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MB Sample ID: SA078302
| Local Sample ID: | Prednisolone-d0-P4 |
| Subject ID: | SU001204 |
| Subject Type: | Mammal |
| Subject Species: | Canis lupus familiaris |
| Taxonomy ID: | 9615 |
| Genotype Strain: | Beagle |
| Age Or Age Range: | 8 to 83 months |
| Weight Or Weight Range: | 12.2 to 18.9 kg |
| Gender: | Male and female |
| Animal Animal Supplier: | In-house breeding |
| Animal Feed: | Standard pellet/kibble maintenance diet |
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Sample Preparation:
| Sampleprep ID: | SP001212 |
| Sampleprep Summary: | Plasma lipids were extracted using a single-phase butanol/methanol extraction method (Alshehry et al, Metabolites 2015 with modifications). After thawing on ice, a 10 µL aliquot of each plasma sample was transferred into a 2 mL polypropylene tube (Eppendorf, Germany). Subsequently, 1 µL BHT (2,6-di-tert-butyl-4-methylphenol, 10 mmol/L in ethanol) and 90 µL of 1-butanol:methanol (1:1, v/v) containing internal standards was added to each sample. Following internal standards were added at 50 ng/mL final concentration in the extraction solvent: ceramide d18:1/17:0 (Avanti 860517P), Glucosylceramide (Matreya 1533), Lactosylceramide d18:1/16:0 D3 (Matreya 1534), LPC 20:0 (Avanti 855777P), LPE 14:0 (Avanti 856735P), PI 12:0/13:0 (Avanti LM-1500), PE 14:0/14:0 (Avanti 850745P), PS 14:0/14:0 (Avanti 840033P), SM d18:1/12:0 (Avanti 860583P), and at 100 ng/mL: DG 12:0_12:0 (Avanti 800812P), PC 14:0/14:0 (Avanti 850345P), TAG 16:0_16:0_16:0 d5 (CDN Isotopes D5815). Samples were then vortexed (30 sec) and sonicated in an ultrasound water bath for 30 min (20°C). After centrifugation (14,000 g, 10 min, 4°C), 90 µL of supernatant were transferred into 1.5 mL polypropylene tubes (Sarstedt, Germany) and dried under a nitrogen stream at 37°C. Dried extracts were stored at −80°C until LC-MS analysis. Dried samples were reconstituted with 90 µL 1-butanol:methanol (1:1, v/v) and sonication in an ultrasound water for 10 min. After centrifugation for 10 min at 20,800 g (4°C), supernatants (80 µL) were transferred into autosampler vials with glass inserts (Agilent Technologies, USA). For the analysis of sphingosine-1-phosphate (S1P), lipid extracts were derivatized according to the method described by Narayanaswamy et al. (Anal. Chem., 2014). To 50 µL lipid extract (see above), 50 µL 13C2D2–S1P d18:1 internal standard solution (Toronto Research Chemicals, Canada; 20 ng/mL in 1-butanol:methanol 1:1 [v/v]) was added. Derivatization was performed by adding 20 µL TMS-diazomethane (2 mol/L in hexanes; Acros Organics, Thermo Fisher Scientific, USA) and subsequent incubation for 20 min at 25°C and 700 rpm (Thermomixer, Eppendorf, Germany). To stop the reaction and inactivate TMS, 1 µL acetic acid 100% (glacial) was added. After centrifugation for 10 min at 20,800 g (7°C), supernatants were transferred into autosampler vials for LC-MS analysis. Process Quality Control (PQC) samples were generated by pooling equal volumes of plasma samples within an experimental group. For Blank samples, plasma was omitted. PQC and Blank were processed together with the study plasma samples with the same procedures. |
| Extraction Method: | Liquid–liquid extraction using butanol:methanol |
| Extract Storage: | -80℃ |
