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Quantification of Duloxetine in the Bacterial Culture and Medium to Study Drug-gut Microbiome Interactions

Prasad B. Phapale Prasad B. Phapale
SB Sonja Blasche
KP Kiran R. Patil
TA Theodore Alexandrov

Published: Vol 11, Iss 21, Nov 5, 2021 DOI: 10.21769/BioProtoc.4214 Views: 2778

Edited by: Juan Facundo Rodriguez Ayala Reviewed by: DURAI SELLEGOUNDER

Original research article

The authors used this protocol in:

http://pubmed.ncbi.nlm.nih.gov/34497420/

Sep 2021

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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 search LC–MS/MS search Biotransformation search Bioaccumulation search Drug quantification search Drug-gut interactions search

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

Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbial biochemistry

Microbiology > Microbial metabolism

Biochemistry

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