SCFAs were measured in 100 mg of dehydrated fecal samples using PerkinElmer-Flexar (Waltham, MA, USA) high performance liquid chromatography (HPLC) equipment as previously reported [5 (link)]. Mobile phase consisted of two solutions; 80% of (A) 20mM NaH2PO4 (Sigma-Aldrich Cat. #S8282, St. Louis, MO, USA) pH 2.2 adjusted with phosphoric acid (J.T. Baker, Estate of Mexico, Xalostoc, Mexico, Cat. #0260-05), and 20% of (B) Acetonitrile (J.T. Baker, Cat. #9012-03, Estate of Mexico, Xalostoc, Mexico), using a 1.0 mL/min flow rate in a 15 cm C-18 column [34 (link)]. All chromatographic data were processed using Chromera (v4.1.2.6410, PerkinElmer, Waltham, MA, USA)—HPLC Flexar Software (PerkinElmer, Waltham, MA, USA).
Chromera
Manufactured by PerkinElmer
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Chromera is a chromatography data system software developed by PerkinElmer. It provides a comprehensive platform for controlling and managing chromatographic instruments, acquiring and processing data, and reporting results.
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4 protocols using chromera
1
HPLC Analysis of Fecal SCFAs
2
HPLC Analysis of 5-HT in Urine
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HPLC (electrochemical method) analysis was carried out as previously described [40 (link)]. Briefly, the analysis of 5-HT content in the samples was carried out using the 3030 Reagent kit for HPLC analysis of this compound in the urine. The calibration curve was generated with concentrations between 1–1000 nM of 5-HT (y = 493146x; R² = 0.9963). The decade electrochemical detector contained a glassy carbon electrode programmed to a potential of 50 mV. Empower Pro software 3 (Waters Corporation, Milford, MA, USA) was used for controlling the produced current For uHPLC procedure, samples (20 µL, 4 °C) were subjected to the uHPLC, and the separation was carried out at a flux of 2 mL/min. Optical density of all the tested compounds was recorded at 280 nm. Quantification was performed based on standard curves for L-Trp (y = 750,542x; R² = 0.9971), 5-HTP (y = 804,860x; R² = 0.9986), 5-HT (y = 782,455x; R² = 0.9977), and 5-HIAA (y = 595,569x; R² = 0.9919). Results were analyzed on using Chromera® software, version 3.2.0, Perkin Elmer (Waltham, MA, USA).
3
HPLC Analysis of Carbohydrates and Lactic Acid
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HPLC equipment for the analysis of carbohydrates and lactic acid consisted of a quaternary pump, an on-line degasser, UV-visible detector (Series 200), a refractive index detector and a column oven (Series Flexar) (Perkin Elmer, Norwalk, USA). Data were collected and processed on a computer with the software Chromera® (Perkin Elmer, Norwalk, USA).
The analysis of GOS, lactose, glucose and galactose in the incubation experiences of lactose solution with the β-galactosidase enzyme were made on an Aminex HPX-87N column (300 x 7.8 mm) equipped with a cation Na + microguard cartridge (Bio-Rad Laboratories, Norwalk, USA). Chromatographic separation was performed using HPLC water as mobile phase at a flow rate of 0.3 mL / min, maintaining the column at 85 ºC.
Aliquots of reaction mixtures were appropriately diluted with distilled water, filtered through 0.45 µm membranes (Millex, Millipore, São Paulo, Brazil) and injected into the chromatograph, using a loop of 20 µL.
The analysis of GOS, lactose, glucose and galactose in the incubation experiences of lactose solution with the β-galactosidase enzyme were made on an Aminex HPX-87N column (300 x 7.8 mm) equipped with a cation Na + microguard cartridge (Bio-Rad Laboratories, Norwalk, USA). Chromatographic separation was performed using HPLC water as mobile phase at a flow rate of 0.3 mL / min, maintaining the column at 85 ºC.
Aliquots of reaction mixtures were appropriately diluted with distilled water, filtered through 0.45 µm membranes (Millex, Millipore, São Paulo, Brazil) and injected into the chromatograph, using a loop of 20 µL.
4
HPLC-IR Quantification of Sugars
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The identification and quantification of sugars: lactose, glucose, galactose and GOS, was performed by HPLC with refractive index detection (HPLC-IR). A Perkin Elmer (Norwalk, USA) chromatograph, equipped with a Aminex HPX-87H column (300 × 7•8 mm) and a cation H + microguard cartridge (Bio-Rad Laboratories, Hercules, USA), a quaternary pump, an on-line degasser, a refractive index detector and a column oven (Series Flexar), and Chromera® software (Perkin Elmer, Norwalk, USA) were used for the analyses. Sugars were eluted with 10 mM H 2 SO 4 , at a flow rate of 0•6 ml/min, at 65 °C. Before injection, the samples 2•5 g were diluted with 10 mM H 2 SO 4 to 25 ml, homogenised and centrifuged at 15 000 g/20 min/4 °C. The supernatant was filtered through 0•45 µm membrane (Millex, Millipore, Brazil) and injected into the equipment, using a loop of 60 µl (Vénica et al. 2015) . Quantification was performed by external calibration using suitable standards (Sigma-Aldrich, Saint Louise, USA).
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