A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test
Background: Heterogeneous laborious analytical methodologies for the determination of urinary lactulose and mannitol limit their utility in intestinal permeability testing. Methods: We developed an assay using a Shimadzu HPLC system, an Aminex HPX87C column, and refractive index detection. The test...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-04-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/27/9/2677 |
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| author | Ivana R. Sequeira Marlena C. Kruger Roger D. Hurst Roger G. Lentle |
| author_facet | Ivana R. Sequeira Marlena C. Kruger Roger D. Hurst Roger G. Lentle |
| author_sort | Ivana R. Sequeira |
| collection | DOAJ |
| description | Background: Heterogeneous laborious analytical methodologies for the determination of urinary lactulose and mannitol limit their utility in intestinal permeability testing. Methods: We developed an assay using a Shimadzu HPLC system, an Aminex HPX87C column, and refractive index detection. The test was calibrated using a series of dilutions from standard stock solutions of lactulose and mannitol ‘spiked’ into urine samples. The utility to quantify urinary excretion during the dual sugar absorption test over 6 h was also determined. Results: Lactulose and mannitol were eluted isocratically at 5.7 and 10.1 min, respectively, with water as a mobile phase at a flow rate of 0.3 mL min<sup>−1</sup>, 858 psi, 60 °C. The calibration curves for both sugars were linear up to 500 µg mL<sup>−1</sup> with a limit of detection in standard solutions at 4 µg mL<sup>−1</sup> and in ‘spiked’ urine samples at 15 µg mL<sup>−1</sup>. The intra-assay and inter-assay CVs were between 2.0–5.1% and 2.0–5.1% for lactulose and 2.5–4.4% and 2.8–3.9% for mannitol. The urinary profiles of the 6 h absorption of lactulose and mannitol showed similar peak-retention times to standard solutions and were well-resolved at 5.9 and 10.4 min, respectively. Conclusions: The assay was easy to automate, using commonly available equipment and convenient requiring no prior laborious sample derivatization. The simplicity, reproducibility, and robustness of this assay facilitates its use in routine clinical settings for the quantification of intestinal permeability. |
| first_indexed | 2024-03-10T03:54:32Z |
| format | Article |
| id | doaj.art-4a6c02fe7af24a9ea866a2dd035bd753 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T03:54:32Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-4a6c02fe7af24a9ea866a2dd035bd7532023-11-23T08:47:56ZengMDPI AGMolecules1420-30492022-04-01279267710.3390/molecules27092677A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption TestIvana R. Sequeira0Marlena C. Kruger1Roger D. Hurst2Roger G. Lentle3Human Nutrition Unit, School of Biological Sciences, University of Auckland, Auckland 1024, New ZealandSchool of Health Sciences, College of Health, Massey University, Palmerston North 4472, New ZealandFood Innovation Portfolio, The New Zealand Institute for Plant & Food Research Ltd., Palmerston North 4474, New ZealandSchool of Health Sciences, College of Health, Massey University, Palmerston North 4472, New ZealandBackground: Heterogeneous laborious analytical methodologies for the determination of urinary lactulose and mannitol limit their utility in intestinal permeability testing. Methods: We developed an assay using a Shimadzu HPLC system, an Aminex HPX87C column, and refractive index detection. The test was calibrated using a series of dilutions from standard stock solutions of lactulose and mannitol ‘spiked’ into urine samples. The utility to quantify urinary excretion during the dual sugar absorption test over 6 h was also determined. Results: Lactulose and mannitol were eluted isocratically at 5.7 and 10.1 min, respectively, with water as a mobile phase at a flow rate of 0.3 mL min<sup>−1</sup>, 858 psi, 60 °C. The calibration curves for both sugars were linear up to 500 µg mL<sup>−1</sup> with a limit of detection in standard solutions at 4 µg mL<sup>−1</sup> and in ‘spiked’ urine samples at 15 µg mL<sup>−1</sup>. The intra-assay and inter-assay CVs were between 2.0–5.1% and 2.0–5.1% for lactulose and 2.5–4.4% and 2.8–3.9% for mannitol. The urinary profiles of the 6 h absorption of lactulose and mannitol showed similar peak-retention times to standard solutions and were well-resolved at 5.9 and 10.4 min, respectively. Conclusions: The assay was easy to automate, using commonly available equipment and convenient requiring no prior laborious sample derivatization. The simplicity, reproducibility, and robustness of this assay facilitates its use in routine clinical settings for the quantification of intestinal permeability.https://www.mdpi.com/1420-3049/27/9/2677high-performance liquid chromatographyintestinal permeabilitylactulosemannitoldual sugar absorption test |
| spellingShingle | Ivana R. Sequeira Marlena C. Kruger Roger D. Hurst Roger G. Lentle A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test Molecules high-performance liquid chromatography intestinal permeability lactulose mannitol dual sugar absorption test |
| title | A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test |
| title_full | A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test |
| title_fullStr | A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test |
| title_full_unstemmed | A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test |
| title_short | A Simple, Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test |
| title_sort | simple robust and convenient hplc assay for urinary lactulose and mannitol in the dual sugar absorption test |
| topic | high-performance liquid chromatography intestinal permeability lactulose mannitol dual sugar absorption test |
| url | https://www.mdpi.com/1420-3049/27/9/2677 |
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