Development of a capilary liquid chromatography method for determination of adenosine in stem cell culture supernatants

Abstract

A reversed phase capillary liquid chromatography (cLC) method using large volume injection (column switching) has been developed the determination of low concentration of adenosine in aqueous samples. The injection volume was 750 µL. Trapping was performed on a 0.3 mm i.d. x 35mm, 5µm Hypercarb column with a mobile phase (pH = 3.3) consisting of acetonitrile(10%), water (89.9%) and acetic acid (0.1%). Elution was performed on a 0.3 mm i.d. x 50 mm, 3 µm Hypercarb analytical column by using a 20 minutes gradient starting at 10% and increasing to 100% acetonitrile at pH 3.3. Quantification was carried out with a UV detector at 278 nm. The calibration curve was linear in the range 0.05-1ng /µL. The within day (n = 3) and the between-day (n = 3) precision of the retention time was in the range 0.6 - 1.2% and 0.6 - 1.1% respectively. The within-day (n = 3) and the between-day (n = 3) precision of the peak area was in the range 1.0 - 13.5% and 4.4 - 16% respectively. When cell culture supernatants were analyzed, several interfering compounds precluded the quantification of adenosine. Different approaches for removing the interfering compounds were investigated, including solid phase extraction, however, none of them was found to be successful.