Empower chromatography data software

Urinary levels of major pteridine derivatives, including pterin-6-carboxylic acid, neopterin, biopterin, xanthopterin, isoxanthopterin and pterin, were measured by HPLC using an alliance HPLC system with a 2475 fluorescence detector and Empower chromatography data software (Waters Corporation, Milford MA, USA). The temperature of the column (COSMOSIL 5C18 packed column, 4.6 × 250 mm, Nacalai Tesque, Kyoto, Japan) was kept at 40 degrees. Each urine sample was diluted to a ratio of 1:10 with distilled water and filtrated with a 0.22 μm filter, and then an aliquot (5 μl) of the sample was injected into the HPLC system. Elution was performed at a flow rate of 0.8 ml/min using a solution consisting of phosphate buffer (10 mM, pH 6.5) and methanol at a ratio of 92 (phosphate buffer): 8 (methanol). Pteridines in an eluent were detected using fluorescence at excitation and emission wavelengths of 380 nm and 460 nm, respectively, and were quantified by using commercial standards of pterin-6-carboxylic acid (Sigma-Aldrich Japan, Tokyo, Japan), neopterin (Cayman Chemical), biopterin (Cayman Chemical), isoxanthopterin (Sigma-Aldrich Japan), xanthopterin (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan) and pterin (Sigma-Aldrich Japan).

ACQUITY^TM Ultra Performance Liquid Chromatography (UPLC) system equipped with ACQUITY^TM UPLC Photodiode Array Detector (PDA; Waters Corp, Milford, MA), and ACQUITY^TM BEH C₁₈ column (1.7 μm, 2.1 × 100) were used for UPLC analysis. In addition, Empower Chromatography Data software (Waters Corp, Milford, MA) was used to analyze the results. The sample was extracted with ultrasonicator (Branson Ultrasonics, Danbury, CT). The reagents, methanol (Junsei Chemical Co. Ltd., Tokyo, Japan), acetonitrile (BAKER, Centre Valley, PA), and water (tertiary distilled water) were all HPLC grade. The standard preparations were purchased from Extrasynthese (Genay Cedex, France) or Sigma-Aldrich (St. Louis, MO).