A Vancomycin HPLC Assay for Use in Gut Microbiome Research

ABSTRACT The human microbiome project has revolutionized our understanding of the interaction between commensal microbes and human health. By far, the biggest perturbation of the microbiome involves use of broad-spectrum antibiotics excreted in the gut. Thus, pharmacodynamics of microbiome changes in relation to drug exposure pharmacokinetics is an emerging field. However, reproducibility studies are necessary to develop the field. A simple and fast high-performance liquid chromatography-photodiode array detector (HPLC) method was validated for quantitative fecal vancomycin analysis. Reproducibility of results were tested based on sample storage time, homogeneity of antibiotic within stool, and concentration consistency after lyophilization. The HPLC method enabled the complete elution of vancomycin within ~4.2 min on the reversed-phase C18 column under the isocratic elution mode, with excellent recovery (85% to 110%) over a 4-log, quantitative range (0.4–100 μg/mL). Relative standard derivations (RSD) of intra-day and inter-day results ranged from 0.4% to 5.4%. Using sample stool aliquots of various weights consistently demonstrated similar vancomycin concentrations (mean RSD: 6%; range: 2–16%). After correcting for water concentrations, vancomycin concentrations obtained after lyophilization were similar to the concentrations obtained from the original samples (RSD less than 10%). These methodologies establish sample condition standards for a quantitative HPLC to enable vancomycin pharmacokinetic studies with the human microbiome. IMPORTANCE Research on antibiotic effect on the gut microbiome is an emerging field with standardization of research methods needed. In this study, a simple and fast high-performance liquid chromatography method was validated for quantitative fecal vancomycin analysis. Reproducibility of results were tested to standardize storage time, homogeneity of antibiotic within stool, and concentration consistency after lyophilization. These methodologies establish sample condition standards for a quantitative HPLC to enable vancomycin pharmacokinetic studies with the human microbiome.

The manuscript represents premature work. The Material and Methods section does not adequately provide information on how vancomycin was extracted from the stool sample. This is critical because the significant mass percentage of stool is represented by bacterial cells. Hence, orally administered vancomycin bound to the cell wall of bacteria present significant portions that are not detected by the method. Furthermore, the administered vancomycin affects the number of bacteria present in the gut and thereby altering the unbound free vancomycin concentration.
Staff Comments:

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Thank you for submitting your paper to Microbiology Spectrum.  Table 3 at days 2 and 32 **This is an interesting observation, especially day 2. There is only a 10 day dosing interval so the day 32 sample not having detectable vancomycin is understandable. Day 2 is a little less intuitive but is likely dependent on transit time of the vancomycin capsule to be excreted in the feces. Have mentioned both of these observations in the discussion.
3-Considering that this study offers a newly developed method for fecal vancomycin quantification, I will recommend for validation purposes to provide discussions/ examples of additional fecal extract chromatograms ( e.g supplemental data) **Have added additional chromatograms as supplemental data as requested.

Reviewer comments:
Reviewer #1 (Comments for the Author): Comment 1: Page 1, line 1: In the title, a vancomycin HPLC-PDA assay was presented. In my opinion, only a single wavelength 205nm was chosen for measuring vancomycin levels. I would remove "PDA" from the title. **Removed PDA as requested.
Comment 2: Page 2, line 25: The authors used "isostatic" elution mode. The "isocratic" elution mode is the correct term in HPLC. **Yes, good spot. Thank you.

Comment 3:
In the section of materials and methods, the extraction of vancomycin from fecal samples was not provided and detailed. **Chenlin Comment 4: Page 6, line 129: The authors stated "Water concentration averaged 78 percent for lyophilized stool samples. This sentence did not make sense, for we know that after the lyophilization process, the final residual water content in the stool is less than 5%. **Have changed this sentence to improve clarity Comment 5: The authors should use an internal standard method by adding an internal standard to the stool samples prior to extracting vancomycin from the samples. In this way, human errors during the correction of water concentrations would be largely reduced.
**This is an interesting thought. In this study, we either used known, spiked vancomycin stool samples or clinical samples from healthy subjects who only took oral vancomycin as part of an ongoing phase I study. For these reasons, we did not feel the need to add an internal standard. In addition, our correction for water concentrations allowed us to be quite close to the spiked concentrations (or our spiking experiments). We did contemplate on adding an internal standard for future studies in patients in which other small molecules may interfere with the HPLC readings.
Reviewer #2 (Comments for the Author): The manuscript represents premature work. The Material and Methods section does not adequately provide information on how vancomycin was extracted from the stool sample. This is critical because the significant mass percentage of stool is represented by bacterial cells. Hence, orally administered vancomycin bound to the cell wall of bacteria present significant portions that are not detected by the method. Furthermore, the administered vancomycin affects the number of bacteria present in the gut and thereby altering the unbound free vancomycin concentration. **Thank you for these comments. We have added a section on how vancomycin was extracted from the stool sample based on this comment and comments from reviewer #2. Your manuscript has been reviewed and there is still an important point to address notably in what concerns the methodology as highlighted by Rev1. In addition, I would like to apologize for the time that this revision has taken, I have been waiting for Rev 2 critics. The decision is based on Rev 1 and my revision. I would be happy to review your final version.
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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Comment 1: Although vancomycin is a hydrophilic drug, the authors should compare the extraction solvent used in the current method with a previous extraction solvent published in the International Journal of Antimicrobial Agents 34 (2009) 555-560. The published report showed that there was a wide variation in free to total vancomycin serum concentrations in patients ( range 12-100%) treated for gram-positive infections. This is critical because vancomycin bound to the wall of bacteria present significant portions that could not be detected by the current method.

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To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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Thank you for submitting your paper to Microbiology Spectrum.

Editor Comments
Comment 1: Although vancomycin is a hydrophilic drug, the authors should compare the extraction solvent used in the current method with a previous extraction solvent published in the International Journal of Antimicrobial Agents 34 (2009) 555-560. The published report showed that there was a wide variation in free to total vancomycin serum concentrations in patients ( range 12-100%) treated for gram-positive infections. This is critical because vancomycin bound to the wall of bacteria present significant portions that could not be detected by the current method.
**Have added an extra experiment comparing the two techniques. Thanks for this suggestion. I`m please to inform that your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.