Pre column securityguard

For the targeted analysis of 188 metabolites and their ratios, the AbsoluteIDQ p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) was used according to the manufacturer’s instructions. For further analysis, Agilent Zorbax Eclipse XDB C18, 3.0 × 100 mm, 3.5 μm with Pre Column SecurityGuard, Phenomenex, C18, 4 × 3 mm was used on a 1260 series HPLC (Agilent, Santa Clara, CA, USA) in tandem with a QTRAP 4500 (ABSciex, Framingham, MA, USA) mass spectrometer.
Lyophilized skin samples were thawed on ice, dissolved in 85% methanol/15% water according to their previous weight (15–25 μL added solvent) and pipetted to the filter plate along with 10 μL internal standards. The samples were derivatized using phenylisothio cyanate, dried, and metabolites were extracted using 40% methanol solution in water. All reagents were HPLC grade (Sigma Aldrich, Darmstadt, Germany).
The serum samples were thawed on ice, pipetted onto a 96-well plate (10 μL per sample) and derivatized using phenylisothiocyanate. A combination of flow injection analysis and liquid chromatography through a C18 column was used to determine the concentrations of metabolites.

AbsoluteIDQ p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) was used for the targeted analysis of 188 metabolites. An Agilent Zorbax Eclipse XDB C18, 3.0 × 100 mm, 3.5 µm with Pre-Column SecurityGuard, Phenomenex, C18, 4 × 3 mm was used on a 1260 series HPLC (Agilent, USA) in tandem with a QTRAP 4500 (ABSciex, USA) mass-spectrometer. The exact protocol is detailed in the user’s manual of the AbsoluteIDQ p180 kit. Briefly, the lyophilized samples were thawed on ice, the two lyophilized phases were both dissolved in 85% methanol/ 15% water according to their previous weight (15–25 μl of added solvent) and both phases were added to the filter plate of the kit. Subsequently 10 µl of internal standards were added. The samples were derivatized using phenylisothiocyanate, dried and metabolites extracted using 40% methanol in water. Acetonitrile, chloroform, formic acid (FA), methanol and water were all HPLC grade and purchased from Sigma-Aldrich (Germany).

An Agilent Zorbax Eclipse XDB C18, 3.0 × 100 mm, 3.5 μm with Pre-Column SecurityGuard, Phenomenex, C18, 4 × 3 mm was used with the AbsoluteIDQ p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) for the targeted analysis of 188 metabolites, measured on a QTRAP 4500 (ABSciex, USA) in tandem with a 1260 series HPLC (Agilent, USA). The exact protocol for the preparation of the samples is detailed in the AbsoluteIDQ p180 kit’s user manual. In short, the serum samples were thawed on ice, pipetted onto the included 96-well plate (10 μl per sample), internal standards added and derivatized using phenylisothiocyanate. The concentrations of numerous metabolites including acylcarnitines, biogenic amines, amino acids, hexose, sphingolipids and glycerophospholipids were determined using a combination of flow injection analysis and liquid chromatography through a C18 column.