Oasis hlb spe cartridge

Extraction of oxylipins from 500 µL human plasma was carried out as described [38 (link)]. Briefly, after addition of internal standard and antioxidant solution, samples were diluted with the same volume of methanol/water (5/95, v/v) acidified with 0.1% acetic acid, and extracted on Oasis HLB-SPE-cartridges (3 mL, 60 mg, 30 µm particles; Waters, Eschborn, Germany) using 0.5 mL methanol followed by 1.5 mL ethyl acetate for elution. LC-MS analysis of oxylipins was carried out as described [38 (link)]. Lipid mediators and deuterated standards used in this study were purchased from Biomol, Germany. Samples for oxylipin analysis were prepared within 1 day and analyzed within 3 days. In QC plasma samples, 74 analytes exceeded LLOQ and the intraday variance of most analytes (84% of quantified analytes) was <15%. Intraday variance for all 74 oxylipins was ≤30%. Concentrations of oxylipins are shown as pmol/L in plasma.

We added 900 μl of HPLC grade water to the 100 μl sample of 10 μl of hemolymph + 45 μl methanol + 45 μl iso-octane and then vortexed the solution. For the 20ul hemolymph samples we used 90 μl methanol, 90 μl iso-octane, and 800μl of water. We then used Waters Oasis HLB SPE cartridges (1cc cartridge Part # 186000383) in an extraction manifold (Waters Part# WAT200677) to separate the hormone-containing methanol layer from the iso-octane layer. Before adding our hormone samples, we primed the HLB cartridges with 1000 μl methanol and then 1000 μl HPLC grade water. We then added the 1000 μl hormone-containing sample to the HLB cartridge, but did not collect the elution. Then, we added 1000 μl of HPLC grade water to the HLB cartridges and also did not collect the elution. We repeated this step three times to wash the cartridge. We then added 50 μl of methanol to the HLB cartridge to elute the hormone from the cartridge. The elution was placed at -20°C until hormone measurement.

Serum and milk samples were analyzed for concentrations of ceftiofur, desfuroylceftiofur, and related metabolites by reduction and derivatization to desfuroylceftiofuracetamide (DCA) [14 (link)]. Ceftiofur and desfuroylceftiofur-related metabolites were extracted from serum and milk following a reduction step through the addition of 1,4-dithioerythritol solution (20 mg/mL in 0.1 M ammonium acetate, pH = 8.9). An internal standard consisting of a cefotaxime solution (200 μg/mL) was incorporated in this step. Following a 30-min incubation step at 50 °C to fully reduce the thioester bond in ceftiofur and desfuroylceftiofur-related metabolites, the resulting desfuroylceftiofur was captured on a C18 solid-phase extraction columns (Oasis HLB SPE cartridges, Waters, Barcelona, Spain) and further derivatized with iodoacetamide to create desfuroylacetamide (DCA). DCA was removed from the column with 30:70 acetonitrile: 0.01 M ammonium acetate with 0.1 % trifluoroacetic acid providing a final injection equal to isocratic HPLC conditions (15 % acetonitrile: 85 % 0.01 M ammonium acetate (0.1 % trifluoroacetic acid)). The HPLC separation was performed using a reverse-phase Kinetex ^TM PFP C18 column (250 × 4.6 mm; 5 μm) with an injection volume of 100 μL. Ultraviolet detector was set at 240 nm.

All solvents used for sample preparation were of HPLC grade and purchased from Sigma-Aldrich (Schnelldorf, Germany). All solvents used for sample analysis, water (H₂O), methanol (MeOH), and acetonitrile (ACN), were of LCMS grade and purchased from Sigma-Aldrich. Formic acid (≥96%) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). H₂O for SPE was purified on a Milli-Q system (Merck Millipore, Billerica, MA, USA). Iturin A2 (>95%) was purchased from Sigma-Aldrich and the stock solution (20 μg·mL⁻¹) was prepared in HPLC grade MeOH. Sodium bicarbonate (NaHCO₃), 98% ¹³C-labelled, was purchased from Sigma-Aldrich. Fluorescence derivatisation was achieved with AccQ-Fluor reagent WAT052880 (Waters; Milford, MA, USA). The mixture of fumonisin B₁ (49.9 μg·mL⁻¹) and fumonisin B₂ (50.6 μg·mL⁻¹) in H₂O/ACN (1:1, v/v) was purchased from Romer Labs (Tulln, Austria). External standards valinomycin and chloramphenicol were purchased from Sigma-Aldrich. Five different, 3 mL, 30 mg, SPE cartridges, Strata-X, Strata-SCX, Strata-WCX, Strata-SAX, Strata-MAX, were purchased from Phenomenex (Torrance, CA, USA). Three millilitre, 30 mg Oasis-HLB SPE cartridges were purchased from Waters.

Extraction of lipid mediators from 500 µL human plasma was carried out as described [23 (link),24 (link)]. In brief, the plasma samples (500 µL) were spiked with an internal standard solution. In order to extract the free oxylipins in serum, solid phase extraction (SPE) without subsequent protein precipitation was performed. In brief, SPE was carried out as follows: Oasis HLB-SPE-cartridges (3 mL, 60 mg, 30 μm particles; Waters, Eschborn, Germany) were cleaned with one column volume of ethyl acetate (EA) and one of methanol (MeOH). After the samples were loaded on preconditioned SPE columns, the columns were washed with two column volumes of 5/95 ACN/water acidified with 0.1% acetic acid. After drying the SPE column by applying low vacuum (0.2 bar), the analytes were eluted using 0.5 mL methanol followed by 1.5 mL ethyl acetate. The eluted samples were evaporated to dryness, reconstituted in 50 μL of methanol, and analyzed by LC-MS/MS as described [23 (link)]. Lipid mediators and deuterated standards used in this study were purchased from Biomol, Germany. Concentrations of lipid mediators are shown as pmol/L in plasma.