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

Prasad B. Phapale; Sonja  Blasche; Kiran R. Patil; Theodore  Alexandrov

doi:10.21769/BioProtoc.4214

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Peer-reviewed

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

Prasad B. Phapale email

SB Sonja Blasche

KP Kiran R. Patil

TA Theodore Alexandrov

发布: 2021年11月05日第11卷第21期 DOI: 10.21769/BioProtoc.4214 浏览次数: 2602

评审: Juan Facundo Rodriguez AyalaDURAI SELLEGOUNDERAnonymous reviewer(s)

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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

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)
Eppendorf Polypropylene DNA LoBind Tubes (2.0 ml) (Thermo Fisher Scientific, catalog number: 10051232)
WebSeal 96-Well Plate, Round Well, Barcoded (Thermo Fisher Scientific, catalog number: 601 80-P206B)
Corning^® 96 well Polypropylene Deep Well Plate (Sigma-Aldrich, catalog number: CLS3957)
Corning^® 96 Round Well Microplate Storage Mat III (Sigma-Aldrich, catalog number: CLS3080)
Zone-Free^TM Sealing Films (Sigma-Aldrich, catalog number: Z721646)
Glassware purchased from DURAN (DWK Life Sciences, Wertheim, Germany)
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]
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)
LC–MS LiChropur^® grade formic acid (Merck KGaA, catalog number: 5330020050)
Standards of Duloxetine (Sigma-Aldrich, Merck KGaA, catalog number: SML0474)
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)
Pierce^TM Triple Quadrupole Calibration Solution, Extended Mass (Thermo Fisher, catalog number: 88340)
mGAM medium: Gifu Anaerobic Medium Broth, Modified (HyServe, catalog number: 05433)
GMM (gut microbiota medium)
DMSO solvent
Extraction solvent (see Recipes)
1 µM Duloxetine stock solution preparation (see Recipes)
1 µM Fluoxetine stock solution preparation (see Recipes)
Mobile phase buffer preparations (see Recipes)

Equipment

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)
Vanquish UHPLC–MS/MS system coupled to a Q-Exactive plus HRMS (Thermo Scientific, MA, USA; catalog number: IQLAAAGABHFAPUMZZZ)
ACQUITY UPLC HSS T3 column (100Å, 1.8 µm; 2.1 × 100 mm) (Waters, catalog number: 186003539)
S-Series Heated Ultrasonic Cleaning Water Bath (Fisherbrand^TM, catalog number:10162372)
Eppendorf MixMate for vortexing (Eppendorf, catalog number: 5353000510) or equivalent
Hamilton^TM 1700 Series Gastight^TM Syringe 500 μl (Thermo Fisher Scientific, catalog number: 365JLT41)
Centrifuge 5418 R, refrigerated (Eppendorf, catalog number: 5401000013)
Vinyl Anaerobic Chambers Type C (Coy Laboratory Products, Inc., USA)
Water bath sonicator; Fisherbrand^TM S-Series heats ultrasonic cleaners (Thermo Fisher Scientific, catalog number: 10162372)

Software

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

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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.