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A Hydrophilic interaction ultraperformance liquid chromatography (HILIC–UPLC) method for genomic DNA methylation assessment by UV detection

Sotgia, Salvatore and Zinellu, Angelo and Pisanu, Elisabetta and Pinna, Gérard Aimé and Deiana, Luca and Carru, Ciriaco (2010) A Hydrophilic interaction ultraperformance liquid chromatography (HILIC–UPLC) method for genomic DNA methylation assessment by UV detection. Analytical and Bioanalytical Chemistry, Vol. 396 (8), p. 2937-2941. eISSN 1618-2650. Article.

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DOI: 10.1007/s00216-010-3565-3

Abstract

A hydrophilic interaction chromatography-based method, in combination with 1.7 μm ethylene bridged hybrid particle packed column (100 mm×2.1 mm I.D.) and ultraperformance liquid chromatography, has been developed to measure cytosine (C) and methylcytosine (mC) in order to evaluate the extent of DNA methylation. Separation of cytosine and methylcytosine was achieved with good resolution and in fairly short times (5.5 min) by using isocratic elution with a mixture of 97:3 (v/v) acetonitrile/ 10 mM ammonium acetate as a mobile phase. The determination coefficients of C and mC were high (R2> 0.999) within the range tested. The %RSD for intraday and interday were respectively 2.2% and 2.5% for C and 3.5% and 3.8% for mC. The limit of detection was 0.52 μM (0.52 fmol on-column) both for C and mC while the limit of quantification was 1.72 μM (1.72 fmol on-column) both for C and mC. The smallest amount of purified DNA that yielded a measurable level of C and mC was 10 μg. On the whole, this method is simple, rapid, sensitive, and precise.

Item Type:Article
ID Code:5388
Status:Published
Refereed:Yes
Uncontrolled Keywords:DNA methylation, hydrophilic interaction chromatography (HILIC), ultraperformance liquid chromatography (UPLC), 1.7 μm ethylene bridged hybrid (BEH) particle
Subjects:Area 05 - Scienze biologiche > BIO/12 Biochimica clinica e biologia molecolare clinica
Area 03 - Scienze chimiche > CHIM/08 Chimica farmaceutica
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
002 Altri enti e centri di ricerca del Nord Sardegna > Porto Conte Ricerche, Alghero
002 Altri enti e centri di ricerca del Nord Sardegna > National laboratory of the national institute of biostructures and biosystems, Osilo
001 Università di Sassari > 01 Dipartimenti > Farmaco, chimico, tossicologico
Publisher:Springer
eISSN:1618-2650
Deposited On:03 Feb 2011 09:30

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