AMK was percolated with 95% ethanol, and 1 gram of AMK extract was concentrated from 15.87 grams of crude drug. AMK was analyzed using high-performance liquid chromatography (HPLC) on a Waters 600E system (Waters, Milford, MA, USA) equipped with ultraviolet (UV) absorbance, refractive index detectors and a C18 column (Kromasil, Sweden). The mobile phase was methanol-water-acetic acid (70:30:1), and the flow rate was 1.0 mL/min. Working solutions for the gavage of AMK and OM (Huayi Pharmaceutical, Yiwu, Zhejiang, China) were prepared in 0.5% sodium carboxymethylcellulose (Solarbio life science, Beijing, China). Aristolochic acid sodium salt (AA-Na) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). CYP 1A1 antibody was purchased from BioWorld (Beijing, China). Sodium bicarbonate (SB) was purchased from Solarbio life science (Beijing, China).
Waters 600e system
Manufactured by Waters Corporation
Sourced in United States
The Waters 600E system is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. It features a modular design, allowing for the integration of various detectors and components to meet specific analytical requirements. The core function of the Waters 600E system is to separate, identify, and quantify components within complex sample mixtures.
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5 protocols using waters 600e system
1
HPLC Analysis of AMK Extract
2
Quantification of Amaranthus Extracts
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AMK extracts were obtained from root crude drug as previously reported (Wang et al. 2016 (link)). Briefly, AMK was percolated with 95% ethanol, 15.87 g of crude drug was concentrated to 1 g of AMK extracts. AMK extracts were analysed using high-performance liquid chromatography (HPLC) on a Waters 600E system (Waters, Milford, MA, USA) equipped with ultraviolet (UV) absorbance, refractive index detectors, and a C18 column (Kromasil, Sweden). 1.09% AA I was detected in the AMK extracts.
3
Antioxidant Component Separation by HPLC
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Separation of the antioxidant active components by HPLC (Waters 600E system, Waters Co., Milford, MA, USA) was performed as follows: The fractions demonstrating antioxidant activity according to secondary column chromatography were concentrated under reduced pressure, after which antioxidant components were isolated from the concentrated solution using HPLC and μ-Bondapak C18 column chromatography (ID 30 × 300 mm, 15–20 μm, Waters Co., Milford, MA, USA). The mobile phase was a gradient-elution system consisting of 0.1% formic acid (A) and methanol (B), with a linear gradient from 0 to 100% B, a flow rate of 15 mL min−1, and injection volume of 0.5 mL.
4
HPLC and GPC Analysis of Sugars
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Sugars were quantified by high-pressure liquid chromatography (HPLC) using a Waters 600E system controller (Waters Corp. Milford, MA, USA) equipped with a refraction index detector (Waters 410) using a Prevail Carbohydrate ES column (250 × 4.6 mm) at 30°C. The HPLC system was eluted with a mobile phase consisting of a 75:25 (v/v) mixture of acetonitrile and water at a flow rate of 1.0 mL min−1. The molecular weight of the Inulin was determined by gel permeation chromatography (GPC) using a serial set of Ultrahydrogel (UG 500 and linear) columns at 30°C. The columns were eluted with a mobile phase sodium nitrate 100 mM at a flow rate of 0.8 mL min−1. Oligosaccharides were separated and analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex), using a CarboPac PAD-200 column (250 × 2 mm). The following gradient was used: eluent A at 100% (0 min), 99% (0.5 min), 80% (25 min), 20% (85 min) and 100% (95 min). Eluent A was 150 mM sodium hydroxide and eluent B was 150 mM sodium hydroxide in 500 mM sodium acetate.
5
Carbohydrate Analysis by HPLC and HPAEC-PAD
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Carbohydrates were quantified by high-pressure liquid chromatography (HPLC) using a Waters 600E system controller (Waters Corp. Milford, MA, USA) equipped with a refractive index detector (Waters 410) using a Prevail Carbohydrate ES column (250 × 4.6 mm) at 30¡C. The HPLC system was run with a mobile phase consisting of a 75:25 (v/v) mixture of acetonitrile and water at a flow rate of 1.0 mL/min -1 . The molecular weight of inulin was determined by Size Exclusion Chromatography (SEC) using a serial set of Ultrahydrogel (UG 500 and linear) columns at 30¡C with a refractive index detector (Waters 410). The columns were run with a mobile phase sodium nitrate 100 mM at a flow rate of 0.8 mL/min. Oligosaccharides were separated and analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex), using a CarboPac PAD-200 column (250 × 2 mm, Dionex). The following gradient was used: eluent A at 100% (0 min), 99% (0.5 min), 80% (25 min), 20% (85 min) and 100% (95 min). Eluent A was 150 mM sodium hydroxide and eluent B was 150 mM sodium hydroxide in 500 mM sodium acetate.
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