Analytical reversed-phase HPLC or UHPLC were performed on C18 columns (4.6 × 50 mm, 3 μm; Phenomenex) in LC2010A or LC2040C3DPlus instruments (Shimadzu Corp., Kyoto, Japan). Linear gradients (15–50%) of solvent B (0.036% TFA in MeCN) into A (0.045% TFA in H2O) were used for elution at 1 mL/min (HPLC) or 0.6 mL/min (UHPLC) flow rate, with UV detection at 220 nm. LC-mass spectrometry was performed in an LC-MS 2010EV instrument (Shimadzu, Kyoto, Japan) fitted with an XBridge column (4.6 × 150 mm, 3.5 μm; Waters, Cerdanyola del Vallès, Spain), eluting with a 15–50% linear gradient of B (HCOOH/MeCN 0.08% v/v) into A (HCOOH/H2O 0.1%, v/v) over 15 min at 1 mL/min with UV detection at 220 nm.
Lc 2040c 3d plus
Manufactured by Shimadzu
Sourced in Japan
The Shimadzu LC-2040C 3D Plus is a high-performance liquid chromatography (HPLC) system. It is designed for a wide range of analytical applications, providing reliable and efficient separation and detection of various compounds. The system features a three-dimensional detector that enables simultaneous measurement of multiple wavelengths, ensuring comprehensive data acquisition.
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4 protocols using lc 2040c 3d plus
1
Analytical HPLC/UHPLC Protocol for Compounds
2
HPLC Analysis of Tyrosine Kinase Inhibitors
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An integrated HPLC system with PDA detector LC‐2040C 3D Plus (Shimadzu Corporation) was used with a Cadenza CX‐C18 analytical column (3.0 μm, 250 mm × 3 mm, IMTACT, Japan). The column temperature was 40°C. The injection volume of the prepared sample was 20 μl. The autosampler temperature was 4°C. Mobile phase A was 0.5% KH2PO4 (pH 3.5) – methanol (80:20). Mobile phase B was acetonitrile–methanol (80:20). The flow rate was 0.5 ml/min. The binary gradient program was 30%–70% B at 0–3 min, held 70% B at 3–4.5 min, 70%–30% B at 4.5–4.51 min, held 30% B at 4.5–10 min. The ultraviolet detection wavelengths for PDA were 233 nm for dasatinib, 261 nm for nilotinib, 267 nm for bosutinib, and 285 nm for ponatinib. The wavelengths were individually set by considering the maximum absorbance wavelengths and the suppression of the influence of interfering components.
3
Kinetic Analysis of Click Reaction
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To a 1.5 mL tube, 420 μL of Dulbecco's phosphate buffer saline (–) (DPBS(–)), 34.5 μL of dimethyl sulfoxide (DMSO), 10 μL of 100 mM 2-(4-((bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid (BTTAA) in DMSO, 5 μL of 100 mM CuSO4 in H2O, 0.5 μL of 10 mM Cy5-PEG4-azide in DMSO, and 5 μL of 10 mM alkyne (Pimo-yne or propargyl alcohol) in DMSO were added in this order with vortexing after each addition. Then, 25 μL of 100 mM sodium ascorbate (NaAsc) in DPBS(–) was added followed by vortexing to start the reaction. The final concentrations of the reagents were 10 μM Cy5-PEG4-azide, 100 μM alkyne, 1 mM CuSO4, 2 mM BTTAA, 5 mM NaAsc, 10% v/v DMSO, in a total volume of 500 μL. At each time point (2, 5, 10, 30, and 60 min after starting the reaction), 50 μL of 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8.0) was added followed by vortexing to quench the reaction. For t = 0 min, EDTA was added before the addition of the NaAsc solution. The solution was analyzed by UHPLC on LC-2040C 3D Plus (Shimadzu) with a Shim-pack Velox C18 column (#227-32007-02, Shimadzu).
4
Potency Analysis of Cannabinoids by HPLC
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Sample extracts were analyzed using a Shimadzu model LC-2040C 3D Plus (Shimadzu Corporation, Kyoto, Japan) reverse phase high-performance liquid chromatography (HPLC) equipped with diode array detector and a NexLeaf CBX for Potency, superficially porous particle (SPP) 2.7 μm C18 4.6x 150 mm column (Shimadzu Corporation, Kyoto, Japan). The mobile phase was 0.085% phosphoric acid in acetonitrile (E1) and 0.085% phosphoric acid in distilled water (E2). Elution was performed by the following gradient: t0 min = 70% (vv-1) E2; t3 min = 70% (vv-1) E2; t8 min = 85% (vv-1) E2; t10 min = 95% (vv- 1) E2; t11.01 min =70% (vv-1) E2. The flow rate was 1.6 mL per min. A reference standard containing the cannabinoid compounds of interest (Cayman Chemical Inc., Phytocannabinoid Mixture 11 (CRM)) was used to prepare the external calibration standards. All compounds were analyzed at 220 nm. CBDA had a retention time of 3.262 min; CBD had a retention time of 3.901 min; THCA had a retention time of 7.604 min; Δ9-THC had a retention time of 6.427 min; CBGA had a retention time of 3.519 min; and CBG had a retention time of 3.726 min. Minor cannabinoids were excluded from the analysis due to low concentrations. CBD, THC, and CBG equivalents (CBDeq, THCeq, and CBGeq) were calculated as described by Westmoreland et al. (2021) (link).
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