Organic acids in the samples were separated on a dC18 column (250 × 4.6 mm, 5 µm, Waters, Atlantis, MA, USA) as described previously [23 (link)] with some modifications. The mobile phase was 0.02 M KH2PO4 (pH 2.8) with a flow rate of 0.7 mL/min and the column temperature was 35 °C. The injection volume was 10 μL. Samples were detected with DAD (Shimadzu, Japan) at a wavelength of 210 nm in the same HPLC system. This method was termed as HPLC method 2.
Dc18 column
Manufactured by Waters Corporation
Sourced in United States
The DC18 column is a laboratory instrument used for high-performance liquid chromatography (HPLC) analysis. It is designed to separate and purify a wide range of chemical compounds, including small molecules, peptides, and proteins. The core function of the DC18 column is to provide efficient and high-resolution separation of analytes based on their hydrophobic interactions with the stationary phase.
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Lab products found in correlation
Tissuelyzer, by Qiagen (1 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions)
Empower 3, by Waters Corporation (1 mentions)
Uplc h class system, by Waters Corporation (1 mentions)
Ecd model 2465, by Waters Corporation (1 mentions)
Cmb 20a communications bus module, by Shimadzu (1 mentions)
Lc 20ad pump, by Shimadzu (1 mentions)
Sil 20a ht autosampler, by Shimadzu (1 mentions)
Spd m20a dad detector, by Shimadzu (1 mentions)
Dgu 20a5 degasser, by Shimadzu (1 mentions)
Microcon ym 30 separators, by Merck Group (1 mentions)
5 protocols using dc18 column
1
Organic Acid Separation by HPLC
2
Quantification of NAD+ in Tissue
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A solution of 0.5 N perchloric acid four times the weight of the DRG tissue was added to the frozen tissue. These samples were homogenized using a Qiagen tissue lyzer. A 10 μL aliquot of the internal standard solution (50 µg/mL in water) was added to the tissue homogenate. The sample was centrifuged at 1600× g for 10 min at room temperature. Next, 50 μL of supernatant was transferred to a clean 96-deep-well plate and diluted further with 150 μL of 5 mM ammonium formate and the NAD+ level was quantified following the protocol of Liang et al. [79 (link)]. Briefly, NAD+ analysis in tissue samples was carried out with QTRAP® 5500 mass spectrometer (AB Sciex, Framingham, MA, USA) equipped with a turbo-electrospray interface in positive ionization mode. The aqueous mobile phase was water with 0.1% formic acid and the organic mobile phase was acetonitrile with 0.1% formic acid. The gradient was 0% formic acid for the first 0.1 min, and then increased to 30% formic acid in 0.9 min, decreased to 0% formic acid within 0.1 min, and maintained at 0% formic acid for another 0.4 min. The flow rate was 0.8 mL/min and the cycle time (injection to injection including instrument delays) was approximately 1.8 min. A volume of 1–3 μL of the final extract was injected onto the analytical dC18 column (100 × 2.1 mm, 3 μm, Waters, Milford, MA, USA).
3
Quantifying Catecholamine Levels in Mice
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Mice were anesthetized by inhalation of isoflurane, and blood was collected from the heart in potassium‐EDTA tubes. Plasma catecholamines were measured by an ACQUITY UPLC H‐Class System fitted with an Atlantis dC18 column (100A, 3 µm, 3×100 mm) and connected to an electrochemical detector (ECD model 2465; Waters, Milford, MA). The mobile phase (isocratic: 0.3 mL/min) consisted of phosphate‐citrate buffer and acetonitrile at 95:5 (vol/vol). The data were analyzed using Empower 3 software (Waters). Catecholamine levels were normalized with the recovery of the internal standard 3,4‐dihydroxybenzylamine. Catecholamines were expressed as nanomole per liter (plasma) or nanogram per milligram protein (rat pheochromocytoma PC12 cells) or picograms per milligram protein (kidney).
4
Quantification of Caffeic Acid Phenethyl Ester in Propolis
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Identification and quantification of CAPE in propolis extract were carried out according to Escriche and Juan-Borrás (2018) (link). Individual stock solutions of CAPE standard were prepared in methanol at 10 mg/mL and stored at -20 °C. The working standard mixture solutions were made by diluting the appropriate amount of each stock standard solution to obtain 5 calibration levels (final concentrations of 5, 10, 20, 200, and 1,000 μg/mL).
CAPE was identified according to the method described by Escriche and Juan-Borrás (2018) (link). The HPLC analyses of propolis extracts were carried out on a Shimadzu HPLC system (LC20AD pump, SPD-M20A DAD detector, SIL-20A HT autosampler, CTO-2OAC column oven, DGU-20A5 degasser, and CMB-20A communications bus module; Shimadzu Corporation, Kyoto, Japan). A total of 1 μL of sample extract was automatically injected into a reverse phase Atlantis dC18 column (250 mm × 4.6 mm, 5 μm particle size; Waters, Dublin, Ireland). The mobile phases were deionised water with 5% formic acid (A) and acetonitrile (B) following a gradient profile of 0–3 min, 10–15% B; 3–18 min, 15–40% B; 18–24 min, 40% B; 24–27 min, 40–66% B; 27–33 min, 66–70% B; 33–40 min, 70–80% B; and then returned to the initial conditions. The column was operated at room temperature, and the flow rate was 0.8 mL/min. The chromatograms were recorded at 295 nm.
CAPE was identified according to the method described by Escriche and Juan-Borrás (2018) (link). The HPLC analyses of propolis extracts were carried out on a Shimadzu HPLC system (LC20AD pump, SPD-M20A DAD detector, SIL-20A HT autosampler, CTO-2OAC column oven, DGU-20A5 degasser, and CMB-20A communications bus module; Shimadzu Corporation, Kyoto, Japan). A total of 1 μL of sample extract was automatically injected into a reverse phase Atlantis dC18 column (250 mm × 4.6 mm, 5 μm particle size; Waters, Dublin, Ireland). The mobile phases were deionised water with 5% formic acid (A) and acetonitrile (B) following a gradient profile of 0–3 min, 10–15% B; 3–18 min, 15–40% B; 18–24 min, 40% B; 24–27 min, 40–66% B; 27–33 min, 66–70% B; 33–40 min, 70–80% B; and then returned to the initial conditions. The column was operated at room temperature, and the flow rate was 0.8 mL/min. The chromatograms were recorded at 295 nm.
5
Serum IS and PCS Quantification by LC-MS/MS
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Serum IS and PCS were measured with LC-MS/MS (4000 QTRAP, Framingham, MA, USA). In brief, serum samples were prepared and deproteinized by heat denaturation. The free concentrations of IS and PCS were measured in serum ultrafiltrates, obtained using Microcon YM-30 separators (Millipore, Billerica, MA, USA). HPLC was performed at room temperature using a dC18 column (3.0 × 50 mm, Atlantis, Waters, New Castle, DE, USA). The buffers used were:(A) 0.1% formic acid and (B)1 mM NH4OAc + 0.1% formic acid in 100% acetonitrile. The flow rate was 0.6 mL/min with a 3.5-min gradient cycling from 90% A/10% B to 10% A/90% B. Under these conditions, both PCS and IS were eluted at 2.73 and 2.48 min, respectively. Standard curves for PCS and IS were set at 1, 5, 10, 50, 250, 500, and 1000 μg/L. The serum samples for both PCS and IS were processed in the same manner, and they correlated with the serum samples with average r2 values of 0.996 ± 0.003. These samples were diluted if the IS or PCS concentrations exceeded the standard curve. Quantitative results were obtained and calculated in terms of their concentrations (mg/L). The sensitivity of this assay was 1 μg/L for PCS and 1 μg/L for IS.
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