The total RNA was applied to a reversed-phase LC column (PLRP-S 4000 Å, 4.6 × 150 mm, 10 μm, Agilent Technologies), and the rRNAs were eluted with a 120-min linear gradient of 10.8%–13.2% (v/v) acetonitrile in 100 mM TEAA (pH 7.0) and 0.1 mM diammonium phosphate at a flow rate of 200 μL/min at 60°C while measuring the eluate at A260 (Yamauchi et al., 2013 (link)). The 5.8S, 18S, and 25S rRNA fractions of the eluate were used directly for the liquid chromatography/tandem mass spectrometry (LC-MS) analysis. The 5S rRNA fraction was further purified by reversed-phase LC using a column (PLRP-S 300 Å, 2.1 × 200 mm, 3 μm, Agilent Technologies) with a 120-min linear gradient of 11.8%–14.2% (v/v) acetonitrile in 100 mM TEAA (pH 7.0) and 0.1 mM diammonium phosphate at a flow rate of 100 μL/min at 60°C.
Plrp s 300
Manufactured by Agilent Technologies
PLRP-S 300 Å is a reversed-phase HPLC packing material designed for the separation of small to medium-sized molecules. It features a polystyrene-divinylbenzene polymer matrix with a 300 Å pore size. This product is suitable for a variety of analytical and preparative applications.
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2 protocols using plrp s 300
1
Purification of Ribosomal RNAs for LC-MS
2
Quantitative Analysis of A-967079
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All reagents and solvents were of at least HPLC grade, unless otherwise specified. Ammonium formate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetone, ethanol, and methanol (LC–MS grade) were purchased from Fisher Scientific (HanoverPark, IL, USA). A-967079 (>98% purity), supplied by Dr. Carl W. White (Pediatrics-Pulmonary Medicine, University of Colorado-Denver, Denver, CO, USA), was purchased from Med Chem 101 (Plymouth Meeting, PA, USA; 98% purity; Lot X101075). A polymeric reversed phase column, PLRP-S 300 Å (50 mm × 2.1 mm, 3.0 μm, part #:PL1912-1301) was purchased from Agilent (Santa Clara, CA 95051). Water was purified to 18.2 MΩ·cm using a Lab Pro polishing unit from Labconco (Kansas City, KS, USA). A-967079 was dissolved in ethanol:water (1:1,v/v) to make a stock solution of 10 mM concentration, which was further diluted to 5 mM in mobile phase A (pH∼4), and stored at 4 °C. The stock solution of A-967079 (5 mM, in mobile phase A) was further diluted to 0.025–500 μM with either mobile phase A or plasma to produce working standard solutions.
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