参见作者原研究论文

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Sep 2021
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Quantification of Duloxetine in the Bacterial Culture and Medium to Study Drug-gut Microbiome Interactions
在细菌培养和培养基中定量度洛西汀以研究药物-肠道微生物组的相互作用   

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Abstract

Expanding our understanding of drug-gut bacteria interactions requires high-throughput drug measurements in complex bacterial cultures. Quantification of drugs in the cultures, media, and cell pellets is prone to strong matrix effects. We have developed a liquid chromatography–high resolution mass spectrometry (LC–HRMS) method for quantifying duloxetine from high-throughput gut-drug interaction experiments. The method is partially validated for its reproducibility, sensitivity, and accuracy, which makes it suitable for largescale drug screens. We extensively used this method to study biotransformation and bioaccumulation of duloxetine and other drugs in several species of gut bacteria.

Keywords: LC–HRMS (液相高分辨高精密度质谱), LC–MS/MS (液相色谱串联质谱), Biotransformation (生物转化), Bioaccumulation (生物蓄积), Drug quantification (药物量化), Drug-gut interactions (药物与肠道的相互作用)

Background

Several recent clinical studies showed the impact of drugs on the gut microbiome. Such interactions can have a considerable impact on drug biotransformation, pharmacokinetics, efficacy, and toxicity of the administered drug (Falony et al., 2016; Scott et al., 2017). To study bacteria-drug interactions, systematic profiling of such interactions in human gut bacterial strains is a powerful approach (Maier and Typas, 2017; Zimmermann et al., 2019; Forslund et al., 2015). Such experiments usually involve large scale screening of several gut bacterial species with targeted drugs and require high-throughput quantification of drugs and their metabolites. Also, the complexity of bacterial cultures and gut microbiota media (GMM) used in such experiments generate strong background signals that interfere with drug analysis. The further study of drugs biotransformation and bioaccumulation requires their quantification in total culture medium, supernatant, and cell pellet, which is prone to strong matrix effects. We addressed these challenges by developing and validating an LC–HRMS method for the quantification of duloxetine. We studied duloxetine, a widely used antidepressant, and found it to be bioaccumulated by several gut microbial species. This LC–HRMS method serves as a validation of the results obtained from large screening of several hundreds of bacteria-drug pairs (Klünemann et al., 2021).


Generally, drug quantification is performed using triple quadrupole instruments due to their sensitivity and specificity. The reported methods for duloxetine quantification are mostly done in blood plasma samples (Senthamil Selvan et al., 2007; Waldschmitt et al., 2007; Chae et al., 2013; Zhang et al., 2017). However, during quantification of duloxetine in bacterial cultures using the reported methods, we found that the specificity of analysis was compromised by the background matrix of the bacterial culture. The matrix effect hampered the accurate quantification of duloxetine, especially from incubated cultures (for 48 h). The method we developed is accurate, specific, and sensitive for quantifying duloxetine from total culture medium, supernatant, and pellet.


The study of drug-gut interactions is an emerging field that is expanding our knowledge about this fascinating research area. Our method can be a useful tool for drug quantification in gut microbiome studies.

Materials and Reagents

  1. Pipette tips (Rainin, METTLER TOLEDO, USA)

    Tips GP-LTS-A-1,000 µl-768/8 (Rainin, catalog number: 17014338)

    Tips GP-LTS-A-250 µl-/F-960/10 (Rainin, catalog number: 30389288)

    GP-LTS-A-10 µl-960/10 (Rainin, catalog number: 30389284)

  2. Eppendorf Polypropylene DNA LoBind Tubes (2.0 ml) (Thermo Fisher Scientific, catalog number: 10051232)

  3. WebSeal 96-Well Plate, Round Well, Barcoded (Thermo Fisher Scientific, catalog number: 601 80-P206B)

  4. Corning® 96 well Polypropylene Deep Well Plate (Sigma-Aldrich, catalog number: CLS3957)

  5. Corning® 96 Round Well Microplate Storage Mat III (Sigma-Aldrich, catalog number: CLS3080)

  6. Zone-FreeTM Sealing Films (Sigma-Aldrich, catalog number: Z721646)

  7. Glassware purchased from DURAN (DWK Life Sciences, Wertheim, Germany)

  8. Bacterial species used: Clostridium saccharolyticum; type strain, WM1; tax ID: 610130; internal database ID: NT5037. [Storage: 20% glycerol in mGAM medium, stored at -80°C]

  9. Optima® LC–MS grade acetonitrile (Fisher Chemical, catalog number: A955-212)

    Methanol (Fisher Chemical, catalog number: A456-212)

    Water (Fisher Chemical, catalog number: W6-121)

  10. LC–MS LiChropur® grade formic acid (Merck KGaA, catalog number: 5330020050)

  11. Standards of Duloxetine (Sigma-Aldrich, Merck KGaA, catalog number: SML0474)

  12. Internal standards Fluoxetine hydrochloride (Sigma-Aldrich, Merck KGaA, catalog number: F132)

    Sulfamethazine (Sigma-Aldrich, Merck KGaA, catalog number: S5637)

    Sulfamethizole (Sigma-Aldrich, Merck KGaA, catalog number:S5632)

  13. PierceTM Triple Quadrupole Calibration Solution, Extended Mass (Thermo Fisher, catalog number: 88340)

  14. mGAM medium: Gifu Anaerobic Medium Broth, Modified (HyServe, catalog number: 05433)

  15. GMM (gut microbiota medium)

  16. DMSO solvent

  17. Extraction solvent (see Recipes)

  18. 1 µM Duloxetine stock solution preparation (see Recipes)

  19. 1 µM Fluoxetine stock solution preparation (see Recipes)

  20. Mobile phase buffer preparations (see Recipes)

Equipment

  1. Pipettes (Eppendorf AG, Hamburg, Germany)

    Eppendorf Multipette® M4 (Eppendorf, catalog number: 4982000012)

    Eppendorf Research plus pipette (100-1,000 µl)

    Multi-channel 8-channel, 30-300 µl (Eppendorf, catalog number: 3122000051)

    Multi-channel 8-channel, 0.5-10 µl (Eppendorf, catalog number: 3122000019)

  2. Vanquish UHPLC–MS/MS system coupled to a Q-Exactive plus HRMS (Thermo Scientific, MA, USA; catalog number: IQLAAAGABHFAPUMZZZ)

  3. ACQUITY UPLC HSS T3 column (100Å, 1.8 µm; 2.1 × 100 mm) (Waters, catalog number: 186003539)

  4. S-Series Heated Ultrasonic Cleaning Water Bath (FisherbrandTM, catalog number:10162372)

  5. Eppendorf MixMate for vortexing (Eppendorf, catalog number: 5353000510) or equivalent

  6. HamiltonTM 1700 Series GastightTM Syringe 500 μl (Thermo Fisher Scientific, catalog number: 365JLT41)

  7. Centrifuge 5418 R, refrigerated (Eppendorf, catalog number: 5401000013)

  8. Vinyl Anaerobic Chambers Type C (Coy Laboratory Products, Inc., USA)

  9. Water bath sonicator; FisherbrandTM S-Series heats ultrasonic cleaners (Thermo Fisher Scientific, catalog number: 10162372)

Software

  1. Thermo Xcalibur software version 4.0.27 (Thermo Scientific, MA, USA) used for data acquisition (Q-exactive plus tune version 2.11) and qualitative (Qual Browser) and quantitative (Quan Browser) data analysis

Procedure

  1. Bacterial Culture inoculation with duloxetine

    1. Inoculate Clostridium saccharolyticum from stock culture (20% glycerol in mGAM medium, stored at -80°C) in 5 ml mGAM and allow it to grow overnight. Then passage it to 5 ml GMM (gut microbiota medium) and grow overnight to get the preculture. Use the preculture to inoculate the 96 deep well plate (total volume between 100 and 800 µl depending on the plate size). Measure the inoculation OD (should be at ~0.01).

    2. Grow the culture plate for 48 h and measure OD (should be between 0.8 to 1).

    3. Prepare drug containing medium (GMM) at a 2× concentration (100 µM duloxetine). Prepare the medium containing the bacterial species (Clostridium saccharolyticum) also with 2× inoculum from Step A2 (OD for 2× is 0.02). Mix the final medium according to the different sample requirements, e.g., 1× drug plus 1× bacteria (in this case, a mixture of 50 µl of 2× drug in GMM and 50 µl of 2× bacteria). The final concentration is then 1× bacteria and 1× drug.

    4. Grow the culture plate for 48 h at 37°C in a vinyl anaerobic chambers type C, in a nitrogen atmosphere containing 12% CO2 and 1% hydrogen (anoxic).


  2. Sample preparation for drug extraction

    1. After 48 h of incubation, remove the culture plates from the anaerobic chamber and transfer 10 μl aliquots of ‘total’ culture (medium + bacteria) to a new 96-well plate. Spin down the remaining culture at 4,000 × g for 10 min to harvest the supernatant. Aliquot 10 μl of ‘supernatant’ in another 96-well plate.

    2. Add 1 ml of cold (-20°C) extraction solvent (see Recipes) to 10 μl aliquots of both total and supernatant samples and vortex for 5 min (100-fold dilution) in the culture plates (deep 96-well plates). Then, further dilute 25 μl with 500 μl of extraction solvent (2,000-fold dilution) in polypropylene deep 96-well plates.

    3. Close the plates with a lid, incubate for 15 min at -20°C, and centrifuge at 14,000 × g for 10 min at 4°C.

    4. Remove 100 μl of extracted supernatant from the above plate and transfer to WebSeal 96-well plate for LC–MS analysis.


  3. Standard calibration curve and Quality Control (QC) sample preparation

    1. Prepare 1 μM stock of duloxetine and internal standards in DMSO (see Recipes).

    2. Serially dilute duloxetine stock to prepare the following calibration using 2× dilutions at each level in extraction solvent.

    3. Standard solutions in ‘total culture’: incubate drug concentrations of 0, 10, 20, 30, 40, 50, and 70 μM in a bacterial culture. Add 1 ml of cold extraction solvent in the total culture inoculate of each concentration level separately. Prepare these standards in the same manner as actual experimental conditions for samples (as mentioned in Step B3 of Sample preparation for extraction of drugs).

    4. Standard solutions in ‘supernatant’: prepare supernatant standard solutions from above ‘total culture’ by centrifugation at 14,000 × g for 10 min at 4°C. Add 1 ml of cold extraction solvent in the supernatant of each concentration level separately. Prepare these standards in the same manner as the actual experimental conditions for samples (as mentioned in Step B3 of Sample preparation for extraction of drugs). Refer to Figure 1 for an overview of sample preparation.



      Figure 1. Overall workflow for sample preparation for LC–HRMS analysis. For a description of total culture, refer to Procedure A.


    5. Prepare QC sample at 40 μM of duloxetine separately for ‘total culture’ and ‘supernatant’ as mentioned above in Steps C3 and C4.


  4. LC–MS/MS analysis

    Preparation of mobile phases:

    1. For buffer solvent A and B preparations, see Recipes.

    2. Mix each buffer solvents thoroughly by shaking and degas both the buffers in the water bath sonicator with loose caps for at least 10 min.

    3. Set the flow rate of 0.3 ml/min.

    4. Maintain the column at 40°C.

    5. Set LC gradient for 10 min as follows: start at 10% of solvent B for 2 min, ramp up to 90% for 2 min, and hold for 2 min; next, allow 4 min of equilibration to the initial condition (10% of solvent B).


  5. Vanquish UHPLC–MS/MS system preparation

    1. Put up both mobile phase buffers on the UHPLC system and purge it with the mobile phase buffers for 5 min.

    2. Set up the UPLC column and start the flow at a rate of 0.3 ml/min with both mobile phase buffers at a ratio of 90:10 (A:B).

    3. Equilibrate the column for at least 15 min after the column pressure is stable.


  6. MS parameter preparation and Q-exactive plus HRMS calibration

    1. Use extended mass calibration solution and perform ‘custom calibration’ for the mass spec with Xcalibur tune software.

    2. Set the tune MS parameters to the ESI positive mode as shown in Table 1:


      Table 1. MS method parameters for Q-exactive plus

      MS parameter value/unit
      Spray voltage 4 kV
      Sheath gas 30
      Auxiliary gas 5
      S-Lens 65 eV
      Capillary temperature 320°C
      Vaporization temperature 250°C
      MS resolution 35,000 or 70,000
      AGC target 3e6
      Maximum IT 100 ms
      Scan range 60 to 900 m/z
      Spectrum data type Profile


  7. LC–MS injection sequence analysis

    Inject 2 μl of each sample in the following manner:

    1. Solvent blank: Inject extraction solvent as Blank to check the instrument background.

    2. Medium blank: Extract GMM medium as described above in Procedure B and inject to check matrix background from medium.

    3. Incubate bacterial culture blank (without drug): Same a 0 μM level mentioned in Procedure C to check matrix background after incubation.

    4. Inject QC sample at least five times the (same as 40 μM standard solution from Procedure C) to stabilize the LC–MS system.


  8. Inject six standard solutions from 10 to 70 μM prepared in supernatant (see Procedure C).

    1. Inject QC sample.

    2. Run ‘total culture’ samples (randomize the sequence of samples).

    3. Inject QC sample after every ten samples.

    4. Repeat the same sequence from Step G3 for supernatant samples.

    5. At the end of each sequence, run QC sample followed by standard calibration curve.

Data analysis

  1. Evaluation of raw data

    1. Get the extracted ion chromatograms (XIC) for duloxetine, fluoxetine, sulfamethazine, and sulfamethizole for their exact masses with a 10 ppm accuracy window, as shown in Table 2. Use Xcalibur Qual browser for raw data visualization and quality check.


      Table 2. Name, molecular formula, and exact mass of protonated ions (M + H) duloxetine and internal standards used to obtain respective XICs

      Name Molecular formula Exact mass (M + H)
      Duloxetine C18H19NOS 298.1265
      Fluoxetine C17H18F3NO 310.1418
      Sulfamethazine C12H14N4O2S 279.0965


    2. A representative chromatogram of duloxetine and its internal standards are shown in Figure 2.



      Figure 2. >Representative extracted ion chromatograms of duloxetine and its internal standards


    3. Confirm the duloxetine identity using exact mass as well as MS/MS spectra (shown in Figure 3), which can be interpreted from the chemical structure of duloxetine (shown in Figure 4). Refer to EMBL-MCF spectral library (http://curatr.mcf.embl.de) for more MS/MS data and protocols (Palmer et al., 2018).



      Figure 3. MS/MS spectra of duloxetine standard



      Figure 4. Chemical structure of duloxetine


  2. Quality control evaluation with internal standards

    1. Get the peak area from the XICs of duloxetine and its internal standards using Xcalibur Quan browser.

    2. Calculate the concentration of QC samples injected throughout the sequence. The deviation of known and calculated concentrations should not be >10%.

    3. Calculated the variation in peak areas internal standards across all samples in analysis sequence, which should not be >10% (shown in Figure 5).

    4. Use peak areas of fluoxetine internal standard for normalization of respective duloxetine peaks areas. Use the slope of the standard calibration curve to calculate concentrations of duloxetine from samples.



      Figure 5. Plot of peak areas/intensities of internal standards from 94 bacterial culture samples. The percentage of CVs for sulfamethizole, sulfamethazine, and fluoxetine were 6.88, 6.85, and 7.54, respectively.

Recipes

  1. Extraction solvent

    1. Mix equal volumes of acetonitrile and methanol in a glass bottle (total volume 1,000 ml).

    2. Prepare 1 ml of 1 µM of internal standard stock solutions for sulfamethizole (270 mg), sulfamethazine (278 mg), and fluoxetine (309 mg) in the extraction solvent (equal mix of acetonitrile and methanol).

    3. Add an internal standard stock solution of sulfamethizole (100 µl), sulfamethazine (40 µl), and fluoxetine (2.5 µl) into 1,000 ml of extraction solvent to get final concentrations of 100 nM, 40 nM, and 2.5 nM, respectively.

  2. 1 µM Duloxetine stock solution preparation

    Dissolve 297.4 ± 2 mg of duloxetine standard in 1 ml of DMSO solvent in an amber color glass vial.

  3. 1 µM Fluoxetine stock solution preparation

    Dissolve 309.3 ± 2 mg of fluoxetine standard in 1 ml of DMSO solvent in an amber color glass vial.

  4. Mobile phase buffer preparations

    1. Preparation of buffer A: add 1 ml (0.1 %) formic acid to 1,000 ml of water.

    2. Preparation of buffer B: add 1 ml (0.1 %) formic acid to 1,000 ml of methanol.

Acknowledgments

We thank EMBL Metabolomics and other core facilities, as well as EMBL Alxandrov team members, for their support during this project.

The sample preparation protocol was partially adapted from Zimmermann et al. (2019).

Competing interests

There are no competing interests.

References

  1. Chae, J. W., Baek, H. M., Kim, S. K., Kang, H. I. and Kwon, K. I. (2013). Quantitative determination of duloxetine and its metabolite in rat plasma by HPLC-MS/MS. Biomed Chromatogr 27(8): 953-955.
  2. Falony, G., Joossens, M., Vieira-Silva, S., Wang, J., Darzi, Y., Faust, K., Kurilshikov, A., Bonder, M. J., Valles-Colomer, M. and Vandeputte, D. (2016). Population-level analysis of gut microbiome variation. Science 352(6285): 560-564.
  3. Forslund, K., Hildebrand, F., Nielsen, T., Falony, G., Le Chatelier, E., Sunagawa, S., Prifti, E., Vieira-Silva, S., Gudmundsdottir, V. and Pedersen, H. K. (2015). Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. Nature 528(7581): 262-266.
  4. Klünemann, M., Andrejev, S., Blasche, S., Mateus, A., Phapale, P., Devendran, S., Vappiani, J., Simon, B., Scott, T. A. and Kafkia, E. (2021). Bioaccumulation of therapeutic drugs by human gut bacteria. Nature 597(7877): 533-538.
  5. Maier, L. and Typas, A. (2017). Systematically investigating the impact of medication on the gut microbiome. Curr Opin Microbiol 39128-135.
  6. Palmer, A., Phapale, P., Fay, D. and Alexandrov, T. (2018). Curatr: a web application for creating, curating and sharing a mass spectral library. Bioinformatics 34(8): 1436-1438.
  7. Scott, T. A., Quintaneiro, L. M., Norvaisas, P., Lui, P. P., Wilson, M. P., Leung, K. Y., Herrera-Dominguez, L., Sudiwala, S., Pessia, A. and Clayton, P. T. (2017). Host-microbe co-metabolism dictates cancer drug efficacy in C. elegans. Cell 169(3): 442-456e418.
  8. Senthamil Selvan, P., Gowda, K. V., Mandal, U., Sam Solomon, W. D. and Pal, T. K. (2007). Determination of duloxetine in human plasma by liquid chromatography with atmospheric pressure ionization-tandem mass spectrometry and its application to pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci 858(1-2): 269-275.
  9. Waldschmitt, C., Vogel, F., Maurer, C. and Hiemke, C. (2007). Measurement of duloxetine in blood using high-performance liquid chromatography with spectrophotometric detection and column switching. Ther Drug Monit 29(6): 767-772.
  10. Zhang, M., Li, H., Ji, Z., Dong, D. and Yan, S. (2017). Clinical study of duloxetine hydrochloride combined with doxazosin for the treatment of pain disorder in chronic prostatitis/chronic pelvic pain syndrome: An observational study. Medicine(Baltimore) 96(10): e6243.
  11. Zimmermann, M., Zimmermann-Kogadeeva, M., Wegmann, R. and Goodman, A. L. (2019). Mapping human microbiome drug metabolism by gut bacteria and their genes. Nature 570(7762): 462-467.

简介

[摘要]扩大我们对药物-肠道细菌相互作用的理解需要在复杂的细菌培养物中进行高通量药物测量。培养物、培养基和细胞颗粒中的药物定量容易产生强烈的基质效应。我们开发了一种液相色谱-高分辨质谱 (LC-HRMS) 方法,用于从高通量肠道-药物相互作用实验中量化度洛西汀。该方法的重现性、灵敏度和准确性得到了部分验证,使其适用于大规模药物筛选。我们广泛使用这种方法来研究度洛西汀和其他药物在几种肠道细菌中的生物转化和生物积累。


[背景]最近的几项临床研究表明药物对肠道微生物组的影响。这种相互作用会对药物的生物转化、药代动力学、功效和毒性产生相当大的影响(Falony等人,2016 年;Scott等人,2017 年)。为了研究细菌-药物相互作用,对人类肠道细菌菌株中的这种相互作用进行系统分析是一种强大的方法(Maier 和 Typas,2017;Zimmermann等,2019;Forslund等,2015 )。此类实验通常涉及使用靶向药物对多种肠道细菌进行大规模筛选,并且需要对药物及其代谢物进行高通量定量。此外,此类实验中使用的细菌培养物和肠道微生物群培养基 (GMM) 的复杂性会产生强烈的背景信号,干扰药物分析。药物生物转化和生物积累的进一步研究需要在总培养基、上清液和细胞沉淀中对其进行量化,这容易产生强烈的基质效应。我们通过开发和验证用于度洛西汀定量的 LC-HRMS 方法解决了这些挑战。我们研究了度洛西汀,这是一种广泛使用的抗抑郁药,发现它被多种肠道微生物物种生物蓄积。这种 LC-HRMS 方法可验证从数百种细菌-药物对的大规模筛选中获得的结果(Klünemann等人,2021 年)。
通常,由于其灵敏度和特异性,使用三重四极杆仪器进行药物定量。已报道的度洛西汀定量方法主要在血浆样品中进行(Senthamil Selvan等,2007;Waldschmitt等,2007;Chae等,2013;Zhang等,2017)。然而,在使用报告的方法对细菌培养物中的度洛西汀进行定量期间,我们发现分析的特异性受到细菌培养物背景基质的影响。基质效应阻碍了度洛西汀的准确定量,尤其是来自孵育培养物(48 小时)。我们开发的方法准确、特异且灵敏,可用于从总培养基、上清液和沉淀中定量度洛西汀。
药物-肠道相互作用的研究是一个新兴领域,它正在扩展我们对这一迷人研究领域的认识。我们的方法可以成为肠道微生物组研究中药物定量的有用工具。

关键字:液相高分辨高精密度质谱, 液相色谱串联质谱, 生物转化, 生物蓄积, 药物量化, 药物与肠道的相互作用


材料和试剂
 
移液器吸头(Rainin,梅特勒-托利多,美国)
提示 GP-LTS-A-1,000 µl-768/8(Rainin,目录号:17014338)
提示 GP-LTS-A-250 µl-/F-960/10(Rainin,目录号:30389288)
GP-LTS-A-10 µl-960/10(Rainin,目录号:30389284)
Eppendorf 聚丙烯 DNA LoBind 管(2.0 毫升)(Thermo Fisher Scientific,目录号:10051232)
WebSeal 96 孔板,圆孔,条形码(Thermo Fisher Scientific,目录号:601 80-P206B)
康宁® 96孔聚丙烯深孔板(Sigma-Aldrich公司,目录号:CLS3957)
Corning ® 96 圆孔微孔板存储垫 III(Sigma-Aldrich,目录号:CLS3080)
Zone-Free TM密封膜(Sigma-Aldrich,目录号:Z721646)
玻璃器皿购自 DURAN (DWK Life Sciences, Wertheim, Germany)
使用的细菌种类:Clostridium saccharolyticum ;类型菌株,WM1;税号:610130;内部数据库 ID:NT5037。[储存:mGAM 培养基中的 20% 甘油,-80°C 储存]
Optima ® LC-MS 级乙腈(Fisher Chemical,目录号:A955-212)
甲醇(Fisher Chemical,目录号:A456-212)
水(Fisher Chemical,目录号:W6-121)
LC-MS LiChropur ®级甲酸(Merck KGaA,目录号:5330020050)
度洛西汀标准品(Sigma-Aldrich,Merck KGaA,目录号:SML0474)
内标盐酸氟西汀(Sigma-Aldrich,Merck KGaA,目录号:F132)
磺胺二甲嘧啶(Sigma-Aldrich,Merck KGaA,目录号:S5637)
磺胺二甲嘧啶(Sigma-Aldrich,Merck KGaA,目录号:S5632)
Pierce TM三重四极杆校准溶液,扩展质量(Thermo Fisher,目录号:88340)
mGAM 培养基:岐阜厌氧培养基肉汤,改良(HyServe,目录号:05433)
GMM(肠道微生物群培养基)
DMSO溶剂
萃取溶剂(见配方)
1 µM 度洛西汀储备溶液制备(见配方)
1 µM 氟西汀储备溶液制备(见配方)
流动相缓冲液制备(见配方)
 
设备
 
移液器(Eppendorf AG,汉堡,德国)
Eppendorf Multipette ® M4(Eppendorf,目录号:4982000012)
Eppendorf Research plus 移液器 (100-1,000 µl)
多通道 8 通道,30-300 µl(Eppendorf,目录号:3122000051)
多通道 8 通道,0.5-10 µl(Eppendorf,目录号:3122000019)
Vanquish UHPLC-MS/MS 系统与 Q-Exactive plus HRMS(Thermo Scientific,MA,USA;目录号:IQLAAAAGABHFAPUMZZZ)耦合
ACQUITY UPLC HSS T3 色谱柱(100Å,1.8 µm;2.1 × 100 mm)(Waters,目录号:186003539)             
S系列加热超声波清洗水浴(Fisherbrand TM ,目录号:10162372)
用于涡旋的 Eppendorf MixMate (Eppendorf,目录号:5353000510)或同等产品
Hamilton TM 1700 Series Gastight TM Syringe 500 μl(Thermo Fisher Scientific,目录号:365JLT41)
离心机 5418 R,冷藏(Eppendorf,目录号:5401000013)
乙烯基厌氧室 C 型(Coy Laboratory Products, Inc.,美国)
水浴超声波仪;Fisherbrand TM S 系列加热超声波清洁器(Thermo Fisher Scientific,目录号:10162372)
 
软件
 
Thermo Xcalibur 软件版本 4.0.27(Thermo Scientific, MA, USA)用于数据采集(Q-exactive plus tune version 2.11)和定性(Qual Browser)和定量(Quan Browser)数据分析
 
程序
 
度洛西汀细菌培养接种
将Clostridium saccharolyticum从原种培养物(mGAM 培养基中的 20% 甘油,储存在 -80°C)中接种到 5 ml mGAM 中,并使其生长过夜。然后将其传给 5 ml GMM(肠道微生物群培养基)并过夜生长以获得预培养物。使用预培养物接种 96 深孔板(总体积在 100 和 800 µl 之间,具体取决于板尺寸)。测量接种 OD(应为 ~0.01)。
将培养板培养 48 小时并测量 OD(应在 0.8 到 1 之间)。
以 2 倍浓度(100 µM 度洛西汀)制备含药物培养基 (GMM)。制备含有细菌种类(溶糖梭菌)的培养基,也用步骤 A2 中的 2x 接种物(2x 的 OD 为 0.02)。根据不同的样品要求混合最终培养基,例如,1x 药物加 1x 细菌(在这种情况下,GMM 中 50 µl 2x 药物和 50 µl 2x 细菌的混合物)。最终浓度为 1x 细菌和 1x 药物。
在 37°C 的 C 型乙烯基厌氧室中,在含有 12% CO 2和 1% 氢气(缺氧)的氮气氛中培养培养板 48 小时。
 
药物提取的样品制备
孵育 48 小时后,从厌氧室中取出培养板,并将“总”培养物(培养基 + 细菌)的 10 μl 等分试样转移到新的 96 孔板中。将剩余的培养物以 4,000 × g 的速度旋转10 分钟以收获上清液。在另一个 96 孔板中分装 10 μl 的“上清液”。
将 1 ml 冷(-20°C)提取溶剂(参见配方)添加到 10 μl 总样品和上清样品的等分试样中,并在培养板(深 96 孔板)中涡旋 5 分钟(100 倍稀释)。然后,在聚丙烯深 96 孔板中用 500 μl 提取溶剂(2,000 倍稀释)进一步稀释 25 μl。
盖上盖子,在 -20°C 下孵育 15 分钟,然后在 4°C 下以 14,000 × g离心10 分钟。
从上述板中取出 100 μl 提取的上清液并转移至 WebSeal 96 孔板进行 LC-MS 分析。
 
标准校准曲线和质量控制 (QC) 样品制备
在 DMSO 中制备 1 μM 度洛西汀和内标库存(参见食谱)。
连续稀释度洛西汀原液以准备以下校准,使用提取溶剂中每个浓度的 2 倍稀释液。
“总培养”中的标准溶液:在细菌培养物中孵育 0、10、20、30、40、50 和 70 μM 的药物浓度。在每个浓度水平的总培养接种物中分别加入 1 ml 冷提取溶剂。以与样品的实际实验条件相同的方式制备这些标准品(如药物提取样品制备的步骤 B3 中所述)。
“上清液”中的标准溶液:通过在 4°C 下以 14,000 × g离心10 分钟,从“总培养物”上方制备上清液标准溶液。各浓度水平的上清液中分别加入1ml冷提取溶剂。以与样品的实际实验条件相同的方式制备这些标准品(如药物提取样品制备的步骤 B3 中所述)。有关样品制备的概述,请参阅图 1。
 
 
图 1. LC-HRMS 分析样品制备的总体工作流程。有关总培养的描述,请参阅程序 A。
 
如上文步骤 C3 和 C4 所述,分别制备 40 μM 度洛西汀的 QC 样品,用于“总培养”和“上清液”。
 
LC-MS/MS 分析
流动相的制备:
对于缓冲溶剂 A 和 B 制剂,请参见配方。
通过摇动和脱气在水浴超声仪中用松盖至少 10 分钟的缓冲液彻底混合每种缓冲溶剂。
将流速设置为 0.3 毫升/分钟。
将色谱柱保持在 40℃.
如下设置 LC 梯度 10 分钟:从溶剂 B 的 10% 开始 2 分钟,斜升至 90% 2 分钟,并保持 2 分钟;接下来,允许 4 分钟平衡到初始条件(溶剂 B 的 10%)。
 
Vanquish UHPLC-MS/MS 系统制备
在 UHPLC 系统上放置两个流动相缓冲液,并用流动相缓冲液将其清除 5 分钟。
设置 UPLC 柱并以 0.3 ml/min 的速率开始流动,两种流动相缓冲液的比例为 90:10 (A:B)。
柱压稳定后平衡柱至少 15 分钟。
 
MS 参数准备和 Q-exactive 加 HRMS 校准
使用扩展质量校准解决方案并使用 Xcalibur 调谐软件对质谱进行“自定义校准”。
将调谐 MS 参数设置为 ESI 正模式,如表 1 所示:
 
表 1. Q-exactive plus 的 MS 方法参数
 
LC-MS 进样序列分析
按以下方式注入 2 μl 的每个样品:
溶剂空白:将提取溶剂作为空白注入以检查仪器背景。
培养基空白:按上述步骤 B 中所述提取 GMM 培养基并注入以检查培养基中的基质背景。
孵育细菌培养空白(不含药物):与程序 C 中提到的 0 μM 水平相同,以检查孵育后的基质背景。
注入 QC 样品至少 5 倍(与程序 C 中的 40 μM 标准溶液相同)以稳定 LC-MS 系统。
 
注入在上清液中制备的 10 到 70 μM 的六种标准溶液(参见程序 C)。
进样 QC 样品。
运行“总培养”样本(随机化样本序列)。
每 10 个样品后注入 QC 样品。
对上清液样品重复步骤 G3 中的相同序列。
在每个序列结束时,运行 QC 样品,然后运行标准校准曲线。
 
数据分析
 
原始数据评估
获取度洛西汀、氟西汀、磺胺二甲嘧啶和磺胺甲二唑的精确质量数提取离子色谱图 (XIC),精度为 10 ppm,如表 2 所示。使用 Xcalibur Qual 浏览器进行原始数据可视化和质量检查。
 
表 2. 用于获得相应 XIC 的质子化离子 (M + H) 度洛西汀和内标的名称、分子式和精确质量
 
度洛西汀及其内标的代表性色谱图如图 2 所示。
 
图 2. 度洛西汀及其内标的代表性提取离子色谱图
 
使用精确质量和 MS/MS 光谱(如图 3 所示)确认度洛西汀的身份,这可以从度洛西汀的化学结构(如图 4 所示)中得到解释。有关更多 MS/MS 数据和协议,请参阅 EMBL-MCF 谱库 ( http://curatr.mcf.embl.de )。(帕尔默等人,2018 年)。
 
 
图 3. 度洛西汀标准品的 MS/MS 谱图
 
图 4. 度洛西汀的化学结构
 
使用内部标准进行质量控制评估
使用 Xcalibur Quan 浏览器从度洛西汀及其内标的 XIC 中获取峰面积。
计算整个序列中注入的 QC 样品的浓度。已知浓度和计算浓度的偏差不应大于 10%。
计算分析序列中所有样品的峰面积内标变化,不应大于 10%(如图 5 所示)。
使用氟西汀内标的峰面积对各个度洛西汀峰面积进行归一化。使用标准校准曲线的斜率计算样品中度洛西汀的浓度。
 
 
图 5. 来自 94 个细菌培养样品的内标峰面积/强度图。磺胺二甲嘧啶、磺胺二甲嘧啶和氟西汀的 CV 百分比分别为 6.88、6.85 和 7.54。
 
食谱
 
萃取溶剂
在玻璃瓶中混合等体积的乙腈和甲醇(总体积 1,000 毫升)。
在萃取溶剂(乙腈和甲醇的等量混合)中为磺胺二甲嘧啶 (270 毫克)、磺胺二甲嘧啶 (278 毫克) 和氟西汀 (309 毫克) 制备 1 ml 1 µM 的内标库存溶液。
将磺胺二甲嘧啶 (100 µl)、磺胺二甲嘧啶 (40 µl) 和氟西汀 (2.5 µl) 的内标储备溶液加入 1,000 ml 萃取溶剂中,最终浓度分别为 100 nM、40 nM 和 2.5 nM。
1 µM 度洛西汀原液制备
在琥珀色玻璃小瓶中将 297.4 ± 2 mg 度洛西汀标准溶解在 1 ml DMSO 溶剂中。
1 µM 氟西汀原液制备
在琥珀色玻璃小瓶中将 309.3 ± 2 mg 氟西汀标准溶解在 1 ml DMSO 溶剂中。
流动相缓冲液制剂
缓冲液 A 的制备:将 1 ml (0.1%) 甲酸加入 1,000 ml 水中。
缓冲液 B 的制备:将 1 ml (0.1%) 甲酸加入 1,000 ml 甲醇中。
 
致谢
 
我们感谢 EMBL 代谢组学和其他核心设施,以及 EMBL Alxandrov 团队成员在该项目中的支持。
样品制备协议部分改编自 Zimmermann等人。(2019)。
 
利益争夺
 
没有相互竞争的利益。
 
参考
 
Chae, JW, Baek, HM, Kim, SK, Kang, HI 和 Kwon, KI (2013)。HPLC-MS/MS 定量测定大鼠血浆中度洛西汀及其代谢物。生物医学色谱27(8): 953-955。
Falony, G., Joossens, M., Vieira-Silva, S., Wang, J., Darzi, Y., Faust, K., Kurilshikov, A., Bonder, MJ, Valles-Colomer, M., Vandeputte, D.,等。(2016)。肠道微生物组变异的群体水平分析。科学352(6285):560-564。
Forslund, K., Hildebrand, F., Nielsen, T., Falony, G., Le Chatelier, E., Sunagawa, S., Prifti, E., Vieira-Silva, S., Gudmundsdottir, V., Pedersen,香港等。(2015)。解开人类肠道微生物群中的 2 型糖尿病和二甲双胍治疗特征。自然528(7581):262-266。
Klünemann, M., Andrejev, S., Blasche, S., Mateus, A., Phapale, P., Devendran, S., Vappiani, J., Simon, B., Scott, TA, Kafkia, E., et艾尔。(2021)。人类肠道细菌对治疗药物的生物蓄积。自然597(7877):533-538。
Maier, L. 和 Typas, A. (2017)。系统地研究药物对肠道微生物群的影响。Curr Opin Microbiol 39:128-135。
Palmer, A.、Phapale, P.、Fay, D. 和 Alexandrov, T.(2018 年)。Curatr:用于创建、管理和共享质谱库的 Web 应用程序。生物信息学34(8):1436-1438。
Scott, TA, Quintaneiro, LM, Norvaisas, P., Lui, PP, Wilson, MP, Leung, KY, Herrera-Dominguez, L., Sudiwala, S., Pessia, A., Clayton, PT等。(2017)。宿主-微生物共代谢决定了秀丽隐杆线虫的抗癌药物功效。单元格169(3):442-456 e418。
Senthamil Selvan, P., Gowda, KV, Mandal, U., Sam Solomon, WD 和 Pal, TK (2007)。液相色谱-大气压电离-串联质谱法测定人血浆中度洛西汀及其在药代动力学研究中的应用。J Chromatogr B Analyt Technol Biomed Life Sci 858(1-2): 269-275。
Waldschmitt, C.、Vogel, F.、Maurer, C. 和 Hiemke, C. (2007)。使用具有分光光度检测和柱切换的高效液相色谱法测量血液中的度洛西汀。药物监测29(6): 767-772。
Zhang, M., Li, H., Ji, Z., Dong, D. 和 Yan, S. (2017)。盐酸度洛西汀联合多沙唑嗪治疗慢性前列腺炎/慢性盆腔疼痛综合征疼痛障碍的临床研究:一项观察性研究。医学(巴尔的摩)96(10):e6243。
Zimmermann, M.、Zimmermann-Kogadeeva, M.、Wegmann, R. 和 Goodman, AL (2019)。通过肠道细菌及其基因绘制人类微生物组药物代谢图。自然570(7762):462-467。
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引用:Phapale, P. B., Blasche, S., Patil, K. R. and Alexandrov, T. (2021). Quantification of Duloxetine in the Bacterial Culture and Medium to Study Drug-gut Microbiome Interactions. Bio-protocol 11(21): e4214. DOI: 10.21769/BioProtoc.4214.
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