Method for detecting concentration of josamycin in plasma

Abstract

The invention belongs to the technical field of medical examination, and particularly relates to a method for detecting the concentration of josamycin in plasma. A to-be-detected sample is treated by adopting a protein precipitation method, operation is simple, plasma dosage is small, and requirements of sensitivity detection are met. An isotope internal standard method is adopted for detection, influences of instruments, environments and other factors on analysis results are reduced, therefore, experimental errors are reduced, and the stability of the analysis results is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of medical testing, and in particular relates to a method for detecting the concentration of josamycin in plasma. Background Art

[0002] Josamycin is a single-component 14-carbon macrolide antibiotic with a chemical formula of C 42 H 69 NO 15 Its pharmacological characteristics and clinical applications are similar to those of midecamycin and spiramycin. It is suitable for pharyngitis and tonsillitis caused by Streptococcus pyogenes, sinusitis, otitis media, acute bronchitis and oral abscess caused by sensitive bacteria, pneumonia caused by Mycoplasma pneumoniae, skin and soft tissue infections caused by sensitive bacteria, and can also be used for infections of Staphylococci resistant to penicillin and erythromycin. It mainly binds to the 50S ribosomal subunit of bacteria, inhibits the synthesis of bacterial proteins by blocking transpeptidation and mRNA translocation, and thus exerts an antibacterial effect. It has good antibacterial effects on meningococci, Bordetella pertussis, Peptococci, Streptococcus aureus, Propionibacterium, Eubacterium, etc. When the blood concentration of josamycin is greater than 5.0 mg / L, the toxic and side effects are enhanced, which can cause gastrointestinal reactions such as nausea, vomiting, loss of appetite, abdominal distension, and diarrhea. Since the therapeutic window of VCMC is narrow and there are large individual differences in the metabolic process in the body, monitoring its blood concentration is an important reference for adjusting dosage and reducing adverse reactions, and is of great significance in clinical medication.

[0003] In the prior art, microbial culture operation is cumbersome, blood sample dosage is large, pretreatment is complicated, overall detection time is long, accuracy and reproducibility are relatively poor, and the lower limit of quantification is high. How to solve the above technical problems, improve the detection sensitivity and stability of josamycin in blood, facilitate operation, and reduce the amount of detection samples and analysis time has become a technical problem that technicians in this field need to solve urgently. Summary of the invention

[0004] In order to solve the problems and shortcomings in the prior art, the present invention provides a method for detecting the concentration of josamycin in plasma, which uses a protein precipitation method to pre-treat the sample to be tested and adopts an isotope internal standard for detection, thereby ultimately improving the stability and sensitivity of the detection, being simple and convenient to operate, and reducing the amount of test samples and the analysis time.

[0005] Specifically, the present invention provides a method for determining the concentration of josamycin in plasma, wherein the preparation method comprises the following steps:

[0006] Weigh josamycin and dissolve it in the first solvent to prepare a stock solution of the substance to be tested;

[0007] Weigh josamycin- 13 C-d3 is dissolved in the first solvent to prepare an internal standard stock solution;

[0008] Using a second solvent, diluting the analyte stock solution into a standard curve working solution and a quality control working solution;

[0009] Using a second solvent, diluting the internal standard stock solution into an internal standard working solution;

[0010] The standard curve working solution is added with blank plasma for dilution to obtain a standard curve sample, and the quality control working solution is added with blank plasma for dilution to obtain a quality control sample; an anticoagulant is added to the blank plasma;

[0011] The plasma samples (the standard curve samples and the quality control samples) were pre-treated by protein precipitation method, and after precipitation and centrifugation, the supernatant was taken for liquid chromatography-mass spectrometry analysis.

[0012] Optionally, the first solvent is dimethyl sulfoxide;

[0013] The second solvent is a 50% methanol aqueous solution.

[0014] Optionally, the concentration of the analyte stock solution is 1.00 mg / ml;

[0015] The concentration of the internal standard working solution is 1.00 ng / ml;

[0016] The concentration range of the standard curve working solution is 1.00-500 ng / ml;

[0017] The concentration range of the quality control working solution is 3.0-400 ng / ml.

[0018] Optionally, the concentration range of the standard curve samples is 50.0 to 25000 pg / ml;

[0019] The concentration range of the quality control sample is 150.0-20000 pg / ml.

[0020] Optionally, the precipitant is pure methanol.

[0021] Optionally, the liquid phase conditions are as follows: phase A is 0.1% formic acid aqueous solution, phase B is pure methanol, the chromatographic column is a Waters C18 chromatographic column, the injection volume is 5 ul, and gradient elution is performed.

[0022] Optionally, the gradient elution conditions are:

[0023]

[0024] Optionally, the mass spectrometry conditions are:

[0025] Detection ion pairs: josamycin: 828.3 / 174.1; josamycin-13 C-d3:832.5 / 178.2.

[0026] Optionally, the single needle detection time is 2 to 5 minutes.

[0027] In the present invention, a protein precipitation method is used to pre-treat the sample to be tested, and then the isotope internal standard method is used to quantitatively measure josamycin in the sample to be tested. Not only is the operation simple, the amount of plasma used is small, and the error of the test result is small, and the analysis result has strong stability and reproducibility. Therefore, the sensitivity and stability of josamycin detection in plasma are improved.

[0028] Based on the following detailed description of specific embodiments of the present invention in conjunction with the accompanying drawings, those skilled in the art will become more aware of the above and other objects, advantages and features of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Hereinafter, some specific embodiments of the present invention will be described in detail in an exemplary and non-limiting manner with reference to the accompanying drawings. The same reference numerals in the accompanying drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the accompanying drawings:

[0030] Figure 1 The figure is a flow chart of a method for detecting the concentration of josamycin in plasma according to one embodiment of the present invention. DETAILED DESCRIPTION

[0031] Refer to the following Figure 1 To describe a method for detecting the concentration of josamycin in plasma of an embodiment of the present invention. In the description of the present embodiment, it should be understood that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined as "first" and "second" can explicitly or implicitly include at least one of the features, that is, include one or more of the features. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. When a certain feature "includes or contains" a certain or some of the features it covers, unless otherwise specifically described, this indication does not exclude other features and may further include other features.

[0032] In the description of the present embodiment, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0033] Isotope internal standard method is a technique used to trace organic substances in analysis. It uses specific natural elements (isotopes) as tracers and adds them to the substance to be traced. These isotopes have specific chemical and biological markers and can be detected more easily. During the detection process, the tracer element is extracted and measured by mass spectrometry or other techniques to determine its quantity in the substance being traced and the characteristics of its dynamic changes. Isotope internal standard method can improve the accuracy, sensitivity and comparability between different samples of mass spectrometry analysis.

[0034] Protein precipitation is a common method for pre-treatment of biological samples during target substance analysis. For protein-rich samples, a large amount of protein precipitation that interferes with the determination must be removed during separation and extraction, so that the target substance to be measured remains in the solution. In addition, the protein precipitation method also has a high concentration multiple, which can increase the concentration of the target substance to a certain extent.

[0035] Figure 1 FIG. 4 is a flow chart of a method for detecting the concentration of josamycin in plasma according to an embodiment of the present invention. Figure 1 As shown, the present invention provides a method for detecting the concentration of josamycin in plasma, and the above detection method comprises the following steps:

[0036] Step S10, weighing josamycin and dissolving it in a first solvent to prepare a test substance stock solution;

[0037] Step S20, weighing josamycin- 13 C-d3 is dissolved in the first solvent to prepare an internal standard stock solution;

[0038] Step S30, using a second solvent to dilute the analyte stock solution into a standard curve working solution and a quality control working solution;

[0039] Step S40, using a second solvent to dilute the internal standard stock solution into an internal standard working solution;

[0040] Step S50, adding blank plasma to the standard curve working solution to dilute to obtain a standard curve sample, and adding blank plasma to the quality control working solution to dilute to obtain a quality control sample; an anticoagulant is added to the blank plasma;

[0041] Step S60, pre-treating the plasma samples (standard curve samples and quality control samples) by protein precipitation method, adding a precipitant for precipitation, centrifuging, and taking the supernatant for liquid chromatography-mass spectrometry detection and analysis.

[0042] In this embodiment, the protein precipitation method is used to treat the sample to be tested, which is simple to operate, uses less plasma, and meets the requirements of low sensitivity detection. The isotope internal standard method is used for detection to reduce the influence of factors such as instruments and environment on the analysis results, thereby reducing experimental errors and increasing the stability of the analysis results.

[0043] In some embodiments of the present invention, the first solvent is dimethyl sulfoxide, and the second solvent is a 50% methanol aqueous solution.

[0044] In this example, josamycin- 13 C-d3 was dissolved in dimethyl sulfoxide to prepare an internal standard stock solution. Dimethyl sulfoxide, as a "universal solvent", has excellent solubility and also has the advantages of high polarity, high boiling point and good thermal stability. In this embodiment, dimethyl sulfoxide was selected as the solvent to make josamycin and josamycin- 13 C-d3 is fully soluble in dimethyl sulfoxide without destroying its own structure.

[0045] In some embodiments of the present invention, the second solvent is a 50% methanol aqueous solution. That is, the analyte stock solution and the internal standard stock solution are dissolved in methanol respectively, and finally the standard curve working solution, the quality control working solution and the internal standard working solution are obtained.

[0046] In this embodiment, because methanol has the advantages of good solubility and low price, methanol is selected as the solvent so that the analyte stock solution and the internal standard stock solution can be evenly diluted and the cost investment can be reduced.

[0047] In some embodiments of the present invention, the concentration of the analyte stock solution is 1.00 mg / ml, the concentration of the internal standard working solution is 1.00 ng / ml, the concentration range of the standard curve working solution is 1.00-500 ng / ml, and the concentration range of the quality control working solution is 3.0-400 ng / ml.

[0048] Specifically, the analyte stock solution was diluted with 50% methanol aqueous solution, and different dilution times were used to obtain the standard curve working solution and the quality control working solution. The internal standard stock solution was diluted with 50% methanol aqueous solution to obtain the internal standard working solution.

[0049] In this embodiment, methanol is selected as the solvent for dilution, which can achieve a good dissolution effect, save costs, and does not affect the chromatographic behavior of the detection method.

[0050] In some embodiments of the present invention, the concentration range of the standard curve sample is 50.0 to 25000 pg / ml, and the concentration range of the quality control sample is 150.0 to 20000 pg / ml. Specifically, the standard curve working solution is diluted with blank plasma added with an anticoagulant to obtain the standard curve sample, and the quality control working solution is diluted with blank plasma added with an anticoagulant to obtain the quality control sample. The dilution multiple can be any value not less than 20, such as 20 times, 30 times, 40 times, 50 times, 65 times, etc., preferably 20 times. The dilution multiples of the standard curve working solution and the quality control working solution can be the same or different.

[0051] In some embodiments of the present invention, the precipitant is pure methanol. That is, when the plasma sample is pre-treated by protein precipitation, pure methanol is selected as the precipitant.

[0052] In this embodiment, pure methanol is used as a precipitant, which can not only remove impurities and solvents to obtain a relatively pure polymer, which is convenient for subsequent detection, separation, purification and other treatments, but also reduce costs.

[0053] In some embodiments of the present invention, the liquid phase conditions are as follows: phase A is 0.1% formic acid aqueous solution, phase B is pure methanol, the chromatographic column is a Waters C18 chromatographic column, the injection volume is 5 ul, and gradient elution is performed.

[0054] Gradient elution is particularly widely used in liquid chromatography. By changing the polarity, ionic strength or pH of the mobile phase, the relative retention value of the analyte can be changed, thereby improving separation efficiency and shortening analysis time. In addition, gradient elution can also improve peak shape, reduce tailing, and increase sensitivity, although it may sometimes cause baseline drift.

[0055] In this embodiment, gradient elution during detection can shorten the analysis cycle and improve the separation degree and sensitivity.

[0056] In some embodiments of the present invention, the gradient elution conditions are:

[0057]

[0058] In some embodiments of the present invention, the mass spectrometry conditions are: detection ion pairs: josamycin: 828.3 / 174.1; josamycin- 13 C-d3:832.5 / 178.2.

[0059] In this embodiment, josamycin: 828.3 / 174.1; josamycin- 13 C-d3: 832.5 / 178.2 is used as the detection ion pair, which has high response to the compound, low baseline, and no ion channel interference.

[0060] In some embodiments of the present invention, the single needle detection time is 2 to 5 minutes. That is, the single needle detection time can be 2 minutes, 2.4 minutes, 3.2 minutes, etc., any time within 2 to 5 minutes, preferably 3 minutes.

[0061] In the present invention, a protein precipitation method is used to pre-treat the sample to be tested, and an isotope internal standard method is used to quantitatively measure josamycin in the sample to be tested. Not only is the operation simple, the amount of plasma used is small, but also the error of the test result is small, and the analysis result has strong stability and reproducibility. Thus, the sensitivity and stability of josamycin detection in plasma are improved.

[0062] At this point, those skilled in the art should recognize that, although multiple exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or derived based on the content disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and recognized as covering all these other variations or modifications.

[0063] The present invention is specifically explained below using several embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0064] Example 1

[0065] A method for detecting the concentration of josamycin in plasma comprises the following steps:

[0066] Step S110, weighing 0.1 g of josamycin and dissolving it in 100 ml of dimethyl sulfoxide to prepare a 1.00 mg / ml analyte stock solution;

[0067] Step S120, weigh 0.1 g of josamycin- 13 C-d3 was dissolved in 100 ml of dimethyl sulfoxide to prepare an internal standard stock solution with a concentration of 1.00 mg / ml;

[0068] Step S130, using 50% methanol aqueous solution, dilute the analyte stock solution with a concentration of 1.00 mg / ml into a standard curve working solution and a quality control working solution; wherein the concentrations of the standard curve working solution are 1.00, 2.00 ng / mL, 5.00 ng / mL, 20.0 ng / mL, 100 ng / mL, 200 ng / mL, 440 ng / mL, and 500 ng / mL, respectively, and the concentrations of the quality control working solution are 3.00 ng / mL, 15.0 ng / mL, 150 ng / mL, and 400 ng / mL, respectively.

[0069] Step S140, using 50% methanol aqueous solution to dilute the internal standard stock solution with a concentration of 1.00 mg / ml into an internal standard working solution with a concentration of 1.00 ng / ml;

[0070] Step S150, adding blank plasma anticoagulated with heparin sodium to the standard curve working solution to dilute to obtain standard curve samples, the concentrations of the standard curve samples are 50.0pg / mL, 100pg / mL, 250pg / mL, 1000pg / mL, 5000pg / mL, 10000pg / mL, 22000pg / mL, and 25000pg / mL respectively; adding blank plasma anticoagulated with heparin sodium to the quality control working solution to dilute to obtain quality control samples, the concentrations of the quality control samples are 150pg / mL, 750pg / mL, 750pg / mL, and 20000pg / mL respectively.

[0071] Step S160, take 50uL of plasma sample, add 50uL of 1.00ng / ml internal standard working solution, add 400ul of pure methanol for precipitation, centrifuge at 4000 rpm for 10min, take 200uL of supernatant and place it in a 96-well plate for analysis in LC-MS / MS system. The liquid chromatograph is Shimadzu 40, and the mass spectrometer is AB 6500+. Phase A is 0.1% formic acid aqueous solution, phase B is pure methanol, the chromatographic column is Waters C18, and the detection ion pairs are josamycin: 828.3→174.1 and josamycin- 13 C-d3: 832.5→178.2; single needle detection time 3min. Gradient elution conditions are 0-0.5min, 75% of 0.1% formic acid aqueous solution, 25% of pure methanol; 0.5-1.5min, 0.1% formic acid aqueous solution decreased from 75% to 5%, pure methanol increased from 25% to 95%; 1.5-2.2min, 5% of 0.1% formic acid aqueous solution, 95% of pure methanol; 2.2-2.7min, 0.1% formic acid aqueous solution increased from 5% to 75%, pure methanol decreased from 95% to 25%; 2.7-3.0min, 75% of 0.1% formic acid aqueous solution, 25% of pure methanol.

Claims

1. A method for detecting the concentration of josamycin in plasma, characterized in that: The steps include: Weigh josamycin and dissolve it in the first solvent to prepare a stock solution of the substance to be tested; Weigh josamycin- 13 C-d3 is dissolved in the first solvent to prepare an internal standard stock solution; Using a second solvent, diluting the analyte stock solution into a standard curve working solution and a quality control working solution; Using a second solvent, diluting the internal standard stock solution into an internal standard working solution; The standard curve working solution is added with blank plasma for dilution to obtain a standard curve sample, and the quality control working solution is added with blank plasma for dilution to obtain a quality control sample; an anticoagulant is added to the blank plasma; The plasma samples (the standard curve samples and the quality control samples) were pre-treated by protein precipitation method, and after precipitation and centrifugation, the supernatant was taken for liquid chromatography-mass spectrometry analysis.

2. The method for detecting the concentration of josamycin in plasma according to claim 1, characterized in that: The first solvent is dimethyl sulfoxide; The second solvent is a 50% methanol aqueous solution.

3. The method for detecting the concentration of josamycin in plasma according to claim 2, characterized in that: The concentration of the analyte stock solution is 1.00 mg / ml; The concentration of the internal standard working solution is 1.00 ng / ml; The concentration range of the standard curve working solution is 1.00-500 ng / ml; The concentration range of the quality control working solution is 3.0-400 ng / ml.

4. The method for detecting the concentration of josamycin in plasma according to claim 3, characterized in that: The concentration range of the standard curve samples is 50.0-25000 pg / ml; The concentration range of the quality control sample is 150.0-20000 pg / ml.

5. The method for detecting the concentration of josamycin in plasma according to any one of claims 1 to 4, characterized in that: The precipitant is pure methanol.

6. The method for detecting the concentration of josamycin in plasma according to claim 5, characterized in that: The liquid phase conditions were as follows: phase A was 0.1% formic acid aqueous solution, phase B was pure methanol, the chromatographic column was a Waters C18 chromatographic column, the injection volume was 5 ul, and gradient elution was performed.

7. The method for detecting the concentration of josamycin in plasma according to claim 6, characterized in that: The gradient elution conditions are:

8. The method for detecting the concentration of josamycin in plasma according to claim 5, characterized in that: The mass spectrometry conditions were: Detection ion pairs: josamycin: 828.3 / 174.1; josamycin- 13 C-d3:832.5 / 178.

2.

9. The method for detecting the concentration of josamycin in plasma according to claim 5, characterized in that: The single needle test lasts 3 minutes.

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