All archived samples of the discovery cohort were sent to the Department of Laboratory Medicine at the Samsung Medical Center, and vitamin D levels were determined from a single baseline serum sample. Serum levels of 25(OH)D2 and D3 were determined by high‐performance liquid chromatography‐tandem mass spectrometry, with an Agilent 6460 Triple Quad mass spectrometer equipped with a 1200 liquid chromatography system (Agilent Technologies, Waldbronn, Germany).12 The intra‐assay and inter‐assay imprecisions were <10% of coefficients of variation, and the combination of 25(OH)D2 and D3 < 10 ng/mL was defined as vitamin D deficiency. Although various cutoff values for vitamin D deficiency have been used in previous studies, such as 20 ng/mL of 25(OH)D for follicular lymphoma and 8 ng/mL of 25(OH)D3 for diffuse large B‐cell lymphoma,4 , 5 we used a cutoff of 10 ng/mL given the high prevalence of vitamin deficiency in the Asian population. In the validation cohort, the serum level of 25(OH)D was measured at the Shanghai Rui Jin Hospital laboratory, and the same cutoff value (10 ng/mL) was used.
1200 liquid chromatography system
Manufactured by Agilent Technologies
Sourced in Germany, United States
The Agilent 1200 Liquid Chromatography System is a high-performance liquid chromatography (HPLC) instrument designed for the separation, identification, and quantification of chemical compounds in various samples. It features a modular design, allowing for the configuration of components to meet specific analytical requirements.
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4 protocols using 1200 liquid chromatography system
1
Vitamin D Deficiency Determination
2
Measuring Nucleotide Levels in Enzyme Reactions
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eATP, eADP, eAMP and eADO in enzyme reaction solutions of nondistended and distended preparations were measured using a reverse-phased gradient Agilent Technologies 1200 liquid chromatography system equipped with a fluorescence detector (Agilent Technologies, Wilmington, DE, USA) as described previously [9 (link),34 (link),82 (link)]. Areas of HPLC peaks were calculated and calibrated to individual etheno-derivatized purine standards of eATP, eADP, eAMP and eADO.
3
Quantification of 1,N6-Etheno-Derived Nucleotides
4
Peptide Stability Analysis via LC-MS
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10mM phosphate buffer (pH adjusted to 7.4 using 0.5M HCl or NaOH) was used to generate 1 mM peptide solutions. Peptides were incubated in a stationary heat block (37 °C), and samples were analyzed via LC-MS every 24h. 30 μL of the incubated solution was added to 270 μL of a 50% acetonitrile in water solution containing 0.1% FA. The analysis of samples was performed on an LC-MS system comprised of an Agilent 1200 Liquid Chromatography system coupled to an Agilent 6340 ion trap mass spectrometer. A Phenomenex Luna Omega column (C 18 , 50 × 2.1 mm) was employed to inject samples (4 μL) using a 2.5-95% acetonitrile in water (0.1% formic acid) gradient. A flow rate of 0.300 mL/min for 7.5 minutes was set-up, followed by a wash step with 95% acetonitrile for 1 minute.
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