Ammonium dihydrogen phosphate

Ceftaroline dihydrochloride was kindly supplied by Pfizer Inc. Acetonitrile HPLC gradient (ACN) and methanol were purchased from Scharlau (Barcelona, Spain), and ammonium dihydrogen phosphate was purchased from Sigma-Aldrich Chemie Gmbh (Steinheim, Germany). The ultrapure water was obtained from the Mili-Q^® Plus apparatus (Millipore, Burlington, MA, USA).
Blank plasma from healthy donors, used to prepare calibration standard and quality control (QC) samples, was provided by the Basque Biobank (www.biobancovasco.org, accessed on 21 May 2021) and was processed following standard operation procedures with appropriate approval from the Ethical and Scientific Committees (Code CES-BIOEF 2020-34).
Plasma from critically ill patients was processed following a protocol previously approved by the Basque Clinical Research Ethics Committee (EPA2018019 (SP)). Samples and data from these patients were provided by the Basque Biobank (www.biobancovasco.org, accessed on 21 May 2021) and were processed following standard operation procedures with appropriate ethical approval.

The high-performance liquid chromatography (HPLC) system (Waters, Milford, MA) consisted of a series 600 controller, a series 717 plus autosampler, and a series 996 photodiode-array detector, connected to a cartridge column (GL Sciences, Tokyo, Japan; average particle size of 5 μm) and a Cosmosil packed 5C₁₈-MS-II column (Nacalai, San Diego, CA; internal diameter of 4.6×250 mm; average particle size of 5 μm). The mobile phase consisted of buffer A (2.5% methanol in 0.01 M ammonium dihydrogen phosphate, pH 5.3) and buffer B (20% methanol in 0.01 M ammonium dihydrogen phosphate, pH 5.1). Elution started with 100% buffer A and consisted of the following linear gradient steps: 0–10 min, 0–25% buffer B; 10–20 min, 25–40% buffer B; 20–60 min, 40–100% buffer B. A flow rate of 0.9 ml/min and an injection volume of 20 μl was used. Temperature of the column was maintained at 25°C. Detection was done at a wavelength of 260 nm. Deionised water used for preparation of the HPLC mobile phase and sample dilution was prepared with the Milli-Q purification system (Millipore, Bedford, MA). Nitrogenous bases, nucleosides, HPLC-grade methanol, and ammonium dihydrogen phosphate were obtained from Sigma-Aldrich (St. Louis, MO).

Propan-2-one, methanol, 1-butanol, LC-MS grade water, primuline yellow dye, ammonium dihydrogen phosphate, 3,5-Di-tert-4-butylhydroxytoluene (BHT) and ammonium formate were from Sigma-Aldrich (Saint Louis, MO, USA). Ethanol and high performance liquid chromatography (HPLC)-analytical grade chloroform (CHCl₃) were respectively from J.T. Baker (Center Valley, PA, USA) and Carlo Erba (Cornaredo, MI, Italy). N-lignoceroyl-D-erythro-sphingosine (Cer C24:0) 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC), Cardiolipin (CL), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE), D-glucosyl-ß-1,1′-N-stearoyl-D-erythro-sphingosine-d5 (HexCer), N-lauroyl-D-erythro-sphingosine, N-lauroyl-D-erythro-sphinganine, N-lauroyl-D-erythro-sphingosylphosphorylcholine N-lauroyl-D-erythro-sphinganylphosphorylcholine, D-glucosyl-ß−1,1′-N-lauroyl-D-erythro-sphingosine, D-lactosyl-ß-1,1′ N-lauroyl-D-erythro-sphingosine and C17 d-erythro-dihydrosphingosine-1-phosphate lipids standards were from Avanti Polar Lipids (Alabaster, Alabama, USA). SL standard mixture, containing SM and Sulfatides (SLF) was from Matreya LLC (Pleasant Gap, PA, USA).