To produce 20,24(OH)2D3 for NMR analysis, a large scale incubation (30 mL) of rat CYP24A1 with 20(OH)D3 was carried out, as described previously [33 (link)]. The 20,24(OH)2D3 and other products were purified by HPLC, as outlined before [33 (link)], using a Grace Alltima column (as above) and a 45% to 58% (v/v) acetonitrile in water gradient over 25 min followed by a 10 min gradient from 58% (v/v) acetonitrile in water to 100% acetonitrile, ending with 20 min at 100% acetonitrile, all at a flow rate of 0.5 mL/min (HPLC Program C). A further HPLC purification step was carried out using the same column employing a 45 min gradient from 64% (v/v) methanol in water to 100% methanol, followed with 15 min at 100% methanol, all at a flow rate of 0.5 mL/min. Collected products were pooled and dried under nitrogen, dissolved in ethanol and the amount of purified secosteroid was measured spectrophotometrically using an extinction coefficient of 18 000 M−1cm−1 [39 (link)].
Alltima column
Manufactured by Grace Bio-Labs
Sourced in United States
The Alltima column is a high-performance liquid chromatography (HPLC) column designed for the separation and purification of a wide range of chemical compounds. It features a stationary phase that provides efficient separation and excellent peak resolution, making it a versatile tool for various analytical and preparative applications.
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2 protocols using alltima column
1
Purification of 20,24(OH)2D3 for NMR Analysis
2
Metabolite Identification of Saussurea involucrata
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The qualitative identification of possible metabolites (main components of Saussurea involucrata) was performed using LC/MSn-IT-TOF equipped with an ESI ion source. An Alltima column (250 × 4.6 mm, 5 μm, GRACE, Chicago, IL, USA) was used for component separation. The flow rate was 0.4 mL/min, and the column temperature was 40 °C. The injection volume was 20 μL. Mobile phase: 0.2% acetic acid/water (mobile phase A) and methanol (mobile phase B). Gradient elution (B%): 0.01 min, 20% → 10.00 min, 30% → 15.00 min, 60% → 25.00 min, 80% → 30.00 min, 80% → 40.00 min, stop. The autosampler temperature was set to 4 °C. The detection wavelength for PDA was 254 nm. The mass spectrometry conditions were set as follows: CDL temperature, 160 °C; heating block temperature, 200 °C; nebulizing gas flow rate, 1.5 L/min; detector voltage, 1.76 kV; and collision energy, 70%. Automatic detection mode was used for fragmentation, with a primary m/z ratio ranging from 50 to 800 and a secondary m/z ratio ranging from 50 to 800.
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