Chromolith fastgradient rp 18e hplc column

Acyl-CoA measurements were made using a previously described method^{19 (link)}. HepG2 cells were plated in 10 cm plates (one million cells each) and cultured for 24 h. Cells were then treated with either 0.1% DMSO, 2 µM simvastatin, 2 µM hymeglusin, 2 µM simvastatin + 2 µM hymeglusin, or 2 mM Metformin. 24 h later cells were washed twice with cold PBS, before 1 mL of 0.3 M Perchloric acid was added. Plates were scraped and lysates transferred to 1.5 mL tube on ice. Stored at −80 °C. For mouse tissue preparation, ~65 mg of frozen liver was weighed out before adding 0.3 M perchloric acid at a ratio of 1 mg tissue/19 µL acid. A glass bead was placed in the tube and samples were disrupted using a Tissuelyser (Qiagen) set to 30 Hz for 2 min. Samples were transferred to a clean tube leaving the bead behind before being stored at −80 °C. The extracts are spiked with ¹³C2-Acetyl-CoA (Sigma, MO, USA), centrifuged, and filtered through the Millipore Ultrafree-MC 0.1 µm centrifugal filters before being injected onto the Chromolith FastGradient RP-18e HPLC column, 50 × 2 mm (EMD Millipore) and analyzed on a Waters Xevo TQ-S triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC system (Waters, Milford, MA).

Malonyl- and acetyl-CoA were extracted with 0.3 M perchloric acid and analyzed by LC-MS/MS using a method based on a previously published report by^{67 (link)}. The extracts were spiked with ¹³C₂-Acetyl-CoA (Sigma, MO, USA), centrifuged, and filtered through the Millipore Ultrafree-MC 0.1 µm centrifugal filters before being injected onto the Chromolith FastGradient RP-18e HPLC column, 50 × 2 mm (EMD Millipore) and analyzed on a Waters Xevo TQ-S triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC system (Waters, Milford, MA).

Liver acyl CoA esters were analyzed as previously described (93 (link), 94 (link)) by flow injection analysis using positive electrospray ionization on Xevo TQ-S, triple quadrupole mass spectrometer (Waters) employing methanol/water (80/20, v/v) containing 30 mM ammonium hydroxide as the mobile phase. Spectra were acquired in the multichannel acquisition mode monitoring the neutral loss of 507 amu. Heptadecanoyl CoA was employed as an IS. The endogenous CoAs were quantified using calibrators prepared by spiking liver homogenates with authentic CoAs (Sigma) having saturated acyl chain lengths C2–C18. Corrections for the heavy isotope effects, mainly ¹³C, to the adjacent m + 2 spectral peaks in a particular chain length cluster were made empirically by referring to the observed spectra for the analytical standards.
MalCoA was extracted with 0.3 M perchloric acid and analyzed by LC–MS/MS using a previously published method (95 ). The extracts were spiked with ¹³C₂-AcCoA (Sigma), centrifuged, and filtered through Millipore Ultrafree-MC 0.1 μm centrifugal filters before being injected onto a Chromolith FastGradient RP-18e HPLC column, 50 × 2 mm (EMD Millipore) and analyzed on a Waters Xevo TQ-S triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC system (Waters).