Overall experimental procedure followed the previous literature in which observed that culture conditions under serum or not, and passage critically affect RA metabolism and induction of 4-hydroxylase activity [19 (link)]. Briefly, HaCaT was seeded and cultured in serum-containing medium for 48 h. Cells at confluency were treated with RA 1 µM (Sigma-Aldrich, St. Louis, MI, USA) and methanol (Sigma-Aldrich, St. Louis, MI, USA) and incubated for additional 48 h. Cultured media and cell lysate were freeze-dried and resuspended in methanol. This methanol-based solution was analyzed by HPLC (Shimadzu, SCL-40, LC-40DXR, CTO-40C etc., Kyoto, Japan). Detailed procedure followed the previous research [20 (link)]. 60 mM ammonium acetate (Sigma-Aldrich, St. Louis, MI, USA) adjusted to pH 5.75 with acetic acid (Sigma-Aldrich, St. Louis, MI, USA) and methanol (Sigma-Aldrich, St. Louis, MI, USA) were used as HPLC buffers. The flow rate was 1 mL/min. The gradient conditions were (1) 15% of methanol at the time of injection, (2) linear increase to 99% of methanol at the 30 min, and (3) maintenance of 99% of methanol for 15 min. UV detection was carried out at 340 nm.
Cto 40c
Manufactured by Shimadzu
Sourced in Japan
The CTO-40C is a temperature-controlled column oven designed for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. The core function of this product is to precisely control the temperature of the analytical column, ensuring stable and reproducible chromatographic separations.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Scl 40, by Shimadzu (1 mentions)
Methanol, by Shimadzu (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Lc 40d xr, by Shimadzu (1 mentions)
Fcv 20ah2, by Shimadzu (1 mentions)
Sil 40c xr autosampler, by Shimadzu (1 mentions)
Lcms 8045, by Shimadzu (1 mentions)
Securityguard c18, by Phenomenex (1 mentions)
Nexera lc 40, by Shimadzu (1 mentions)
Sil 40c xs autosampler, by Shimadzu (1 mentions)
Carnosic acid, by Selleck Chemicals (1 mentions)
Lcms 8050, by Shimadzu (1 mentions)
Dgu 20a5r, by Shimadzu (1 mentions)
Ascentis express rp c18 column, by Merck Group (1 mentions)
Liquid chromatography system, by Shimadzu (1 mentions)
Esi source, by Shimadzu (1 mentions)
Spd m40, by Shimadzu (1 mentions)
Sil 40c, by Shimadzu (1 mentions)
5 protocols using cto 40c
1
RA Metabolism and 4-Hydroxylase Activity Assay
2
Quantification of Pyrrolizidine Alkaloids Using LC-MS/MS
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The reduction of the PA-N-oxides and the clearance of their parent PAs were quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The analysis was performed using a UHPLC system (Shimadzu SIL-40C XR autosampler) coupled with a column oven (Shimadzu CTO-40C) and a valve unit (Shimadzu FCV-20AH2) combined with an MS system (Shimadzu LCMS-8045, Kyoto, Japan). A 1 µL injection volume of sample was examined with a reverse phase C18 column (Phenomenex 1.7 μm 2.1 × 50 mm) at a flow rate of 0.3 mL/min. Milli-Q water and acetonitrile were used as mobile phases, each containing 0.1 % v/v formic acid. The measurement started with 0 % to 100 % ACN for 6 min. 100 % ACN was kept for 1 min after which it changed to the starting condition of 0 % ACN within 1 min. Prior to the next injection, this percentage was kept for 4 min. The PAs and their PA-N-oxides were analyzed with a positive ionizing mode and quantified in the multiple reaction monitoring (MRM) mode; detailed information on [M + H]+ of precursors to products m/z, collision energy (CE) and retention times (RT) can be found in Table 1S of the Supplementary Material . Calibration curves of these compounds were defined using commercially available reference compounds in a range of 0.001–1 μM, and employed to quantify the concentrations of PAs in the samples using the total ion chromatogram (TIC).
3
HPLC Analysis of Carotenoids in Lymphocytes
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HPLC analysis of carotenoids was performed on a Nexera LC-40 (Shimadzu, Kyoto, Japan) UHPLC system equipped with a diode-array detector SPD-M40, FCV-0607H high-pressure flow-line selection valve, CTO-40C column oven, SIL-40C XR autosampler, and LC-30D XR pump. The isocratic separation was carried out on a reversed-phase Kinetex (Phenomenex Inc, Torrance, USA) core–shell column, 150 mm × 3 mm i.d., particle size 2.6 µm (Phenomenex Inc, Torrance, USA), equipped with a Security Guard C18 3 × 4 mm i.d. (Phenomenex Inc, Torrance, USA). The autosampler was set to 4°C, and the column was thermostated at 30°C. The mobile phase consisted of methanol and methyl tert-butyl ether (95:5, v:v). Injection volume was 5 µl, the flow rate 0.8 mL/min, and runtime 15 min. The peak spectra were scanned from 300 to 800 nm.
The isolation of carotenoids from B and T lymphocytes was carried out as follows. The cells were pelleted 1,000 × g, 4°C, 2 min. The cell pellet was homogenized and carotenoids extracted using 2 mL of ice-cold acetone. The extract was filtered using a 0.22 µm PTFE syringe filter. The acetone extract was dried under a steady stream of argon. Dried samples were redissolved in 96% ethanol and immediately injected into the HPLC system.
The isolation of carotenoids from B and T lymphocytes was carried out as follows. The cells were pelleted 1,000 × g, 4°C, 2 min. The cell pellet was homogenized and carotenoids extracted using 2 mL of ice-cold acetone. The extract was filtered using a 0.22 µm PTFE syringe filter. The acetone extract was dried under a steady stream of argon. Dried samples were redissolved in 96% ethanol and immediately injected into the HPLC system.
4
UPLC Quantification of Carnosol and Carnosic Acid
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Carnosol and carnosic acid were quantitated with an UPLC system
(Shimadzu Corporation, Kyoto, Japan) equipped with a C18 phase column
(Acquity UPLC BEH C18, 15 cm, 1.7 μm, Waters, Milford, MA, USA).
A sample volume of 4 μL was injected into the system, and chromatographic
separation was performed with a 0.3 mL/min flow rate over 15 min.
Eluents were (A) 1% acetic acid in Milli-Q water and (B) 1% acetic
acid in acetonitrile. Eluent gradient conditions were 95% A and 5%
B at 0 min, followed by a gradual decrease of A to 25% during 4 min
and then to 20% at 9 min and to 5% at 11 min; this last ratio was
maintained until 13 min and then set to 95% A to the end of the run
(15 min). The temperature of the samples (SIL-40C XS Autosampler,
Shimadzu) and column oven (CTO-40C, Shimadzu) were set at 5 and 50
°C, respectively. Detection was performed using a PDA UV detector
at a scanning 190–500 nm wavelength. carnosic acid (17108689,
Selleck Chemicals, Houston, TX, USA) and carnosol (17205690, Selleck
Chemicals, Houston, TX, USA) analytical standards (16–500 ppm)
were used for method development, peak detection, and quantification.
The UPLC chromatogram of carnosol and carnosic acid and standard curves
are shown inFigure S2 .
(Shimadzu Corporation, Kyoto, Japan) equipped with a C18 phase column
(Acquity UPLC BEH C18, 15 cm, 1.7 μm, Waters, Milford, MA, USA).
A sample volume of 4 μL was injected into the system, and chromatographic
separation was performed with a 0.3 mL/min flow rate over 15 min.
Eluents were (A) 1% acetic acid in Milli-Q water and (B) 1% acetic
acid in acetonitrile. Eluent gradient conditions were 95% A and 5%
B at 0 min, followed by a gradual decrease of A to 25% during 4 min
and then to 20% at 9 min and to 5% at 11 min; this last ratio was
maintained until 13 min and then set to 95% A to the end of the run
(15 min). The temperature of the samples (SIL-40C XS Autosampler,
Shimadzu) and column oven (CTO-40C, Shimadzu) were set at 5 and 50
°C, respectively. Detection was performed using a PDA UV detector
at a scanning 190–500 nm wavelength. carnosic acid (17108689,
Selleck Chemicals, Houston, TX, USA) and carnosol (17205690, Selleck
Chemicals, Houston, TX, USA) analytical standards (16–500 ppm)
were used for method development, peak detection, and quantification.
The UPLC chromatogram of carnosol and carnosic acid and standard curves
are shown in
5
Comprehensive LC-MS Analysis of Plant Extracts
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The hydroalcoholic extracts (Em-L, Em-A, Es-L, Es-A, and Cv-L) were analyzed through the LC–MS technique using a Shimadzu liquid chromatography system (Kyoto, Japan), composed of a CBM-20A controller, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20A5R degasser, a CTO-40C column oven, a SIL-40C autosampler, an SPD-M40 photo diode array detector, and an LCMS-8050 mass spectrometer, through an ESI source (Shimadzu, Kyoto, Japan).
Separation analyses were performed on a 150 × 4.6 mm; 2.7 µm Ascentis Express RP C18 column (Merck Life Science, Merck KGaA, Darmstadt, Germany). The mobile phase was composed of two solvents, water (solvent A) and acetonitrile (solvent B), both acidified with formic acid at 0.1% v/v. The flow rate was set at 1 mL/min and a simplified linear gradient of elution program was followed: 0–5 min, 0–30% B, 5–30 min, 30–100% B, 35 min, 100% B. PDA range: 200–400; λ = 280 nm (sampling frequency: 40.0 Hz, time constant: 0.08 s).
The applied mass spectrometry conditions were as follows: scan range, m/z 100–1200; scan speed, 2500 amu/s; event time, 0.3 s; nebulizing gas (N2) flow rate, 1.5 L/min; drying gas (N2) flow rate, 15 L/min; interface temperature, 350 °C; heat block temperature, 300 °C; DL (desolvation line) temperature, 300 °C; DL voltage, 1 V; interface voltage, −4.5 kV.
Separation analyses were performed on a 150 × 4.6 mm; 2.7 µm Ascentis Express RP C18 column (Merck Life Science, Merck KGaA, Darmstadt, Germany). The mobile phase was composed of two solvents, water (solvent A) and acetonitrile (solvent B), both acidified with formic acid at 0.1% v/v. The flow rate was set at 1 mL/min and a simplified linear gradient of elution program was followed: 0–5 min, 0–30% B, 5–30 min, 30–100% B, 35 min, 100% B. PDA range: 200–400; λ = 280 nm (sampling frequency: 40.0 Hz, time constant: 0.08 s).
The applied mass spectrometry conditions were as follows: scan range, m/z 100–1200; scan speed, 2500 amu/s; event time, 0.3 s; nebulizing gas (N2) flow rate, 1.5 L/min; drying gas (N2) flow rate, 15 L/min; interface temperature, 350 °C; heat block temperature, 300 °C; DL (desolvation line) temperature, 300 °C; DL voltage, 1 V; interface voltage, −4.5 kV.
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