ZORBAX SB C182.1 × 100mm, 3.5 μm, mobile phase A of 0.1% formic acid aqueous solution, mobile phase B of acetonitrile, sample introduction of 5 μ L, column temperature of 30 deg.C, flow rate of 0.4m L/min, and mobile phase gradient shown in the following table 1:
TABLE 1 mobile phase gradiometer
The following mass spectrometry conditions were used:
the ion source mode IS ESI + mode, TEM 450 deg.C, IS 4000V, CUR 20L/min, CAD 6L/min, GS1 45L/min, GS2 15L/min.
Compared with the prior art, the invention has the beneficial effects that:
the tedizolid medicine is not listed in China, is not included in the 'Chinese pharmacopoeia' 2015 edition, and has no relevant quality standard, the invention establishes a method for testing the tedizolid medicine, improves the quality supervision requirements of the medicine market on the medicine, and simultaneously has high sensitivity, strong specificity, good reproducibility and short analysis time for detecting the tedizolid medicine for resisting drug-resistant bacteria infection.
Drawings
FIG. 1: a matrix blank mass spectrum, the matrix being a blank plasma sample. In fig. 1, the abscissa is retention time and the ordinate is response value.
FIG. 2: a blank mass spectrum of a solvent, wherein the solvent is methanol and acetonitrile involved in the method. The abscissa in fig. 2 holds time, and the ordinate is the response value.
FIG. 3: the tedizolid chromatogram is shown in fig. 3 with retention time on the abscissa and response on the ordinate.
FIG. 4: the chromatogram of tedizolid-d 3 is plotted on the abscissa for retention time and on the ordinate for response in FIG. 4.
Detailed Description
The present invention is further illustrated by the following examples, in which reagents and equipment are commercially available, and conventional methods in the art are not specifically described in the examples.
Reagent and main instrument
(I) reagent
Nitrogen gas: the purity is 98 percent;
pure water with the specification of 4.5L, the cat number of 20181123H and the Drech.
Methanol HP L grade C, CAS number 67-56-1, cat # UN1230, Merk, Specification 4L.
Acetonitrile HP L grade C, CAS number 75-05-8, cat number A998-4, Fisher, specification 4L.
Formic acid HP L grade C, CAS number 64-18-6, cat number A117-50, Fisher, Specification 50m L.
Tedizolid standard: the purity is more than or equal to 99 percent, and the molecular formula is as follows: C17H15FN6O3, molecular weight: 370.34, CAS number: 856866-72-3, cat number: t127021, Aladdin, specification: 5 mg.
Isotopically internal standard tedizolid-d 3: the purity is more than or equal to 99 percent, and the molecular formula is as follows: C17H15D3FN6O3, molecular weight: 373.36, CAS number: 856866-72-3, TrC, Specification: 1 mg.
0.1% aqueous formic acid solution 600. mu.l formic acid was pipetted into a 600m L pure water mobile phase glass vial using a 1m L pipette, shaken well and sonicated for 3 min.
(II) Main instrument and equipment
Detection equipment of American AB L C-MS/MS 4500MD
Detecting environmental requirements: the humidity is 20-80%; the temperature is 15-28 DEG C
The detection reagent has no special requirement, and the following reagents are all in chromatographic purity.
The invention provides a liquid chromatography tandem mass spectrometry quantitative detection method for drug resistance bacteria infection drug concentration, which comprises the following steps:
s1, preparing a standard solution, namely precisely weighing 5mg of a drug-resistant bacteria infection resisting tedizolid standard substance by using a balance, dissolving the drug-resistant bacteria infection resisting tedizolid standard substance by using a methanol solution, diluting the drug-resistant bacteria infection resisting tedizolid standard substance to 10m L to obtain a stock solution A (namely the standard solution) with the concentration of 500 mu g/m L, and diluting the stock solution A into standard working solutions of the tedizolid solution with the concentrations of 0.48825 mu g/m L, 3.90625 mu g/m L, 15.625 mu g/m L, 31.25 mu g/m L, 62.5 mu g/m L, 125.0 mu g/m L, 250.0 mu g/m L and 500.0 mu g/m L, wherein the diluent is methanol.
S2, preparing an internal standard solution, namely precisely weighing the internal standard tedizolid-d 31mg of the isotope of the drug-resistant bacteria infection resisting drug, dissolving the internal standard solution with a methanol solution, diluting the internal standard solution and fixing the volume to 10m L to obtain stock solution B (namely the internal standard solution) with the concentration of 100 mu g/m L, and diluting the stock solution B into the internal standard working solution of the internal standard tedizolid-d 3 with the concentration of 20.0 mu g/m L, wherein the diluent is methanol.
S3, biological sample treatment, namely thawing a plasma sample at room temperature, adding 10 mu l of isotope internal standard tedizolid-d 3-1.5 m L centrifuge tube, sucking 100 mu l of plasma sample, adding 0.6m L methanol, carrying out vortex mixing and oscillation for 5min, centrifuging for 15min, taking 200 mu l of supernatant, carrying out blow-drying with nitrogen, redissolving with 100 mu l of acetonitrile, carrying out vortex mixing and oscillation for 1min, centrifuging for 5min, taking 90 mu l of supernatant, and carrying out L C-MS/MS analysis.
S4: and drawing a standard curve, and drawing the standard curve by taking the concentration ratio of the standard working solution to the internal standard working solution as an abscissa and the area ratio of the standard peak area to the internal standard peak area as an ordinate.
And S5, calculating concentration, namely, carrying out L C-MS/MS analysis on the processed biological sample in the step S3, and quantifying by using the standard curve in the step S4 to calculate the content of tedizolid in the sample to be detected, wherein the related mass spectrum conditions comprise an ion source mode of ESI + mode, TEM 450 ℃, IS:4000V, CUR: 20L/min, CAD: 6L/min, GS 1: 45L/min and GS 2: 15L/min.
In order that those skilled in the art will better understand the technical solutions of the present invention, the following description will be further described with reference to specific examples.
The embodiment is suitable for quantitative determination of tedizolid in heparin sodium whole blood. The whole blood sample, the detection equipment and the detection reagent are prepared before the test, and the preparation comprises the following steps:
collecting a whole blood sample: before the whole blood sample is collected, the nervous psychology of a patient needs to be eliminated, the operation needs to be standardized during blood collection, and the whole blood sample is mixed in time.
The type and the quantity of the sample, namely the sample to be tested is extracted from 2m L blood of a patient by a heparin sodium green cap blood sampling vacuum tube.
Selecting a specimen container: sterile disposable vacuum blood collection tubes (bidi medical devices (shanghai) ltd.).
The chromatographic conditions were as follows:
a chromatographic column: brand name: agilent; the model is as follows: ZORBAX SB C182.1 mm, 3.5 μm
Mobile phase A: 0.1% aqueous formic acid solution
Mobile phase B: acetonitrile
Sample size 5 mu L
Column temperature: 30 deg.C
Flow rate of 0.4m L/min
The mobile phase gradients are as in table 1 below:
TABLE 1 mobile phase gradiometer
The mass spectrometry conditions were as follows:
ion source mode: ESI +
TEM：450℃
IS:4000V
CUR：20L/min
CAD：6L/min
GS1：45L/min
GS2：15L/min
DP：60V
CE:23eV
MRM parameter setting table 2:
TABLE 2 MRM parameter setting Table
Methodology validation
1 anti-interference experiment
1.1 purpose of the experiment
And (3) inspecting the influence of the matrix and the pretreatment reagent on the analyte and the internal standard peak of the whole blood sample.
1.1 Experimental methods
Selecting 5 blank plasma samples of healthy people from different sources, pretreating and analyzing the blank plasma samples according to the method of S3 (adding 10 mu l of isotope internal standard tedizolid-d 3 to be PBS solution), and simultaneously respectively preparing a low-concentration quality control product (5 mu g/m L) and an internal standard working solution sample added into the blank plasma as references, wherein chromatograms are shown in figures 3 to 4.
And (4) experimental conclusion: as can be seen in fig. 1-4, the whole blood sample matrix and reagents do not interfere with the tedizolid target peak and the internal standard peak.
Day to day precision within 2 days
2.1 purpose of the experiment
Stability of the investigation experiment
2.2 Experimental methods
Respectively adding 250.0 mu g/m L standard working solution 20 mu L, 250.0 mu g/m L0 standard working solution 160 mu L1 and 500.0 mu g/m L standard working solution 400 mu L into a 2ml centrifuge tube, respectively correspondingly adding 980 mu L, 840 mu L and 600 mu L of healthy human blood, respectively carrying out vortex mixing, respectively preparing low-concentration (5 mu g/m L), medium-concentration (40 mu g/m L) and high-concentration (200 mu g/m L) labeling parallel samples of each 5 parts, carrying out pretreatment and analysis according to the method of S3, measuring the in-day sample injection, and continuously measuring three days to calculate the precision of the in the day
2.3 results of the experiment
Table 3: results of determination of method precision
2.4 conclusion of the experiment
The RSD of the experimental result is less than 15 percent, and the requirement of biological sample determination is met.
3 recovery rate of added standard
3.1 purpose of the experiment
The accuracy of the experimental results was investigated by the recovery of the spiked samples.
3.2 results of the experiment
Table 4: experimental results of recovery
3.3 conclusion of the experiment
According to experimental results, the recovery rates of the three standard adding concentrations are all 85-120%, and the relative standard deviation RSD is less than 15%, so that the requirement of methodology verification is met.
4 standard curve
4.1 purpose of the experiment
Verification of the Linear Range of the method
4.2 Experimental methods
A standard curve is established according to the method of the invention, and 4 batches are analyzed in total by sample injection analysis.
4.3 results of the experiment
Table 5: standard curve verification results
4.4 conclusion of the experiment
The linear range of the standard curve is in the range of 0.488 mu g/m L to 250.0 mu g/m L, the linearity is good, and r is2Are all larger than 0.995, and meet the requirements related to methodology.
Lower limit of 5 quantitative
5.1 purpose of the experiment
Examining the accuracy of detection of a sample at around the lower limit of quantitation
5.2 Experimental methods
With a 10-fold signal-to-noise ratio (10S/N) as a limit of quantitation (L OQ), a batch of low-concentration samples was prepared, pretreated as described in S3 and analyzed by injection, and each batch of samples was assayed in parallel five times.
5.3 conclusion of the experiment
The quantitative limit (L OQ) is 0.48 mu g/m L, the linear range is 0.488 mu g/m L to 250.0 mu g/m L, the linearity is good, and the requirement of methodology verification is met.
At present, the method for detecting the concentration of the tedizolid in human plasma by using a mass spectrometry is not reported at home, and has no quality standard related to the method, the method is favorable for establishing a method for detecting the medicine of the tedizolid, so as to improve the quality supervision requirement of the medicine market on the tedizolid medicine, and by combining the verification experiments, all technical indexes of the method meet the requirements of China qualified assessment national committee for approval (CNAS), namely the precision judgment limit in the day is less than 1/4TEA (6.25%), the precision judgment limit in the day is less than 1/3TEA (8.33%), the liquid chromatography tandem mass spectrometry is used for detecting the content of the medicine of resisting drug-resistant bacteria infection, the stability is good, the detection precision is high, and meanwhile, the method uses an internal standard method for quantification, eliminates the system error, so that the analysis of a target compound is more accurate, the analysis time is short (5.25 min is required for each sample detection), the sensitivity is high (the quantification limit is 0.48 mu g/m L), the specificity is strong (the characteristic isotope ion of the tedizolid ion is 370.3, 156.1, the tedizolid), the maximum value of the plasma ion → 36.373), and the clinical isotope ion monitoring requirement of the plasma concentration is met, so as the research on the plasma pharmacokinetics and the plasma pharmacokinetics.
Claims (5)
1. A liquid chromatography tandem mass spectrometry quantitative detection method for drug resistance bacteria infection drug concentration is characterized by comprising the steps of taking a standard substance in a standard solution as a tedizolid standard substance, taking an internal standard substance in an internal standard solution as an isotope internal standard tedizolid-d 3, drawing a standard curve by taking the concentration ratio of a standard working solution to an internal standard working solution as a horizontal coordinate and the area ratio of a standard substance peak area to an internal standard substance peak area as a vertical coordinate, carrying out L C-MS/MS analysis on a processed biological sample, carrying out quantification through the standard curve, and calculating the content of the tedizolid in a sample to be detected.
2. The method of claim 1, wherein:
preparing a standard solution, namely precisely weighing 5mg of a drug-resistant bacteria infection resisting tedizolid standard substance by using a balance, dissolving, diluting and fixing the volume to 10m L by using a chromatographic pure methanol solution to obtain a stock solution A with the concentration of 500 mu g/m L;
preparing an internal standard solution, namely precisely weighing the isotope internal standard tedizolid-d 31mg by using a balance, dissolving, diluting and fixing the volume to 10m L by using a chromatographic pure methanol solution to obtain a stock solution B with the concentration of 100 mu g/m L;
and (3) biological sample treatment, namely unfreezing a plasma sample at room temperature, adding 10 mu l of internal standard isotope internal standard tedizolid-d 3 to 1.5m L centrifuge tube, sucking 100 mu l of the plasma sample, adding 0.6m L chromatographic pure methanol, carrying out vortex mixing oscillation for 5min, centrifuging for 15min, taking 200 mu l of supernatant, drying by blowing with nitrogen, redissolving with 100 mu l of chromatographic pure acetonitrile, carrying out vortex mixing oscillation for 1min, centrifuging for 5min, taking 90 mu l of supernatant, and carrying out L C-MS/MS analysis.
3. The method of claim 2, wherein:
the standard working solution is a tedizolid solution standard working solution which is prepared by diluting the stock solution A into 0.48825 mu g/m L, 3.90625 mu g/m L, 15.625 mu g/m L, 31.25 mu g/m L, 62.5 mu g/m L, 125.0 mu g/m L, 250.0 mu g/m L and 500.0 mu g/m L, and the diluent is chromatographically pure methanol;
the internal standard working solution is Tedizolid-d 3 internal standard working solution diluted to 20.0 mu g/m L by the stock solution B, and the diluent is chromatographic pure methanol.
4. The method of claim 2, wherein:
the blood collection tube for collecting the plasma sample is a green cap blood collection tube containing heparin sodium.
5. The method of claim 1, wherein the L C-MS/MS analysis uses the following chromatographic conditions:
the chromatographic column comprises ZORBAX SB C182.1 × 100mm, 3.5 μm, mobile phase A of 0.1% formic acid aqueous solution, mobile phase B of acetonitrile, sample introduction of 5 μ L, column temperature of 30 deg.C, flow rate of 0.4m L/min, and mobile phase gradient shown in the following table 1:
TABLE 1 mobile phase gradiometer
The following mass spectrometry conditions were used:
the ion source mode IS ESI + mode, TEM 450 deg.C, IS 4000V, CUR 20L/min, CAD 6L/min, GS1 45L/min, GS2 15L/min.
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| CN114858932A (en) * | 2022-03-15 | 2022-08-05 | 昆明和合医学检验所有限公司 | Method for determining nervonic acid content in dry blood filter paper sheet by high performance liquid chromatography tandem mass spectrometry |
| CN114858932B (en) * | 2022-03-15 | 2024-04-26 | 昆明和合医学检验所有限公司 | Method for measuring content of nervonic acid in dry blood filter paper sheet by high performance liquid chromatography-tandem mass spectrometry |
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Application publication date: 20200728 |