Oasis hlb solid phase extraction column

Five-milliliter sterile syringes (BD), gavage needles (16-gage, 80 mm, curved needle), 1.5-ml/2-ml centrifuge tubes (Axygen, United States), 50-ml/15-ml centrifuge tubes (Corning, United States), 96-well cell culture plates (Corning, United States), 10 kD filters (Pall, United States), Oasis HLB solid phase extraction column (Waters, United States), 1-ml/200-μl/20-μl pipette tips (Axygen, United States), a BCA kit (Thermo Fisher Scientific, United States), high pH reverse peptide separation kit (Thermo Fisher Scientific, United States), and iRT (indexed retention time, BioGnosis, United Kingdom) were used.

The samples were lysed in lysis buffer (8M Urea in 50mM HEPES pH 8.0) and sonicated briefly. Samples were reduced with 10 mM TCEP and alkylated with 25 mM iodoacetamide. Protein concentrations were determined by BCA protein assay (Thermo Scientific). Fifty μg of protein was taken from each sample for digestion. Sequencing grade protease Lys-C was added with 1:250 ratio and sample was digested overnight at room temperature with mixing. A second digestion was performed by diluting the sample with 50 mM HEPES to lower the urea concentration to 1M and trypsin was added with 1:100 ratio for a further 12 hour digestion at room temperature with mixing. Digests were acidified with formic acid and subjected to Oasis HLB solid phase extraction column (Waters).

Endocannabinoids and oxylipins were isolated from 250 µL of plasma using solid phase extraction and quantified by liquid chromatography tandem mass spectrometry as previously described [1 (link)]. Briefly, plasma was thawed on ice, and mixed with deuterated endocannabinoid and oxylipin internal standards in the head space of 60 mg Oasis-HLB solid phase extraction column (Waters Corp., Milford, MA, USA), where they were up-diluted to 20% methanol/0.1% acetic acid, and gravity loaded onto the columns, followed by vacuum to remove solvent. The columns were then wetted with 0.2 mL methanol and gravity eluted with 1.5 mL ethyl acetate. Solvent was removed by vacuum and residues were reconstituted in 50 µL methanol containing the internal standard 1-cyclohexyl-3-dodecyl-urea (Sigma, Aldrich, St. Louis, MO, USA). The resulting samples were filtered and analyzed by UPLC-(ESI)MS/MS by back-to-back (+)-mode/(−)-mode injections for endocannabinoid and oxylipin levels, respectively.

The following equipment was used in this study:
A UPLC1290-6540B Q-TOF; a 1290 ULTRA high-pressure liquid chromatography system; a 6540B Q-TOF quadrupole tandem TOF-MASS spectrometry system with AJS, ESI, and APCI sources; a Mass Hunter workstation, and Metabolites ID software (Agilent Technologies, Ltd.) (Beijing, China).
A UPLC 1290-6470A ultrahigh performance liquid chromatography-triple quadrupole mass spectrometer equipped with an ultrahigh-pressure binary gradient pump, an ultra-efficient automatic sampler, an ultra-efficient column temperature chamber, and triple quadrupole mass spectrometer (Agilent Technologies Co., Ltd., Beijing, China).
A Waters UPLC (I-class) ultrahigh performance liquid chromatographer with a PDA detector (Waters Corporation, Milford, MA, USA) (Beijing, China).
A Waters Xevo TQ-S Micro with a Tipped XBOBbie Dynamic Range (XDR) detector and an electrospray ion source (ESI) source.
An Oasis HLB solid-phase extraction column (200 mg/3 mL; Waters Corporation, Milford, MA, USA).

AS1350 and the serum samples were pre-treated as described above: 4 μl phosphoric acid was added to a 200 ul sample and then vortexed for 60 s. The mixed solution was applied to a pre-actrbate Oasis HLB solid phase extraction column (Waters, USA) which was washed with 1 ml of methanol and 1 ml of water. Then, after 1 ml of 100% water was washed through, the mixed solution was eluted by 100% methanol and the elute collected and dried under a stream of nitrogen gas at 45 °C. Each dried sample was thawed in 100 μl of 80% methanol and centrifuged at 13,000 rpm for 10 min at 4 °C, and then filtered through a 0.22 μm membrane before UPLC/MS analysis.

In order to assess the stability of the SSRIs over the 5-day exposure period chosen for the static exposure water tank renewal period, a nominal concentration of 1.5 ng/mL of each SSRI was monitored over the 5-day period (n = 3 tanks per SSRI). The 20 mL samples were removed from 3 L tanks daily and the SSRIs were extracted using Oasis HLB solid phase extraction columns (Waters Canada, Mississauga, ON, Canada) following the manufacturer’s instructions. SSRIs were eluted from the columns with 100% methanol and eluates were analyzed on an ACQUITY UPLC BEHC18 column (2.1 × 100 mm, 1.7 µm) using an UltiMate^TM 3000 LC pump coupled to an Exactive™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The limit of quantitation (LOQ) was found to be 0.1 ng/mL. Mixtures were not tested for SSRI stability since loss (which is either due to degradation, or adsorption to tank material, or both) is assumed to be independent of the presence of other SSRIs. It was found that all three of the compounds were stable over the exposure period with the maximum reduction of 20% found for paroxetine at day 5 (Supplemental Scheme S1).

Cells were washed three times with cold PBS and then harvested using a cell scraper in lysis buffer (8M urea, 0.1 M Tris-HCl, pH 8.5) containing Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland). The lysates were sonicated and the supernatant was carefully collected after centrifugation at 14,000 g at 4°C for 10 min. The concentration of proteins was determined by Bradford assay. Extracted protein samples from heavy labeled NCI/ADR-RES cells and light labeled OVCAR8 cells in forward SILAC labeling, light labeled NCI/ADR-RES cells and heavy labeled OVCAR8 cells in reverse SILAC labeling were combined at a 1:1 ratio separately. In-solution digestion was performed with the following protocol. Protein mixtures were reduced with 10 mM 1,4-dithiothreitol (DTT) at 37°C for one hour, subsequently alkylated in 40 mM iodoacetamide (IAM) for 30 min at room temperature in the dark and then Lys-C was added and digested at 37°C for 6 hours. After diluting the concentration of urea to 1M with 25 mM NH₄HCO₃, sequence grade trypsin (Promega, Madison, WI) was added at an enzyme/protein ratio of 1:50 and digested at 37°C overnight. Digestion was stopped by adding formic acid to a final concentration of 1‰. The digested peptide mixtures were collected by centrifugation at 14, 000 g for 10 min. All digested products were desalted on Waters Oasis^® HLB solid phase extraction columns.