Method for detecting novel pleuromutilin in chicken plasma and lung tissue through liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting novel pleuromutilin in chicken plasma and lung tissues through liquid chromatography-tandem mass spectrometry, and belongs to the technical field of biological analysis and detection. The method comprises the following steps: pretreating a chicken plasma or lung tissue sample, and detecting the concentration of the novel pleuromutilin P14-1 in the sample by adopting liquid chromatography-tandem mass spectrometry. The method provided by the invention is good in precision and high in accuracy, indexes such as matrix effect, recovery rate, dilution linearity and the like meet requirements of quantitative analysis of biological samples, and preclinical and clinical pharmacokinetic researches of novel pleuromutilin P14-1 can be supported.

Description

Technical Field

[0001] The invention relates to the technical field of biological analysis and detection, in particular to a method for detecting novel pleuromutilin in chicken plasma and lung tissue by liquid chromatography-tandem mass spectrometry. Background Art

[0002] Mycoplasma gallisepticum is one of the most significant pathogens causing economic losses in poultry farming. M. gallisepticum is a serious problem in the poultry sector worldwide, causing chronic respiratory disease in broilers, laying hens, breeder chickens, and infectious sinusitis in turkeys. It also reduces egg production, affecting the economic benefits of the poultry industry. In the most serious cases, M. gallisepticum infection can aggravate viral infections and secondary bacterial complications, and reduce the effectiveness of avian virus vaccines.

[0003] Pleuromutilin antibiotics have a broad antibacterial spectrum and a unique antibacterial mechanism. They do not produce cross-resistance with most antibiotics and have good antibacterial effects on Gram-positive bacteria, mycoplasmas, etc. Currently, tylosin and valnemulin, which have been successfully launched on the market, are mainly used as feed additives to prevent and treat mycoplasma and Gram-positive bacterial infections in pigs and poultry. Retapamulin and lefamulin, which are approved for human use, are mainly used to treat coccal infections and acquired bacterial pneumonia. With pleuromutilin as the lead compound, it is expected that veterinary antibiotics that can be used to treat Mycoplasma gallisepticum infections will be developed. However, the current methods for detecting pleuromutilin have problems such as low precision and accuracy and low work efficiency. Therefore, it is urgent to develop a new method for detecting pleuromutilin to provide a new method for detecting more types of pleuromutilins and provide more raw material sources for veterinary antibiotics for the treatment of Mycoplasma gallisepticum infections. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting novel truncated pleuromutilin in chicken plasma and lung tissue by liquid chromatography-tandem mass spectrometry to solve the problems existing in the above-mentioned prior art. The method is simple and quick to operate, has good reproducibility, high sensitivity and fast analysis speed, can quickly and efficiently detect the concentration of novel truncated pleuromutilin in chicken plasma and lung tissue, can be used for drug metabolic kinetics research, and provides a method reference for preclinical and clinical research on novel truncated pleuromutilin.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for detecting novel pleuromutilin in chicken plasma and lung tissue by liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0007] The plasma samples or lung tissue samples of the tested chickens are pre-treated, and then the novel pleuromutilin in the chicken plasma or lung tissue samples is detected by liquid chromatography-tandem mass spectrometry.

[0008] Preferably, the pretreatment of the chicken plasma sample comprises the following steps: thawing the chicken plasma sample to be tested at room temperature, then fully mixing it with acetonitrile, centrifuging it, taking the supernatant, and filtering it with a filter membrane.

[0009] Preferably, the volume ratio of the chicken plasma sample to the acetonitrile is 1:3.

[0010] Preferably, the pre-treatment of the chicken lung tissue sample comprises the following steps:

[0011] After homogenizing the chicken lung tissue sample, acetonitrile and anhydrous sodium sulfate were added for extraction, vortexing (5 min), ultrasonication, centrifugation, the supernatant was taken, acetonitrile was added for repeated extraction, and the supernatants were combined to obtain the acetonitrile extract;

[0012] The acetonitrile extract is mixed with water, added into an HLB solid phase extraction column for elution and purification, and the eluate is collected; the eluate is blown dry, then re-dissolved in an acetonitrile-water solution, vortexed, and ultrasonicated, and then n-hexane is added to remove fat, vortexed, the lower layer of liquid is taken, and then filtered with a filter membrane.

[0013] Preferably, the ratio of the chicken lung tissue sample, acetonitrile and anhydrous sodium sulfate is (0.5-1) g: 4 mL: (0.5-1) g.

[0014] Preferably, the volume ratio of the acetonitrile extract to water is (1-2):6.

[0015] Preferably, the elution purification step comprises: passing the solution obtained by mixing the acetonitrile extract and water through a column, then rinsing with water, draining, eluting the target substance with acetonitrile, and collecting the eluate, wherein the volume ratio of the solution, water and acetonitrile is (7-8): (3-4): (5-7).

[0016] Preferably, the target compound is the novel pleuromutilin P14-1, and its chemical structure is shown below:

[0017]

[0018] Preferably, the liquid chromatography conditions in the liquid chromatography-tandem mass spectrometry detection include: the chromatographic column is a HypersilGOLD chromatographic column, the filler is C18, the particle size is 5μm, and the specification is 150mm×2.1mm; the mobile phase A is a 5mM ammonium acetate aqueous solution, the mobile phase B is acetonitrile, and the elution is performed by gradient elution, and the gradient elution program is: 0.0~0.5min, the volume fraction of the mobile phase B changes from 15% to 95%; 0.5~3.0min, the volume fraction of the mobile phase B is 95%; 3.0~3.5min, the volume fraction of the mobile phase B changes from 95% to 15%; 3.5~9.0min, the volume fraction of the mobile phase B is 15%; flow rate: 0.25mL / min; column temperature: 40℃; injection volume: 5μL.

[0019] Preferably, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry detection include: ionization mode: electrospray ionization, triple quadrupole tandem mass spectrometry; scanning mode: ESI + ; Detection mode: multiple reaction monitoring mode; curtain gas flow rate: 35L / min, nebulizer gas flow rate: 60L / min, auxiliary nebulizer gas flow rate: 55L / min; ion source temperature: 540℃; spray voltage: 4500V; quantitative ion pair: 605.4 / 303.2; dwell time: 200ms; declustering voltage: 140V; collision energy: 23eV.

[0020] The present invention discloses the following technical effects:

[0021] The method provided by the present invention can shorten the extraction time of tissue samples and reduce the amount of organic reagents by pre-treating chicken plasma or lung tissue samples and then detecting the concentration of the novel pleuromutilin P14-1 in the samples by liquid chromatography-tandem mass spectrometry. The method is used to detect the novel pleuromutilin P14-1 in chicken plasma or lung tissue samples with good precision and high accuracy. Indicators such as matrix effect, recovery rate, and dilution linearity meet the requirements of quantitative analysis of biological samples, and can support preclinical and clinical pharmacokinetic studies of the novel pleuromutilin P14-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 It is the molecular structure formula of the novel pleuromutilin P14-1;

[0024] Figure 2A novel pleuromutilin P14-1 13 C-NMR spectrum;

[0025] Figure 3 A novel pleuromutilin P14-1 1 H-NMR spectrum;

[0026] Figure 4 This is the blank matrix chromatogram of chicken plasma;

[0027] Figure 5 This is the matrix chromatogram of chicken blank lung tissue;

[0028] Figure 6 Chromatograms were spiked with 2 ng / mL for chicken plasma;

[0029] Figure 7 For chicken lung tissue, 5 μg / kg chromatogram was added;

[0030] Figure 8 is the standard curve of the novel pleuromutilin P14-1;

[0031] Fig. 9 This is the chicken plasma working curve;

[0032] Fig.10 This is the working curve of chicken lung tissue. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The present invention provides a method for detecting a novel pleuromutilin P14-1 in chicken plasma and lung tissue by liquid chromatography-tandem mass spectrometry, wherein the target detection substance involved is pleuromutilin P14-1, and the standard pleuromutilin P14-1 used in the method is donated by Professor Tang Youzhi of South China Agricultural University. Pleuromutilin P14-1 is obtained by modifying the nitro group at the C14 position to an amino group on the basis of the pleuromutilin P14 mother nucleus, and the compound exhibits a good antibacterial effect against Mycoplasma gallisepticum. The chemical structure of pleuromutilin P14-1 is as follows Figure 1 shown.

[0039] The pleuromutilin P14-1 was subjected to nuclear magnetic resonance detection, which was completed by a biological detection company. 13 C-NMR and 1 H-NMR Figure 2 and Figure 3 shown.

[0040] 13 C NMR (151MHz, DMSO-d6) δ217.61, 168.26, 168.19, 150.24, 141.17, 138.73, 130.38, 129.14, 128.87, 121.16, 116.85, 115.70, 115.57, 115.1 3, 73.12, 70.13, 60.24, 57.73, 45.43, 44.43, 44.15, 41.93, 36.83, 34 .47, 30.57, 28.92, 27.06, 24.94, 21.22, 16.59, 15.01, 14.55, 12.00.

[0041] 1H NMR (600MHz, DMSO-d6) δ10.05 (s, 1H), 7.72 (d, J = 8.8Hz, 2H), 7.62 (d, J = 6.5Hz, 1H), 7.36 (d, J = 8.7Hz, 2H), 7.21 (t, J = 7.6Hz, 1H), 6.7 7 (d, J=8.4Hz, 1H), 6.62–6.58 (m, 1H), 6.34 (s, 2H), 6.13–6.07 (m, 1H), 5.54 (d, J=8.3Hz, 1H), 5.05–5.01 (m, 2H), 4.50 (d, J=6.1Hz, 1H ), 4.03 (q, J=7.1Hz, 1H), 3.79–3.69 (m, 2H), 2.37 (s, 1H), 2.09–2.05 (m, 2H), 1.99 (s, 2H), 1.67–1.60 (m, 2H), 1.49–1.38 (m, 2H), 1.36 (s, 3H), 1.30–1.23 (m, 2H), 1.19 (d, J = 7.1Hz, 1H), 1.14 (d, J = 15.9Hz, 1H), 1.04 (s, 3H), 0.82 (d, J = 6.9Hz, 3H), 0.61 (d, J = 7.0Hz, 3H).

[0042] Example 1 A method for detecting novel pleuromutilin P14-1 in chicken plasma and lung tissue by liquid chromatography-tandem mass spectrometry

[0043] 1. Sample pretreatment

[0044] 1.1 Pretreatment of plasma samples: After the plasma sample is thawed at room temperature, shake it well, accurately pipette 200 μL of plasma into a centrifuge tube, add 600 μL of acetonitrile, vortex thoroughly for 1 min, centrifuge at 12000 rpm for 20 min, take the supernatant and filter it with a 0.22 μm needle filter membrane, and analyze it on the machine.

[0045] 1.2 Pretreatment of lung tissue samples: This pretreatment includes two steps: extraction and purification. Extraction step: Accurately weigh 0.5g of chicken lung tissue in a 10mL polypropylene centrifuge tube, homogenize the tissue, add 4mL of acetonitrile, then add 0.5g of anhydrous sodium sulfate, oscillate for 5min, ultrasonicate for 5min, centrifuge at 8000r / min for 8min, take the supernatant, add 2mL of acetonitrile to repeat the extraction, and combine the supernatant; then take 1.5mL of acetonitrile extract, add 6mL of aqueous solution, mix well, and prepare for purification; Purification step: Take 5mL of methanol to activate the HLB solid phase extraction column, and balance it with 5mL of aqueous solution. Take 7.5 mL of the above solution and pass it through the column, rinse with 3 mL of water, drain, elute the target with 6 mL of acetonitrile, collect the eluate, blow it with nitrogen at 37 ° C, blow it dry, add 1 mL of 50% acetonitrile-water solution (i.e., the volume ratio of acetonitrile to water is 1:1) for re-dissolution, vortex for 3 min, ultrasonic for 5 min, add 1 mL of n-hexane to remove fat, vortex for 15 s, take the lower layer of liquid, pass it through the membrane, and detect it on the machine.

[0046] 2. Liquid chromatography-tandem mass spectrometry detection

[0047] Take 5 μL of the sample solution to be tested for liquid chromatography-tandem mass spectrometry detection. The liquid chromatography conditions and mass spectrometry conditions are as follows:

[0048] 2.1 Liquid chromatography conditions: The detection instrument used a high performance liquid chromatography coupled to a triple quadrupole mass spectrometer; a Hypersil GOLD column was selected, the filler was C18, the particle size was 5 μm, and the specification was 150 mm × 2.1 mm; the mobile phase A was 5 mM ammonium acetate aqueous solution, the mobile phase B was acetonitrile, and a gradient elution method was adopted; the flow rate was 0.25 mL / min; the column temperature was 40 °C; the gradient elution program and the mobile phase volume ratio at each time are shown in Table 1.

[0049] Table 1 Mobile phase gradient scheme

[0050]

[0051] 2.2 Mass spectrometry conditions: ionization mode: electrospray ionization, triple quadrupole tandem mass spectrometry; scanning mode: ESI + ; Detection mode: multiple reaction monitoring mode (MRM); curtain gas: 35 L / min, nebulizer gas: 60 L / min, auxiliary nebulizer gas: 55 L / min, ion source temperature: 540°C, spray voltage: 4500 V, ion information is shown in Table 2.

[0052] Table 2 Multiple reaction monitoring ion situation

[0053]

[0054] Note: The ion pairs with * are quantitative ion pairs. The quasi-molecular ion of the parent ion is [M+H] +.

[0055] 3. Methodological assessment and results

[0056] 3.1 Specificity and specificity

[0057] Figure 4 and Figure 5 These are the blank sample chromatograms of chicken plasma and chicken lung tissue, Figure 6 and Figure 7 The chromatograms of plasma and lung tissue with added quantitative limit concentration (2ng / mL and 5μg / kg) are shown respectively. In the blank plasma chromatogram of chicken, there is no interference of impurities near the peak, the peak shape is sharp, and the separation is good, which can meet the analysis requirements; in the blank matrix chromatogram of chicken lung tissue, there is impurity interference near the peak, and the peak area is less than 20% of the quantitative lower limit, which does not affect the quantitative analysis of chicken lung tissue.

[0058] 3.2 Sensitivity

[0059] Blank plasma and lung tissue were taken, and drugs were added to make the drug concentrations 1, 2, 5 and 10 ng / mL (lung tissue unit: μg / kg), and the samples were treated according to the pretreatment method, with 5 replicates for each concentration. When the signal-to-noise ratio (S / N) was greater than or equal to 3, the corresponding concentration was the minimum detection limit, and when the signal-to-noise ratio (S / N) was greater than or equal to 10, the corresponding concentration was the minimum quantification limit. The detection limit of plasma samples was 1 ng / mL, and the quantification limit was 2 ng / mL; the detection limit of lung tissue samples was 1 μg / kg, and the quantification limit was 5 μg / kg.

[0060] 3.3 Standard curve

[0061] Take an appropriate amount of the new pleuromutilin standard stock solution, dilute it with 50% acetonitrile water to 5, 10, 20, 50, 100, 200, 500 ng / mL standard working solution, and detect it by liquid chromatography-tandem mass spectrometry. Repeat the measurement 5 times a day for each concentration, and repeat the operation for 5 days. Perform linear regression between the obtained peak area mean and the corresponding concentration, draw a standard curve, and obtain the linear regression equation and correlation coefficient.

[0062] like Figure 8 As shown, the standard curve equation is y=36200x-10600, and the linear relationship is good in the range of 5-500 ng / mL, with a correlation coefficient R of 0.9984.

[0063] 3.4 Working curve

[0064] Take 200 μL chicken plasma, add the appropriate concentration of standard solution, so that the drug concentration in each sample reaches 5, 10, 50, 100, 200, 500 and 1000 ng / mL; take 0.5 g chicken lung tissue, add the appropriate concentration of standard solution, so that the drug concentration in each sample reaches 5, 10, 50, 100, 200, 500 and 1000 μg / kg. Process according to the sample pretreatment method, and detect by liquid chromatography-tandem mass spectrometry. Linear regression is performed between the obtained peak area mean and the corresponding concentration, and the regression equation and correlation coefficient of the working curve in chicken plasma and lung tissue are calculated.

[0065] like Fig. 9 As shown, the chicken plasma working curve equation is y=10300x+5590, and the linear relationship is good in the range of 5-1000ng / mL, with a correlation coefficient R of 0.9994; Fig.10 As shown, the equation of the lung tissue working curve is y=9440x+31000, and the linear relationship is good in the range of 5-1000 μg / kg, with a correlation coefficient R of 0.9984.

[0066] 3.5 Accuracy and precision

[0067] Appropriate concentrations of the new pleuromutilin standard solution were added to 200 μL chicken plasma and 0.5 g lung tissue, respectively, to make the drug concentration reach 5, 10, 500 and 750 ng / mL (μg / kg), covering the high, medium and low concentrations of the quantitative range. The samples were prepared according to the sample treatment method and then subjected to liquid chromatography-tandem mass spectrometry detection. Each concentration was repeated 5 times on the same day, and the average value was taken to calculate the intra-day coefficient of variation; each concentration was repeated for 3 days, and the inter-day coefficient of variation was calculated. The recovery rate and precision of the new pleuromutilin in chicken plasma and lung tissue are shown in Table 3 and Table 4, respectively. The plasma recovery rate was between 82.4% and 124.3%, and the intra-batch and inter-batch coefficients of variation were less than 10%; the lung tissue recovery rate was between 73.0% and 96.4%, and the intra-batch and inter-batch coefficients of variation were also less than 10%, which met the methodological assessment requirements.

[0068] Table 3 Recovery rate and coefficient of variation of plasma

[0069]

[0070]

[0071] Table 4 Recovery rate and coefficient of variation of lung tissue

[0072]

[0073]

[0074] 3.6 Matrix Effects

[0075] Six batches of plasma and lung tissue blank matrices from different sources were used, and drug concentrations of 10 and 750 ng / mL were added; for each batch of matrix, the peak area in the presence of the matrix was calculated, and the peak area ratio of the standard solvent without the matrix was calculated to calculate the matrix factor of the sample. The matrix effects of plasma and lung tissue are shown in Table 5. The peak area ratio of the blank matrix spiked with the standard solution solvent was about 90%, and the matrix effect was small. The coefficient of variation of the matrix factors of the six batches of both was less than 10%.

[0076] Table 5 Plasma and lung tissue matrix effects

[0077]

[0078] 3.7 Dilution linearity

[0079] Six mixed samples of the new pleuromutilin with a concentration of 10000 ng / mL were prepared from blank chicken plasma and blank lung tissue matrices, and then diluted to 1000 ng / mL with blank matrices to prove the reliability of the detection after the sample was diluted 10 times. By verifying the dilution linearity of the detection method, the detection range of this method can be expanded. The dilution reliability results of chicken plasma and lung tissue are shown in Table 6. The ratio of the sample diluted 10 times to the undiluted sample is close to 100%, indicating that the dilution is reliable.

[0080] Table 6 Plasma and lung tissue dilution reliability

[0081]

[0082] The above experimental results show that the method established by the present invention is successfully applied to the determination of novel pleuromutilin in chicken plasma and lung tissue, and the recovery rate and coefficient of variation can meet the national standard requirements for precision and accuracy of analytical methods. It has high sensitivity, simple operation, short experimental time, and can well improve work efficiency.

[0083] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting novel pleuromutilin in chicken plasma and lung tissue by liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: The plasma samples or lung tissue samples of the tested chickens are pre-treated, and then the novel pleuromutilin in the chicken plasma or lung tissue samples is detected by liquid chromatography-tandem mass spectrometry.

2. The method according to claim 1, characterized in that The pretreatment of the chicken plasma sample includes the following steps: thawing the chicken plasma sample to be tested at room temperature, then fully mixing it with acetonitrile, centrifuging it, taking the supernatant, and then filtering it with a filter membrane.

3. The method according to claim 2, characterized in that The volume ratio of the chicken plasma sample to the acetonitrile was 1:

3.

4. The method according to claim 1, characterized in that The pre-treatment of chicken lung tissue samples included the following steps: The chicken lung tissue samples were homogenized, extracted with acetonitrile and anhydrous sodium sulfate, vortexed (5 min), sonicated, and centrifuged, the supernatant was taken, acetonitrile was added again for repeated extraction, and the supernatants were combined to obtain the acetonitrile extract; The acetonitrile extract is mixed with water, added into an HLB solid phase extraction column for elution and purification, and the eluate is collected; the eluate is blown dry, then re-dissolved in an acetonitrile-water solution, vortexed, and ultrasonicated, and then n-hexane is added to remove fat, vortexed, the lower layer of liquid is taken, and then filtered with a filter membrane.

5. The method according to claim 4, characterized in that The ratio of the chicken lung tissue sample, acetonitrile and anhydrous sodium sulfate is (0.5-1) g: 4 mL: (0.5-1) g.

6. The method according to claim 4, characterized in that The volume ratio of the acetonitrile extract to water is (1-2):

6.

7. The method according to claim 4, characterized in that The elution purification step includes: passing the solution obtained by mixing the acetonitrile extract and water through a column, then rinsing with water, draining, eluting the target object with acetonitrile, and collecting the eluate, wherein the volume ratio of the solution, water and acetonitrile is (7-8): (3-4): (5-7).

8. The method according to claim 7, characterized in that The target compound is the novel pleuromutilin P14-1, and its chemical structure is shown below:

9. The method according to claim 1, characterized in that The liquid chromatography conditions in the liquid chromatography-tandem mass spectrometry detection include: a chromatographic column is a Hypersil GOLD chromatographic column, a filler is C18, a particle size is 5 μm, and a specification is 150 mm×2.1 mm; mobile phase A is a 5 mM ammonium acetate aqueous solution, mobile phase B is acetonitrile, and elution is performed in a gradient elution manner, and the gradient elution program is: 0.0 to 0.5 min, the volume fraction of the mobile phase B changes from 15% to 95%; 0.5 to 3.0 min, the volume fraction of the mobile phase B is 95%; 3.0 to 3.5 min, the volume fraction of the mobile phase B changes from 95% to 15%; 3.5 to 9.0 min, the volume fraction of the mobile phase B is 15%; flow rate: 0.25 mL / min; column temperature: 40° C.; injection volume: 5 μL.

10. The method according to claim 1, characterized in that The mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry detection include: ionization mode: electrospray ionization, triple quadrupole tandem mass spectrometry; scanning mode: ESI + ; Detection mode: multiple reaction monitoring mode; curtain gas flow rate: 35L / min, nebulizer gas flow rate: 60L / min, auxiliary nebulizer gas flow rate: 55L / min; ion source temperature: 540℃; spray voltage: 4500V; quantitative ion pair: 605.4 / 303.2; dwell time: 200ms; declustering voltage: 140V; collision energy: 23eV.

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