Simultaneous quantification of tetrahydrobiopterin, dihydrobiopterin, and biopterin by liquid chromatography coupled electrospray tandem mass spectrometry

A simple and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method was developed for the quantification of tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), and biopterin (B) in human umbilical vein endothelial cells (HUVECs). Freshly prepared cell samples were treated with a mixture consisting of 0.2 M trichloroacetic acid (TCA) and a cocktail of various antioxidants in order to precipitate proteins and other cellular components and to stabilize red/ox conditions in the lysates. Chromatography of the cell lysates was performed on a Poroshell 120 SB-C18 column (2.7 μm, 150 × 2.1 mm) using a stepwise gradient elution made from two mobile phases. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) at positive ion mode. Total chromatographic run time was 23 min. The method was validated for analysis in HUVECs, and the limits of quantification were 1 nM for BH4 and BH2 and 2.5 nM for B. Standard curves were linear in the concentration ranges of 1 to 100 nM for BH4 and BH2 and 2.5 to 100 nM for B. The current study reports a novel method for the simultaneous and direct quantification of BH4, BH2, and B in a single injection.