New Method to Quantify Antibiotic For Gut Microbiome Studies


A team developed and validated a new analytic method to quantify omadacycline and its epimerization in stool to facilitate microbiome research.

Omadacyline (Nuzyra, Paratek Pharmaceuticals) is a tetracycline-class antibiotic that was FDA approved for the treatment of adults with community-acquired bacterial pneumonia (CABP) and acute skin and skin structure infections (ABSSSI). The antibiotic is also being studied for a variety of potential indications.1 Investigators from the Garey lab at the University of Houston explored omadacycline as it relates to the microbiome and potential application for its use for the healthcare-associated infection, C difficile infection (CDI). In a recent study by Garey et al, they investigated omadacycline for possible utility in this indication as compared to vancomycin, and they found omadacycline had high fecal concentrations with a distinct microbiome profile compared to those in the vancomycin group.2

They concluded that omadacycline was safe for “patients at high risk for C diff infection.”2

In addition, researchers from the Garey lab also explored finding a novel method to quantify omadacycline and its epimerization in stool. The results of this research were published in a recent issue of Journal of Chromatography B. 3

Previous research showed the ability to extract and quantify omadacycline in plasma and urine using high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), but there was serious limitations when it came to fecal analysis, the investigators reported. “Only a single study reported using LC-MS/MS to quantify human fecal omadacycline, however, it involved multi-step extraction and lacked validation of matrix effect, precision, and accuracy.”3

As such the team developed and validated a new method with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and were able to quantify omadacycline and its epimerization in stool to facilitate microbiome studies.3

Study Methods
They had 8 healthy individuals take oral omadacycline for 10 days. The participants had stool samples collected at baseline, during therapy duration, and at follow-visits. The team extracted the antibiotic in a methanol-water-ethylenediaminetetraacetic acid (ETDA) solvent containing deuterated omadacycline as internal standard, followed by dilution. In an optimal gradient elution mode, omadacycline and its C4 epimer were separated within 5 min on reversed-phase C18 column.3

In terms of results, they were able to find a simple solution to quantify the antibiotic. “The method showed a broad working range of 0.1–200 ng/ml with a limitation of detection (LOD) of 0.03 ng/ml, little fecal matrix effect, good intra-day and inter-day accuracy (90–101 %), precision (2–15 %), and recovery rate (99–105 %),” the investigators wrote. “The method was sufficiently sensitive to quantify omadacycline in human fecal samples (n = 82) collected during a 10-day therapy course and at follow-up (day 13 and day 30) that ranged from 1 to 4785 µg/g. Further analysis revealed that ∼9 % of omadacycline was epimerized in fecal matrix control while, on average, 37.4 % was epimerized in human fecal samples.”3

“This study revealed that on average, 37.4% of omadacycline transformed to a C4 epimer form in fecal samples with a wide variability noted between samples. The mechanism of epimerization is unknown and will require further study. However, concentrations of native omadacycline well above minimum inhibitory concentration for pathogenic bacteria including C difficile were noted in all samples,” the investigators concluded.3


1. Fleming M. FDA Approves Omadacycline for ABSSSI and CABP. ContagionLive. October 3, 2018. Accessed March 8, 2024.

2. Brooks A. Omadacycline Demonstrates Safety, Possible Utility for Healthcare-Associated Infection Compared to Vancomycin.

3. Hu C, Wang W, Jo J, Garey KW. Development and validation of LC-MS/MS for quantifying omadacycline from stool for gut microbiome studies. J Chromatogr B Analyt Technol Biomed Life Sci. Published online February 28, 2024. doi:10.1016/j.jchromb.2024.124057

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