A Vancomycin HPLC Assay for Use in Gut Microbiome Research

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.