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Application News No. L516 High Performance Liquid Chromatography Applying an Organic Acid Analysis System to Intestinal Flora Research LAAN-A-LC-E294 In recent years, it is becoming clear that intestinal flora in the gut contribute to the maintenance and preservation of the health of their hosts. Therefore, when examining the effects of intestinal flora on the host, the metabolites produced by intestinal flora can be considered to be related to these benefits. *1 While LC-MS may be used for analysis due to the large number of substances present in these metabolites, formic acid and acetic acid cannot be detected since LC-MS employs these organic acids in the mobile phase. In contrast, the post-column pH buffered electric conductivity detection method, which combines the ion exclusion mode and post-column reaction detection method performed using HPLC, employs neither formic acid nor acetic acid in the mobile phase, and this enables both highly sensitive and selective detection of organic acids including formic acid and acetic acid. This article introduces an example of extracting metabolites produced by intestinal flora from mouse feces and measuring the short-chain fatty acids contained in the metabolites using an organic acid analysis system. Y. Oka Analysis of Standard Sample Solution Table 1 lists the analysis conditions. Fig. 1 shows the result of injecting 10 μL of standard solution (20 mmol/L each). Table 1 Analysis Conditions Column : Shim-pack SCR-102H (300 mmL. × 8.0 mm I.D.) × 2 Guard column : Guard column SCR-102H (50 mmL. × 6.0 mm I.D.) Mobile phase : 5 mmol/L p-toluenesulfonic acid Flow rate of mobile phase : 0.8 mL/min pH buffering solution : 5 mmol/L p-toluenesulfonic acid 20 mmol/L Bis- Tris 0. 1 mmol/L EDTA Flow rate of pH buffering solution : 0.8 mL/min Column temp. : 45 °C Detection : Electro conductivity detector (CDD-10Avp) Injection vol. : 10 μL Chromatogram of Standard Solution Calibration Curve Fig. 2 shows the calibration curve of propionic acid created through analysis with the conditions in Table 1. The calibration curve was created over the ranges of 0.1 to 2.0 mmol/L and 1.0 to 20 mmol/L. Favorable linearity was obtained with a coefficient of determination (R 2 ) of 0.999 or higher for each component. Linearity of Calibration Curve
Application News No. L516 First Edition: Apr. 2017 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. Company names, product/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation or its affiliates, whether or not they are used with trademark symbol “TM” or “£”. Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. © Shimadzu Corporation, 2017 www.shimadzu.com/an/ Pretreatment Fig. 3 shows a flowchart of the pretreatment process used to extract metabolites from the mouse feces. *1 Feces were collected from a C57BL/6J mouse bred under standard conditions. 50 mg of fresh feces were added to 450 μL of phosphate buffered saline and agitated. The supernatant from the agitated mixture was then separated by centrifugation and ultrafiltrated to obtain the measurement sample. Note Analysis of Mouse Fecal Extract Fig. 4 shows the result of injecting 10 μL of mouse fecal extract. Seven components were detected that include acetic acid and formic acid, which are difficult to detect using LC-MS. Table 2 lists the quantitative result for each component. Table 2 Quantitative Results No. Compounds : Quantitative Value (mmol/L) 1 Phosphoric acid : 7.88 2 Succinic acid : 0.07 3 Lactic acid : 0.26 4 Formic acid : 0.13 5 Acetic acid : 2.69 6 Propionic acid : 0.59 8 Butyric acid : 0.61 Pretreatment Flowchart Chromatogram of Mouse Fecal Extract References *1 M. Matsumoto, R. Kibe, T. Ooga, Y. Aiba, S. Kurinara, E. Sawaki, Y. Koga, Y. Benno: Scientific Reports 2, 223 (2012). *2 Seiko Hoshi, "Methods for Measuring Organic Acids in the Contents of the Large Intestine" in Nutritional Physiology and Enteric Bacteria of the Digestive Tract, 11-25 (2011). Note: If feces cannot be agitated uniformly, pulverize them with a mortar and pestle for homogenization and then perform agitation with a vortex mixer. * The sample used in this measurement was provided by Mr. Matsumoto from Kyodo Milk Industry Co., Ltd. Filtration Take supernatant Centrifugate (4 °C × 10000 G) Take supernatant On ice (2 min) Intense mixing with vortex mixer (1 min) On ice (5 min) Intense mixing with vortex mixer (1 min) Add 450 μL phosphate buffered saline to 50 mg mice fecal
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