Lactose, glucose, lactate, SCFA (acetate, propionate, and butyrate), and branched-chain fatty acids (BCFA) (isobutyrate and isovalerate) were measured in fecal samples of 40 infants at 6, 10, 12, 18, and 24 months by HPLC as described previously (15 (link)). Fecal samples were mixed with 1 ml 0.15 mM H2SO4, homogenized, and centrifuged at 4°C at 9,000 × g for 20 min. Clear supernatants were passed through a 0.45 μl filter (Infochroma AG, Zug, Switzerland) before injection. HPLC analysis (Thermo Fisher Scientific Inc. Accela, Wohlen, Switzerland) was performed using a SecurityGuard Cartridges Carbo-H (4 ×3.0 mm) (Phenomenex, Torrance, CA, USA) connected to a Rezex ROA-Organic Acid H+ (300 × 7.8 mm) column (Phenomenex, Torrance, CA, USA) at a flow rate of 0.4 mL min−1 at 40°C and 10 mM H2SO4 as eluent solution. Data were expressed as μmol g−1 feces.
Rezex roa organic acid h 300 7.8 mm column
Manufactured by Phenomenex
Sourced in United States
The Rezex ROA-Organic Acid H+ (300 × 7.8 mm) column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of organic acids. The column features a stationary phase that is suitable for the separation of various organic acid compounds.
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4 protocols using rezex roa organic acid h 300 7.8 mm column
1
Fecal Metabolite Profiling of Infant Gut
2
Quantifying Cell Dry Weight and Metabolites
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To quantify the cell dry weight (CDW) concentration, 4 × 1 ml of biosuspension was centrifuged with 14,000 rpm for 5 min at 4 °C, washed twice with demineralized water, transferred into pre‐weighed glass vials (1.5 ml, VWR International, Radnor, Pennsylvania) and eventually dried at 105 °C for 24 h. The weight was determined using a micro balance (XP26 Delta Range®, Mettler Toledo, Gießen, Germany). Enzyme kits (r‐biopharm AG, Darmstadt, Germany) were applied to quantify the organic acids, D‐glucose and D‐gluconic acid in the supernatant. 2‐ketogluconic acid was measured using isocratic HPLC equipped with the RI detector (1200Series, Agilent, Santa Clara, CA, USA) and a Rezex ROA‐Organic Acid H+ (300 × 7.8 mm) column (Phenomenex, Aschaffenburg, Germany) at 50 °C. 5 mM H3SO4 was used as a mobile phase at a rate of 0.4 ml min−1.
3
HPLC Analysis of Carbohydrate and Glycerol Profiles
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Carbohydrate profiles (maltose, maltotriose, glucose, dextrin) and glycerol content were analyzed by high-performance liquid chromatography (HPLC) [17 (link)]. Samples were degassed and centrifuged with a laboratory centrifuge MPW-351R (10 min, 5000 rpm). Then the samples were diluted with water and filtered through 0.22 µm filters. A 5-fold dilution for wort and a 2-fold dilution for beer samples was used. Separation was made using Rezex ROA—Organic Acid H+ (300 × 7.8 mm) column (Phenomenex, Torrance, USA). The HPLC method, using Prominence (Shimadzu, Kyoto, Japan) was used. Measurement parameters: injection volume—0.02 mL, flow rate—0.6 mL/min, temperature of separation—60 °C, mobile phase—0.005 mol/dm3 H2SO4. A refractometric method of detection was used. All measurements were performed in triplicate. Results were presented as g/L of wort or beer.
4
Quantification of Microbial Biomass and Metabolites
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4 × 1 mL of biosuspension was centrifuged with 20,000 g for 5 min at 4°C, washed twice with demineralized water, transferred into pre‐weighed glass vials (1.5 mL, VWR International, Radnor, Pennsylvania) and eventually dried at 105°C for 24 h. The weight of the remaining biomass was determined using a micro balance (XP26 Delta Range®, Mettler Toledo, Gießen, Germany). The organic acids, glucose, and gluconate in the supernatant were quantified using enzyme kits (r‐biopharm AG, Darmstadt, Germany). 2‐ketogluconic acid, 2‐ketoisovalerate, and isobutanol were measured using an isocratic HPLC equipped with the RI detector (1200Series, Agilent, Santa Clara, CA, USA) and a Rezex ROA‐Organic Acid H+ (300 × 7.8 mm) column (Phenomenex, Aschaffenburg, Germany) at 50°C. 0.4 mL min−1 of 5 mM H2SO4 was used as mobile phase for the separation of the HPLC analytes. The extracellular concentrations of l ‐valine, l ‐isoleucine, and l ‐leucine were determined according to the amino acid detection protocol described by Buchholz et al. [36 ].
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