Hplc system

In plasma and isolated lipoprotein fractions, the content of Sa (d18:0), So (d18:1), sphinganine-1-phosphate (Sa1P; d18:0P), So1P (d18:1P), and Cer (d18:1 Cer) was determined by the HPLC method according to the previously described protocol by Min et al. [26 (link)]. Briefly, 250 μl of plasma samples were homogenized with ultrasonication in ice-cold water. Then, with the addition of internal standards (30 pmol C17-sphingosine-1-phosphate and 10 pmol of C17-sphingosine; Avanti Polar Lipids, Inc.; Alabaster, AL, USA) and acidified methanol, lipids from obtained homogenates were extracted by adding chloroform, 1 M NaCl, and 3 N NaOH. The upper alkaline aqueous phase containing Sa1P and So1P was then transferred into a new tube. The residual phosphorylated sphingoid bases in the chloroform phase were re-extracted twice with a 1:1 (v/v) solution of methanol and 1 M NaCl, and then all the aqueous fractions were combined. Next, into this combined solution we added alkaline phosphatase (Sigma Aldrich; Saint Louis, MO, USA), in order to dephosphorylate the sphingoid base-1-phosphates into sphinganine and sphingosine, which will enable indirect determination of their amount. To improve the extraction yield of released So, some chloroform was carefully placed at the bottom of this reaction tube. In the next step, the primary chloroform fractions containing free sphinganine and sphingosine as well as tubes with dephosphorylated sphingoid bases were washed with alkaline water and subsequently evaporated under a nitrogen stream. After that, the dried lipid residues were re-dissolved in ethanol and converted to their o-phthalaldehyde derivatives, which were transferred into separate inserts and determined by the use of HPLC system (Agilent Technologies; Santa Clara, CA, USA) equipped with a C18 reversed-phase column Omnisphere 5 (4.6 × 150 mm; Varian Inc., Lake Forest, CA, USA) and fluorescence detector.
Simultaneously, a small aliquot (50 μl) of the chloroform phase containing extracted lipids was taken from the primary tube and then was placed in a fresh tube containing C17-base (N-palmitoyl-D-erythro-sphingosine) as an internal standard.. Next, the samples were evaporated in a stream of nitrogen and then were subjected to alkaline hydrolysis in 1 M KOH in 90% methanol and heated for 60 minutes at 90 °C to deacylate ceramide into sphingosine. After that, the amount of sphingosine liberated from Cer was determined by means of HPLC as described above. Since the chloroform extract used for the Cer assay contains trace amounts of free sphingoid bases, the Cer concentration was corrected for the content of free sphingosine determined in the same sample. The datasets of sphingolipids concentrations obtained in our study are provided in the Supporting Information file S2 Data.
In brief, the HPLC technique was validated to ensure reliable and repeatable results in accordance with the International Council for Harmonization (ICH) guidelines. Peak height analyses included the measurement of the signal-to-noise ratio (S/N), with a threshold of three for the limit of detection (LOD) and an S/N > 10 for the limit of quantification (LOQ). For HPLC procedures, a blank and a placebo extract were applied as reference materials, known as a “cocktail” and retention marker solution, to ensure precise results in evaluating an analyte in the sample; the synthetic precursors, enantiomers, and excipients were applied to enhance the method’s selectivity. The standard deviation (SD) or relative standard deviation (RSD) was calculated from a suitable number of homogenous sample aliquots to establish a reliable technique. Multiple injections, at least five replicates, of the same reference solution were used to evaluate the precision, and the acceptable peak area precision value was required for the quantitative analysis of this method [27 ]. The average between-run variations (%CV) for the selected plasma sphingolipids measured by the aforementioned method are as follows: Sa 10.7%, So 3.9%, Sa1P 9.1%, So1P 5.8%, and Cer 0.8%. The within-run variations are as follows: Sa 9.8%, So 5.0%, Sa1P 8.2%, So1P 5.2%, and Cer 4.1%. The linear dynamic range of the above method for Sa and So is from 3 to 2000 pmol/ml of plasma, for Sa1P and So1P is from 6 to 2000 pmol/ml, and for Cer it is from 150 to 30,000 pmol/ml. The limit of detection in plasma is approximately 1 pmol/ml for Sa and So, approximately 2 pmol/ml for Sa1P and So1P, and approximately 30 pmol/ml for Cer [28 (link)].

The analysis of Sunset Yellow was performed using reversed-phase high-performance liquid chromatography in conformance with the method described in [48 (link)]. The studies were performed with the use of a Varian (Palo Alto, CA, USA) HPLC system equipped with a diode-array detector (DAD, type 335), high-pressure gradient systems composed of two pumps (type 210), and an autosampler module (type 410) with a column thermostat. Separation was obtained with a chromatographic column Gemini C18 (150 × 4.6 mm; 3 µm), connected with a pre-column Gemini C18 4 × 3 mm (Phenomenex, Torrance, CA, USA) using 0.02 mol/L aqueous solution of ammonium acetate with a pH of 4.5 adjusted by adding acetic acid (solvent A) and methanol (solvent B) at a constant flow rate of 0.6 mL/min. The gradient (v/v) was generated, decreasing from 85% of solvent A to 20% in 6 min, followed by increasing to 65% in 10 min and to 85% in 15 min. Before each injection, the system was stabilized for 5 min with the initial A/B ratio (85:15, v/v). Before use, the mobile phase was filtered through a 0.45 µm micro-pore filter membrane. The injection volume was 20 µL. Chromatograms were recorded at 484 nm and a column temperature of 30 °C. The concentration of Sunset Yellow in the sample was determined from the calibration curve equation plotted based on the analysis of standard solutions. The identification of Sunset Yellow was performed based on the retention time and the UV spectrum of the reference substance.

Lyophilized samples were dissolved in 100 µL water and centrifuged at room temperature for 3 min. The supernatant was transferred into a glass vial with micro insert. Ion-pairing-LC-MS measurement was performed by using an Agilent HPLC system, consisting of a degasser, a quaternary pump, and an autosampler [41 (link)]. The system was coupled to a Bruker micrOTOF mass spectrometer (Billerica, MA, USA). The chromatic separation was performed using a RP-C₁₈ column (3.5 mm × 150 mm × 4.6 mm) with a C₁₈ pre-column. The mobile phase composition was: (A) 5% methanol and 95% water, containing 10 mM tributylamine as ion-pairing reagent, 15 mM acetic acid for pH adjustment to pH 4.9 and (B) 100% methanol, as previously described [44 (link)]. The mass spectrometer operating in ESI negative mode was used over a mass range from 50 to 3000 m/z. Metabolite identification was carried out by a comparison of retention time and mass spectra with an in-house database and HMDB [45 (link)]. Quantification of metabolite signals was done by QuantAnalysis 2.0. For normalization, the internal standard camphor sulfonic acid was used. Detection limit was usually in the nmol range. The energy charge (EC) was calculated with absolute concentrations of ATP, ADP and AMP. For this purpose calibration curves were used. The curve fitting was done by a 1/x weighting using a quadratic calibration mode. The following equation [46 (link)] was used: EC = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]).

Samples were analyzed on Agilent HPLC system. Separation was carried out through column 20RBAX ECLIPSE, XDB-C18, (5 μm; 4.6 × 150 mm, Agilent USA) with UV-VIS Spectra-Focus detector, injector-auto sampler. Solvent A (0.05% trifluoroacetic acid) and solvent B (0.038% trifluoroacetic acid in 83% acetonitrile (v/v) with the following gradient: 0-5 min, 15% B in A, 5-10 min, 70% B in A, 10-15 min, 70% B in A are used for separation The flow rate was 1 ml/min and injection volume was 10 μl. Eleven standard compounds including rutin, myricetin, vitexin, orientin, hyperoside, isovitexin, isoquercetin, luteolin, apigenin, kaempherol, and luteolin-7-glucoside were run for comparative detection and optimized. The calibration curves were defined for each compound in the range of sample quantity 0.02-0.5 μg. All samples were assayed in triplicate.