Development and application of a multi-sugar assay to assess intestinal permeability

Abstract

Aim: Bioanalytical assays to measure rhamnose, erythritol, lactulose and sucralose in human urine and plasma were developed to support an indomethacin challenge study for intestinal permeability assessment in healthy participants. Methods: The multi-sugar assays utilized 5-μl sample matrix and a simple chemical derivatization with acetic anhydride, followed by RPLC-MS/MS detection. Results: Rhamnose and erythritol quantification was established between 1.00–1,000 μg/ml in urine and 250–250,000 ng/ml in plasma. For lactulose and sucralose, dynamic ranges of 0.1–100 μg/ml (urine) and 25–25,000 ng/ml (plasma) were applied for biological measurements. Conclusion: This work helped overcome some of the common analytical challenges associated with the bioanalysis of mono- and disaccharides and achieved improved limits of quantification.

Article highlights

Background

• Permeability ratios of lactulose to rhamnose, as well as sucralose to erythritol are important biomarkers for assessing intestinal permeability modulation.

• Accurate quantification of lactulose, rhamnose, sucralose and erythritol in both urine and plasma was achieved using a simplified sample procedure and RPLC-MS/MS detection.

Methods

• The direct addition of acetic anhydride and pyridine onto 5 μl urine or plasma matrix enabled efficient derivatization of the sugar analytes and straightforward separation by RPLC and MS/MS detection.

• For urine analysis, a 20-min RPLC method was optimized for the four sugar derivatives to ensure selectivity and limit isomer/isobaric interferences, while a 16-min run time was applied for plasma.

• Technical considerations such as MS tune settings and mobile phase selection were important for driving favorable ammonium adduct formation with sugar molecules.

Results & conclusion

• Using the final RPLC-MS/MS methods, the disaccharides – lactulose and sucralose could be measured down to 0.1 μg/ml in urine and 25 ng/ml in plasma using only 5 μl matrix and limited sample handling/enrichment – a vast improvement for this type of method.

• Overall, these reproducible and precise (within 20%) analytical methods enabled robust assessment of intestinal barrier permeability and can help provide confidence in disaccharide to monosaccharide ratio end points with modest changes.

• The concepts of this RPLC-MS/MS work may be broadly applied to other bioanalysis requests involving saccharides.

Keywords: :

• ammonium adducts
• bioanalysis
• carbohydrates
• derivatization
• LC-MS/MS
• saccharides

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17576180.2024.2374168

Acknowledgments

The authors thank John T Mehl, Mike Wright, Benjamin Dodgers, Hans van Eijk, Enric Bertran, Claire Allan, Omer Omer, Tim W. Sikorski, and Naidong Weng for their support, input, and critical review of this work.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

All authors were employees of GSK during the course of this work. N Schneck, C Teague and HL Perez are currently shareholders. The authors have no other competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript apart from those disclosed.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval for all human investigations. The study protocol (GSK Study Register 218678), informed consent and other information that required pre-approval were reviewed and approved by the London-Riverside Research Ethics Committee (REC) in accordance with the ICH GCP and applicable country-specific requirements. In addition, written informed consent has been obtained from all the participants involved.